isoforms. FEBSJ.Accepted AuthorManuscript. http://dx.doi.org/10.1111/febs.14367 D. A.andErvasti, J.M.(2018),Impairedmuscle relaxationandmitochondrial fissionassociatedwith genetic ablationofcytoplasmic O'Rourke, A.R., Lindsay,A.,Tarpey,M.D., Yuen,S.,McCourt,P.,Nelson, D.M.,Perrin,B.J.,Thomas, D.D.,Spangenburg,E.E., Lowe, This istheauthor's manuscriptofthearticle publishedinfinaleditedform as: ______endoplasmic reticulum kinase long-chain-fatty-acid (PERK), bindingenzyme 1(IRE1α), protein (BiP), immunoglobulin kinase RNA respiratory exchange(RER), protein disulfide inositol- isomerase(PDI), ratio ryanodine receptor (RYR),oxygen species(ROS), untranslated region(UTR), controlrespiratory controlratio (RCR), reactive (CT), response unfolded (UPR), protein ATPase - (SERCA), knockout (KO), plasmic muscle specific(ms), Ca2+ reticulum Mitochondrialdigitorumlongus (EDL), (MAMs),sarco-/endo associatedmembranes transmissionEmbryonicMEF), Fibroblast microscopy (TEM),extensor electron EndoplasmicSarcoplasmic Reticulum(ER),Abbreviations: reticulum Mouse(SR), Article Original : Article type Role Running of Title: Indiana, 46022 5 MN, Minnesota,Minneapolis, 55455 4 University, Greenville,NC,27858 3 55455 2 MN,Minneapolis, 55455 1 Spangenburg McCourt O’Rourke R. Allison Impaired muscle relaxation andassociatedImpaired muscle mitochondrial withgenetic ablation relaxation fission of Department of Biochemistry, Molecular Biology, and Biophysics University of of University Biophysics and Biology, Molecular Biochemistry, of Department East Carolina Diabetes and Obesity Institute, Department of Physiology, East Carolina East Physiology, of Department Institute, Obesity and Diabetes Carolina East Department of Biology Indiana University-Purdue University Indianapolis, Indianapolis, Indianapolis, Indianapolis, University University-Purdue Indiana Biology of Department eatet f eaiiain Medicine, Rehabilitation of Department Accepted Article Minnesota, of University Development and Biology Cell Genetics, of Department 4 D’anna , 3 , Dawn, Lowe A.

M. 1 , Angus Lindsay Angus , β Nelson - - and γ 2 cytoplasmic actin isoforms. actin cytoplasmic - muscle. 4 , and James M. Ervasti and M. , James , ejmn . Perrin J. Benjamin 2 , Michael D. Tarpey D. Michael , nvriy f inst, inaoi, MN, Minneapolis, Minnesota, of University 5 4 , ai D Thomas D. David 3 – , CoA ligase 4(FACL4), CoA ligase Samantha Yuen Samantha 4 re , -like quiring 4 Espen E. E. Espen , Preston Preston , - This article isThis article by protected copyright. All rightsreserved. Accepted theobserved providing for . apotentialmechanism pathological while mice aged in changes morphological SR observed the of development presage muscle month 12 of muscles in slowed significantly was contraction fou month we Instead, 12 mice. knockout from fibers muscle skeletal in similar was consumption oxygen as affected measurably not was muscle in respiration lacking fibroblasts embryonic mouse primary in events fission mitochondrial in decrease significant a with associated morphologies mitochondrial interconnected and elongated significantly measured also and mitochondria between interface sarco the represent which preparations, membrane β found Articleag in reticulum sarcoplasmic the and mitochondria the both in abnormalities morphological identified we microscopy, myopathy progressive non of knockouts specific Abstract: Keywords: Send to: correspondence ATP sy ubiquinol cReductase Cytochrome inositol (UQCR), 5 1,4, - nolsi reticulum endoplasmic hl α While - nthase subunitalpha (ATP5A). nthase cyto pcfc ncot ie Or aa ugs ta ipie Ca impaired that suggest data Our mice. knockout specific

- β

and γ and - actin, γ sk - ci ioom peoiae n dl srae msl, kltl muscle , striated adult in predominate isoforms actin cyto - - actin, actin to be enriched in isolated mitochondrial associated associated mitochondrial isolated in enriched be to proteins actin a ffected by an unknown mechanism. Using transmission electron electron transmission Using mechanism. unknown an by ffected

- [email protected] muscle

isoforms ed muscle ed

important in signaling and mitochondrial dynamics. We We dynamics. mitochondrial and signaling in important

β d ht h mxml ae f eaain fe isometric after relaxation of rate maximal the that nd

cyto cytoplasmic β cytoplasmic , - mitochondrial

n/r γ and/or - specific β specific

- l muscle old cyto cyto cyto - - dynamics.

ci. neetnl, mitochondrial Interestingly, actin.

- or γ or

and γ and cyto - pcfc β specific - - cyto actin trisphosphate (IP3R

- actin knockout mice. We mice. knockout actin - l β old

each cause a mild, but but mild, a cause each cyto cyto 2+ -

-

re n γ and

n γ and - uptake may may uptake - cyto cyto 3), - - actin actin -

This article isThis article by protected copyright. All rightsreserved. detectable any without but myofiber significant weakness, organe intracellular or lattice costameric the of perturbations with muscle myopathy and progressive degeneration/regeneration a by characterized phenotypes Prins γ either muscle generated previously we muscle, skeletal in in organelles several muscle. of function the to contribute cytoplasmic sarcop with mice in disorganization associated myopathy skeletal protein (FHL1) 1 protein γ to due from presumably myofibrils of force uncoupling contractile of loss and the from displacement heav clathrin of Knockdown coupling. contraction peri the localization (Tmod3) the extra of Knockout the at and tropomyosin the Z subsarcolemmal sk adult development myoblasts muscle muscle striated adult in filaments thin sarcomeric of constituents essential Introduction: Acceptedcyto Article - ci ws fo was actin - disk t al. et While abundantly expressed skeletal ( skeletal expressed abundantly While To more directly test the functional importance of non of importance functional the test directly more To that shown have studies Several

eletal muscle and localize to an to localize and muscle eletal cyto - yolsi atn ( actins cytoplasmic Z , -

- ik lmns ih soitd eet i tid opooy n excitation and morphology triad in defects associated with elements disk [3,4] and mitochondria 2011) , actin [5

– n hv be ipiae i svrl e ses f myogene of steps key several in implicated been have and 8] ( und in an isolated isolated an in und Actg1 . After myogenes After . Tee w single two These . as a result of result a as - [23] sO Sonnemann msKO, [22] . .

[9 [9,13 β

– wie ee ncot f H1 ass progressive a causes FHL1 of knockout while , Collectively, these studies suggest that suggest studies these Collectively, sfr TM5NM1 isoform cyto 12] - –

, 16] (Tmod1) knockout (Tmod1) 1 tropomodulin and re is, very low levels of levels low very is, gions of the sarcoplasmic reticulum (SR) flanking flanking (SR) reticulum sarcoplasmic the of gions complex containing containing complex . gene

extra γ γ cyto cyto [21] - α - - actin colocalizes with isoforms of , ankyrin, of isoforms with colocalizes actin ci) r te rdmnn ioom in isoforms predominant the are actin) knockouts developed qualitatively similar similar qualitatively developed knockouts t al. et sarcomeric cytoskeleton sarcomeric sk . - acmrc cytoskeleton sarcomeric - A recent proteomic study reported that that reported study proteomic recent A amc eiuu ad mitochondrial and reticulum lasmic actin) and cardiac ( cardiac and actin) y chain was shown to drive drive to shown was chain y [19] 2006) ,

o dsuto o toooui 3 tropomodulin of disruption or , - pcfc ncot ie lackin mice knockout specific β o β or , cyto four lles in mice 3 mice in lles - muscle cytoplasmic actins actins cytoplasmic muscle -

- and and and cyto - α - γ a actin ca cyto - af I domain LIM half [20] - extra that includes the the includes that ac - actin remain in remain actin - [13,14,17,18] adult adult tin) actins are are actins tin)

6 months old old months 6 ( both perturb both Actb - sarcomeric [1,2] sis during during sis γ cyto - skeletal msKO, , - actin actin non g - - .

This article isThis article by protected copyright. All rightsreserved. physiological function extra l their while fission mitochondrial in β defect a either causes fibroblasts embryonic of loss specific that showing by them extend studies previous with consistent are data present our Collectively, Actg1 d Actb intact or bundles ROS or respiration mitochondrial in deficit significant no fission, of rates β for ablated γ fibroblasts and/or embryonic primary mouse While fractions. membrane β both Actb and msCT age to compared reticulum sarcoplasmic vesiculated muscles Actg1 skeletal that show and (TEM) microscopy electron other actin knockoutof β both of age of months 6 until apparent not were mice knockout [12,24] ecrease in the maximal relaxation rate in rate relaxation maximal the in ecrease Accepted Articlecyto -

- sarcomeric cytoskeleton of skeletal muscle mildly perturbs sarcopla perturbs mildly muscle skeletal of cytoskeleton sarcomeric - - n γ and cyto msKO and Actb and msKO Actb and msKO Here, we examined muscle ultrastructure as a function of age using transmission transmission using age of function a as ultrastructure muscle examined we Here, Ntby utatutrl eet i te sarco the in defects ultrastructural Notably, .

Actg1 cyto a maue i ioae Actg1 isolated in measured was -

and γ and - cyto ci ehbtd akdy nrae mtcodil egh ad decr and lengths mitochondrial increased markedly exhibited actin - - sO n Actb and msKO cis a so te eeomn o te phe the of development the slow may actins - cyto msCT mice) msCT - actin co actin .

- - msKO mice msKO - associated extra proteins inthe - sO ie xii misshape exhibit mice msKO msKO mice. Finally, we measured a small but significant significant but small a measured we Finally, mice. msKO

- enrich with biochemically isolated mitochondrial isolated biochemically with enrich , or younger knockout animals. We also demonstrate demonstrate also We animals. knockout younger or , - msKO

which was not linked to a defect in SERCA function SERCA in defect a to linked not was which

suggests that partial functional redundancy of of redundancy functional partial that suggests - extensor digitorum longus ( longus digitorum extensor sO r Actb or msKO

- - matched d sarcomeric cytoskeleton [14,20]

cyto mitochondria and dilated and and dilated and mitochondria plasmic reticulum of Tmod1 Tmod1 of reticulum plasmic - sO kltl uce fiber muscle skeletal msKO -

r γ or . The progressive nature nature progressive The . oye osre after observed notypes oto aias (Actg1 animals control rm 22 from ,

cyto oxygen consumption oxygen

- [20,23,25 ci fo mouse from actin EDL - smic 5 ot old month 25 oss from the from oss )

- muscles of muscles associated – reticulum reticulum 27] [14,20] eased , and and , that that cyto . - - . . ,

This article isThis article by protected copyright. All rightsreserved. in maintained be to appear morphology cristae and density electron Mitochondrial knockoutsamples and control between unchanged is morphology which morphology. mitochondrial in change overall an of suggestive is section, cross in ratio width to length the in change three are mitochondria and images dimensional th Although Q). 1 (Figure observation qualitative J 1 Figure age in observed com in L) K, 1 Figure arrows, mitochondria of subset J 1 (Figure age of months mit abnormal M; I, normal Z P E, i arrow white a with (denoted reticulum sarcoplasmic the of dilation displayed a white a with (denoted 12most month from Actg1 architecture, muscle studies 22 and 12, 6, at animals Actg1 from ultrastructure muscle the examine to TEM utilized extra the with associated proteins binding actin of ablation genetic after mitochondria and reticulum sarcoplasmic Aged in Morphologies Mitochondrial KOCytoplasmic Actin Skeletal Muscle and Reticulum Sarcoplasmic Altered Results: Accepted Article . t 22 At ). - iue 2 Figure msCT or Actb or msCT eea o Several [12,24] - disk alignment, but presented with sarcoplasmic reticulum dilation (Figure 1G dilation (Figure reticulum sarcoplasmic presented with but alignment, disk - L). ochondrial morphology in Actg1 in morphology ochondrial - 5 ots f g, l Actg1 all age, of months 25 analysis ,

