A Thesis Present.Ed to the Faculty of University of Manitoba in Partíal

ENERGY METASOLISM IN PROGRESSIVE MUSCIILAR DYSTROPHY-- STI]DIES ON OXIDATIVE PHOS?HORY].ATION OF GENETICALLY DETERMINED MUSCIILAR DYSTROPHY ]N MICE AND HAMSTERS.

A Thesis Present.ed to the Faculty of Graduate Studies and Research of the University of Manitoba

In PartÍal Fulfillment of the Requirements for the Degree of Doctor of Philosophy

Klaus trnlrogemann Department of Biochemistry, Faculty of Medicine

r969

c Klaus lnlrogemann 1969 1. TAsLE OF CONTENTS

PAGE

TABLE OF CONTENTS 2

LIST OF TABLES 9

LIST OF FIGURES T2

ACKNOT^TLEDGEMENTS t3

ABSTRACT 15

ORGANIZATION OF THE THESIS ... L9

GLOSSARY 20

ÏNTRODUCTION . 23

LITERATURE REVIEI^I 2B

Tíssue high energy phosphate content 29

Glycolytic capacity and glycolytic enz)¡me activity in dystrophÍc muscle .. 32

Studies on mitochondrial structure and functÍon in muscular dystrophy . 34 SLudies on oxídative phosphorylation in muscular dystrophy 31

MATERIALS AND METHODS .. 39

A. MATERIALS

(a) Chemicals 40

(b) Animals 44

B. METHODS

(a) Assessment of the stage of the disease 45

X. Mice . 45

Hamsters 45 Iß. 1. Age. 45

2. Body weight, heart weight, heart weight body weight ratio 45 -2- TABLE OF CONTENTS (CONT'D)

PAGB

J, Macroscopic description of heart and skeletal muscle pathology . 46

His topathology 46

5. Serum creatine phosphokinase determination . 47

6. Protein, non-collagen protein, collagen protein in skeletal muscle 48

7. Acid phosphatase activíty in skeletal mus cle 49

8. f'gLucuronídase activity in skeletal mus cle 49

9. Cathepsin activity in skeleËal muscle 50

10. DNA and RNA content in skeleËal- muscle 51 (b) Dissection of animals and preparation of mitochondria . 52

L. Iulouse skeletal muscle mitochondria . 53

2. Mouse skeletal muscle mitochondria prepared in the presence of albumin . ,. s4

J, Hamster heart mitochondr|a . s4

4. Hamster skeletal muscle mitochondria: Standard Proteinase Preparation ...... ;. 54

5. Hamster skeletal muscle mitochondria: Standard Proteinase Preparatíon wÍt.h l% albumin ... 55

6. Hamster skeletal muscle mitochondria: Modífied Proteinase Preparation .. 55 7. Hamster skeletal muscle mitochondria: Lochner Preparation .. 55

(.) Polarographic measurements 56

1. Apparatus 56

2. Measurements 56

-3- TABLE OF CONTENTS (CONT'D)

PAGE

3. Oxygen calibration .. 59 4. Materials added durÍng the polarographic experiments 59

5. Calculations 59

(d) Polarographic measurements in the presence of a hexokinase trap . 60

(e) Manometric measurements 62 t. Oxidative phosphorylation with pyruvate/malate as substrate . 62 ) Respiration rates with palmitate as substrate . 64

3. Respiration rates with palmityl-L- carnitine as substrate . 64

(f) Assay procedures ... 65 1. ProteÍn estimation o¿,. direct 65 indirect Iß. 65

2. NADH concentration 66

3. ADP concentration 66

4. Acetyl-L-carnítine concentration 67

5. Palmityl-L-carniiine coneentration 67

(g) Substrate preparations 67

1. Acetyl-L-carnitine. 67

2, Palmityi--L-carnitine .. 67

3. Potassium palmitate 67 (h) Tissue mitochondrial content determinations 68 (i) Electron microscopic examination of isolated mitochondrLa , 69

-/,+ TABLE OF CONTENTS (CONT'D)

PAGE

(j) Calculations and statistics ... 69

(k) Preparation of distilled water .. . .. 7A

RESIILTS 7L

Outline of the Results 72

A. OxídaÈive phosphorylatíon by skeletal muscle mitochondria from normal and dystrophic mice . 73

The effect of albumin . 80

Conclus ions B6

B. Oxidative phosphorylation by heart and skel-etal muscle mitochondria from normal and dystrophic hamsters 89

(a) HamsËer hearË mitochondrLa . 89

Characteristics of the dystrophic hamsters ... 90 Oxidative phosphorylation by heart mitochondria of dystrophic hamsters with a mean age of 110 days 90

Respiration with DL-ct-glycerophosphate and NA-DH as substrate 9B Oxidative phosphorylation by heart mitochondria from hamsters with a mean age of 160 days 1-05

The effect of a hexokinase trap L07 Oxidative phosphorylation by normal and dystrophic hamsËer mitochondria--isolation of the organelles by a different person 110 Oxidalive phosphorylation by heart mitochondria from normal and dystrophic hamsters more than 200 days old . IL4 The effect of higher reaction temperature LL4 1, With the standard substrate combination pyruvate/malaËe LL4 2. i{ith Dl-l3-hydroxybutyrate LLl Oxidative phosphorylation with palmityl-L- carnitine as substrate . L22

-5- TABLE OF CONTENTS (CONT'D)

PAGE

Mitochondrial yields . L24

Sunmary of the results obtaÍned from hamster heart mitochondria . .... L26

(b) Hamster skeletal muscle mitochondria . ... . L27

Oxídative phosphorylaEion by skeletal muscle mitochondria from normal and dystrophic hamsters with a mean age of 110 days ..., LZB

Respíration with Dl-ct-g lycerophosphate as substrate . 135

Respiration wÍth NADH as substrate .. 138

Tissue mitochondrial content of normal and dystrophic hamster skeletal muscle ... 138

Oxidative phosphorylation by skeletal muscle mitochondria from hamsters with a mean age of 160 days. Isol-ation of the organelles by a different person ..... 1"42

The effect of a hexokínase trap .. .. . L43

Oxidative phosphorylaËion by skeletal muscle mitochondria from severely affecled dystrophic hamsters. Comparison of different methods of mitochondrial preparations .. .. . L46

1. Standard Proteinase Preparation . .. L46 Recoupling of uncoupled oxidative phosphorylation in dystrophic hamster mitochondria by NI9CL2 L49 ) Standard Proteinase Preparation withalbumin. .L54

J. Lochner Preparation .. L57

Comparison of polarographic and manomeEric techniques for the determination of oxidative phosphorylation parameters ... .. L66 Oxídative phosphorylation by hamster skeletal muscle mitochondria prepared by the Modified Proteinase Procedure .. LlL

-6- TA3LE 0F CONTENTS (CONT!D)

PAGE

Reproduction of the manometrÍc experiments of Lochner using hamsters with a mean age oÍ. 2L5 days L7 4 Respiration with palmitate and palmityl-L- carnitine as substrate . L75 Confirmation of the manometrically detected mitochondrial respíration defect by polarographic techniques ... L79

A single or a t\'/ofold defect? LB2 The localízatíon of the respiration defecl t84

1. Impaired respiration wiLh acetyl-L-carnitine LB4

2. Normal respiratíon with succinate 185

3. Normal respiration with NADH . T87 Is the observed mitochondríal respiration defect also present in viri-g? 189 Studies on the tissue compositíon of normal and dystrophic muscle L92

1, Collagen, non-collagen protein, DNA, RNA L92

2. Hydrolytic enz)¡rne activities ... 194

Sunrnary of the results obËained from hamster skeletal muscle 198

DISCUSSION ... 200

On theoretical AnP/0 ratios and the relation between ADP/O and respiratory control ratios .. zOL

Oxidative phosphoryLaËion by skeletal muscle mitochondria from normal and dystrophic mice .. 208 Oxidative phosphorylation by heart mitochondria from normal and dystrophic hamsters ...... 2L0

Oxidative phosphorylation by skeletal muscle mitochondria from normal and dystrophic hamsters ... 2L4

-7 TABLE OF CONTENTS (CONT'D)

PAGE

LIST OF REFERENCES .,. 222

APPENDIX 247

-B- II. LIST OF TABLES

TABLE PAGE

Characteristics of dystrophic mice and their littermate controls ... 74 Characteristics of mice used in the preparation of mitochondria in the presence of albumin ... 75

3a RespÍration and oxidative phosphorylation by mouse muscle mitochondría . 79

4a Respiration and oxidative phosphorylation by muscle mitochondria prepared from 4 control mice and the effect of ATP 81

5a Respiration and oxidative phosphorylation by mouse muscl-e mitochondría prepared in the presence oî. L% albumin B3

6a Yield of mouse muscle mitochondria prepared in the presence of. L% albumin 87

7a Characteristics of the hamster Group t ... 9L

Ba Respíration and oxidative phosphorylation by heart mitochondria from hamsters of Group I (subsÈrate added prior to firsE ADP) 93

9a Respiration and oxidative phosphorylation by heart mitochondria from hamsters of Group 1 (indígenous substrate depleted) 97

10 Multiple comparison of the means of the different periods of Tables 8 and 9 99

lla Respiration by heart mitochondria from hamsters of Group I (substrates: DL-ct-glycerophosphate and NADH) 100

L2a Characteristics of the hamster Group 2 ... 106

l3a Respiration and oxidative phosphorylation by heaÉ mitochondria from hamster Group 2 . . . 1-08

L4a Characteristics of Ëhe hamster Group 3 ... 111

15a Respiration and oxidative phosphorylation by heart mitochondria frsm hamsters of Group 3 ... 113

L6a Characteristics of the hamster Group 6 .., 1l-5

-9- Lrsr 0F TABLES (CONT'D)

TABLE PAGE

L7 a Respiration and oxidative phosphorylation by heart mitochondria from hamsters of Group 6 (substrate: pyruvate/malare) 116

18a Respiration and oxidative phosphorylation by heart mitochondria from hamsters of Group 6 (substrate: Dl-p-hydroxyburyrare) ll8

L9a Respiration and oxidative phosphorylation by heart mitochondria from hamsters of Group 6 (substrate: palmityl-L-carnÍtíne -l- malate) L23

20a Yield of hamster hearr mitochondría . LZs

ZLa Respíration and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 1 (substrate added príor to first ADp) 131

22a Respiration and oxídative phosphorylation by skeletal muscle mitochondria from hamsters of Group 1 (indigenous substrate depleted) 133

23 Multiple comparison of the means of the dífferent periods of Tables 2L and 22 L34

24a Respiratíon by skeletal muscle miËochondria from hamsters of Group I (substrates: Dl-ct-glycerophosphate and NADH) 136 25a Succinic dehydrogenase activíty and tissue mitochondrial content in skeletal muscle of hamsters of Group I 140 : . 26a Yield of hamster skeletal muscle mitochondría , , (Proteinase Preparations) I4I

27 a Respiratíon and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 2 ... L44 2Ba , i;:T:i:i';T":î:;îl::;:ff.T1';lH'il*:l::." of Group 3 (Standard Proreinase Prepararion) I47

29a Respiration and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 3 (Standard Proteinase preparation with 1% albumin)..... 156

-10- LTST OF TABLES (CONTID)

TABLE PAGE

30a Respiration and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 3 (Lochner Preparation) L63 3la YieId of hamster skeletal muscle mitochondria (Lochner Preparation) 165

32a CharacterisEícs of the hamster Group 4 ... L67 33a Respiration and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 4 ... 168

34a Characteristics of the hamster Group 5 ... L72

35a Respiration and oxidative phosphorylation by skeletal nnrscle mÍtochondria from hamsters of Group 5 ... L73

36a Respiration and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 6 (Manometric experiments) L76

37a Respiration and oxidative phosphorylation by skeletal muscle mítochondria from hamsters of Group 6 (substrate: pa1-mityl-L-carnitine) 180

38a Respiration and oxidative phosphorylation by skeletal muscle mitochondria from hamsters of Group 6 (substrate: pyruvate/malate) l-BI

39a Respiration rates with succinate and NA-DH by skeletal muscLe mitochondria from hamsters of Group 6 ... 186

40a RNA, DNA, and protein content in skeletal muscle hamsters of Group 6 ... I93

4La Lysosomal enz)rme activities in skeletal muscle of hamsters of Group 6 .. . W5

- lt - III. LIST OF FIGURES

FIGIIRE PAGE I Schematic representation of a typicaL polarograp hi c experiment 5B

2 PoLarographic records of oxygen consumption of mice No. 16 and 17 7B 3 Electron mícrographs of normal and dystrophic mouse skeleLal- muscle mitochondrla . 85 4 Electron micrographs of normal and dystrophíc hamster heart mitochondria . 95

5 Oxidation of NADH as a function of rhe concentration of the initial heart muscle homogenate .,. ... LO4

6 Polarographic records of oxygen consumption of heart mitochondria from hamster No. 77 with Dl-/3-hydroxybutyrate as substrate LzL 7 Electron micrographs of normal and dystrophic hamster skeletal muscle mitochondría . .... 130 8 Polarographic records of oxygen consumption of normal and dystrophic hamster skeletal muscle mitochondria and the ef f ect of lvþCl Z ...... L5Z 9 ElecËron mícrographs of normal hamster skeletal muscle mitochondria. Comparison of the Lochner PreparaËion with the Standard Proteinase Preparation .. .., 160 10 Histopathology of normal and dystrophic hamster skeletal muscle ... LgL

11 Scatter plots of ADP/O versus RCR and ADP/O versus l/nCn .... 203 L2 Theoretical and experimental relation between ADP/O and RCR ..... 206 13 Hypothesis on the sequence of events in the dystrophy process in Syrian golden hamsters of strain BT0 14.6 ...... 2ZO

-L2- IV. ACKNOIÁTLEDGEMENTS

It has been my privilege to have had the guidance and supervision of Dr. Marcel C. Blanchaer Èhroughout the work of this thesis. His constant interest and guidance and the fact that he was available at any time r¡¡ere of invaluable help f or me.

I wish to thank lvlr. Brian Holl for his inspiring companionship and good spirits in joint experiments.

I wish to thank Miss Beryl Jacobson for her expert technical assistance and advice and for the Lireless efforts in typing the rough drafts of the thesis.

I wish to thank Dr. Nenman L. Stephens for his help with the statistical treatment of the experimental data and for his interest in discussing various experímental problems.

I am greatly Índebted to the Department of Pathology, University of Manitoba, for the generous help in preparing the histological slides, examining the mitochondrial preparations r^Iith the electron microscope, and preparing the photographs for this thesis. In partÍcular I am grate- ful to Dr. Drunnnond H. Bowden, Dr. Catherine E. Thomas, Dr. Ian Y.R.

Adamson, Miss l{artha M.J. Melville, Mrs. Erika Neuendorff, Miss Sabine

Gagelmann, Miss Charmaine Hedgecock, Mrs. Carolyn MaGee, and Mr. Gordon

McLaren.

I wish to thank Mr. Michael Rosenbluth and Mr. Siewnarine Siewsankar for their technical assistance.

I am indebted to many mernbers of the Department of Biochemistry for their advice and heI-p during the course of this project.

-13- I gratefully acknowledge the help of the Computer Department for HeaIth Sciences, University of Manitoba.

The Muscular Dystrophy Assocíation of Canada generously provided a Postdoctoral Fellowship and further financial support through grants- in-aÍds.

-L4- V. ABSTRACT

The literature has been reviewed in respect to energy metabolism in muscular dystrophy. From this it seemed possible lhat an impairment of energy production might play a primary role in the dystrophy process.

Therefore oxidative phosphorylation \Àras studied in isolated mÍtochondria from skeletal muscle of dystrophic mice and from heart and skeletal muscle of dystrophic hamsters. Oxidative phosphorylatíon was first studied in mitochondría from normal and dystrophic skeletal muscle of mice of the Jackson Laboratory slrain L29/Re. The dystrophic animals, aged 37 -77 days, were in a moderately advanced stage of the disease. The sEudy revealed no differences between the diseased and control animals in any of the parameters studíed.

These were the rate of oxygen uptake (0rrate), the ADP/O ratio, the respiratory control ratio (RCR), and the phosphorylation rate (f rate). Bovine serum albumin increased the ADP/0 and respiratory control raLios, but the values for the control and dystrophic preparations again did not díffer significantly in the presence of albumin. It was concluded that an abnor- rnality in mitochondrial oxidative phosphorylation per sg could not be regarded as a primary cause of the disease. However, the possibility \.{as considered that mitochondrial oxidative phosphorylation in vivo m:ight be impaired due to a decreased amount of substrates from glycolysis being available for the mitochondria.

Oxidative phosphorylation was also studied in isolated heart mitochondria from rrormal and dystrophic hamsters of the strain BI0 14.6.

All dystrophic animals were affected by the disease, as confirmeà by macroscopic pathology, hístopathology, and determinations of serum creatine

15- phosphokinase acEivities. The dystrophic animals, aged f.rom 75-275 days,

generally were in a stage with true heart hypertrophy, but none was in

termÍna1 congestive heart failure. Mítochondria from these animals were

of excellent quality, as shown by electron micrographs, low respiration

rates with NADH as substrate, and high values of all the oxidaËive phosphorylation parameters. Mitochondria from dystrophic hearts were indis- tínguíshable from the control organelles ín any of the parameters tested.

This was true both at ZBOC and 37oC and with different substrates, including pyruvate/L-malate, DL-oc-glycerophosphate, DL-¡3-hydroxybutyrate, and palmityl-L-carnitine. Normal oxidatíve phosphorylation was also observed in the dystrophic animals in the presence of a glucose-hexokinase trap for high energy phosphate. The mitochondrial yield was similar in normal and dystrophic animals only ín the youngest hamsters. From older dystrophic animals significantly less mitochondria r¡rere isolated than from the control animals. Since considerable subjective judgement is requíred during the isolation procedure, it was gratÍfying to find that the above results could be reproduced by persoris other than the author. Nevertheless, under a variety of conditíons, oxidative phosphorylation by dystrophíc heart mitochondrÍa gave no indication at all of a mitochondrial def ect. However, very young animals r¡rere not studied, and the possibility remains Ehat a mÍtochondríal defect in early life d.ue to a magnesium deficiency might pLay a role ín the process of the hamster cardíomyopathy.

Hamster skeletal muscle mitochondría were studied in 91 anímals.

The dystrophic hamsters, ranging in age fuom 75-275 days, all were aff ected to various degrees by the disease. Depending on the age of animals, the severity of disease, the isolation techniques, and on the methods used to

-L6- measure oxidative phosphorylation, the results differed considerably. However, normal oxidative phosphorylation parameters !Íere obtained by at Least one method in each animal group studied, except in very old animals. The most surprising finding was that with certain dystrophic animals, normal as well as abnorur,al results could be obtained, either by using different isolatíon procedures for the organelles or different techniques to measure the oxidatíve phosphorylation parameters. These contradicting findíngs raise some doubts as to the relevance of such in vitro experiments to the in vivo situation.

trtlhile normal oxidative phosphoryl-ation parameters r¡7ere obtained r,¡ith most of the dystrophic animals, old dystrophíc hamsters exhibiËed a defect of respiration with palmitate, palmityl-L-carnitine, acetyl-L- carnitine, and pyruvate/L-malate as substrates, although respiration with succinate and NADH was normal. The defect(s) was (were) detected by both manometric and polarographic techniques. It seemed likely that a defect in the conunon pathway for the above 4 substrates could explain all the observed abnormalities. The most líkely site for such a defect would be in the tricarboxylic acid cycle. However, since this defect was observed only in very old animals, it is not regarded to be of primary importance in initiating the disease process. once agaín it is possíble that the resul-ts actually did not reflect the in vivo situation, but were caused by the changes in tissue composition of the dystrophic muscle, with many

ínfiltrating cells rich in lysosomal enzymes. The latter could have adversely affected the quality of the mitochondria during the isolatíon procedure.

In several groups of younger dystrophic hamsters another defect was found in which all parameters of oxídative phosphorylatíon \¡rere

-L7 significantly decreased, when measured by standard polarographic

technique, The defect was undetectable, however, when Ëhose mitochondrial preparations T¡/ere tested polarographically or manometrically

in the presence of a hexokinase trap. rt seems likely that magnesíum added with the hexokinase trap to the test medium lras responsible for

the difference. In a few experiments it actually was shown that lvISClr: when added to the standard polarographic reaction medium, normaLLzed, the oxidative phosphorylation parameters. This finding suggested. a hypothesis on the sequence of events in the dystrophy process of Br0 14,6 hamsters which could serve as a guide for future stud.íes.

-18- VI. ORGAN]ZATION OF THE THESTS

The main body of the thesis ís divided into three chaprers: Literature Review, Resu1ts, and Discussion.

The Literature Review deals only wíth aspects directly related to energy metabolism in muscular dystrophy. Publications on oxidative phosphorylation in muscular dystrophy are mentioned in this section, but are crÍtically evaluated in the Discussion. The Results contaÍn the experimental observations, usually ín the sequence in which they were actually made. For reasons of ci-arity, the tables in this chapLer present only the mean values of parameters obtaíned and their statistical evaluation. These tables are designated with anrtarrafter the table number. The individual data of the e:cperi- ments surnrnarized in these tables are listed in tables which are identi- cally numbered, but are designated wÍËh trbrr, in the Appendix, page 24L.

Terms and abbreviations appearing in the tables and text are explained in the Glossary, page 20.

In general, Ëhe significance of the results of each set of experíments is discussed under Results, inrnedÍately after Ëhe findings are reported. The formal Discussion is therefore confined to the principal findings. VÏI. GLOSSARY

ADP- Adenosine-5' -diphosphate

ADP/O- The ratio of molecules of ADP esterfied per atom of oxygen utilized. It is determined polarographically, and under ideal experimental conditions is equal to the P/0 ratio.

llo(rl - The level of statisEical significance. A critical level of c¿ =0.05 has been chosen. I ATP- Adenosine- 5 -triphosphate

CoA- Coenzyme A

CPK- Creat ine phosphokinas e

DNA- Deoxyribonucleic acid

DNP- 2 ,4-dlni-trophenol

DTT- Dithiothreitol (C1-e1-and's Reagenl)

EDTA- Ethylenediamine tetraacetíc acid

GDP- Guanosine - 5 ' -diphosphate

GTP- Guanos ine- 5 r -triphosphate

Heart Disease- A table heading under rvhich the macroscopÍc pathology of the cardiomyopathy is summari-zed, using an arbitrary scale from 0 (no sÍgns present) to 4t (severe hypertrophy with some indication of congestion). lrNtl- Number of animals

NAD- Nicotinamide adenine dinucleotíde

NADH- Nicotinamide adenine dinucleoËide (reduced) N.S.- Not significant (p>-0.05)

0rrate- Respiration rate of standard polarographic or manometric experiments expressed infmoles 02 per min. per g protein. 02 Orate- Respiration rate of polarographic experiments in the presence of a hexokinase trap.

-20- rf prf _ Itprobabilityrr that the means of the two sample populations under comparison are equal. A value of p<0.05 is regarded as statistical evidence that the tT¡ro means under comparison are signif Ícantly diff erent.

PCA- Perchloric acíd Period- In this thesis a cycle of State 3 - State 4 respiration of polarographic experiments.

Pi- Inorganic phosphate 32p/ o- The molecules of phosphate esterified to ATP per atom of oxygen utilized, when determined polarographically with radioactive phosphorus ín the presence of a hexokinase trap.

P rate- Phosphorylation rate measured in standard polarographic or manometric experiments. Phosphorylation rate is a measure for the capacity of mitochondria to produce ATP (132), expressed as ¡r,moles ADP phosphorylated per min. per g protein. I 32P ,^t"- The phosphorylation rate (P rate) obtained in polarographic experiments Ín the presence of a hexokinase trap.

RCR- Respiratory control ratio. The ratio of respiration rates of State 3 to following State 4, calculated according to Chance and WíllÍams (128).

RNA- Ribonucleic acid S.E. - Standard error of estimate

SDH" Succinic dehydrogenas e

State 3- Respiration raLe in the presence of phosphate accêptor (t28)

State 4- Respiration rate in the absence of phosphate acceptor (128)

Streaks - A table heading under which the amount of necrosis in hamster skeletal muscle is surnrnarLzed, using an arbitrary scale from 0 (no streaks) to 4+ (several muscles completely necrotic) tttll - t-value obtained from t-tests (L49)

-zL- Tris- Tri s (hydroxyrnethyl ) aminomethane rrlrr - Arithmetíc mean value (f49)

-22- VIII. INTRODUCTION -24-

VIII. ]NTRODUCTÏON

The term tdystrophia muscularis progressivat, progressive muscular dystrophy, ù/as introduced by Erb in l8B3 (I) and r8B4 (2) to

distinguish a group of priunry myopathies from progressÍve muscular atrophies of spinal origin. Today the definition of muscular dystrophy as a "genetically- determined, primary, degenerative myopathytr proposed by Walton in

1958 (3), has been widely accepted. As the term suggests, it covers a group of ínheritable diseases, the principal finding of which is the occurrence of muscle wasting. Thís is not caused by neurogenic or nutritional defects, but the degenerative process develops primarily in the muscle fibers themselves (4), There are wide differences among muscular dystrophy cases in respect to the mode of inheritance, time of onseL of the disease, the preference of primarily affected muscle groups, and in the rate of progress of the disease (5). trrIalton has classif ied the varieties of progressive muscular dystrophy on clinical as well as on genetic grounds (6).

I shall only mentionthe mosË contrnon and most lethal type, the

Duchenne type muscular dystrophy. It is characterLzed by an early onset of the disease--usually during early childhood--with progressive muscular wasting (4). Muscle fibers become necrotic and then are replaced by connective tissue and fat (7), the latter giving rise sometímes to the false impression of hypertrophic muscles (6). Therefore the s¡monym pseudohypertrophic dystrophy is often used instead of Duchenne type (6).

As the disease progresses physical disabilíties become more prominent, the connective tissue tends to shrink, sometimes causing bízarre skeletal -25- distortions and muscle contractures. Death usually occurs in the second decade from respiratory infecËion or cardiac failure (6), since the heart is also involved in most, Íf not all cases of Duchenne type of muscular dystrophy (8, 9).

The cause of the disease is virËually unknown. As Pearson stated it during a recent syurposium: tttrrle do not have so far a clear clue about which of the many complex systems of the muscle fiber is principally involved in the dystrophíc díseasesr' (10).

Since there is no therapy avaÍlable which definitely has proved to be effective (11) the fate of these patients is desperate.

In Canada about 5,000 people are suffering fromrtruetmuscuLar dystrophy, most of them children who are affected by the fatal Duchenne type (12).

Muscular dystrophy therefore is also a great socíal problem, which may explain the generous supporL of research by muscular dystrophy associ- ations in all major countries of the western hemisphere.

Although it is generally not feasable to study human muscular dystrophy directly because of the difficulties in obtaining dystrophic t.issue, researchers in the field are fortunate no\À7 to be able to study a variety of animal muscular dystrophies, These generally show many features in common with human muscular dystrophy (13, 14) , alËhgugh it is impossible at present to decide røhether the genetÍc defect in all these muscular dystrophies is the same (15).

For many investigators the laboratory animal of choÍce is the dysErophic mouse, which was first described by Míchelson et al ín 1955 (16)

ThÍs dystrophy arose as an apparently spontaneous mutation in the inbred strain L29 of. mice of the Jackson LaboraLory, Bar Harbor, Maine(15). Unfortunately, certain biochemical studies could not be performed with -26-

these animals because of their small muscle mass. This problem has been partially solved by lhe discovery of a genetically determined muscular dystrophy in the chicken ín 1956 (30), of a genetically determined polymyopathy in the Syrian golden hamster ín L962 (14) and of hereditary muscular dystrophÍes in the white Pekin duck in 1961 (I7 , 18) and in the domestic turkey in 1967 (19). Hereditary myopathies of the dystrophy type have also been reported in the goat, dog, cow, and kangaroo (20). In contrast to .n"Z#.TTtiå"ne type muscutar dystrophy, which is and usually sexlinked,/recessively t.ransmitted (5) , all the above mentioned animal dystrophies seem to be of autosomal Ínheritance, which is recessive in the mouse (f5) , chicken (2L), hamster (f3) , and turkey (19) and possibly autosomally domÍnant in the ducirc (22).

I{hile many authors point out the similarities between human and animal muscular dystrophies (L4, L3,23), others stress that exact

rcopiest of the human disease in anímals are unlikely (24,25). In spite of thís reservation the elucidation of the disease process in one specÍes might well cast light on the mechanisms involved in other species and in human muscular dystrophy as well (20, 27, 26). It is not a very original idea to think that in a disease vrith muscular r^Teakness and muscle wasting an impairment of energy production might be involved Ín the mechanism of the disease. There is ample evidence--direct and indirect--in the literature that there exist alterations of energy metabolism. However, many of these reports are at variance with others, as r¿ill be pointed out in the folLowing chapter.

In view of the uncertainties in the literature, as to whether a defect in energy production could have a causal relationship to the disease process, an extensive investigation of energy metabolism in _27 dystrophic muscle was undertaken.

The main fuel sources for heart and skeletal muscle are fatty acÍds and carbohydrates (28). Since all the energy from fatty acÍd oxidaLion and up to 95% of the energy from carbohydrate utilization is produced in the mitochondria (29) , the topic of this study narrows down to the question: rrls there a defect ín mitochondrial oxidative phosphorylation in muscular dystrophy?rr This simplificat.ion, of courseris only justifiable under the assumptíon that an inadequate substrate supply for the mitochondria (e.g. from glycolysis) ís not the cause of the possible defect ín energy production.

In spite of the broad title of the thesís, the experimental part of this study will therefore deal only with mitochondrial oxidative phosphorylatíon which was studied in skeLeLal muscle of dystrophic mice of strain 129 (16) and in both heart and skeletal nn¡scle of Syrian golden hamsters of straÍn BI0 14.6 (13). The ínformation available on this subject in the literature at the time Lhis study \^7as started as well as more recent reports witl be summarized in the followíng chapter. IX. LITERATTIRE RNVIEtr{ -29-

IX. LITERATI]RE REVIEI^I

In this chapter I shaLl review the literaËure on direct aspecËs of energy metabolism in muscular dystrophy, i.e. glycolysís and mitochondrial oxidative phosphorylation. Information on the ultrastructure of mÍtochondria, steady state levels of high energy phosphates, and tissue activities of enzJ¡mes of energetic pathways are included.

Other biochemical clianges, whÍch ultimately might also be caused by a disturbance of energy production will not be mentioned. Since workers in Lhe fÍeLd of muscular dystrophy study a great variety of human rmrscular dyst.rophies as well as several species of the animal kingdom, one is always tempted to draw analogies between the different species and to assume findings in one specíes apply to the disease of others. This is certainly bound to lead ín some instances to erroneous conclusions. However, in view of lhe relatively little information available on each of the various types of muscular dystrophy, findings on energy metabolism in the ttrue' human muscular dystrophies

(4) and the muscular dystrophies in animals (see Introduction) witl be presented together.

Tissue hieh enersy phosphate content

Because of their central role in cellular energetics, labile phosphorylated compounds, such as phosphocreatine and adenosíne triphosphate (ATP) have been studied extensively in rmrscular dystrophy (31). As early as 1936 CoLLazo et al (32) had reported an average decrease of 30% of phosphagen (phosphocreatine) in skeletal muscle of 6 patients with -30-

Duchenne type muscular dystrophy. In t93B Reínhold et al (33) confirmed these results in an extensive study on the muscle composition oî. 2L patients with progressive muscular dystrophy. Apart from decreased creatine phosphate concentrations, they also found the ATP levels of muscle to be decreased. This could be explained only partially by the infiltration of fat tissue, since decreased values were also observed, when expressed on a fat-free basis. The authors also tried to assess the contribution to the muscle weight by infiltrative connective tissue.

I{hen the phosphorus results \¡zere corrected for the presence of fat and connective tissue, the labile phosphorylated compounds were still lower in dystrophic muscle. However, the authors conceded that their estimatíon of the connective tissue by histological examination needed improvement by suitable quantitative measurements.

Thus, the problem of a lack of an absolute reliable reference base for the expression of various constituents in dystrophic muscle has been known for a long time. Recently it has become widely accepted to exPress the results per nortcollagen nitrogen (34, 35-37). tr^Ihen thís is done, many of the abnormalities in composition of dystrophic muscle become less striking and, indeed, may disappear (31). Thus Bonetti et al found

ATP concentrations in human dystrophic skeleËal muscle decreased when expressed per fresh muscle weight, but no differences between normal and dystrophic tissue concentrations \,,r'ere present when the results \,{ere expressed per noncollagen nitrogen (35). Ronzoni et al found significantly decreased phosphocreatine content in human dystrophic muscle, but normal

ATP concentratíons using chromatographic techniques for the separation of the nucleotide (37). In view of the reversíbi1íty of the creatine phosphokinase (38) reaction " one is surprised to see a decrease of phosphocreatine -31 -

without a concomitant decrease of ATP concentration. However, from the measured equilibríum constant of the creatine phosphoryl transfer

reaction in nmscle (39) one actually expects and finds a much greater

decrease of phosphocreatine than ATP in a situation of decreased energy production (40-42).

Zymaris detected decreased ATP levels in dystrophic mouse

muscle, which probably represented a rtruet finding, since t.he concen-

trations were expressed on a noncollagen basis (36). This decrease

apparenLly \^/as not due Ëo increased activíties of myof ibritlar ATpase, adenylate kinase, or adenylate deaminase (43). Lochner et al studied the high energy phosphate content in

skeletal muscle (44) and hearts (45) of dysËrophic hamsrers. The ATp and phosphocreatine levels \,/ere not different from normal in several skeletal

muscles. Hor^rever, in the m. biceps f emoris signif icantly less ATp was

found in dystrophic animals, but the same muscles contained more phospho-

creatine, a fÍnding which was significant at the 0.5% level. Thus, the

total content of high energy phosphates in this muscle definitely r¡zas not decreased. However, it is difficult to understand the distortion of the ATP-phosphocreatine pattern in view of the equílibrium constant of the

creatine phosphoryltransferase reaction (39). In the dystrophic hamster heart Lochner et al (44) found decreased phosphocreatine levels, but un- changed ATP concentrations.

Since all the above experiments dealt with steady state measurements of high energy phosphate compounds, they gave no indication of the high energy phosphate turnover. An attempt to assess this parameter of phosphorus metabolism was made by Zymaris et al (46) who injected no11nal and dystrophic mice rvith radíoactive phosphorus and measured the specifíc -32- activities of various nucleotides 2 hours later. Their experíments showed greatly increased specifÍc activities of A-DP and GDP, but the specific activities of ATP and GTp were also increased significanrly (p < 0.05). Thus, the observed decreased steady state levels of ATp in dystrophic mice do not necessarily reflect a decreased energy prod.uction. They are merely the net result of the balance between production and consumpËion. It should be remembered that energy consumption might be increased in dystrophic muscle for reasons other than changes in amount of muscular work, í.e. increased protein s5mthesis (48, L79) and fatty acid synthesis

(49) could account for increased energy requirements and thus great.er high energy phosphate turnovers,

In spit.e of a number of interesting observations on high energy phosphate metabolísm Ín dystrophic muscle noted Ín the above reporÊs, no consístent Pattern suggestive of a specific defect ís apparent from this earlier work. If one considers the great diffÍculties of determining accurately the nucleotide composition in tissues (50), one may have some reservations as to whether the reported levels of high energy phosphates actually represent in situ concentraËíons. According to Bucher (50) such metabolícally labile compounds cannot be determined by direct methods, since an ischemic state for only a few seconds during the interval that the tissue is being frozen can cause drastic changes in the nucleotide 1eve1s,

Glycolytì-c capacity and glycolytic enzyme activities in dystrophic muscle

There is good evidence and general agreement that the glycolytic activity (51) and various glycolytic enzJ¡me actÍvit.íes are decreased in *ìystrophic -33-

human skeletal muscre (51, 31 , 52-54, 55). These abnormalirÍes still persist after correction for the partial- replacement of the muscle fibers

by fat and connective tissue (53). Similar results have been obtained

from skeletal muscle of dystrophic mice (56-60). However, sundermeyer et al (6f) inferred from indirect evidence an increased activity of the

glycolytic pathway in the hearts of patienËs with muscular dystrophy and

Kleine et al (62) found increased aldolase activity in dystrophíc human

heart.s .

Lochner et al (45) found glycol_ysis in hearrs of dysrrophic hamsters to be significantly increased, but the activities of several

glycolytic enz)¡mes were normal. OnIy lactic dehydrogenase showed a significant decrease. The authors ascribe the increased glycolysis rates

to elevated tissue levels of inorganic phosphate, which would increase the activity of phosphofructokinase (63). Qualitatively similar results \¡/ere rePorted by Lochner et al (44) f.or the skeletal muscle of dystrophic hamsters. Thus, the findings in dystrophic hamsters are at variance with those from human and mouse skeletal muscles with progressive muscular dystrophy, The studies on human dystrophic heart.s are too few to rn/arrant any conclusíons as yet.

While it is possible Èhat the observed decreased glycolytic activiLies in man and mice could lead to a lack of adequate energy supply and thus accelerate the course of the disease, this defect is not regarded as the primary cause of the disease (31). It is generally found that those muscle enz)¡mes present in the highest activities in the serum of dystrophíc subjects exhibit the greatest decreases in the muscle (59). Thus, the abnormalities of lhe glycolytic enzyme pattern in dystrophíc muscle can be most easily explained by a leakage of these cytoplasmicalty localized -34- enzJrmes (64) into the serum.

It also should be pointed out that mosË of the above studies deal with tissue enz)rme activÍties determined under optimal conditions after complete disruption of the tissue sLructure. However, it is well known that such measurements alone give no indication of the metabolic fluxes through the pathways concerned (65). This latter aspect wíll have to be examined before any conclusion can be drawn as to Ëhe metabolic consequences of decreased glycolytic enzJ¡me leveIs in dystrophic muscle.

Studies on miËochondrial structure and function in muscular dystroohv

Since ATP under aerobic conditíons is produced largely by oxidative phosphorylation in the mitochondria, a possible defect in thi s process could be responsible for severeenergy impairment. A myopathy, caused by a defect in mitochondrial oxidative phosphorylation has been reported by Ernster et al (66-69). The subject, a \^/oman, suff ered from a severe hypermetabolism with increased basal metabolic rate, increased body temperature, perspiration, polydipsía, and polyphagia. In spite of a large food intake she suffered from'muscular weakness and wastÍng, but her thyroid function $/as normal. The cause of the dísease I^ras found to reside in her skel-etal muscle mitochondria.

They were increased in size and number and in cytochrome oxidase content per gram mitochondrial protein. Their respÍratory activity was normal" but this \¡Ias not inhibited by oligomycin. I,trhile the P/O ratios were only slightl-y subnormal, the mitochondria exhibited no respiratory control.

Thus, respiration in these mitochondria proceeded at maximal rates whether lhere I¡Ias energy requirement or not. Irihen the energy requirement was low, -35- the excess energy from the presumably uncontrolled oxidation of food was dissipated as heat, This mitochondrial defect of loosely coupled oxidative phosphorylation could explain all the observed clÍnical s¡rmptoms.

To the authorrs knowledge there exists no other muscle disease in which a mitochondrial defect can explain the clinical sympLoms as satisfactorily as in the above patient. However, there are many indications in the literature that mitochondria might be abnormal in muscular dystrophy. Although a functional defect need not be associaËed wíth morphol-ogÍcal changes, it is of interest that many mitochondrial abnormalities have been reported ín erectron microscopic studies. Thus, Van Breemen (70) reported lobulation and disintegration of mitochondria of dystrophÍc human skeletal muscle. Fisher et al (71) observed mitochondrÍal hypertrophy, swelling, distortion, pallor of the matrix, and abnormal cristae arrangements in Duchenne dystrophy. Indeed,

Pearce (238) observed mitochondrial abnormalities in the preclinical stage of human muscular dystrophy.

Símilar mitochondrial abnormalities have been reported to occur in dystrophic mice (73, L5), and appear in the newborn animals some weeks before the overt signs of the disease manifest themselves (74). On the other hand, Ross et aL (73) stress the fact that mítochondria often persist in dystrophic myofibríls until all the other cellular structures have under- gone complete necrosis.

It seems noteworthy that in both human and muríne dystrophic muscle fibers mitochondria seem to accunulate in the subsarcolenunal space (70,75).

However, in spite of the many reported mitochondrial abnormalities there seemsto be no consistency and no typical- pattern in these results. -36-

This may be contrasted with the very definite mitochondrial changeswhich occur in muscl-e within 24 hours after denervation (16). rt has been suggested that the mitochondrial changes in dystrophic muscle descríbed above are secondary and merely reflect the t'notorious sensitivÍty of mitochondría to noxj-ous stimuli't (7f ). Furthermore, üIeinbach et aL (77) studied the mitochondrial morphology in the coupled, uncoupled, and recoupled states and found no direct correlation between biochemical function and morphology. On the other hand, Hackenbrock (78, 79> c1-ear1-y demonstraÈed a relatÍonship between mitochondrial morphology and various rnormal-t functional states of the organelles. (This aspect of the problem is also considered under Results).

During the last few years several reports have appeared on a new kind of myopathy, so called tmitochondrial myopathiest (72, 80-85). These were ín most instances discovered by hÍstochemical and electron microscopical studies. The muscles exhibited an unusual intense staining for mitochondrial- oxidatíve enz)¡mes. Electron microscopy showed numerous mitochondria which usually \,rere accumulated in the subsarcol-enrnal space.

Many of the organelles were abnormal in size and shape and some contained

ínclusions. However, none of the cases exhibited a truly Íncreased basal metabolic rate, as did the case of Ernster eË al (66-69) described above. The cases of Hulsmann et aL (82, 83) were investigated for theÍr mitochondrial function. The ísolated nnrscle organelles exhibited loose coupling of oxidative phosphorylation. However, Hulsmann et al (86) have also isolated such loosely coupLed mitochondría from normal rat muscle and in his latest publication Hulsmann (87) concludes that loosely coupled mitochondria cannot be regarded as Ëhe cause of the disease. At present it is not known whether Ëhe 'mitochondrial myopathies' -37- represent a single form of an inherited disease or whether the mitochondrial changes are non-specific consequences of these myopathies (88). The mitochondrial abnormalities just described are usually not seen in the Duchenne type of muscular dystrophy or the dystrophies of mice and hamsters, studied in this work.

I,rÏhile some of the reports revieqred above suggest a possible impairment of mitochondrial function in muscular dystrophy, the findings are often contradictory and do not allow any definite conclusÍons. It therefore seemed appropriate to study oxÍdative phosphorylatÍon in muscular dystrophy on isolated mitochondría. The chapter on Resul-ts in thís thesis deals exclusively with this aspect of dystrophy.

