Involvement of Protein Degradation, Calpain Autolysis and Protein

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2009 Involvement of protein degradation, calpain autolysis and protein nitrosylation in fresh meat quality during early postmortem refrigerated storage Wangang Zhang Iowa State University

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Involvement of protein degradation, calpain autolysis and protein nitrosylation in fresh

meat quality during early postmortem refrigerated storage

Wangang Zhang

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Meat Science

Program of Study Committee: Elisabeth Huff-Lonergan, Major Professor Steven Lonergan Joseph Sebranek Patricia Murphy Richard Robson

Iowa State University

Ames, Iowa

2009

TABLE OF CONTENTS

ABSTRACT

GENERAL INTRODUCTION 1

INTRODUCTION TO LITERATURE REVIEW 7

Structure of skeletal muscle 8

Myofibrillar proteins 9

Sarcoplasmic proteins 31

Stromal proteins 34

Muscle contraction 37

Fresh meat quality 39

Water holding capacity 39

Drip loss formation 44

Meat tenderness 46

Muscle fibers types and meat quality 50

Calpain family 53

Calpains 53

Calpain activity assay 59

Calpain autolysis 61

Calpastatin 64

Protein modification and meat quality 66

Protein oxidation 66

Protein nitrosylation 70

Summary 76

iii

Literature cited 78

METHODS TO DETERMINE THE ACTIVITY OF CALPAIN PROTEINASE 116

Abstract 116

Introduction 116

Assays and discussion 119

Summary 128

Literature cited 130

CONTRIBUTION OF POSTMORTEM CHANGES OF INTEGRIN, DESMIN AND µ-CALPAIN TO VARIATION IN WATER HOLDING CAPACITY OF PORK 134

Abstract 134

Introduction 135

Materials and methods 138

Results and discussion 142

Conclusions 150

Acknowledgement 150

Literature cited 151

Tables and Figures 155

S-NITROSOGLUTATHIONE AFFECTS THE ACTIVITY AND AUTOLYSIS OF µ- AND m-CALPAIN 161

Abstract 161

Introduction 162

Materials and methods 163

Results 170

Discussion 177

Conclusions 184

Literature cited 186

Tables and Figures 190

COMPARISON OF PROTEIN NITROSYLATION BETWEEN POSTMORTEM BEEF

LONGISIMMUS DORSI AND PSOAS MAJOR 196

Abstract 196

Introduction 196

Materials and methods 198

Results and discussion 203

Conclusions 208

Literature cited 210

Tables and Figures 214

GENERAL SUMMARY 220

ACKNOWLEDGEMENTS 224

ABSTRACT

Fresh meat quality including meat tenderness and water holding capacity are important for consumer acceptance and industry profitability in the US. Exploring the basic mechanism that regulates the development of meat quality attributes is critical for delivering consistently high quality fresh meat to consumers. The overall hypothesis of this study was that biochemical factors that influence proteolysis during the antemortem or the early postmortem period can contribute to variations in fresh meat quality. The objective was to determine the extent to which protein modifications including protein degradation, protein nitrosylation and calpain autolysis affect the development of fresh meat quality.

The first experiment was designed to test the hypothesis that protein degradation and calpain activation could influence water holding capacity of fresh pork. Intensity of intact desmin was positively correlated with moisture loss, while intensity of intact integrin was negatively correlated with moisture loss during postmortem storage. Significant correlations were found between different calpain autolysis products and intensity of intact desmin and moisture loss. These results support the hypothesis that proteolysis of desmin contributes to greater water holding capacity. The second experiment hypothesized that the properties of calpains could be regulated by a nitric oxide donor S- nitrosoglutathione. S-Nitrosoglutathione could further nitrosylate µ-calpain in both the absence and the presence of calcium especially at pH 6.5. The combination of S- nitrosoglutathione and calcium affected the activity of m- and µ-calpain and regulated the rate of µ-calpain autolysis. In the third experiment, the hypothesis that the muscle fiber type could be related to the levels of protein nitrosylation in postmortem beef was tested.

Beef longissimus dorsi muscle showed higher percentage of type IIA/X and lower percentage of type I myosin heavy chain isoforms than psoas major muscle. The intensity of protein nitrosylation in longissimus dorsi muscle was greater compared to psoas major muscle in samples prepared 1 d postmortem. In conclusion, biochemical and biophysical reactions during preslaughter and postmortem storage can mediate the development of fresh meat quality during postmortem aging.

GENERAL INTRODUCTION

Lack of consistency in tenderness and water holding capacity are two major problems facing the meat industry. Variations in these fresh meat attributes largely influence the satisfaction of consumers and ultimately the profitability of meat producers (Barbut et al.,

2008; George, Tatum, Belk and Smith, 1999; Stetzer and McKeith, 2003). For example, 24% of US retail beef loin steaks have been categorized with values lower than slightly tender. At least 50% of pork in the US market has been found to have high drip or purge loss

(Kauffman, Cassens, Scherer and Meeker, 1992). Thus, it is of particular importance to explore basic biochemical processes that govern meat quality including meat tenderization and water holding capacity.

Skeletal muscles have complex structures to allow them to function. Muscle proteins on average account for 18-19% of muscle weight (Aberle, Forrest, Gerrard and Mills, 2001). In postmortem muscle, proteolysis of myofibrillar and myofibril-associated proteins can change the structure of skeletal muscle and thus influence meat quality during postmortem storage

(Anderson and Parrish, 1989; Davey and Gilbert, 1966, 1969; Davis, Sebranek, Huff-

Lonergan and Lonergan, 2004; Goll, Henderson and Kline, 1964; Huff-Lonergan and

Lonergan, 1999, 2005, 2007; Koohmaraie, 1992; Kristensen and Purslow, 2001; Melody,

Lonergan, Rowe, Huiatt, Mayes and Huff-Lonergan, 2004; Rowe, Huff-Lonergan and

Lonergan, 2001; Taylor, Geesink, Thompson, Koohmaraie and Goll, 1995). Integrin plays a role in the adhesion between cell cytoskeleton and cell extracellular matrix. During postmortem aging, degradation of integrin has been found to be associated with the formation of drip channels and increased drip loss in pork (Lawson, 2004; Straadt, Rasmussen, Young,

and Bertram, 2008; Zhang, Lonergan, Gardner and Huff-Lonergan, 2006). Another protein,

desmin is one of the intermediate filament proteins that connect adjacent myofibrils and also

links the cell membrane to myofibrils. Proteolysis of desmin can remove connections

between adjacent myofibrils and thus could prevent myofibril shrinkage to be transferred to

the level of the entire muscle cells (Offer and Trinick, 1983). Several studies have shown that

desmin degradation is associated with improved tenderness and better water holding capacity

(Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Olson and Robson, 1996; Kristensen and

Purslow, 2001; Melody et al., 2004; Rowe et al., 2001; Zhang et al., 2006).

Calpains are a family of protease enzymes that have been shown to be involved with many cellular processes. However, the model of its activation and control is still poorly understood in vivo. One of the obstacles is lack of appropriate methods to monitor the status

of calpain. In postmortem muscle, the calpain system is the major factor for meat

tenderization during postmortem aging (Goll, Taylor, Christiansen and Thomson, 1992;

Huff-Lonergan et al., 1996). Therefore, finding some sensitive and continuous activity assays

for calpain is critical to control variations of fresh meat quality. Currently, 14 members of

cysteine proteases have been found in calpain family (Huang and Wang, 2001). The calpain

system was originally composed of two cysteine proteases m- and µ-calpain and their

inhibitor calpastatin (Goll, Thompson, Li, Wei and Cong, 2003). Both calpains need calcium

for their proteolytic activation. Calcium is also required for the interaction between

ubiquitous calpains and their inhibitor calpastatin (Cottin, Vidalenc and Duscastaing, 1981).

At the same time, m- and µ-calpain can autolyze in the presence of calcium (Suzuki, Tsuji,

Ishiura, Kimura, Kubota and Imahori, 1981a). Autolysis can decrease the calcium

concentration required for proteolytic activation of m- and µ-calpain (Suzuki, Tsuji, Kubota,

Kimura and Imahori, 1981b). However, complete autolysis can destroy their integral structure and thus lead to loss of their proteolytic activity (Elce, Hegadorn and Arthur, 1997).

In postmortem muscle, autolysis of µ-calpain has been shown to be parallel to its proteolytic activation (Saido et al., 1994; DeMartino, Huff and Croall, 1986). Thus, the extent of µ- calpain autolysis is an effective index for the levels of protein degradation resulted from

calpain (Gardner, Huff-Lonergan and Lonergan, 2005). Calpastatin is the specific inhibitor of

m- and µ-calpains. Variation in calpastatin activity can be used to explain some of the

differences in the rate of protein proteolysis and meat tenderization in different species and

muscles (Geesink and Koohmaraie, 1999; Koohmaraie and Geesink, 2006; Ouali and

Talmant, 1990; Shackelford, Koohmaraie, Miller, Crouse and Reagan, 1991).

Postmortem muscles have decreased ability to maintain their reducing environment.

This can result in increased accumulations of reactive oxygen species and reactive nitrogen

species (Renerre, Dumont and Gatellier, 1996). These compounds may have adverse effects

on meat quality through oxidation and nitrosylation reactions with muscle proteins and lipids.

Protein oxidation can inactivate calpain and thus arrest protein degradation and meat

tenderization during postmortem aging (Carlin, Huff-Lonergan, Rowe, and Lonergan, 2006;

Rowe, Maddock, Lonergan, and Huff-Lonergan, 2004b). Oxidation induced protein

aggregation and polymerization could influence physical properties and nutritional values of

meat products (Liu and Xiong, 2000; Srinivasan and Xiong, 1996). Nitric oxide is an

endogenous radical produced by many mammalian cells and can influence the redox state of

skeletal muscles (Stamler and Meissner, 2001). Protein functions can be modified through S-

nitrosylation by nitric oxide and other reactions with some secondary products produced from nitric oxide. These reactions may be involved in muscle metabolism and meat quality

(Cottrell, McDonagh, Dunshea, and Warner, 2008; Michetti, Salamino, Melloni, and

Pontremoli, 1995; Zhang, Lonergan and Huff-Lonergan, 2007).