B The measured mitochondrial length to width ratios confirmed the the confirmed ratios width to length mitochondrial measured The hr rus ae bevd age observed have groups ther - ). ace Actg1 matched - - old animals were also not different from from not different wereold also animals E eaiain f kltl uce n rs scin revealed section cross in muscle skeletal of examination TEM including Z including msCT which appeared rounded in the two the in rounded appeared which - f ogtdnl etos rm month 6 from sections longitudinal of rwed n iue D F, 1D, Figure in rrowhead 25 months of age (Figure (Figure age of months 25

-

ihr magnification Higher sarcomeric actin cytoskeleton cytoskeleton actin sarcomeric control - , ) Actg1 Q). L, parison to the more prevalent elongated mitochondria mitochondria elongated prevalent more the to parison - disk alignment disk - sT r Actb or msCT mice (Figure 1 A 1 (Figure mice

- sO n Actb and msKO - sO n Actb and msKO - msKO and Actb and msKO

that ese measurements were made on two on made were measurements ese - dimensional structures, the observed observed the structures, dimensional - - images reveal internal mitochondrial mitochondrial internal reveal images eedn mrhlgcl eet in defects morphological dependent sT uce wie ro heads, arrow (white muscle msCT

was not different from age from different not was - C, 1 ). P P , n Actg1 one ), osset ih u previous our with Consistent ; - Figure 2 Figure - msKO muscle exhibited a a exhibited muscle msKO Actg1 dimensional images dimensional [14,20,23] - msKO muscles displayed displayed muscles msKO - msKO muscles at 22 at muscles msKO - sO n Actb and msKO - - old animals showed showed animals old msCT or Actb msCT or A ). While muscles muscles While ). .

- (Figure 1M (Figure msKO sample sample msKO Therefore n Figure 1 1 Figure n - matched -

- msKO msCT msCT (white (white we , - - O) 25 25 - - . This article isThis article by protected copyright. All rightsreserved. which mitochondria, perturbations β inmorphology when and reticulum sarcoplasmic co MAM the to localized enzyme lipid a FACL4, of by (identified fraction isol the in mitochondrial enriched most and C), cytochrome crude the in present weakly immunoreactivity), β and isoform each to specific antibodies β containing fractions from MAM, isolated biochemically we dynamics mitochondrial and metabolism, lipid signaling, death cell Mitoch sarco the apposition. and mitochondria between close interface important of functionally a are site (MAMs) membranes their at organelles both with Actg1 or FHL1 Tmod1 of ablation genetic with observed previously than manifest to longer take which Actg1 of mitochondria and reticulum sarcoplasmic [14,20,23] C 3 (Figure localization and levels similar at expressed was which , in changes Actg1 by impacted in phenotype morphology mitochondrial mitochondrial observed proper for important be to known muscle. knockout

Accepted- Article nih ih mmrn fato togt o ersn te interface the represent to thought fraction membrane a with enrich wildtype - msKO and Actb and msKO opooia dfcs n oh h srolsi rtclm n mit and reticulum sarcoplasmic the both in defects Morphological cyto [14,20,23] - or E dt rva age reveal data TEM our , - /endo ci wr peet n h ctslc rcin vrfe b by (verified fraction cytosolic the in present were actin

os liver mouse - plasmic reticulum involved in mitochondrial respiration, Ca respiration, mitochondrial in involved reticulum plasmic . nte ctseea poen te inte the protein, cytoskeletal Another

cyto - msKO muscles suggested that β that suggested muscles msKO -

and γ and

- [35] E) relative to control muscle. control to relative E) cyto

and performed western blot analysis to identify the the identify to analysis blot western performed and

- mitochondrial, and endoplasmic reticulum fractions fractions reticulum endoplasmic and mitochondrial, actin immunoreactivity immunoreactivity actin cyto ( Figure -

or γ - dependent dependent cyto ated MAM fraction verif fraction MAM ated 4 ) -

actin areknockedout(Figure 1). - [24,12,36,37] sO n Actb and msKO [38,39] - msKO and Actb and msKO

. Thus, both β both Thus, . ultra each cyto rmediate filament desmin is is desmin filament rmediate Consistent with Consistent using previously established established previously using -

structural and γ and A epce, oh γ both expected, As . ipa age display [28,29] - cyto sO s iey not likely is msKO - [30 msKO muscle msKO ied by the presence the by ied nra associated ondrial - cyto actin may interact may actin – defects in the the in defects 34] - Hwvr the However .

(Figure 3 (Figure

and γ and other ochondria of of ochondria b . Therefore, . 2+ etween the the etween - dependent dependent crosstalk, crosstalk, cyto

studies studies s, -

actin actin A cyto but - B) -

This article isThis article by protected copyright. All rightsreserved. AcceptedA 6 Figure (Fis1; 1 fission mitochondrial we however, dynamics protein of abundance the in decrease a to linked (Actg1 to compared MEFs KO Actb/Actg1 and Actb, Actg1, in decreased significantly ( analyzed pools MEF all different across not was events fusion of frequency the While quantified. were events fusion were mitochondria Therefore, fusion. mitochondrial in increase an or fission, mitochondrial of size rate the in decrease a be would mitochondrial in increase measured the for explanations network per branches of number the and J) controls to compared MEFs knockout in greater is branch with mitochondria of number the While I). 5 (Figure mitochondria β either lacking MEFs plug Fiji the using measured as H) 5 (Figure branches without Actb or (Actg1 control to compared MEFs KO Actb/Actg1 and Actb, Actg1, in Mitochondria area surface Article both or actin, gene alleles Actg1 or Actb ( fibroblasts embryonic mouse primary in mitochondria in fibers muscle skeletal roles adult in measure to key challenging are play to shown been recently mitochon have actin and reticulum endoplasmic fusion and fission of processes opposing the by controlled dynamics Actin Cytoplasmic in Dynamics Embryonic Fibroblasts Mitochondrial of Perturbation

or adenovirus or - Abnormalities in mitochondrial morphology imply a defect in mitochondrial mitochondrial in defect a imply morphology mitochondrial in Abnormalities CT or Actb or CT - T MF hv smlr ubr o idvda mtcodi, r mito or mitochondria, individual of numbers similar have MEFs CT) ra fission drial actins oprd to compared - CT) - 5 GFP as a control a as GFP 5 displayed

which were treated with adenovirus with treated were which n ay el types cell many in cyto MEFs ( MEFs

-

actin found that proteins involved in mitochondrial fission, fission, mitochondrial in involved proteins that found iohnra sz in size mitochondrial individual mitochondria which were which mitochondria individual

Figure or

γ Figure cyto

5 - live actin

- J).

[36] B) or fusion, mitofusin2 (MFN2) and dominant dominant and (MFN2) mitofusin2 fusion, or B) [25,26,41] - 5 imaged and the frequencies of fission and and fission of frequencies the and imaged

The observed decrease in fission could be be could fission in decrease observed The

(Figure 5 K) is not different. different. not is K) 5 (Figure . ae n nrae n ewr o branched or network in increase an have ) te rqec o fsin vns were events fission of frequency the I),

MEFs ablated for either β either for ablated MEFs s known to be critical for critical be to known s ,

the length of the branches (Figure 5 (Figure branches the of length the

, however, mitochondrial dynamics dynamics mitochondrial however, , oto MF ( MEFs control MEFs) homozygous for floxed floxed for homozygous MEFs) - 5 CRE to knockout the target the knockout to CRE 5 [42] . Therefore, we imaged imaged we Therefore, . - in MiDA in

or network formation formation network or significantly larger significantly Figure

[40] The most likely most The [43] es (Figure 5 I) I) 5 (Figure es cyto - mit ul Mouse Null - Bt the Both . . However . actin, γ actin, ochondrial chondria chondria 5

control control A - - cyto G CT by by ). - , This article isThis article by protected copyright. All rightsreserved. ( muscles gastrocnemius isolated in consumption oxygen of measurements our with Consistent in expenditure Actb and months β group each from bundles respiration II and I Complex during Super (RCR via respectively, II, complex s and control IV or II Complex in detected differences Actg1 II and in detected were differences no to correspond protein old fiber muscle inter MitochondrialNormal Respiration both β fission mitochondrial in actin controls. to muscle relative C 6 Figure (OPA1; atrophy1 optic Acceptedcyto Article Actg1 - linked actin ) - - cyto

msCT

As defects in mitochondrial morphology were most evident in aged Actg1 aged in evident most were morphology mitochondrial in defects As C perturbations Because

respiration n culn efficiency coupling and . components of components

; - - -

sO uce (iue 1 (Figure muscles msKO maue of measure a and msKO or γ or Figure and γ [44 , bundles from the from bundles

ciae control uccinate cyto – or 46] cyto

- 22

actin actin Actb 8 ewe fbr ude ioae fo age from isolated bundles fiber between - we , actin isoformsnon actin ) and primary mouse embryonic fibroblasts fibroblasts embryonic mouse primary and ) - measureable 5 month 25 Actb - does not lead to perturbations in mitochondrial respiration at 12 at respiration mitochondrial in perturbations to lead not does msKO electron transport chain transport electron

investigate . Taken together these data indicate that the absence of either either of absence the that indicate data these together Taken . nopee hneig f lcrn truh h Q the through electrons of channeling incomplete - msKO mice msKO Our results are consistent with the previously defined role for defined role for previously the with results Our areconsistent Complex I Leak I Complex [25,26]

in h Q the : red portion of the of portion red : and

-

old indicator h prin f eprto atiue to attributed respiration of portion the m - ifrne in differences itochondria F) were not deplete not were F) d

Actb -

in Cytoplasmic Actin KO Skeletal Musclein Cytoplasmic KOSkeletal Actin and extend previ extend and Actb ucin ( junction ( - whether mitochondrial respiration was altered in in altered was respiration mitochondrial whether J Figure Figure redundantly support mitochondrialredundantly support fission. - .