Studies on oxidative phosphorylation in muscular dvstrophv

At the time the present study was starred (May, T966) there existed only one short report on oxidative phosphorylation in muscular dystrophy: Opie et al (89) observed decreased P/O ratLos in homogenaËes of hearts from dystrophic hamsters. Af ter r,ve had f ound oxidative phosphorylation in dystrophic mice to be normal (90) we were anxious to see whether we could detect in isolated mitochondria the defect reported by

Opie et al in homogenates. This would not have necessarily been expected, since the abnormality reported by opie et al (89) could also have been caused by the presence of uncouplers, non-mitochondrial ATPase, or, more un1ikely, glucose-6-phosphatase in t.he whole heart homogenates. Our initial studies gave rlormal results for al1 the oxidative phosphorylation

Parameters of both heart and skeletal muscle of dystrophic hamsters (91, 92). Meanwhile, Lochner et al had described loosely coupled mitochondria -38- in heart (45) and skeletal muscle (44) of dystrophic hamsters. Schr^rartz et al (93) and Lindenmayer et al (94) observed loosely coupled oxídative phosphorylation in heart mitochondria from hamsters in terminal hearÈ failure, but normal oxídative phosphoryl.ation parameters in animals at earlier stages of the disease (94). March et al (95) found increased respiration rates in muscle mitochondria from dystrophic chicken, Ì.n/hereas Lin et ù (96, 49) and Strickland et aL (97) observed decreased oxid.atíon rates with palmitaLe and pyruvate in skeletal muscle mítochondria of dystrophíc mice.

Oxidative phosphorylation in human muscular dystrophies have been studied by several workers. ronasescu et g! (98, 99) reported loosely coupled mitochondria in Duchenne type of dystrophy and partially uncoupled mitochondria in other forms of dystrophy. olson er al (100) found normal P/O ratios in various hunran dystrophies. Essentially normal_ oxidative phosphorylation parameters in human muscular dystrophies were also reported recently by Hulsmann et al (87 ) and peter er al (101). The above sunmarizes the literature on oxídative phosphorylation, but only in outline form. A critical evaluation of these findings and their relevance to the findings in this study will appear in various sections of the Results and in the Discussion. X. }.{ATERIALS AT{D METHODS -40-

X. MATERIALS AND METHODS

A. MATERIALS

(a) Chemicals

All chemicals were of reagent grade, unless otherwise stated.

The f ollowing chemicals rnrere obtained f rom the sigma chemical

Company:

Adenosine-5' -trÍphosphate (ATp) from equine muscle, disodium salt (crystatline)

Coenzyme A from yeast Fumaric acid Glucose-6-phosphate, dipotassium salt L-glutamic acid

Dl-oc-glycerophosphate, disodium salt, grade X Hexokinase Type III from yeast Hemoglobin, bovine powder, Type II DL-p-hydroxybutyrate, sodium salt Lactic dehydrogenase from rabbít muscle, crystalline

suspension in ammonium sulphate, Type I L-malic acid D-mannitol

Nicotinamide adenine dinucleotide (reduced), disodium salt, grade III

Phenolphthalein-mono-p-glucuronate, sodium salt

Tris (hydroxymethyl) amÍnomethar:re rttTrizma basetr Trís (hydroxyrnethyl) aminomethane hydrochlorÍde, I'Trizma-HCln -4L-

The followíng chemicals were obtained from The Britísh Drug llouses Ltd.:

Acetone Acetyl chloride Citric acid

CuprÍc Sulphate Ethylenediamine tetraacetic acíd (EDTA), disodium salt Glacial acetic acid

Magnesium chloride

Phenol PerchlorÍc acid Potassium chloride Potassium hydroxide

Sodium acetate

Sodium bicarbonate

Sodium carbonate

Sodium sulphite

Sodium tartrate

Succinate, sodium salt

Sucrose Sulphuric acid Trichloroacetic acid

The following chemicals were obtained from Fisher ScientífÍc

Company:

1 -Amino- 2-naphthol-4-sulf oni c acid

B enz ene

Diphenylamine -42-

Di sodium phenylphosphat e

Hydroxylamine-HCl

Is obutanol 0rcinol Palmitic acíd

Sodium fluoride

The following chemicals were obtained from Mallinckrodt

Chemical trrtorks Ltd.:

AceËaldehyde

An¡nonium molybdate

Glucose

Potassium cyanide

Potassium phosphate, mono and dibasic

Sodium bisul-phite

The following chemicals were obtained from Baker Chemical

Company:

Phenolphthalein

Potassium f erricyanide

Sodium hydroxide

From Mann Research Laboratories were obtained:

L- (-) -carnitine chloride

Deoxyríbonucleic acid (DNA) , sodium salt, from calf Ehymus Glycíne

RÍbonucleic acid (RNA) , sodium salt, from yeast Tyrosine -43-

From Armour Pharmaceutical Company were obtained:

Bovine albumin powder, Fraction V, from bovine plasma Bovine plasma albumin, crystalline

From Calbiochem were obtained: Cytochrome c from equíne heart Dithiothreitol

From P-L Biochemicals, Inc. were obtaíned: Adenosine-51 -diphosphate, sodium salt

Nicotinamide adenine dinucleotide (NAD)

From Hartman-Leddan Co. Inc. were obtained:

Nesslerts Reagent

Triton X-100

The following chemicals were obtained from the companies indicated:

4-Amino-antipyrine (Eastman Organic Chemicals)

Creatine phosphokinase, U.V. Test Combinations,

(Boeringer Mannheim Corporation)

2,6-DichLorophenolindophenol (Hoffman LaRoche, Inc. )

2,4-Dinitrophenol (Matheson, Coleman and Be11) Ethanol, absolute (Canadian Industrial Alcohols) Nagarse, crystalLized lyophilized bacterial proteinase,

(Nagase and Co. Ltd., Osaka, Japan) Palmitic acid chloride puriss. (Fluka A.G. Switzerland)

32Phosphorus, as phosphate in d.ilute HCl (Atomic Energy

of Canada Ltd.) -44-

Rotenone (K+K Laboratories)

(b) Animals

1. Mice. --411 míce were obtained from Jackson Laboratory,

Bar Harbor, Maine. Dystrophic mice were of the I29/ Re-dydy straín. Control animals were either of the I29/Re-Dydy strain

(heterozygous), or of the L29/Re-DyDy srrain (homozygous).

Only male mice were used. All mice were fed Purina Lab Chow pellets and water ad lib.

2, Hamsters.--Dystrophic hamsters of the straÍn BI0 14.6

were obtaíned from the BI0-Research Institute, Cambridge, Mass.. This institute also supplied the control animals of the N.I.H"

random bred strain and of the LSH strain (London School of

Hygíene). Additional control animals were of the Lakeview

random bred strain obtained from Lakeview Hamster Colony,

Newfield, New Jersey. Some animals of the BIO 14.6 and the

LSH strain r¡rere bred locally by random mating. Animals in

Group 3 and 5 (see under Results) consisted of males and females;

all other groups consisted of male animals only. Hamsters of

Group I were fed Purina Lab Chow and water ad lib; all other

hamsters received in addition peanuts, sunflower seeds, and lettuce. -4s-

D. METHODS

(a) Assessment of the stage of the disease

c<. Mice

The stage of the disease process ín dystrophic mice was assessed by the age of the animals, their body weights, and by an arbitrary grading of the typical signs of the disease (16). Details are given under Results.

B. HamsËers All offspring of BT0/L4.6 hamsters are affected by rhe polymyopathy (47). This was also confÍrmed in the present study. However, since the physical appearance and behavior of the myopaËhic hamsters remains normal for a long period of their Lif.e (47 ), the presence of the disease l¡7as ascertained by various mearì.s. The particular involvement of both heart and skeletal muscles were ínferred from some of the following parameters:

l. 4€9. --The age of the animals \,{as recorded in days. It can give useful ínformation in regard to the stage of the heart disease, since this develops rather consistently at a certain age (102) . However, this is not true for the disease process in skeletal muscle. Here different animals can show great differences in the kind and degree of muscle involvement at the same age (i03).

2. Body weight, heart weieht, heart weieht:bodv weieht ratio.-- During the first half of their l-ives the myopathic hamsters are smaller in size and lighter in weight than control animals (47). In later stages the body weight of the diseased hamsters will surpass considerabty that of the controls because of fluid retentíon (edema) and congestion of ínternal -46- organs (LOz). None of the animals of this study were in this late stage of the disease. It was therefore possible to use the body weight as a reference for the heart weight to demonsErate hearË hypertrophy by an increase in the ratio heart weight (mg):body weight (e).

3. Macroscopic description of heart and muscle pathology. --The macroscopic pathology of the hearts is suurnarized in some Tables under tHeart Diseasetwith an arbitrary scale from 0 (no signs present)to 4f (severe hypertrophy with some indicaËion of congestion). Typical signs v/ere heart hypertrophy, scars and spots of calcification of the heart muscle, and occasionally true signs of congestion such as a swollen or hardened liver and fluid retention in the various body cavities.

The macroscopíc pathology of the skeletal muscles is summarized in some Tables underrstreaksr. This refers to areas of necrosis, which, if they are large enough, can be seen in the skeletal muscle in the form of white streaks. The extent of the streaking was recorded under an arbitrary scale from 0 (no streaks) to 4+ (several muscles completely necrotic).

4, HisËopathology. --Four muscle samples l¡rere saved f rom each animal for hisLological examinations. These were from the apex of the heart, the m. biceps femoris, the m. gluteus, and the back muscle (m. erector spÍnae). These biopsies were taken in the cold room at 0-4oC about 5 min. after decapitation of the animal. The skeletal muscle samples

T^rere stretched on a piece of cardboard and fixed in cold (0-4oC) LO% buffered formalin, pH 7.0. The heart biopsies were fixed without stretch- ing. The fixed samples were embedded in paraffin, sectioned, stained with hematoxylin-eosin and examined under the tight microscope. -47

5. Serum creatine phosphokinase (CPK) determinations.--Bl-ood

\.üas collected after decapitation from the severed vessels of the neck.

After the blood had stood 20 min. at room temperature, the serum \ÀIas separated from the clot in a clinical centrifuge (ful1 speed, l0 min.).

Serum creatine phosphokinase activity was determined at 25oC in the forward direction with pyruvate kinase as auxiliary enzlmre and

laclic dehydrogenase as indicaEor enz)ryne ("Biochemica Test Combination",

Boehringer Mannheim Corporation, New York). Serum was diluted for the assay 1:10 with 0.9% NaCl. In some tests 0.8 nM dithíothreitol (ltt) was added as a reducing reagent (104). Activities were expressed as ¡rmoles substrate converted per min. per 1. Unexpectedty high activities in normal hamsters \,üere observed by this method, when the values r¡/ere compared to those of Eppenberger et al (f05). However, when human venous blood \^zas Ëested, the same test procedure yielded Lhe expected normal values.

It was found t.hat the high values obtained from normal hamsters probably resulted from a contamination of the I true serum enz¡zmet by tissue enz)nne from the severed neck muscles. This inevitably happens when blood is collected from the decapitation wound. However, it coul-d be demonstrated that the serum enz)¡me increases dramatically, when blood is

collected from the wound for more than a few seconds. Thus, when blood from the drípping wound was collected for several minutes, serum CPK activities of over 1000 units r¡rere found, even in normal- animals. In

contrast, the normal mean values reported in this study, where blood was

colLected for a few seconds on1y, ranged from 20-105 units. In the same animals blood obtained by heart puncture showed activities of an order of magnitude smaller. However, this source of blood for CPK estimations could -48- not be used because r,üe needed the hearts of these animals undamaged. Also no big veins are easily accessible in Lhese hamsters. It proved necessary to continue to use the blood collected from decapitation. However, the sampling time was kept minimal and constant within each Group of hamsters studied.

The possibility that the observed increased CPK activiËies in sera of dystrophic hamsters \¡rere caused by a greater ratio of the 'tissue enz¡rmet : ttrue serum enzymet due to unnoticed differences in the blood sampling lechnique could be excluded, as will be explaíned in the next paragraph.

For some years it has been knorm that serum CPI( activities can be increased several times by the presence of reducing reagenLs in the test

(f06-110). However, fresh muscle tissue CPK, purified or in crude extracts, is not activated by reducing reagents (106, 107, 111). Therefore, we measured all serum CPK activities in the absence and presence of the reducing reagent dithÍothreitol (DTT) and recorded the percentage of activation. If the higher activities found in dystrophic hamster serum had been caused by a greater rcontamination' of the blood with 'tissue CPKi , one would have expected a significantly lower percentage of activation of the CPK in dystrophic animals. However, this r¿as not Ëhe case, since in alt 6 Groups of hamsters in this study Ëhe percentage of activation was either equal in the normal and dystrophic animals or higher in the latter.

6. Protein, noncollagen rotein. collaqen protein in skeletal muscle. --One g of muscle mince which had been kept frozen at -20oC was thoroughly homogenized with 4 ml of 0.9% NaCl in a rKonLes' all glass homogenizer. Aliquots were diluted to desired concentrations and incubated -/,o-

in 1 N NaOH overnight aE room temperature. Protein was then estímated by the method of Lowry et al (LL2) and called total protein (T.p").

Collagen protein (C.P.) was precipitated at room temperature by

diluting the homogenate 1 in l0 r¿ith 0.05 N NaoH for 18 hours (34,113).

The precipitate was sedimented in an Eppendorf MicrocenËrifuge 3200 at

121000 x g for 2 min. The protein in the supernatant r¡ras assayed as ouË- lined before and termed noncolragen protein (N.c.P.). collagen proteín

(C.P.) was calculated as the dífference between total protein (T.P.) and noncollagen protein (N.C.P. ).

7. Acid phosphatase actívitv in skeletal muscle. --This enzyme was estimated by the method of Kind and King (114). Aliquots of the muscle homogenate described under (6. ) above were placed on ice in the

presence of 0.1% Triton X-100 for 15 minutes. The homogenaËe was then

Íncubated with gentle shaking at 37oC for t hour in a system containing

5 uM disodium phenyl phosphate and 0.1 M citrate buffer, pH 4.9. The

control had no homogenate present during the incubation. The reaction was

terminated by the addition of L66 mM NaOH, after which homogenate r^ras added

to the control, The color reaction for the liberated phenol was obtained

after the addition of 83 mM NaHCO. , 0.L% 4-amino-antípyrine, and 0.4% J I("Fe(CN)-. After 10 min. the absorbance was read at 500 mt¿ on a Beckinan r -b I DU spectrophotometer. The readings r¡rere compared with Lhose from a standard

curve prepared with knor,¡n amounLs of phenol. The results rÀrere expressed as

gg phenol liberaEed per mg protein per hour. I

8. ¡lGlucuronidase activity in skeletal muscle. --Thís enz)¡me

assay vTas a combination of varÍous features of three similar published -50- meËhods (115-117). The enzyme Ín a muscle homogenate treated with Triton X-100 (see under (7.)) was assayed in 0.08 M acetate buffer, pH 4.5 with 1.25 trM phenolphthalein-p-glucuroníde as substrate. After I hour incubation at 37oC with gentle shaking the reaction was terminated by the addition of 0.2 M glycine-NaoH buffer, pH 10.7. The suspension was then filtered twice through tr{hatman No. L fÍlter paper. The absorbance of the red color of the filtrate was read Ín a Becknan DU spectrophotometer at

550 ny and compared with that of a phenolphthalein standard at the same pH.

Enzyme controls were incubated without subsLrate and a substrate control was incubated without homogenate. Both control readings were subtracted from the test reading and the result.s \,Íere expressed as flg phenolphthalein liberated per mg protein per hour,

9. Cathep.sin activity in skeletal muscl-e. --This assay was done according to Pollack et al (1i5). It is based on the method of Anson (118) as modified by Bird et al (1f9). The activity was determined in a Triton

X-100 treated homogenate (see under (7.)) in 0.225 NI acetate buffer at pH 3.5. As substrat-e L% acid denatured hemoglobin was used (see below).

The test was incubated at 37oC Íor 30 min. in a shaking water bath. The reaction was terminated by the addition of lBB mM cold Ërichloroacetic acíd.

(TCA). The deproteinized mixture \Áras allowed to stand on ice for 15 min. and Lhen spun for l0 min. in a clinical centrifuge. Aliquots of the clear supernatant were used to obtain a color reaction wÍth Folin-Ciocalteau reagent as described by Park et aI (120). A control was set up by addíng homogenate after the addition of TCA and treated as above. A second (substrate) control was incubated without homogenate to determine Èhe extent of nonenzJ¡matic breakdown of hemoglobin. The absorbance of the resulting colors was read aE 660 mf on the Beckrnan DU spectrophotometer. The absorbance -51 -

obtained by subtracting both control readÍngs from the test was compared with a tyrosine standard which contained the same final concentration of

TCA as the test. The as results Irere expressed ft7 tyrosíne liberated per mg protein per 30 minutes.

Hemoglobin r¡/as denatured by incubating a 3% solution at pH 1.3 at 37oC for one hour. The pH was then increased to 3.5 and the solution stored at OoC.

10. DNA and RNA content Ín skeletal muscle. --The extractíon and separaEion of ríbonucleic acid (RNA) from deoxyribonucleic acid (DNA) is based on the procedure of Schmidt and Tannhauser (LzL). Two ml of the muscle homogenate, described under (6.), were mixed with perchloric acid

(PCA) to a final concentration of 0.7 N. After it had sËood for 15 min. on ice ít was centrifuged at 12,000 x g at 0-2oC for 10 minutes. The supernatant was discarded and the precipitate washed tr^ro more times in a total volume of 7 ml with 0.7 N PCA. The final precipitate \,ras suspended in 4 ml 1N NaOH and incubated in the shaking \^raler bath at 37oC f.or I hour.

By adding 4 mI 2 N PCA, the solution was made 0.5 N with respect to PCA, mixed, and spun at 121000 x g for 10 minuEes. The supernatant was reserved as 'hydtoLyzed RNA'. The precipitate r¡ras washed once with 0.5 N PCA and after centrifugation the supernalant was added to the thydroLyzed RNA].

The 'hydroLyzed RNAÍ solution was neutraLLzed by the addition of 3.25 mL of. 2.0 N NaOH and diluted to 20 mL total volume. Determination of RNA content was done by the orcinol procedure, see below.

The above precipÍtate r^ras suspended in 5 ml of l0% PCA and incubated o aL 70 C for 15 minutes. The suspension r,^ras cenËrifuged at L2,000 x g for 15 min. and the precipitate washed once more in 5 ml of 10% PCA. The combined supernatants were saved as 'hydroLyzed DNAr. -52-

DNA r¡as assayed ín the rhydrolyzed DNAI sol-uËion by the method of Burton (L22) as modified by Giles et al (L23). Calf thymus DNA was used as standard. The results \,üere expressed as fg DNA per g muscle. RNA was assayed Ín the 'hydroLyzed RNA| solution by the orcinol reaction. To 0.2 ml were added 4.8 ml of water, 5 ml of 0.02%FeCLrLn conc. HCl, and 0.3 ml 10% orcinol in ethanol. The mixture was boiled for

30 min., cooLed down to room temperature and the absorbance read in the

Beckman DU spectrophotometer at 665 n/L. Standards \^/ere prepared from yeast RNA. The results hTere expressed as fC RNA per g muscle.

(b) Dissection of animals and preparation of mitochondria

The following media were used for Ëhe preparation of mitochondria:

Stock Medium: 0.21 M I"iannitol

0.07 M Sucrose

0.1 mM EDTA

pH 7.4

Homogenizing Medium: 0.2L M Mannitol

0.07 M Sucrose

0. I mI'{ EDTA

0.01 M Tris-phosphate 0.5 mg/ml Proteinase (Nagarse)

pH 7.6

Suspending Medium: O.2L M Mannitol

0.07 M Sucrose

0.1 mM EDTA

0.01 M Tris - chloride pH 7.4 -53-

Lochner Medíum: 0.25 M Sucrose

lmM EDTA

pH7 4

l. Mouse skeletal muscle mitochondria. --Ivlouse skeletal muscle mitochondria were prepared by a modification of the procedure developed by Hagihara (L24). After the mice had been decapitated and skinned, the whole body was placed in ice-co1d Stock Medium for 5 minutes. The remaÍn- der of the procedure was completed at 0-4oC. Muscle was dissecLed from the hínd limbs, pelvic girdle, and triceps portíon of the forelimbs.

After removal of as much as possible of the extraneous tissues, the muscle was minced with scalpels into cubes of about 1 nrn3 síze. The resulting mince, weighing 0.6-1.0 g, \.47as incubated r¡rith 40 volumes of

Homogenizing luiedium f or 10 min. with occasional stirring. The suspension was then homogenized gently for 1 min. at 100 r.p.m. in a glass-Teflon homogenízer (sLze C, A.H. Thomas Co., Philadephia) wíth a loosely fitting pestle of 0.66 rmn clearance (clearance = difference between inside tube diameter and pestle diameter). After the homogenate had stood on ice for

10 min. it was diluted with an equal volume of Stock Medium and then homogenized with a tighter pestle of 0.20 nw clearance. The preparatíon was then centrifuged for 5 min. at 400 x g and Ëhe supernatant removed, carefully avoíding contamination with the loosely packed upper layers of the pellet of nuclei and cellular debris. The supernatant r,¡as centrifuged at L2,000 x g*to yield a mitochondrial pellet whích was rinsed thoroughly wit.h Stock Medium to remove a white layer of debris that was thicker in preparations from dystrophic muscle. Failure to remove this material completely, resulted in a final preparation which was partially uncoupled. xfor 10 min. -54-

The rinsed pellet was suspended in l0 ml of Suspending Medium and re- centrifuged at 8,000 x g for 5 minutes. The final pellet was suspended in Suspending Medium to give a mitochondrial protein concent.ration of

10 mg/ml.

2. Mouse skeletal muscle mitochondria prepared in Ëhe presence of albumin. --The procedure was nearly identical in all parts to that outlined above (1), except that the Suspending Medium contained L% fraction

V bovine albumin that had been dialyzed agaínst I rM EDTA.

3. Hamster heart mitochondria. --Hamster heart mitochondria \¡/ere prepared by a modification of the procedure developed by Hagihara (L24).

The heart \^Ias removed as quickly as possible after decapitation of the hamster, and placed into ice-co1d Stock Medium f.or 5 minutes. It was freed from all nonmuscular tissue and rinsed again in Stock Medium. A fine mince was incubated for I min. ín 66 volumes of Homogenizing Medium. The further procedure was identical Eo that described under (1), except that the second period of íncubation was also reduced to B min. and the protein concentration of the final suspension \,{as 20 mg/mt.

4. Hamster skeleLal muscle mitochondria : Standard Proteinase Preparation, --Dissection--Hamsters were decapitated and the blood ejecËing duríng the first seconds was collected for CPK determinations. The chest

.I¡ras opened and the heart removed and placed into ice-cold Stock Medium. All further steps were performed at 0-4oC. The hind part of the animal was skinned and inrnersed into ice-cold Stock Medium. Next, bíopsies for histology were taken. After this, the lower portion of the back muscles, all the muscles of the pelvic girdle, and of the upper portion of both -55- hind legs were dÍssected. (In animals of Group L the described nuscles of only one side were dissected). The muscles were thoroughly cl-eaned of extraneous tissues, such as fat, fascia, and tendons, and were minced finely with scalpels. This resulted in a fairly uniform muscle mince mixture of all the dissected muscles. A portion of this was sometimes used for DNA and other determínations as descríbed under item (a), (p),

(6- 1o) .

PreparaLion of mitochondria--Another portion of the muscle mince was incubated in 20 volumes of Homogenizing Medium and then carried through the procedure exactly as outlined under (l), page 53. However, Ehe final pellet \¡/as suspended to yíeld a proteÍn concentration of 20 mg/mL.

5. Hamster skeletal muscle mitochondria : Standard Proteínase Preparation with l% albumiq. --This procedure differed from that outlined above, item (4) , only by the composition of the Suspending Medium, whích contained L% f.raction V bovine albumin that had been díalyzed against 1 mM

EDTA.

6, Hamster skeletal muscle mitochondría : Modified Proteinase Preparation. --This procedure differed from the standard procedure outlined under item (4), in thaÈ the A.H. Thomas homogenizer with the tight pestle was replaced by a Tri-R homogenizer S 37 with a tight glass-reinforced

Teflon pestle vrith 0.15 mm clearance. Also, the two incubation times were reduced from 10 to 5 min. each.

7. Hamster skeletal muscle mitochondrLa i Lochner Preparatíon (44\

--An aliquot of the muscle mince (see under item (4)) was finely homogenized with 10 volumes of Lochner Medíum in a Kontes all glass homogenizer, size D, -56- for about 2 min. at approximately 300 r.p.m.. The homogenate was centrí- fuged at 700 x g for l0 minutes. The supernatant was saved and the pellet resuspended in an equal volume of Lochner Medium and centrifuged as before.

The supernatant of this step \nzas combined with that from the first centrifugation and the mitochondria were sedímented at 10,000 x g for 10 minutes.

The upper layers of the resultant pellets Ì^rere thoroughly rinsed with

Lochner Medium, although no actual separate layers were discernable. The pellet was then suspended in Lochner Medium to yield a final protein concentration of approximately 20 mB/ml.

l. Apparatlrs. --Polarographic measurements r,rere made at 28oC on a Model KM Oxygraph (Gilson Medical Electronics, G.M.E.). The Oxygraph was equipped with a vibrating platinum el-ectrode G.M.E., which fírst had been proposed by Chance (L2-5). It was coated with collodion, as proposed by Hagihara (126). A polarLzLng voltage of 0.6 volts was applied. This electrode was used for polarographic determinations of all mouse experiments and the hamster experiments of Group I (see below). For all other polarographic determinations a Clark-type electrode

(L27) was used (Yellow Springs Instrument Co.) with a Teflon membrane. A poLarízing voltage of 0.8 volts was applied. The reaction medium i,.ras constantly mixed with a magnetic stirrer.

The total test volume varied between L.6 and 2.2 mL.

,) Measurements. --The polarographic reaction medium contained:

0.23 M Mannitol

0.07 M Sucrose -57

0.02 M Tris-chloride

0.02 mM ÐTA

5 rnM K-phosphate

pH 7.2

A typical polarographic experímenË is shown schemaEíca1ly in Fig. l with arbitrary numbers inserted. The react.ion medium containing Pi pre-equilibrated T,,lith aí.r at 2BoC was placed into the reaction vessel . When mitochondria were added (M) a slow oxygen consumption ensued. After the addition of a limited amount of ADP a short fast period of respiraËíon started, Índicating the presence of substrate in the mitochondria. The rate declined as the indigenous substrate was used up. At this point Ëhe substrate to be tested (tike pyruvate/malate (Pyr) ) was added. This resulted in a fast rate of coupled state 3 respíration (i2B). upon completion of the phosphorylation of the added ADP, t.he oxygen uptake rate dropped sharply to a slower rate, called State 4 respiratíon (128). A Second Period of coupled respiration could be initiated by a further addition of ADP, which resulted in another cycle of state 3-state 4 respiration. This could be repeated several Ëimes until all the oxygen of the medium \,ras exhausted. From such an experiment one can calculate: i, the respiraËion rate (0, rate)

ii. Ehe ADP/0 ratio

íii. the respiratory control ratio (RCR) iv. the phosphorylation rate (P rate) as will be outlined under (5). Unless otherwise stated, aLL these parameters were calculated from the Second Period of State 3-State 4 respiration in order to avoid the interference from indÍgenous substrate durÍng the First Period. -58-

Pi orJ ( zlopl¡) { State 3 rate 240 RCR = = State 3 --- State 4 rate -3î ---* 240 \ \\ ( 3:opu¡

1 - --ADP/0 330pM ADP Sopli o, = B@æ t\ o2

#nr )

0^=Zefo time ( min) ¿

Fígure 1"--SCFIH,ÍATIC RæRESEFTIATION 0F A TypICAt

.PC,IÀRO GiLA*ÐHI C E}PERTMENT The numbers along the curve ind.icate the 0, uptake in ¡imole= 02 per min" per g protein. -59-

3. Oxvgen calibration. --The oxygen conceritration at 2BoC in the reaction medium T,vas calculated from the data for Lhe solubility of oxygen in Ringer's Solution (L29) with correction for the prevailing barometric pressure. I{hen the oxygen concentration ín the medium T,üas tilrated (130) with enz¡rmatically assayed NADH using sonicated míto- chondria, 9% less oxygen was found than by the above calculatíon. However, this difference was disregarded and the calculated solubility was used throughout.

4. Materials added during the polarographic experiments. --

Mitochondria containing protein of. 400-L200 f.g were added. However, in a given series the concentration was kept as constanL as possible (+ L57.)

ADP additíons varied between 230 and 330 ¡M. Other concentrations: PyruvaLefmalate: 5 mI,I/1 nM

DL-oc-glycerophosphate: 9.5 rnM

NADH: 110 FM in Group l; 500 yM in Group DL-p-hydroxybutyrate: 9. 5 nM

Palmityl -L - carnitine/ma late: 20 N 300 pM Acetyl-L-carnitine/maLaEe: 1. 2s ñI/ 300 yM

NIICLT: 5 nrM

Cytochrome c¡ L6 ytrr

5. CalculaLions. --(i) The respiration rate (02 rate) was calculated from the oxygen consumption during State 3 respiration and vras expressed as ¡.moles 0, uptake per min. per g protein. (ii) The ADP/O ratio was calculated according to Chance gE el (4) as the ratio of ADP concentration added over the concentration of oxygen in g atoms/l required to phosphorylate the ADP (see Fig. 1, page5g). The Anp/O -60- ratio is a measure of the efficiency of phosphorylatÍon with regard to oxygen uptake.

(íii) The respiratory control ratío (RCR) was calculated according to Chance g! al(128)as the ratio of State 3 respiration over the following

State 4 respiration (see Fig. 1, page 58). Thís ratío is regarded as one of the most sensitive parameters for the intactness of mitochondría (L28, 131).

(iv) The phosphorylation rate (P rate) was calculated according to Klingenberg (I32) by multiplying the ADP/0 ratio by the State 3 respiration rate expressed as fg atoms oxygen uptake per min. per g protein. The P rates Ì^rere expressed as ¡moles ADP phosphorylated per mÍn. per g protein. They served as a measure of the capacity of the mitochondria to produce ATP.

(d) Polarographic measurements in the presence of a hexokinase trap

The reaction medium contained:

0.23 M Mannitol

0.07 M Sucrose

0.02 M Trís-chloride

0.02 mM ÐTA

200 ADP f.M

5 mM MSCI,

10 mM Glucose

5 mlui Pyruvate

1 mM L-Malate

pH 7.2 -6L-

The Oxygraph reaction vessel was fílled with 1.49 mL of this medium

air at 2BoC. Then 50 a 1% hexokinase solution eqnilibrated with lo| "t in 0.1% glucose \^rere added bef ore mitochondria containing approximately

500 ¡rg protein. State 3 respiration l^zas started by the addition of an I 84 mM 32n-pho"phate solution to yield a final concentratíon of 5 mM phosphate. At the moment the oxygen in the reaction medium was exhausted,

200 yI 50% TCA were added and the total mixture transferred Ëo ice-cold test tubes. Control experimenÈs rnrere performed by adding TCA prior to the mítochondria. Extraction of glucose-6-phosphate from the test and control

TCA-suspensions was done according to the procedure of Nielsen and Lehninger

(i33) with minor modif ications. One ml of the TCA-suspension T¡ras transferred to a stoppered test tube. After addítion of 1.5 ml acetone and 10 mixing, the suspension \^ras allowed to stand for minutes. Fifty ¡1-

80 mM glucose-6-phosphate \¡rere added as carrier and 1ml of the acid mo1-y- bdate reagent of Martin and Doty (f34) , (5% anmonium molybdate in 4 N HZS04), to complex inorganÍc phosphate. After gentle shaking by hand 2. 15 ml of r¡7ateï saturated !üith 1:l isobutanol:benzene were added, followed by 5.0 ml l:1 isobutanol:benzene saturated with Í¿ater. The tubes i¿ere shaken vigor- ously by hand for 30 seconds and the two layers then separated by cenLrÍ- fugation in a clinical centrifuge, The organic (upper) Layet was discarded and the T¡Iater layer, containing glucose-6-phosphate' vTas passed through a trtlhatman No. 50 paper. After Lhe addítion of lO Ê 0.05 M KHZPO4 as carrier, another phosphomolybdate extraction \^ras perf ormed with another 5 ml isobutanol:benzene. Aliquots of 100 yL ot the water layer were p1-ated on filter paper in steel planchets and the radioactivity counted in a

Nuclear Chicago ModeI Cl15 Low Background Automatic Sample Changer connected -62-

to a Model 1814 Decade Scaler. 32Pí The number of mole" of incorporated via ATP into glucose-

6-phosphate r,ras measured and divided by the gram atoms of oxygen consumed. The quotient was called 32t/O ratio. This quotient was obtained from the f ormula:

c.p.m. test - c.p.m, control x 79.L48 mM phosphate 32p/ total c.p.m. added tñIOrx2 o

The factor 79.148 resul-ted from the various volumes and dilutions of the procedure.

The respiratíon rate in these experiments is listed r" O, * 32p/O rate and the phosphorylation rate, whích is the product of raËio x

0Z rate x 2, ís listed 32P rate. (The factor 2 converts gram moles Hf ^r of O, to gram atoms).

Phosphate was determined by the method of Fiske and SubbaRow (135) 1a H3--P04 was obËained in dilute HCI from the Atomic Energy of

Canada Ltd.. It was heated for I hour at 100oC to convert any metaphos-

phoric and pyrophosphoric acids to orthophosphoric acid. It r,vas then

d.íluted with potassium phosphate buffer to yield an 84 o,U 32e-phosphate

solution with a specific activity of approxÍmaLely 7.5 x 104 counts per (c.p.m.).per minute ¡¿tom P. Under the counting conditÍons used, 1 mc of 32P to about 3.5 x 108 c.p.m. "orr"sponded

(e) Manometri c measurement s 1. 0xidative phosphorvlatÍon with pvruvate fumarate as substrate.

--The respiration rates (02 rates) and P/0 ratLos r¡¡ith this substrate r^rere determíned in a tr'Iarburg apparatus by a method sÍmilar to that of Lochner et aI (44) -63-

Mitochondria containing 700 þE Vroteín \À/ere added to a medium containing: 93 mM Sucrose

56 mM 1(C1

22.3 nú"L KH^PO ------''¿- 4 I0 m¡M EDTA

12.9 mM NaF

536 uM NAD t s37 pM ADP 18 t'uM Cvtochrome c 5.4 mM Fumarate

5.4 mM Pyruvate 8.6 rnM MsCl"2 pH 7.4

The total volume wiEh these constituents I^7as 2.8 mL' 0.2 ml 20% KOH was placed into the center well- which contained a folded filter paper. The flasks with two sidearms were equilibrated for B mín. at either ZBoC ot 37oC. 0, uptake was followed after the addition of 0.2 ml 0.5% hexokÍnase (Sigma type III) in 0.25 M glucose. At this time the reaction in a conËrol flask identicaL to the testT,úas stopped by the addition of 0.2 ml concentrated

HCl04 from the second sidearm. The Lest reaction was terminated in an identical way after 20 minutes. Phosphate was determined in the deproteinized tests and controls by the method of Fiske and Subba Row (135). The P/0 ratios were cal-culated according to Slater, extrapolating the oxygen uptake curves to the time base (136). For the manometric measurements with skeletal muscle mitochondria of hamsters of Group 6 the EDTA concentration was increased to L7B yVI. -64-

) Respiration rates with palmitate as substrate. -- Mítochondria containing I mg protein were added to a system containing:

100 mM Sucrose

400 fM EDTA 8 mM K-phosphate buffer

2.5 mM NAD

80 mM KCl

5 mM MgCl^

40 ¡M CoenzSrme A 5 mM ATP

I mM L-carnitine

L26 yM Succinate L7 N Cytochrome c

zLa yNI K-palmitate in 5% albumin (molar ratio 7.4:L)

The total volume was 3.0 ml plus 0.2 ml 20% ]KOH in the center rvell_ with folded filter paper.

The f lask containing the complete test sysLem \¡ras equilibrated for 6 min. at 37oC before the stopcocks were closed. Oxygen consumption v/as measured for one and a hal-f hours. During the first 20 min. the oxygen uptake steadily accelerated until a linear rate of oxygen uptake was attained. The oxidaËíon rates r¡rere calculated from the linear part of

Ehe oxygen uptake curve and expressed as 0, uptake per g protein per ¡moles mínute.

3, Respiration rates with palmityl-L-carnitine as substrate.--

In these experiments the system described under (2) was used, except Co- enz)¡me A, ATP, L-carnitine, and K-palmitate \,,/ere omitted and replaced by -65-

1. I mM palmityl-L-carnitine and the hexokinase trap described under (l)

(f) Assav procedures 1. ProEein estimation

c(. DÍrect.--Proteín \¡ras determined by the method of Lorøry et al (LL2) after dissolving suspensions of mitochondria or homogenates over night ín 1 N NaOH (137). The absorbance of the developed color was read at 500 m¡ on the Beckrnan DU spectrophotometer and compared to standards prepared from crystalline bovíne plasma albumin (Armour Pharma- ceutÍcal Co. , Chicago).

Indirect. --The above method vras not suitable for determining the mitochondrial protein concentration of suspensions which contained albumin, since repeated washÍng of the mitochondria in albumin-free medium would riot remove all the albumin originally added (138). The mitochondrial protein content of albumin containing suspensions was therefore determined indirectly as foll-ows.

The protein content of mouse mítochondria prepared in the presence of albumin r¡ras measured turbidimetrically. The absorbance of diluËe mitochondrial suspensions I¡/as measured at 625 m¡ ín Lhe Beckman DU spectro- pholometer and compared with a sLandard curve. The standard curve i,¡as obtained by splitting the 400 x g supernatant (see page 53 ) of a mouse muscle preparation into two equal portions. The mitochondria of the two superî.a- tants were isolated under exactly the same conditions, except that the pellets of one part r,7ere suspended wíLh I% albumin in the Suspending Medíum and the pellets of the other part with no albumin. It was assumed that both suspensions contained equal amounts of mitochondrial protein. Protein was -66- measured by the method of Lowry et al (see under l. c<) only in the albumin- free suspension of mitochondria. The absorbance measurements of various concentrations of the mitochondrial suspension exhibited a linear relationship between mitochondriaL protein concentration and absorbance. The absorbances of mitochondrial suspensions \.üíth albumin, which supposedly contained the same amount of mitochondrial protein, differed as much as

! L5% from the absorbance of the albumin-free mitochondrial suspensions, The protein contents of Group 3 hamster skeletal muscle mitochondria suspensions containing albumin, were determined by sp1-itting the preparations into equal portÍons, one prepared wíth albumin and one without. It was assumed for each animal that the two preparations contained equal amounËs of mitochondrial protein, which was determined only in the albumin-free suspensions by the method of Lowry et al (see under I. o(,).

2. NADH concentration, --NADH l'ras assayed enzymatically wíth lactÍc dehydrogenase. The test conËained in a total volume of 3 ml 100 mM

Tris-chloride, 650 fe lactic dehydrogenase (Sigma) , 66 pM NADH and 10 mM pyruvate. The absorbance was read at 340 m¡, in the Becknan DU spectrophotometer before and 5 min. after the addition of pyruvate against a blank containÍng no NA-DH. From the difference in absorbance the NADH concentration was cal-culated using a millimolar extinction coefficient of 6.22 (139). At pH7.2 Lhe equilibrium of the reaction lies far towards the production of lactateo so that 99.6% of the NADH should be oxLdLzed at this pH (r40).

3. ADP concentration. --The concentration of ADP used for the polarographic determinations of ADP/0 ratios was estimated by ul'travíolet absorbance on the Becknan DU spectrophotometer at 260 ny. Samples were -67 diluted ín 0.02 M potassium phosphate buffer pHl.z, and read against a blank containing buffer only. The ADP concentration was calculated from the data on the ultraviolet absorption spectrum provided by P-L Biochemi- cals Inc. (i41).

4. Acetyl-L-carnitine concentration. --The purity of the acetyl-

L-carnitine slmthesized in this laboratory (see below) was estimated by the method of HestrLn (L42).

5. Palmitvl-L-carnitine concentration. --The purity of the palmityl-L-carnitine synthesized in this laboratory (see below) was estimated by the method of Skidrnore g! aL (L43),

(e) Substrate preparations

1. Acetyl-L-carnitine. --Acetyl-L-carnitine was synthesízed by the method of Brendel and Bressler (L44). Eighty-six "A of the producr proved to be ester according to the test of Hestrin (L4Z).

2. Palmitvl-L-carnítine. --Palmityl-L-carnitine was synthesized by the method of Bremer (145). Ninety-five per cenË of the prod.uct proved to be ester according to the test of Skidmore et al (143).

3. Potassium palmitate. --An equimolar amount of potassium carbonate in 50% ethanol was added to palmitíc acid. The suspension was mixed until all the palmític acid had been dissolved, The solution was then gently heated in a boiling water bath to drive off the ethanol.. The palmitate solution was made to a final concentration of 5.3 mM with 5% albumin present (molar ratio of palmitate to albumin 7.4:L under the -68- as Sumpt ion of a molecular weight of 69,000 for albumin)

(h) Tissue mitochondrial content determinations

This procedure r¡ras proposed by Kleitke et al (L46). The proËein

concentration and succinic dehydrogenase activity were determined Ín the final mitochondrial preparation and also in a portion of the tissue from which the mitochondria were isolated. Since succinic dehydrogenase is localized entirely within the mitochondria (L47), the mitochondrial conËent per gram of tissue could be calculated with the formula:

mg miËochondrial protein/g muscle = SDH/mg homogenate protein Ä@-, mg homogenate protein

Succinic dehydrogenase activíty was determíned by a modífícation of the method of Príce et al (148). The test cuvetËe contained l0 mM KCN,

L5 mM 2,6-dichlorophenolindophenol, and 20 mM sodíum succínate. Phosphate buffer (100 mM, pH7.6) was added to give a final volume of 0.3 rnl.. In the blank the substrate was replaced by 9.5 mM malonate to block the cuvettes contained 7 rotenone oxidaEion of índigenous succinate. Both ¡rM to inhibit the oxidation of malate generated duríng succinate oxidation. Prior to testing, the mitochondria and homogenates r^7ere treatedin a Virtis

45 homogeníz,er at 45,000 r.p.m. f or two 15 sec. periods at 4oC. The resulting suspensions were added to both the test and blank cuvettes. The reaction was follovred on a Beckrnan DB spectrophotometer at 600 m¡r, and 25oC. The reaction rate tended to increase during the first five minutes and became approximately linear afterwards. Activities r¡rere calculated from the nearly -69-

linear period of the reaction using an extinction coefficient of 19.9 x 106 *o1e-1 "*2 Í.or 2,6-dichlorophenolinodphenol aL pH I .6 (t<1-eitke et al (146)).

(i) Electron microscopi_q- examination of isolated mitochond.ría

Final mitochondrÍal pellets or concentrated mitochondríal suspensions r¡/ere fixed in buffered osmium tetroxide (pH 7.4), dehydrated, and embedded in a polyesrer resin. Thin sections (approximately 800 i) were stained by lead hydroxide and examined in a Philipps EM 200 electron mi cros cope.

(i) Calculations and statistics

All major calculatÍons were done with a desk computer (Olivetti

Underwood Programna 101) carrying the values to 4 decimals through each steP. However, the final values were truncated as follows for presentatíon in the tables: t-values to 3 decimals; ADp/0 ratios and respíratory control ratíos to 1 decimal, and 0, rates and P rates are given in whol_e numbers only.