Animal muscles have big variations in fiber type compositions by expressing different proteins and metabolic enzymes (Hunt and Hedrick, 1977; Schiaffino and Reggiani, 1996).

Muscle fiber characteristics have been found to be correlated with metabolic rate and meat quality in postmortem porcine and bovine muscles (Ashmore, 1974; Ryu and Kim, 2005).

These studies support that large fast glycolytic fibers are associated with unfavorable meat attributes (Calkins, Dutson, Smith, Carpenter and Davis, 1981; Crouse, Koohmaraie and

Seidemann, 1991; Ryu and Kim, 2005). Different fiber types are variable in the amount of nitric oxide synthase which is the enzyme catalyzing the production of nitric oxide in skeletal muscle (Kobzik, Reid, Bredt and Stamler, 1994). Protein nitrosylation may cause protein polymerization or aggregation by disulfide bonds and influence the susceptibility of proteolysis to protease (Klatt, Molina, Perez-Sala and Lamas, 2000). In addition, nitric oxide induced S-nitrosylation can inactivate m- and µ-calpain and thus affect the extent of protein degradation (Michetti et al., 1995). Therefore, muscle fiber differences may contribute to variations of fresh meat quality among muscles.

In conclusion, meat tenderness and water holding capacity are two important attributes that influence the profitability of the meat industry. It is known that postmortem biochemical and biophysical processes including postmortem proteolysis contribute to the development of fresh meat quality. Many muscle proteins including desmin and

integrin that can be degraded during early postmortem aging are substrates of calpains.

The overall hypothesis of this study was that biochemical factors that influence proteolysis during the antemortem or early postmortem period can contribute to variations in fresh meat quality. The objective was to determine the extent to which protein modifications including protein degradation, protein nitrosylation and calpain autolysis affect the development of fresh meat quality.

Due to the different functions and location of integrin and desmin in skeletal muscle, we hypothesized that proteolysis of these proteins and activation of calpain can influence fresh meat quality including water holding capacity (the first paper “contribution of postmortem changes of integrin, desmin and µ-calpain to variation in water holding capacity of pork” published in Meat Science, 200674:578-585). In postmortem muscle, the cellular environment changes dramatically and it is very important to define how these changes regulate the calpain system. Increased accumulation of reactive nitrogen and oxygen species in postmortem muscle can cause protein oxidation and S- nitrosylation. S-Nitrosylation can regulate protein function and cysteine enzyme activity including calpain. Therefore, we hypothesized that calpain properties including proteolytic activities and autolysis may be mediated by nitric oxide (the second paper “S- nitrosoglutathione affects the activity and autolysis of µ- and m-calpain” prepared for submission to Journal of Animal Science). Nitric oxide synthase catalyzes the nitric oxide production in skeletal muscle and the distribution of nitric oxide synthase is muscle fiber type dependent. Therefore, we tested the hypothesis that postmortem beef muscles with different composition of muscle fiber type may have different extent of protein

nitrosylation (the third paper “comparison of protein nitrosylation between postmortem beef longissimus dorsi and psoas major” prepared for submission to Meat Science).

INTRODUCTION TO LITERATURE REVIEW

The focus of this dissertation was to examine some of the biochemical processes involved in regulating fresh meat quality during postmortem storage. It is known muscle structure and redox environment within muscles are changed during aging. The hypothesis was that biochemical reactions including protein degradation, µ-calpain autolysis and protein nitrosylation could contribute to variations of meat quality during postmortem aging. The topics in this literature review include skeletal muscle structure, fresh meat quality, calpain family, and protein modifications including oxidation and nitrosylation.

I. Structure of skeletal muscle

Myofibrillar proteins

Sarcoplasmic proteins

Stromal proteins

Muscle contraction

II. Fresh meat quality

Water holding capacity

Drip loss formation

Meat tenderness

Muscle fiber types and meat quality

III. Calpain family

Calpains

Calpain assay

Calpain autolysis

Calpastatin

IV. Protein modifications and meat quality

Protein oxidation

Protein nitrosylation

V. Summary

VI. Literature cited

Structure of skeletal muscles

Skeletal muscles have a highly organized structure in order to transmit contractile forces

that originate from the myofibrils. The cells that make up skeletal muscles are known as the

muscle fibers. Muscle fibers contain myofibrils made up of protein filaments which are responsible for the muscle's contractile ability and are responsible for the striated bands under the electron microscope. Myofibrils have light I bands and dark dense A bands. These bands have different protein densities. The I band is made up of thin filaments, while the A band contains both thick filaments and thin filaments. The M-line bisects the A-band and is responsible for maintaining the thick filament lattice. The Z-disk is located at the middle of the I band. The Z-disk is an attachment point and a mechanical link between thin filaments in adjacent sarcomeres. The structure between adjacent Z-lines containing one A-band and two half I bands is called a sarcomere. The sarcomere is the basic unit for muscle contraction and relaxation.

The ultrastructure of skeletal muscle is characterized by complex protein-protein interactions. Muscle proteins approximately account for 18-19% of muscle weight (Aberle et al., 2001). The muscle proteins can be broadly divided into the myofibrillar proteins (soluble in concentrated salt solutions), the sarcoplasmic proteins (soluble in water or dilute salt solutions) and the stromal proteins (insoluble in neutral aqueous solvents). Other proteins include membrane associated proteins, especially proteins composed of the sarcoplasmic reticulum and dihydropyridine receptors which play important roles in the rate of metabolism during the conversion of muscle to meat.

Myofibrillar proteins

Myofibrillar proteins compose or are associated with myofibrils and classified as contractile, regulatory and cytoskeletal proteins based on their locations and functions. The predominant proteins in the thick and thin filaments are myosin and actin. Other proteins function as scaffolds, regulators of muscle contraction and transmission of tension from sarcomeres to whole myofibrils (Au, 2004).

Myosin and thick filament associated proteins

Myosin is the major protein that makes up the thick filament. Muscle myosin contains two heavy chains (molecular weight of 220 kDa) and four light chains including two essential light chains and two regulatory light chains. Structures of myosin include the myosin S1 head, the regulatory domain and the C-terminal rod domain. The S1 head is composed of the N-terminal region of each myosin heavy chain and two light chains including essential and regulatory light chains (Milligan, 1996). The myosin head has

ATPase enzyme activity and contains the binding sites for actin and ATP (Rayment et al.,

1993). The regulatory domain is located at the neck region of myosin consisting of a

regulatory light chain, an essential light chain and a portion of heavy chain. The C-terminal

regions of the myosin heavy chain make up the myosin rod region that forms the backbone of

the thick filament.

Other proteins associated with the thick filament include myosin binding proteins C-

protein and H-protein and a giant protein titin. Titin is the largest protein in nature and the

third most abundant protein after myosin and actin in skeletal muscle (Wang, McClure and

Tu, 1979). One titin molecule spans half of the sarcomere with the C-terminal ending in the

M-line and the N-terminal being located in the Z-disk (Au, 2004). Titin is believed to play

key roles in the structural organization, elasticity and integrity of myofibrils. Titin contains a

sequence repeat known as the Z-repeat in the Z-disk areas. Z-repeat interacts with the C-

terminus of the Z-disk protein α-actinin to contribute to the sarcomeric stability and the Z- disk thickness (Gautel, Goulding, Bullard, Weber and Furst, 1996; Ohtsuka, Yajima,

Maruyama and Kimura, 1997). I-band region of titin functions as an elastic connection between the Z-line and the thick filament. The spring-like elements in this region of titin can produce passive forces and influence the levels of myofibril stiffness (Granzier and Labeit,

2004). In the A-band, titin is associated with light meromyosin portion of myosin and possibly interact with myosin binding protein-C and AMP-delaminase (Soteriou, Gamage and Trinick, 1993; Wang, Jeng and Sun, 1992). The A-band region of titin is involved in the regulation of the assembly and the length of the thick filament during myofibrillogenesis

(Whiting, Wardale and Trinick, 1989). M-line titin interacts with muscle specific RING-

finger protein-1 and M-line proteins including M-protein and myomesin. These interactions

form an integral part of the protein network responsible for the stability of the sarcomeric M-

line region (McElhinny, Kakinuma, Sorimachi, Labeit, and Gregorio, 2002). In conclusion,

titin plays critical roles in the alignment of the sarcomere, the generation of passive tension

and the regulation of the thick filament length. Due to these structural roles of titin in living

cells, it is believed that postmortem degradation of titin weakens the sarcomeric structure and

disrupts the stability of muscle. Thus, degradation of titin may contribute to meat

tenderization during postmortem storage (Huff-Lonergan et al., 1996; Taylor et al., 1995).

Myosin binding proteins H- and C-protein are located at crossbridge-containing C zones

(the inner two-thirds of the A-band) of striated muscle sarcomere. They are composed of

globular immunoglobulin-like and fibronectin type III-like domains. Three isoforms of C-

proteins are identified known as skeletal fast, skeletal slow and cardiac isoforms in adult

muscle (Reinach, Masaki, Shafiz, Obinata and Fischman, 1982). H-protein has sequence

homology with C-proteins and only one isoform of H-protein has been identified (Vaughan,

Weber, Ried, Ward, Reinach and Fischman, 1993). C-protein contains two myosin biding sites including a myosin light meromyosin-binding site and a myosin S2 binding site

(Okagaki, Weber, Fischman, Vaughan, Mikawa, Reinach, 1993). A titin-binding site is located in the C-terminus of C-protein that makes C-protein interact with titin (Gilbert, Kelly,

Mikawa and Fischman, 1996). Based on these interactions with titin and myosin, C-protein may play a role in stabilizing the myosin molecules and aligning the thick filament in the A- band. In the early stage of muscle development, C-protein may be involved in regulation of the assembly and the thickness of the thick filament by interaction with titin and myosin

(Gilbert et al., 1996). In addition, C- and H-protein can modulate muscle contraction by

controlling the velocity of muscle shortening and the range of myosin movement (Winegrad,

1999).