L, - mediated - s N - msCT

msCT Q o change o f iohnra coupling mitochondrial of

, e lo esrd hl bd energy body whole measured also we ), or ADP or l 8

shape, shape,

Figure

(ETC) (ETC) J

gastrocnemius muscle from 12 month 12 from muscle gastrocnemius animals )

respiratory n Actb and respiration ( respiration were were

- was detected was stimulated ous studies by demonstrating that demonstrating by studies ous dynamics 8 complexes I complexes d in Actg1 in d

also F ( - Figure ) respiratory G), - - ace Actg1 matched sO ie ( mice msKO function o dfeet among different not Figure , Complex I Complex

and - msKO or Actb or msKO

8 - in the abundance of abundance the in [36] IV

A 8 (

Seiial, no Specifically, . function are often are function Figure (

- tee a no was there , Figure D C - ) E ope I or I complex . Nor were were Nor . or Complex I I Complex or control ratio ratio control ) - . msKO Figure Complex I I Complex 8 7

- )

junction H

, - -

msKO msKO msKO - which

I

, E ), fiber fiber and and 9 ). - - This article isThis article by protected copyright. All rightsreserved. fromlonger different Actbsignificantly in relaxation of rate maximal Actg1 the in time relaxation muscles rel force produce Actg1 following force of fatigue recovery as well as protocol fatigue a during force of rate maximal force, specific that showed Actb from Actg1 or Actb 9 Impaired impairment inmitochondrial function. a with by associated not are caused 3) 1, (Figures dynamics Actb or and Actg1 of ablation morphology genetic mitochondrial in alterations the that indicate B). 10 (Figure muscle knockout and control in similar also non is production, of increase percent the is which index, scavenger controls to compared muscle knockout in different significantly non and buffered that indicated fibers muscle skeletal oxygen reactive of mitochondria of aspect another muscle, wholeof function( respiratory body aged between difference ) to explain the mild, but progressive myopathy caused by muscle by caused myopathy progressive but mild, the explain to )

Accepted month 12 in slowed significantly was axation Article -

12 month 12 sT and msCT - msKO and Actb and msKO huh eprto ws’ dtcil prubd n Actg1 in perturbed detectibly wasn’t respiration Though a of absence the In protocol

oprd o g matched age to compared

Relaxation

- [12,24] old Actg1 old [47

were not different (Table 1, (Table different not were Actb – pce (O) Eaiain f h RS pce H species ROS the of Examination (ROS). species 49]

- in CytoplasmicActin MuscleSkeletal KO we , - msKO msKO muscles to respond to electrical stimuli electrical to respond to muscles msKO

were - msCT and and msCT Actb measurable - characterized msKO

mice - Actb largely msCT

. Figure - 1, (Table

msCT

ute a Further Actg1

Actg1 - and Actb and msKO muscles(Table 1,

unaffected deficit in mitochondrial function ( function mitochondrial in deficit function which could be affected is the is affected be could which function

9 otatl function contractile - muscles slowed to the point that it was no no was it that point the to slowed muscles - msKO animals, a animals, msKO ) sT n Actb and msCT contraction . Figure Figure

ayi o ttnc smti contractions isometric tetanic of nalysis - msKO mice in in mice msKO Itrsigy te maximal the Interestingly, . 11

- 11 old

A , D). By 22 By D). - force development time development force - Actg1 ufrd H buffered C), - ufrd vr ufrd H buffered over buffered - nd indicating that the ability of of ability the that indicating sT muscles msCT any measured parameter measured any of -

sO n Actb and msKO Figure 12 and 22 and 12

- - sltd D muscles EDL isolated

25 months of age, th age, of months 25

sO r Actb or msKO Fgr 10 (Figure 2 - O , specific ablation of ablation specific release Ca release 2 11 Figure emission

2 a submaximal submaximal a D). - O These re These 25 2 measurable a ws ½ was as , in isolated isolated in

month A). 8 emission , loss of loss , , ae of rate - -

2+ Figure msKO msKO msKO is not not is , and , sults sults The The - 2 old O e 2

This article isThis article by protected copyright. All rightsreserved. Actg1 1, (Table None of abundance stress cellular of result a as (UPR) Ca of Dysregulation Actb between significant activity no SERCA found We in inhibitors. differences of absence or presence the in muscle knockout phenotype. SERCA in deficit a be to unlikely SERCA1a B D Actg1 between abundance in change Anky small muscle skeletal in expressed SERCA of isoform primary Actb in and msCT, relaxation immunoreactivity of levels muscle similar revealed analysis for responsible primarily is (SERCA) ) -

Accepted Articleobserve we phenotype SR the that suggesting E), s iey u t S dilation SR to due likely is - - the msKO andmsKO Actb hl Ca While - less, the dilated sarcoplasmic reticulum (Figure 1) and slowed relaxation relaxation slowed and 1) (Figure reticulum sarcoplasmic dilated the less, Figure as well as small Ankyrin 1 in control and knockout samples knockout and control in 1 Ankyrin small as well as i 1 rin We also measured SERCA ATPase activity in SR isola SR in activity ATPase SERCA measured also We several UPR proteins in Actg1 an Actg1 in proteins UPR several ( 2+ Figure 11

- msKO bt sget ht acpamc Ca sarcoplasmic that suggest both ) 2+ reuptake by the the by reuptake -

msKO skeletalmsKO muscles. can result in the activation of the unfolded protein response response protein unfolded the of activation the in result can 12

compared to compared

A - C )

[51] ahr hn R proliferation SR than rather

viaiiy r activation or availability [52 . - msKO or Actb or msKO Calsequestrin –

Act 55] sarco b . However, . - ms -

/endo - msKO (Figure 13 13 (Figure msKO d Actb KO skeletal muscle (Figure (Figure muscle skeletal KO Actb d CT

in Act in skeletal muscle lysates against lysates muscle skeletal

-

- by TEM by lsi rtclm Ca reticulum plasmic msKO and control SR (Figure 12 (Figure SR control and msKO also

we observed no change in the in change no observed we g1

did 2+ and a regulator of SERCA, SERCA, of regulator a and - rate msKO compared to Actg1 to compared msKO

that that (Figure 1 G 1 (Figure r glto i ipie in impaired is egulation not show any detectible detectible any show not .

h smlr ees of levels similar The [48,50] causes ted from control and and control from ted A - B indicate that it is is it that indicate ad controls. and ) ,

- I, M; Figure 2 Figure M; I, the observed observed the western blot blot western 2+ - ATPase ATPase

14 the the ). -

This article isThis article by protected copyright. All rightsreserved. Ca to addition in functions of array large a for important is which microenvironment γ death both cell of enrichment initiate even and rates respiration elongated mitochondria actasaprotective may feature. emission respiration detectable with paired not were dynamics mitochondrial individually β found and dynamics study to model MEF a utilized we cells, a dynamics retic fusion undergo regularly structures surrounding are dynamics mitochondrial of aspects multiple in [57,58] mitochondrialandfunctional role in dynamics. a link a and supporting betweenγ fission, of knockout muscle skeletal localizes SR subsarcolemmal muscle skeletal in structures include muscle skeletal adult in localized been has actin cytoplasmic which to cellularstructures of array diverse a to localize to shown muscle skeletal Discussion: Acceptedcyto Article - ulum distributed ad obe ci K MF, niaig ht γ that indicating MEFs, KO actin double and , huh ny conig o a for accounting only Though At the mitochondrial the At T bioenergetics maintaining for critical muscle are skeletal in Dynamic mitochondria . As a consequence of the high energy demand of skeletal muscle, perturbations muscle, skeletal of demand energy high the of consequence a As . he . have been implicated in the fission process fission the in implicated been have

hs idn supports finding This or observed alterations in mitochondria morphology and and morphology mitochondria in alterations observed important for mitochondrial fission. e rtcl n l cl tps n ocr ih ihr rqec i non in frequency higher with occur and types cell all in critical re

γ [13 cyto an alteration in redox balance redox in alteration an

[56] cause - in skeletal muscle to enable maximal functional connectivity with with connectivity functional maximal enable to muscle skeletal in – costameres

16] r β or , . non

cyto cyto a s However [30,60,61] - - -

muscle [57] id bt rgesv myopathy progressive but mild, actin - n β and SR/ER interface SR/ER

[9 . i i sil unclear still is it , – causes n non In 12]

cyto cytoplasmic cytoplasmic

h ie i the in idea the nldn ohr mitochondria other including , -

mitochondria actin with the MAM. MAM. the with actin iue fraction minute cyto -

uce el, b cells, muscle perturbation in in perturbation -

or β or , specifically reactive oxygen species (ROS) species oxygen reactive specifically , , C

can

γ a cyto

cyto

2+ - how - be fatal in fatal be [60,64

signaling [9]

actin localization at the mitochondria localizationat actin

and β and mitochondrial dynamics dynamics mitochondrial [25,26,63]

cyto and the peri the and

ablation

f h ttl ci po in pool actin total the of t atn n the and actin oth both - –

cyto n β and 66]

differences

[12

can influence mitochondria influence can -

mice The MAM forms a small small a forms MAM The

. decreased fission in fission decreased mitochondrial actin isoforms have been have isoforms actin ,24]

. Because mitochondrial mitochondrial Because . Here, we demonstrate demonstrate we Here, of cyto measured changes in changes measured [62] [46,59] . - γ - Z

cyto Here we show that that show we Here - ci ioom are isoforms actin disk region where region disk

- ih hc they which with in

or . Mitochondria Mitochondria . mitochondrial mitochondrial

endoplasmic endoplasmic β morphology cyto il that field - - . Some Some . ci in actin muscle muscle

γ adult adult cyto 2+ 2+ - ,

This article isThis article by protected copyright. All rightsreserved. role inactincytoskeleton the the modifications and dynamics polymerization differing have changes acid amino similar biochemically 4 and sequences 3’UTR the by only differ sequences and conserved the that likely is overlap partial this some in compensate likely can but important knocked are either when displayed phenotypes modest but acceleratemay aging skeletal in muscle 22 ms [76] aging with sarcopenia dysregulation β Ca muscle skeletal defective perturbations observed the S of cause the be may membrane SR Ca that suggesting 13) Figure and 12 (Figure SERCA dysfunction. through Ca with Problems themitochondrialfrom MAM the of malformations structural by MAM. which the organelles form lipogenesis and signaling cyto eln o te R smlr o ht e bevd ee hs en en alongside seen been has here, observed we what to similar SR, the of welling

Accepted- Article O kltl muscle skeletal KO 25 months 25 . phenotypes -

The ci seea muscle skeletal actin Ca The partial redundancy of the cytoplasmic actin isoforms, illustrated by the similar similar bythe illustrated isoforms, actin the cytoplasmic redundancy of The partial ,

the the 2+ decreased relaxation rate relaxation decreased

including but not limited to limited not but including , which , lead

signaling

[77,78] oee w dd o osre hne i te bnac o atvt of activity or abundance the in changes observe not did we However Y, EC, h mmrn, or membrane, the SERCA, RYR, of age of Emk n Dve, 2002) Davies, and (Ermak s

observed here observed

[25,26,61,67 to SR Ca SR to

has been linked to alterations in Ca in alterations to linked been has 2+ . Despite the high degree of sequence of degree high the Despite.

sequestration ( is critical to critical is - Figure SR/ER may impairinter interface preceded 2+ O eaain defect relaxation KO .

2+ – 11 leak 72] suggest release release D) Dysregulationthe signalingcasc of

. . , regulating skeletal muscle skeletal regulating similar a

, suggesting that knockout knockout that suggesting ,

further further We observed alterations in the morphology of both both of morphology the in alterations observed We can occur at a number of points including: leak leak including: points of number a at occur can seen here here seen :

mitochondrial fission, ER stress, ROS signaling, signaling, ROS stress, ER fission, mitochondrial s . [20]

[73 that each isoform does isoform each that ROS elevation elevation ROS , supporting a role for Ca for role a supporting , erae in decrease Oe uh tes s uce aging, muscle is stress such One . – 75]

aspects for the other. An explanation for explanation An other. the for aspects in . The . . 12 month old month 12

of

C

lua srsos a idc Ca induce can stressors ellular 2+ refore, loss of loss refore, yndn receptor ryanodine 2+ 2+ γ cyto -

leak through RYR or the dilated dilated the or RYR through leak organelle flow Actb and ultimately loss of loss ultimately and -

[77,79,80] identity - n β and out, indicates that both are are both that indicates out, [76] - of either cytoplasmic actin cytoplasmic either of msCT

Actg1 play a unique and unique a play .