Statistical operations were done according to Dixon and Massey

(L49) and the "tr'-tables of Documenta Geigy (150) were used. The level of signif icance r^7as chosen at o( = 5/. (L49). The p-values, listed in Tables, of 0.05,0.025,0.02,0.01,0.00.5, and 0.001, always mean thar the p-value is smaller than the listed value. - 70 -

(k) Preparation of distilled water

Tap viater r¡ras passed through an Illco-trrlay Research Model ion exchange coluum (f51) and then distilled in a Corning all glass still.

This water was used to prepare all media and reagents. SJTNSSE 'ÏX - 72 -

XI. RESULTS

Outlíne of the Results

This chapter deals with oxídatíve phosphorylation in two different dystrophic animal species: A. mice and B. hamsters. In the latter, both heart (("), page 89) and skeletal muscle ((b), page L27) were studied. Skeletal muscle mitochondria of dystrophic míce were studied in only tr¡/o groups of animals (90). The rásumá is given on page 86. A more extensive investigation \¡/as done on mítochondria of striated muscle of dystrophic hamsters, since the results init.ially obtained (91, 92) were at variance with those of other workers (89, 45,93,44).

A great deal of this study \¡/as devoted to an attempt to explain these apparent discrepancies. I^lhile this was not actually achieved, the sËudies on dystrophic hamster heart mitochondria gave a very clear picture, as sumnarLzed on pageL7;. The skeletal muscle mitochondria exhibited various results, depending on the stage of the disease in the animals, the techniques used to isolate the organelles, and the methods used to measure the oxÍdative phosphorylation parameters. The results on hamster skeletal muscle are summarized on page 198. Major sections of this chapter are preceded . by an under- lined headíng, which also can be found in the List of Contents: page - 73 -

A. 0xídative phosphorylation by skeletal muscle mitochondria from

normal and dystrophic mice

þstrophic mice of the I29/Re strain of the Jackson LaboraËory were used in this study. The animals were of various ages and showed the typical signs of the disease (16), including dragging of the hind 1egs, nodding of the head, muscular atrophy, kyphosis, and ruffling of the fur. These characteristics were judged on an arbitrary grading

of the severity from 0 to 4* as recorded in Tables I ar,d 2. Also listed

are the ages and body weights of Ëhe animals. The significantly lower body weight of the dystrophic mice is also a feature of the disease (16). Various muscles hlere secLioned and examined histochemically for succinic dehydrogenase activity (L52) and showed the picture described b¡z others in dysËrophic mice. This included the tendency of the different fiber types toward uniform staining (153), rounding of t.he fiber contours, inequality of fiber síze, and an increased amount of interstitial Lissue between the fibers (16).

Various parameters of oxidative phosphorylation were examined polarographically in mitochondrial preparations from normal and dystrophic skeletal muscle of mice. The parameters determined r¡/ere the respiratory control ratios (RCR) , the ADP/O ratios, the respiration rates (0, rates), and the phosphorylation rates (P rates). (These terms are defined in the Glossary). Pyruvat.e plus malate at final concentrations of 5 rnl"l and 1rnI"I respectively were chosen as substrate combination because in preliminary experiments mouse skeletal muscle mitochondria exhibited higher respiratory activity with this substrate combinaLion than with succinate, glutamate, or DL-c(-glycerophosphate. - 74 -

Table 1

CHARACTERISTICS OF DYSTROPHIC MICE AND THEIR LITTERMATE CONTROLS

Control mice:k Dystrophic mice

Age I,ie ight Signs of Age We ight Signs of days g disease days 0 d ise ase

L2 4s 18 0 13 45 I2 #

I4 47 22 0 t5 47 I6 +

T6 51 2L 0 I7 5l 13 #

1B 56 25 0 L9 56 13 #

Jc Heterozygous littermate control mice - 75 -

Tabl,e 2

CHARACTERISTICS OF I'{ICE USED IN THE PREPARATION OF MITOCHONDRIA IN THE PRESENCE OF ALBI]MIN

Control mice Dystrophic mice

Age Weight Signs of No. Age trrïe ight Signs of days C d ise ase days 6 d ise ase

I 321Y 24 7 37 I6 +

2 39r( 24 8 50 I6 #

I JQ*:k 24 9 51 I4 #

4 52r'<2'r 24 10 58 13 #

J pQ:kJr 28 11 77 I4 #+

6 10 4:t 29

Homozygous normal mice

Control mice, genotype not specified by the supplier, except that they were either heterozygous littermates or homozygous normal animals of the same strain. - 76 -

Mitochondria were isolated from mice aged 45 to 56 days (Table I, pageT/¡). Control animals were heterozygous littermates

with no signs of the disease. The organelles from a dystrophic mouse and its littermate control \¡/ere prepared and tested together in order to eliminate day to day experimental variation. A typical polarographic experiment is shown in Fig.2 f.or the pair No. 16 and 17. The ordinate represents the oxygen concentration and the abscissa time. Mitochondria

(ì4) were added after the substrate (P/M) to the cuvette medium cont.aining phosphate since t.hese mitochondrial preparations contained no recognizable

indigenous substrate. The oxygen uptake in the absence of ADP was slow,

but the respiration rate stimulated by ADP resulted in a rate of coupled St.ate 3 respiration (see Glossary) double that found with most vertebrate muscle mitochondria (132). Upon completion of the phosphorylation of

the added ADP, Ehe 02 uptake rate dropped sharply (State 4 rate) , yielding respiratory control ratios and ADP/O ratios somewhat lower than those obtained from intact pigeon heart mitochondria in this laboratory (154). This cycle of a State 3 and following State 4 respiration rate will be called the First Period. A Second Period of coupled respiration, initiated by a further ADP addition, resulted in a faster State 3 respiration and higher RCR and ADp/O ratios. However, the mitochondria isolated from the dystrophic animals were indistinguishable by these parameters from organelles isolated from the control animal s .

Such experiments done on four animal pairs are suÍìmarized in Table 3a. (the inaividual data from these experiments can be found in

Table 3b in the Appendix). Except f.or a slightly faster O, rare in rhe -77

Figure 2 POLAROGRAPHIC RECORDS OF OXYGEN CONS1MPTION OF

MICE NO. 16 AND 17

To 2.1 ml reaction medium (described under Methods, pageJ6 ) was added 5 mM pyruvate and I mM L-malate (PM), mitochondria (M), and

33f ¡rM ADP. The dystrophic and normal mitochondria contained 509 and

533 ltC protein respectively. The numbers along the curves indicate the rates of 02 uplake in ¡rmoles 02 per min. per g protein. o z = 25oPM

LDP/O = 1.9 me/o = 2.1 RCR = 5.8 RCR = 5.'l

30 ADP

ADP/o = 2.2 ADP/o = 2.2 245 RCR = 7"0 RCR = 7.5 1 min.

Dyst r op hic No rrnql

( No.l7 ) ( N o.l6)

02 = zero Figure 2 - 79 -

Table 3a

RESPIRATION AI{D OXIDATIVE PHOSPHORYLATION BY MOUSE MUSCLE I'{ITOCHONDRIA,{ Substrate: pyruvate/malate.

lst Period 2nd Period

ADP/O Orrate P rate ADP/O Orrate P rate

Con- ; 5.1 2.3 133 599 6.6 2.4 22r LO47 tro I S.E 0.5 0.1 14 48 0.8 0.2 27 B4

Dys- x 5.1 2.0 1sB 637 6 2.5 237 113 9 trophic S.E 0.6 0.1 18 92 0 0.2 26 B4

N" S.

Data from 4 animal pairs. Values are given as means and st.andard errors.

I I Based on a t-test for paired observations (149). -80- dystrophic mitochondria during the First Period, no significant differences could be detected between mitochondria from normal and dystrophic mice. During the Second Period the 02 rates also showed no significant difference. Thus, skeletal muscle mitochondria from nor- ma1 and dystrophic mice exhibited equally tight coupling, as demonstrated by similar RCRrs and ADP/O ratios. They were indistinguishable in their oxidation rates and, as shown by similar phosphorylation rates, they had the same capacity to phosphorylate ADP. However, paired statistical analysis shows that, except for the ADP/O ratios, aIl parameters l{ere signif icantly increased during the Second Period, both in the normal and the dystrophic group. To determine whether the improvement of these parameters possibly could have been an effect of the ATP generated during the First Period, mitochondria r^iere test.ed as before, but, in an additional experiment, also in the presence of 250 pM ATP. The results obtained with 4 mice are summarized in Table 4a. As can be seen from a comparison of the means, all parameters tended to be higher in the presence of ATP. Although these animals were of considerably older age, this is not thought to have inf luenced the effect of ATP. However, the effect \^/as signif icant in only one parameËer in each Period. The possibility Lherefore remains that other factors may also have been responsible for the increased values during the Second Period.

The effect of albumin

Other workers who studied mitochondrial function in hearË failure (155) have stated that the abnormaiities they found were more clearly demonstrated the better the qualíty of their control mitochondria was -81 -

Table 4a

RESPIRATION AND OXIDATIVE PI{OSPHORYLATION BY MUSCI,E MITOCI{ONDRIA PREPARED FROM 4. CONTROL }4ICE AND THE EFFECT OF ATP Animals were 256-281 days o1d; substrate: pyruvate/malate.

lst Period 2nd Period

RCR ADP/O Orrate P rate ADP/O 0rrate P rate noi 4.0 r .9 r25 49r 5.2 2.r L51 648 ATP S.E. 0.4 0. r L4 B0 0.6 0.1 18 9L

2s0 i 4.9 2.0 L44 562 6 2 2A6 875 pM ATP S.E. 0.7 0.1 17 62 0 0 25 r28

N. S. N.S" N. S. 0 .05 tç Statistics based on a t-test for paired observations (L49) -82-

Since albumin is known to improve the tightness of coupling of oxidation to phosphorylation due to its abiliry ro bind uncouplers (f56-160), mouse muscle mitochondria \,üere also prepared in the presence of 1% albumin. Animals described in Table 2, page 75, were used for this study. Although the animals of the normal and dystrophic group were not of identical age and varied over a fairly wide age range, no correlation

\¡/as apparent betvleen age of the animals and the results in either group.

This can be seen from the individual data shown in Table 5b, Appendix.

The results are summarized in Table 5a. I¡Ihen these results are compared with those in Table 3a, page 79, the beneficial effect of a-l-bumin is quite apparent: all parameters \nrere raised Ëo higher levels. The increase was significant for all values of the First Period, except for the O, rates and P rates in the dystrophic group. In the presence of albumin the differences between the First and Second Period \^7ere no longer seen) since optimal values \¡/ere apparently already obtained during the First Period. However, even with these tighLly coupled preparations of mitochondria, no significant differences were demon- strable between the oxidative phosphorylation parameters of normal and dystrophic mouse mitochond.ria.

Mitochondrial preparations of the above group were examined under the electron mícroscope to check for possible contaminations of the mitochondria by other cel1 particles and to see whether any of the various reported abnormalities of mitochondrial structure (161-163)

\¡/ere more prevalent in the pellets from the dystrophic animals. No gross morphological differences could be detected bet\,zeen mitochondrial pellets from normal and dystrophic míce (Fig. 3). As can be seen from the electron micrographs, the mitochondria are morphologically intact -83-

Table 5a

RESPIRATION AND OXIDATIVE PITOSPHORYLATION BY MOUSE MUSCLE MITOCHONDRIA PREPARED IN THE PRESENCE OF t% ALBUMIN

Substrate: PJ'ruvâte /malate.

lst Period 2nd Per iod

ADP/O O2rate P rate ADP/O Orrate P rate

10.1 2.7 223 LL75 10.1 2.8 250 L337 Con- Ërol S.E. 0.9 0.0 26 113 0.9 0.1 138

N 6644 6 6 4

x 11.1 2.7 220 1153 9.3 2.7 246 L296 Dys- tro- S .E. 0.9 0.1 66 311 0.6 0.1 68 313 phic N 5544 5 5 4 4

* Based on a t-test for comparison of the means of ungrouped data (149). -84-

Figure 3. -- ELECTRON MICROGRAPHS 0F NORMAL AND DYSTROPHIC

}.,IOUSE MITOCHONDRIA

Preparations with 1% albumin in the suspending medium. Fixed as pe1lets. Magnification 18,000 x. Upper picture: normal mítochon- dria; lower picture: dystrophic mitochondria. - 85-

: .i3: ,*,j .t ll

.{, ,å.

##-#-'Ã' .'s'

flø ôr -86- and largely free of ce1lu1ar debris. The purity of these preparations resembles that of pigeon heart miËochondria isolated in this laboratory, except that the mouse organelles are somewhat less tightly packed with cristae.

Since the mitochondria of the dystrophic animals might be more susceptible to damage during the isolation procedure, it seemed possible that such broken mitochondria might escape isolation and not appear in the final mitochondrial pe1let. Therefore, the yield of mitochondrial protein per gram of muscle was calculated as surunarized in Table 6a. The facË that the amount of mitochondrial protein isolated from dystrophic animals \.vas not decreased, ruled out a loss of trabnormalt' dystrophic mitochondria, provided the total number of mitochondria in situ did not differ in the normal and dystrophic animals. However, it should be pointed out that this result is not very informative due t.o the great variability of the yields (see individual yields in Table 6b) and due to the fact that this method of preparation is not designed for a quantitative isolation of all the mitochondria present, but rather for good quality. Thus, the amount of mitochondria isolated might be only around 15% of the total mitochondria present in situ, if one assumes the mitochondrial concentration in skeletal muscle of mice to be similar to that deËermined in hamster skeletal muscle (see later). The problem of mitochondrial concentration in dystrophic mouse muscle de- serves further investigation by a more satisfactory method, such as that of Kleitke et al (f46).

Conc 1us ions

The results of the above studíes of oxidative phosphorylation _87

Table 6a

YIELD OF MOUSE MUSCLE MITOCHONDRIA PREPARED IN THE PRESENCE OR ABSENCE OF ALBUMIN t

Values expressed in mg mitochondrial protein per g muscle

No Albumin triíth Albumin

* Contro I 1 .2+ 0.1 (8) L.6 + 0.2 (s)

Dys trophic 1 .4! 0.2 (4) 1.7 + 0.5 (5) p + N.S. N.S

These are the yields obtained from mice No. 1-19 and from some control mice used in preliminary experiments.

Means + S.E. with the number of animals in parentheses.

+ Based on a t-test for comparison of the means of ungrouped data. -88- in mitochondria of dystrophic mice thus revealed no abnormalities.

However, although in most animals the disease vras only at a moderately advanced stage, the disease process certainly was there. It must therefore be concluded that a mitochondrial malfunction cannot be regarded as the primary cause of the dystrophic process in dystrophic mice. -89-

B. 0xidative phosphorylation by heart and skeletal muscle mitochondria

from normal and dvstrophic hamsters

Dystrophic hamsters of the BIO 14.6 strain were used in

Ëhis study. These animals develop a myopathy both in the heart. and skeletal muscle (13, L4,164,165) which is very similar to the disease process of human muscular dystrophy (13). The cause of the disease and the questíon as to whether the same primary defect causes the myopathies in both heart and skeletal muscle is unknown (47).

Oxidative phosphorylation was studied both in heart and skeletal muscle mitochondria of these animals to see whether a malfunction of the energy producing organelles could be considered as a possible cause for the disease in either organ. Meanwhile, other workers have already reported impairments of oxidative phosphorylation in heart (45, 89, 93) and skeletal muscles (44) of dystrophic hamsters.

(a) Hamster heart mitochondria

Since most of the BI0 14.6 hamsters eventually become so severely affected by the cardiomyopathy that they develop heart failure from which they ultimately die (47 , 166), it was of special interest to deLermine whether a malfunction of oxidative phosphorylation could be the primary cause of the heart disease.

Heart mitochondria T¡iere isolated by the method used success- fully in this laboratory (154) for pigeon heart mitochondria, which involves the incubation of the homogenate with a proteinase. The heart mitochondria studied were from 4 groups of hamsters. In all -90- groups the hamsters r,{ere in an earlíer stage of the disease which was manifested by heart hypertrophy but not cardiac failure. The selection of this non-terminal stage of the disease process seemed advisable in order to avoid any secondary effects on the mitochondria due to congestion and possible poor oxygenation of the tissue during failure.

Characteristics of the dvstrophic hamsters

Although the incidence of the polymyopathy in the BIO 14.6 hamsters is 100% (47), its presence Tras confirmed in three \.üays.

(i) By the occurence of the changes in body, heart, and liver weights expected in the disease. (ii) By the histological examinations of biopsies taken from the apex of each heart.. Although my experience in heart histopathology is limited, I was able to recogníze cardiomyopathy Ln 92% of the dystrophic hamsters by the characteristic histological findings (47), viz. enlarged nuclei and areas of ce1lular infilrration, overt necrosis, and occasionally calcification. (iii) By the determinatÍon of serum creatine kinase activities. These were significantly increased in the dystrophic animals of all hamster groups test.ed: Although other workers seem to imply that this reflects mainly leakage of this enzyme from the heart (105) there is no reason why a leakage of this enzyme from skeletal muscle also should not contribute as much or more to the increased enzyme activity in the serum.

0xidative phosphorylation by heart mitochondria of dystrophic hamsters with a mean age of 110 days

The characteristics of the harnsters of the First Group are summarized in TabLe 7a. The animals had a mean age of 110 days, the -9L-

Table 7a

CHARACTERISTICS OF THE HAI"ISTER GROUP 1

CPK Age Body wt. mg heart mg liver -DTT +DTT Percentage g gbody gbody ac tivation

i 110 Lr7 2.65 37.9 72 r58 220 Nor- matT s.E. 5 3 o.o5 2.5 1t 22 4 N66 55

i 109 8s 2.98 36.4 456 680 2L2 Dys - tro- S.E. 5 3 0.08 1.3 135 186 23 phic N6664655

pr. N. S. 0 . 001 0 .005 t Not*.l hamsters were of the N"I.H. random bred strain.

:l Based on a t-test for comparison of the means of ungrouped data. -92- time up to which areas of necrosis are most frequently seen in heart muscle of these hamsters (103). The animals exhibited some hearL hypertrophy as indicated by the significantly increased heart weLght/ body weight ratios. However, no signs of congestion were present as suggested by the normal liver weight/body weight ratios. The lower body weights of the dystrophic animals also indicate that no congestion

\,7as present, since later stages with congestion result in hydrops and concomitantly increased body weights of the BIO 14.6 hamsters (47). Heart mitochondria isolated from these animals were polar- graphically tested for their oxidative phosphorylation parameters in a r¡/ay identical to that described above for mouse skeletal muscle mitochondria, i."., to the phosphate-containing reaction medium the other constituents \,/ere added in the sequence: substrate (pyruvate/ malate), mitochondria, ÆP. This resulted in a State 3 -- State 4 cycle, called the First Period. A Second Period \¡Ias started by the addition of another limited amount of ADP. The results obtained ln this way are summarLzed in Table Ba. It is apparent from the values in both periods that these mitochondria rrere excellent in quality, as demonstrated by high RCRrs, oxidation rates (0, rates), and phosphorylation rates (P rates). The significance of the ADP/O ratios which did not reach maximal theoretical values (167) is presented in the

Discussion. The general intactness of the mitochondria is also demonstrated in Fig. 4, which shows high magnification electron micrographs of the normal and dystrophic heart mitochondria. The intact double membrane structure can be clearly seen and 1ittle non-miËochondrial material is present. The large empty areas between the mitochondria are caused by the method of fixation employed for these pictures: a -93-

Table Ba

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HA},ISTERS OF GROUP lYC

Substrate (pyruvaEe/malate) added prior to lst ADP.

lst Period 2nd Period RCR ADP/O Orrate P rate RCR ADP/O Orrate P rate

Nor- x 8.7 2.2 285 L269 8.9 2 4 274 r304 mal S.E 0.7 0.1 32 L45 0.7 0 0 30 L47

Dys- 9.8 2.2 280 I24B 27L L27 3 trophic 0.8 0.0 28 r29 20 97

N. S. N. S. N.S" iç Values obtained from 6 normal (N.I.H. strain) and 6 dystrophic hamsters t Based on a t-test for comparison of the means of ungrouped data. -94-

Figure 4. -- ELECTRON MICROGRAPHS 0F NORMAL (upper) AtiD

DYSTROPHIC (lower) HEART MITOCHONDRIA

Fixed as suspensions. Magnification 28,000 x. ffi -96-

concentrated mitochondrial suspension raÈher than a tightly packed pe11et, \^ras fixed in osmium tetroxide. This is in contrast to the previously described mouse preparations in which pellets were fixed. The quality of the mitochondria judged by the four oxidative phosphorylation parameters measured (Table Ba) was good in both the normal and the dystrophic hearts and indistinguishable in the tr.vo preparations.

It has been suggested (168) that the respiratory control and

ADP/O ratios may be favourably affected by the presence of indigenous substrates in the mÍtochondria. Since these ratios were determíned in the experiments just described with some indigenous substrate present, these parameters \n/ere also determined in a fresh lot of organelles after they had been depleted of indigenous substrate by the addition of ADP. This produced a brief burst of respiration which subsided in less than I min. upon exhaustion of the indigenous substrate. Residual ADP \,\7as removed by the addition of pyruvate/malate.

A second aliquot of ADP was then added to obtain the Second Period measurements summarized in Table 9a. Statistical analysis of these results also revealed no significant differences between the normal and dystrophic hamsters in any of the parameters t.ested.

I^Iith the above procedure, similar amounts of indigenous substrates were detected in both normal and dystrophic heart mitochondria.

Although the amount vras only sufficient to cause an oxygen consumption of about 100 m ¡moles O, per g of mitochondrial protein, the effects of these indigenous substrates on the results r..rere tested by a statistical analysis. A multiple comparison of the means was performed from the First and Second Period of Tables 8, which were obtained with the -97

Table 9a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HA}4STERS OF GROUP l>K

Substrate: pyruvate/malate. Second period of experiments in which indigenous substrate was depleted by addition of ADP prior to pyruvate/malate during the First period.

RCR ADP/O O rr ate P rate

Nor- ; 8.8 )? 277 L278 ma1 S.E. 0.7 0.0 4l 181

Dys- ; o7 2.3 27r 1250 trophic S.E. 0.7 0.0 15 B2

p+ N" S. N"S" N" S. N. S.

Values obtained from 6 normal (N. I.H" strain) and 6 dystrophic hamsters.

Based on a t-test for comparison of the means of ungrouped data. -98-

indigenous substrate present and from the just mentíoned Tables 9, in which the indigenous substrates had been d.epleted prior to the oxidative phosphorylation measurements. The comparison was done by utiliz]rng the q-statistic (r49). As can be seen from Table 10, rhe only differences between the 3 categories rdere found in the ADp/o ratios of

both normal and dystrophic animals. When tested for differences between the individual category means, these were found to be significant only between the First and Second Period of mitochondria not depleted from indigenous substrates. The means obtained in Tables 9 from mito-

chondria free of indigenous substrate \.,/ere not dífferent from the means obtained during the First and Second period of the experiments repres-

ented in Tables 8. Thus, it was found that indigenous substrate, except

for a minor effect on the ADP/O ratios, did not influence the results of the oxidative phosphorylation parameters. It was therefore decided for subsequent experiments to list only the results of the Second Period of respiration in which indigenous substrate had been depleted.

Respiration with DL-o(-glycerophosphate and NADH as substrates

In hamsters of Group 1 two additional substrates were tested,

DL-oc-glycerophosphate and reduced nicotinamideadenine dinucleotide (NADH) as shov¿n in Tables 11. The respiration rates with e{-glycerophosphate were very low, in accordance with the known low capacity for oc-glycerophosphate oxidation in the hearr (169). Although coupled respiration might be expected with this substrare, yielding p/o values of L-2 (167), the addition of ADP did not stimulate the respiration. Phosphorylation, undetectable by this polarographic method, mây have occurred, but the coupling was clearly 1oose. A similar observation by Hatefi et al (170) -99-

Table 10

MULTIPLE COMPARISON OF THE MEANS OF THE DIFFERENT PERIODS OF TABLES B AND 9

The q statistic was used (I49) with an o( = 0.05 critical region. Means are equal, if observed q is smaller than theoretical q.

Normal DysËrophic

q ob- theo- ob- theo- i served retical * served retical

1st Period 8.7 9.8 Tb1. B RCR 2nd Period 8.9 0.28 3.67 8.9 1.40 3-67

2nd Period Tbl. 9 8.8 9.7

lst Period 2.2 2.2 Tb1. B ADP/O 2nd Period 2.4 4.53 3.67 2.3 3.69 3.61 2nd Period Tb1. 9 2.3 2.3

lst Period 285 280 Tb1.8 Orrate 2nd Period 27 4 0.32 3.67 2lL 0.42 3.67

2nd Period Tb1. 9 277 271

lst Period L269 L248 Tbl. 8 P rate 2nd Period 1304 0.22 3.67 1273 0.24 3.67

2nd Period Tbl. 9 L278 L2sO -100-

Table l1a

RESPIRATION BY HEART MITOCHONDRIA FROM HAT4STERS OF GROUP

Substrates: DL-oc-glycerophosphate and NADH.

9 .5rnl4 DL-oc- glycerophosphate

Before ADP +245y1{, A,DP Homo . -:k 11O¡rM;NADH concn.

i l1 B 309 48 Nor- malt s.E. 1 I 7 1

N 5 5 5 5

Ë 13 9 253 31 Dys - tro- S.E. I 2 10 4 phic N 2 2 4 4

+ PI N.S. N.S. 0.005 0 .005

Concentrations of the heart homogenates, from which the mitochondria were isolated, in mg he ar t / 20m1 homogen izíng medium.

I Normal hamsLers were of the N.I.H. random bred strain.

+ Based on a t-test for comparison of the means of ungrouped data. -101- with succinate, another FAD-linked substrate, r¡7as interpreted as indicating that respiratory control \,,/as exerted only at the first phosphorylation site in the respiratory chain. However, the present rates obtained were always considerably lower than the state 4 rates with pyruvate/malate. It therefore is also possÍble that Lhe rates with

DL-oc-glycerophosphate were too low to a1low a build up of high energy intermediates to a level sufficient to slow the respiration in the absence of ADP (167). Alternatively, in terms of the chemiosmotic theory of oxidative phosphorylation (171), one must assume that at this lovr respiration rate the transmembrane potential cannot be main- tained at a high enough level for the stimulatory effect of ADP to be seen. In fact, ADP addition signifícantly decreased the already low respiration rate vüith DL-c(-glcerophosphate. Inhibition of respiration by ADP has also been described with other substrates in loosely coupled or uncoupled liver mitochondria and mitochondrial particles and has been termed trreverse acceptor controlr' (I72-174). Nevertheless, the obtained rates both with and without lÐP vrere not significantly different between the normal and dystrophic heart mitochondria. It is widely held that intact mitochondria do not utilize externally added NADH (175). However, it has been shown that the membrane integrity influences the oxidation of added NADH by heart mitochondria (L54, L76, L77). The finding of a high rate of NADH oxidation is therefore suggestive of mechanical or osmotic damage during the isolation of the organelles (L76). Mitochondrial abnormalities, if associated ioith the dystrophic process, might be expected to increase the susceptability of the organelles to such damage and therefore increase the rat.e of NADH oxidation. -toz-

The rates with NADH rrere slo\{ (Tables 11, pagelOO), amounting

to a maximum of L7% of those exhibited with pyruvate/malate (Tables Ba

and 9a, pages 93and97 ). Upon disruption of the mitochondria by

ultrasonication the NADH rates were increased by more than an order of magnitude. However, the intact isolated mitochondria of the diseased

hamsters unexpectedly showed a significantly lower o, uptake with NADH than the organelles from the control animals (Table rla). This is

clearly an indication t.hat the dystrophic mitochondrial membranes l{ere

as rrintactU as Ëhe isolated control organelles or actually were perhaps even less permeable to NADH. There are tr,/o possible explanations for

this phenomenon: (i) It has been found in our laboratory that the

NADH oxidation of heart mitochondria prepared by the Nagarse procedure

is influenced critically by the concentration of the initial homogenale.

As shown in an experiment with pigeon heart mitochondria (fig. 5), the

NADH oxidation rate progressively decreased in mitochondria prepared

from more dilute heart homogenates. This experiment was done in col- laboration with Brian Holl. There is evidence that the decreased oxi-

dation rate of externally added NADH was due to more intact membranes,

preventing NADH from reaching the respiratory chain (178). I^Ihereas this phenomenon was originally observed on pigeon heart mitochondria,

it is possible that the slightly lower homogenate concentrations, used in the hamster heart mitochondria preparations (Table 11a) of dystrophic hamsters, caused the decreased NADH oxidation rates in these animals. Thís

possibrlity was tested in an experiment in which 2 homogenates \^rere prepared which \.{ere similar in concentration to those shown in Table l1a, page100. Hearts from control animals were used on1y. Mitochond.ria prepared from the more concentrated homogenate (325 ng/20 nL) oxidized NADH -103-

Figure 5 -- OXIDATION 0F NADH AS A FUNCTION OF THE CONCEN-

TRATION OF THE INITIAL HEART MUSCLE HOMOGENATE

l4.itochondria were prepared from pigeon heart at five different homogenate concentrations according to the procedure described under

Iviethods for hamster NADH hearts. concentration: 150 FM. I 60 à L.0 e. q' 't

Ø d) o 40 E z. I a H F- o & I ä 2A v(ñ Õ C) ñt I I ö I I 0 0 50 100 CONCENTRATION (rng myocardium/ ml)

Figure 5 -105-

at rate 47 a of Émo1es Or/mín./g protein, whereas mitochondria pre-

pared from a more dilute homogenate (233 ng/20 m1) respired with NAÐH as substrate ât a rate of 30 ¡rmoles or/mirr'./g protein. Although the exact fit of this experiment with the data in Table lla might be some-

!.7hat coincidental , it appears that the decreased NADH oxidation observed is not specific for the dystrophic mitochondria, but was rather caused by an artefact during the mitochondrial isolation procedure. In subsequent experiments therefore, the heart homogenate concentration was always adjusted to 300 ng/20 ml homogenLzLng medium. (ii) The other possible explanation'is that an experimenter might uncensciously have favored a normal result from dystrophic preparations. Thus, a more careful homogenization of dystrophic muscle might leave the mitochondria from this tissue relatively more intacË than the organelles from the control tissue. The latter would therefore exhibit a faster respiration rate with NADH. Although such a bÍas seemed un1ike1y, it is nevertheless true that the isolation procedure for the mitochondria requires a number of rather subjective decisíons to be made by the experimenter, which could influence the mitochondrial quality. Therefore it was decided to have the heartx of the second hamster Group isolated by another person.

Oxidative phosphorylation by heart mitochondria from hamsters with a mean age of 160 days

Hamsters of Group 2 (Table I2a) were somewhat older, with a mean age around 160-165 days. The dystrophic animals had a pronounced increase of the heart weight/body weight ratio. The serum creatine phosphokinase activities in these animals were high and were significantly

*mitochondria -106-

Table 12a

CHARACTERISTICS OF THE HAMSTER GROUP 2

CPK Age Body wt. mg heart -DTT +DTT Percentage g g body ac tivation

x 165 118 2.58 105 180 L74 Nor- malt s.E. B 2 0 .06 10 13 L7

5 4

* 160 109 3 .63 384 1025 268 Dys- tro- S.E. 6 0.45 48 15s 23 phic 5 5

p". N.S. N.S. 0.0s 0.00s 0.005 0 .02

Ï Normal hamsters were of the LSH strain.

Js Based on a t-test for comparison of the means of ungrouped data. -L07-

more activated by the reducing reagenr dithiothreitol (DTT) (104). rt

is known that fresh tissue CPK is not activated by the reducing reagents, whereas serum cPK enzyme, whÍch is probably aged in the circulatory system, is activated by such reagents (106) . Thus, the higher per-

centage of cPK activation in dystrophic animals probably is due to a

greater amount of rtruer serum errzyme relative to the enzyme which

leaks out from the muscle tissue as a result of the decapitation through

the neck musculature. (For more details, see under Methods , page 47 ). Hamsters of Group 2 descríbed above were tested for their oxidative phosphorylation parameters with pyruvate/malate as substrate. Although these heart mitochondria were prepared by a different person than those from Group I (Tables B and 9, pages )3and97), the four parameters measured, RCR, ADP/O, orrate, and P rate showed again (Tables 13) the high quality of the mitochondria, but no differences were detected between the normal and the dystrophic group.

The effect of a hexokinase Lrap

Since other workers studying the cardiomyopathy in these hamsters (89, 45) had used a manometric technique in which the ATP formed is immediaLely converted to glucose-6-phosphate and ADP by an excess of glucose and the enzyme hexokinase (such a system is commonly ca1led a thexokinase trap'), r" used this system for the mit.ochondrial preparations of Group 2 to see whether this different. technique would yield the same results. Different values between our standard polarographic technique and the hexokinase system would be expected, if the preparations contain high ATPase activitíes. Inlhereas the presence of this enzyme would tend to lower the ADP/O ratios obtained by the polarographic -108-

Table 13a

RESPIRATION AT{D OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HA},ISTERS OF GROUP 2* Substrate: pyruvate/ma1ate.

32p/O 32p RCR ADP/O Orrate P rare O, *rare t^t"

Nor- ; 11.8 2.6 262 1336 2.4 398 Ig2L ma1 s.E. 1.6 0.1 18 79 0.1 26 1s9

Dys- i 10.3 2.5 280 1424 2.4 368 L76g trophic S . E . 0 .5 0.0 16 82 0.0 22 I20

p T N.s. N"s" N.s" N.s. N.s. N.s. N.s.

J.' Values from 5 normal (LSH) and 5 dystrophic hamsters. t Based on a t-test for comparison of the means of ungrouped data. -109- method, it should have litt1e effect ln the presence of an excess of hexokinase and thus should not lower the P,/0 ratios obtained by this system.

In this system the oxygen uptake rras measured polarographi- ca1ly, as before, but the ATP formation r^/as determined using 32p in the cuvette medium, which during phosphorylation was incorporated into ATP and consequently glucose-6-phosphate. Thus, glucose-6-phosphate formation, stoichiometrically equal to the synthesized ATP, was measured as the radioactivity which remained in the reaction medium 32P. after extraction of the irrorgrni" rne 32p/o ratios, respiration

(OZ rates ,tra rates) and phosphorylation rates (32P rates) thus obtained are also listed in Tables 13, page l0B. Once again with this techníque no significant differences \¡rere obtained between normal and dystrophic hamster hearts in any of these 3 parameters.

A statistical comparison between the values obtained with our standard polarographic technÍque and those found with the hexokinase trap present revealed no differences between the ADP/O ratios una 32p/O ratios and between the two kinds of phosphorylation rates (P rates and ') ¡) "P rates). This is a clear indication that these mitochondrial preparations were free of ATPase activity under the measuring conditions, sÍnce ATPase activity would tend to lower the ADP/O ratios of our standard polarographic technique. However, a significant increase (p<0.05) of the oxidation rates in both the normal and dystrophic group was observed in the presence of the hexokiase trap (Table 13a, 02 rate vs. 0, rate). Since the conditions of the systems differed "* by the additional presence of 5 mM l"lgC1r, 10 mM glucose, and the hexokinase itself in the hexokinase trap medium, the effects of these -110- constituents on the oxidation rates l{ere tested individually as well as in various combinations on a normal hamster heart mitochondrial preparation. In this experiment the oxidation raËe \..ras increased by

42% in the complete hexokinase trap system. The rate was increased by 14% when ADP r¿as added in moderate excess (750 ¡rt"t) so that no transitíons from State 3 to State 4 rates would occur due Lo depletion of ADP. An B% increase occurred when the subst.rate (pyruvate/malate) was added prior to the mitochondria, as r,{as always done in the trap experiments to avoid the possibly deleterious effects of substrate depletion on the mitochondria. A 9% increase of the respiration rate was observed after the addition of 5 mM Msclr. The further addition of hexokinase and glucose in the presence of Mgcl, caused another 1l% increase of the rate. This experiment thus indicated that the increased respiratory activity in the presence of the hexokinase trap

\^Ias caused by a combination of several factors. Nevertheless, the increased raLes \^/ere noL seen in normal hamst.er hearts only, but in the dystrophic as we11.

Oxidative phosphorylation by normal and dvstrophic hamster heart mitochondria -- isolation of the organelles by a different person

Hamsters of Group 3 (Tables 14) were similar in age to the previous Group 2, but the animals in general \.{ere more severeLy aÍ.- fected by the disease. The serum creatine phosphokinase activities in the dystrophic animals vrere particularly high. The newly introduced column rHeart diseaset covers all macroscopic signs of heart disease which are summarLzed by an arbitrary scale from 0 (no signs present) to 4* (severe hypertrophy with some indication of congestion). The - 111

Table 14a

CHARACTERISTICS OF THE HA]4STER GROUP 3:K

,J- CPK Age Body mg heart heartf Streaks* -DTT +DTT Percenrage v¡t. g body disease activation

,r- x L47 110 2 .87 0 0 45 92 2t6 rl s .E. 18 5 0.09 9 16 20

's- i L63 115 3.26 æ2+ 669 2o7g 37 9 'o- =3+ ric S.E . 18 6 0. 16 25I 57 6 55

p+ N.s. N.s. N.s. 0.02s 0 .00s 0.02

Values from 7 normal and 6 dystrophic hamsters. 6 normal hamsters were of the LSH strain; one r¡ras a Lakeview hamster. See these parameters for the individual animals on Table 14b, page 252.

Based on a t-test for comparison of the means of ungrouped data. -TLz- signs rÀ/ere hearL hyperLrophy, macroscopically visible scars, and spots of calcification of the heart muscle and occasional signs of true congesEion in the 1iver, or edema vrith fluids in the different body cavities. The column 'streaksr will be dealt with later under rSkeletal luluscle Resultsr. The oxidative phosphorylation parameters r¡rere studied exactly as in the previous group, except that the mitochondria were prepared by a different person. Although the quality of these mitochondria

(Tables 15) was not as good as in t.he previous group, i."., the RCRts and the ADP/O ratios were lower, there was again no significant difference detectable between normal and dystrophic hamster hearts by any of the seven different parameters of oxidative phosphorylation measured.

An interesting result was obtained from dystrophic hamster

No. 32 of Group 3 (Table 15b). Mitochondria from this heart, when measured by our standard polarographic technique, vrere completely uncoupled and respired very slowly. This abnormality \,/as not found with heart mitochondrÍa from any of the other 24 dystrophic and 26 normal hamsters studied. However, this same mitochondrial preparation, when tested with the hexokinase trap system, gave completely normal results; the low oxidation rate 73 protein (Orrate) was of ¡mo1es Or/mín./g increased 4 fold to 275 units (02 Hf rate). This finding strongly suggests that something \nrent \nrrong with the standard polarographic assay. However, with no real proof on hand, the author believes that the magnesium present in the hexokinase trap system caused this bene- fÍcial effect. This view is somewhat supported by the studies of

Bajusz (166), who finds a marked decrease in magnesium concentration in the hearts of BI0 14.6 hamsters. However, his findings were obtained - 113 -

Table 15a

RESPIRATION AND OXIDATIVE PHOS?HORYLATION BY HEART MITOCHONDRIA FROM HAMSTERS OF GROUP 3:K Substrate: pyruvate/ma1ate.

t'r/o 32p RCR ADP/O orrate P rare o2'Krare t^t.

Nor- i 6.5 2.2 27I 1168 2.5 284 L404 mal s.E. 0.6 0. 1 10 42 0.1 13 68

Dys- x 5 .9 I .8 225 926 2.5 257 1266 trophic S.E. I.2 0.4 32 204 0.0 12 56

e+ N.s. N.s. N.s. N"s" N.s. N.s. N.s"

)k Values from 7 normal (6 LSH, 1 Lakeview) and 6 dystrophic hamsters. I I Based on a t-test for comparison of the means of ungrouped data. -TL4- from very young hamsters (mean age 29 days); no information is available regarding the hamsters around 160 days of age. It also should be pointed out that the results from these heart mitochondria of hamster

No. 32 are strikingly similar to those reported below for some skeletal muscle mitochondria preparations, where a sími1ar defect could be shown to be at least partly due to an apparent magnesium deficiency.

Oxidative phosphorylation by heart mitochondria from normal and dystrophic hamsters more than 200 days o1d

Since no abnormality in heart mitochondrial function \^ras detected in animals of Groups 1, 2, and 3, a considerably older hamster group rras investigated (Tables 16) . Consistent with a mean age greater than 200 days was the finding that most of the dystrophic hearts \¡rere considerably hypertrophied and several hamsters showed signs of a beginning congestion, but norre r¡/as in the terminal stage of congestive heart failure.

The effect of a higher reaction temperature

l. I^fith the standard substrate combination pyruvate/malate

-- Mitochondria from the hearts of certain hamsters in Group 6 were also studied with pyruvate/malate as substrate, at the reaction temperature usually employed by us, and also at 37oC, since other workers

(89, 93) measured at this temperature. No decrease r¡/as found in any of the 4 parameters in the dystrophic animals at either temperature

(Tables 17) . Indeed, the 0, rates and P rates \^rere slightly but significantly increased above normal in the dystrophic group at 28oC. However, since this apparent superiority of the dystrophic mitochondria -115-

Table 16a

CHARACTERISTICS OF THE HA}4STER GROUP 6

CPK Age Body mg heart Heart S tre aks t -DTT +DTT Percentage \^It. g body d isease:t ac t ivat ion

222 L34 2.76 24 48 2L8 cr- alf S.E. 15 4 0.06 4 5 18

N l3 13 l-) 11 11 11

N X 2LL L23 3.45 aJ- 511 r47 6 516 ys- -Ll ro- S.E. 13 4 1.11 205 436 L47 ric N L2 L2 L2 11 11 11

P+ N.S. N.S" 0. 001 0.05 0 .00s N.S.

See these parameters for the indívidual animals on Table L6b, page254.

10 LSH hamsters and 3 of the Lakevier^r random bred strain.

Based on a t-test for comparison of the means of ungrouped data. -T16-

Table 17 a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HNART MITOCHONDRIA FROM HA}4STERS OF GROUP 6 Substrate: pyruvate/ma1ate.

28oc 370C

ADP/O 02rate P rate ADP/O orrate P rate

X 7 .4 2.3 209 956 7.8 2.3 409 L897 Nor- mal?k S.E. 0.3 0.0 7 34 0.5 0.1 10 77

N 8888 6 6 6 6

; a1 ^ 8.1 2.3 227 1054 8.2 449 205L Dys - tro- S.E. 0.4 0.0 4 29 0.6 0.0 L9 106 phic N 8888 7 7 7 7

* 2 Lakeview and 6 LSH hamsters. II B"sed on a t-test for compari-son of the means of ungrouped data. - LLl

did not appear at 37oC and was less than 10%, it r¿as considered to be of no great importance physiologically.

It is interesting to compare the temperature effects in the normal and dystrophic groups (Table L7a). A t-test for paired observations did not reveal any significant differences in RCR's and ADP/O ratios between 28oC and 37oC results. However, the oxidation and phosphorylation rates were significantly higher at 37oC (P<0.001) than at 2BoC. The rates \^rere nearly doubled at this temperature, i.e. in the normal group at 37oC the 02 rate was 196% that of the 28oC rate and the P rate was 198% that of the 28oC rate. In the dystrophic group the corresponding values were 198% and 195% respectively. There- fore, in this respect the dystrophic heart mitochondria were again indistinguishable from the control organelles. Although it is not per- tinent to the present study, it is interesting how similar the Q10 value of pyruvate/malate oxidation (=Z) r¡ras to most of the reported

Q1g values of single enzyme reactions (29), although a great complex of subsequent and interlocking reactionsis involved in mitochondrial oxidative phosphorylation.