Actin and thin filament associated proteins

Thin filaments anchor in the Z-disk and extend toward the middle of sarcomere and are

approximately 1 µm long. The fast growing barbed end and the slow growing pointed end of

actin are capped by CapZ in the Z-line and tropomodulin respectively (Babcock and Fowler,

1994; Papa et al., 1999). Other proteins associated with thin filament including nebulin,

troponin and tropomyosin run along the length of thin filament. Nebulin modulates the

assembly of the thin filament, while troponin and tropomyosin complex controls the interaction of actin and myosin (Lehman, Vibert, Uman and Craig, 1995). In thin filament region, titin contains elastic elements to maintain the sarcomeric integrity and regulate the muscle stiffness.

Actin is the major component of thin filament and accounts for approximately 20% of myofibrillar proteins (Aberle et al., 2001). One of the properties of globular actin (G-actin) is to polymerize and form fibrous actin (F-actin). The growth of actin filament is different in slow-growing pointed end and fast-growing barbed end (Svitkina and Borisy, 1999). The actin monomers are continually translocated from the barbed end to the pointed end of actin filament known as treadmilling (Neuhaus, Wanger, Keiser and Wegner, 1983). ATP can accelerate the polymerization from G-actin to F-actin and increase the association of actin filament in the barbed end (De La Cruz, Mandinova, Steinmetz, Stoffler, Aebi and Pollard,

2000).

Actin monomer contains large and small domains and each of the domains has two

subdomains. The cleft between two domains is the sites for the binding of ATP or ADP

nucleotides and a divalent cation (Steinmetz, Stoffler, Hoenger, Bremer and Aebi, 1997). The

small domain is comprised of subdomains 1 and 2 and the large domain is comprised of

subdomains 3 and 4. The interaction between the subdomain 1 of actin and the head of

myosin activates myosin ATPase activity.

Tropomyosin and troponin are major regulatory proteins located in the grooves of the

actin helix in thin filaments. They interact with each other to regulate actin-myosin

interaction and muscle contraction. Tropomyosin spans seven actin monomers and runs along

the grooves of actin filament (Phillips, Fillers and Cohen, 1986). Tropomyosin is expressed with two genes Tm1 and Tm2 (α and β) and the alternative splicing of the two genes results in isoform diversity (Lees-Miller and Helfman, 1991). In skeletal muscle cells, tropomyosin

is the mixture of αβ heterodimer and αα or ββ homodimer (Bronson and Schachat, 1982;

Lehrer, 1975). In relaxed muscle, tropomyosin blocks the binding site for the myosin heads on the thin filament and thus blocks the interaction between myosin and actin known as the steric blocking model (Hitchcock, Huxley and Szent, 1973; Lorenz, Poole, Popp, Rosenbaum and Holmes, 1995). In addition to regulating muscle contraction, tropomyosin functions in stabilizing the thin filament. Tropomyosin can strengthen the thin filaments, inhibit their fragmentation and decrease the polymerization and depolymerization at the pointed ends

(Broschat, 1990; Wegner, 1979).

Troponin (80 kDa) is a complex of three noncovalent subunits named according their function as TnC, TnI and TnT. TnC is the subunit for calcium binding which changes the

conformation of InI and initiates the muscle contraction. TnI is the inhibitory subunit which

inhibits actomyosin ATPase activity in vitro and holds the troponin-tropomyosin complex in

position by binding thin filament (Leavis and Gergely, 1984). TnT can interact with

tropomyosin, TnC and TnI to regulate the troponin-tropomyosin complex and muscle

contraction (Perry, 1998). The TnC, TnI and part of TnT form the 8 nm globular troponin

head. The rest of TnT makes up the 17 nm long tail of troponin to anchor the troponin

complex on the thin filament by interacting with tropomyosin (Flicker, Phillip and Cohen,

1982). The 17 kDa TnC has four divalent cation biding sites and C-terminal sites III and IV

are always filled with divalent cations (Herzberg and James, 1985). The major function of

troponin is to modulate the interaction of actin and myosin. The binding of Ca2+ to the N- terminal sites I and II changes the conformation of TnC and alters its interaction with TnI.

TnI has decreased affinity for actin and increased affinity for TnC and TnT in this condition

(Potter and Gergely, 1974). The alteration of these affinities can result in conformational change of troponin complex and the conformational change alters the interaction between

troponin and tropomyosin. Tropomyosin shifts its position and exposes the binding site for

myosin on actin allowing the interaction between actin and myosin (Zot and Potter, 1987).

Both troponin and tropomyosin are substrates for calpain protease and 30 kDa products from

troponin-T degradation have been used as an indicator for meat tenderness due to proteolysis

for many years (McBride and Parrish, 1977; Olson and Parrish, 1977; Penny and Dransfield,

1979). Degradation of troponin-T may disrupt the integrity of the thin filaments and change

the interactions between actin and myosin resulting in the fragmentation of the myofibrils

(Taylor et al., 1995).

Nebulin is a cytoskeletal protein with molecular weight of 500 to 900 kDa depending on

its isoform in different skeletal muscles. Nebulin runs along the thin filament by binding

subdomain 1 of actin (Shih, Chen, Linse and Wang, 1997). Its C-terminus interacts with α- actinin, myopalladin, cap-Z and the intermediate filament protein desmin to anchor the thin filament to the Z-disk (Bang et al., 2001; Goll, Robson and Stromer, 1984; Pappas,

Bhattacharya, Cooper and Gregorio, 2008). The N-terminus of nebulin extends to the pointed end of actin and interacts with the capping protein tropomodulin (Wang and Wright, 1988).

Cloned human nebulin fragments could connect myosin heads to actin filament myosin in vitro (Root and Wang, 1994). This provides the possibility that nebulin may be involved in

regulation of muscle contractions by influencing actomyosin interaction. In developing

muscle, nebulin can maintain the stability of thin filament and define its length during

myofibrillogenesis (McElhinny et al., 2005; Moncman and Wang, 1996). Nebulin acts as a

regulator for the length of the thin filament in mature skeletal muscles evidenced by the

correlation between molecular masses of nebulin and the length of thin filament (Kruger,

Wright and Wang, 1991; Trinick, 1994). Due to its structural functions, postmortem

degradation of nebulin is believed to weaken the structure of thin filament and thus

contribute to the meat tenderization (Huff-Lonergan, Parrish and Robson, 1995; Huff-

Lonergan et al., 1996; Taylor et al., 1995).

Tropomodulin is the only known protein that caps the pointed ends of the thin filaments.

Erythrocyte and skeletal muscle tropomodulin are two tropomodulin isoforms that have been

indentified in vertebrate muscle (Cox and Zoghbi, 2000). The N-terminus of tropomodulin

binds to the tropomyosin, while the C-terminus interacts with actin and nebulin (McElhinny,

Kolmerer, Fowler, Labeit and Gregorio, 2001). The interaction with actin functions in

maintaining the length of thin filament. Interaction between tropomodulin and tropomyosin

can stabilize the thin filament (Gregorio, Weber, Bondad, Pennise and Fowler, 1995). These

interactions with tropomyosin and actin aid in the full capping activity of tropomodulin for

the thin filament (Weber, Pennise, Babcock and Fowler, 1994). Hence, tropomodulin plays

important roles in thin filament arrangement and control the concentration of barbed end of actin filament (Weber, Pennise and Fowler, 1999).

M-line proteins

The M-line is a transverse band at the center of A-band. The M-bridges within M-line are responsible for the cross-linking of a thick filament to its six neighbors. M-lines function in stabilizing the thick filament lattices and positioning them within the sarcomere in an ordered manner (Luther and Squire, 1978; Vaughan, Weber, Ried, Ward, Reinach and Fischman,

1993). Some proteins including spectrin, ankyrin and obscurin have been reported to connect

M-band to the sarcolemma or the neighboring myofibrils (Bagnato, Barone, Giacomello,

Rossi and Sorrentino, 2003; Flick and Konieczny, 2000; Wang and Ramirez-Mitchell, 1983).

Three specific M-line proteins have been identified including M-protein, myomesin and a muscle specific creatine kinase. Other M-line associated proteins contain the C-terminus of titin, myosin and skelemin (Agarkova and Perriard, 2005).

Five M lines have been identified with M1 located at the center. M4´/M4 lines are located at each side of M1 line with 22 nm distance. The additional lines are M6´/M6 lines which are 22 nm further out of M4´/M4 lines in both sides of M1 line. The distributions of M lines are muscle fiber type dependent. The M4´/M4 lines are present in all muscles, while the

density of M1 and M6´/M6 are correlated with different muscle types. Type II fibers have three lines (M1, M4 and M4´), while type I fibers show four strong lines (M4, M4´, M6 and

M6´) (Carlsson and Thornell, 1987). In addition to M-bridges, M-filaments running parallel to the thick filament laterally link M-lines together and these filaments possibly are composed of titin and myomesin (Luther and Squire, 1978; van der Ven, Obermann, Weber and Furst, 1996).

Creatine kinase functions to regenerate ATP by catalyzing the reversible reaction using phosphocreatine (also called creatine phosphate) and ADP as substrates (Kenyon and Reed,

1983). Five isoforms of creatine kinase have been found including three cytosolic isoforms

(brain type, muscle type and a heterodimer of two forms) and two mitochondrial isoforms

(ubiquitous and sarcomeric isoform) (Eppenberger, Dawson and Kaplan, 1967; Schlegel,

Wyss, Eppenberger and Wallimann, 1990). Muscle type creatine kinase has been found to contribute the M4´/M4 bridges indicating its structural role in the sarcomere (Turner,

Wallimann and Eppenberger, 1973). In early postmortem stage, muscle creatine kinase utilizes phosphocreatine to generate ATP through the re-phosphorylation of ADP (Aberle et al., 2001). Thus, the amount of phosphocreatine and the activity of creatine kinase in skeletal muscle may affect the rate of muscle metabolism during the conversion of muscle to meat.