Oxidation of the RYR RYR the of Oxidation signaling. cyto , γ , ades can be caused be caused ades can

γ cyto kltl muscle skeletal n eaain rate. relaxation in . The presence of of presence The . - 2+ - cyto

msKO and Actb and msKO ci ae highly are actin - in the γ the in

and relaxation and - and β and

or

r SERCA or

β cyto cyto cyto

muscle muscle - -

- actin actin actin

vital vital and or or at 2+ - .

This article isThis article by protected copyright. All rightsreserved. Accepted2 mice pern = mouse. genotype and n=10fibersper ImageJ using adjusted were contrast and Brightness u was sum projection ImageJ slices software. accompanying the using objective immersion oil 60× the with USA MA, Prolo using slides on mounted were Fibers visualization. for used was 488 Fluor Alexa to coupled anti with incubated 24 for 7.5) pH NaCl, mM hours 150 and Na2HPO4, mM 42 X NaH2PO4, mM 8 (PBS: PBS Triton in paraformaldehyde (wt/vol) 0.1% 4% in postfixed further were Muscles paraformaldehyde. and DMSO, (vol/vol) USA MA, Danvers, 13 from harvested were perfusion muscles (EDL) longus digitorum Extensor MuscleImaging Fiber Institution Minnesota Care and of University the to according for cared Article were animals male. were examined HSA lacking HSA for hemizygous and alleles, floxed 1999) al., et Miniou (HSA promoter actin to crossed cre was expressing line mice each mice, knockout generate To generations. five of minimum were previously described as ( Mice Mouse Lines Methods: Mus musculus Mus while rotating. Single muscle fibers were mechanically teased apart and and apart teased mechanically were fibers muscle Single rotating. while - t vsaie uli Fbr wr iae o te lmu FuVe FV1000 FluoView Olympus the on imaged were Fibers nuclei. visualize to ) Use CommitteeUse policy. fixed mice and immunostained for desmin (D93F5 desmin for immunostained and mice fixed g od niae egn wt DP (el inln Technology Signaling (Cell DAPI with reagent antifade Gold ng - cre

ht r rfre t as to referred are that - ). Perfusion media (50 mM Pipes, mM (50 media Perfusion ). desmin overnight with rotation. Goat anti Goat rotation. with overnight desmin . Knockout animals were homozygous for either the Actb or Actg1 or Actb the either for homozygous were animals Knockout . ) containing the conditional floxed floxed conditional the containing ) - - Cre mice were provid were mice Cre recombinase under the skeletal muscle specific human skeletal skeletal human specific muscle skeletal the under recombinase

e t cmie w Z two combine to sed All genotype. confirm to used was analysis PCR Standard

each backcrossed on to the C57BL/6 background for a a for background C57BL/6 the to on backcrossed each

- r, otos were controls cre, ihr Actb either ed by Judith Melki, INSERM, Evry, France Evry, INSERM, Melki, Judith by ed - 100, pH 6.8) contains 4% (wt/vol) (wt/vol) 4% contains 6.8) pH 100, - stacks for a total 0.8 microns depth. depth. microns 0.8 total a for stacks

[83] 5 mM EGTA, 2 mM MgSO4, 10% 10% MgSO4, mM 2 EGTA, mM 5 - sT r Actg1 or msCT Actb . Images are representative of of representative are Images . ;

- Cell Signaling Technology Signaling Cell [81] rabbit secondary antibody antibody secondary rabbit age

- and ace floxed matched Actg1 - msCT - 4 ot old month 14

[24] , Danvers, Danvers, , . al Animal Animal al All mice mice All

alleles

mice mice , ; This article isThis article by protected copyright. All rightsreserved. Accepted fusion or fission mitochondrial any events. MEFs fro for frame by frame examined then were images time The 2min. for 10s every imaged were cells events fusion and fission quantify t present number the for assessed a then binary, to it converted used was Fiji for plugin toolset (MiNA) Analysis Network Mitochondria the formation network and mitochondria indi all of area surface the determine use ( was UK) mitochondria Ireland, Northern (Belfast, Bitplane by software USA). AZ, (Tucson, camera HQ Coolsnap protocol num 60x a with UK) Chalfont, Buckinghamshire, (Little system Microscopy Deconvolution DeltaVision Precision Applied GE Adfection Opti CMXR MitoTracker 0.01nM Adenovirus previously as blot western by published protein of knockout complete showed cells Articleinfection Core’s ( seventh the On the (http://www.medicine.uiowa.edu/vectorcore). Ad5 following with treated explicitly USA) IA, City, (Iowa was Core Vector Viral Iowa of University The from purchased group Ad5 other adenovirus control the the hrGFP) with treated was pool One pools. two into split and counted, USA), CA, Carlsbad, Invitrogen Fungizone; 0.5ug/mL Pen/Strep, CO % 5.0 in 37°C at cultured were previously described as embryos mouse M Primary Cell Culture andImaging - MEM with 10%FBS for 15 min. Media was replaced and cells were imaged using a using imaged were cells and replaced was Media min. 15 for 10%FBS with MEM e egh n nme o branches of number and length he [36] F wr clue fo E35 Actb E13.5 from cultured were EFs . Once knockout was confirmed by western blot, MEFs were stained with with stained were MEFs blot, western by confirmed was knockout Once . vrapn mtcodi were mitochondria overlapping m least at different 3individual genotype per wereexamined. embryos Actb L/L [43,83] Actg1 ,

of mitochondria with or without branches. If branches were were branches If branches. without or with mitochondria of

os (ThermoFisher Scientific M7512; Scientific (ThermoFisher os

mitochondrial skeleton was defined was skeleton mitochondrial . Briefly, MiNA defined the mitochondrial network and and network mitochondrial the defined MiNA Briefly, . L/L 2 in DMEM media (supplemented with 10% FBS, 1% 1% FBS, 10% with (supplemented media DMEM in Es o nint or MEFs) iul mitochondrion. vidual erical aperture 1.42 objective and Photometrics Photometrics and objective 1.42 aperture erical [36,84]

sn a z a Using e mtcodin ee quantified. were mitochondrion per L/L .

Briefly: MEFs from individual embryos embryos individual from MEFs Briefly: Actg1 , xldd rm hs analysi this from excluded ( h

- r ( Cre Actb - tce image stacked L/L L/L n dul Actb double and Ad5CMV To assess the overlapping overlapping the assess To /Actg1 , this skeleton was then then was skeleton this , Waltham, MA, USA MA, Waltham, to d L/L -

Cre - MEFs) day post post day MEFs) define individual individual define F (Ad5CMV GFP Imaris Imaging Imaging Imaris - GP virus eGFP) L/L /Actg1 s) and and s) - lapse ) in ) To L/L L/L

- This article isThis article by protected copyright. All rightsreserved. fat and tissue connective microscope, dissecting MgCl 6.56 phosphocreatine, - i to added immediately and weredissected gastrocnemius muscle described been previously has bundles fiber muscle permeabilizing and preparing for technique state native their in mitochondria of assessment the for allows approach this as respiration mitochondrial assess to used were bundles fiber muscle studies, these In Mitochondrial Respiration was t measuredallowingfor Z from measured was width. for axis widest using measured the and length for axis longest the on ratio USA. PA, Hatfield, Sciences, Microscopy quantification. of overlap prevent st from free non contained which field every in acquired were Images 120kV. at USA) OR, Hillsboro, BioTWIN, Spirt l and were muscles ethanol, of concentrations in embedded increasing with dehydration After buffer. pos 2.5% of fix secondary Na 0.1M a in Glutaraldehyde in 4°C at overnight incubated and dissected were muscles r Na M 0.1 in glutaraldehyde 1.5% and PFA 4% intra underwent mice 22 or 12, 6, at Mice Transmission ElectronMicroscopy Accepted Articlehours. 2 for 4°C at solution same the in fixed further and emoved .3 K 7.23 t -

fixed in 1% in fixed ead citrate and visualized on the transmission electron microscope (FEI Technai Technai (FEI microscope electron transmission the on visualized and citrate ead was done using Fiji’s measurement tool measurement Fiji’s using done was

2 [86] GA 27 CaK 2.77 EGTA, aining debris and free from section rips, fields were not repeat imaged to to imaged repeat not were fields rips, section from free and debris aining Epon 812 resin. 65 nm ultrathin sections were stained with uranyl acetate uranyl with stained were sections ultrathin nm 65 resin. 812 Epon . 12 month old mice were anesthetized and red portions of the the of portions red and anesthetized were mice old month 12 .

Fiji OsO -

25 months were anesthetized using avertin. Once anesthetized the the anesthetized Once avertin. using anesthetized were months 25 as performed by Gokhin et al. et Gokhin by performed as - 4 peritoneal perfusion with PBS, followed by perfusion fixation with with fixation perfusion by followed PBS, with perfusion peritoneal -

disk to Z to disk in 0.1 M Na M 0.1 in -

he determination shiftin of Z Cacodylate. Muscles were washed in Na in washed were Muscles Cacodylate. 2 ·6H 2 - GA 2 iiaoe 2 tuie 5.7 taurine, 20 imidazole, 20 EGTA, disk the offset of Z of offset the disk 2 , 0 E, H ., 9 mosmol/kgH 295 7.1, pH MES, 50 O, - cacodylate for 1 hour, and rinsed in rinsed and hour, 1 for cacodylate

l chemicals All and Quantification

Quantification of mitochondria length:width length:width mitochondria of Quantification [83] - cacodylate buffer. The lower limb was was limb lower The buffer. cacodylate h smls ee de were samples The , briefly mitochondria were measured were mitochondria briefly , ee eoe fo te e portions red the from removed were [83,20] -

disks from neighboring neighboring from disks were - disks.

- briefly, the length sarcomere the briefly, overlapping muscle sections sections muscle overlapping ucae fo Electron from purchased

Z ce - Tibialis Anterior Tibialis disk - cold b - cacodylate buffer, buffer, cacodylate

alignment was was alignment - Na 2 identified and and identified ) Udr a Under O).

uffer - ATP, 14.3 14.3 ATP, cacodylate cacodylate [85]

X

. . ([mM]

(TA) The This article isThis article by protected copyright. All rightsreserved. Assay Red Ultra Amplex The added. were [100µM] (BCNU) carmustine and [1µM] (AF) solut buffered the for and [10µM] succinate Then determined. h incubated and saponin 30ug/ml above described as is bundles fiber muscle permeabilizing and H preparing for technique induced The production. succinate assess to used were bundles fiber muscle studies, these In MaximumSuccinate Sigma from purchased Louis, were reagents and chemicals All MA). Beverly, C Cahn (Orion weighed and USA) MO, City, Kansas (Labconco, Follow freeze H2O, distilled in addition. washed were bundles c muscle protocol, the cytochrome of completion exogenous to response in respiration in increase >10% a produced that bundles muscle excluding by confirmed was integrity membrane Tetramethyl A antimycin µM, 10 rotenone µM, 5 c cytochrome mM, 5 succinate mM, 2.5 ADP mM, 5 glutamate mM, 2.5 4mM, malate pyruvate of concentration final a at and order following the in substrates the of creatine 20mM with supplemented bl and 25µM monohydrate Z buffer in conducted were Experiments 2014). al. et (Ryan described previously as oxygen ~350µM of concentration oxygen starting Oxygraph OROBOROS mosmol/kgH K ice to transferred were bundles fiber Muscle 4°C. at rotation continuous with min 30 for saponin µg/ml 30 containing X buffer in permeabilized then were bundles fiber Muscle wt. wet mg 1 ~ of bundles fiber into muscle separating before orseradish peroxidase [1U/ml] was added to the fibers and background was was background and fibers the to added was [1U/ml] peroxidase orseradish -