2. tr^lith DL-/: -hydroxybutyrate. -- It has been known for many years that ketone bodies are utilized by the heart (28). More recently it has been shown (iBO) that acetoacetate and p-hydroxybutyrate, if available ) are preferred to glucose as substrate. One of the ketone bodies , DL-p-hydroxybutyrate> i,Ías tested under the same conditions as pyruvate/malate and the results are summartzed in Table 18a. l{ith Dl7-hVdroxybutyrate t.oo, oxidative phosphorylation of heart mitochondria was identical in normal and dystrophic animals. However, the - 118 -

Table 18a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART }.,IITOCHONDRIA FROI'{ HA},ISTERS OF GROUP 6

Substrate z DL-p-hyd.roxybutyrate.

28oc 37o c

AD?/O Orrate P rate RCR ADP/O Orrate P rate

x 6.4 2.r 168 7L5 3.1 2.0 r52 Nor- ma1:k S.E. 0.8 0.0 19 83 0.1 0.1 9

N 6666 6 6 6

x s.6 2.r L52 642 2.9 2.0 r50 s99 Dys- tro- S.E. 0.6 0.0 20 86 0.3 0.1 15 76 phic N 8888 7 7 7 7

Normal hamsters \,/ere of the LSH straín. f Based on a t-test for comparison of the means of ungrouped data. - 119 -

RCRrs and ADP/O ratios r¿ere lower than those obtained with pyruvatef malate. Also, in contrast to the findings with the latter substrate,

the oxidation and phosphorylation rates with DL-p-hydroxybutyrate \dere

lower at 2BoC and were not increased at 37oC. A partial explanation for this discrepancy is suggested by the results in Fig. 6, which shows typical polarographic tracings with Dl-p-hydroxybutyrate at

both temperatures. It will be noted that the respiration rates de-

creased considerably with each successive cycle of Stale 3 - State 4

respiration and that these decreases !üere much more pronounced at 37oC. Since ín Tables 18 only the Second Periods are represented, it is understandable why no increase in the respiration and phosphory-

lation rates had been observed at 37oC.

According to Hatefi et al (181) the observed oxygen con-

sumption with ¡3-hydroxybutyrate by heart mitochondria is only partially accouni:ed for by the /6-hydroxybutyrate dehydrogenase step; a considerable portion is accounted for by the oxidation of acetoacetate.

The rate limiting step in t.he oxidation of this compound seems to be the formation of acetoacetyl CoA, the formaËion of which involves

succinyl CoA. Hatefi et al showed that all metabolites which would

directly or indirectly lower the mitochondrial succinyl CoA concen-

trations, also decrease acetoacetate oxidation and vice versa. Thus,

Pi, AI"IP, ADP, GDP, DNP inhibited, whereas ATP and citric acid cycle intermediates activated acetoacetate oxidation. The latter effect can be explained by an increased synthesis of succinyl CoA, together with a facilitation of enhanced acetyl CoA oxidation. How the decline of. p-hydroxybutyrate oxidation is caused in our preparations has not

been explored. However, it seems possible that a decrease of indigenous -L20-

Figure 6 -- POLAROGRAPHIC RECORDS OF OXYGEN CONSUMPTION 0F

HEART MITOCHONDRIA FR0I"1 ANIMAL N0. 77 I,,IITH DL-F-HYDROXYBUTYRATE AS

SUBSTRATE

At 28oc to 1.60 ml of reaction medium (described under Methods, page 56 ) were added mitochondria (M) containing 840 lt! protein, 250 yM ADP each, and 9.5 mM DL-p-hydroxybutyrate. The experiments at 37oC ï,/ere similar, except that only 504 Pg protein of the mitochondrial suspension vere added at (I"1) . The numbers along the curves indicate the O, uptake rates in ¡moles 0, per min. per g protein. ADP f -F-oue7"""---- 02 = 252 pM me/o = 2.2 150 RCR - 4.6 ADP L - -P:'HB oz = 216 PM :¡ *DP

tnp/o 2.2 top/o 2.2 RCR 3.9 RCR 5.5 64

I ag@c H 57@ü NJ H

I mp/o = 2.1 Anp/o 2.1 173 RCR = 3.2 RCR 4.7 1 13 Ma].ate Malat e 55 I ,ADP 241 1 min.

193

zero 0^¿ = Figure 6 -L22-

substrate contributes to this effect (see the small amount of indigenous substrate detected during the First Period in Fig. 6, which could not

be depleted completely by the initial ADP addition, since the rate tends

to level of f asymptotically) . Addition of 0.3 rnlvi L-malate bef ore the

Fourth Period caused an increase of the subsequent State 3 rate (Fig. 6) but could not restore the rate to the values obtained during the First

Period. It also is possible that the intramitochondrial Pi and nucleo-

tide concentrations changed during the course of the experiments due

to exchange with the reaction medium and due to metabolization. This

phenomenon was not explored further, since it occurred both in the normal and dystrophic preparations to the same extent.

3. 0xidative phosphorylation with palmityl-L-carnítine as

substrate. -- Since fatty acids are Ëhe major fuel for the working heart (183) a substrate of this group was also included to test the

oxidative phosphorylation parameters of the above hamster heart group.

The substrate chosen lvas palmityl-L-carnitine. A small amount of malate

(0.3 rnl4) had to be added to f acilitate maximal oxidation rates with this

substance (182). The rates caused by malate alone were always less

than l0% of the State 3 rates obtained with palmityl-L-carnitine and were disregarded in the calculations of the rates. Otherwise the test

conditions r¡/ere identical to those with pyruvatefmalate at 28oC (Tables 17)

Experíments r,vith palmityl-L-carnitine vrere performed only at this tem-

perature. The results are summari-zed in Table 19a. I,Jith this substrate

al1 the oxidative phosphorylation parameters, RCRts, ADP/O ratios,

oxidation and phosphorylation rates \^/ere once again indistinguishable between normal and dystrophic hamster hearts. The results T¡iere practically -L23-

Table l9a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY .,- HEART MITOCHONDRIA FRO}4 HA}.,ISTERS OF GROUP 6 " Substrate: palmityl-L-carnitine * malate.

RCR ADP/O O2rate P rate

Nor- i B. o 2.r 220 923 mal s.E. 0.4 0.0 5 2L

Dys- x 8.8 2.L 229 965 LL U- phic S.E. 0.6 0.0 7 42

pt N.s. N.s. N.s. N.s. tr Values from 6 normal (LSH) and 5 dystrophic hamsters. t nased on a t-test for comparison of the means of ungrouped data. -L24- identical to those obtained for this hamster group with pyruvate/ma1ate,

(compare Tables L7 and 19, pages ll6and 123) except that the ADP/O ratios were signif icantly lower with palmityl-L-carnitine. This \^7as not unexpected, however, if one remembers that one of the two dehydrogenation steps of the fatty acLd þoxidation cycle is FAD-linked (29), i.e. the reducing equivalents from this step are expected to yield only 2 ATPrs per hydrogen pair rather than the expected 3 ATPrs from hydrogen pairs of NAD-linked substrates.

Mitochondrial yields

In Tables 20 the mitochondrial yields obtained from all the heart preparations are presented. The mitochondrial yield from the dystrophic hearts was similar to that from the control animals in Group 1, but. this was not the case in the anímals of the other three groups. Two explanations suggest themselves for the lower yield in the older dystrophic animals. The first of these is related to the fact that the present procedure yields only about half of the total mitochondrial content of the muscle in situ, as determined in a few animals by the method of Kleitke et aI (L46). It is therefore possible that extraneous factors might result. in a disproportionally lower yield from the dystrophic myocardial tissue. Alternatively, it is possible that the mitochondrial concentration in the hypertrophied hearts of the dystrophic animals was decreased. If Lhis were the case, it could lead to a decreased capacity for oxidative energy production, which in turn could accelerate the heart failure appearing eventually in these animals. 0n the other hand, it ís equally feasable that the postulated decrease in mitochondrial concentration is secondary to the hypertrophy -L25-

Table 20a

YIELD OF HA}.{STER HEART MITOCHONDRIA

Values in mg mitochondrial protein per g heart muscle.

Group 1' Group 2 Group 3 Group 6

; lB.1 L2.4 14.6 17 .2 Nor- mal S. E. 1.1 0.5 1.0 0.9

N 6 5 7 B

i 17.8 9.8 1r.2 L2.5 Dys- tro- S.E. L.4 0.5 1.0 0.6 phic N6 5 6 7

P'k N.s. 0 .01 0 .05 0 .001

Jc Based on a t-test for comparison of the means of ungrouped data. -L26-

This view is supported by the normal yields obtained from hamster hearts of Group 1, whích represented the youngest animals. Unfortunately

no choice can be made at present between these possibilities, but this should be possible with a method such as that of Kleitke et al (146) for measuring the true in situ mitochondrial concentration.

Summary of the results obtained from hamster heart mitochondria

Oxidative phosphorylation has been studied in normal and dystrophíc hamster hearts. The dystrophic animals all were affected by the disease process, which ultimately leads to death by congestíve heart failure, but none of the animals vrere in the terminal stage of the disease. Mitochondria from these animals were indistinguishable

from the control organelles in the four parameters of oxidative phos-

phorylation studied, i.e. - the respiratory control ratio, ADP/O ratio, oxidation rate, and phosphorylation rate. This was true both at

2BoC and 37oC and with three different substrates: pyruva]efmalate, p-hydroxybutyrate, and palmityl-T.-carnit.ine. Normal oxidative phos-

phorylation T¡ras also observed in the dyst.rophíc animals in the presence

of a hexokinase trap. The mitochondrial yield from myocardium was similar in normal and dystrophic hearts only in the young hamsters of

Group l. Significantly fewer mitochondria were isolated from the

dystrophic animals of hamster Groups 2, 3 , and 6 than from the control animals. Since considerable judgement is required during the ísolation

procedure; it was gratifying to find that the above results could be reproduced by persons other than the author. - L27

(b) llamster skeletal muscle mitochondrÍa

Oxidatíve phosphorylation by skeletal muscle mitochondria was

studied in 6 different groups of hamst.ers. The animals of Group I have been described already in TabLe 7a, page 91 , in relation to the ínvolve- ment of the heart ín the disease. These animals, whÍch had a mean age

of about ll0 days, showed significantly elevated serum CPK activities, which is suggestive of enz)¡me leakage f rom the heart and/ or skeletal muscle. However, no distinction can be made between the involvement of

these organs by this method. Therefore, muscle specimens from each anímaI v/ere examined histologically. The biopsies were taken from the lower portion of the back muscle (m. erector spinae) , from the m. gluteus, and the m. biceps femorís. In agreement with the findings of others (165)

Ít was observed that different muscles were affected to dÍfferent degrees by the disease. The typical histological findings (165) were rowing of nuclei in longitudinal sections, areas of necrosis, and, in cross sections, increased ínequalíty of fiber diameters and an increased number of central nuclei. Although the author is not an experienced pathologist, he was able to recognize the dystrophic process in all dystrophic anímals studied in at least two of the biopsies taken. In the back muscle and the gluteus the disease v/as evident in all cases and in the biceps femoris muscle 96% of the specimens of dystrophic animals were judged as being affected by the dísease. Because of variabilíty of the disease, both in extent and in severity, from muscle to muscle, it \¡/as not possible to correlate, even in a qualitative way, the mitochondrial oxidative phosphorylation results with the histological findings. LzB -

Oxidative phosphoryl-ation by skeletal muscle mÍtochondria from normal

and dystrophic hamsters with a mean age of 110 days

Mitochondria were isolated from hamsters of Group 1 using the

Standard Prot.einase Method. Electron micrographs of both a normal and a dystrophÍc preparation (FÍ9. 7) showed again no differences in gross morphology and also indicated that the organelles \^rere largely intact and free of contamination by non-mitochondrial materíal.

The various parameters of oxidative phosphorylation were Ëested polarographically exactly as has been described above for heart mito-

chondria (page 92). Table 21a sunrnarizes the results of the lst and 2nd

Periods of respiratíon in which the substrate, pyruvate/malate, r¡/as added príor to the first lot of ADP. Judged by the respiratory control ratios, high values of which are regarded as the most sensítive indícator for the intactness of mitochondria (L28, L32, L3L), these preparatíons \¡/ere of good quality and compare well with those described by other workers.

An explanation of the relatively low ADP/O ratios will be given below in the Discussion. The somewhat better quality of the mouse skeletal muscle mitochondria described above appears to be due to species differences, since mouse mitochondria prepared during the course of the hamster experiments T¡/ere consistently of a better quality. The mouse tíssue seemed to be of less tough consistency and this probably facilitated the isolation of organelles of greater integrity.

Statistical analysis of the data from Tables 21 revealed, except in one instance, no significant dífferences (p>0.05) between skeletal muscle mítochondrial oxidative phosphorylation of normal and dystrophic hamsters as reflected in the respiratory control ratios , ñP/ 0 ratios, -L29-

Fig. 7.--ELECTRON MICROGRAPHS OF NORIvIAL (upper)

AND DYSTROPHIC (IowCT) HAMSTER SKELETAL MUSCLE

MITOCHONDRIA

Standard Proteinase PreparaËion. Fixed as a suspension. Magnification 28,000 x.

-131-

TabLe 2Ta

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROUP 1

Substrate (pyruvate/malate) added príor to 1st ADp. Standard Proteinase preparation

1st PerÍod 2nd Period

ADP/0 )rrate P rate RCR ADP/0 O2rate P rate

; 4.8 2.t 2L4 879 5.5 2.3 228 I057 Nor- malf s. e. 0.6 0.1 L4 75 0.4 0.0 7 37

N 6666 5 5 6 5

X 4.6 2.0 190 7 59 Dys - tro. S. E. 0.5 0. I 27 116 phic N 6666

0. 005 N. S.

t Normal hamsters were of the LSH strain. J. Based on a t-test for comparison of the means of ungrouped data. -L32-

oxygen uptake, and phosphorylation rates. However, during the Second

Period, the mean ADP/O ratio was 9% lower in the dystrophic animals, and this proved to be statistically significant (p<0.005).

Addition of ADP to the Oxygraph cuvette before the substrate

resulted in no acceleration of 0, uptake, índicating the absence of

indigenous substrate in Ëhe mitochondria. trrlhen a Second Period of

respiration uTas then initiated by ADP in the presence of added pyruvate/ malate, the results obtained (Table 22a) were similar to Ëhose in Table ZLa. Again, no differences were found between the normal and dystrophÍc animals.

As a further test of whether the different sequ.ence of substrate and ADP additions in the experiments in Tables 2L and Tables 22 had any

effect, a multiple comparison r¡/as made of the means of all the oxidative phosphorylation parameters between the First and Second Periods of Tables

2L and the Second Period oî. TabLes 22. The result of applying the q- statistic (L49) shown in TabLe 23 indicates that the means of the RCR's,

ADP/O's,'¿ 0" rates, and P rates T¡/ere not signÍficantly different in Ëhese three periods, except for the ADP/O ratios in the normal animals. trnlhen the individual period means r¡rere tested, it was found that a difference existed only between the Fírst and Second Period of mitochondria not depleted from indígenous substrates. However, the means from the Second Period experiments with mitochondria depleted of indigenous substrates did not differ from the means of any other period both in normal and dystrophic animals. It was therefore decided in future experiments to list only the values obtained during the Second Period of experiments in which the organelles were freed of indigenous substrates. -133-

TabLe 22a

RESPIRATION AND OXIDATIVE PHOSPHORYT,ATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROI]P 1 Substrate: pyruvate/malate.

Only the Second Period of respiration is shown. During the First Period indigenous substrate was depleted by addition of AJP prior to pyruvate/ malate.

SËandard Proteinase Preparatíon.

RCR ADP/O }rrate P rate

F 4.9 2.L 2L7 907

Nor- 0.1 7 46 L t.t. 0.5 mal I N6 6 6 6

r 4.8 2.2 2L4 950 Dys - tro- S. E. 0.4 0.1 22 L37 phic N5 5 5 5

P" N"s. N.S. N. S. N. S.

-L I Normal hamsters were of the LSH strain. :k " Based on a t-test for comparison of the means of ungrouped data. -r34-

TabLe 23

MIILTIPLE COMPARISON OF THE MEANS OF THE DIFFERENT PERIODS OF TABLES 2L Æ\D 22.

The q statistic was used (L49) with an c¿=0.05 critical region. Ivleans are equal , if the observed q ís smaller than theoretícal q. Animals No. 1 and 2 were left out, because data were not obtained for all periods.

Normal Dystrophic

q q theo- theo- ob- reti- ob- retí- ; served cal X served cal

lst Period .n*,LvL' LL,., 5.3 5.0 RCR 2nd period 5.5 0.53 3,71 5.5 z.3L 3.77 2nd Period TbL.22 5.4 4.8

lst Period _.,ruL' LL.,, 2.L 2.0 ADP/O 2nd period 2.3 6.L4 3.77 z.L 3.62 3.17 2nd Period TbL.22 2.2 2.2

lst Period 224 ZL5 -.,LDL' ¿L^" orrate 2nd period z3z L.L4 3.17 226 0.76 3.77 2nd Period TbL.22 22L 2I4

lst Period ,",*-*'-- )1 934 864 P rate 2nd Period L057 2.69 3.77 952 0.99 3.77 2nd Period TbL.22 945 950 -r35-

Respíration with DL-o(-glycerophosphate as substrate

Skeletal muscle Ís composed of at least two different fiber types, red and white (184). These differ strikingly in their biochemical properties, showÍng an inverse relationship between glycolytic and aerobÍc oxidative activities (185-187) , glycolysis being more predominant in the i,¡hite f ibers (186). There is evidence that the two f iber rypes might be involved differently in the disease process of muscular dystrophy. hlhíte fibers seem to be affected first by the disease (f8B) and glycolysis is decreased (52-54, 56). Since the mitochondria of whÍte fibers oxidize

L-o{-glycerophosphate 5-6 tÍmes more rapidly than the organelles from red fibers (189), it seemed possible that. the respiration of the present mitochondría1 preparations with this substrate might reveal a change in the dystrophic muscle. If the metabolic processes of the mitochondria of the white fibers \À/ere suppressed by the dystrophic process in the same r¡7ay as glycolysis is in these fibers, one would expect a lower rate of c<-glycerophosphate oxidation by the organelles isolated from dystrophic muscle. A similar result would be obtained íf the disease had altered the muscle by a selective destruction of Ëhe whiËe fibers, followed by theír replacement by red fibers, or if the disease process would lead to an interconversion of white fibers to a fiber type with more properties of the red type. The results with *glycerophosphate are shown in Tables 24. It is apparent that Lhe rates were only slightly increased by the addition of a limited amount of ADP, but no transition from a StaÈe 3 to a State 4 rate r,Ías observed. Therefore no ADP/0 ratios and phosphorylation rates were obLaÍnable. This apparent uncoupling of the mitochondria with this substrate may well have been an artefact, since the 0, uptake rates with -136-

Table 24a

RESPIRATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROI]P 1

Substrates: Dl-oc-glycerophosphate and NADH Standard Proteinase ?reparation

9. 5 mM DL-c<'glycerophosphate

110 NADH Bef ore ADp +245 y.Nr ÃDp ¡LM

^ 57 62 25 J. Normal- S . E. 4 4 2

N 4

X 66 73 22

Dystrophic S. E. 7 7 1

N 5 5 4

pt N.S. N"S " N.S.

Normal hamsters r¿ere of the N.r.H. random bred strain. t Based on a t-test for comparison of Ëhe means of ungrouped data. - L37

this substrate were quite low. It has been our experience that coupling with other substrates, such as pyruvate/ma1ate, which normally are well

coupled, mâY be loose or absent when the rates are sufficiently slow.

Lehninger (190) has produced a similar artefact by the slow addition of ADP. Under these circumstances, 32Pi .rpt"ke revealed normal coupling of

oxidation to phosphorylation, but this was not detectable polarographi-

cally, since no clear transition from state 3 to 4 occurred. (This phenomenon is also discussed under oc-glycerophosphate oxidation in heart miLochondria, page 98 ). As can be seen from the statistics in Table 24a, the skeletal muscle ot-glycerophosphate rates were not significantly differenË in the myopathic hamsters and the control animals. However, the means \,{ere slightly higher in the dystrophic group. This seems to be consistenË with the results of Lochner et al (44) who find glycolysis in skeletal muscle of dystrophic hamsters of older age to be íncreased. These findings would appear to point t.or^rard a relative increase in the dystrophic hamster in white fiber numbers or in the type of metabolic actÍvities usually associated with this fiber type. This also seems to be supported by the macroscopic appearance of these muscles which usually look paler than the red muscles of control animals. However, no direct evidence is available so far for a change toward an increase in white fibers. If this should prove to be the case, it would be just opposite to the findings reported in the inherited dystrophíes of mice and men (52-54, 56,188) -138-

Respiration with NADH as substrate

For the same reasons as r¿ere outlined above for heart mitochondria of Group 1, page 101, the skeletal muscle mÍtochondria of thís

hamster group were tested for their permeabiliËy to NADH as measured by

the respiration rates with externally added NA-DH as substrate (Tables 24).

The mean values for normal and dystrophic animals were and 22 25 ¡nnoles 02/mín./g protein respectively. These low rates r.dere consistent with the structural integríty of these organelles seen in the electron micrographs (f ig. 7, page130 ). I¡lhen these organelles were disrupted by ultrasonication, the NADH oxidation rates increased more than 15-fold.

Since our polarographic measurements of mitochondríal oxygen uptake represent the average behavior of a huge number of single organelles, it should be pointed out that the observed oxidation rates with

NADH are quíte compatible with the concept that intact mitochondria are essentially impermeable for NAJH (L75). Thus, the observed rates coul-d mean either that the average mitochondrion had been slightly damaged during the isolation procedure or that most of the mítochondrial membranes were íntact and impermeable for NADH, but a few were extensively damaged.

Tissue mitochondrial content of normal and dystrophic hamster skeletal mus cle

The method of isolating mitochondria used for the foregoing experiments I¡/as designed to yield preparations which were largely free of broken organelles and cellular debris. However, the successive centri- fugations necessary to remove the debris entailed the loss of much of the original mitochondrial- content of the tissue. The extent of these losses \¡ras assessed by a method described by Kleitke et al (L46). In this -L39-

procedure the proteín concentrations and succinic dehydrogeriase actívities are determined in the final mitochondrial preparation and also in a por-

tíon of the tissue from which the mítochondria are isolated. Since suc- ciníc dehydrogenase is located entirely within the mitochondria (191), it is possible from the above measurements and the tissue weight to calculate

the mitochondrial protein content per gram of tissue. The results are

surnrnarized in Table 25a, The succinic dehydrogenase activity expressed

per mitochondrial protein was similar in mitochondria of normal and dystrophic skeletal muscle, a finding which agrees well with Lhe literature (53, 54). Also it may be seen from Table 25a that the mitochondrial content of skeletal muscle was identÍca1 in normal and dystrophic animals.

This result therefore does not support the possibility mentioned above that the disease process brÍngs about an íncrease in the red to white fiber ratio. If this were the case one would have expected an increase in the calculated mitochondrial content, since muscles containing predominant- ly red fíbers have a greater mitochondrial content (189). Since our isolation procedure for the organelles gave a rather

1o¡¿ mitochondrial yield (only about L8% of. the total mitochondrial Ëissue content could be recovered in the final suspension of the organelles), it

is possible that abnormal mitochondrÍa might have selectively escaped Í-so-

lation. However, the essentially identical yÍeld obtained from normal and dystrophic animals of Group I hamsters (Tables 26) does not support this possibility. In suurnary, aLL the oxidation and phosphorylatíon results with pyruvate/malate, oc-glycerophosphate, and NA-DH were essent.ially identical in the nor:rnal and dystrophic animals. Sínce the disease \^ras histologically recognLzable in at least two muscles of each of the dystrophic animals, it -r40-

Table 25a

SUCCINÏC DEH\DROGENASE (SDH) ACTIVITY AND TISSIIE MITOCHONDRIA CONTENT IN SKE'LETAI MUSCLE OF HA.IVISTERS OF GROUP 1

Calculated mitochon- SDH activíty dríal tissue content (pmoles/g mit.prot./min) (mg mit.prot./g tissue)

x S.E. Nor- t LLe!5 9.r t 1.0 :k mal N 4 4

Dys- x t S.E. 122L2 9.L ! L.I trophic N 4 5 pt N"S. N.S.

" Normal hamsters'were of the N.I.H. random bred strain. t g"s"d on a t-test for comparison of the means of ungrouped data. -L41-

Table 26a

YIELD OF HAMSTER SKELETAL MUSCLE MITOCHONDRIA

Values in mg miËochondríal proteín per g muscle. Proteinase Preparationsx

Group I Group 2 Group 3 Group 4 Group 5

E 1.5 L.4 L,4 1.0 1.5

No5- s. o.z 0.2 0.1 0.1 ma-L u. 0.1

N6 5 6 6 5

x L.6 1.1 1.5 0.9 1.3 Dys - tro- S. E. 0. 1 0.2 0.0 0.1 0.1 phic N6 5 5 5 9

J- pr N.S. N.S. N"S. N. S. N.S"

:k In Group I-4 the Standard Proteinase Preparation was used; in Group 5 the ModÍfíed Proteinase Preparation has been employed. t Based on a t-test for comparison of the means of ungrouped data. -L42-

wasconcluded that the dysËrophic process in skeletal muscle of the poly- myopathic hamsters of strain BI0 14.6 is not caused by an impairment of mitochondrial oxidative phosphorylation. This conclusion was, however, not consistent with the findings

of Lochner et a1 (44) who reported partially uncoupled skeletal muscle mitochondria in dystrophic hamsters of the related BI0 1.50 strain (13).

It r¡as therefore decided to investigate some of the possible reasons for this discrepancy.

In additíon to using hamsters of a strain (Bl0 1.50) which differed from ours, Lochner and her co-worker s (44) employed techniques for ísolating the mitochondrÍa and for testing them whích were quÍte un- like those employed in Ehe present work. Thus, they used no Nagarse proteinase in preparing the organelles (terrned the Lochner Preparation in this thesis) and measured oxidative phosphorylation by a manometric technique at a temperature (37. 5oC) higher than the 2BoC used in our standard polarographic procedure.

Since a number of subjective decisÍons are requíred from Ëhe experimenter in the course of the isolation procedures of mitochondria, the organelles of the following group were prepared by a different person. oxidative phosphorylation by skeletal muscle mitochondria from hamsters with a mean age of 160 days. Isolation of the oreanelles bv a different pers on

The animals of hamster Group 2 (Tables 12, page 106 ) had a mean age of about 160 days similar to that of the animals used by Lochner et al (44). The present dystrophíc animals were all affecred by rhe disease and had elevated serum creatine phosphokínase activities and hearË -L43-

hypertrophy' From these anímals muscle mítochondría were prepared by the SÈandard Proteinase Preparation as before, but by a different experimenter.

The results are sur¡rnarized in the firsË four columns of TabLe 27a. Judged

by the respiratory control ratios, these preparations \^/ere inferíor in

quality to the preparations from the First Group (Tables zL and zz, pages 131 and 133 ). This could not be attributed solely to the different experimenter, since the other experimenter (the auËhor) also was not able

to isolate mitochondría with higher RCR's from these hamsters. Since mouse muscle mitochondría prepared wíth Ëhe same technique at the same time gave high respiratory control ratios (:l ), Ehe rather poor values in Table 27a cannot be attríbut.ed to technical errors or inadequacies. Thus, it would appear that the discrepancy between the quality of the organelles in Table 27a and those in Tables 2L and 22, pages 131 and 133 , were due to some unidentified differences between the two groups of animals.

The respíratory control and ADP/0 ratios were similar in the organelles of the dystrophic and norrn¿Il animals (Table 27a). However, the respiration rates were significantly lower in the dystrophic animals (p<0.02), Since the 0, rates is a factor which contríbutes to the calculation of the phosphorylation rates (L32> the latter \¡/ere also decreased in the dystrophic animals (p<0.05). The lowered respiration rates suggest a defect somewhere between the entry of substraËe into the dystrophic organelles and the final acceptance of electrons from the respiratory chain by oxygen.

The effect of a hexokinase trap

However, it is apparent from the last three colurnns oÍ. TabLe 27a -r44-

Table 27 a

RESPIRATION AND OXIDATWE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FRoI"l HAMSTERS OF GR0UP 2 Substrate: pyruvaËe/malate Standard Proteinase preparation

Polarographíc Method Hexokinase Method J¿P/o 32P ADP/ O O2rate P rate O2gKrate ,utu

Nor- f 2.9 2.0 2t3 857 2.0 196 774 ma1 S. E. 0.2 0.0 11 37 0.1 13 76

Dys- ï 2.9 2.L L48 629 2.3 247 TL52 t ro- phíc 9.6. 0.2 0.1 t7 87 0.1 23 105 J- Pr N. S. N. S" 0.02 0.05 0 .025 0 .02

Values from 5 normal (LSH) and 5 dystrophic hamsters. T I Based on a t-test for comparison of the means of ungrouped data. -L45-

that the above defect \^/as not seen when the oxidative phosphoryl-ation parameters T¡lere tested in the presence of the hexokinase trap (descrÍbed

in connection with the heart mitochondria of hamsters of Group 2, page 107 ). The same dystrophic organelles which in the standard. polarographic method showed abnormal 0, and P rates exhibited significantly higher 32p/O ratios as well as beËter phosphorylation rates than the cont.rol mitochon-

dria. It is interesting that in the normal group the results obtained by the two methods did not differ significantly, r.{hereas ín dystrophic aní- mals respiration and phosphorylation rates T,rere considerably higher when measured in the presence of the hexokinase trap (p<0.01). Unfortunately, Lhe all important question, as to whích of the opposing results obtained with the dystrophic organelles is relevant in vivo, cannot be answered.

rt can be said, however, that. a dífference exists between normal and dystrophic muscle, since mitochondria Ísolated from the two different tissues behave differently in four of the seven parameters of Table 27a. clearly, the reaction medium used in our standard technique, whÍch was designed to give good results with miËochondria from normal muscle, did not give optimal values with mitochondria isolated from dystrophíc tissue.

Conversely, the reaction medium which included the hexokinase trap seemed to have a composition better suited for measuring oxidative phosphorylation in miEochondria from dystrophic muscle than ín organelles from normal muscle. The most likely constituent responsible for these dÍfferences l-s the MgCl2 added with Ëhe hexokinase trap, buË which is not present in the standard reaction medium. supporting evidence for this view will be presented below,

As was the case with Group 1, the mÍtochondrial yield from the above hamsters of Group 2 did not differ significantly in the normal and -L46- dystrophic animals (Tables 26, pagell¡l )

Oxidative phosphorylation by skeletal muscle mitochondria from severely affected dystrophic hamsters. Comparison of differen.t methods of mitochondrial preoarations

llamsters of Group 3 (Tables 14, page 111) were similar Ín age to those in Group 2. However, Ëhe skeletal muscle T¡ras more severely affected by the disease. This l^ras macroscopically visible in the forn of white streaks, which T¡/ere seen within the muscles and did not have the consistency of fat tissue. No specifíc muscl-e groups appeared to be prefer- entially involved. Bajusz (103) has stressed that these streaks are not limited to animals of a particular age. Histologically these areas appear to be severely necrotic with numerous infiltrative cel1s, but contain very little connective tissue (see Fig. 108, page 191). The majority of these infiltrative ceLls appear to be macrophages (L92).

For comparative purposes, the extent of the streaking r¡/as recorded f or all animals, using an arbitrary scale of 0 (no streaks) to 4+ (several muscles completely necrotic). The average grade assigned to the dystrophic animals of Group 3 (Tables 14, page 111 ) was 3*. Apart from having more severely affected skeletal muscles, these animals were similar to those in Group 2.

1. Standard Proteínase PreparatÍon

The oxidative phosphorylation parameters of mitochondría prepared by the Standard Proteinase Preparation are surnrnarízed in Table

ZBa. They were tested both by the standard polarographic procedure and -L47-

Table 28a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKE]fiTAL MUSCLE I'{ITOCHONDRIA FROM HAMSTERS OF GROUP 3T Substrat.e: pyruvate/malate Standard ProteÍnase Preparation.

Polarosraphic Method Hexokinase Method 32p/o 32P ADP/O Orrate P rate . o rate rate 2HK

x 2.5 r.8 191 679 L.9 L77 673 Nor- S.E. 0.1 0.1 14 78 0.1 5 38 maltk N 66s5 6 5 5

X 1.6 0.6 104 193 2.r r95 813 Dys- tro- S.E. 0.4 0.4 22 LzI 0.0 8 45 phic N 555s 5 5 5

0.05 0.02 0.02 0.01 N. S. 0 .05

f _), I The values obtained from anímal No. 30 (See Table 28b) \^/ere not included in the statistical calculations.

:k In the normal group were 6 LSH and 1 Lakeview hamster.

T Based on a t-tesË for comparison of the means of ungrouped data. -L4B-

in the presence of the hexokinase trap. The four polarographically

pararneters obtained: RCR, ADP/0, 02 rate, and p rate T¡rere all significantly decreased in the dystrophic animals. However, in the presence of

the hexokinase trap this d.ifference \¡/as absent (32p/o and 0, rate) and, ¿ ,.,,T]K the phosphorylation rates 132P rates) were actuarry slightly higher in

mitochondrÍa from the dystrophic animals than in those from the normal hamsters.

It should be noted thar in 3 of the 5 dystrophic animals,

oxidative phosphorylation was completely uncoupled (Table z9b). I4lith

these 3 animals (No. 26, 27, and 34) oxygen uptake \^zas noË inf luenced by

the presence or absence of Ëhe phosphate acceptor ADp. However, more

strikingly, the respíration rates in these animals r¡/ere greatly decreased

below normal, It therefore is possible that the apparent uncoupling was at least in part caused by the low respÍration rate, í,e. phosphorylation may well have occurred, but the respiration rate vTas too slow to cause accumulation of a sufficient concentration of high energy intermedÍates

to slow the respiration rate down. It was also observed that the rate of oxygen uptake decreased during the course of each polarographic determination (4-10 min. ) in these uncoupled dystrophíc miLochondria. This is

suggestive eÍther of a rapid deterioration of the complex organell-e structure or of a leakage of one of the more soluble compounds, such as cytochrome c or NAD (193, L47 ) from the mitochondria. However, neither cytochrome c nor NAD added during the course of a polarographic experiment improved the respiration rates appreciably. -L49-

Recoupling of uncoupled oxidative osphorylatíon in dystro ic hamster

mitochondria bv l4eC1

since the respiration rates of animals No. 26, 27, and 34 were within the normal range in the presence of a Mg+2-containing hexokinase

trap system (Table 28b), the eff ect of 5 mM tutgclZ was tesred. Addirion

of MgClt during the course of a polarographic experiment to uncoupled dystrophic mitochondria usually caused a slight increase in the respiration rate. However, in spite of many trials, it was never possíble Lo reach the high oxygen uptake rates obËained when these mÍtochondrÍa were tested in the presence of the hexokinase trap. In two dystrophic animals,

2 r4"dintErophenol stimulated the Iow respiration rates of the uncoupled mítochondria, although phosphate and ADP added earlier in these experiments did not stimulate the respiration rate. Thus, in one of these animals (No. 56, Table 34b, page 273), the low respiration rate in the

Presence of phosphate and ADP (33 ¡rmoles O2/mi-n./g mitochondrial protein) was increased to 125 ¡moles/min. /g by the addirion of 15 FM 2,4-dLnL- trophenol. In terms of the chemical theory of oxidative phosphorylation, dinitrophenol uncouples oxidation from phosphorylation by causing the hydrolysis of high energy intermediates and thus results in a respíration that can proceed ín the absence of phosphare and ADp (L67). since ín animal No. 56 dinitrophenol increased the respiration rate after phosphate and ADP had failed to do so, it would appear thaE the earlier steps in the coupling between oxidation and phosphorylation actually were intact but that the final step in ATP formation which is ug+2 dependenr (lg4) was limiting.

As noted above, the addition of MgCl, during the course of the -150-

polarographic determination produced a definíte but rather slight

increase in respiration. In consideríng lhe possible reasons for the

much more striking effect obtained in the presence of the hexokinase

trap, it was noted that in the latter experiments, l¿gclz was added Ëo

the medium before the mítochondria. This situation therefore was

simulated with our standard polarographic technique in an experiment

shown in Fig. 8. Part A shows the Oxygraph tracing with mitochondria from a LSH hamster. The vaLues obtaÍned are within the range of the normal hamsters of Group 3. Part B shows the respiratory behavior of mitochondria from a dystrophic hamsterr T9 days old, with streaks rated aL 2+. The oxygen uptake curve in the latter \¡/as very similar to that found with uncoupled mitochondria of animals No. 26, 27, and 34 (Table 28b) i. e. the respiration rate during Ëhe First Period was slow, no distinct

cutoff occurred at the point when ADP should have been depleted (compare with curve A). ALso, the further addition of ADP was without effect, the respiration raLe decreased progressively, and addition of 5 mM MgCl2 caused only a moderate increase of the respiration rate but did not restore the rate to normal. Curve C shows an identical experiment with mitochondria from the dyst.rophic anímal. However, in thÍs case t"lgclz was added prior to the mitochondría (M) Ëo simulate the situation in hexokinase trap experiments. The eff ect r,ras striking. Apart from a slightly poorer First Period, the behaviour of the dystrophic mitochondria was indistinguishable from that of the control mitochondría

(curve C -VS. curve A). In curve D the ef f ect of adding WCLZ prior to the mitochondria was tested to see whether this would also improve the performance of the control organelles. This was not the case. On the contrary, the ADP/0 and respiratory control ratios were lower (curve D -151-

Fig. B. --POLAROGRAPHIC RECORDS OF OXYGEN CONSUMPTION OF NORMAL

AND DYSTROPHIC HAMSTER SKELETAL MUSCLE MITOCHONDRIA

AND THE EFFECT OF MgCl2

A. To 1,55 ml of reaction medíum (see under Methods, page 56 ) 1318 FC protein of normal mitochondrial suspension (M) were added, 2L6 ¡rM ADP at indicated points, and 5 ûì14/1 nM pyruvate/malate (PM).

B. Experimental conditions as in A. , except LL7 5 pg protein of a dystrophic mitochondrial suspension was added.

C. Experimental condítions as in 8., except the reaction medium contained 5 mM MgC12.

D, Experimental conditions as in 4., except the reaclion medium contained 5 mM MgClr.

The numbers along the curves indicate the rates oÍ. 02 uptake in ¡rmoles 02 per min. per g protein. The ADP/O and respiratory control ratios (RCR) for the First, Second, and Third Period (I, II, III) are listed below each curve. ADP .t I Á.DP PM **Sa-U ADP_pur -?LBpWoa--- 2

122

A. CONTROI B. DYSTROPHIC C. DYSTROPHIC t H + MgCl,, (Jl usclN lv I 1 min. 162 -¿_ 0^ Z1TO 57 ---¿ = A. r . ADP/o 1.7 B. uncoupled. c. r . mp/o 1.4 D. r . tnp/o r.3 RCR 2.6 RCR 2.1 RCR 1.9 rr. ADP/O 1.8 rr. tor/o r.B rr. me/o t.6 RCR 3.0 RCR 2.8 RCR 2.4 rrr. ÃDP/o r.B rrr. tw/o 1.8 RCR 3.3 RCR 2.9

Figure B -153-

yq. curve A), indicating a slightty looser coupling. Thís was consistent

with the higher State 4 respiration rates of 64 and 61 units vs. 54 and,54 units in curves D and A respectively. The results shown in FÍg. B, which have been confirmed in several additional uncoupled mitochondrial preparations from dystrophÍc animals, allow three conclusions:

(i) There seems to be r Mg+2 defÍciency in dystrophic miro-

chondría , relative to the control organelles, when the organelles are

isolated by the Standard Proteínase Procedure.

(ii) This relative Mg+2 deficiency in dystrophic mitochondria seems to be responsibl-e for the discrepancy between the results obtained with our standard polarographic technique and those found in the presence of the hexokinase trap (Tables 27 arrd 28).

(iii) i-e+2 def iciency limiting the rate of the f inal step of oxidative phosphorylatíon in which ATP is formed, could explain the observation that dínitrophenol increased the respiration rate in some of the apparently uncoupled preparations from dystrophic anímals. Not all the effects seen with lrtg+2 can be explained in Lhis way, since stimulation of respiratíon by dinitrophenol was not consistently seen viith all uncoupled mitochondrial preparations. However, al1 such organelles responded

L') well to Mg'" if it was added prior to the mÍtochondria. This has been confirmed in dystrophic animals between 72 and 2L3 days of age. It is uncertain why various preparations of uncoupled mitochondria respond diff erently to dinitrophenol , r.vhí1e all are st.imulated by tfg+2. Perhaps the dinítrophenol response is dependent upon the severity of the tutg+2 lack.

In a personal communication, Bajusz (103) has stated that he has -Ls4- observed a decreas.d Mg+2 concentration in skeletal muscle of BI0 L4.6 hamsters. It therefore seems possible that a defect in Ugf2 metabolism could lead to loose coupling or uncoupling of mitochondrial oxidative phosphorylation. The failure of sufficient -P formatíon might then resulË in the necrotic areas which are visible macroscopically as white streaks, However, in his personal corrumrnication Bajusz has not revealed at what stage the Mg+2 concentration drops. It also is still uncertain whether the mitochondrial defecË which is detectable in vitro and whích

L, is cured by l4g'' addition, does occur at all in vivo. Furthermore, it is not known at present at which level of metabolism the l,tg+2 aeticiency might produce a defect.' For example, the mitochondria, if involved, might be only secondarily affected by low ltg+2 levels in the cell. Alternatively, mitochondria themselves might be defective and unable to accumulate sufficient MgT''ô to meet the needs of the íntramitochondrial enz)¡mes requíríng this ion.

2, Standard Proteinase Preparation with Albumin

DefecËs of mitochondrial oxidative phosphorylation similar to those described above could theoretically also be caused by the presence of uncouplers. These could be localized extramiLochondrially in the ceI1 in situ and might exert their effects only after the tissue structure r¡ras disrupted during the homogenization steps of the isolation procedure. To examine this possÍbílity, albumin, which is known to bind uncouplers such as fatLy acids (159, 160) , was added during the final 2 steps of the Standard Proteinase Procedure. In such experiments, a portion of the mitochondrial preparation was completed wit.hout albumin and this provided the control observations already seen in Tabl-es 28. Albumin was added to -155-

a concentration of L% to anoËher portion of these preparatíons. The

resulting findings are shown in Tables 29. As expected (f56-f60), the addition of albumin raised all seven parameËers to higher values both in normal and dysËrophic animals. Hor¡ever, the differences between normal and dystrophic animals observed in Table 28a, pageL47, persisted in the first four parameters obtained by the standard polarographíc technique. In the presence of the hexokinase trap tne 32p/O ratios (Tables 29) of the albumin.treaËed dystrophic mitochondria were improved and approached

in activity that of the organelles shown in Table ZBa, page L47. The respiration rates also increased considerably in the presence of Lhe hexokinase trap. However, these improvements hTere seen in the albumin- treated organelles from both normal and dystrophic animals and consequently

the difference in respiration rates persisted (Table 29a, p10.005) . Although the "P/0?,) ratios r¡rere simílar in the normal and dystrophic animals, the lower respiration rates in the dystrophic organelles

(co1-umn 6, Table 29a) resulted in significantly lower phosphorylation

¡) rates (t'P') rates, p<0.01). Thus, pretreatment wíth albumin was able to eliciL a difference between the normal and diseasedanimals ín the

presence of the hexokinase trap (Tables 29) which T^ras not detectabLe when albumin was omitted (tables 28, pagel4T ).