Supplementation of creatine monohydrate in finishing pig increased the 24 hour firmness and decreased the drip loss after 14 days of postmortem storage in longissimus muscle (James et al., 2002). In another study, dietary supplementation with 50 g creatine monohydrate per day increased 30 and 45 minute pH and decreased 48 hour drip loss and juiciness in Duroc pork

(Young, Bertram, Rosenvold, Lindahl and Oksbjerg, 2005). Feeding creatine monohydrate

prior to slaughter may slow down postmortem anaerobic metabolism and lactate

accumulation. Delayed lactate formation can postpone pH decline and improve the water

holding capacity during postmortem aging (Bendall and Swatland, 1988).

Myomesin and M-protein are related proteins which share approximately 50% sequence

homology. Both M-protein and myomesin contain 12 immunoglobulin-like and fibronectin-

like domains in the same order. The differences between these two proteins are in the N- and

C-terminal regions (Masaki and Takaiti, 1974). M1-bridges are composed of M-protein and

are present in fast (type II) fibers which have three M-lines including M1, M4 and M4´

(Pask, Jones, Luther and Squire, 1994). Myomesin is present in all muscle fiber types and is

the candidate for the structural roles of M4/M4’ bridges in M-line (Obermann, Gautel,

Steiner, Van der Van, Weber and Furst, 1996). M-protein and myomesin bind to the C-

terminus of titin and the light meromyosin region of myosin (Obermann, Plessmann, Weber and Furst, 1995). These interactions indicate the structural role of M-protein and myomesin in linking the thick filament into assembling sarcomeres (Ehler, Rothen, Hammerle,

Komiyama and Perriard, 1999).

Z-disk proteins

Z-disk is an essential component for the integrity of the sarcomere and for the tension transition of the muscle contraction. The protein complex in the Z-disk serves as the attachment point for the thin filament and the cytoskeletal proteins including nebulin and titin. Both the actin filament and titin overlap within the Z-disk, while nebulin ends in the edge of the Z-disk. Intermediate filament proteins are located in the periphery of the Z-disk to connect adjacent myofibrils. The Z-disk is also the anchoring site for the costameric

proteins via other proteins that link the myofibrils to the sarcolemma (Craig and Pardo,

1983). These linkages enable contraction forces to be transmitted from myofibrils to the

entire cells and the surrounding connective tissues. In addition to structural roles, the Z-disk

may be involved in signal transduction and muscle homeostasis. A large number of Z-disk

associated proteins are signaling components and dynamically distribute between the Z-disk

and other subcellular locations such as nucleus (Clark, McElhinny, Beckerle and Gregorio,

2002).

α-Actinin is the major component of the Z-disk which interacts with both actin and titin.

In addition to α-actinin, numerous proteins have been mentioned in the area of Z-disk

including amorphin, myozenin, myotilin, myopalladin, myopodin, the enigma family

proteins, telethonin, obscurin, cap-Z, calcineurin and γ-filamin (Clark et al., 2002). Most of

these proteins position in Z-disk by interacting with α-actinin. α-Actinin is composed of three major domains containing a N-terminal actin-binding domain, a central rod domain with four spectrin repeats and a C-terminal domain. The rod domain of α-actinin forms antiparallel dimers to crosslink the thin filament and titin from opposite half-sarcomeres (Djinovic-

Carugo, Young, Gautel and Saraste, 1999). Most of other Z-disk associated proteins bind to

α-actinin in the area of the Z-disk. The interactions among these proteins function in providing the tensile integrity of the Z-disk (Faulkner, Lanfranchi and Valle, 2001). Four isoforms of α-actinin have been identified. α-Actinin-1 and α-actinin-4 are nonmuscle isoforms, while α-actinin-2 and α-actinin-3 are muscle specific isoforms located at the Z-disk

(Honda et al., 1998; North and Beggs, 1996).

Cap-Z interacts with α-actinin within the Z-disk to cap the barbed end of the thin

filament and anchor the thin filament in the Z-line (Casella, Craig, Maack and Brown, 1987).

During myofibrillogenesis, Cap-Z can regulate the addition and the loss of actin monomers

and organize the barbed ends of the thin filaments (Schafer, Hug and Cooper, 1995). Cap-Z

is a heterodimer composed of α and β subunits and each of subunits has two isoforms to bind actin differently (Casella and Torres, 1994). The different combination of Cap-Z isoforms may determine the properties of the thin filaments among different muscles.

Filamin was discovered from chicken smooth muscle and has a molecular weight of 250 kDa (Wang, Ash and Singer, 1975). Three isoforms (α, β and γ) have been identified and γ- filamin is the muscle-specific filamin isoform (Hock, Davis and Spencer, 1990). Filamin is located at the periphery of the Z-disk and some filamin is at the costameres (Price, Caprette and Gomer, 1994; Thompson et al., 2000). There is an N-terminal actin-binding domain and

24 immunoglobulin-like domains in the filamin molecular structure. Filamin can interact with many muscle proteins including thin filament protein actin, Z-line proteins including myotilin and FATZ-family proteins and cell-matrix protein integrin (Faulkner et al., 2000;

Loo, Kanner and Aruffo, 1998; Thompson et al., 2000; van der Ven et al., 2000). These interactions indicate that filamin may link the membrane with the cytoskeleton, stabilize the muscle cytoskeleton and be involved in the myofibrillogenesis (Hauser et al., 2000; van der

Flier et al., 2002).

Desmin and intermediate filament associated proteins

The cytoskeleton of vertebrate muscle cells is typically composed of three major filament systems: 7 nm diameter microfilaments, 10 nm intermediate filaments and 23 nm

microtubules (Robson, 1989; Robson et al., 1997). Intermediate filaments are especially important to maintain the overall structural and functional integrity of muscle and transfer the mechanical forces among adjacent myofibrils (Boriek et al., 2001; Robson, 1989). The 53 kDa desmin is the major constituent of the intermediate filaments in mature skeletal muscle.

In addition to desmin, intermediate filament and intermediate filament-associated proteins include synemin, vimentin, syncoilin, plectin and filamin (Robson, 1989). All intermediate filament proteins contain three common domains including an N-terminal head domain, a C- terminal tail domain and a conserved central rod domain (Meng, Khan and Ip, 1996). The intermediate filament runs perpendicularly along the long axis of myofibrils and skeletal muscle cells (Robson et al., 1984). Three major roles of desmin-containing intermediate filaments are connecting adjacent myofibrils laterally at the periphery of the Z-disk areas, linking myofibrillar Z-lines to subcellular organelles including nuclei and mitochondria, and integrating the connections between myofibrils and cell membrane skeleton (Bellin, Huiatt,

Critchley and Robson, 2001; Richard, Stromer, Huiatt and Robson, 1981; Milner, Mavroidis,

Weisleder and Capetanaki, 2000; Robson, 1995).

Synemin (molecular weight of 230 kDa) and plays important cytoskeletal roles in linking intermediate filaments to other structures. Synemin can not form intermediate filaments by itself but coassemble with desmin and vimentin (Bilak et al., 1998). The rod domain of synemin binds desmin and vimentin and its tail domain interacts with vinculin and

α-actinin (Bellin, Sernett, Becker, Ip, Huiatt and Robson, 1999). With these interactions, synemin aids intermediate filaments in linking adjacent myofibrils and interacting with costameres (Bellin, Huiatt, Critchley and Robson, 2001).

Figure 1. The structure and protein composition of costameres in striated muscle relative to Z- disks and the myofibrillar lattice (Taylor et al., 1995).

Syncoilin is not associated with desmin to form filaments, but it is involved in

anchoring the intermediate filament network to Z-lines and to the sarcolemma of skeletal

muscle cells (Poon, Howman, Newey and Davies, 2002). Syncoilin interacts with desmin and

a dystrophin associated protein, α-dystrobrevin, at the membrane of muscle cells. With these

bindings, syncoilin functions in linking the dystrophin associated protein complex and the

intermediate filament (Moorwood, 2008). These structural functions provide the hypothesis

that syncoilin plays important roles in muscle integrity and the contractile force transmission.

Syncoilin may be also involved in the muscle regeneration by mediating the desmin

filaments during myoblast fusion (Newey et al., 2001).

Plectin contains three domains: an N-terminal actin-binding domain, a central coiled-

coil rod domain and an intermediate filament-binding domain (Steinbock and Wiche, 1999).

Its C-terminal domain interacts with desmin and β-synemin, while its N-terminal domain

binds actin and α-dystrobrevin (Hijikata et al., 2008). Plectin C-terminal domain also links at

both ends to β4-integrin and other junction proteins (Rezniczek, de Pereda, Reipert and

Wiche, 1998; Steinbock and Wiche, 1999). With these interactions, plectin plays key roles in connecting intermediate filaments to their membrane attachment sites contributing to the mechanical integrity of the muscle cells (Wiche, 1988).

The 53 kDa desmin is the primary protein for the intermediate filament and desmin filaments are present throughout the skeletal, cardiac and smooth muscles. Only one gene coding for desmin is indentified which is located at the chromosome 2q10 (Capetanaki, Ngai and Lazarides, 1984; Quax, Meera, Quax-Jeuken and Bloemenda, 1985). Hence, desmins in all vertebrates are similar with slight difference due to posttranslational modifications.