Accepted Article 5K EGTA, 1 KCl, 35 MES,

MO, USA).MO, esrmns f high of Measurements - 2 p O) andwashed 15min O) for - phenylenediamine dihydrochloride). Preservation of mitochondrial mitochondrial of Preservation dihydrochloride). phenylenediamine

[86] 0.2 . H 2 - K Oooo Instruments, (Oroboros 2K O ebbistatin. Mitochondrial respirati 2 -

production .m dry 0.3mg 5 µM, ascorbic acid 2 mM and TMPD 0 TMPD and mM 2 acid ascorbic µM, 5 -

resolution O2 consumption were made using the the using made were consumption O2 resolution 30 minutes at 4°C. Amplex Red Assay Buffer [25µM], [25µM], Buffer Assay Red Amplex 4°C. at minutes 30 2 HPO 4 3 MgCl 3 ,

at 4°C withcontinuous rotation. weight of mouse muscle was combined with with combined was muscle mouse of weight 2 ·6H

Innsbruck, Austria) at 37°C with a a with 37°C at Austria) Innsbruck, 2 O, 5mg/ml BSA, pH 7.4, 295 295 7.4, pH BSA, 5mg/ml O, on was assessed by deliveryassessedon was by - cold buffer Z ([mM] Z buffer cold ion inhibitors auranofin auranofin inhibitors ion - 35, Thermo Electron, Thermo 35, .5 mM (N,N,N’,N’ mM .5

- Aldrich (St. (St. Aldrich – - dried

110 2 ing O 2 - This article isThis article by protected copyright. All rightsreserved. 30min for 20,000xg mitochondria supernatant 5min, pestle and mortar glass and Teflon a using homogenized model 75 mannitol, 225mM and mannitol, 225mM by followed HCL, 75 mannitol, (225mM buffer dislocation cervical al et Wieckowski in protocol the to according fractionated was animals male month 12 from liver Mouse MAM Fractionation continuou ANCOVA as weight body with manufacturer, analyzed and the Kcal/h by as provided expressed formula the with calculated was expenditure Energy EchoMRI an using content water m quantitative body and tissue, lean fat, body total for analyzed x the on measured was ratio exchange respiratory Instruments including activity 4 = (n days habi 22 calorimetryIndirect andbody composition 1 second.of time integration an with seconds, 10 for 565/600 of excitation/Emission with 37°C at run was

Accepted Article- 5 ot od m old month 25 tuated to the metabolic chamber for 1 day 1 for chamber metabolic the to tuated ad nry expenditure energy and , supernatant supernatant h Oya Cmrhnie a Aia Mntrn Sse (Columbus System Monitoring Animal Lab Comprehensive Oxymax the Clmu, H USA OH, Columbus, ,

- was collected and collected was 7). ( agnetic resonance system resonance agnetic the

xgn osmto (O) n cro doie rdcin (VCO2) production dioxide carbon and (VO2) consumption Oxygen

[35] supernatant supernatant The . l m ale te superna the ,

a cletd n rsu udr h sm conditions. same the under respun and collected was .

Briefly: the mouse was anesthetized using avertin using anesthetized was mouse the Briefly:

- ie ws immed was liver ice were individually housed for metabolic testing and were were and testing metabolic for housed individually were ice -

M urs, n 3m Tris 30mM and sucrose, mM (RER) were calculated from the gas the from calculated were (RER) n z and - M urs, .% S, .m ET ad 0M Tris 30mM and EGTA 0.5mM BSA, 0.5% sucrose, mM

spun at 9,000xg for 10min, the 10min, for 9,000xg at spun containing the and ER ER and cytoplasm the containing

- ee esrd using measured were xs ih h ue f nrrd em. beams. infrared of use the with axes at a su fr h a 1h for spun was tant ). 75

O (oue 2, C2 vlm C2, and CO2), (volume VCO2 O2), (volume VO2 - mM sucrose, 0.5% BSA, and 30mM Tris 30mM and BSA, 0.5% sucrose, mM

(Echo Medical Systems Medical (Echo aey eoe and removed iately

prior to prior

the - metabolic monitoring systems systems monitoring metabolic HCL) at 4° at t 100,000xg, the the 100,000xg, t collection of data over 2 over data of collection . pellet contains the crude the contains pellet C - successively successively exchange data. Activity Activity data. exchange The liver was minced, minced, was liver The , and , , Houston, TX, USA TX, Houston, , were peitr i an in predictors s

spun at 740g for for 740g at spun

, centrifuged sacrificed by sacrificed Mice washed resulting resulting

- HCL, were were [87] The

at at .5 in in ). ). - . ,

This article isThis article by protected copyright. All rightsreserved. UK), 2 (mAb Danvers, USA), MA, Technology, Signaling Cell (C50B12; BiP UK), Anti Cambridge, Milton, 100v. Abcam, at 1h were for Samples membrane 150v. at PVDF 1h onto for transferred gel polyacrylamide 10% a on run was lysate protein of 40ug immunoreactivities, relative of comparison For USA). NanoDr MA, Waltham, Scientific; a on A280 by determined was concentration Protein fraction. insoluble the remove to min 3 for 20,000xg at centrifuged Sigma 1mM; PMSF 0.79 Benzamide 100uM, using disruption disruption tissue mechanical or fat any and whole The dislocation. cervical 22 or 12 Western Blotting USA.MO, u Beckman a in fraction MAM at centrifuged was supernatant MAM The collected. min 10 for 6,300xg at centrifuged MAM (SW rotor swinging b done was media gently was pellet washed The mitochondria crude the containing cytoplasm the is supernatant Accepted Articleltra

high ) asqeti (3516; calsequestrin

.

- and lower band band lower and h fatoain f h cue iohnra no pure into mitochondria crude the of fractionation The 4; as published in in published as 4; - - 25 month old male mice were anesthetized using avertin and sacrificed by by sacrificed and avertin using anesthetized were mice male old month 25 speed

y centrifuging the the centrifuging y β

cyto (rotor 50.2 Beckman 50.2 (rotor

JS -

oncie ise a rmvd Poen a then was Protein removed. was tissue connective 1%SDS buffer in 1xPBS with a cocktail of protease inhibitors (Aprotinin inhibitors protease of cocktail a with 1xPBS in buffer 1%SDS ci AC Actin centrifuge (over 20,000xg). (over centrifuge

- HS 32 - Aldrich, St. Louis, MO, USA). Lysate was boiled for 5 min and and min 5 for boiled was Lysate USA). MO, Louis, St. Aldrich,

Beckman h igh gm, E mg/mL,

- 5 A41 Sigma (A5441; 15 ba, itn Cmrde UK Cambridge, Milton, Abcam, ( - [37] ue mitochondria pure speed percoll layered percoll c fraction ic ), Cytochrome ), - i hmgnzto in homogenization via q Coulter ooeie i bfe ad l and buffer in homogenized uadriceps femoris uadriceps

. The pellet from the pure mitochondrial sample was sample mitochondrial pure the from pellet The . - etiue 2,0x ad ne) r Beckman or under) and (20,000xg centrifuge was washed and spun at 10,000xg for 10min twice. 10min for 10,000xg at spun and washed was Coulter, Brea, CA, USA) CA, Brea, Coulter, - 64 10nM, Leupeptin 10uM, Pepstatin 0.1mg/mL, 0.1mg/mL, Pepstatin 10uM, Leupeptin 10nM, 64 , Brea, CA, USA). The resulting upper band upper resulting The USA). CA, Brea, ,

n te elt s h ER the is pellet the and p ND op

All reagents from Sigma from reagents All suspension at 95,000xg for 30min using a a using 30min for 95,000xg at suspension

- Aldrich, C (ab13575; Abcam, Milton, Cambridge, Milton, Abcam, (ab13575; C )

ee ah olce, ahd and washed, collected, each were - 1000 Spectrometer (Thermo Fisher Fisher (Thermo Spectrometer 1000

100,000xg for 1h, the pellet the 1h, for 100,000xg muscle was muscle t Lus M, USA MO, Louis, St.

liquid nitrogen and chemical chemical and nitrogen liquid ois to bodies , i1 (HPA017430 Fis1 ), . All spins were done at 4°C 4°C at done were spins All yrd n o o percoll of top on ayered

mitochondria and MAM MAM and mitochondria immediately dissected immediately

fraction

- ATP5A (ab14748; (ab14748; ATP5A xrce through extracted Aldrich, St. Louis, Louis, St. Aldrich, ) , γ ), . The pellet pellet The . Sigma ; cyto was

- L8 Actin Actin

(

the the the - M -

This article isThis article by protected copyright. All rightsreserved. Acceptedtime Maximal plateau a reached muscle the until contraction tetanic isometric minutes, two Every contraction. tetanic isometric an by later 30s followed later 30s elicited was twitch second A V. 150 at pulse S48 (Grass USA). RI, 150Hz at 200ms for muscle Warwick, Telefactor, Grass unit; isolation stimulus SIU5D the a through delivered stimulator stimulating by plateaued testin until min for 2 protocol a beginning before (P min contractions tetanic isometric Maximal 5 function. contractile for bath the in quiescent remained myotend (L length ˚C. 25 O at maintained was % that buffer (95 oxygenated with filled and assembly bath mL (300B system 22 and 12 from removed Diamondback USA). BW; AZ, mg/kg Socttsdale, (100 Drugs, pentobarbital sodium with anesthetized were Mice ArticleIn Vitro Contraction concentrations.MembranesLicor a wereimaged on UK), Cambridge, Milton, Abcam, (ab110252; UQCR USA) CA, Diego, UK), Cambridge, Milton, Cambridge, Milton, Abcam, Abcam, (ab14715; SDHA USA), MA, Danvers, Technology, Signaling Cell (D11A8; PERK USA), MA, Danvers, Technology, Cell (C81H6; PDI USA), MA, Danvers, Technology, Signaling Cell (14C10; (Sigma GAPDH UK), Cambridge, Milton, Abcam, (D2D10; MFN2 Aldrich,

trace t Lus M, USA MO, Louis, St. i os ucin o myotend to junction nous rates of rates o s bsd n etn tnin Msl lnt ws hn esrd from measured then was length Muscle tension. resting on based ) . P

w mnts f et ee olwd y ftge rtcl of protocol fatigue a by followed were rest of minutes Two a tic fre was force twitch eak - LR; Aurora Scientific Inc., Aurora, ON, Aurora, Inc., Scientific Aurora LR;

- Aldrich, el inln Tcnlg, avr, A USA MA, Danvers, Technology, Signaling Cell contraction and relaxation were derived from the slope the from derived were relaxation and contraction T ,

bln 52 T04 Sigma (T6074; B512 ubulin St. Louis, MO, USA MO, Louis, St. - 25 month old mice and mounted and mice old month 25 UK), , P1 620; D isine, a Js, A USA), CA, Jose, San Biosciences, BD (612606; OPA1 ),

sAmk1 (ARP42566 sAmk1 d xesr iioim Longus Digitorium Extensor esmin (D1033; Sigma (D1033; esmin measured i os ucin sn dgtl aies Muscles calipers. digital using junction nous Muscles were adjusted to adjusted were Muscles f force of ), IP3R ),

y tmltn te muscl the stimulating by tre contractions (three - Aldrich, -

3 (610312; BD Biosciences), Biosciences), BD (610312; 3

- Odyssey (Lincoln,NE,USA). T100; Aviva Systems Biology, San San Biology, Systems Aviva T100; were used at the recommendation the at used were