Two explanations suggest themselves for the different behaviour of the organelles in Tables 28, pagel4T and Tables 29, in the presence of the hexokinase trap:

(i) In mitochondria prepared in the Presence of albumin, the appeared to be caused by defect(s) seen in the dystrophíc animals ^W*2 deficiency which the Mg+2 in the hexokinase trap medium did not correct completely. - 156 -

Table 29a

RESPIRATION AND OXIDATIVE ?HOSPHORYLATTON BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROI]P Substrate: pyruvate/malate Standard Proteinase Preparation v/irh l-% albumin

Polarographíc Method Hexokínase Method i2 32 ADP/ O O2rate P rate --P/0 O2"rarate "-P rate

:; ^ tL.g 2.4 249 L1B2 2.5 315 1 586 Nor- + Þ.^ -t1 . 1.8 0.1 13 s0 0.1 15 89 mall N 7766 7 6 6

x 5. 3 L.7 t57 65L 2.5 243 L204 Dys - tro- S. E. L.7 0. 4 33 L69 0.1 10 50 phic .l\-f 6655 6 5 5

0.025 N"S. 0.025 0.01 0.005 0.0r t The normal group consísted of 6 LSH and I Lakeview hamster. Based on a t-test for comparison of the means of ungrouped data. -L57-

(ií) In addítion to the mg+2 deficiency a second different defect \^ras present, which v¡as elicited oni-y at the higher respiration

and phosphorylation rates seen when the mitochondria r.,Jere prepared in the presence of albumin.

No experimental evidence is at hand which permits a choice between these explanations. If, as suggested above, albumin merely afforded protection from uncouplers which are active only in vitro, then it seems likely

that a second defect, in addition to a possibl" Mg+2 deficiency, might have been present in the dystrophic organelles. However, it is probably an oversimptifícation to assume that albumin acts only by removing un-

couplers, since under these circumstances one would not have expected the higher 0, uptake rates in the organelles pre-treated with albumín, as seen Ln Table 29a. For these reasons, it is diffícult to assess the relevance of these findings in vivo, although they point out the importance of examining further the possibility of the two defects suggested by a comparison of the findings in Tables 28a anð. 29a.

3. Lochner Preparation

The above abnormal findings wíth dystrophic hamsters confirm the decreased P/0 ratios found by Lochner et aI (44) in hamsters of strain BI0 1.50 related to the one used in this work (BI0 f4.6). However, theír results were obtained with animals whích, judging from their report, may not have been affected as seriously as some of the present animaLs of

Group 3. Thus, it seemed possible t.hat the method which these workers used to prepare mitochondria might in some way have been able to reveal -IsB-

abnormalíties at an earlier stage than the Nagarse procedure used to isolate the organelles for the experiments reported above. In the

experiments which f ollow, the mitochondria \¡Iere prepared by a method identical wíth that of Lochner et al (44), in as far as it was possible to work from the description of their method. It differs from the Standard Proteinase Procedure (see Methods, page J{ ) in the following r¡rays: (Í) an a1l-glass homogenizer replaces a Teflon-glass homogenizer,

(ii) the composition of the homogenizing medium differs in that mannitol

and the Nagarse are omitted and sucrose and EDTA are used in higher

concentrations, (iii) the whole procedure is 35 mín. shorter than the

Standard Proteinase Preparation which takes about B0 minutes. The details

of the Lochner Procedure may be found in the Methods section.

Fig. 9A shows an electron micrograph (10,200x magnification) of a section of a skeletal muscle mitochondrial pellet obtaíned by the

Lochner Procedure from an LSH control hamster. This may be compared with

a pellet made by the Standard Proteinase Procedure using muscle from the

same animal (nig. 98, at the same magnification). Two things are strik- ingLy different in these preparations:

(i) The Lochner Preparation (¡'ig. 9A) shows a hígh degree of

contamination by non-mitochondrial material, whereas very little is seen

in the Standard Proteinase Preparation (Fig. 9B).

(ii) The average mitochondrion is considerably smaller in the

Lochner Preparation, although the organelles were isolated from the same

muscle mince of the same animal and an ídentical magnification is shown. Only a few of the larger organelles of the Lochner Preparation are comparable in size to the average in Fig. 98. It is possible thal the lesser average mitochondrial díameter in FÍg. 9A is the result of -L59-

Fig. 9. --H,ECTRON MICROGRAPHS OF. NORMAL HAMSTER

SIGLETAL MUSCLE MITOCHONDRIA. COMPARISON

0F THE LOCIil\ER PREPARATION (4, upper) trrIITH

THE STANDARD PROTEINASE PREPARATION (8, lower) Fixed as pellets, I4agnificatÍon 10,200 x. - 160-

& ,ffi *@

#,P wwe ffiww ;&e w* -L6L-

reformation of smaller t'organellesil from fragments produced by the

intense trauma to which the tissue is subjected by glass-on-glass homo-

genization in the Lochner method. This Lendency of mitochondrial mem-

branes to reform organeLle-Iike vesicles after disruption is known Ëo occur after ultrasonication (195). Another interesting observation, best seen in Fig. 98, Ís that

the majority of the organelles shor,^/ very electron-dense inner membranes and cristae with clear areas in between, which represenE the inter-

membranal and intracristal space. Also, the outer membrane of such

organelles seem to be separated by a greater space than ís usual1-y seen

in familíar pictures and diagrams (see for example ref. 131). A few

organelles showed yet another picture. Their appearance r¡ras more uni-

formly grey and they have no clear areas. Ho$zever, cristae are clearly

recognLzable and the outer membrane always closely surrounds the organell-e.

It is now known from the work of Hackenbrock (78,79) that these trúo different morphological appearances represent mitochondria in different

metabolic states and both are trnormalrr. Thus, the eLectron dense organelles described above are seen in respiration State 3 (128) and are

called by Hackenbrock (79) the rcondensed' conformation, whereas in State

4 one sees maínly the uniformly grey type, which Hackenbrock names the

rorthodox' conformation. Since these variations have only recently been recognized as being normal variants, this raises the possibility that some of the reported morphological 'rabnormalities't of mitochondria

reported previously in dystrophic muscle (70,74) may have been due to a failure to recognize the different conformational states possible in normal organelles.

The Lochner Procedure r^7as used to isolate mitochondria f rom -L62-

Group 3 hamsters for oxidative phosphorylation studies. The characËer-

istícs of these animals are shown in Tables 14, page 11I. The seven

parameters of oxidative phosphorylation measured are surtrnarízed in Table 30a. These were clearly not significantly different in the dystrophic

group from those of the norrnal animals. Surprisingly, the apparent Mg+2

deficiency which r¿as detected by the Proteinase Preparation in dyst.rophic

hamsters of this Group r.^zas not observed Ín these organelles isolated by

the Lochner Procedure. The reason f or Ëhis is not understood. Horrrever,

it is possíble that the omission of a washing step from the Lochner Procedure, as compared with the Standard Proteinase Procedure, is respon-

síbte for the observed dífference in apparent Mg+z deficiency in the latter preparations. Considering the rough treatment the mitochondria receíve in the glass-on-glass homogenizer in the Lochner Procedure, these organelles

exhibited surprisíngly high respiratory control ratios. On the other

hand, the ADP/O ratios were very lovr. However, in the presence of the

hexokinase trap these values were raised to somewhat better levels. This finding might be explained by the presence of a relaLively active ATPase

in these preparations. This would tend to lower the ADP/O ratíos obtained with the standard polarographic technique because the newly synthesized

ATP would be hydrolyzed to generate more ADP, with the result Ehat a

considerably larger 02 consumption would result from a given initial ADP addition. However in the presence of the hexokinase trap, the ATP formed would largely be converted into glucose-6-phosphate and so escape hydrolysis by ATPase. The respiration rates ín Table 30a appear to be very low, when compared, for example, wíth the rates found with normal organelles prepared with the Proteinase Procedure (TabLe 28a, page L47). However, the rates - 163 -

Table 30a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKEI,ETAL MUSCLE MITOCHONDRIA FROI4 HAMSTERS OF GROUP 3

Substrate: pyruvate/malate. Lochner Preparation.

Po l arogr aphic lule thod Hexokinase Method 32P ADP/O Orraxe P rate 0 rate t^t" 2HK

i 4.3 L .7 r27 487 2.2 186 827 Nor- rnalf S . E. 0 .7 0.3 20 96 0.1 L9 108

N 7777 7 7 7

X 4.0 L .7 rr7 471 ?2 163 767 Dys- tro- S.E. 0 .8 0.3 23 82 0.1 4L 224 phic N 66s5 6 6 6

t In the normal group were 6 T,SH and I Lakeview hamster.

Based on a t-test for comparison of the means of ungrouped data. -L64-

in both cases are expressed per gram proEein. IË is apparent from the electron mícrographs of the Lochner Preparation (Fig. 94, page 160) that these organelle pellets contain a considerable amount of non-mitochondrial material. This material, if unable to respire, would contribute protein

and therefore would result in false low oxidation rates when these are expressed per gram protein. In spite of these peculiarities associated

with the Lochner Procedure, the oxidative phosphorylation parameters were

very similar ín the normal and dystrophic animals. Therefore it was concluded that the discrepancy between our earlier findings and those of

Lochner et al (44) aLso could not be ascribed to the different methods of mitochondrial preparalion.

Nevertheless, three findings in the dystrophic hamsters of

Group 3 seem to be important: (i) Mitochondria prepared by the Proteinase Preparation

exhibited a relalive deficiency of Mg+Z. (ii) It was possible to obtain normal and abnormal oxidative

phosphorylation parameters from the same mitochondrial preparat.ions when

these parameters hTere measured by Lwo different methods.

(iíí) Normal as well as abnormal oxidative phosphorylation

could be demonstrated with the same dystrophic muscle samples when the mitochondria were isolaËed by two differenË procedures. These also resulted in different miËochondrial yields. I{hereas the Proteinase

Preparation gave the same yield from normal and dystrophic animals (Tables 26, page L4I) , wíth the Lochner Procedure significanlly less mitochondria were isolated from the dystrophic animals (Tables 31).

The above suflìrnary demonstrates rather clearly that one cannot attach too much significance to results obtained in vitro with a single - 165 -

Table 31a

YIELD OF HAI4STER SIGLETAL MUSCLE MITOCHONDRIA

Values given in mg mitochondrial protein per g muscle. Lochner Preparation.

Group 3 Group 6

Nor- i+ S.E. 1.1+ 0.1 2.2 t 0.1 ma1 N 7 13

Dys- X+ S.E. 0.9 ! 0.1 1.5 0.1 tro- t phic N 6 11

prk 0 .025 0.001

* Based on a t-test for comparison of the means of ungrouped data. -L66- method only. Also, the discordant results,although obtained with procedures corrsidered by their proponenEs as satisfactory, raise considerable doubts as to the relevance of such findings to the situation in vivo.

Comparison of polaroqraphic and manometric tecþnlques for the determination of oxidative phosphorylation paraqe-lers

There remained one additional aspect of the general methodology of Lochner et al (44) which differed from that of this laboratory. That was their use of a manometric technique to determine the oxidative phos- phorytation parameters. It was felt that dystrophic mitochondria uright be more susceptíble to an aging effect than the normal organelles and that this difference might not be detected during our short polarographic experimenrs (4-10 min.) at 28oc, but might appear during the 30 min. incubarion at 37.5oC used in the Inlarburg technique of Lochner et al (44). It was therefore decided to compare oxidative phosphorylation wíth the same organelles simultaneously with polarographic technique and manometri ca I ly.

The experiments hTere done on hamsters of Group 4 (Tables 32). This was the youngest animal group tesËed and had a mean age of about 85 days. Although heart disease was barely detectable, the skeletal muscles of the dystrophic animals showed more white streaks than those of any other grouP.

The results on the measurements of oxídative phosphorylation are summarized ín Table 33a. AtI the dystrophic mitochondria appeared to be uncoupled r¿hen measured by the polarographic method. Also, the -167 *

Table 32a

CHARACTERISTICS OF THE HAI4STER GROUP 4T

CPK Age Body mg heart Heart Streaks:k -DTT +DTT Percentage r¡/t. g g body d isease:k activation

Nor- x B6 118 2.7 9 20 81 450 ma1 S.E. 2 3 0.10 418 106

Dys- i 83 104 2.84 :L+ p1-L 3r4 IT22 412 trophic S . E. 2 3 0 .03 72 1.5 1 89

p+ N. s. o .02 N.S. 0.00s 0.001 N.S.

T Values from 6 normal and 5 dystrophic hamsters. iç See these parameters for the individual animals in Table ILb, page 249.

T g"""d on a t-test for comparison of the means of ungrouped data. - 168 -

Table 33a

RESPIRATION AND OXIDATIVE PHOS?HORYLATION BY SKEI,ETAL MUSC],E MITOCHONDRIA FROM HAI4STERS OF GROUP 4 Substrate: pyruvate/malate in polarographic experiments; pyruvate/fumarate in manomeLric experiments. Standard Proteínase Preparation.

Polarographic Iulanome tr ic

28oc 28oc 37oc

RCR ADP/O Orrate P rate P/O Orrate P rate I P/O Orrate P rate

X 2.6 r.7 158 542 2.4 325 1568 2.6 405 2068 lor- ral I S.E. 0.2 0.1 26 107 0.1 18 L46 0.2 4L 2rO

N 4444 444 444

i 1.0 0.0 59 0 2.6 262 1353 2.4 356 1722 )ys- :ro- S.E. 0.0 0.0 9 0 0.1 7 58 0.1 30 126 ¡hic N 555 5 555 444

Ptk 0.001 0.001 0.01 0.001 N. S " 0.01 N" S. N.S" N.S" N.S. t Normal animals were of the Lakeview random bred strain.

þ Based on a t-test for comparison of the means of ungrouped data. -169-

respiration rates of these organelles were very low and decreased continuously during the course of the measurements, in a manner similar to that shown in Fig. BB, page L52, which illustrates the respiration

pattern seen in the apparerrt Mg+2 deficiency described in the dystrophÍc

hamsters of Group 3. The effect of 5 mM MgCl2 r^/as tested with one

animal of the present group in the same r¡/ay as shown in Fig. BC, page 152.

The presence of Mg+2 .u"rrlted ín moderately coupled mitochondria. The beneficial effect of this ion was also indicated by the results of the

manometrÍc experiments ín which i"tg+2 i, added together with the other

ingredients of the hexokinase trap (Tables 33)

The abnormal findings delected polarographically in the dystrophic organelles r¡Iere not seen in the manometric resultso except for

a persístently low respiration at 2Bo (column 6, Table 33a). However,

the respiration rates of dystrophic mitochondria at 37oC decreased during

the lalter half of the 30 min. manometric incubatíon tíme, whereas this v¡as seen less often in the control mitochondria and Ít never occurred at

2BoC in either group. In a separate experiment it was found that the phosphorus uptake decreased in a manner parallel with the oxygen uptake. Thus, the non-linearity did not interfere with the P/0 determination.

(In reviewing the medium constituents used in these particular experiments, it would appear that the accidental use of an excessively low EDTA

concentration may have been the cause of the falling off of activity with

time. However, this was not proved by direct experíment). Although these experiments again did not confirm the findings of Lochner et al (44), they nevertheless offered further indirect support for the idea that the organelles from these dystrophic hamsters also were

L') Mg' - deficient, at least ín vitrg, and that this may have caused the - L70 - defective oxidative phosphorylation seen in the polarographic experiments,

in which Mg+2 t"" not added

It may have been notíced that the quality of mitochondria obtained by the Proteínase Method from Group 1 (Tables 2L and 22, pagesL3L and 133 ) was better than that of any of the later groups described in Tables 27, 28, 33 (pages L44, L47, f6S). This applied to the controL animals as well as the dystrophic ones. Repeated attempts ü7ere made to identify the reason(s) for this decline in the quality of the organelles isol-ated. These v/ere unsuccessful. However, in a revier¡I of the probl-em, two things rrere noted:

(i) Inlhenever we applied the same Standard Proteinase Prepara-

tion technique to mouse skeletal muscle, \¡/e l^rere always able to obtain organelles, the quality of which equalled or surpassed thaÈ of the mito-

chondria described earlier Ín Tables 3, pa1e 79. (ii) The hamsters of Group I were fed 'Purina Lab Chowt plus i,rater ad. lib. , whereas all the subsequent groups received peanurs,

sunflower seeds, and lettuce in addition to Lab Chow. A study on dystrophic animals at. 190 days of age revealed no differences in oxidative phosphorylation parameters, whether they were fed the cournerciat diet only or wheEher they received the above dietary

supplements as well. However, in order to exclude a dielary influence completely such studies would have to be extended, especiatly to animals of younger age and to control hamsters as well.

The author believes that the reason for the decrease in quality of the isolated mitochondria lies in the animals, but there is no proof

for this on hand. One possibility is that there are seasonal changes in

oxidative phosphorylation. This is noE at all unlikely since hibernators - L7L - such as hamsters show other seasonal metabolic changes (196). Unfortu- nately, time did not allow investigation of this possibility.

During the above investigations of the reasons for the apparently poor quality of the muscle mitochondria, some modifications were introduced into the procedure which resulted in the isolation of organelles of better quality. This tModified Proteinase Preparationl differed mainly from the Standard Proteinase Preparation, used in the earlier studies Ín thís thesis, ín thai the incubation time with proteinase I¡Ias reduced from 20 min. to 10 minutes. (Further details are given under Methods).

Oxidative phosphorylation by hamster skeletal muscle mitochondria prepared bv the Modified Proteinase Procedure

Muscle mitochondrÍa rdere isolaled from another set of hamsters

(Group 5, Tables 34) using the Modifíed Proteinase Procedure. The hamsters of this Group were the oldest so far tested, with a mean age of about 185 days. They exhibited severe signs of thecardiomyopathy, but the skeletal muscles showed fewer areas of macroscopically visible necrosis (streaks) than the two prevíous groups. The mitochondria were tested, as before, polarographically and manometrically. The results are sumnarized in Tab1e 35a. It is evidenË that the quality of the mitochondria, as judged by the four polarographically determined parameters, were improved over those shown in Tables 27, 28, 33, pages I44, I47 , 168. The manometrically determined results (Tables 35) were qualitatively similar to those in the previous group (tables 33, page 168 ).

However, they were performed only on a few animals of Group 5 and, as -L72-

Table 34a

CHARACTERISTICS OF THE HA}{STER GROU? 5I

CPK Age Body mg heart Heart S tre aksJc -DTT +DTT Percentage r¡/t. g g body disease:t ac t ivat ion

Nor- i L93 LlB 2.BB 40 85 1/,O mal S .E. 13 Ja 0.11 13 19 33

Dys- i L82 L24 3 .56 ¿3* 326 1 108 459 tro- -2+ phic S . E. 6 2 0.16 86 219 72

p+ N.s. N.s. o.o2 0 .0s 0.01 N.S.

+__- I Values from 5 normal (LSH) and 9 dystrophic hamsters.

:k See these parameters for the individual animals on Table L2b, page 259. I_ T Based on a t-test for comparison of the means of ungrouped data. - t-73 -

Table 35a

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HÆ,ISTERS OF GROUP 5 Substrate: pyruvate/malate in polarographic experiments; pyruvate/fumarate in manometric experiments. Modified Proteinase Preparation.

Polarographic lvlanome tr ic

2BOC 2BOC 37oc RCR Orrate P rate P rare p/O Orrate P rate ADP/O P/O Orrate I

i 3.9 1.8 L99 723 2.5 27 9 L373 2.2 382 1684 Ior- ral:k S . E. 0.2 0.1 15 70 0.3 L2 198 0.1 28 797

N 555s 222 222

x 3 .9 L.6 17 4 667 2.5 200 993 2.4 27 6 1273 )ys- :ro- S.E. 0.6 0.3 27 L39 0.1 28 r22 0.1 55 217 ¡hic N 9999 444 444

pï N. S" N"S. N. S. N.S. N.S. N.S.

< Normal animals were of the LSH strain. f Based on a t-test for comparison of the means of ungrouped data. - L74 - mighE be expected, statistical analysís revealed no signifícant díffer- ences between normal and dystrophic animals. The above observation that the respiration rates of dystrophic miEochondria prepared by the Standard

Proteinase MeLhod tended to decline at 37oC in the Warburg experiments was also observed in Group 5. Looking at the individual results in Table 35b, it can be seen that 2 dystrophíc anímals, No. 56 and No. 58, appeared uncoupled when tested polarographically. (The behaviour of the mitochondría of. animal No. 56 has already been described above on page I49 Ln relation to the st.imuLation of their low uncoupled respiration rate by dinitrophenol). A possible Mg-'L') deficiency is suggested in this animal, since in the manometric determination with MgCl2 present, these mitochondria gave the highest respiration rate in the group. Animal No. 58 also apparently was Mg+2 deficient, since in this case coupling of oxidation to phosphorylation could be produced by adding MgCIZ to the polarographic reaction medium prior to mitochondria. However, the most important conclusion to be drawn is that oxidatíve phosphorylation in this rather old group of dystrophic animals !üas not significantly different from that of the control anímals.

Reproduction of the manometric experiments of Lochner u-sing hamsters wíth a mean age of 2I5 days

From the foregoing muscle mitochondria experiments, it is apparent that it had not been possible to identify the reason for the discrepancy between the present findings and those of Lochner et al (44).

However, our approach in the experiments described so far r,üas to change one variable at a time. It was therefore decided to see whether the - L75 - findings of Lochner might be reproduced if a combination of all their experimental conditions were employed together. This was done on hamsters of Group 6, the characteristics of which have been described already ín connection with the heart experiments (Tables 16, page 115 ). These hamsters vrere the oldest animals tested by us, with a mean age of

215 days. Ilowever, the skeletal muscles did not show as extensive macroscopic necrosis (streaks) as those of Groups 3 and 4. Nevertheless, all animals were affected by the disease, as demonstrated by their elevated serum creatine phosphokinase activities (Tables 16, page 115 ). The skeletal muscle also showed definíte biochemical changes which will be discussed further below.

The results of manometríc experiments performed wÍth pyruvatef fumarate as substrate according to Lochner et aI (44) are summarized in the last two columns of Table 36a. The signíficantly lower P/0 ratios in the dystrophic preparations agree with the findings of those workers.

However, the respiration rates in dystrophic animals \,/ere not affected in Eheir experiments with pyruvate/fumarate, whereas in ours they were decreased significantly. No decline in the respiratíon rates l¡ras seen here during the course of the incubation time in contrast to the fall- in respiration described above in dystrophic animals of Groups 4 and 5 at

37oC. This may have been due to the use of higher EDTA concentrations in the reaction medium in the present experiments on Group 6 animals.

Respiration with palmitate and palmityl-L-carnitine as substrates

Most of our studies on oxidative phosphorylation in dystrophic muscle described so far were done with pyruvatefmalate or pyruvatef fumarate - 176 -

Table 36a

RES?IRATION AND OXIDATI\TE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HA}4STERS OF GROUP 6 Values obtained from manometric experiments with pyruvate,/fumarate, palmitate, and palmityl-1,-carnitine as substrates at 37oC. Lochner Preparat.ion

Palmi t at e Pa lmity I - L- c arn it ine Pyruvate /Fumar ate

Orr aLe O rr ate orrate P/o

X 93 tr4 204 2.L Nor- I malI S.E. 5 13 9 0.1

N 9 3 77

X 54 72 r25 r.9 Dys- tro- S.E. 3 7 5 0.1 phic N 9 3 66

Pr. 0 . 001 0 .05 0 .001 0.05 t g lStl and I Lakeview hamster.

;k Based on a t-test for comparison of the means of ungrouped data. - r77

as subsËrate. The activíty seen with these substrates represents the disposal of a product of glycolysis (pyruvate) in the presence of

catalyt,ic amounts of a member of the tricar/b oxylic acid cycle (malate or fumarate). However, a consíderable amount of energy in muscle is derived from the oxidation of fatty acids (28, L97-I99). Since the intracellular site of fatty acid oxidation is the mitochondrion (64),

it was decided to study also the oxidaËion of palmitate by the mito-

chondria of Group 6 hamsters. A defect of palmitate oxidation in dys-

trophic mouse muscle has been described by Lin et al (96). In preliminary experiments it was found that palmitate

oxidation was extremely slow in mitochondria prepared by our Standard

Proteinase Procedure, although all the necessary cofactors (200) were

present, including carnitine, ATP, coenz)¡me A, and Mg+z. However, this

problem \¡ras overcome when the Lochner Procedure for the isolation of mitochondria was used. trrlith such preparations palmitate oxidation rates were obtained which were only slightly lower than those found with palmityl-L-carnitine as substrate (Tables 36). Why should Ëhe two mito-

chondrial preparations have behaved so differently? Since palmityl-L- carnitine was oxidized well by both kinds of preparations, the difference presumably lay in the ability of the two preparations to synthesize palmityl-L-carnitine f rom palmíEate. There are t\^/o enzyrnes involved in this synthesis, a thiokinase and an acyl-transferase (64). The intra-

cellular LocaLizaLion of these enzJ¡mes in muscle is not known exactly, but it is possible that the thiokinaseis largely extramitochondrial.

Since there r¡ras a much greater contamination in the Lochner Preparatíon by extramitochondrial material (¡'ig. 9A., page 160 ), as compared to Lhe Standard Proteinase Preparation (¡'ig. 98), it is possible that activation -L7B-

of palmitate by this 'tdebrisrr in the Lochner Preparation accounts for the ability of these organelles to utilize the free fatty acid. Another possibility is that the proteinase used ín preparing mitochondria by the

Standard Proteinase Procedure adversely affected the two enzymes. Bode et al (lB2) also found no palmitate oxidation in muscle mitochondria

prepared in the presence of a proteinase. However, Lin et at (49)

recently demonstrated palmitate oxidation in mouse skeletal muscle mito-

chondria prepared by a proteinase procedure, but their rates r^zere somewhat lower than those obtained here with the Lochner Preparat.ion.

The manometrically determined respiration rates vrith palmitate

differed significantly in normal and dystrophic hamster skeletal muscle

mitochondria prepared by Èhe Lochner Procedure (Tables 36). In the

díseased animals the rates amounted to only 58% oÍ. the control values. In the last three such experimenËs the respiration rates vtere also

determined with palmityl-L-carnitine to examine the possibility that a

defect in the synthesís of this compound might explain the poor oxidation of palmitate (Table 33a). Although only tested in a few animals in the present group, the rates with palmityl-L-carnitine were also significantly

lower in dystrophic hamsters by a similar percentage (63% of the control

rate). A similar decrease in oxidation of palmitate and pyruvate has been reported by Lin et al (49) in muscle mitochondria from dystrophic mice. Sínce the oxidation rales with pyruvate/fumarate, palmitate and palmityl-L-carnítine all were decreased by a similar percentage ín Table

36a, Lt is possible there may be a defect in the portion of their metabolic pathways that these compounds have in conrnon.

Since MgCL, v/as present in the reaction medium of all manometric experiments in Table 36a, the decreased values in the dystrophic - L79 -

animals are probably not explainable by M{2 def íciency of the mito- ^ chondria that can be reversed in vitro by mg+2 addition. It Ís of

interest that the Lochner Preparation used here was also employed with

the animals of Group 3,discussed above, and here too vt{z defíciency " was not detected.

Confirmation of the manometrically detected mitochondrial respiratíon defect by polaroqraphic techniques

Since it seemed possible that the abnormalities in TabLe 36a rnight be associated with the longer reaction time needed in the manometric procedure, we tested the respiration rrrith palmityl-L-carnitine and pyruvate

(plus malate in each case) with our standard polarographic technique to see whether the manometrically detected defect would persíst. The shorËer

reaction time of this procedure in theory would be expected to minimíze the effect of agíng of mitochondria of both the normal and diseased animals.

The oxidative phosphorylatÍon parameters were measured both at 37oC, the reaction temperature used in the manometric experiments, ancl at 2BoC, the

temperature ordinaríly used in our standard polarographic technique. The results with palmityl-L-carnitine are suiffnarized in Table 37a and those with pyruvate/malate in Table 38a. At 2BoC all four oxidative phosphorylation parameters were significantly decreased with both substrates. The fact that some parameters at 3loc were statistically not different in normal and dystrophic animals may have been due to a greater variance of the data at this temperature, which \4ras caused by a poorer response of the Clark electrode (see Methods, page 56 ) at this temperature and lower oxygen concentration in the reaction medium. However, at both temperatures -180-

Table 37a

RESP]RATION AND OXIDATIVE ?HOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROI"I HAMSTERS OF GROUP 6 substrate' * malate' J:åïå:'l;i;:::îï:;:"

2Boc 37oc

RCR ADP/O Orrate P rate RCR ADP/O 0^rate P rate

x 2.9 L.6 86 284 3.0 t.B L42 498 Nor- f malr S.E. 0.2 0.0 3 t7 0.3 0.1 8 40

N 10 10 10 10 8 Õ 8 8

X 2.0 L.4 6s 180 )) 1) 100 27r Dys- tro- S.E. 0.2 0.1 6 24 0.3 0.2 11 64 phic N 8888 9 9 9 9

0.005 0 .005 0.005 0 .005 0.01 0 .02 t Normal hamsters were of the LSH s train.

J. Based on a t-test for comparison of the means of ungrouped data. - 181 -

Table 3Ba

RESPIRATION AND OXIDATIVE ?HOSPHORYLATION BY SKEIETAL MUSCLE MITOCHONDRIA FROM HA}.4STERS OF GROUP 6

Subst.rate: pyruvate/malate. Lochner Preparat.ion.

28oc 37oc

RCR ADP/O Orrate P rate RCR ADP/O Orrate P rate

i 3.2 r.B 96 346 3.7 10 178 695 Nor- a mall S.E. 0.1 0.0 4 L7 0.3 0.1 10 65

N 13 13 13 13 7 7 7 7

x 2.2 L.7 73 240 2.6 L.7 131 Dys- tro- S.E. 0.2 0.0 4 14 0.4 0.1 13 phic N tl 11 11 11 7 7 7

0.001 0.01 0 .001 0.001 0 .05 0 .02 0 .05 t 10 LSH and 3 Lakeview hamsters.

Based on a t-test for comparison of the means of ungrouped data. -TB2- and $títh both substrates the respiration rates decreased to a similar extent in the dystrophic organelles. The mean 0, uptake with palmityl-L- carnitine in dystrophic animals amounted to 75"/. of Lhe control mean value at 28oC and to 7O% of the control mean value at 37oC. The corresponding values with pyruvate/malate as substrate were 76% and 74% respectively. Thus, these results are ín good agreement wiËh the manometric experiments

(Tables 36, page L76 ). However, faster aging of the dystrophic mitochondria may nevertheless have occurred in the manometric experiments, since the percentage decrease in the respiraÈion rates in the l{arburg experiments r¡ras slightly higher.

A sinsle or a two-fold defect?

The results of Tables 37 and 38 showing decreased respiratory control ratios and ADP/O values on the one hand and decreased respiration rates on the other, suggest a two-fold defect in these dystrophic mitochondria. I{hereas the f ormer t\^ro parameËers indicate a def ect in the mechanism of coupling phosphorylation Lo respiration, the decreased respiration values indicate that the oxidatíve mechanism was defectíve as well.

A good indicator of the tightness of coupling is the State 4 rate, i.e. the respiration rate in the absence of phosphate acceptor (I2B).

In tiàhtly coupled mitochondria this rate is very low, whereas in completely uncoupled mitochondria this rate is much more rapid and, at least ín theory, is identical with the State 3 rate in which phosphate acceptor is present, The State 4 raLes are not listed in the Tables. Ho\n/ever, they can be easíly calculated by dividing the respiration rate, whích is - 183 -

the State 3 rate, by the respiratory control ratio. The State 4 rates

in the dystrophic animals (Tables 37b and 38b), were found to be

statístically indistinguishable from the control values. Now, if the

StaLe 4 rates had remained constant and on1-y lhe State 3 raÈes had

decreased, the respiratory control ratios would have been smaller. Thus,

the decreased respiratory control ratios are simply a consequence of the decreased State 3 respiration rates observed in the dystrophic mito-

chondrÍa. Does this also apply to ADP/0 ratios? As wilL be shown below in the DiscussÍon, there existed a significant correlation between res-

piratory control ratios and ADP/0 ratios. Thus, a decreased ADP/O value can also be expected in mitochondria that have decreased State 3 respiration rates but constant State 4 rates. Such a correlation between respíration rates and ADP/O ralios at constant State 4 rates can be predícted from the following theoretical considerations. IÁIith absolutely tight coupling and no ATPase actívity present to break down the ATP as it is synthesized, a State 4 rate of. zero would be expected. On the other hand, a measurable State 4 raLe could be caused either by a somewhat loose coupling of oxidation to phosphorylation or by a breakdown of the newly formed ATP by an ATPase or its use by oËher reactions which yietd ADP. Thus, a State 4 rate reflects respiration without net phosphorylation. If one assumes that at least part of such a State 4 rate persists during State 3 respiratíon, one would expect with a given State 4 raEe a decreased ADP/O ratio ín those organelles with a slower State 3 rate.

This follows because for a given amounL of A-DP, such a preparation needs more time to phosphorylate it to ATP, and during Ëhis longer interval the

State 4 respiration will contribute disproportionately to the total oxygen consumption needed to phosphorylate a certain amount of ADP, Consequently - l8/+ -

Ehe ADP/O ratio, with a larger 0, uptake Ín the denomínator, will be srraller.

In view of these considerations, it can be seen that there really is no conclusive evidence that the mitochondrial coupling of phosphorylation to respiration in these dystrophic animals was defective. However, they definiLely exhibited a defect in the oxidation of palmitate, palrnilyl-L-carnitine, and pyruvate/malate.

The localÍ-zatiorr of the respiratÍon defect

Impaired respiration with acetyl-L-carnitíne, --After the experiments planned for this thesis research had been completed, the substrate acetyl-L-carnitine was tested in anoËher group of old hamsters. The results (201) were qualitatívely ídentical to those reported with palmityl-L-carnitine. It therefore ís concluded further that the defect in palmitate oxidation probably did not lie in the slmthesis of palmityl- L-carnitine or ín its entrance into the mÍtochondría or in the p-oxidation (64) of this subsËrate. However, the oxidation of palmitate, palmityt-L- carnitine, acetyl-L-carnitine, and pyruvate all end in a conrnon final pathway via acetyl-CoA and the citric acid cycle. Thus, it is possible that the same mitochondrial defect is responsible for all the decreased respiration rates with these substrates in dystrophic mitochondria.

In an attempt to localize the defect in respiration more accurately, two additional substrates \,/ere tested in these mitochondrial preparations, succinate and NADH. Succinate dehydrogenation is FA-D- linked and feeds the reducíng equivalents into the respiratory chaín at the flavoprotein level (64). NADH is directly oxidízed by the respiratory -185-

chain (131). The results are suÍtrnarized in Table 39a and índicate that

succinate and NADH are oxidized by normal and dystrophic mitochondría

equally wel1.

2. Normal respiration with succinate. --The respÍration rates with 'succinate alone' (Tabes 39) were measured in the absence of ADP, because upon addiLion of A-DP the mitochondria exhibited an abnormal

pattern of respiration, which has been described previously by Chance

et al (202). Upon addition of ADP these workers observed a brief period

of accelerated respiralion, which subsided within a few seconds and was followed by a rate much slower than the initial respiration rate. tr{e

observed the same phenomenon in these muscle mitochondria. Chance el al

(202) explained the brief acceleration upon ADP addition by Ehe oxidation of indigenous substrates and the inhibition phase by the accumulation of oxaloacetate, which is a powerful inhíbitor of succinic dehydrogenase

(L32, 203). The accumulation of oxaloacetate itself, shortly after ADP additíon, is according to Chance (202) associated with oxidation of NADH to NAD, which would al1ow a slight shift of the oxaloacetate-malate poÍse towards oxaloacetate. The inhibition by oxaloacetate can be relieved by the addition of glutamate (2OZ), which will remove some of the oxaloacetate by transamination (204). The accumulatÍon of oxaloacetate and its inhibitory effect can be prevented, if the oxidation of reduced NAD is prevented by the addition of amytal (204) or rotenone. As has been mentioned, the respiration rates with succinate r¡/ere measured in the absence of ADP and thus represent only State 4 respiration rates. This was done Ín order to avoid the rabnormalr -186_

Table 39a

RESPIRATION RATES I^IITH SUCCINATE AND NADH BY SKELETAL MUSCIE MITOCHONDRIA FROM HAMSTERS OF GROUP 6 Lochner Preparation. P/M=pyruvate/malate; cyt. c=cytochrome c.

Succ inate NADH

alone af ter PlM -cyt.c *cyt. c

2}oc 3loc 284 c 37o c z}oc 37oc 2}oc aToc

Lr3 186 62 L4L 190 286 393 648 Nor- " malf S.E. 51827 10 17 19 T4

N 85134 86/+ ¿+

; 138 zLL 6t 130 r82 264 374 Dys- tro- S.E. L221313 7 18 3 phic N 6 s 11 4 at 5 3

N"S" N.S N. S.

+ 10 LSH and 3 Lakeview hamsters.

Based on a t-test for comparison of the means of ungrouped data - r87

respiratíon pattern caused by this nucleotide described above. The rates observed in normal animals were quite similar to the State 3 rates with pyruvate/malate (Table 3Ba, page 181 ) and palmityl-L-carnítine (Table 37a, page 180 ). However, in dystrophic animals the succínate respiration did not parallel the decreased respiration of those substraËes, but equalled the rates of the control animals. One should, however, perhaps not try to read too much into this result, since these rates were observed in the absence of ADP and with a substrate the oxidation of which is greatly influenced by small intramitochondrial changes of oxaloacetaLe concentrations, However, what should be stressed is that the rates wÍth succinate, although not maximal State 3 rates, are about twice as fast as the State 3 rates with pyruvatefmal.ate and palmityl-L-carnitíne in the muscle mitochondria of the dystrophic aninals. This is an indication that the defective oxidation with pyruvate and palmityl-L-carnitine was probably not caused by a defective, and thus rate limiting, electron transport between the flavoprotein level and oxygen. The respiration rates with tsuccinate after pyruvatefmalate¡

(Tables 39) were used only to assess rapidly the protein concentration of each mitochondría1 suspensíon, because these inhibited succinate rates were empirically found to be very constant Ín both normal and dystrophíc mitochondria, By this method it was possible Eo keep the mitochondrial protein, added to Warburg experíments, constant within L L0/".

3. Normal respiration with NADH. --In order to assess the capacity of the total mitochondrial respiratory chain wíthout involvement of any tricarboxylic acid cycle enzymes and íntermediates, -188- respiration raËes \^rere also tested wíth externally added NADH as substrate. Intact mitochondria are probably impermeable to this substrate (175) and oxídíze it only after disruption of the membranes. Hor¡ever, the preserit organelles isol-ated by the Lochner Procedure were probably topened' by the vígorous homogenization with the all glass rKontes' homogenLzer.

This resulted in very fast NADH oxidation (Tables 39, page 186 ) and this could not be íncreased further by sonicating the mitochondria. Addition of cytochrome c on the other hand increased these rates substantially, which might indiate that some of the cytochrome c content of the organelles had been l-ost during isolation. The stimulation by cylochrolne c, however, r^7as similar in normal and

The above conclusion decreases the number of possible sites of the defect. Since a failure of the fatty acid activation and part of the p-oxidation have been excluded, the def ect presumably lies somei¡Ihere between the formation of acetyl-CoA and the entrance of reducing equivalents into the respiratory chain, i.e. in the tricarboxylic acíd cycle. However, our findings also do not exclude the possibility that the capacity of the mitochondria to phosphorylate was Ímpaired. Since the respiration of all of the above substrates is coupled to phosphorylation, a decreased capacity to phosphorylate would also cause the observed impaired respíration. -L89-

Thus, it is not clear whether the observed decreased phosphorylation rates \^Iere caused by impaired respiration or whether a possibly impaired

capacity to phosphorylate caused the decreased respiration rates and consequently decreased phorphorylation rates. In either case, the out-

come would be the same, viz. the phosphorylation rates would be decreased and thus energy production in these muscles would suffer. This could be even more serious t.han the values suggest, since significantly fewer mitochondria per weighË of muscle were isolaEed from the dystrophic animals (Table 3la, page L65), suggesting that the miLochondrial content of the dystrophic muscles in situ may have also been decreased.

Is the observed mitochondrial respiration defect al-so present in vivo?

The above deductions of course are based on the assumption that the defects demonstrated in the dystrophic hamsters of Group 6, also were present in vivo. It must be admitted that no clear ansr¡ler can be given to thís important problem. In this regard iL is worth emphasizíng thaE the dystrophic muscle is really quite a different tissue from the normal- muscle. In Fig. 10 these tr,\io extremes are shown. Photo A illustrates a section of a normal muscle and photo B a section of a dystrophic muscle which macroscopically showed white streaks. In the latter the rrormal muscle fibers are almost completely destroyed and there is massive infÍ1- tration by macrophages. hlould one really expect to isolate mitochondria of identical properties from two such different tissues? Inlould it not actually be surprising, if the isolated organelles rrere identical? Since the properties of the organelles obtained from the two tissues were in fact dífferent, four explanations suggest themselves: -190-

Fig IO. --HISTOPATHOLOGY OF NORMAL AND DYSTROPHIC

HAMSTER SKELETAL MUSCLE

A. The upper picture was taken from the m. biceps femoris of the normal hamster No. L6.

B. The lower picture was taken from the m. biceps femoris of the dystrophic hamster No. 27

Hematoxylin eosin staining. MagnificaËion 150 x. Note, the disintegration of the muscle fiber bundles of the upper normal picture is due to an artefact. W ffiry W-

W ,rW

-T6I. -L92-

(i) The mitochondría ín the fibers of normal and dystrophic muscle had dÍfferent properties in situ.

(ii) The abnormal findings in dystrophic animals were due to the presence of tr¿o different mitochondrial populations, those of muscle and those of infÍltrating ce11s. (iií) The muscle mitochondria in dystrophic muscle in situ r¡/ere normal , but became defective during the isolation procedure due to contact during and af ter homogenization with unkno!,irì non-mitochondrial substances, which act as inhibitors or uncouplers. (iv) Any combination of the above 3 situations is also possible and more likely than any single one alone.

Studies on the tissue composition of normal- and dvstrophic muscl-e

In a preliminary examination of some of the above explanations, the composition of the Lwo tissues (normal and dystrophic)was investigated.