Desmin contains three domains including a highly conserved α-helical rod domain, a highly variable non-α-helical amino-terminal head and a carboxyl-terminal tail domain (Geisler and

Weber, 1982). The rod domain is responsible for polymerization by lateral association and tail domain for the filament integration and interaction between proteins and organelles (Bar,

Strelkov, Sjöberg, Aebi, and H. Herrmann, 2004). Desmin is usually present as phosphorylated forms and the phosphorylation regulates its polymerization and change the structure of desmin filament (Huang, Foster, Lemanski, Dube, Zhang and Lemanski, 2002).

Desmin is one of the earliest muscle-specific proteins to appear during myogenesis detected in somites and myoblasts (Herrmann, Fouquet and Franke, 1989; Kauffman and Foster,

1988). Mutation of desmin expression showed that desmin may be involved in regulation of myofibrillogenesis (Weitzer, Milner, Kim, Bradley and Capetanaki, 1995). In mature skeletal muscle, desmin is located at the periphery of the myofibrillar Z-disk (Richardson, Stromer,

Huiatt and Robson, 1981). Desmin filament functions in interlinking adjacent myofibrils in

the Z-disk and integrating myofibrils with sarcolemma, nuclei and mitochondria (Robson,

1985, 1989).

Most of intermediate filament proteins including desmin, synemin, paranemin and plectin are substrates of calpain which is a family of cysteine protease (Baron, Jacobsen and

Purslow, 2004; Bilak et al., 1998; Huff-Lonergan et al., 1996). Degradation of these proteins may change the linkages between adjacent myofibrils and between myofibrils and the sarcolemma of muscle cells (Huff-Lonergan et al., 1996; Taylor et al., 1995). Degradation of desmin has been reported to be associated with fresh meat quality for both water holding capacity and tenderness (Huff-Lonergan and Lonergan, 2005; Huff-Lonergan et al., 1996;

Kristensen and Purslow, 2001; Melody et al., 2004; Rowe et al., 2001; Taylor et al., 1995).

The degradation of desmin in postmortem muscle may disrupt the lateral linkage among myofibrils and connections between the cell cytoskeleton and cell membrane. This disruption may disrupt the integrity and influence the whole organization of muscle architecture to increase meat tenderness during postmortem aging. This hypothesis has been supported by many studies. In the study of Huff-Lonergan et al. (1996), western blots results showed that desmin was degraded to three different products with molecular weight of 45, 38 and 35 kDa in bovine longissimus thoracis during postmortem storage. Interestingly, postmortem samples with lower shear force values showed faster rate of desmin proteolysis and earlier appearance of desmin degradation products. In another study by Wheeler and Koohmaraie

(1994), shear force was reduced from 8.66 kg to 4.36 kg during postmortem storage between

24 to 72 hours. This decrease in shear force was consistent with the postmortem degradation process of desmin. The authors concluded that postmortem proteolysis was the major factor

for the improved tenderness during aging. In agreement with this, the extent of protein

degradation including desmin was suggested to be partly responsible for the significant

difference of meat tenderness between porcine longissimus dorsi and semimembranosus at

48 and 120 hours postmortem (Melody et al., 2004). For example, the intensity of intact

desmin in semimembranosus was 21% higher than the intensity of longissimus dorsi at 120

hour postmortem. However, Warner-Bratzler shear force of semimembranosus was

significantly higher than longissimus dorsi at both 48 and 120 hour postmortem.

As for the involvement of desmin in drip loss of postmortem muscle, two reviews have

been published recently by Huff-Lonergan and Lonergan (2005 and 2007). When the desmin

and other costameric proteins remain intact during rigor process, the shrinkage of myofibrils could potentially be translated into the shrinkage of whole muscle cells (Offer and Cousins,

1992). The whole cell shrinkage can decrease the volume of muscle cells to limit the

available space for water to be held within muscle cells (Huff-Lonergan and Lonergan, 2005).

The consequence is to drive the water out of intro- and extramyofibrillar space to the

extracellular space to be lost as drip from drip channels. In a study with pork muscle, the

degradation products of desmin were detected as early as 45 min in psoas major and psoas

major had greater desmin degradation than longissimus dorsi at both 45 min and 6 hour

postmortem. Consistent with this, longissimus dorsi had more average drip loss after both 24

and 96 hours of postmortem storage than psoas major muscle (Melody et al., 2004). This

indicates that less desmin degradation may be associated with higher levels of drip loss. This

conclusion was supported by another study which showed that degradation of desmin could

both influence water holding capacity and tenderness of pork (Rowe et al., 2001). The

correlation was as high as 0.437 between 24 hour drip loss and desmin degradation. In

another study, different biochemical parameters were used including pH at 24 hours,

temperature decline, desmin degradation and µ-calpain autolysis to predict water holding

capacity of fresh pork (Gardner, Huff-Lonergan and Lonergan, 2005). They found that the

intact desmin after 24 hours of postmortem storage was the first variable to enter the stepwise

regression analysis model to predict purge loss and that high intact desmin is associated with

greater purge loss in longissimus dorsi muscle.

Based on many previous studies, the extent of desmin degradation seems to play critical

roles in fresh meat quality for both drip loss and tenderness during postmortem aging.

Therefore, controlling the variation of desmin proteolysis is very critical for improving the

consistency of fresh meat quality.

Costameric proteins and cell-matrix proteins

Costameres are referred as protein assemblies that connect muscle myofibrils to muscle

sarcolemma (Craig and Pardo, 1983). Costameric proteins contain vinculin, talin, ankyrin,

dystrophin, β-spectrin, γ-actin, β1-integrin and some intermediate filament proteins including desmin and vimentin. Most of these proteins are located at the periphery of the Z-disk of the

I-band in skeletal muscle (Pardo, Siliciana and Craig, 1983). A second costameric complex is also identified to link the myofibrils in the M-line region to the sarcolemma (Porter,

Dmytrenko, Winkelmann and Bloch, 1992). The dystrophin associated protein complex and

integrin adhesions are two families of proteins involved in connections between the cell

cytoskeleton and the extracellular matrix. Dystrophin associated proteins and integrin are

responsible for lots of physiological processes including cell adhesion, migration,

proliferation, apoptosis and homeostasis (Hynes, 1992; Hynes, 2002). Dystrophin is located

at the sarcolemma where it interacts with other proteins including α-dystrobrevins to form dystrophin associated protein complex in skeletal muscle (Blake, Nawrotzki, Peters,

Froehner and Davies, 1996). These connections maintain the sustaining sarcolemmal integrity during muscle contraction (Ervasti and Campbell, 1993).

Integrins are a family of cell adhesion molecules that link the extracellular matrix to the intracellular cytoskeleton in muscles. Due to their special location, integrins are critical for the transmission of signals from extracellular environment to different signal cascades, cell- cell interaction mediating cell migration, differentiation, apoptosis, wound healing, cell shape and proliferation (Arnaout, Mahalingam and Xiong, 2005; Hynes, 1992; Luo, Carman and

Springer, 2007; Luo and Springer, 2006; Pellinen and Ivaska, 2006). These signals can be transmitted by integrins bidirectionally across the sarcolemma. Mutations in integrins have been reported to be associated with several diseases. For example, expression pattern of integrins can be disrupted in tumors and this alteration may cause changes in cell adhesion and migration for primary tumor invasion (Juliano and Varner, 1993; Varner and Cheresh,

1996). Muscular dystrophy has been linked to the mutation of α7 subunit and decreased level

of α7β1 integrin (Hayashi et al., 1998; Hodges, Hayashi, Nonaka, Wang, Arahata and

Kaufman, 1997). Integrins are heterodimeric proteins which are composed of α and β subunits. Until now, eighteen α-subunits and eight β-subunits have been identified with twenty four different combinations in vertebrates (Arnaout, Goodman and Xiong, 2007).

Each subunit is made up of a cytoplasmic domain, a transmembrane helix and an N-terminal extracellular domain (Hynes, 2002). Different combinations of α and β chains can determine

their ligand specificity which is an integrin binding surface containing exposed amino acids and coupling to signal pathways for recognizing extracellular proteins. Generally, β chains of integrins are responsible for the linkage of the cell membrane to the cell cytoskeleton

(Cukierman, Pankov and Yamada, 2002; Higgins et al., 2000; Wiesner, Legate and Fassler,

2005). This attachment is through the interaction between integrin and other signaling proteins including talin, filamin, vinculin, α-actinin, focal adhesion kinase, zyxin and paxillin

(Bakolitsa et al., 2004; Garcia-Alvarez et al., 2003; van der Flier and Sonnenberg, 2001;

Kiema et al., 2006; McCleverty, Lin and Liddington, 2007; Ziegler, Gingras, Critchley and

Emsley, 2008). Among these proteins, talin plays critical roles in the entire structure of integrin adhesion complex on the cell surface, integrin activation and the linkage of integrin to the actin cytoskeleton (Nayal, Webb, and Horwitz, 2004; Ulmer, Calderwood, Ginsberg and Campbell, 2003).

Talin can recruit other proteins such as vinculin, paxillin, tensin and α-actinin to reinforce the integrin-cytoskeleton at the early stage of focal complex formation (Giannone,

Jiang, Sutton, Critchley and Sheetz, 2003). Calpain induced proteolysis of talin has been shown to be important for the turnover of focal adhesion between the cell and the extracellular matrix (Franco et al., 2004). These interactions can be regulated by posttranslational modification of integrin including phosphorylation, alternative splicing, proteolytic cleavage and glycosylation (Bellis, 2004).

Several papers reported calpain induced degradation of integrin may be involved in the regulation of physiological functions (Inomata, Hayashi, Ohno-Iwashita, Tsubuki, Saido and

Kawashima, 1996; Sato and Kawashima, 2001). Earlier studies found that β3-subunit of

integrin could be cleaved by both m- and µ-calpain. This cleavage may influence the

interaction between the cell membrane and the cell cytoskeleton (Aoyama et al., 2004; Du,

Saido, Tsubuki, Indig, William and Ginsberg, 1995; Schoenwaelder, Yuan, Cooray, Salem

and Jackson, 1997; Yan, Calderwood, Yaspan and Ginsberg, 2001). In following study,

Pfaff, Du and Ginsberg (1999) presented that other β subunits including β2, β7, β1A and β1D in

cytoplasmic domains of integrin were also susceptible to m-calpain at NPXY/NXXY motifs

in vitro. In contrast, the cytoplasmic domain of αIIB was not cleaved by µ-calpain (Du et al.,

1995).