Canada) with 5 with Canada) - Aldrich, 2 on a dual a on Kes igr bicarbonate Ringer Krebs ) t Lus M, USA MO, Louis, St. o ) were performed every performed were )

their anatomic optimal optimal anatomic their ), (EDL) St. Louis, MO, USA MO, Louis, St. - AL (ab155282 FACL4 SERCA1 (ab2819; (ab2819; SERCA1

mode muscle lever lever muscle mode s were performed were s ihn 0. within - 0 suture in a 1.5 1.5 a in suture 0 wt a 0.5 a with e

muscles were were muscles s

of the force the of submaximal submaximal Signaling Signaling 8 mN 98

) IRE1α , and and , - ms ms ), ). g - ;

This article isThis article by protected copyright. All rightsreserved. Plus (Molec microplate spectrophotometer skeletalvesicles SRconductedpriorto adding inSpectraMax was MgATPa andread room temperatureat assaywiththe compound/DMSO, mix and mentioned above, each keeptheto wellto fina usedfinal inthe assaywells. and wasdissolvedto Thapsigargan adjusted 100the in times DMSO concentration Inhibition volume 200μL. total to (MolecularSunnyvale,Devices, CA microplate spectrophotometer (MgATP)5mMandtriphosphate concentration atafinal of readinaSpectraMax Plus eachThe well.assayw [Ca2+] free to the desiredvalues. lactateIU/mL calciumdehydrogenase, ionophoreA23187, set andCaCl2 addedto 7uM EGTA,0.2 m 96 NADHcoupled, SR muscle werepurifiedprocedure fractions from the outlinedabove. using SERCA Activity monitored byisometric was recovery Fatigue 5min. over 200ms for 7.5s every contractions isometric (60Hz)

Accepted Article- well micro of SERCAof activitywasevaluatedmouseskeletal SR in vesicles. M NADH,10M pyruvate1 mM phosphoenol pyruvate, IU/mL 10 kinase, - plates. - linked ATPase wasusedmeasurelinked assay to SERCAATPaseactivity in

Each MOPS100 KCl, wellcontainedmM (pH7.0), mM 50 1 mM

as started upon the additionadenosine uponthe magnesiumas started of tetanic

l [DMSO] at1%(v/v)during assay. l [DMSO] the

Then,either2uL compound added of DMSO onlywas or contraction

Then,1.0 μg mouseskeletalvesicles usedin SR was s ularDevices,CA Sunnyvale,

(200ms 150 at Hz)

every 5min for 30min. , USA ,

A 20minincubation , USA, ), bringing), the

An enzyme ).

- This article isThis article by protected copyright. All rightsreserved. Accepted James M. Ervasti DawnLowe A. Espen E.Spangenberg David ( Thomas D. (Conceptionand Editing) design, ( Nelson D’anna Preston McCourt( Samantha Yuen( MichaelTarpey D. Angus Lindsey Data, Figure Preparation of Acquisition design, and (Conception O’Rourke R. Allison ArticleAuthor Contributions: AR057220 E. a sources: funding Our analysisdesignSERCA of and activitydata. of University the at Core Physiology and Biology Integrated the at Bartolomucci Alessandro Minnesota and Razzoli Maria of University the preparation. at Facility Characterization The Acknowledgements: E. Spangenburg, a Pilot and Feasibility grant to B.J. to grant Feasibility and Pilot a Spangenburg, E. , and a NIH training grant AG029796 D.M. andaNIHtraining grant to Nelson ,

(Conception and design, Editing, Supervision)design,(Conception andEditing, (ConceptionAcquisition ofData,Editing) anddesign, Conception and design, Acquisition of Data, Editing Data, of Acquisition design, and Conception

Conception and design, Acquisition of Data, Editing Conception design, of and Acquisition (Conception design, ofManuscript, Editing, Drafting and Supervision)

Conceptiondesign, and , Conception design, and Editing,Supervision (Conception design,Data, Editing)of Acquisition and Drafting ofManuscript

(Conception anddesign, NIH grant AR049899 to J.M. Ervasti, a NIH grant AR066660 to to AR066660 grant NIH a Ervasti, J.M. to AR049899 grant NIH Minnesota for whole body respirometry. body whole for Minnesota

, Editing, Acquisition of Data, Editing Acquisition of

Editing, Supervision) Editing, )

Perrin

awarded through NIH P30 NIH through awarded . )

Ji Li for experimental for Li Ji

) )

Benjamin J. Perrin J. Benjamin o TM sample TEM for )

Analysis

f Data of

,

This article isThis article by protected copyright. All rightsreserved. Accepted9 CraigSW&PardoJ actin,V (1983) Gamma , andintermediate filament PJ (2009)8 Duan De R&Gallagher Nahirney7 Nowak SJ, PC,Hadjantonakis A 6 PeckhamM (2008) Engineering amulti & G 5 Lloyd C,Schevzov comprehensiveK &LessardJ(1991)A 4 McHugh K,Crawford the analysis of Article3 Schwart Flick2 CrawfordK, R,CloseA&LessardJ(2002) R,BoveK,Kumar Shelly L,D,Paul A,1 Kumar CrawfordK,CloseLorenz MadisonDoetschman Pawlowski L, J, T, M, S, Bibliography: sites. costameres,myofibril vinculinproteins colocalizewith at and myosinIIA. nonmuscle 3282 mammalian myoblastfusion. actin remodelingfor isessential cytoskeleton. endogenous .from actin gamma rat. developmental andtissue the chickenactinmultigene family. expressionof diethe neonataldeficitsand duringperiod. Mice lacking skeletal muscle actin showreducedmusclestrength and growth Proc.Natl.Acad.Sci.U.S.A Rescuecardiac a of Duffy J,Neumann Robbins J, Boivin&LessardJL(1997) BA J, O’Toole GP, Dev. Biol. – z R & Rothblum K (1981)Geneswitchingdifferentialz R&Rothbluminmyogenesis: Cell Motil. 3293. - actin myoblastselicitsfeedback regulatory responses genesinto different

J. Microsc. 148

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This article isThis article by protected copyright. All rightsreserved. Accepted19 VlahovichN,KeeAJ, VanderPoelKettleHernandez C,E, 18 Almenar Davis17 AyalonJQ, G, Scotland PB& 16 NakataT, NishinaY&YorifujiH(2001)Cytoplasmicactin gamma Z asa LeinonenKaakinen H,KaistoT,&Metsikkö15 Papponen S,M K(2009)Evidencefor 14 GokhinDS,McKeownLewisR a, CR,NowakLieber RB,KimNE,Littlefield RL RS, Article13 KeeSchevzov Nair AJ,G, Call a,12 PrinsKW, J(2011)Quadriceps LoweD& ErvastiJM amyopathycausedby Patel11 Rybakova IN, JR&E CA,10 Otey & Bulinski KalnoskiM JC(1988)Immunolocalizationmuscleand of J. Biol.J. Chem. capsactinskeletal tropomodulin,that filament functionalfor organizationdystrophin of . and protein. 218 gamma skeletalcapping muscle and physiology. & Fowle results inmusculardystrophy. novelintropomyosin toacytoskeletalskeletalmuscle nonmuscle compartment LinMR, JJ skeletal muscle Biol. andcostameric stronglinkmechanically betweenthesarcolemma actin. Cytoskeleton ofactininmyogenicisoforms nonmuscleand adultskeletal cells muscle. –

25. 150 - - Queralt A & Lee A (1999) Identification of a novel tropomodulin isoform, A Queralt & LeeA(1999)Identificationnovel a tropomodulin of isoform, r VM (2010) Tropomodulin filamentpointedVM isoformsregulate r thin Biochem.Biophys.Res. Commun. actina Zdisccomponentin as sk , 1209 , - C, WeinbergerR,Gunning PW&

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This article isThis article by protected copyright. All rightsreserved. Accepted26 ManorU,Bartholomew S, Golani ChristensonKozlovHiggs G, E, & H,Spudich M, J Ramabhadran25 KorobovaF,V&Higgs (2013) Anactin HN 24 SonnemannFitzsimons KJ,DP,PatelJR,Liu Y,Schneider MossRL&Ervasti MF, AK,23 DomenighettiAA,ChuPH,SheikhPeter F,GokhinLT,CuiWu T, DS,Guo Z, ArticleLi Miao J,ZhangWu D,ZhangYuan22 WangL,Z, L, Z&Sun Y,Peng K(2013) 21 (2011)20 GokhinDS VM Cytoplasmic &Fowler gamma VassilopoulosGentil C,Lainé Buclez S,J, P 464 mitochondrial th mediated fission by 97. itsdevelopment butaprogressive absenceto myopathy. leads (200 JM Genet. withassociatedmyofibrillar disorganization andintermyofibrillar in mice. (2014) L SheltonV., LieberJ FA,Seidman CE,GomesA Seidman GD, RL&Chen JG, DC,Parfenov Christodoulou JM,LiDY,Banerjee MG, Gorham I,LaiWitzmann X, IIB by analysisprotein of an FHL1of proteincomplexcontainingIdentification gamma sarcomere clathrin(2014) Actinscaffoldingskeletal byheavychain for isrequired muscle GarciaR, HeuserJE,BrodskyFM, G,VoitT, Piétri Bonne L, 194 linkisoforms to the sarcoplasmicin reticulum skeletalmuscle fibers. skeletal muscle. normtropomyosin Tm5NM1isrequiredfor Lynch GS,Parton& Hardeman RG,Gunning EC(2009)Cytoskeletal PW

, 105 , – 7.

23 6) Cytoplasmic gamma oss of FHL1oss of induces anage – , 209 20. organization.

– 225. Mol. Biol. Cell

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20 - actinrequired isnot skeletal muscle for - protein interactions. protein interactions. , 400 , e ER

- 205 dependentskeletal musclemyopathy – 9. , 377 , - associated INF2. formin

al excitation - O, Franck A, Ferry A, Précigout FranckFerry O, A, G, Roth – 93.

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8 , e79551., Science J. CellJ. Biol. Hum.Mol.

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– This article isThis article by protected copyright. All rightsreserved. Accepted AgnolettoBononi34 Patergnani De JM, C, E,Giorgi M, S,Suski Marchi C, A,Bonora 33 RaturiA&Simmen T(2013) BiochimicaBiophysicathe endoplasmic et Acta Where 32 BritoOMDe&Scorra Pinton R&DuszynJ(2006)Overexpression31 WieckowskiMR, P,Rizzuto adenine of Carrington FayFS,30 RizzutoPinton W, FogartyKE, R,Tuft P, LifshitzLM, RA & ArticleWittigL, 29 WinterI,PeevaEggersHeidler V,B,ChevessierJ, Kley F, K, RA,Barkovits Mavroidis M,Weisleder 28 MilnerN&CapetanakiY (2000)Desmincytoskeleton DJ, 27 VlahovichN,KeeAJ,Poel KettleHernandez CVanDer, E, Marchi S,MissiroliPoletti & Pinton S,F,Rimessi A,Duszynski Wieckowski MR P J, membran reticulumand mitochondrion knot : the the Themitochondria tie mitochondria. mitochondria. 2+ signalbetween ERnucleotidereduces Ca the translocasetransmission and Mitochondrial Responses. of Ca2+ Pozzan Endoplasmic Contactswiththe Determinants T(1998)Reticulum Close as in skeletal mitochondrial functiondesmin substantially , andmaintenance morphology perturbs V,HerrmannStrecker C, Berwanger H,Marcus K&KornblumC (2016)Mutant 150 linked muscle to respiratoryfunction. mitochondrialdistributionand Skeletal Tropomyosin Required Normal Tm5NM1Is for Excitation Lynch GS,Parton& Hardeman RG,Gunning EC(2009)Cytoskeletal PW nucleating spire mitochondrial proteinregulates d Lippincott , 1283,

Muscle. Muscle. e MAM( ) - – schwartz J (2015)schwartz J A mitochondria muscle tissue. 1297. Nature Biochem.Res.Commun. Biophys.