L. Col-l-agen, non-collagen protein, DNA, RNA. --The collagen protein and non-collagen protein levels shown in Tables 40 agree wíth those reported by Lochner et al (44). Of special interest ís the finding of relatively 1ittle collagen protein in dystrophic hamster muscle, which is in marked contrast to the findings in murine (15) and human (53) muscular dystrophy. However, these low collagen levels agree qualitatively with the hístological sections whÍch show relatively 1iÈt1e connectíve tíssue. Also, as might be expected from the microscopíc picture Ín Fig 108, page 191, shoT¡ring great numbers of nucleated infiltrative cells, the DNA content in dystrophic muscle was almost double that of the control tissue - 193 -

Table 40a

RNA, DNA, AND ?ROTEIN CONTENT IN SKET,ETAL MUSCIE OF HAI'{STERS OF GROUP 6

mg pgr g muscle ug per g muscle

N. C. P. RNA DNA

; 164.8 158.6 6.7 105 7 540

Nor-a mall S.E. 3.0 3.4 2.4 32 14 - N 999 9 9

; 159.0 151.8 7.2 1208 r_003 Dys- tro- S. E. 2.4 2.8 l.s 46 74 phic N L2 T2 12 I2 L2

N. S. N" S. N" S. 0.025 0.001

T.P. = total protein N.C.P. = non-collagen protein C.P. = collagen protein t 8 rro.*al hamsters \n/ere of the LSH strain and one T¡/as a Lake- vier,¡ hams ter .

:k Based on a t-test for comparison of the means of ungrouped data. -r94-

(Tables 40), Since in a gíven species the DNA content is thought to be

constant per nucleus (205), the raísed DNA levels reflect an increased

number of nuclei. These presumably include the increased number of nuclei per muscle fiber seen as "rowing" of nuclei (165), plus the nuclei of the great number of infiltratíve cells. This finding as well as in-

creased RNA content agrees with the observaËion of Srivastava on dys-

trophic mice (48,206), who also observed concomitant with the increased

DNA and RNA content in dystrophic muscl-e an increased incorporation of l4C-l"tr"ine into protein (48). Srivastava (206) regarded the determina-

tion of DNA content in dystrophic muscle as a useful tool to determine the degree to which the disease has progressed. This seems to apply also for dystrophic hamsters. Since there \^ras no overlapping of the DNA values between the normal and dystrophic muscles in hamsters of Group 6, this also confirms that the disease \¡/as present ín skeletal muscle of all the dystrophic hamsters of the Group.

2. Hvdrolv.tic enzvme activities.--According to an experE pathologistrs opiníon (192) the majority of the infiltratíve cells in dystrophic hamster muscle, such as seen in Fig. 108, page LgL, consist of macrophages, which are known to be rich in lysosomes (207). The activities of certain lysosomal enzymes (208) including acid phosphatase, cathepsin D, and p-Blucuronídase \^iere measured in the muscle homogenates.

As suunnarízed Ln Table 4la, the activities of all three enz)¡mes r.^rere signifícantly different in normal and dystrophic muscle. Unexpectedly, the acid phosphatase activities were decreased rather than elevated in the dystrophic anímals, although the difference between Ëhe means was only 10%. trnle do not know the significance of this finding, since the - 195 -

Table 41a

LYSOSOMA], ENZYME ACTIVITIES IN SKELETAL T4USCLE OF HA},ISTERS OF GROUP 6

Incubation temperature 37oC.

Acid Phosphatase:k Cathepsi¡ þ:k:k p-gLucuro¡id¿ss:k:k:k

X 12.8 4.s4 0.18 Nor-a malr S.E. 0.¡ 0.24 0 .02

N 9 9 9

^ 11 .5 6.78 0.61 Dys- tro- S.E. 0.3 0 .35 0 .06 phic N I2 L2 L2

pf 0 .01 0.001 0 .001

:k ¡rg phenol liberated per mg protein per hour.

;kJr fÀB tyrosine liberated per mg protein per 30 minutes. rkJr:k ¡.tg phenolphthalein liberated per mg protein per hour.

f 8 r,.ot*"l hamsters r¡rere of the LSH strain and one r^/as of the Lakeview random bred strain.

.1--¡- Based on a t-tesË for comparison of the means of ungrouped data. -L96-

two other lysosomal enz)¡mes had greatly increased activities in the dystrophic muscles. This peculiar result may explain the failure of

other workers to report on this enz)¡me, although it is consídered the

typical lysosomal marker enz)¡me (208). However, the increased actÍvítíes

of other hydrolases in dystrophic tissue is a well known fact (113, L20,

209-2Lr) .

While it is not certain that these lysosomal enz¡¡mes actually

come from macrophages, this explanation has been suggested for the in-

creased lysosomal enz)¡me levels found in dystrophic mice and chickens

(2tt). Nevertheless, this finding together with the above measurements of nucleic acids clearLy indicates the quiEe different tissue composition

of dystrophic muscle.

The significance of the elevated lysosomal enzJ¡mes in relation

to the abnormalities in oxidative phosphorylation here becomes apparent from a recent report by Mellors et al (zLZ). They demonstrated that

lysosomes, which \¡rere treated by repeated f.reezi-ng and thawing to free

the enzymes, uncoupled oxidative phosphorylation of liver and heart mito-

chondria. It also is of interest that the three lysosomal enz)¡mes measured in muscle homogenate in the present \.^/ork showed no latency, i.e. they had at this stage already been released from t.he lysosomes (2I3).

Presumably the lysosomal membranes had been disrupted by the homogen-

ízatLon procedure. Since the homogenLzalíor's for both the enz)¡me deter- minatíons and the mitochondríal isolations were done inrKontesr all glass homogenizers, it is conceivable that lysosomal enz¡¡mes, freed by the homogenization, could have acted on the mitochondria during the early steps of the isolation procedure. Thus, an abnormality of mitochondrial oxidative phosphorylation might be produced in viLro that possibly did - L97

not exist in the íntact tissue, provÍded the 1-ysosomes r¡/ere intact in the dystrophic muscle prior to homogenization. However, we have no results

to either prove or disprove thís possibility. The two facts should be kept in mind though: oxÍdative phosphorylation in these dystrophic hamsËers was defective in vitro and the tissue composiËion of the skeletal muscle differs greatly between diseased and control animals in DNA and RNA

content and in lysosomal enz)¡me activities. As mentioned before, the relevance of these findings to the in vivo situation cannot be assessed at present. However, the decreased phosphorylation capaciËy of dystrophic mitochondria from Group 6 is unlikely to be of causal relationship to the dÍsease process, since this abnormality r¡/as observed in old animals in advanced stages of the disease. It is very likely that the observed mitochondrial malfunction ín this

Group is a secondary effect of the diseaser orr as also suggested, an in vitro artefact. This view is supporLed by the fact that from younger animals, already suffering from the disease, normal oxidative phosphorylation parameters \¡rere obtained in dystrophic animals (Group 1 with the Standard Proteinase Preparation, page L28 ; Group 3 with the Lochner Preparation, page 55 ; and Group 5 with the lulodíf íed Proteinase Prep- aration, page L7L ). Another area also needs further expLoration. That is the possibí1íty of a magnesium deficiency causing a miLochondrial malfunction.

Indications of an apparent magriesium deficiency of dystrophic mitochondria

ín vitro were seen in varíous animals from Groups 2-5, Bajusz (f03) recenËly corurunicated that magnesíum deficiency in muscle of BI0 14.6 hamsters was only very transitory. This could explain why a mitochondríal magnesium def iciency r¡/as not seen in all dystrophic animals. Further -198-

speculation appears unr/arranted at present. However, ít is clear that a

systematic study in these dystrophic hamsters of the magnesium metabolism

and the mitochondrial requirement.s for this ion is urgently needed,

Sun¡nary of the results obtained from hamster skeletal muscle

Oxídative phosphorylation has been studied in normal and dystrophic mitochondria of 6 different groups of hamsters. Normal as well

as abnormal results were obtained from dystrophic animals, aLI of which

def initely \¡7ere aff ected by the disease process. Animals of Groups 1 and 5 exhibited normal oxidatíve phosphorylation parameters. Dystrophic

hamster mitochondrLa of. Groups 2-4 showed defects in various oxídative

phosphorylation parameters, which disappeared when measured in the presence of a hexokinase trap either polarographicalLy or manometri caI|y. In separate experiments it could be demonstrated that therhealing' effect

of the hexokinase trap could be achieved also by MgC12, indicating some magnesíum deficiency in these mitochondria. However, this defect could not be observed when mitochondria from the same animals (Group 3) were isolated by the Lochner Procedure.

Dystrophic mitochondria from very old hamsters (Group 6) exhibited a respiration defect with palmitate, palmityl-L-carnitine, acetyl-L-carnitine, and pyruvate/malate. Since oxidatíon of NADH and succinate were not impaired, the possÍble common defect could lie in the citric acid cycle. This defect probably was not due to the magnesium deficiency seen in Groups 2-4, sínce the mitochondria were prepared by the Lochner Procedure and the defect T^ras also present when the mitochondria rrere tested manometrically in the presence of a hexokinase trap. -L99-

The muscles of dystrophic animals of Group 6 contaíned

elevated amounts of DNA and RNA and exhibíted higher activities of the lysosomal enz)¡nes cathepsin D and p-glucuronidase than the control tissues.

The bearing of all these in vitro resulËs on the situation

in J¡ivo has been discussed. It has been concluded that in view of the discrepant results obtained by different methods, these results allow no definite conclusions as regards the situatíon in vivo. However, it should be stressed that at least by one method, each of the oxidative phosphory:

latíon parameËers of dystrophÍc hamsters from Groups 1-5 did not dÍffer

from those of the control animals. The possibility that the apparent magnesium deficiency may play a primary role in the dystrophic disease process will be dealt wíth further in the Díscussion. XII. DISCUSSTON -20L-

KII. DISCUSSION

On theoretical ADP/0 ratios and th-e relation between ADP/0 and respiratorv control ratios

It has been mentioned at several places ín the Results that

the ADP/0 ratios obtained did not reach theoretical levels. I shall

deal now with the questions: Are our measured ADP/O values too low? Ilhat Levels should be expecled? Are the conclusions in this study ín- fluenced by possibly too low ADP/0 levels?

According to current belief, maximal ADP/0 raËios with NADH Iinked substrates, such as pyluvate/maLate, should approach the value of 3 (131, 167). In thís study such high values \,7ere not f ound, aL-

though in many groups high respiratory control ratios vlere observed. Since the latter is regarded as a more sensitive parameter for the Ín- tegrity of mitochondria (L28, 131, L32) , the quality of the mitochondrial

preparations exhibiting high RCRr s was considered acceptable. Generally the mítochondrial preparatÍons with good RCRIs gave

hígher ADP/0 ratios than preparations with poor RCR's. This was statistically verífied. A correlation between ADP/0 and respiratory control-

ratios of 487 periods of polarographic experiments proved to be highly significant. However, a scatter plot of the data (Fig. flA) indicated that the relatíonship was non-linear, but followed a polynomial function. The line of best fit in the portions of the scatter plot containing most of the data approximated a rectangular hyperbola.

Such a hyperbolic relationship between ADP/0 and respÍratory control ratios can be predicted, if the following assumptions are made:

(i) The maximaL ADP/ 0 ratio of three is associaËed with a -202-

Fig. 1l_. --SCATTER PLOTS OF mp/0 VERSUS RCR (A) AND ADP/O

vERSUS I/RCR (B).

Computer plots of data paírs f.rom 487 polarographicatly

obtained periods. In curve A the circles shor¿ the line of best fit drawn by the computer; in curve B the circles show Ëhe best firring straight line. This line is also

drawn in Fig. L2, curve C page 206 -203-

3.0

€ ftP{

3"0

ô ÊÊ{ "4

1"5

0"0 o"5 1/RCR Figure 1n -204-

RCR of infinity (with pyruvate/malate as substrate).

(ii) The highesË respiratory control ratio is to be expected,

when State 4 respiration ís zero, i.e. the RCR will have the value of infinity (cf. Fig. 1). (iii) State 4 is an rruncoupled" respiration which contributes a constant amount to Ehe preceding State 3 rate. In other \¡/ords, the

measured State 3 rate consists of a tcoupledt rate plus an 'uncoupledt rate. The 'coupledr rate by itself would give rise Ëo an ADP/O ratio of three, whereas the contribution of the 'uncoupled' State 4 respiration

to the measured State 3 rate would determine, how much the ADP/O ratio would deviate from the theoretical value. Thus, e.g. a RCR of 2 would

índicate that half the measured State 3 consisted of 'coupledr rate and

the other half of runcoupledr respiration, This would therefore give an

ADP/O of 1.5.

(iv) At a RCR of one, the ADP/O Ls zero.

Making these assumptions the relationship between ADP/0 and

RCR shovrn in Fig. LZA, \,ras constructed. The curve had some resemblance to the experi-mentally obtained curve (Fig. 114). As noted above, the function of Fig. I2A proved to be a hyperbola, since it could be converted

ínto a linear curve by plotting ADP/O versus I/RCR (Fie. LZB). When Ëhe same \¡/as done with the 487 experimenËal data pairs of Fig, 114, page 203 , the scatter pLoË shown as Fig. 118, page 203 , riras obtained. The best fitting straight line was plotted and is also included in Fig. 12, curve

C, with the standard error of estímates within curves d and e.

It will be noted that the theoretical maximum ADP/O ratio of 3 r^/as not observed in the present work. In part thÍs may have been due to the fact that the estimate of the 02 concentratj-on in the polarographic -205-

Fig. I2.--THEORETICAI AND EXPERIMENTAL RELATION BETI,IEEN AOP/O AND RCR

Curve A: ADP/0 versus RCR (theoretical)

Curve B: ADP/0 versus I/RCR (theoretical)

Curve C: ADP/0 versus l/nCn (experÍmenta1, taken from Fig. llB, page 203 ).

Curves d and e represent t I S.E. of curve C. A--- \¿\/ \ /^\ \ vso 1/RCR (theor"); \ù.-./ \B /ADPlo y =-3,0 1/x + 3"0 ar/ / I / I ¡ ADP/O vs. 1/RCR (exper.); y = -2.12 1/x + Z.5j i

Á,DPlo / I N) ! y = -1.89 1/x + 2.49 O ¡ Or J !r I Br\ j \a¡plo vs R.CR ( theor" ) y = -2.35 1/x + 2"60 ! " f f

Figure 12

RCR 0.5 1/RcR 1.O - 207 medium, obtained from physical tabl-es, may have been too high by 9%. (see Methods, page 59 ). Correction of the observed ADP/O ratios by this amount would have yielded in Fig. LZC an extrapolated maximum

ADP/O of 2.8, which, within the error of experimental methods, agrees reasonably well with the theoretical value of three.

Comparíson of the theoretícal (B) and experimental curves (C) in Fig. L2 aLso reveals a difference in Ëhe slopes. The experímenËal curve is flatter and does not yield an ADP/O value of zero at a RCR of one. This means thaË assumption (iv) has not been fulfilled. Also assumption (iii) appears to be partly r^rrong, since the contribution of the 'uncoupled' State 4 rate to the measured State 3 was lower, especíally in preparations with low RCRrs, Ëhan ËhaË predicted from theoretícal considerations, However, from experimental experience this is not unexpected. Lehninger et al (f90) have demonstrated that phosphorylation can occur during State 4 respiratÍon, if ADP ís added continuously to produce a constant low concentration of the nucleotide. It is clear from the above discussion that our measuring techniques did not yield maximal theoretical ADP/0 ratios, probably, because r¡/e overestimated the initiaL OZ concentration in the polarographic reaction medium. It was tempting to rrcorrectrr the 0, vai-ues, but we have not done so for two reasons. The first was Slaterts (167) recent coÍEnent on the current tendency to "helprr oxidative phosphorylation results to approach theoretical values:

"The concept of 'theoreticalr P/0 ratíos, whích is found frequently in the current literature, introduces an un- necessary and very real difficulty for the beginner. If he is cautious and self-critical, as he should be, he is -208-

Likely to obtaín considerably lower P/0 ratios than the ttheoreticalr . Since a ttheoreticalt?/0 raLio

has become a status symbol, he is irmnediately placed in a disadvantage

A second practical consideration was that a correction, if made, would apply to both the dystrophic and normal animals, and so would not alter the conclusions.

Oxidative phosphorvlation bv skeletal muscle mitochondrÍa Êrom normal and dvstrophíc mice

No abnormalities were detected in dystrophic mice in any of the four oxidative phosphorylation parameters, the ADP/0 ratios, respiratory control ratios, the oxidaÈion rates, and the capacíty of the mitochondrÍa to produce ATP (P rate). This was true whelher the organelles were íso- lated in the presence of albumin or not. These results are in agreemenE with mitochondrial studies on dystrophic mice by PeEer (2L4). However, L4 Lin et ù (49, 96) and SLrickl-and et aL (97) found the production of ,0, from palmitate- t-L4c, pyruvat"-3-14c, and from acet yt-t-L4c-carnitÍne decreased signíficantly in dystrophic mouse skeletal muscle. Although the reason for the discrepancy between tfteir findíngs and Lhose of Ëhe presenË study is not certain, it is likely that the difference in findings is related to the age of the dystrophic animals. While the mice in our first group studied ranged in age from 45 to 56 days (Table 1, page 74 ) and those from the second group f.rom 37 to 77 days (Table 2, page 75 ) the animals studied by the Strickland group ranged in age from 60-98 days and 53-L20 days respectÍvely. I¡trhether thís is the correct explanation -209-

remains to be demonstrated by a systematic study exploring the effects

of the age of the animals and the severity of the disease. However, it should be noLed that simílarly decreased oxidation rates wilh palmitate, palmityl-L-carnitine, acetyl-L-carnitine, and pyruvate were also seen in the present. study in skeletal muscle mitochondría of a group of old dystrophic hamsters (Group 6, see below). Although the skeletal muscle mitochondria of younger dystrophic mice of this study exhibited normal oxidative phosphorylatíon parameters in vitro, it is possible that the mitochondrial energy production coul-d be impaÍred in vivo by a nonmitochondrial defect, ê.g. if the subsËraËe supply to the mitochondria were decreased. This possibilÍty is supported by some evidence. As has been mentioned in the Literature Revíew, page

32 , glycolytic enzymes in dystrophic mouse muscle are decreased. More recently Coleman (57 , 2L5) found an increase of triosephosphates in dystrophic mouse muscle homogenates and a diminished synthesis of pyruvate.

Coleman demonstrated quite conclusively that this abnormality was caused by decreased activities of the enz)¡mes glyceraldehyde-3-phosphate dehydrogenase and L-oc-glycerophosphate dehydrogenase. The defect was demon- strable as early as LÞ, to 2l weeks after birth. This defect could possibly lead to a defect in energy productíon in the dystrophic cell in

3 ways:

(í) Due to decreased substrate level phosphorylation Ín glycolysis.

(ii) Due to decreased production of pyruvate, a major míto- chondrial fuel.

(iii) Due to a possibly impaired function of the L-oc-glycerophosphate shuttle (L32, L75), which is thoughr to transport ìIADH from the -2I0- cytoplasm Ínto the mitochondria. A decreased function of this shuLtle would not only transport fewer reducing equivalents into the mitochondria, but could also inhibit glycolysis by a rise in NADH/NA}| ratio Ín the cytoplasm (57).

There is some indirect evidence from nutrition studies that such an impairment of energy production might be in part responsible for the short life span of dystrophic mice (2L6). When dystrophic mice r,¿ere fed a diet rich in protein or fat, their f-ifespan was considerably longer than when they were fed Purina Lab Chow only (57 , 2L7). Even better results were obtained in a díet enriched with glycine (2L6, 2L7). The catabolic pathways of fatty acids and amino acids all lead ultimately into Ëhe citríc acid cycle in the mitochondria (64) without having to pass the postulated metabolic block at Ëhe triosephosphate level described above. It thus seems possible that the explanation for Lhe results of these nutritional studies is that the enriched diets contained fuels which could be converted into energy without usíng the glycolytic pathway at and above the level of triosephosphate.

If the above explanation is Ërue, it woul-d be an explanation for an interestÍng situation, in which energy production by the mitochondria in vivo woul-d be decreased due to a non-mitochondrial defect. Such a situation would be consÍstent wíth the normal oxidatíve phosphorylation results obtained in this study.

Oxidative phosphorylation bv heart mitochondria from normal and dystrophic hamsters

The results on oxidative phosphorylation in normal and dystrophic -zLL- hamster hearts r¡Iere sufirrlarízed on page L26. The results gave a clear ansl¡rer: although all dystrophic hamster hearts studied were affected by the dystrophy process, all the oxidative phosphorylation parameters vrere normal. This ü/as true for all the various substrates, reaction temperatures and measuring techniques tested. Normal results were also obtained when organell-es were isolated by a person other than the author.

These results aîe aL variance with those obtained by two other groups of rn¡orkers. The group of Lochner et al reported loosely coupled oxidative phosphorylation in dystrophic hamster heart homogenates (89) and also in isolated mitochondria (45). Schwartz et al (93) reported decreased respiratory control in heart mitochondria of strain B I0 14.6 dystrophic hamsters. Lochner et al (45) were able to show some evidence that the defect may have been at the phosphorylaËion step between cytochrome c and oxygen (Site III). Since Lochner et al used a different strain of myopathic hamsters, it is not knor^m whether it is proper to compare our findings with theirs. It is also possible that their animals r¡ere in a terminal stage of the disease with heart failure. In that case,

ít is quite likely lhat the observed mítochondrial defect was secondary to the dÍsease. This view is also supported by the studies of Helinsky et 41 (159) who found in aged liver mitochondría a defect in phosphory- laLion at Site III, but not between the flavoprotein level and cytochrome c (Site II). Thus, the oxidative phosphorylation system at the third phosphorylation site seems to be more susceptible to damage by aging or possibly other effects, such as hypoxia, which may occur during the heart failure produced by the dystrophic process.

The discrepancy between our study and that of Schwartz et_ aL

(93) has been recently clarified by those workers (94). They studíed -2L2- dystrophic hamster heart mitochondria from hamsters in heart hypertrophy

(as studied ín this thesis) and also from hamsters in terminal heart failure, The latter anímals were in a late stage of the disease with a life expectancy of less than 48 hours (94). Only in those moribund hamsters were Schwartz anð. his workers able to find the defect in oxidative phosphorylation by heart mitochondria. The consistently normal results found in this work with heart mítochondría from dystrophic hamsters, all of r¿hich were definitely affected by the dystrophic cardiomyopathy, suggest that the primary disease is not caused by a mitochondrÍal malfunction. This statement is valid only in as far as one can drar¿ conclusions as to the i-q vivo situation from in vítro experiments. Even if such an extrapolation were valid, it would apply only to hamsters of the age range studíed. However, neÍther very young animals nor hamsters in the terminal stage of the disease have been studied.

Changes in mitochondrial function in the later stages of the disease are of interest in that they may have a bearíng on the deveLop- ment of the terminal heart failure, but clearly are not causally related to functional and structural abnormalities that occur in the myocardium at a time when mitochondrial function is normal. On the other hand, a study of heart mitochondria from animals younger than those examined here might be of greater significance in this respect.

Inlhen the hearts of the first Group of hamsters rrere found to exhibit normal oxidatíve phosphorylation, progressívely older groups of animals were studied in the attempt to confirm the abnormalitÍes observed by other Ì^/orkers (45, 89, 93). Although normal results i4rere alr,rays obtained, this does not necessarily allow the conclusion that oxidative -2L3- phosphorylation in younger dystrophic hamster hearts can al-so be expected to be normal.

The youngest dystrophic heart studied was from a hamster 75 days old. However, as mentioned earlíer already, Bajusz et aL (166) found a significant. decrease in magnesium concentration in dystrophic hamster hearts from animals between 23 and 33 days old, but the defect could not be detected ín an animal group with a mean age ot 62 days (range 56-7L days). At present it is not known why this magnesium deficiency is only transient (103). However, Bajusz found that the earliest observable areas of necrosis in heart muscle are preceded by this magnesium deficiency (103). Since mitochondrial oxidative phosphorylation requires magnesium (L94) it is possible that an impaírment of energy production of the mito- chondría caused by a magnesium deficiency might be responsible for the appearance of necrosis. In a recent publ-ication Bajusz emphasized that these minor structural changes may be of etiological signifícance for the development of heart failure (I02). Thus, it seems possible thaË a defect in mitochondrial oxidative phosphorylation present only at an early stage of life in a dystrophic hamster heart could explain the subsequent development of heart dísease, ending in terminal heart failure. possible etiologic role of U{2 deficiency in early lífe The ^ of dystrophic hamsters is further supported by two other recent observations of Bajusz et al:

(i) I{hen myopathic hamsters were f ed a low l,tg-F2 di.t, they invariably díed wíthin eight days showing severe cardiac lesions, whereas the control animals survived and did not show any lesions prior to the 16th day. However, this was observed in hamsters 25-30 days old, but did not occur in animals older than 60 days (218). -2L4-

(ii) Inlhen parabiosis was perf ormed by surgical skín anastomosis between dystrophic and control hamsters, the life span of the dystrophic animals could be nearly tripled. However, this was only achieved when the parabÍosis lras started between 28 and 56 days of age. Parabiosis started at ages of 76 days and over had no life prolongation effect. Thus, these recent studÍes suggest that a critical defect occurs early in life and can be aggravated or prevented by various means. Un- fortunately, as noted above, oxidative phosphorylation has not been studied as yet in dystrophic hearts of hamsters younger thran 75 days.

Such a study Ís indicated in view of the possible consequences of a magnesium deficiency in younger animals described above. A hypothesis of the muscular dystrophy process in hamsters, which íncludes this aspect, will be presented at the end of this chapter. Thus, while the extensive studies in this thesis on heart mitochondria gave no indication at all of an impairment of oxidative phosphorylation and therefore no indication of the cause of the disease to be in the mitochondria, the possibility cannot be excluded that in young dystrophic hamsters an impairment of mitochondrial oxidative phosphorylation exists.

Oxidative phosphorylation by skeletal muscle mitochondría from normal and dystrophic hamsters

The results on. oxidative phosphorylation in skeletal muscLe mitochondria presented in this study seem to be confusing and inconsístent because quite different results were obtained by varíous isolation and measuríng techniques in the various hamster groups. A strict correlation -2L5-

of abnormalitíes to age r,ras not Eo be expected, since the progress of the disease in skeletal muscle can vary greatly from one animal group to another of

the same ages (103), A great deal of effort was spent to reproduce the findings of Lochner et al (44) who found decreased P/0 ratios, but norrnal oxidation rates in isolated skeletal muscle mitochondria of dystrophic hamsters. We r¡/ere able to confírm a defect, but the abnormalities in all

oxidative phosphorylation parameters (Group 6) seemed to be caused by a

defect in respiration rather than in Ëhe coupling of phosphorylation, as

explained in the Results on page L82 . Lochner et aI (44) are the only \,¡orkers , apariu from us, who have studied mitochondrial function of skeletal muscle in dystrophic hamsters. However, it is possible that in spite of

their use of the same species, our results are not directly comparable, sínce Lochner et al used dystrophic hamsters of a different strain (BIO

1. so) .

Essentially three types of results \^rere obtained in our study: (i) Normal oxidative phosphor.ylation parameters. (ií) Impaired oxidative phosphorylation in skeletal muscle of very old dystrophic hamsters.

(iii) rrUncoupledtr oxidative phosphorylation in hamsters aË ages between 72-2L3 days, which apparently was due to a magnesium deficiency.

In regard to point (i), normal oxÍdative phosphorylation was observed in the great majority of dystrophic hamster skeletal muscle preparations. trdith the exception of animals of Group 6, which consisted of very old hamsters, normal results were obtained in all the oËher groups by at least one method. These results are consistent with our findings on dysËrophic mÍce (90). Essentially normal oxidative phosphorylation was also reported in human dystrophic skeletal muscle by Olson et al (100), -216-

Peter et al (101) , and Hul-smann et aL (87). Ionasescu et al (9B) reported abnormalities viz. - decreased RCR's in Duchenne type dystrophy and decreased P/0 ratios in other types of human dystrophies, However, the reliability of their results seems to be somewhat doubtful, since they found a combinaEion of decreased P/O ratLos with normal RCR|s, which cannot be explained on grounds of presently accepted theoríes on oxidative phosphorylation (L67, L7L). Also, the increased respiration rates observed by March et al (95) in dystrophic chicken muscle may have been an in vitro artefact, since Ëhose workers measured the respiration in isolated mitochondria at a much too low phosphate concentration (219) , i. e. --that generated from I mM ATP by a hexokinase trap. A higher concentration of

Índigenous phosphate concentration in dystrophic mitochondria could explain their findings -- and phosphate concentrations are e.g. significantl-y elevated in striated muscle of dystrophic hamsters (44, 45). The decreased oxidation rates of dystrophic mouse mitochondria observed by Lin et aL (49, 96) and Strickland eL al (97,220) mentioned above, probably are characteristic of advanced stages of the disease. Such abnormalities are also reported here in very old dystrophic hamsters and have been seen in advanced stages of Duchenne type dystrophy (2L4). The significance of Lhis finding will be further discussed be1ow. Thus, the overwhelming impression from dÍrect studies of oxidative phosphorylation in muscular dystrophy is that this process is not impaired ín the earlier stages as long as possibly secondary effects of the disease on the mitochondria are not of too great an influence. As indicated in point (ii), page 2l-5 , abnormal results were also obtained with very o1-d dystrophic hamsters. Skeletal muscle mitochondria from Group 6 animals exhibited decreased values of all the oxidaËive -2I7 -

phosphorylation parameters tested, This fínding was observed with four

substrates: palmitate, palmítyl-L-carnitine, acetyl-L-carnitine, and

pyruvate, suggesting a possible cornrnon defect might be responsible for

all the observed abnormalities. As indicated in the Results, page LBz,

all the abnormal oxidative phosphorylation parameters observed can be explained without invoking a defect in the respiratory chain or in the

associated phosphorylation, both of which appear to be normal (see page

185) .

These results are qualitatively in agreement with those obtained by Lin et al (49, 96) and Strickland et al (97 , 220) on dysrrophÍc

mice. These workers also believe Ehat a cormnon defect might be responsible for all the observed abnormalitÍes. The most lÍkely localizaxion

of the defect would be the citric acid cycle (see ref. 220 anð. page 184 ). It should be reiterated at this poínt that in all likelihood these abnormalities observed in advanced stages of the disease are not causally related to the disease. A1so, in view of the quite different

cell population and the increased lysosomal enzyme activities in advanced stages of the disease (see page I94 ) the probability that the abnormal oxidative phosphorylation parameters represent the in vívo siËuation in the remaining muscle fibers seems doubtful (see page 189 ). In the authorrs opinion, the most importanË finding of thís

study is the impairment of oxidative phosphorylation caused by an apparent magnesium deficiency in dystrophic skeletal muscle mitochondria (point iii, page 2f5 ). This occurred in animals considerably younger than those of Group 6 and is best seen in Fig. 88, page152 in a dystrophic hamster only 79 days old. ?olarographically these mitochondria appeared uncoupled, Í.e. no RCR could be demonstrated and therefore the ADP/O ratio \^7as zero. Such -2r8-

defects T,vere present in animals of Groups 2-4. In several cases the beneficial effect of magnesium addition to the polarographic reaction medium was demonstrated, but in most cases thís effect \,ras inferred retrospectively from the respiratory behaviour of the mitochondria ín

Ëhe presence of a hexokinase trap containing magnesium.

These abnormalities, i.e. respiratory control ratios of one and ADP/0 ratios of zero are results obtained in vitro. The expression

"ADP/O equals zero't is of course a technícr, ; and does not mean thaË these mitochondría cannot synthesize ATP. The contrary has been shown in the presence of a hexokinase trap (Groups 2-4). Certainly, if there had in fact been a comparable uncoupling of oxidative phosphorylation in skeletal muscle in vivo, the dystrophic hamsters would noË be likely to live and definitely would not exhibit a fairly normal clinical appearance (22L). It should be noted that the uncoupling of oxidative phosphorylation due to an apparent Mg+2 defíciency shovrn in Fig. 88, page I52 , did not resemble the t.ypical uncoupling pattern as is usually described

(L67). Thus, truly uncoupled mitochondria respire at maximal rates as long as they are offered substrate and oxygen. They do not produce AïP, and the energy is dissipated as heat. Loosely coupled mitochondria, as defined by Ernster et al (66, 68) also respire at maximal rate, but can phosphorylate if the necessary substrates (pi and ADP) were offered. Mitochondria of the first type probably would be incompatible with Iífe. The latter, loosely coupled type, had been described by Ernster et al (66-69) in a mitochondrial myopathy associated with severe hypermetabolism (see Literature Revíew, page 34 ). Due to the relatively great skeletal muscle mass of the body -2r9- a defect of loosely or uncoupled mitochondría as defíned above clearly should be associated with an increase in the basal metabolic rate.

Tyler et al (222> found no change in basal metabolic rate Ín human mus-

cular dystrophy and Danowski et aL (223) observed a mean increase of. L9"/", which was noL significant. From the results obtained in this study, an increase in basal metabolic rate in muscular dystrophy also would not be expected, since rtruly' uncoupled or loosely coupled mítochondria were not observed. The abnormalíties in Group 6 i¿ere probably caused by a defect in respiration rates and the decreased oxidatÍve phosphorylation parameters in mitochondria wÍth apparent Mg+2 defíciencies always exhibited greatly reduced respiratíon rates.

As noted above, it Ís unlikely that the magrresium deficÍent mitochondria exhibit the same poor oxidative phosphorylation in vivo as they did in the polarographÍc experimenls. Nevertheless, these experiments clearly indicated a difference between normal and dystrophic animals, and it is possible thaL this difference might also exist in vivo to various degrees and cause a lack of energy production.

Based on these possibilities, the author wishes Ëo outline his concept on the sequence of events in the muscular dystrophy process in the BI0 L4.6 strain of hamsters. It should be clearly understood that at present this cannot be much more than a personal credo or a working hypothesis for further studies. It is outlined Ín Fig. 13 in form of a scheme illustrating a sequence of events. The primary genetic defect (Í) is still unidentified. A sarco- plasmic membrane defect (ii) as an early event in the dystrophic process was postulated some time ago (224-227 , 228). For (Íii) there is ample evidence in the literature (31, 5l-53 , 55-60, 105 , 107, lll , L46, L66, -220-

I-ig" 1 3 "--HYPOTHtrSIS 0N THE SEQUEI\CE OF Ã\TNNTS IN l.i-iÏi T,YSTRO* PITY FROCESS IN SYRIAN G,OIDEIq HêJ',STERS OF STRAI].] BTO 14 .6

(a) Prímary genetic d.efect

I ü (ri) Sarcop'l ¿smic tnenbrane d"efect

I T ( j_ii ) leakage of ce}l solutes, inclucling eleetrolytes (K*, ¡,rg**) and protei.ns ( glyco1-yti c enzyrûes , CPI( , transamÍnas es )

I s ( iv) l,{itochondrial impaÍrn.r.ent du-e to tlg** d.efj.eíenc;r

I ü (v) Acute energy lack in ind.ivid.ual fibers

I .,þ (vi) Necrosis I

I ..t (vii) InfÍltra*¡"* cells

{/I ( vr].]. ) Connective tissue, fat

( ix) uuru^u,{*r,. in muscle, of heart muscle muscular v'rasting

I .tI heart failu-re -22r-

224-22Ð. Event (iv) has been proven in vitro in this study and in-

directly supported by many findings of Bajusz (I02, LO3, L66,ZLB) as

outlined before. It is also knor^¡n for several years that a nutritional magnesium deficiency causes heart failure with necrosis and uncoupl-ing

of oxidative phosphorylation in heart mitochondria (229, 230). That lack of energy results in vivo, (v), is very diffÍcult Lo prove, but conceivable, It would offer one of the best possible explanations for the occurrerÌce of necrosislvÐIhe subsequent sequence of events (vii-íx), was proposed some tíme ago (7).

As noted in the scheme, the same primary defect leads to the pathologícal events in both heart and skeletal muscle. Although this assumption is of course not proven, the probability of its correcËness seems high, since all BfO L4.6 hamsters are always affected both in heart and skeletal rmrscle (165, 221,23L). The involvement of the hearË in human muscular dystrophy also is being detected with ever increasing frequency (9, 6L, 232-237 ) .

The verifícation or rejection in part or full of the above hypothesis will of course depend on future work. AL the moment iL seems possible that an early magnesium deficiency might impair oxidatíve phosphorylation sufficíently to initiate the se{uence leading to the overt disease. However, if this does not prove to be t.rue, it would then appear that impairment of mitochondrial oxidative phosphorylation is not the primary, or indeed an early secondary factor of this disease. Such abnormalities in oxidative phosphorylation, as have been observed here and by others, occur so late that it is likely they are a consequence rather than the cause of the disease process. XIII. LIST OF REFERENCES -223-

XIII. LIST OF REFERENCES

Erb, i,l . : Tagebl. d. Naturforscherversamml. in Freiburg 1883.

2. Erb, lni.: Uber die trjuvenile Formrr der progressiven Muskelatro- phie und ihre Beziehungen zur sogenannteri Pseudohypertrophie der Muskeln. Dtsch. Arch. klin. Med. 34, 46i (1884). I¡la1ton, J.N.: Muscular dystrophy and Íts relation to the other myopathies. Res. Pub1. Ass. nerv. menr. Dis. 38, 378 (1960)

4. I,r7a1ton, J.N. : Clinical aspects of human muscular dystrophy. In Muscular dystrophy in man and animals. Edited !X G.H. Bourne and Ma. N. Golarz. Hafner eultistringf.o. , -Irrc., N.Y. 1963, p. 263.

trrlalton, J.N.: Muscular dystrophy (c1inica1 , genetic, and pathological aspects). fn Biochemical aspects of neurological disorders. Edited -þy. J.N. Cumings and M. Kremer. Blackwell Scientific Publications. Oxford 1965, p. 1. 6. inlalton, J.N. : Dystrophia musculorum progressiva. In Progressive Muskeldystrophie, Myotonie, Myasthenie. Edited !¿ E. Kuhn. Springer Verlag, N.Y. !966, p. 57.

Pearson, C.M.: Pathology of human muscular dystrophy. In Muscular dystrophy in man and animals. Edited ÞJ G.H. Bourne and Ma. N. Golarz. Hafner Publishing Co., Inc., N.Y. 1963, p. 1.

8. Rossi, E. : Die l"Iyopathien in padiatrischer Sicht. In Progres- sive Muskeldystrophie, Myotonie, l'{yasthenie. Edited !¿ E. Kuhn. Sprínger Verlag, N.Y. L966, p. L32.

q Eckert, H. and R. Kirst: Das Herz bei der dystrophia musculorum progressiva Erb. ZbI. allg. Path. path. Anat. 109, 264 (1966) 10. Pearson, C.M.: History, Erb, dystrophy and beyond. In Progres- sive Muskeldystrophie, Myotonie, Myasthenie. Edited !y E. Kuhn. Springer Verlag, N.Y. L966 , p. 13.

11 Beckmann, R.: Gesprach uber die Therapie der progressiven Muskeldystrophie. In Progressive }luskeldystrophie, Myotonie, Myasthenie. Edited !y E. Kuhn. Springer Verlag, N.Y. L966, p. r45.

T2 In: Muscular Dystrophy Reporter XI, 1 (1968) , published by The Muscular Dystrophy Association of Canada, Toronto.

LJ Homburger, F., C.I^i. Nixon, M. Eppenberger, and J.R. Baker: Hereditary myopathy in the Syrian hamster: Studies on pathogenesis. Ann. N.Y. Acad. Sci. 138, 14 (1966). -224-

14, Homburger, F. , J.R. Baker, C.W. Nixon, and R. trrlhitney: Primary, generalized polymyopathy and cardiac necrosis in an inbred line of Syrian hamsters. lu1ed. exp. 6, 339 (1962).

15. Harman, P.J., J.P. Tassoni, R.L. Curtis, and M..B. Hollinshead: Muscular dystrophy in the mouse. In Muscular dysÈrophy in man and animals. Edited -ry. G.H. Bã-urne and l4a. N. Golarz. Hafner Publishing Co., Inc., N.Y. 1963, p. 407. L6. Michelson, 4.M., E.S. Russel, and P.J. Harman: Dystrophia muscularís: A hereditary primary myopathy in the house mouse. Proc. Nat1. Acad. Sci. USA 41, I079 (i955).

L7 . Rigdon, R.H.: Muscular dystrophy: Spontaneous occurrence in ducks. Texas Rep. Biol. t"Ied. 19, L67 (1961) .

18. Rigdon, R.H.: Spontaneous muscular dystrophy in the r¿hite Pekin duck. Am. J. Path.39,27 (1961).

19. Harper, J.A. and J.E. Parker: Hereditary muscular dystrophy in the domestic turkey. J. Hered. 58, 189 (1967>.

20. Wechsler, W. und hT. Pabelick: Erbliche MuskeldysËrophien beim Tier. In Progressive Muskeldystrophie, Ivlyotonie, Myasthenie Edited !¿ E. I(uhn. Springer Verlag, N.Y. L966, p, 165. 21. Julian, L.M. and V.S. Asmundson: Muscular dystrophy of the chicken. In Muscular dystrophy in man and animals. Edited -Þy. G.H. Boäne and Ma. N. Golarz. Hafner Publishing C"., Inc., N.Y. L963, p. 457.

22 Rigdon, R.H.: Hereditary myopathy in rhe v¡hite Pekin duck. Ann. N.Y. Acad. Sci. 138, 28 (1966).

23 Mclntyre, 4.R., A.L. Bennett, and J.S. Brodkey: luluscle dystrophy in mice of the Bar Harbor strain. Arch. Neurol. Psych. 81, 678 (1ese).

24. GoTarz, M.N. and G.H. Bourne: Some histochemical observatÍons on the muscles of mice with hereditary muscular dystrophy. Acta anat. 43, f93 (1960) .

25 Pearce, G.W. and J.N. Inlalton: A histological study of muscle from the Bar Harbor strain of dystrophic mice . J. PaLh. Bact. 86, 2s (1963). 26. Murphy, E.D.: Discussion. Ann. N.Y. Acad. Scí. 138, 59 (L966).

27 . Milhorat, A.T. : Introductory remarks. Ann. N.Y. Acad. Sci. 138-, 3 (1e66) .

28 . Drummond, G. I. : Muscle metabolism. Fortschr. ZooI. 18, 359 (1967). -225-

)q West, E.S., W.R. Todd, H.S. Mason, and J.T. Van Bruggen: Textbook of Biochemistry, 4th ed.. The Macmillan Company N.Y. L966.

30. Asmundson, V.S. and L.lul. Julian: Inherited muscle abnormality in the domestic fow1. J. Hered. 47, 248 (1956).

31. Dreyfus, J.C. and G. Schapira: Biochemistry of hereditary myopathies. Charles C. Thomas, Publisher, Springfield, 111. L962.

32. CoIIazo, J.4., J. Barbudo, and I. Torres: Der Chemismus des Muskels bei der Dystrophia muscularis progressiva. Dtsch. med. trrlschr. 62, 57 (1936). 33. Reinhold, J.G. and G.R. Kingsley: The chemical composition of voluntary muscle in muscle disease: A comparison of progressive muscular dystrophy with other diseases together with a study of glycine and creatine therapy. J. clin. Invest. L7 , 377 (1938). 34. Lilienthal Jr., J.L., K.L. Zi'er1-er, B.P. Folk, R. Buka, and M.J. Riley: A reference base and system for analysis of muscle constituents. J. biol. Chem. 182, 501 (i950).