Very few studies have been published about the possible involvement of integrin in

regulating water holding capacity of fresh pork (Lawson, 2004; Straadt et al., 2008; Zhang et

al., 2006). Drip channels caused by the detachment between the extracellular matrix and the

cell cytoskeleton were detected in all pork samples after 24 hours of postmortem storage

(Lawson, 2004). In samples with high drip loss, large drip channels appeared as early as after

3 hours of postmortem storage. In comparison, no channels were visible until 9 hours of

postmortem storage and full channel openings were only detected at 24 hours postmortem in

samples with low drip loss. Consistent with this, Zhang et al. (2006) found intensity of intact

integrin at day 1 was negatively correlated with drip loss over 5 days of storage. Negative

correlations were also found between intensity of integrin after 5 days of storage and sirloin

purge loss after 7 days of storage. These studies indicate that integrin degradation may

contribute to decreased water holding capacity. In adult muscle, the most common isoform of

integrins is β1 subunit and α7β1 is the receptor responsible for the laminin binding

(Brakebusch, Hirsch, Potocnik and Fassler, 1997). Since β1 is considered as the most

important isoform for matrix interaction in adult muscle, a β1 integrin antibody has been

utilized to examine extent of integrin degradation in muscle system. Immunostaining of the

cytoplasmic domain of β1-integrin showed that the degradation of this domain was consistent with the time process of the opening of drip channels. However, no degradation of dystrophin and the extracellular domain of integrin were detected in this study (Lawson, 2004). This further shows that the interaction between the cell cytoskeleton and the cytoplasmic integrin may be involved with the drip channel formation in early postmortem pork. To determine whether calpains were responsible for the degradation of integrin, both the cysteine protease inhibitor E-64 and the calpain specific inhibitor PD150,606 were utilized in this study (Lawson,

2004). Only 10% loss of fluorescence intensity of the cytoplasmic domain of β1-integrin was

shown in both inhibitor groups compared with 80% loss of intensity of control groups.

Immunostaining showed that m-calpain was specifically colocalized with β1-integrin

adhesion areas, while µ-calpain had a uniform distribution across whole muscle areas

(Lawson, 2004). This indicates that m-calpain is more possibly involved with the degradation

of integrin. The possible mechanism for the involvement of integrin in drip loss may be the

degradation of integrin allowing drip channel formation between cell membrane and cell

cytoskeleton. When degradation of integrin occurs before the onset of rigor, the shrinkage of

muscle myofibrils may force water out from these drip channels instead of being retained by

connective tissues (Lawson, 2004).

Sarcoplasmic proteins

The sarcoplasmic proteins are soluble in water or dilute salt solutions and account for

30-35% of total muscle protein (Aberle et al., 2001). Hundreds of proteins have been identified in sarcoplasmic fractions of skeletal muscle (Bendixen, 2005). These include some mitochondria proteins comprising the enzymes for tricarboxylic acid cycle and enzymes for anaerobic metabolism. Other sarcoplasmic proteins are enzymes for glycolytic pathways, lipid and protein synthesis, and amino acid metabolism (Scopes, 1964). Two groups of proteins those are closely correlated with meat quality during postmortem aging are muscle pigment protein myoglobin and the muscle proteases including cathepsin, caspase, proteasome and calpain.

Myoglobin is a heme protein which is responsible for the red color of fresh meat. It is comprised of a single peptide protein, globin, and the prosthetic group heme. The center of the heme ring has an iron atom with six coordination sites (Livingston and Brown, 1981).

Nitrogen atoms bind four of these six coordination sites in the plane and other two sites are perpendicular to the plane. One of the perpendicular sites binds to the histidine residue of the globin and another site is free to combine other ligands. The fresh meat color is determined by the molecules bound to the sixth coordination site and the oxidation state of the iron. The three forms of myoglobin are deoxymyoglobin (purple, ferrous iron), oxymyoglobin (red, ferrous iron) and metmyoglobin (brown, ferric iron). Myoglobin distribution is dependent on the species, sex, muscle fiber types, age and physical activities. For example, muscles with relatively high proportions of red fibers have greater content of myoglobin compared to

muscles with high percentage of white fibers. At comparable ages, the male animals contain

more myoglobin than females and castrate animals (Aberle et al., 2001).

Cathepsin is a complex group of peptidases present in the lysosomes of muscle fibers.

These enzymes include cysteine proteases (cathepsins B, L, H, S, F and K), aspartic

proteases (cathepsins D and E) and a serine protease (cathepsin G). Most of these enzymes

are active at acidic conditions and their contribution to meat tenderization has been a

controversial area for many years. Several arguments support that cathepsins have no major

roles in fresh meat quality. Cathepsins are located in lysosomes and thus they need the

breakdown of lysosomes to have accessibility to their myofibrillar substrates (Koohmaraie,

1996). Both myosin and actin are substrates of cathepsins, but little or no degradation

products of myosin and actin have been detected during postmortem aging (Koohmaraie,

Whipple, Kretchmar, Crouse and Mersmann, 1991).

Another muscle protease that has drawn the attention for fresh meat quality in recent years is caspase. The major function of caspase in organism is involved with programmed cell death (apoptosis) that selectively eliminates the excessive, damaged or potentially dangerous cells (Hengartner, 2000; Kerr, Wyllie and Currie, 1972). The substrates of caspase that have important structural roles in skeletal muscle included αII- and βII-spectrin, actin, vimentin and desmin (Byun, Chen, Chang, Trivedi, Green and Cryns, 2001; Chen, Chang,

Trivedi, Capetanaki and Cryns, 2003; Wang, 2000). Caspase-induced cell death may be involved in the regulation of fresh meat quality in the early stage before rigor mortis (Ouali et al., 2006). In postmortem muscle, increased intracellular free radicals may cause dysfunction in mitochondria to start apoptotic cell death (Halliwell, 1991; Machlin and Bendich, 1987).

In addition, electrical stimulation may damage the nervous system of muscle cells and this alteration at the neuromuscular junction may cause apoptosis in postmortem skeletal muscles

(Chrystall and Devine, 1985; Sandri and Carraro, 1999). Caspase 3, 8 and 12 were found to be expressed in several porcine muscles and their levels were variable among muscles including trapezius, psoas, longissimus dorsi and semitendinosus (Kemp, Parr, Bardsley and

Buttery, 2006). The shear force of porcine longissimus muscle was significantly correlated with the caspase activity (caspase 3/7 and caspase 9) and levels of spectrin degradation were induced by caspase during postmortem conditioning (Kemp, Bardsley and Parr, 2006).

However, activated caspase 3 was not detected in bovine longissimus thoracis and sternomandibularis muscle during postmortem aging and thus caspase may not be associated with beef tenderization (Underwood, Means and Du, 2008). Obviously, more studies need to be conducted in this area to determine whether the caspase family is involved in protein degradation and whether it contributes to fresh meat quality during postmortem aging.

Proteasome is a widely distributed protease that has been indentified in most of tissues and cells examined so far. The 20S proteasome exists in a free state or is associated with regulatory complexes including 19S and 11S complexes to form 26S proteasome (Hendil,

Hartmann-Petersen and Tanaka, 2002). The main function of the proteasome is to degrade unneeded or damaged proteins by non-lysosomal proteolysis regulating cellular functions including antigen processing, cell differentiation and apoptosis (Adams, 2003; Baumeister,

Walz, Zühl and Seemüller, 1998; Ciechanover, Orian and Schwartz, 2000). Proteasome only can degrade proteins which are labeled by a regulatory protein ubiquitin. The C-terminal glycine residue of ubiquitin can covalently bind to the ε-amino groups of lysine residues of

substrate proteins. ATP provides energy for the formation of these covalent bonds between

ubiquitin and substrate proteins (DeMartino and Slaughter, 1999; Haas, Warms, Hershko and

Rose, 1982). In the absence of ATP, the 26S proteasome complex can be dissociated into 20S

proteasome (Peters, Franke and Kleinschmidt, 1994). In skeletal muscle, the distribution of

proteasome is muscle fiber type dependent and the oxidative muscles are reported to have high concentration of 20S proteasome (Dahlmann, Ruppert, Kuehn, Merforth and Kloetzel,

2000). Proteasome has been found to be activated and retain most of its activity after 7 days of aging in postmortem bovine muscle (Lamare, Taylor, Farout, Briand and Briand, 2002).

When myofibrils were treated with purified 20S proteasome, proteasome could induce proteolysis and this degradation was associated with changed muscle structure especially in

Z-line and M-line (Dutaud, Aubry, Guignot, Vignon, Monin and Ouali, 2006; Robert,

Briand, Taylor and Briand, 1999; Taylor, Tassy, Briand, Robert, Briand and Ouali, 1995).

The proteins that are possibly degraded by 20S proteasome including myosin heavy chain, nebulin, α-actinin, actin, tropomyosin, troponin and myosin light chain (Houbak, Ertbjerg and Therkildsen, 2008; Matsuishi and Okitani, 1997). From these studies, proteasome can be activated in postmortem muscle and potentially cause protein degradations. These studies support the possible roles of proteasome in meat tenderization during postmortem aging.

Stromal proteins

About 10-15% of total muscle proteins are stromal proteins including connective tissues proteins, noncollagenous proteins and some membrane proteins. The major proteins composed of connective tissue are collagen and elastin (Aberle et al., 2001). Connective

tissues play critical roles in mechanical support for the blood vessels and nerves to serve the whole muscle tissue and each muscle fiber. In developing muscles, cell-matrix interactions are involved in proliferation and growth of muscle cells (Purslow, 2002). Contractile forces are transmitted through cell-matrix interactions and endomysial connective tissues in mature skeletal muscles (Trotter and Purslow, 1992).