20 , 400 , ☆ no L(2008)Mitofusin tethers 2 endoplasmicto reticulum

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This article isThis article by protected copyright. All rightsreserved. 42 41 DeVos AllanGrierson KJ,MP and AJ&Sheetz (2005) Mitochondrial VJ, function 40 Westermann B(2010)Mitochondri 39 Krisans SK& KimJ, 38 LewinVance TM, Granger DA, JE& BoganKornegay MayerU,KaufmanSJ, 37 HanftJN&E LM, DJ, X,O’Rourke MoriarityBS,36 Patrinostro AR,ChamberlainPerrin CM, JM BJ&Ervasti DuszynskiGiorgi J&Pinton35 WieckowskiMR, C,LebiedzinskaM, P(2009)Isolation

Accepted Article PflugerKabra Aichler PT, DG, M,SchrieverVC,SC,Pfuhlmann García Lehti K, M, M, DeLucaM,Molke M, Angelis WalchRollmann Aronow M, A, Perez SM, BJ, Müller TD, KutschkeWeber J, Rozman Elrod M, J, Feuchtinger JW,Hevener AL, Hrabě De A, Biol. actin regulate Publ. Gr. mitochondrial membrane inprotein located twodistinctsubcellularorganelles,peroxisomes, and 24679. inLiverand RatInhibited CanBe Independently *. 1 Isoforms4Synthetase and5Subcellular , , ArePresentinDifferentMembranes dystrophy. Cytoplasmic gamma fibroblasts. βcyto (2017) Relativeimportanceof cells. mitochondriaof Signal.Cell Commun. Mitochond (2011) Calciumsignalingaround

15 Nat. Protoc.

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11 Neuromuscul. Disord.Neuromuscul. Mol. Biol.Mol. Cell

, 872 , Coleman RA (2002) Rat liver RA(2002)Ratacyl Coleman – - 83. associated membrane. - associated membranes and mitochondria from animal andmitochondriaassociated and membranes from tissues

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and γ cyto Arch. Biochem.Arch. Biophys. - dependent mitochondrial fission. –

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276 Tilve D, Jastroch Tilve D,Jastroch , 263 , - CoA , 24674, – 270. Curr. Nat. –

- This article isThis article by protected copyright. All rightsreserved. AcceptedPF, Muriel51 DesmondMarkwardt J,ML,Rizzo & MA Bloch RJ (2015)Identification of 50 OdermattA,Khanna TaschnerP, Busch JableckiBreuning V, H,Karpati G, C, & M &StephensonLamb GD theexcitation49 Stephensonof DG, GMM (1998)Events (1968) Calcium48 EbashiM S&Endocontraction. and ion muscle 47 Brown &HasserEM(1996)Complexity age M of Article SA, Detmer 46 ChenGriffin Ewald EE, H, AJ, SE Fraser DC &Chan (2000)Mitofusins & Chan45 Chen(2005)Emergin DC H (2006) The44 Anesti&Scorrano L relationshipbetweenmitochondrial V and shape Maddalena43 Valente AJ,MoradiJA(2017)Robb EL, LA,F&Stuartsimple A ImageJ (SERCA1)regulatoryprotein inskeletal muscle. smalla novel ankyrin1as Ca2+ reticulum sarco(endo)plasmic disease. skeletal musclesarcoplasmic ATPase, reticulumCa2+ associated are withBrody MacLennan D(1996)Mutationsinthegene musclefibres relevant to fatigue. contraction B Gerontol.A. Biol.Sci.J. Sci.Med. embryonic dev and Mfn1 coordinatelyMfn2 for essentialmitochondrial fusion regulate andare and fission. function andthecytoskeleton. culture. toolmacro foranalyzing mitochondrial morpholo network Elongationwith Metabolism.Energy iol.

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g functions of mammalian of g functions mitochondrialfusion 51 326. J.Cell Biol. Cell Metab. , B117,

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relatedin muscle. change skeletal 160 J. Biol.J. Chem. 22 , 838 , , 189 , - twitch mammalian muscle twitch mammalian muscle

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This article isThis article by protected copyright. All rightsreserved. & G 61 Csordás 60 EisnerCsordas &Hajnoczky (2013) V,G G Interactions betwee Jain Mootha RenS,ClishC,M, 59 ChenV, McCafferyJM&Chan H, DC (2015) mitochondrialDC 58 MishraP&Chan (2016)Metabolicregulationof dynamics. 5 RybakovaPatel IN, 56 Hanft LM, JR,Rafael M(2008)Endoplasmic reticulum55 Schröder stress responses. 54 NiwaM & P Walter decide. (2000) Pausing to 53 MaY& 52 Liang S Accepted (2014)7 EisnerLenaers&HajnóczkyG Mitochondrialisfrequentin V,G fusion Article Calciumand ROS. and reactiveoxygenspecies signaling. reticulumand mitochondria incardiacandskeletal muscle 211 Titration ofmitochondrial Mff fusion rescues Bi 195. skeletal excitation muscleand supports dystrophin Cytoplasmic gamma 862 12396 Cell Biochem.J. immediate expressionof kinase)the stress isrequiredtoactivate ol. , 795 , –

107 212 894. – - Hendershot LM (2001)HendershotThe unfoldingtalethe of unfoldedproteinresponse. H, ZhangW,McGrathDR P&Cavener (2006)PERK BC,Zhang (eIF2alpha 12397. , 827 , , 379 , –

805. - deficient muscle.

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- early genesERhomoeostasis. upondisruptionof calcium Proc. Natl. Acad.Sci.U. S.Proc. Natl. A. - J. CellJ. Sci. - - - contraction coupling. Fortney J a & Ervasti JM (2006) Fortney Ja&ErvastiJM mitochondrial communication: local activated MAPKsandthe induce - -

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– - 90. endoplasmic endoplasmic J.Biol. Cell

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– This article isThis article by protected copyright. All rightsreserved. AcceptedCollins70 KornmannB,Currie E,Nunnari SR,Schuldiner M, Weissman J, JS&Walter 69 Grimm S (2012)TheER 68 Gilady SY,BuiLynes Benson M, EM, WattsR, MD, VanceJE &Simmen T(20 67 BurtéF,CarelliChinneryPF & V,Yu Carrington FayFS,66 RizzutoPinton W, FogartyKE, R,Tuft P, LifshitzLM, RA, Article65 ErmakG& 64 Chami OulèsSzabadkaiTa M, B,G, Csordás &63 SpätA, Szanda(2008) G G, HajnóczkyG High 62 GlancyB,HartnellMalide LM, D,YuZ Screen. P (2009)AnER Biochim.Biophys. Acta membrane associated(MAM). andnormoxicEro1α requiresoxidizing localize conditions to mitochondria the to disorders.dynamics andneurodegenerative Ca2+ responses. withClose the asdeterminants reticulum contacts endoplasmic of mitochondrial BrownNunnari M, Montero MacDonaldRizzuto MJ, J, LJ, R&KassGEN (1998) Fay FS,RM, PozzanHeim JouavilleLS, T, RizzutoR, TsienCarrington WA, RY, PozzanLawrie T,RizzutoR, Pozzan AM, Rutter T,Simpson Dr AWM, GA, death. Mitochondria ERto during Stress. RoleProapoptotic SERCA1 Isoforminthe of Truncated CalciumfromTransfer ER 63. mitochondrial ofsignalling.dependentmechanisms calcium muscle. & Balaban RS (2015)Mitochondrialf reticulum

Mol Immunol Nature Science (80

Davies KJ(2002)Calcium fromandcell stress: oxidativesignalingcell to

- 523 Mitochondria Complex Tethering byaSyntheticBiology Revealed Science (80 , 617,

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44 - ummond , 51 , 10) – - This article isThis article by protected copyright. All rightsreserved. AcceptedSaha79 ZhangS,Shabalina &Kashina SA F, Differential A(2010) Arginylation of multiple78 RubensteinThefunctional of PA(1990) importance actinisoforms. P (1993)Noncoding& Gunning77 Lloydregionsthe gamma C of Reiken DC,MJ, S,Meli76 Andersson Betzenhauser AC,UmanskayaA, AA,Bossy75 KushnarevaYE,Gerencser B,Ju Article74 SingaraveluNelson C,BakowskiBritoOM,Ng K, D,De Chen73 Dayanithi G, AE,72 RusiñolCui Z,ChenMH& Mongillo71 Li G, Chin M, Sequence BioEssays genethe impactofmorphology. onmyoblast and calciumintracellular leak in muscle weakness aging. T, ZalkR,Lacampagne A&MarksARoxidation (2011)Ryanodine receptor causes excitotoxicity.and for homeostasis sensitizes Perkins &BossyMH, G Chem. Ca2+ to the plasmamembranewithdepolarized store in mitochondria. cells migrationScorrano& Parekhtheregulates L AB(2011)Mitofusin2from STIM1 of involvement OPA1 inCa2+ clearance. Characterization Ca2+signalling of inpostnatal mouseretinal ganglioncells: 27494 pre membrane fromrat liverfraction has ahighcapacityand lipid for contains synthesis endoplasmic reticulum stress ERO1 - Golgi secretory proteinsGolgi lipoproteins. secretory nascent including - –

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induced apoptosis. Vance JE (1994)Auniquemitochondria Vance JE Science (80 Ophthalmic Genet. W - Cell Differ. Death - triphosphateactivity in receptor K, White AD,Waggoner Ellisman J,

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This article isThis article by protected copyright. All rightsreserved. Accepted 87 Tschöp SpeakmanJR,Arch MH, JRS,Auwerx Brüning Eckel J, L, JC, Chan RH, 86 LarkDS,Torres MJ, LinC CGR, KanePD 85 Perry DA,LanzaIR&Neufer (2013)Methods forassessing & 84 BunnellTM Article83 Schindelin Arganda J, RoblotP, TizianoD,Frugier82 MiniouN,Meur T, MLe& JM(2010)81 PerrinBJ,Sonnemann KJ&Ervasti β ThiemSM, SE,Zhu 80 Bergeron M, Friderici(2010) Ion KH&RubensteinPA analysis of mouse energy metabolism.analysis ofmouse energy Tho Rahmouni GI, K,Shulman SC,Maratos Farese RV,GalganiHamblyHerman Horvath JE,C,a, M TL, Kahn BB,Kozma C24 muscle myofibers. skeletalpermeabilized quantificationtime mitochondrial phosphorylation of efficiency oxidative in mitochondrialin diabetes. function survival ingrowth and mice. Actg1null biological V, EliceiriTomancakan open P&Cardona A(2012)Fiji: K, SaalfeldPreibischSchmidS, RuedenTinevez B, C, J S, tomousestriatedmusclelineage. restricted maintenance. haircell auditoryrequiredstereociliadispensable development for but actin isoforms. mammalian betweenα dependentpolymerization differences 5.