35. Bonetti, E., N. Frontali Toschi, and tvl. Levi: Acid-soluble phosphorus fraction in progressive muscular dystrophy. Sperimentale 104, 315 (19s4).

36. Zymaris, M.C., N. Epstein, A. Saifer, S.M. Aronson, and B.I^l . Volk: Distribution of acid-soluble nucleotides in hind 1eg muscles of mice \^7ith dystrophia muscularis. A,rn. J. Physiol . L96, 1oe3 (195e).

37 Ronzoni, E., S. Wa1d, L. Berg, and R. Ramsey: Distribution of high energy phosphate in normal and dystrophic muscle. Neurology B, 359 (1958) .

38. Harper, H.A.: A review of physiological chemistry. Lange Medical Publication, Los A1tos, CaLíf.. 1967, p. 474. 39. Carlson, F.D. and A. Siger: The creatine phosphoryltransfer reaction in iodoacetate-poisoned muscle. J. gen. Physiol. 43, 301 (1959).

40. I^iilkie, D.R.: Energetic aspects of muscular contractions. In Symp. Biol. Hung. 8, 207 (1967). Edited Þy E. Ernst and F.B. Straub. Akademiai Kiado, Budapest 1968. 4r. I^iilliamson, J.R. : Glycolytic control mechanisms. II. Kinetics of intermediate changes durÍng the aerobic-anoxic transition in perfused rat heart. J. þiol. Chem. 24I,5026 (L966). -226-

42. Lowry, O.H.: Metabolite 1eve1s as indicators of control mechanisms Fed. Proc. 25 , 846 (1966) .

+J Kaldor, G. and J. Gitlin: ATPase, myokinase, 5'adenylic acid deaminase activity and syneresis of the myofibrils of the dystrophic mouse. Proc. Soc. exp. Bio1. Med. 113,802 (1963).

44 Lochner, A. and A.J. Brink: Oxidative phosphorylation and glycolysis in the hereditary muscular dystrophy of the Syrian hamster. C1in. Sci.33,409 (7967). 45. Lochner, A. , L.H. 0pie, A.J. Brink, and A.R. Bosman: Defective oxidative phosphorylation in hereditary myocardiopathy in the Syrian hamster. Cardiovasc. Res. 3, 297 (1968). 46. Zymarís, M.C., A. Saifer, and B.W. Volk: Specific activities of acid-solub1e nucleotides ín hind-1eg muscles of mice with dystrophia muscularis. Nature 188, 323 (1960). 47. Bajusz, E., F. Homburger, J.R. Baker, and L.H. Opie: The heart muscle in muscular dystrophy with special reference to involvement of the cardiovascular system in the hereditary myopathy of the hamster. Ann. N.Y. Acad. Sci. 138, 213 (1966). 48. Srivastavâ, U.: Biochemical changes in progressive muscular dystrophy. VII. Studies on the biosynthesis of protein and RNA in various cellular fractions of the muscle of normal and dystrophic mice. Can. J. Biochem. 46,35 (1968). 49. Lin, C.H., A.J. Hudson, and K.P. Strickland: Fatty acid metabolism in dystrophic muscle in vitro. Life Sciences 8, 2L (1969).

50 Bucher, Th., K. Krejci, W. Russmann, H. Schnitger, and InI . Wesemann: Metabolite assay in frozen samples of liver tissue. In Rapid mixing and sampling technique in biochemistry. Edited þ B. Chance, R.H. Eisenhardt, Q.H. Gibson, and K.K. Lonberg- Ho1m. Acad. Press. N.Y. L964, p.255.

51 Dreyfus, J.C. et G. Schapira: Glycog6nolyse et phosphoglucomutase du muscle humain normal et myopathique. C.R. Soc. Biol. (Paris) 147, LL45 (1953) .

52. Ronzoni, E., L. Berg, and I^I . Landau: Enzyme studies in progressive muscular dystrophy. Neuromusc. Disord. 38, 72I (1960).

53. Schapira, G. and J.C. Dreyfus: Biochemistry of progressive muscular dystrophy. In Muscular dystrophy in man and animals. Edited _ÞL G.H. Bourne and Ma. N. Golarz. Hafner Publishing Co., Inc., N.Y. 1963, p. 47 .

54. Vignos, Jr., P.J. and M. Lefkowitz: A biochemical study of certain muscle constituents in human progressive muscular dystrophy. J. c1in. Invest. 38, 873 (1959) . -227-

55 Dreyfus, J.C., G. Schapira, and F. Schapira: Biochemical study of muscle in progressive muscular dystrophy. J. c1in. Invest. 33 , 7 94 (i954) .

56. McCaman, l"l.hi. : Dehydrogenase activities in dystrophic mice . Science lI, 62I (1960). 57. Coleman, D.L.: Accumulation of triose phosphates in dystrophic mouse muscle homogenates. Arch. Biochem. 111, 494 (1965). 58. Leonard, S.L.: Phosphorylase and glycogen levels in skeletal muscle of mice with hereditary myopathy. Proc. Soc. exp. Biol. Med. 96, 72O (L957).

59 Richterich, R. : Biochemische Unterschiede zwischen Muskeldys- trophie und Muskelatrophie: Cytoplasmatische Enzyme in der Muskulatur. In Progressive luluskeldystrophie, Myotonie, Myasthenie. Edited Þ E. Kuhn. Springer Verlag, N.Y. 1966, p. 155.

60. Harm, K.: Enzymaktivitatsbestimmungen in Leber und Muskel von lvlausen mit hereditarer Muskeldystrophie. Enzymol. biol. c1in. 9, 205 (1968).

6I . Sundermeyer, J.F., S. Gudbjarnason, V.E. tr^Iendt, P.B. Den Bakker, and R.J. Bing: Myocardial metabolism in progressive muscular dystrophy. Circulation 24,1348 (1961).

62. Kleine, T.0. und H. Chlond: Enzyrnmuster gesunder Skelett-, Herz- und glatter Muskulatur des Menschen sowie ihrer pathologischen Veranderungen mit besonderer Berucksichtigung der Muskeldys- trophie (Erb) . Clin. chim. Acta 15, 19 (1967) . 63. Mansour, T.E.: Studies on heart phosphofructolinase: Purification, inhibition, and activation. J. bio1. Chem. 238, 2285 (1963). 64. In/hite, 4., P. Handler, and E.L. Smith: Principles of biochemistry. 4th ed., McGraw Híll Book Company. Toronto 1968.

65. lless, B. and K. Brand: Enzyme and metabolite profiles. In Con- trol of energy metabolism. E{ited !¿ B. Ch.tr"", n.W.-estabrook, and J.R. l{il1iamson. Acad. Press N.Y. 1965, p. 111.

66. Ernster, L., D. Ikkos, and R. Luft: Enzymatic activities of human skeletal muscle mitochondria: A tool in clinical metabolic research. Nature 184, 1851 (1959).

67. Luft, R., D. Ikkos, G. Palmieri, l. Ernster, and B. Afzelius: A case of severe hypermetabolism of non thyroid origin with a defect in the maintenance of mitochondrial respiratory control: A correlated clinical, biochemical, and morphological study. J. clin. Invesr. 4I, L776 (1962). -228-

68. Ernster, L. and R. Lufr: Further studies orr a population of human skeletal muscle mitochondria lacking respiratory control. Exp. Cel1 Res . 32, 26 (1963) . 69. Ernster, L. and R. Luft: ltitochondrial respiratory control. In Advances in metabolic disorders, Vo1. 1. Edited !X R. Levine and R. Luft. Acad. Press N.Y. 1964, p. 95.

70. Van Breemen, V.L.: Ultrastructure of human muscle. II. Obser- vations on dystrophic striated muscle f ibers . A,rn. J . Path. 37 , 333 (1960).

7L Fisher, E.R., R.E. Cohn, and T.S. Danowski: Ultrastructural observations of skeletal muscle in myopathy and neuropathy with special reference to muscular dystrophy. Lab. Invest. 15, 778 (1966).

72. Shy, G.M. and N.K. Gonatas: Human myopathy with giant abnormal mitochondria. Scíence 145, 493 (1964). 73. Ross , M.H., G.D. Pappas, and P.J. Harman: Alterations in muscle fine structure in hereditary muscular dystrophy of mice. Lab. Invest. 9, 388 (1960).

74. Platzer, A.C. and W.H. Chase: Histological alterations in preclinical mouse muscular dystrophy. Am. J. Path. lç4, 931 (Le64) .

75, trrlechsler, trnI; und W. Pabelick: Erbliche Muskeldystrophien beim Tier. In Progressive Muskeldystrophie, Myotonie, Myasthenie. Edited l¿ E. Kuhn. Springer Verlag, N.Y. 1966, p. 165.

76. Miledi, R. and C.R. Slater: Some mitochondrial changes in denervated muscle. J. Cell Scí.3,49 (1968).

77 Inleinbach, E.C., J. Garbus, and H.G. Sheffield : lulorphology of mitochondria in the coupled, uncoupled, and recoupled states. Exp. Cell Res . 46, I29 (L967) . 78, Hackenbrock, C.R.: Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes r¿ith change in metabolic steady state in isolat.ed liver mitochondria. J. Cell Biol. 30, 269 (1966) 79. Hackenbrock, C.R.: Ultrastructural bases for metabolically linked mechanÍca1 activity in mitochondria. II. Electron transport linked ultrastructural transformations in mitochondria. J. Cell Bíol . 37 , 345 (1968). B0 . Gonatas , N. K. and G.M. Shy: Childhood myopathies with abnormal mitochondria. Excerpt. Med., Intern. Congr. Ser. No. 100, p. 606 (L96s). -229-

81. Shy, G.M. , N.K. Gonatas, and M. Perez: T\do childhood myopathies with abnormal mitochondria. BraÍn 89, 133 (L966).

82. Hulsmann, W.C., J. Bethlem, A.E.F.H. Meijer, P. Fleury , and J.P.M. Schellens: Myopathy with abnormal structure and function of muscle mitochondria. J. Neurol. Neurosurg. Psychiat. 30, 519 (1967).

83. Van laiijngaarden, G.K., J. Bethlem, A.E.F.H. Meijer, Id.C. Ilulsmann, and C.A. Feltkamp: Skeletal muscle disease with abnormal mitochondria. Brain 90, 577 (1967) . 84. DtAgostino, 4.N., F.A. Zíte'r, M.L. Ral1ison, and P.F. Bray: Familial myopathy with abnormal mitochondria. Arch. Neurol. (Chic.) 18, 388 (1968).

B5 Coleman, R.F., A.W. Nienhius, I^I.J. Brown, T.L. Munsat, and C.M. Pearson: New myopathy with mitochondrial enzyme hyper- activity. J. Am. med. Ass. I99, 624 (L967). 86. Hulsmann, W.C., J.W. De Jong, and A. Van Tol: Mitochondria with loosely and tightly coupled oxidative phosphorylation in skeletal muscle. Biochim. biophys. Acta 162, 292 (1968).

87. Hulsmann, W.C., A.E.F.H. Meijer, J. Bethlem, and G.K. Van trriijngaarden: Different mitochondrial species in human skeletal muscle. Excerpt. Med., Intern. Congr. Ser. No. 186, p. 48 (1969).

88. Fardeau, M.: Ultrastructural lesions observed in progressive muscular dystrophies: Critical study of their specificity. Excerpt. Med., Intern. Congr. Ser. No. 186, p. 9 (1969).

89. Opie, L.H., A. Lochner, A.J. Brink, F. Homburger, and C.lnl . Nixon: 0xidative phosphorylat.ion in hereditary myocardiopathy in the Syrian hamster . Lancet 2, L2l3 (7964) .

90. Inlrogemann, K. and M.C. Blanchaer: Oxidative phosphorylation by muscle mitochondria of dystrophic mice. Can. J. Biochem. 45, L277 (L967) .

9I. Wrogemann, K. and M.C. Blanchaer: Respiration and oxidative phosphorylation by muscle and heart mitochondria of hamsters with hereditary myocardiopathy and polymyopathy. Can. J. Biochem. 46, 323 (1968) .

92. Blanchaer, M.C. and K. L{rogemann: Oxidative phosphorylation by mitochondria isolated from hearts of BIO 14.6 myopathic hamsters. Trans. N.Y. Acad. Sci. 30, 949 (1968).

93. Schwartz, 4., G.E. Lindenmayer, and S. Harigaya: Respiratory control and calcium transport in heart mitochondria from the cardiomyopathic Syrian hamster. Trans. N.Y. Acad. Sci. 30, esr (1e68). -230-

94. Lindenrnayer, G.E., S. Harígaya, E. Bajusz, and A. Sch\dartz: Oxidative phosphorylation and calcium transport of mitochondria isolated from cardiomyopathic hamster hearts. Sub- mitted for publication.

95. lularch, 8.E., J. Bie1y, and V. Coates: Respiration rate of muscle mitochondria from genetically dystrophic chickens. Proc. Soc. exp. Bio1. Med. 129, 566 (1968). 96. Lin, C.H. , A.J. Hudson, and K.P. Strickland: Oxidation of palmitic acid 1-'L4C by muscle homogenates and mitochondrial preparations from dystrophic mice (strain I29). Fed. Proc. 27 , 332 (1968).

97 Strickland, K.P., C.H. T-in, and A.J. Hudson: Lipid metabolism in dystrophic muscle . Excerpt. Med., Intern . Cong. Ser. No. LB6, p. 44 (Ie6e).

98 Ionasescu, V., N. Luca, and 0. Vuia: Respirat.ory control and oxidative phosphorylation in the dystrophic muscle. Acta Neurol. Scand . 43 , 564 (L967) . 99. Ionasescu, V., N. Luca, and 0. Vuia: Disturbance of the oxidative phosphorylation in the human dystrophic and denervated muscle. Excerpt. Med., Intern. Congr. Ser. No. 186, p. 16 (L969).

100. Olson, E., P.J. Vignos Jr., J. tr^Ioodlock, and T. Perry: Oxidative phosphorylation of skeletal muscle in human muscular dystrophy. J. Lab. clin. Med. 71, 220 (1968). 101. Peter, J.8., K. Stempel, and J. Armstrong: Biochemical and electron microscopic studies of mitochondria isolated from patients with muscular and neuromuscular diseases. Excerpt. lv1ed. , Intern. Congr. Ser. No. L86 , p. 49 (L969) .

IO2. Bajusz, E., J.R. Baker, C.W. Nixon, and 'F. Homburger: Spontaneous hereditary myocardial degeneration and congestive heart failure in a strain of Syrian hamsters . Ann. N.Y. Acad. Sci. 156, lOs (re6e). r03 Bajusz, E.: Personal communications.

104. C1eland, W.W. : Dithiothreitol (DTT) , a netÀr protective reagent f or SH-groups. Biochemisrry 3, 480 (1964) .

105. Eppenberger, M., C.W. Nixon, J.R. Baker, and F. Homburger: Serum phosphocreatine kinase in hereditary muscular dystrophy and cardiac necrosis of Syrian golden hamsters. Proc. Soc. exp. Biol. Med. 117., 465 (7964).

106 RotLhauwe, H.W. und S. Kowalewski: Aktivierung und Alterung der Serum-Kreatin-Phosphokinase. Kli. Ilschr . 45_, 387 (7967) . -23r-

L07. okinaka, S., f. sugita, H..Momo|, Torokura, T. watargÞu, F..Ebashi, and S. Ebashi: Cystein-stimulatedT. serum creatine kinase in health and disease. J. Lab. clin. Med. 64, 299 (1964). i08. Fleisher, G.A. : Automated method for the determination of serum creatine kinase activity. Clin. Chem. 13, 233 (1967) . i09. Rosalki, S.B.: An improved procedure for serum creatine phosphokinase determination. J. Lab. clin. Med, 69, 696 (1967).

110. Inliesmann, W., J.P. Colombo, A. Adam, and R. Richterich: Deter- mination of cysteine actívated creatine kinase in serum. Enzymol. bio1. c1in. 7, 266 (1966). 111. Vester, J.W., G. Sabeh, R.H. Newton, H.B. Finkelhor, G.H. Fetterman, and T.S. Danowski: Muscle creatíne phosphokinase in primary myopathies. Proc. Soc. exp. Biol. Med, I2B, 5 (1968). IL2. Lowry, 0.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall: Pro- tein measuremenl with the folin phenol reagent. J. bio1. Chem. 193, 265 (1951).

113 . Pennington, R.J. and J.E. Robinson: Cathepsin activity in normal and dystrophic muscle. Enzymol. biol. c1in. 9, 175 (1968).

TL4. Kind, P.R.N. and E.J. King: Estimation of plasma phosphatase by determination of hydrolyzed phenol with amino-antipyrine. J. clin. Path. 7 , 322 (1954). 115. Pollack, M.S. and J.I^/.C. Bird: Distribution and particle properties of acid hydrolases in denervated muscle. Am. J. Physiol. 2L5, 716 (1968).

116. Gianetto, R. and C. De Duve: Tissue fractionation studies. 4. Comparatíve study of the binding of acid phosphatase, P- glucuronidase, and cathepsin by rat liver particles. Bio- chem. J. 59, 433 (1955) .

II7. Levy, G.A. and J. Conchie: Mammalian glycosidases and their inhibition by aldolactones. In Methods Í-n Enzymology, Vo1. 8. Edited E.F. Neufeld and V. Ginsburg. Acad. Press N.Y. Ww Ð stt. 118. Anson, M.L.: The estimation of cathepsin rvith hemoglobin and partial purification of cathepsin. J. gen. Physiol. 20, 56s (1937). 119. Bird , J.W.C., T. Berg, and J.H. Leathem: Cathepsin activity of liver and muscle fraction of adrenalectomized rats. Proc. Soc. exp. Biol. NIed. \27, r82 (re68).

I2O - Park , D.C. and R.J. Pennington: Proteinase activity in muscle particles. Enzymol. bio1. clin. 8, L49 (L967). -232-

IzL. Schmidt, G. and S.J. Tannhauser: A rnethod for the determination of desoxyribonucleic acid, ribonucleic acid, and phos- phoproteins in animal tissues. J. bio1. Chem. 161, 83 (1945>.

L22. Burton, K.! A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315 (1956).

L23. Giles, K.W. and A. Meyers: An improved diphenylamíne method for the estimation of deoxyribonucleic acid. Nature 206, 93 (1965).

L24. Chance, B. and B. Hagihara: Direct spectroscopic measurements of interaction of compounds of the respiratory chain with ATP, ADP, phosphate, and uncoupling agents. Proc. Int. Congr. Biochem. 5th Moscow, I96L, 5, 3 (1963). L25. Chance, B.! Spectrophotometry of intracellular respíratory pig-. ments. Science I20, 767 (1954) . T26. Hagihara, B.i Techniques for the application of polarography to mitochondrial respiration. Biochim. biophys. Acta 46, 134 (1e61). t27 Clark, Jr., L.C., R. lniolf , D. Granger, and Z. Taylor: Continuous recording of blood oxygen tensions by polarography. J. appl. Physiol. 6, 189 (1953).

I2B. Chance, B. and G.R. Williams: The respiratory chain and oxidative phosphorylation. Advanc. Enzymol. L7, 65 (1956).

L29. Umbreit, W.W., R.H. Burris, and J.F. Stauf fer: Manometric techniques, 4th ed., Burgess Publishing Company, Minneapolis 1964. 130. Estabrook, R.W. and B. Mackler: Enzymatic and spectrophotometric studies of a reduced diphosphopyridine nucleotide oxidase preparation from heart muscle. J. biol. Chem. 229, 1091 (1957)

131 Lehninger, A.L. : The mitochondrion. I^I .4. Benjamín Inc. , N.Y. L964.

L32 Klingenberg, M.: Muskelmitochondrien. Ergeb. Physiol. bío1. Chem. exptl. Pharmakol. 55, 131 (1964). f33. Nielsen, S.0. and A.L. Lehninger: Phosphorylation coupled to the oxidation of ferrocytochrome c. J. biol. Chem. 2I5, 555 (1955)

L34. Martin, J.B. and D. Doty: Determination of inorganic phosphate. Modification of the isobutyl alcohol procedure. Ana1. Chem. 2L, 965 (L949) .

135. Fiske, C.H. and Y. Subba Row: The colorimetric determination of phosphorus. J. biol. Chem. 66,375 (L925). - á.4.1 -

136. Slater, E.C.: Manometric methods and phosphate determination. In Methods in Enzymology Vo1. 10. Edited -Þ¿ R.l,/. Estabrook and M.E. Pul1man. Acad. Press, Inc., N.Y. L967, p. 19.

I37 . Layne, E.r Spectrophotometric and turbidimetric methods for measuring protein. In Methods in Enzymology Vol. 3. Edíted !y S.P. Colowick an¿ lt.O. Kaplan. Acad. Press, Inc., il?:- 1957 , p. 447 . 138. Blanchaer, M.C.: Respiration of mitochondria of red and white skeletal muscle. Am. J. Physiol. 206 1015 (1964). L39. Bergmeyer, H.U.: ?rinciples of enzymatic analysis. In Methods of enzymatic analysis. Edited !¿ H.U. Bergmeyer. Acad. Press N.Y. 1963, p. 3.

140. KlingenberB, M.: Reduced diphosphopyridine nucleotide (DPNH). In Methods of enzymatic analysis. Edited !I H.U. Bergmeyer. Ããad. Press N.Y. 1963, p. 531.

L4L. P-L Biochemicals Inc., Circular OR-10, 5th prinLing, 1967. L42. Hestrín, S.r The reaction of acetylcholine and other carboxylic acid derivatives r¡/ith hydroxylamine, and its analytical application. J. biol. Chem. L80,249 (1949).

I43. Skídmore, W.D. and C. Entenman: The determination of esterified fatty acids in glycerides, cholesterol esters, and phosphatídes. J. Lipid Res.3,356 (1962).

L44. Brendel, K. and R. Bressler: The resolution of (!)-carnitine and the synthesis of acylcarnitines. Biochim. biophys. Acta 137, e8 (Le67). f45. Bremer, J.t Long-chain acylcarnitines. Biochem. Prep. 12, 69 (1968). 146. Kleitke, 8., E.G. Krause, und A. Wollenberger:. Zu Bedeutung des Glycerin- 1- Phosphat-Zyklus im Stof fwechse 1 des l¡Iarmbluterherzens . Acta biol. med. germ. 11, 660 (1963).

I47. Cleland, K.W. and E.C. Slater: Respiratory granules of heart muscle. Biochem. J. 53, 547 (1953) .

148. Price, C.A. and K.V. Thimann: The succinic dehydrogenase of seedllngs' Arch. Biochem. 33, L7O (1951) . 149. Dixon, W.J. and F.J. Massey Jr.: Introduction to statistical analysis. lulcGrar¡-Hill Book Company, Inc., N.Y. 1957. 150. Ducumenta Geigy, Scientific Tables 6th edition. Egit"dty K. Diem. Geigy Pharmaceuticals, Montreal, 1962. -234-

151 Smith, A.L.: Preparation, properties, and conditions for assay of mitochondria: slaughterhouse material, smal1 scale. In Methods in Enzymology Vol. 10. Edited !¿ R.lI. EsËabrook and J.R. I^lilliamson. Acad. Press, Inc. , N.Y. L967 , p. 81. L52. Nachlas, M.M., K.-C. Tsou, E. De Sonza, C.-S. Cheng, and A.M. Seligman: Cytochernical demonstration of succinic dehydrogenase by the use of a ne\^r p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5, 420 (L957).

153. Bajusz, E.i Succinic dehydrogenase in muscular dystrophy. An experimental study on secondary changes resulting from disturbance in neuromuscular integrity. E*p. Med. Surg. 23, 169 (196s).

L54. Blanchaer, M.C., C.-G. Lundquist, and T.J. Griffith: Factors influencing the utilizatLorr of reduced nicotinamide dinucleotide by pigeon heart mitochondria. Can. J. Biochem. 44, 105 (re66) .

155. Lindenmayer, G.E., L.A. Sordahl, and A. Schwartz: Reevaluation of oxidatÍve phosphorylation in cardiac mitochondria from normal animals and animals in heart failure. Circul . Res. ry, ß9 (1e68).

156. Lewis, S.E. and E.C. Slater: Oxidative phosphorylation in insect sarcosomes. Biochem. J. 58, 207 (1954). L57. Sacktor, B.: Investigations on the miËochondría of the housefly musca domestica 1. III. Requirements for oxidative phos- ph".yt.ti.tr..i. gen. PhysioI. 37, 343 (1954).

158. I,üeinbach , E. C. and J . Garbus: Restoration by albumin of oxidative phosphorylation and related reactions. J. biol. Chem. 241, r6e (1e66).

159. Helinski, D.R. and C. Cooper: Studies on the action of bovine serum albumin on aged rat liver mitochondria. J. biol. Chem. 235, 3s73 (1e60). f60. ùiojtczak, T,. and A.L. Lehninger: Formation and disappearance of an endogenous uncoupling factor during swelling and contraction of mitochondria. Biochim. biophys. Acta 51, 442 (196i).

161. Platzer, A.C. and tr{.H. Chase: Histological alteration in preclinical mouse muscular dystrophy. Arn. J. Path. 44, 931 (1964).

L62 Ross, M.H. , G.D. ?apas, and P.J. Harman: Alterations in muscle f ine structure in hereditary muscular dystrophy of míce. Lab. Invest. 9, 388 (1960).

163. Fisher, E.R., R.E. Cohn, and T.S. Danowski: Ultrastructural observation of skeletal muscle in myopathy and neuropathy with specíal reference to muscular dystrophy. Lab. Invest. Þ, 778 (1e66). -235-

L64. Homburger, F., J.R. Baker, C .inl . Nixon, and G. tr^Iilgram: Ner¡/ hereditary disease of Syrian hamsters. Arch. intern. Med, 110, 660 (L962 ).

165. Homburger, F. , J.R. Baker, G.F. LrÌi1gram, J.B. Caulf ield , and C"W. Nixon: Hereditary dystrophy-1ike myopathy. Arch. Path. 81, 3o2 0966) .

L66 Bajusz, E. and K. Lossnitzer: A new disease model of chroníc congestive heart failure: S tudies on its pathogenesis. Trans. N.Y. Acad. Sci. 30, 939 (1968).

L67 . Slater, E.C.: Oxidative phosphorylation. In Comprehensive biochemistry, Vo1. 14. Edited !¿ M. Florkin and E.H. Stotz. Elsevier Publishing Company, N.Y. L966, p. 327. r68 01son, M.S. and R.Ini. Von l(orf f : The ef fect of depletion of endogenous substrate on the metabolic behavior of isolated rabbit heart mitochondria. J. biol. Chem. 242, 333 (1966).

169. Klingenberg, M. and W. Slenczka: Atmungsaktivitat von Mitochondrien verschiedener 0rgane mit Glycerin-1-phosphat im Vergleich zu Substraten des Tricarbonsaurezyclus. Biochem. Z. 331, 334 (1e5e).

170. Hatefi, Y., P. Jurtshuk, and A.G. Haavik: Studies on the electron transport system. XXXII. Respiratory control in beef hearE mitochondria. Arch. Biochem. 94, I48 (1961). L7L. Mitchell, P.l Chemiosmotic coupling in oxidative and photosyn- thetic phosphorylation. Biol. Rev. 4L, 445 (L966).

L72. ZLegLer, F.D., L. Vasquez-Colon, W.B. Elliott, A. Taub, and C. Gans: Alteration of mitochondrial function by bungaris fasciatus venom. Biochemisrry 4, 555 (1965).

Il3. Lehninger, A.L. and C.T. Gregg: Dependence of respiration ort phosphate and phosphate acceptor in submitochondrial systems. I. Digitonin fragments. Biochim. biophys. Acta 78, 12 (1963).

174. Gregg, C.T. and A.L. Lehninger: Dependence of respiration on phosphate and phosphate acceptor in submitochondrial systems. II. Sonic fragments. Biochim. biophys. Acta 78, 27 (1963).

775. Boxer, G.E. and T.M. Devlin: Pathways of intracellular hydrogen transport. Science L34, 1495 (1961).

L76. Blanchaer, M.C. and T. Griffith: Control of reduced nicotinamide adenine dinucleotide oxydation by pigeon heart mitochondria. Can. J. Biochem.44, L527 (L966).

L77 . Griff ith, T.J. and i"i.C. Blanchaer: Kinetics of NADH oxidation by pigeon heart mitochondria. Can. J. Biochem. 45, 881 G967). -236-

L7B. Holl, B. , K. trrlrogemann, and M.C. Blanchaer: In preparation. 179. Srivastava, U. and L. Berlinguet: Biochemical changes in progressive muscular dyst.rophy. V. Incorporation of leucine- rac into protein of various tissues of normal and dystrophic mice. Arch. Biochem. II!, 320 (L966).

180 . trnlil l iamson, J . R. and H. A. Krebs : Ace toace tate as fuel of respiration in the perfused rat heart. Biochem. J. 80, 540 (1e61).

181. Hatefi, Y. and T. Fakouhi: Control of p-hydroxybutyrate and acetoacetate oxidation by inorganic phosphate and adenosine 5tdiphosphate in heart mitochondria. Arch. Biochem. 125, Lr4 (1968).

I82. Bode, C. und M. Klingenberg: Die Veratmung von Fettsauren in isolierten Mitochondrien. Biochem. Z. 34I, 27L (1965).

183. Opie, L.H.: Metabolism of the heart in health and disease. ParL II. Am. Heart J. 77, 100 (L969).

184. Gergely, J., D. Pragay, A.F. Scholz, J.C. Seidel, F.A. Sreter, and M.M. Thompson: Comparat.ive studies on white and red muscle. In Molecular biology of muscular contraction. Edited Ð S. Ebashi, F. Oosawa, T. Sekine, and Y. Tonomura. -E-t".ni"t Publishing Company N.Y. L965, p. L45. r85 Dubowitz, V. and A.G.E. Pearse: Reciprocal relationship of phosphorylase and oxidative enzymes in skeletal muscle. Nature 185, 70r (1960) . 186. Domonkos, J.: The metabolism of the tonic and tetanic muscles. I. Glycolytic metabolísm. Arch. Biochem. 95, 138 (1961).

L87. Domonkos, J. and L. LaLzkowitzz The metabolism of the tonic and tetanic muscles. II. Oxidative metabolism. Arch. Biochem. 95, L44 (1961).

188. Fahimí, H.D. and P. Roy: Cytochemical localization of lactate dehydrogenase of the mouse. Science L52 176I (i966). f89. Rigault, Y. and M.C. Blanchaer: Respiration and oxidative phosphorylation by red and white skeletal muscle. Can. J. Biochem. submitted for publication. 190. Carafoli, 8., C.S. Rossi, and 4.T,. Lehninger: Energy-coupling in mitochondria during resting or State 4 respiration. Biochem. biophys. Res. Commun . 19, 609 (1965) . 191. Cleland, I(.I^I. and E.C. Slater: Respiratory granules of heart muscle. Biochem. J. 53,547 (1953) L92. Bray, G.: Personal communication.

193. Purvis, J.L. and J.II. Lowenstein: The relation between intra- and extramítochondrial pyridine nucleotides. J . biol. Chem. 231, 2194 (1961).

194. Penef sky, H.F., M.E. Pullman, A. Datta, and E. Racker: Partial resolution of the enzymes catalyzing oxidative phosphorylation. II. Participation of a soluble adenosine triphosphatase in oxidative phosphorylatíon. J. bio1. Chem. 235, 3330 (1960).

L95. Greville, G.D., E.A. Munn, and D.S. Smith: Observations on the fragmentation of isolated flight muscle mitochondria from calliphora erythrocephala (diptera). Proc. Roy. Soc. L6L, 403 (196s).

L96. Hoffman, R.A.: Hibernation and effects of 1ow temperature. In The golden hamst.er, its biology and use in medical research. Edited U R.A. Hof fman, P.F. Robinson, and H. Magalhaes. Iowa State Univers. Press, Ames 1968, p. 25.

L97. Carlson, L.A.: Lipid metabolism and muscular work. Fed. Proc. 26, 1755 (L967) . f98. Havel , R.J., A. Naimark, and C.F. Borchgrevink: Turnover rates and oxidation of free fatty acids of blood plasma in man during gxercise: Studies during continuous infusion of palmitate-1- r4c. J. clín. rnvesL. 4t,1054 (1963). L99. Fr ttz , I.B" , D.G. Davis, R.H. Holtrop, and H. Dundee: Fatty acid oxidation by skeletal muscle during rest and activity. Am. J. PhysioL. L94, 379 (1958).

200. Frttz, I.B.: Carnitine and its role in fatty acid metabolism. Adv. Lípid Res. 1, 285 (1963).

2OI . Blanchaer, M.C., B. Jacobson, and K. I,rlrogemann: In preparaËion. 202. Chance, B. and B. Hagihara: Activation and inhibition of succinate oxidation following adenosine diphosphate supplements Ëo pigeon heart mitochondria. J. biol. Chem. 237, 3540 (7962).

203. Pardee, 4.8., and V.R. Pot.ter: Inhibition of succinic dehydrogenase by oxalacetate. J. bío1. Chem. 176, 1085 (1948). 204. Chappel, J.B.: Effect of uncoupling agents on mitochondrial oxida- tions. Fed. Proc. 20, 50 (1961).

205. West, E.S., tr^I .R. Todd, H.S. Mason, and J.T. Van Bruggen: Textbook of Biochemistry, 4tLi edition. The Macmillan Company, N.Y. 1966 , p. 355. -238-

206. Srivastava, U., A. Devi, and N. Sarkar: Biochemical changes in progressíve muscular dystrophy. I. Nucleic acid metabolism in normal and dysËrophic rabbit and mouse, liver, brain, and muscle. E*p. Cel1 Res. 29, 289 (1963).

207 Cohn, Z.A. and E. Inliener: The particle hydrolases of macrophages. I. Comparative erìzymology, isolation, and properties. J" exp. lvled. 118 , 99I (1963) . 208. De Duve, C., B.C. Pressman, R. Gianetto, R. trrlat.tiaux, and F. Appelmans: Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue. Biochem. J. 60, 604 (195s).

209. tr^Ieinstock, I.M. and M. Lukacs: Enzyme studies in muscular dystrophy VI. Cathepsin and acid deoxyribonuclease activities during the progression of hereditary muscular dystrophy in the chicken Enzymol. biol. clin. 5, 103 (1965).

2LO. Abdu11ah, F. and R.J. Pennington: Ribonuclease activity in normal and dystrophic human muscle. Clin. chim. Acr.a 20, 365 (i968)

2II. Tappel, 4.1., H. ZaIkin, K.A. Caldwe11, I.D. Desai, and S. Shibko: Increased lysosomal enzymes in genetic muscular dystrophy. Arch. Biochem. 96, 340 (1962) . 2L2. Me1lors, 4., A.L. Tappel, P.L. Sar,rant, and r.D. Desaí: Mitochondrial swelling and uncoupling of oxídative phosphorylation by lysosomes. Biochim. biophys. Acta I43 , 299 (1967) .

2I3. Stagni, N. and B. De Bernard: Lysosomal enzyme activity in rat and beef skeletal muscle. Bíochim. biophys. Acra 170, L29 (1968).

2L4. Pe ter , J . B. : Unpubl ished observation. 215. Coleman, D.L.: Studies on the acetoacetate like compound found in dystrophic mouse muscle homogenates. Arch. Biochem. 111, 489 (1 e6s) .

2L6. trrlest, W.T. , H. Meier, and Il .G. Hoag: Hereditary mouse muscular dystrophy with particular emphasis on pathogenesis and attempts at therapy. Ann. N.Y. Acad. Sci. 138, 4 (1966). 2L7. Coleman, D.L. and Ini.T. trriest: Effects of nutrition on growth, life- span, and histopathology of mice with hereditary muscular dystrophy. J. Nutr. 73, 273 (1961).

2L8. Bajusz, E., F. Homburger, J.R. Baker, and P. Bogdonoff: Dissociation of factors influencing myocardial degeneration and generalized cardiocirculatory failure. Ann. N.Y. Acad. Sci. L56,396 (1969). 2I9. Bygrave, F.L. and A.L. Lehninger: The affinity of mitochondrial oxidative phosphorylation mechanisms for phosphate and adenosine diphosphate. Proc. nat. Acad. sci. usA 57, I4O9 (1967). -239_

220. Strickland, K.P., C.H. Lin, and A.J. Hudson: Lipid metabolism in dystrophic muscle. Submitted for publícation.

22L. Bajusz, E.: HerediËary cardiomyopathy: A new disease model. Am. Heart J. 77 , 686 (L969). 222. Tyler, F.H. and G.T. Perkoff: Studies on disorders of muscle. VI. Is progressive muscular dystrophy an endocrine or metabolic disorder? Arch. intern. Med. 88, L75 (1951)

223. Danowski, T.S., R.M. Bastiani, F.D. McI^Iilliams, F.14. Mateer, and L. Greenman: Muscular dystrophy. IV. Endocrine studies. Am. J. Dis. Child 9L, 356 (1956). 224. ZterIer, K.L.: Aldolase leak from muscle of mice with hereditary muscular dystrophy. 8u11. Johns Hopk. Hosp. 102, L7 (1958).

225. ZierLer, K.L.: Potassium flux and further observations on aldolase flux in dystrophic mouse muscle. Bull. Johns Hopk. Hosp. 108, 208 (1961) .

226. Hazler¿ood, C.F. and J.M. Ginski: Muscular dystrophy: in vivo resting membrane pot.ential and potassium distribution in strain I29 mLce. Am. J. phys. Med. 47, 87 (1968).

227. Hazlewood, C.F. and J.lvl. Ginski: Skeletal muscle electrolytes as a function of age in normal and dystrophic mice of sLrain I29. Johns Hopk. Med. J. I24, L32 (1969).

228. Zdrodovskaya, E.P., E.A. Lotosh, A.I. Podlesnaya, and E.V. Rosenhart: Genetical defect of the membranes of muscle cells in case of heredítary muscular dystrophy. Genetica (Moscow) 4, 111 (1968).

229. Vitale, J.J., M. Nakamura, and D.M. Hegsted: The effect of magnesium deficiency on oxidative phosphorylation. J. bio1. Chem. 228, 573 (L957) .

230. Di Giorgio, J., J.J. Vitale, and E.E. Hellerst.ein: Sacrosomes and magnesium deficiency in ducks. Biochem. J . 82, L84 (1962) . 23I. Caulfield, J.B.: Electron microscopic observations on the dystrophic hamster muscle. Ann. N.Y. Acad. Sci. 138, 151 (L966).

232. Boas, E.P. and H. Lowenbèrg.: The heart rate in progressive muscular dystrophy. Arch. íntern. Med. 47, 376 (1931). 233. ZaluchnL, J., E.E. Aegerter) L. Molthan, and C.R. Shuman: The heart in progressive muscular dystrophy. Circulation 3,846 (1951).

234. Rubín, F. and A. Buchberg: The heart in progressive muscular dystrophy. Am. Heart J. 43, 16l (1952). -240-

235 . tr^Ieisenf eld, S. and W. I4.essinger: Cardiac involvement in progressive muscular dystrophy. Am. Heart J.43, I70 (1952).

236. Storstein, O. and K. Amsterheim: Progressive muscular dystrophy of the heart. Acta med. Scand. 150, 43L (i955).

237. Levin, S., G.S. Baens, and T. Weinberg: The heart in pseudohypertrophic muscular dystrophy. J. ?ediat. 55, 460 (1959). 238. Pearce, G.W.: Electron microscopy in the study of muscul-ar dystrophy. Ann. N.Y. Acad. Sci. 138, 138 (1966). XIV. APPENDIX -242-

Tab1e 3b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY },IOUSE MUSCLE MITOCHONDRIAJT

Substrate: pyruvate/ma1ate.

lst Period Znd Period

RCR ADP/O Orrate P rate ADP/O O rr ate P rate

L2 3 .5 2.4 95 456 4.4 ta L43 829

Con- L4 5.6 2.5 130 650 7 .6 2.3 228 L049 trol L6 5 .7 2.r L52 638 7.0 2.2 245 1 078

r-B 5 .5 2 .L 155 65I 7.5 2.3 268 L233

13 3.4 1.8 103 37r 3.9 )9 160 928

Dys- 15 6.0 2.2 r7s 776 6.8 2.4 273 13 10 trophic I7 5.8 t.9 L75 665 7.5 2.2 248 10 91

L9 5.0 2.r t77 743 6-4 2-3 267 L22B

+ t-valuel 0.132 2.200 -3.206 -0.845 1.368 -1.000 - r .537 -1.515

Experiments done on animal pairs. For pair numbers see Table No. 2.

L I Based on a t-test. for paired observations (149). -243-

Table 4b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY MUSCLE MITOCHONDRIA PREPARED FRO}Í 4 CONTROL MICE AND THE EFFECT OF ATP

Animals were 256-28l days old; substrate: pyruvaEe/malate.

lst Period 2nd Period

No. ADP/O Orrate P raLe ADP/O O 2r ate P rate

20 3 .7 2.2 r52 669 4.7 2.3 r82 837

2I 3.5 1.8 113 407 4.s L.9 r27 483

22 3.6 r.7 93 316 4.8 2.0 L25 s00

23 5.3 2.0 L43 s72 6.9 2-O r93 772

20 4.2 1 .8 L82 655 6.2 2' 275 L2IO

250 2r 5 .1 2.0 140 560 6.4 11 188 790 ¿I{ I ATP 22 3.5 1.9 L02 388 4.9 L.9 r57 597

ô1 ¿J 6 .8 2.r 153 643 8.4 2.2 205 902 t-value Jc -2.L4O -O.I74 -3.44O -2.067 -3.767 -o.577 -2.805 -3.379

* Obtained from a t-test for paired observations (149). -244-

Table 5b

RESPIRATION AND OXIDATIVE PIÍOSPHORYLATION BY MOUSE MUSCLE MITOCHONDRIA PREPARED IN THE PRESENCE OF 1% ALBU{IN Substrate: pyruvate/ma1ate.

lst Period 2nd Period

No. ADP/O Orrate P rare RCR ADP/O Orrate P rate

1 7 .7 2.7 6.8 2.8

2 10.7 2.7 8.1 3.1

Con- 3 Ir.4 2.5 29s 7475 10.5 2.6 300 t5 60 tro 1 4 L3.2 2.7 r73 934 11.5 2.6 185 962

5 9.6 2.6 228 1186 LI.2 2.6 250 1300

6 8. I 2 .B r97 1103 L2.3 2.9 263 L525

7 1r.B 2.9 162 940 7.8 2.6 222 LL54

8 9.1 2.5 413 2065 r0.2 2.5 443 22L5 Dys- tro- 9 11.1 2.7 L23 664 10 .6 3.0 r42 852 phic 10 9.2 2.6 L82 946 8.0 2.7 778 96L

11 r4.2 3 .0 10 .0 ,a

t-valueJç o -676 o.2L3 0 .045 0. 120

Jc Based on a t-test for comparison of the means of ungrouped data (149) -245-

Table 6b

YIEIÐ OF I'{OUSE MUSCLE MITOCHONDRIA PREPARED* IN THE PRESENCE OR ABSENCE OF ALBI]MIN

Values expressed in mg milochondrial protein per g muscle

No Albumin With Albumin Control Dystrophic Control Dystrophic

1.0 L.7 1.9 2.5

L.2 L.6 1.3 2.8

r.4 0.8 L.6 0.7 L.6 1.5 2.r 2.r 1.1 r.2 0.5 0.8

1.3

L.2

t-valuet -L.org - 0.199

These yields were obtained from mice No. 1-19 and from some control mice used in preliminary experiments.

t-values were obtained from a t-test for comparison of the means of ungrouped data. -246-

Table 7b

CHARACTERISTICS OF TT{E HÆ{STER GROUP 1

CPK No. Age Body wt. mg heart mg liver -DTT .I-DTT Percentage Þo g body g body activation

1 95 L22 2.6r

3 100 lL4 , qa 7L L67 235

Nor- 6 108 113 2.58 32.L 89 190 2L4 mal I B 110 L29 2.52 40 .5 55 118 2t5

9 L22 109 2.78 36.6 44 98 223

I2 L25 LI4 2 .80 42.2 102 2L7 2r3

2 97 93 3 .00 870

4 r01 9I 2.72 95 273 289

Dys- 5 LO2 90 2.84 32.7 r27 294 232 t rophic 7 109 79 101 37 .8 796 r225 L54

10 L23 73 3.25 38.3 322 640 L99

11 L2/1 86 3.10 36.6 523 969 185 t-value 0 .098 7 .2r5 -3 .7 29 0.581 -2.56L -2.779 0.351 t Normal hamster were of the N.I"H" random. bred strain.