Muscle fibers are surrounded by fine collagen fibers and these connective tissues are called the endomysium. The endomysium is located outside of the cell membrane of skeletal muscle fibers. Muscle fibers are grouped together to lead to the formation of primary bundles. These primary bundles are further gathered to form secondary muscle bundles. A sheath of collagenous structure named perimysium surrounds both primary and secondary bundles. The epimysium is the connective tissue that surrounds entire muscle and connects tendons to link muscles to bones (Bailey and Light, 1989; Huff-Lonergan and Lonergan,

2005). All of these connective tissues are important for integral structure and mechanical properties of skeletal muscles.

In animal body, collagen is the most abundant protein and is the major protein for connective tissues. Collagen is also the primary component of tendons and ligaments. At the present, 19 different collagens have been identified. Among these isoforms, the fibrous type

I, II and III and the non-fibrous type IV of membrane are the major types (Bailey and Paul,

1998). Despise this variety, glycine-X-Y is the tripeptide repeat in the primary sequence of all isoforms of collagen. Therefore, glycine accounts for one-third of the total amino acids in collagen. Proline and hydoxyproline approximately comprise another one-third of amino acids in collagen. Tropocollagen is the basic unit of collagen fibrils which is stabilized by

hydrogen bonds between glycine in adjacent chains. The intramolecular and intermolecular

cross-linkages within the triple helices make collagen insoluble and increase tensile strength

especially in old animals. From the consideration of meat tenderness, collagen is the main

factor to determine the background toughness of meat. High amounts of collagen and chemically mature collagen are associated with high levels of meat toughness. Even after cooking, some of the cross-linkages in collagen are stable and collagen still retains its physical strength. Only some specific collagenases can degrade collagens and cleave the triple helix to short helical fragments. These fragments can be denatured at a lower temperature and other metalloproteinases including gelatinase can degrade them to amino acids (Aberle et al., 2001).

Elastin is a rubbery protein present in many body tissues including muscle. Aggregation of elastin fibers has a yellow appearance referred to as yellow connective tissue. Similar to collagen, one third of amino acids in elastin are glycine. In contrast to collagen, the primary sequence of elastin has no repeating tripeptides and contains lesser amounts of hydroxyproline (Gallop and Paz, 1975). The formation of cross-links in elastin is initially catalyzed by lysyl oxidase to break down lysyl residues to aldehydes. These aldehydes and schiff base compounds to form complex intramolecular and intermolecular cross-links

(Gallop and Paz, 1975). These cross-links makes elastin resistant to most of digestive enzymes and cooking.

Muscle contraction

Muscle contraction is a multiple step procedure with the involvement of myosin, actin,

ATP hydrolysis and regulatory proteins including troponin and tropomyosin. Muscle

contraction starts with an electrical stimulus from brain or spinal cord. Stimuli are

transmitted from brain or spinal cord to skeletal muscle by motor nerves at the myoneural

junction. Electrical signals can be amplified by a chemical agent, acetylcholine, which is

released from the terminal nerve branches. Acetylcholine can increase the membrane

permeability to Na+ without changing the permeability to K+. Therefore, Na+ can enter into the cells to change the membrane potential between inside and outside of muscle cells in resting state. When these action potentials are transmitted to sarcoplasmic reticulum by transverse tubule system, it can stimulate the calcium release from the sarcoplasmic reticulum to the cytoplasm of muscle cells. The calcium concentration in sarcoplasmic fluid can be increased by 1000 fold from 10-7 to 10-4 moles/liter (Aberle et al., 2001). Calcium ions bind the regulatory protein troponin-C resulting conformational changes of troponin. These changes ultimately cause the movement of troponin I and tropomyosin to expose the myosin binding sites on actin. The myosin head interacts with actin to form actomyosin between thin and thick filament. The myosin ATPase is activated to hydrolyze ATP in this condition. The interaction between myosin and actin results in the release of inorganic phosphate and ADP inducing a conformational change in the myosin molecules. Myosin hinge bending tilts the head through approximately 45° and this motion pulls the thin filaments towards the M line called “power stroke”. The power stroke generates forces to cause the thick filament to slide past the thin filament causing sarcomere shortening (Huxley and Niedergerke, 1954). For

muscle relaxation, the enzyme cholinesterase is released to break down acetylcholine. As a

result, muscle membrane is repolarized and returns the normal electrical potential of its

resting value. Calcium ions are pumped back into the sarcoplasmic reticulum using ATP as

energy to overcome the calcium concentration gradient between the cytoplasm of muscle

cells and the sarcoplasmic reticulum. Lower calcium concentration results in troponin and

tropomyosin to recover their normal conformations. Troponin and tropomyosin interact with

each other blocking the myosin binding sites on thin filament. The re-binding of ATP to the

myosin head can decrease the affinity for the interaction between actin and myosin and

allows them to separate. No tension is available between thin and thick filament in the

absence of actomyosin. The two filaments can passively slide past each other and the muscle

cell returns to its resting conditions. In postmortem muscle, no energy is available when

glucose or glycogen supply is exhausted. In the absence of ATP, actin and myosin can not

dissociate with each other. This leads to the production of permanent actomyosin responsible

for increased toughness that develops within the first 24 hour of refrigerated storage

(Wheeler and Koohmaraie, 1994).

Fresh meat quality

Water Holding Capacity

Water is the major component of lean muscle accounting for approximately 75% of its

weight (Aberle et al., 2001). Water holding capacity is generally referred as the ability of fresh meat to retain both inherent and added water. The water holding capacity can be determined by several methods including filter paper press (Kauffman, Eikelenboom, van der

Wal, Merkus, and Zaar, 1986), centrifugation measurement (Honikel, 1998), gravimetrical

bad method (Honikel, 1998) and nuclear magnetic resonance relaxation methods (Bertram,

Anderson and Karlsson, 2001). Inconsistent and low water holding capacity are major

problems for pork producers. These defects can largely influence the acceptance of

consumers and industry profitability. Pale, soft and exudative (PSE) and dark, firm and dry

(DFD) are two extreme conditions associated with the large variation in drip loss. Several

investigations have reported that at least half of pork produced in the US has unacceptably

high drip or purge loss (Kauffman et al., 1992). Stetzer and McKeith (2003) indicated that as

much as 15.5% of pork was PSE and this inferior quality costs the pork industry in the USA

$100 million annually (Carr, Kauffman, Meeker and Meisinger, 1997). Abnormal drip loss

has a great influence on the quality of meat products including tenderness, juiciness and

sensory perception. Among pork quality traits, high drip loss was found to be associated with

lighter color, lower tenderness scores, less flavor and more off-flavor (Huff-Lonergan, Baas,

Malek, Dekkers, Prusa and Rothschild, 2002). Because of potential economic losses, water

holding capacity has been a significant research area for many years. Many factors have been

reported to affect water holding capacity including genotype, feeding strategy, pre-slaughter

stress, chilling rate, aging, muscle fiber types and even protein oxidation and nitrosylation.

Among these factors, temperature and pH including the rate and extent of pH decline

are critical for water holding capacity. After animals are slaughtered, they lose the ability of their circulatory system to provide nutrients and oxygen for muscles. Muscle metabolism irreversibly shifts to anaerobic metabolism which produces lactic acid. Since lactic acid can not been transported to liver or heart by circulatory system from the postmortem muscle,

lactic acid is retained in the muscles. Accumulation of lactic acid decreases muscle pH from

7.4 of living muscle to around 5.3-5.7 at 24 hours which is the ultimate pH in normal conditions (Briskey and Wismer-Pedersen, 1961). A fast rate of pH decline plus high body temperature can cause denaturation of muscle proteins resulting in the loss of their functionality and decreased water holding capacity. pH induced myosin denaturation can result in the shrinkage of myosin head and this shrinkage can decrease the space between myofibrillar filaments to force the water out of myofibrils and increase drip loss (Offer and

Knight, 1988). Bee, Anderson, Lonergan and Huff-Lonergan (2007) determined that the rate and extent of pH decline influenced fresh meat quality. The porcine longissimus muscles in their study were divided into two groups based on their three hour postmortem pH. Protein degradation including intermediate filament protein desmin and costameric proteins vinculin and talin, µ-calpain autolysis and drip loss were measured during postmortem aging. The high pH group showed greater proteolysis of desmin after 24 and 48 hours of postmortem storage than low pH groups. Extent of talin degradation was greater in the high pH group than in the low pH group in all storage periods. These differences were suggested by the authors to be due to the earlier autolysis of µ-calpain and earlier loss of its proteolytic activity in low pH group. The 76 kDa subunit, the autolyzed products of µ-calpain large 80 kDa subunit, at 24 hours of postmortem storage were negatively correlated with intensity of intact desmin of 7 days of storage. In another study, the percentage of 76 kDa products at 24 hours postmortem were also negatively correlated with drip loss over 5 days in pork (Zhang et al.,

2006). Therefore, earlier activation of µ-calpain tends to be associated with high levels of protein degradation like desmin and decreased drip and purge loss.

Two major genes have been identified to be associated with abnormal pH decline and

high drip loss associated with PSE pork. They are the Halothane (HAL) and the Rendement

Napole (RN-) genes. The Halothane gene is associated with accelerated pH decline and the

RN- is related to low ultimate pH. In HAL gene pigs, arginine is changed to cysteine in the

615th amino acid due to the single substitution of T for C at nucleotide 1843 in the Ca2+

release channel (Ryanodine receptor, RyR1) of skeletal muscle (Fujii et al., 1991). This

mutation disrupts the normal ability of Ca2+ release channel which functions in regulating

Ca2+ release from sarcoplasmic reticulum to sarcoplasm of muscle cells. Under this condition,

the pigs are hypersensitive to halothane gas and to stress to open RyR1 resulting in fast Ca2+

release and high Ca2+ concentration in the sarcoplasm of muscle cells (Mickelson, Gallant,

Litterer, Johnson, Rempel and Louis, 1988). Muscles have accelerated rate of anaerobic metabolism due to the accelerated release of Ca2+ and this leads to a faster rate of lactic acid

accumulation and rapid pH decline (Lundstrom, Essen-Gustavsson, Rundgren, Edfors-Lilja

and Malmfors, 1989). Fast rate of pH decline with high temperature can cause the

denaturation of muscle proteins resulting in the loss of their functionality and high drip loss.