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- T, Ryan TE,Anderson real EJ&NeuferPD (2016)Direct Nat. Methods

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, C239 , – - This article isThis article by protected copyright. All rightsreserved. post Tukey ANOVA with # and Actb Table1: T maximal tetanic isometric maximal Denotes comparison to Actg1 comparison Denotes Maximal ContractionMaximal Rate Force Development Time 1/2 Relaxation Time (ms) RelaxationMaximal Rate Accepted ForceMaximal Recovery Article after Fatigueafter (% of initial max. tetanic iso.max. force) Specific Force (N/cm2) TetanicMaximal Force Fatigue (% of initial - msKO EDLmuscles. etanic isometric contraction control,etanic isometric measurements Actg1 in Genotype force) (mN) (ms) (N/s) (N/s) Age - test. test. Actg1-msCT 491.3 ±11.3 491.3 149.0 ±13 149.0 93.0 ±2.1 93.0 ±2.3 60.8 ±0.7 23.0 ±1.0 17.0 ±1.0 21.5 86 ±12 86

# - or * is 0.01

Actg1-msKO 407.5 ±17.2 407.5 16.2 ±1.0 16.2 90.8 ±4.1 90.8 ±2.1 64.7 ±0.5 13.7 ±0.9 18.0 113 ±3 113 120 ±4 120 # 12mo # Actb-msCT 490.6 ±8.8 490.6 93.7 ±1.4 93.7 ±2.3 60.8 ±0.8 23.1 ±0.7 15.8 ±0.4 22.5 144 ±6 144 87 ±6 87

442.8 ±21.6 442.8 Actb-msKO 17.9 ±1.5* 17.9 92.5 ±1.8 92.5 ±1.8 64.7 ±0.7 14.9 ±0.7 21.0 144 ±8 144 ±8 100 413.8 ±31.3 413.8 Actb-msCT 15.4 ±2.1** 15.4 91.5 ±2.7 91.5 ±2.7 58.3 ±0.6 15.1 ±1.1 20.8 157 ±8 157 86 ± 7 ± 86 - msCT. 1 - msKO, 22-25 mo 22-25

14.6 ±1.2*** 14.6 420.5 ±22.4 420.5 Actb-msKO - way 95.7 ±2.4 95.7 ±2.5 64.6 ±0.8 14.6 ±1.5 21.4 165 ±10 165 86 ±8 86

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. S.E.M. ** significance. determine to performed were test hoc ( cristae. denote heads arrow white structure, internal mitochondria 22 (J SR. 1µm a white SR, normal denote arrowheads White Actg1 Actb and Actg1 sarcoplasmic Altered 1: Figure Q - Accepted Articleratio. length:width mitochondrial of Quantification ) 25 months of age. age. of months 25 .

- ( - msKO, and Actb and msKO,

P L) Transverse sections of TA from control, Actg1 control, from TA of sections Transverse L) Qatfcto o te ecnae f mgs xmnd ht display that examined images of percentage the of Quantification )

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reticulum and mitochondrial morphology in aged aged in morphology mitochondrial and reticulum

(A - - C), 12 (D 12 C), ) Longit I) .

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1 Hg mgiiain mg of image magnification High ) is 0.01

msKO mice at mice msKO cl br = bars Scale ed dilated dilated ed m .

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article way determined nosignificant ANOVA difference across disk alignmentadjacentpairs of and22 sarcomere at6months (A) Figure 2:No inZ shift - disk Alignmentin Actg1 muscle. andActbKOskeletal

genotypes. - 25 months (B). 25 months

Z 2 - - This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article DAPI inblue. isolatedEDLRepresentative musclestained desmin fibersgreenand for localization in hoc testdet ANOVATukey’s withpost normalized to GAPDHand average the relative to ofthesamples. control and Actg1 skeletal muscle Figure 3:Unalteredabundance desmin localization in and - msKO andmsKO Actb Scale bars = Scale bars .

(A - B) Western blotagainst desmininquadricepscontrol muscle from - 20

msKO animalswesternblot, quantificationof and µm .

ermined nosignificant difference.

Actg1 andActbKO

(C 1 - - E) way This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article f membranes associated mitochondrial isolated of preparation the during obtained fractions the from membrane Figure 4 : Enrichment of γ of Enrichment : . hw ae dnia wsen lt lae wt eul mut o protein of amounts equal with loaded blots western identical are Shown rom mouse stained liverto and withantibodies theindicatedprotein

cyto -

and β and cyto - actin in isolated mitochondrial associated associated mitochondrial isolated in actin

.

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article and KO, Actb KO, significance Actg1 CT, MEFs. in dKO frequencies Actb/Actg1 fusion and fission of network (branched) Quantitation a of network off of number branch mean of each The of (K) number mitochondria. length mean The The (J) (I) genotype. per genotype. mitochondria per mitochondria branched) each of area (G) 10µm. = bar Scale (CT). control to compared MEFs dKO Actb/Actg1 and fission decreased and Figure 5 : bain f γ of Ablation * is 0.01

mitochondrion per genotype. genotype. per mitochondrion (A . cyto

1 - - -

) iohnra per lnae i At1 O cb KO, Actb KO Actg1 in elongated appear Mitochondria F) a AOA ih Tu with ANOVA way r β or cyto - actin results in increased mitochondrial area area mitochondrial increased in results actin rnhs n ntok iohnra (L mitochondria. network a on branches

key’s post hoc test determined determined test hoc post key’s (H) The number of individual (non individual of number The (H) re S.E.M.

Surface - M) - . This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article difference. ( OPA1 muscles. (A Fis1 for average control to relative skeletal abundance of quantification KO Actb and Actg1 from isolated mitochondrial in proteins dynamics mitochondrial of abundance Unaltered 6: Figure E - F).

1 - way ANOVA with Tukey’s post hoc test determined no significant significant no determined test hoc post Tukey’s with ANOVA way

- B), MFN2 (C MFN2 B), etr bos and blots Western - D), and D), This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article hoc testTukey’s determinednosignificantdifferen post Actg1 to relative and GAPDH to normalized immunoreactivity protein, V complex a ATP5A, of Quantification (D) mean. GA to normalized immunoreactivity Actb or Actg1 to relative and GAPDH to normalized immunoreactivity protein, II complex muscle. to immunoreactivity skeletal KO Actb and Actg1 Figure 7 - : Unaltered abundance of electron transport chain complex proteins in in proteins complex chain transport electron of abundance Unaltered : sT en () uniiain f QR a compl a UQCR, of Quantification (C) mean. msCT

proteins from complex II, III, and V. (B) Quantification of SDHA, a SDHA, of Quantification (B) V. and III, II, complex from proteins

- sT r Actb or msCT D ad eaie o Actg1 to relative and PDH (A) Representative western blot showing showing blot western Representative (A) -

sT mean. msCT ce.

-

sT r Actb or msCT 1 - way ANOVA with with ANOVA way x I protein, III ex - - msCT msCT msCT This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. 1 CI JO of ratio the is Contr Succinate (JO of ratio I Complex the (F) is TMPD. and control JO acid, ascorbic rate, A, respiration antimycin of IV presence Complex the in (E) measured rotenone. of presence the in measured JO (CII), rate respiration II Complex JO (D) (CI+II), succinate. of rate presence the respiration in measured I+II Complex (C) 3). (state ADP of presence the in malate, JO leak), (CI leak I Complex (A) fibers. muscle. Figure -

Accepted Articleway determineddifference. ANOVAhoc test nosignificant withTukey’spost -

JO 2 8 and glutamate (state 2). (B) 2). (state glutamate and

CI leak)/JO CI Mtcodil ucin s nfetd n cg ad cb O skeletal KO Actb and Actg1 in unaffected is function Mitochondrial : (A - J) Oxygen consumption parameters measured in isolated gastrocnemius gastrocnemius isolated in measured parameters consumption Oxygen J) ol (JO ol 2 CI/JO 2 CI. (J) E (J) CI. 2 CI+II 2 CI leak. (I) Biochemical coupling efficiency is the ratio of (JO of ratio the is efficiency coupling Biochemical (I) leak. CI

2 -

JO - Complex I+II I+II Complex

supercomplex is the ratio of JO of ratio the is supercomplex 2 CI)/JO

Complex I respiration rate (CI), JO (CI), rate respiration I Complex 2 2 CI+II. (H) RCR is the respiratory control ratio it it ratio control respiratory the is RCR (H) CI+II. was measured in the presence of pyruvate, pyruvate, of presence the in measured was -

JO 2

ope II)/JO Complex

2 CI+II/ (JO CI+II/ 2

ope II. (G) I+II. Complex 2 was measured measured was 2 CI + JO + CI

2 2 2 2 CII). was was was was 2 This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article study. O of exchange ratio O (A) ActbKO mice. Figure 2

c 9 onsumption.(B) CO : Unaffected body 22 in whole respiration (A - E)Whole bodymitochondrialof22 respirometry 2 to CO 2

. (E) (F)Body. Ambulation. mice usedcomposition of in 2

consumption.(C) Heatreleased. (D) Respiratory

- 25 monthold muscle - 25 month old25 month mice. - specific specific This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article inhibitorsare present succinateincrease induced in reductase inhibitorBCNU (B) Theindexisthepercent (Carmustine). scavenger presence (AF/BCNU)thioredoxin of AF (auranofin)andglutathione reductaseinhibitor Succinate Figure 10 : Unaltered H stimulated H .

2 O 2 O 2

2

production

Production in Act H 2 O 2

production relativeto

in oldanimals 12 month g1

and Act

H b 2

O KO skeletal muscle.KO skeletal 2

in absenceor the production when

(A) This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article are S.E.M. significance. relaxation. generated duringmaximalisometrictetaniccontractions.(D) Maximal of rates old Actg1 control, isometricRepresentative contraction tracings force tetanically of Figure 11 : Impairedrelaxationrates in

1 - way wereperformed ANOVAhoc test determine to withTukey’spost #

oris 0.01

- msKO(B Act g1

) EDL) muscles.Peak (C) specificforces and

Act b d *** isbarsd p<0.001. Error

KO skeletal muscle.KO skeletal - stimulated 12 month month stimulated 12

(A - B) This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article significantdifference. relative control to Quantification(E) protein bywesternblot, calsequestrin of representativeonSRCalsequestrin westernblot fractions normalizedby westernblot, protein load, torelativecontrol to SR isolatedskeletalmuscle fractions from GAPDH to relative skeletal muscle Figure 12 : Unaltered SERCA1 . (

average. A control average ) QuantificationSERCA of to 1immunoreactivitynormalized

1 - way ANOVATukey’s posthoctestdetermined with no and SR protein .

(B) Small(B) Ankyrin 1 . (C)Quantification. small of ankryin1protein

abundance in in abundance

representative westernblot

isolated from skeletal isolatedfrom muscle. normalizedprotein load, to

average Act g1 .

(D)

and Act b

KO

on This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article nosignificantdifference. determined (B) blotfor measured bywestern calcium Vmaxand (pCa5 skeletal muscle. Figure 13:Unaffected SERCA

Activity of SERCAActivityof Actb (A) - pCa8). SERCA SERCApCa8). relative to as activity abundance

ATPaseActg1 ActivitySRfrom in andActbKO

Actb

- msKO. - msKO inmsKO high calcium, low (pCa5) (pCa8)

1 - way A

NOVA withTukey’shoc test post This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article difference. no significant Actg1 I and (C), PERK proteins. pathway stress ER old. months 12 at mice KO Actb specific Figure - msCT or Actb or msCT 14 : ER Stress Response Pathway Proteins are not upregulated in muscle in upregulated not are Proteins Pathway Response Stress ER : RE1α (D). All samples were normalized to GAPDH and relative to to relative and GAPDH to normalized were samples All (D). RE1α - msCT mean. msCT

Quantification of immunoreactivity to PDI (A), BiP (B), (B), BiP (A), PDI to immunoreactivity of Quantification

1 - way ANOVA with Tukey’s post hoc test determined test hoc post Tukey’s with ANOVA way Western blot showing immunoreactivity to immunoreactivity showing blot Western

-