Based on a t-test for comparison of the means of ungrouped data. -247-

Table 8b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HAI'ISTERS OF GROUP 1

Substrate (pyruvate/rnalate) added prior to 1st ADP.

I st Period 2nd Period

ADP/O 0 P rate ADP/O 0 rate P rate 2

I 7 .4 2 .0 2L0 858 6.2 2.3 2L8 10 16

Ja 10 .3 2 .3 3L6 L452 9.0 2.4 237 LLzB

Nor- 6 7 .7 2.2 235 IO42 a) na 223 LOI2 mal t ö 7 .8 2.3 223 1015 7.6 2.5 228 1 140

9 7 .4 2 .3 310 1446 11.1 2.5 358 L7 54

T2 11 . s 2.2 4r7 L7 99 r0. 4 2.3 380 I77 2

2 L2.L 2.3 258 rL99 L0.2 2.4 240 1131

4 11.0 2.2 247 1060 8.2 2.4 239 LL64

Dys- 5 8 .3 2 .2 235 1016 8.5 242 1086 trophic 7 7.6 2.2 222 976 7.8 aa 25L 1141

10 8.6 2.3 319 1450 q) 2.4 294 L425

11 11.1 2.2 399 1788 9.6 2.3 362 L693 t-valueJr - 1 .057 -0 .324 0 .122 0 . 105 0 .000 0.969 0.073 0.172 t Normal hamsters were of the N. I.H. random bred strain.

Based on a t-test r'or comparison of the means of ungrouped data. -248-

Table 9b

RESPTRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HA},ISTERS OF GROUP 1

Substrate: pyruvate/malate. Second Period of experiments in which indigenous substrate \^Ias depleted by addition of ADP prior to pyruvate/malate during the First Period.

No. RCR ADP/O O rr ate P rate

1 6.0 )t 2L3 955

3 9.9 2.4 24L 1163

Nor- 6 8.3 )2 197 884 mal T 8 8.1 2.4 2L5 10 16

9 a) )l! 351 1677

L2 11 .0 2.2 446 r97 0

2 LO.2 2.3 24L 1113

4 8.1 2.3 225 LO28

Dys- 5 13.1 2t 279 L250 trophic 7 9.L 2? 268 L2L6

l0 9.1 2.3 276 L282

l1 8.8 2.4 334 160 9

t- value:k -0.973 0.000 0.153 0.140

I Normal hamsters \^rere of the N. I.H. random bred strain.

Based on a t.- test f or comparison of the means of ungrouped data. -249-

Table llb

RESPIRATION BY HEART MITOCHONDRIA FROM HAI4STERS OF GROUP

Substrates: DL-or-glycerophosphate and NADH.

9 . 5mM DL-oc- glycerophosphate

No. Before ADP +245 l'{ ADP Homo . -:k 110 M NADH concn.

1 11 11 318 49

3 13 7 294 48

Nor-f, mall 6 8 5 29r 50

ö 11 7 325 45

L2 13 11 318 48

2 I4 7 279 4.0

Dys- 4 L2 10 246 37 trophic 5 254 25

7 233 ¿J

a t-value t -r.110 -0.139 4.845 4.349

Concentrations of the heart homogenates, from r¿hich the mitochondria were isolated, in mg he art / 20m1 homogen izing medium.

I I Normal hamsters were of the N.I.H. random bred strain.

+ Based on a t-test for comparison of the means of ungrouped data. -250-

Table lzb

CHARACTERISTICS OF THE HA}ISTER GROUP 2

CPK No. Age Body wt mg Heart -DTT +DTT Percentage ó g body ac t ivat ion

13 L46 L20 2.6r 95 2ro 22L

t6 rs4 115 2.55 89 L46 164 Nor- malj< 17 160 r20 2.38 133 r88 T4L

! 2L 181 I25 2.64 4541 7æI ls5+

23 184 I12 2.72 LO4 L77 L7I

I4 r47 104 2.9r 493 934 189

15 r48 95 5 .38 307 813 265 Dys - tro- l8 160 113 2.95 244 675 277 phic L9 L62 L20 3.43 47r L562 331

22 181 113 3 .50 405 LI43 282

-I t-value+ 0.557 L.929 -2.3r0 -s .095 - 4. 811 -3.163

Normal hamsters were of the LSH strain. t CPK values of hamster No. 2I were disregarded, because the animal was decapitated through the mouth.

Based on a t-test for comparison of the means of ungrouped data. -25r-

Table 13b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HAMSTERS OF GROUP 2 Substrate: pyruvate/malate.

32P RCR ADP/O OrraEe P rate r ate t^r.

13 9.3 2 .7 269 L453 2.4 367 17 62

L6 9 .6 2.s 293 1465 2.2 342 1 505 Nor- mal",c 17 16.9 2.4 306 L468 ,L 387 185 8

2L 8.9 2.4 227 1090 2.5 404 2020

23 L4.5 2.8 2r5 1204 2.5 492 2460

L4 10.2 2.6 284 L477 ¿.J 304 13 98

15 11 .3 2.6 290 1s0B 327 r57 0 Dys- tro- 18 IO.2 2.5 311 1555 )\ 397 1 985 phic I9 8.5 2.5 220 1100 2.4 402 I 930

22 lL .2 2.5 296 1480 409 L963 t-value f 0.9r7 0.237 -0.765 -0.777 0 .000 0.9r2 0.764

Normal animals were of the LSH strain. t Based on a t.-test for comparison of the means of ungrouped data -252-

Table 14b

CHARACTERISTICS OF THE HAI'TSTER GROUP 3

CPK No. Age Body mg heart I{e ar t S tre aks -DTT +DTT Percentage \n/t . g body disease ac t ivat ion

24 rB8 L22 2.66 0 0 30 88 29L

28 53 85 3.15 0 0 64 154 24r

29 198 101 3. 18 0 0 40 r02 254 r- l:k 30 148 I05 3 .00 U 0 35 65 LB7

31 148 L23 2.63 n 0 84 LL7 139

33 148 115 2.6s 0 0 10 23 235

36 r49 L20 2.80 0 0 55 92 L66

25 189 101 3.64 + # 6I6 rB07 293

26 190 113 3 .81 -# +# 226 13 16 582

S- 27 75 97 2.77 0 # 185 9 47 62 256 o- ic 32 174 L20 3.02 # # 228 645 353

J4 175 L32 3 .31 -l-# -{*# 390 L990 510

35 175 L28 3 -O2 # # 693 t946 2BT valuef -0.617 -0.642 -2.r95 -2.702 -3.770 -2 .957

Normal hamsters were of the LSH strain, except No. 28, which was a Lakeview random bred animal.

Based on a t-test for comparison of the means of ungrouped data. -253-

Table 15b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HAIVISTER OF GROUP 3 Substrate: pyruvate/ma1ate.

32 RCR ADP/O OrraLe P rate P rate

24 7 .7 2.4 2s8 L23B 2\ 3s8 L7 90

28 9 .I 2.2 290 L27 6 2.3 272 L25L

29 6 .s 2.3 262 L205 2.3 289 1329 Nor- ! mall 30 6 .9 2 .0 315 L260 2.6 274 L425

31 4.2 2.I 275 1155 2.5 263 13 15

33 5 .l 2.r 235 987 2.6 256 133 1

36 5 .5 2.0 263 L052 2.5 278 r3 90

¿), 9 .7 2 .3 285 1311 t1! 278 r334

26 B .4 2 .3 286 1316 1t, 278 133 4

Dys- 27 6. 1 2.4 248 1190 2.4 269 T29I trophic 32 1.0 0.0 73 0 2.6 275 143 0

J4-l s.4 I.9 245 931 2.5 229 IL45

35 5 .0 I .9 2r2 806 2.5 2L2 1060 t-value * o .443 t .032 r .467 L .263 0 .081 L -549 t.554

Normal hamsters were of the LSH strain, except No. 28, which was a Lakeview random bred animal.

Based on a t-test for comparison of the means of ungrouped data. -254-

Table 16b

CHARACTERISTICS OF THE HAI'{STER GROUP 6

CPK No. Age Body mg heart Heart S tre aks -DTT +DTT Percentage \nit. g body disease activation

64 119 118 2.62 0 0 65 187 L52 2.30 0 0 - 67 r5B r65 2.6L 0 0 1; 29 264 69 254 L44 2.63 0 0 1B 40 222 70 259 r42 2.78 0 0 28 42 150 or- 73 269 136 2.72 0 0 4s 81 180 alr 74 273 I25 2.76 0 0 T7 37 2I8 76 280 L28 2.17 0 0 B 30 375 tó 289 r27 3.07 0 o 34 60 176 B1 181 IL9 2.90 ô 0 29 55 190 B2 198 107 3 .08 0 n 18 42 233 B4 r99 r42 2.60 0 0 t6 J+ 213 B7 2r5 tL2 2.97 0 0 4T 73 L78

JI11 2L3 132 3.10 # # 66 224 L34 3.10 ++ 0 25; 15 1; sB4 68 200 134 3.36 #+ -].# 81 528 652 7I 243 I23 3.82 + 0 456 1649 362 rys- 7 2 219 r22 3 .6s # -l-H 29 553 L907 ro- 75 257 L29 3.33 # -+ 100 273 273 'hic 7 7 265 I2I 4.09 # + 200 1016 s08 79 275 L24 3.23 + # 401 742 185 BO L44 113 2.gt 0 -l-# 2297 5444 237 B3 155 L34 3 .04 H # 331 10 99 JJ¿ 85 L65 L22 3 .66 # + 222 102 1 460 86 17I 9I 4.06 + o L244 2393 L93

-va1ue* 0.543 r.97s -s .539 -2.380 -3 .293 -2.025

Hamsters No. 64,65, arrd 6l were of the Lakeview random bred strain. The other normal hamsters were of the LSH strain. l- Based on a t-test for comparison of the means of ungropued data. - 25tt

Table L7b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HAMSTERS OF GROUP 6 Substrate: pyruvate/malate.

280c 370C

No. RCR ADP/O Orrate P rate ADP/O 0 rate P rate

64 7 .5 2.2 207 gLI 65 7.2 2.3 241 1109 69 6.9 2.3 L73 796 a.r 2.3 is tl tø Nor- 7O 8.4 2 .3 207 952 8.0 )2 407 L7 9l malf 73 8.1 2.3 20L 925 7.3 2.2 4L3 7B17 74 8.2 2.2 207 911 9.r 2.3 400 1840 76 6.5 2.4 2tB 1046 5.7 2t! 4L2 r978 7B 6.3 2.3 2L6 994 8.4 t\ l+/+8 2240

37 7 .6 2.4 230 1104 6.7 2.4 389 1867 66 8.2 2.4 249 1195 11.3 2.4 5L2 2458 68 9.6 2.2 2rs 946 Dys- 7L 9 .7 2.4 220 10s6 8.8 )? 462 2125 tro- 72 7 .9 2.4 232 LLL4 7.L 2.2 409 1800 phic 75 7 .5 2.3 220 LOr2 6.9 2.r 402 16BB 77 7 .3 2.3 223 T026 7.5 2.3 458 2707 79 7 .3 2.2 223 981 9.3 )? 503 23r4 t-value;k -r.678 -0.987 -2.307 -2.237 -0.s66 0 .502 T.76I -r.r45

Animal s No. 64 and 65 rvere of the Lakeview random bred strain, other normal hams ter s were of the LSH strain.

Based on a t-test for comparison of the means of ungrouped data. -256-

Table 18b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HA]4STERS OF GROUP 6

Subs trate : DL-p- hydroxybutyra te .

o 28oc 37C

No. ADP/O Orrare P rate ADP/O 0 rate P rate

69 4.0 2 .L 99 4t6 2.9 1.8 I23 T++J 70 5 .5 2 .0 L34 536 3.0 7.9 131 498 Nor- 73 6 .2 2.2 161 708 3.1 L.9 L69 642 mal:'c 7 4 7 .0 2.r r97 827 3.5 2.0 rB4 736 76 5.8 2.I 20s 861 2.9 a2 159 731 7B 10.0 2.2 2r4 942 2.9 L.g L47 559

a1 JI 6.0 2.2 163 7r7 ?2 2.2 140 616 66 4.s 2 .r r2B 538 3.0 2.r L52 638 Dys- 68 9.6 2.r 278 1168 tro- 7I 5 .6 2.1 t22 5I2 ).+ L.9 150 570 phic 72 4.4 2.0 105 420 2.0 L.7 109 37L 75 4.r 2 .0 105 420 r.9 L.6 108 345 77 s.s 2.2 r49 656 3.2 2.L 173 727 79 4.7 2 .r 168 7 06 3.9 2.r 22L 928 t-valuet 0.8s6 0.41s 0.s73 0.593 0 .341 0.083 0.095 0 .023

Normal hamsters were of the LSH straÍn.

Based on a t-test for comparison of the means of ungrouped data. -257-

Table 19b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY HEART MITOCHONDRIA FROM HA},ISTERS OF GROUP 6

Substrate: palmityl-L-carnitine * malate.

No. RCR ADP/O O^rate P rate ¿

69 8-3 2.2 206 906

70 9.L 2.r 216 907

Nor- 73 9.0 2.1 207 869 L mal I 74 7.6 2.0 222 888

76 79 2.L 238 r000

7B 6.9 2.L 23t 970

7L 10.9 2.2 ,LO r0 96

72 7.8 2.L 2L5 903 Dys- tro- 75 B.B 2.0 214 856 phic 9.L 2.L 229 962

79 7.4 2.L 240 1008

L.-va1ue:k -T,T32 0 .000 -1.107 -0.948

Normal hamsters were of the LSH s train .

Based on a t-test for compar ison of the means of ungrouped data. -258-

Table 20b

YIELD OF HA}ÍSTER HEART MITOCHONDRIA

Values in mg mitochondrial protein per g heart muscle.

Group 1 Group 2 Group 3 Group 6

No . Yie ld No . Yie ld No . yie 1d No . yie ld

I 15.28 13 13.s1 24 L5.77 64 L9.76 3 20.77 L6 10.81 28 11.89 65 14.96 Nor- 6 2L.03 L7 L2.31. 29 10.94 69 L9.46 mal I 19.14 21 11.59 30 17 .90 tO 20.46 9 14.78 23 L3.57 31 14.56 73 17.s0 L2 17 .7r 33 16.95 7 4 L4.33 36 74.L4 76 14.58 78 16.54

2 L7.47 L4 II.74 25 11.50 37 15.26 Dys- 4 18.51 15 8.92 26 9.42 66 13.05 tro- 5 22.72 18 10.17 27 11.60 7t 12.84 phic 7 20 .I4 19 9 .80 32 15 .34 7 2 L2 .26 10 13.1s 22 8.49 34 8.55 75 10.70 11 L4.72 35 10.84 77 10.61 79 12.69

t-value* 0. 185 3.476 2.476 4.316

:k Based on a t-test for comparison of the means of ungrouped data. -259-

Table 21b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAI MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROUP 1

Substrate (pyruvate/malate) added príor to lst ADP. Standard Proteinase Preparation.

lst Period 2nd Period

No. RCR ADP/O }rrate P rate RCR ADP/O }rrate P rate

t 2.3 1.8 L66 604 2L0 3 5.8 2.0 r8l 72L 6.3 2.3 22L rão: Nor- 6 4.7 2. L 252 L064 4.7 )) 229 r01 B -.l- mall 8 6.2 2.L 248 1030 5.2 2.4 233 LL34 9 4.L 2.L 2L2 880 4.8 2.2 2L8 973 L2 5.9 2.2 226 97 6 6.4 2-2 259 I 158

2 2. 3 L.7 68 232 76 4 5.0 2.0 202 819 5.0 2.L 2L2 906 Dys- 5 5.5 2.L L77 7 47 6.0 2.L 2L0 892 tro- 7 4.8 2.0 198 7Br 5.4 2.0 2L8 874 phic t0 5.1 2.L 254 L076 6.2 2.I 244 LO46 11 4.1 L.9 243 899 4.8 2.L 247 1043

t -va lue 0. 350 1 . 00r 0.77 6 0. 87 4 0.000 4.072 L.0L4 L.97 6 t Norrnal hamsters were of the LSH strain. J. Based on a t-test for comparison of the means of ungrouped data. -260-

TabLe 22b

RESPIRATION AND OXIDATIVE PHOSPHORYLATTON BY SIGLETAL MUSCLE MTTOCHONDRIA FROM HAMSTERS OF GROUP Substrate: pyruvate/malate.

Only the Second Period of respiration is shown. During the FÍrst Period indigenous substrate was depleted by addition of ADP prior to pyruvat e/ma late. Standard Proteinase Preparation

No. RCR ADP/ O }rrate P rate

1 2.7 1. B 198 7LB q,o 3 2.2 191 B2L Nor - 6 4.4 2,L 225 937 mal f 8 5.8 2.L ¿JJ 990 9 4.6 2.2 22L 972 L2 o. z )) 233 t003

2 Dys - 4 5.9 2.5 280 tlzt tro- 5 4.6 2.r r68 7L2 phic 7 5.2 )1 22L 932 t0 4.8 2.2 237 1030 tt .1..) 2.0 163 6s4

t -value" 0.245 -0.7 68 0. L42 -0.325

t Normal hamsters were of the LSH strain. Based on a t-test for comparison of the means of ungrouped data. -26r-

TabLe 24b

RESPIRATION BY SKELETAI MUSCLE MTTOCHONDRIA FROM HAMSTERS OF GROUP 1

Substrates: DL-cc-glycerophosphate and NADH. Standard Proteinase Preparatíon

9. 5 mM DL-o<-glycerophosphate 110 NADH No. Before ADP +245 yM ÃDP ¡M

I 54 53 22 3 46 5B 23 Nor- 6 54 59 26 mal" I s4 68 ,? 9 78 78 L2 56 56

2 54 51 20 4 63 82 24 Dys- 5 55 66 ¿J tro- 7 64 74 phic 10 l1 9¿+ 94

I t -va lue I -L.091 -r.464 L.7 22

>k Normal hamsters were of the N.I.H. random bred straín. t Based on a t-test for comparison of the means of ungrouped data. -262-

TabLe 25b

SUCC]NIC DEHYDROGENASE (SDH) ACTTVITY AND TISSUE MITOCHONDR]AL CONTENT IN SKELETAI MUSCLE OF HAMSTERS OF GROU? 1

Cal cul-ated mitochon- SDH activity drial tissue content (¡.tmoles/g mit . prot. /min) (mg mir. pror. /g rissue)

J LL4 8.6 Nor- 6 108 6.6 mal " 8 L2L 9.6 9 131 LL.4

4 130 6.I Dys - 5 118 6.8 tr o- 7 119 10. 1 phic l0 L23 10. 9 11 119 r1.5

! t -va lue I -0.6s8 -0.020

Normal hamsters were of the N.I.H. random bred strain. t Based on a t-test for comparison of the mearrs of ungrouped d.ata. TabLe 26b

YIELD OF HAMSTER SKELETAI MUSCLE MITOCHONDRIA

Values l_n mg miËochondrial protein per g muscle

Proteinas e Preparat ionstk

Group I Group 2 Group 3 Group 4 Group 5

I 0.65 13 2.23 24 1. 08 3B 1-. 09 50 1. 68 a J 1. 63 L6 1. 13 2B L.4L 39 0. 85 54 1. B0 Normal 6 t. 80 L7 1. 38 29 1.11 43 r. 05 55 1. 51 B L.7 4 2L l-. 13 30 L.44 46 0.7 5 57 L.27 9 1,.52 23 1. 13 31 47 1. Ll 59 L.4L L2 L.63 JJ 1. 55 49 0. B8 36 L.69 I N] (,O) 2 L.93 L4 0,99 25 L.56 40 0.77 51 1. 10 I 4 L. 56 15 0. B0 26 L.45 42 1. 1_1 52 1. 03 Dystrophic 5 l_. Br 1B L.25 27 1. 38 44 0. 84 53 L.6L 7 1. 88 L9 1. 61 32 45 l-.03 56 1. 39 l0 L. L6 22 o. 82 34 1. 53 48 0.7 L 5B 1. Ll 11 r-. 40 35 L.59 60 L.77 6L 1. r0 62 L.29 63 L.04

t-valuet -0.60 2 L.L7o -t. o7o 0.658 1. 886 t'Gto.rp 1-4, Stand.ard Proteinase Preparation; Group 5, Modified Proteinase Preparation. t ¡ased on a t-test for comparison of the means of ungrouped data. -264-

Table 27b

RESPIRATION AND OXIDATTVE PHOSPHORYT,ATÏON BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROUP 2 Substrate: pyruvate/malate Standard Proteinase Preparation

Polarographic Method llexokinase Method

ADP/0 }2raLe P rate P/0 0 P rate

13 3.0 2.0 2L7 868 2.L L73 727 Nor- L6 3.2 2.L 22L 928 t.7 L82 6L9 _ tí Ll 2.6 2.L L1L 7L8 1.8 L76 634 mal ZL 3.3 L.9 240 9L2 2.L 243 LO2L 23 2.4 2.0 2L5 860 2.L 207 869

L4 2.9 2.2 L26 554 2.2 264 LL62 Dys- 15 2.8 2.I L02 428 2.7 L93 LO42 tro- 18 3.0 2.L 150 630 2.3 306 1408 phic 19 3.6 2.3 206 948 2.4 277 1 330 22 2.L L.9 L54 585 2-L L95 819

I t -valuel 0. 068 I. 319 3.L48 2.425 -2.82L -L.938 -2.9L9

Normal animals were of the LSH strain. t Based on a t-test for comparison of the means of ungrouped data. -265-

Table 28b

RESPIRATION AND OXIDATWE PHOSPHOR\T.ATION BY SKELETAL MUSCLE MITOCHONDRIA FROM I{AMSTERS OF GROUP Substrate: pyruvate/malate Standard Proteinase Preparation

Polarographíc Method Hexokínase Method

32p/o 32p No, RCR ADP/O 02rate P rate o2roarare ,^r"

24 2.7 2.0 226 904 2.0 L93 772 28 2.9 1. 8 203 7 3L 1. B 183 659 Nor- 29 2.5 1. B 2L0 7 56 L.6 168 538 mal^ 3Ùl- 1.0 0.0 7s+ o L.6 105 t:, 31 2. 5 L.7 2.0 JJô1 2. L L.7 150 510 2.L L67 70L 36 2.2 I.s 164 492 2.0 174 696

25 2.7 L.7 168,. 57L 2.0 L75 700 Dys- 26 1.0 0.0 3æ 0 2.L 209 878 tro- 27 1.0 0.0 101+ 0 2.L 180 756 phic 34 1.0 0.0 83+ 0 2.0 L95 780 35 2.4 1. 5 131 393 2.2 2L6 950

a t -va luel 2.329 3.064 3.277 3.365 -1.838 928 367

No. 28 was a Lakeview hamster; all the other normal animals were of the LSH strain. I + The values obtained from this animal were not included in the statistical calculations. I + These values were obtained during t.he Fírst period since no Second Period could be obtained. I I Based on a t-test for comparison of the means of ungrouped data. -266-

Table 29b

RESPIRATION AND OXIDATIVE PHOS?HORYLATION BY SKELETAL I'{USCLE MITOCHONDRIA FROM HA},ISTERS OF GROUP 3

Substrate: pyruvate/ma1ate. Standard Proteinase Preparation with 1% albumin.

Polaroqraphic Method Hexokinase lvle thod 32P No. ADP/O O aLe P rate o rr 2Hr(t ttu rate

24 10.9 2.4 2L9 1051 2.6 3Bs 2002

28 r9.9 2.3 278 1279 2tr 30r r445

29 15.6 2.3 294 L352 ?) 3L6 13 90 Nor- ma1:k 30 7.6 2.6 227 1180 2.7 287 L5L7

31 6.4 2.3 2.5

33 10.3 2.6 228 1186 2.6 309 L607

36 r2.3 2.L 249 L046 2.6 299 1555

25 72 .0 2 .4 263 1262 2.4 279 1339

26 3.0 2.0 92 368 2.3 232 L067

Dys- 27 3.3 2.0 92 368 2.5 239 1195 tro- phíc 32 1.0 0.0 2.8

34 3 .9 1.8 138 497 2.6 2L7 IT2B

35 8.8 r.9 200 760 2.6 248 L290

_! t-value I 2 .641 2 .106 2 .801 3 .290 -0.201 3.839 3 .538

No. 28 was a Lakeview hamster; all the other normal animals were of the LSH s train.

Based on a t-test for comparison of the means of ungrouped data. -267-

Table 30b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAL I'{USCLE MITOCHONDRIA FROM HA}4STERS OF GROUP 3 Substrate: pyruvate/ma1ate. Lochner Preparation.

Polarographic Method Hexokinase Method ADP/O Orrare P rate o2HKt*t" 32P t^t"

24 5.3 2.L 8s 357 2.3 I25 575

28 3.8 2.r r43 601 1.8 r44 518

29 5 . s 2.0 r47 588 L.9 140 532

30 3 .3 2 .O 109 436 2.r 220 924

31 4.9 2.0 189 756 21! 214 LO27

33 1.0 0.0 43 0 )1! 202 970

36 6.6 I.9 176 669 2.4 259 12/+3

25 3.4 2.0 81 324 2.0 B5 340

26 4.8 2.3 78 3s9 2.L 109 4s8

Dys- 27 3.0 2.0 84 336 L.9 66 25L trophic 32 1.0 0.0 2.6 324 1685

34 s .2 2.0 I57 628 2.3 163 750

J) 6 .7 t.9 186 707 2.4 233 1118

L-valrrel 0.308 0.064 0.341 0.119 -0.2L5 0.540 0.25¿+

:k No. 28 was a Lakeview hamster; al1 the other normal animals were of the LSH strain.

tBased on a t-test for comparison of the means of ungrouped data. -268-

Table 31b

YIELD OF HA]4STER SKELETAL MUSCLE MITOCHONDRIA

Values given ín mg mitochondrial protein per g muscle. Lochner Preparation.

Group 3 Group 6

No. Yie 1d No. Yield

t/, L.2l 64 1.36 2B I.4I 65 1.52 29 1 .00 67 2.TB 30 L.2L 69 2.48 31 T.L2 70 2.79 33 1 .09 73 2.78 Normal 36 0.89 74 2.58 76 2.02 78 2.47 81 2.54 82 2.LT 84 2.27 87 2 .00

25 0.97 66 r .44 26 I.I4 68 r.42 27 1 .01 7T 1.01 32 0.76 72 1 .58 J+ o.7L 75 1 .60 Dystrophic 35 0 .66 77 1.15 79 I .58 BO 2.24 83 1 .68 85 1.38 B6 r.77 t- value:k 2.643 4.400

:k Based on a t-test for comparison of the means of ungrouped data. -269-

Table 32b

CHARACTERISTICS OF THE HAMSTER GROUP 4

CPK No. Age Body mg heart Heart Streaks -DTT +DTT Percentage v/t. g body disease ac t ivat ion

JÕ 81 II4 2.98 0 0 23 r52 658 39 B1 108 2.73 0 0 L2 105 864 Nor- 43 B3 116 3.02 0 0 30 1ô 254 mal:k 46 89 IT4 2.98 0 ô 25 7L 283 47 90 130 2.38 0 0 6 26 427 t, l) 90 723 2.64 0 0 26 56 2I2

40 79 103 2.82 0 +# 206 672 326 Dys- 42 81 98 2.86 + -|.H 26L 1500 575 tro- 44 81 105 2.76 + l-++ 586 L285 2L9 phic 45 87 101 2.92 0 190 L27 2 67L 48 B8 115 2.83 + # 328 880 268

-! t-value I 0.9s4 2.992 -0.428 -4.506 -7 .582 0.266

:k Normal hamsters were of the ]-akeview random bred strain.

I Based on a t-test for comparison of the means of ungrouped data. Table 33b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROUP 4 Substrate: pyruvate/malate in polarographic experiments; pyruvateffumarate in manometric experiments. St.andard Proteinase Preparation.

Pol arographic Manome tric

28oc 28oc 37oc

No. RCR ADP/O O ate P rate P/O 0 rate P rate P/O 0 rate P rate rr 2 2

38 1.4:k II2r( 3L4 2.s 373 1865 2.4 sl1 2453 I Nor- 39 2.6 L.9 LT4 433 2 I47 2 2342 w .3 320 3.2 366 -t malt 43 2.4 1.9 210 798 2 .7 320 17 28 2.3 423 1946 49 3.2 1.6 195 624 2.L 287 L205 2.4 319 1531 I

40 1.0 0.0 J ¿+" 0 2.9 254 1473 2.4 3L4 1507 Dys- 4¿ 1.0 0.0 49,',, 0 2.7 27 9 L507 2.4 32t L54r tro- 44 1.0 0.0 oo^ 0 2.6 249 1295 2.3 445 2047 phic 45 1.0 0.0 5 6'k 0 2.3 27 9 1283 2 .6 3¿+5 L7 9¿+ 48 1.0 0.0 88Jr 0 2.4 25r 1205 t-value t 8.443 ls.843 3.96L 5 .803 -1.087 3.590 I.493 0 .686 0 .947 L .4L5

I Uormal animals were of the Lakeview random bred strain. Jc These values were obtained during the lst Period. No 2nd Period was performed or could be obtained properly in these experiments.

I { Based on a t-test for comparison of the means of ungrouped data. -271-

Table 34b

CHARACTERISTICS OF THE HA}.,ISTER GROUP 5

CPK No. Age Body mg heart Heart Streaks -DTT +DTT Percentage v/t. g body d ise ase ac tivat ion

50 175 L25 2 .88 0 0 T2 38 315 1't1 Nor- 54 776 TI9 0 0 OJ 100 158 mal:k 5 5 r67 L20 2.79 0 0 29 93 318 57 232 119 2.67 0 0 79 t44 184 59 2L5 109 3 .30 0 0 18 50 27L

51 177 L32 3 .88 ].# + 96 s65 587 52 177 723 3 .85 -+# # 236 649 276 53 778 124 3.23 + # 764 L260 165 Dys- 56 L77 TL6 3.94 .+# + 336 I2I5 362 tro- 58 2L3 r24 2.65 + r.# 659 2600 394 phic 60 2L4 r15 4.L6 # # 500 15 15 303 6I 164 I2L 3 .60 # + 93 627 677 62 165 133 3.20 ++ # 176 923 525 63 169 L32 3 .50 j:-H # 73 6L6 844

I t-va1ue 0.906 -1.683 -2.963 -2.4L9 -3.426 -2 .O7 2

* Normal hamsters were of the LSH strain.

I Based on a t-test for comparison of the means of ungrouped data. Table 35b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKEI,ETAL MUSCLE MITOCHONDRIA FROM HA]4STERS OF GROUP 4 Substrat.e: pyruvate/malate in polarographic experiments ; pyruvate/fumarate in manometric experiments. Standard Proteinase Preparation.

Polarographic Manome tr ic

2BOC 2Boc 37oc

No. RCR ADP/O O ate P rate Plo O ate P rate P/O 0 rate P rate rr rr 2

50 3.2 1.5 151 453 I Nor- 54 3.6 2.0 Ì\) r92 768 2.7 zét wtt 2.3 409 1881 -l ma1'"- 55 4.3 1.8 229 824 2 .2 267 II7 5 2.L 354 1487 t\l 57 4.2 L.9 L95 74L I 59 4.4 1.8 230 828

51 5.5 1.8 2r7 78L 2.6 L4B 770 2.7 209 Lr29 52 5.4 2.0 203 8I2 2.5 I59 7 95 2.3 158 727 53 3.7 2.L 22I 928 2.6 227 1180 2.4 353 L694 Dys- 56 1.0 0.0 33 0 ,:t ,27 r22B 2.0 385 1s40 tro- 58 1.0 0.0 50 0 phic 60 3.0 1.8 140 504 6L 5.5 )2 206 906 62 4.5 1.8 246 886 63 5.7 2.4 2l+8 1190 t-value I 0 .020 0.5s7 0.665 0.281 -0.269 L.829 r.728 -0.668 7.25L 1.183

>k Normal animals were of the LSH strain.

T Based on a t-tesË for comparison of the means of ungrouped data. - ¿1J -

Table 36b

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HAMSTERS OF GROUP 6 Values obtained from manometric experiments with pyruvate/fumarate, palmiËate, and palmityl-L-carnitine as substrates at 37oC. Lochner Pre par at ion .t

No. Palmitate Pañity1-L- carnit ine Pyruvate /Fumar ate

O ate O ate 0 rate Plo rr rr 2

67 I17 235 2.3 69 99 208 )) 70 9B 2L0 2.L Nor- 73 92 L7B I.9 malJc 7 4 100 205 )t 76 101 224 2.0 7B 82 88 rlo ,_o B1 73 724 82 76 130

66 46 68 62 L25 I.7 7L 45 I2I 2.0 Dys- 72 67 108 1.9 tro- 75 51 L2L I.9 phic 77 55 131 r.9 79 70 OJ ,:t ,_, 80 46 72 83 42 60

l- t-va I lle I 6-79s 2 .880 7 .6s9 2.396

Values represent the means of duplicates.

Normal hamsters were of the LSH strain, except for No. 6l , which was a Lakeview hamster.

* sase¿ on a t-test for comparison of the means of ungrouped data. - 274 -

Table 37b

RESPIRATION AND OXIDATIVE PHOSPHORYI.ATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HA},ISTERS OF GROUP 6

Substrate: palrnityl-L-carnitine * malate. .Lochner Preparation.

o o 28C 37C

RCR ADP/O Orr ate P rate RCR ADP/O Orrate P rate

69 3.3 L.7 89 303 70 3.2 r.7 89 303 : 73 2.5 1.6 78 250 1.6r. rir ¡is 74 2.6 1.6 79 253 2.4 1.5 148 444 Nor- 76 2.4 1.5 96 2BB )? 1.7 t49 507 matt 7 B 2.5 L.6 74 237 ?7 1.8 109 392 8l 3 .0 L.6 77 246 3.6 I.9 r37 52L 82 3.4 L.7 BB 299 4.L 2.0 t64 656 B4 3.9 L.9 110 4r8 4.L r.9 L74 66L 87 2.2 l.s 82 246 3.0 7.6 L4L 45L

7I 1.0 0.0 68'k U 72 1.8 r.2 53 r27 1 .5:'r 1 .4)k 7 8:, 2L8 75 1 . 6:k I . l)k 61:k I34 1.0 0.0 7 6i, 0 Dys- 77 1.9 L.4 67 188 1.8* r.4* 93), 260 tro- 7 9 2.T l.s 83 249 2 /!'tt 1 Á:k 115?k 368 phic 80 I .2r1 1 .0rç 4l'tc 82 2.L L.4 7i r99 83 2 .2 7.6 50 160 3.3 1.9 97 369 85 2.6 L.6 87 278 3.3 L.7 164 558 86 2.7 1.s 7s 225 3.4 L.7 138 469

f t-value I 3.567 3.309 3.361 3.657 T.937 1.988 2.983 2.925

Normal hamsters were of the l,SH strain.

These values \¡/ere obtained during the lst period.No 2nd period \{as performed or could be obtained properly in these experiments.

Based on a t-test for comparison of the means of ungrouped data. - 275 -

Table 3Bb

RESPIRATION AND OXIDATIVE PHOSPHORYLATION BY SKELETAL MUSCLE MITOCHONDRIA FROM HA}4STERS OF GROUP 6 Substrate: pyruvate/ma1ate. Lochner Preparation.

2Bo c 37o c

No. RCR ADP/O Orrate P rate ADP/O O rrate P rate

64 2.9 L.7 82 279 65 2.7 1.8 75 270 67 3.3 1.9 rr9 452 69 3 .5 L.9 92 350 70 3.6 1.8 9L 328 Nor- 73 3.s L.7 87 296 3.2 I.7 t+t +lg malf 7 4 3.2 I.7 92 313 )q L.7 156 s30 76 2.7 L.7 111 377 2.9 I.7 L69 575 78 2.4 1.8 80 288 81 3.4 1.9 94 357 4.3 2.L 182 764 B2 3.6 r.7 110 374 4.6 ) '), L92 883 84 4.0 2.0 118 472 4.7 2.L 2r8 916 87 2.9 1.7 10r 343 3.5 L.9 189 778

66 r.7 1 .8 57 205 68 2.4 r.6 69 22r 7I 1 .8 L.6 68 2r8 72 2.0 1.5 62 186 Dys- 75 L.7 2.0 7L 284 rl +,* L .21) rõ¡r. z+l tro- 77 L .9 r.6 B0 256 1.8 1.8 115 474 phic 79 2.3 1.6 79 2s3 I .8'k L .4j, 100:t 280 BO 2.I r.7 69 235 2.8 r.9 113 429 B3 r.9 1.5 s4 162 3.8 2.r 133 559 85 2.6 L.6 99 3r7 2) 1.8 190 684 86 3 .7 L.6 93 298 3.4 2.0 164 656

I t-vatue I 4.8L4 2.926 4.057 4.664 2.400 I.zTL 2 .936 2.485 f Hamsters No. 64, 65, and 67 were of the Lakeview random bred strain. The other normal hamsters were of the LSH strain.

These values were obtained during the lst Period. No 2nd Period was performed in these experiments.

J. t Based on a t-test for comparison of the means of ungrouped data. RESPIRATION RATES I^IITH SUCCINATE AND NADH BY SKELETAL MUSCLE MITOCHONDRIA FROM HA]4STERS OF GROUP 6

Lochner Preparation. P/lul=pyruvate/ma1at.e; cyt. c=cytochrome c .

Succ inate NADH alone after P/I'l -cvt.c +cvt. c 28oc- 37oc zs%-----zoc 28uc 37uc 2g"c 37o c

64 59 65 54 67 67 69 58 70 6I Nor - 73 88 f 59 159 mall 74 93 62 L97 76 140 74 L64 237 I 78 L76 52 L67 232 NJ 81 96 67 240 311 437 623 ! o\ B2 L23 68 215 331 472 683 I 84 119 6B 186 308 359 659 87 L27 62 194 297 365 627

66 50 6B 60 7I 59 72 tr: 6L r66 Dys- 75 63 155 ,?, tro- 77 60 159 phic '7o. 140 59 200 80 L23 57 L82 23s 546 B3 97 50 195 254 373 s19 85 180 B2 200 326 369 699 86 ts4 70 198 279 380 591

L-value:k 0 .447 0 .7 23 0 .703 0 .883 0 .860 L .407

Hamsters No.64r 65, and 67 were of the Lakeview random bred strain; the other normal animals were of the LSH strain. Based on a t-test for comparison of the means of ungrouped data. - 277 -

Table 40b

RNA, DNA, A}]D ?ROTEIN CONTENT IN SKELETAL }4USCLE OF HA}4STERS OF GROUP 6

mg per g muscle þg per g muscle

T.P" N.C.P" C. P" RNA DNA

67 176.8 L73.7 3.1 944 484 69 168 .0 L62.7 s .3 1003 558 70 L64.3 152.5 1r.B LT7 3 s56 73 17 Nor-I 4.L 151.0 23.I 1l98 540 malr 7 4 L47 .7 144.5 3.2 975 555 76 r7I.5 L70.4 1.1 993 478 78 L60.2 151.0 9.2 986 s34 84 L64.2 160 .8 3 .4 1151 s36 B7 1s6 .8 Ls7 .6 0 r0 93 62L

37 L7s.7 r7r.4 4.3 1075 839 66 15s.3 r44.r Lr.2 LO46 851 68 r47 .6 141.0 6.6 1 483 1242 7T L67 .4 1s0.9 L6.5 r 148 767 Dys- 72 L55 .2 L52.s 2.7 L37 9 L243 tro- 75 r51 .8 L42 .9 8 .9 110 9 9L7 phÍc 77 Is7.s 158.1 0 I 133 789 79 156 . s r43 .2 13 .3 TL44 809 80 Ls4.7 1s1.9 2.8 1033 855 83 L63 .6 r53 .2 10.4 TL69 87I 85 169.0 L67.8 r.2 7455 L349 B6 1s3.6 144.8 8.8 1327 1505 t-va1ue:k L.546 1.533 -0.199 -2.5L4 -5.342

T.P. = total protein N.C.P. = non-co11agen protein C.P. = collagen protein t Normal hamsters were of the LSH strain, except for No. 67 which was a Lakevíew hamster.

:k Based on a t-test for comparison of the mearis of ungrouped data. -278-

Table 41b

LYSOSOMAL ENZYME ACTIVITIES IN SKELETAL MUSCLE OF HA}4STERS OF GROUP 6

Incubation temperature 37oC.

No. Ac id Phosphatase)k CaËheps in Dtrrk p- glucuronid¿se:krk>k

67 11 .3 2.90 ô ?5 69 L2.3 4.39 0. 16 70 12.7 4.7 9 0. 15 Nor- 73 T2.L 4.55 0. 16 matl 7 4 L4.L s .35 0.72 76 L2.9 L 17 o.L2 78 13 .6 4. 88 0. 13 84 L2.B 4.96 0.22 87 L3.7 4.9L 0.28

37 11 .3 7 .02 o.69 66 9.4 s.r4 0.58 68 1t o 9.s4 0. 98 7I 10 .5 5.22 0.43 Dys- 72 t2.9 7.49 0. 93 tro- 75 7L.9 6.89 0 .38 phic 77 10. 9 5.62 0.37 79 11 .8 6.6r 0 .53 80 10. 9 6.20 0.59 83 11.9 7 .54 0 .78 B5 TL.9 7 .Os 0.lL 86 T2.T 7 .00 0 .33 t.-va1u 3 -r11 -4.970 l¿c p rated per mg prote per nour.

:t:k fag tyrosine liberated per mg proËein per 30 minutes.

:k:k>k f¿B phenolphthalein liberated per mg protein per hour.

t Normal animals were of the l,sH straín, except for No. 67 which was a Lakeview hamster.

* na""d on a t-test for comparison of the means of ungrouped data.