Compared with Halothane gene, muscles in RN- gene pigs have higher glycogen

potential for extended glycolysis compared to normal pigs. Glycogen potential is defined as

the concentration of glycogen, glucose, glucose 6-phosphate and lactate in postmortem muscle indicating muscle’s capacity for postmortem glycolysis. At slaughter, it can be used

as an index of the extent of pH decline (Monin and Sellier, 1985). The initial study found that

Hampshire breed had two to three times of glycogen level than Chester White and Poland

China breeds (Sayre, Briskey and Hoekstra, 1963). Subsequent studies confirmed that greater

glycogen stores were responsible for extended glycolysis and lower ultimate pH in RN-

carrier pigs (Monin and Sellier, 1985). However, no differences were found in the activities

of glycogen phosphorylase and glycogen debranching enzyme as well as the rate of early

postmortem pH decline in RN- carrier pigs (Estrade, Ayoub, Talmant and Monin, 1994;

Molin and Sellier, 1985). Therefore, RN genotype mutation tends to increase glycogen potential without influencing the rate of glycolysis. Milan et al. (2000) discovered that pigs possessing RN gene have a non-conserved substitution in protein kinase adenosine monophosphate-activated γ3-subunit gene (PRKAG3). The PRKAG3 gene is responsible for encoding muscle specific isoform of regulatory γ-subunit of adenosine monophosphate activated protein kinase (AMPK). During muscle contraction, the increase of ATP consumption changes the ratio of AMP/ATP to activate AMPK resulting in fatty acid oxidation and glucose uptake. (reviewed by Winder, 2001). With the low ultimate pH especially when the pH is close to the protein’s isoelectric point, proteins have decreased ability to hold and attract water and this is associated with low water holding capacity.

To reduce the levels of muscle glycogen stores in normal pigs, fasting preslaughter is a common practice in meat industry. Fasting 24 hours before slaughter result in lower glycogen levels in longissimus dorsi and almost complete glycogen depletion in pig neck semispinalis muscle at slaughter (Bertol, Ellis, Ritter and Mckeith, 2005; Jones, Rompala and Haworth,

1985; Wittmann, Ecolan, Levasseur and Fernandez, 1994). Increased ultimate pH was reported in the same study in longissimus dorsi after 48 hours fasting while after 24 hours fasting in semispinalis.

Feeding animals with supplementing ingredients is reported to be an effective strategy to improve water holding capacity for pork. Vitamin E is lipid-soluble antioxidant which is able to quench reactive free-radicals. It was hypothesized that vitamin E may have a beneficial effect on drip loss through keeping the integral membrane structure of muscle cells by preventing oxidation during postmortem storage (Asghar, et al., 1991). In British

Landrace pigs, feeding 500 mg vitamin E/kg diet for 46 days reduced drip loss by 45% and

54% respectively in longissimus thoracis of Halothane positive and Halothane negative pigs.

Dietary supplement with 1000 mg vitamin E/kg diet significantly decreased the occurrence of

PSE carcass in PSE-prone Landrace x Large White Halothane positive pigs (Chea, Chea and

Krausgrill, 1995). In the same study, supplementation of vitamin E did not influence drip loss of a red muscle masseter. Therefore, effects of vitamin E supplementation on drip loss seem to vary with muscle fiber types. This tendency was also reported in bovine muscles. Dietary vitamin E supplementation could decrease drip loss of semitendinosus muscle while it increased drip loss of psoas major muscle. No significant effects were found in longissimus lumborum muscles by supplementing vitamin E (den Hertog-Meischke, Smulders, Houben and Eikelenboom, 1997). Inconsistent results were also reported that drip loss was not different in porcine loin muscles between control and vitamin E supplementation groups (60 mg of vitamin E/kg feed from 20 to 100 kg live weight) (Dirinck, Winne, Casteels Frigg,

1996). The variation among these studies may be due to different amounts of vitamin E supplementation, different muscle compositions and different stability of mitochondria and sarcoplasmic reticulum caused by oxidation of membrane phospholipids (den Hertog-

Meischke et al., 1997). For example, the supplementation dose of vitamin E was 100 E/kg

feed in Connon’s (1996) study compared to 1000 E/kg supplementation in Chea’s paper

(1995). Chea et al. (1995) suggested that vitamin E could stabilize the membrane of

sarcoplasmic reticulum and inhibit the activity of phospholipase A2 which is present in skeletal muscle, erythrocyte and other tissues (Diplock, Lucy, Verrinder and Zielenlowski,

1977). Phospholipase A2 is the enzyme for the hydrolysis of phospholipids which produces

long-chain unsaturated fatty acid and lyso-derivatives (Nachbaur, Colbeau and Vignais,

1972). These products could induce the uncoupling and swelling of the membrane of

sarcoplasmic reticulum and mitochondria (Chea and Chea, 1981). Therefore, the inhibition of

phospholipase A2 induced by vitamin E could prevent calcium leakage into sarcoplasm to be

associated with lower sarcoplasmic calcium concentration. Lower calcium in sarcoplasm

may be associated with slower rate of pH decline and lower levels of protein denaturation

resulting in increased water holding capacity (Cheah, Cheah, Crosland and Casey, 1984).

Since red and white muscles have different amounts of mitochondria and sarcoplasmic

reticulum, this can explain variations of vitamin E effects on drip loss among different

muscles.

Drip loss formation

The majority of water in muscle is held between thin and thick filaments within

myofibrils in living muscle (Offer and Cousins, 1992). Other compartments for holding water

include: between the myofibrils, between the myofibrils and the cell membrane, between

muscle cells and between muscle bundles (Huff-Lonergan and Lonergan, 2005; Huff-

Lonergan and Lonergan, 2007; Offer and Cousins, 1992). As postmortem muscle undergoes

rigor, the shrinkage of myofibrils can decrease the space for holding water within myofibrils.

When the shrinkage of myofibrils is translated into entire muscle cells, water can be forced out from the myofibrillar structure and the intracellular space into extracellular space to be lost as drip (Offer and Knight, 1988). Degradation of intermediate filament proteins like desmin and costameric proteins such as talin and vinculin could remove inter-myofibrillar linkages and costameric connections. These removals then limit the shrinkage of muscle fibers and entire muscle cells to decrease drip formation. This hypothesis was supported by

Kristensen and Purslow (2001) who reported that water holding capacity decreased in the first 2 to 7 days postmortem and then increased after that in postmortem pork. The increased water holding capacity was evidenced by water loss with 3.9% at day 1, 11.9% at day 3 and

4.5% in the remaining period until day 10 postmortem. In agreement with this research,

Davis et al. (2004) found that pork loins with high purge loss had less extent of desmin degradation, while low purge loss pork loins showed high degree of desmin proteolysis. In another study (Schafer, Rosenvold, Purslow, Andersen and Henckel, 2002), the relationship between drip loss and early postmortem metabolism and structural changes in pork was investigated. Using the analysis of partial least squares regressions, total extracellular space was shown to account for 39% of the variation of drip loss and areas between fibers account for 46% of drip variation. All of these studies support the idea that the extent of postmortem degradation of key cytoskeletal proteins is involved in the formation of drip channels and the amount of drip loss during postmortem storage. In addition, some studies provide the hypothesis that sarcoplasmic proteins may be involved with water holding capacity of postmortem muscle. Wilson and van Laack (1999) studied the influence of sarcoplasmic

proteins on water holding capacity by combining the myofibrils with protein containing and

protein free sarcoplasmic extract. Protein-free sarcoplasmic extract were prepared by heating

sarcoplasmic extract at 80 oC for 30 minutes in a water bath. Myofibrils combined with protein-containing sarcoplasmic extract had higher water holding capacity than myofibrils combined with sarcoplasmic extract with protein removal (Wilson and van Laack, 1999). The exact mechanism is still not clear to explain the possible effects of sarcoplasmic proteins on water holding capacity. Bendall and Wismer-Pedersen (1962) suggested that denatured sarcoplasmic proteins may be attracted by the myofibrils and block the charged groups of myofibrillar proteins that bind water. Another possible explanation that has been suggested is that the presence of sarcoplasmic proteins may influence ionic strength and osmotic value of meat to contribute the variation of water holding capacity (Wison and van Laack, 1999).

Meat tenderness

Meat tenderness has been reported as the most important quality attribute for consumers for decades (Huffman et al., 1996). Inconsistency and variability in meat tenderness have been known as a major problem for the meat industry (Morgan et al., 1991). In the survey by

Jeremiah (1982), toughness was found to be the main unacceptable attribute for beef that influences acceptability and selection by consumers. Toughness has also been found to be a common problem in pork and lamb in this study. Tenderness defects have been estimated to cost the beef industry over $216,000,000 annually and these are with conservative estimates

(Morgan, 1995). Three major factors are believed to determine final values of meat tenderness: background toughness including the amount and the solubility of connective

tissues; toughening process induced by sarcomere shortening during rigor development; and tenderization step resulted from proteolysis during postmortem aging (Koohmaraie, Doumit and Wheeler, 1996; Wheeler and Koohmaraie, 1994).

The background toughness is largely determined by the amount and the characteristics of connective tissues. Connective tissues can be divided into endomysium (surrounding muscle cells/fibers), perimysium (surrounding muscle fiber bundles) and epimysium

(surrounding entire muscle). Among these connective tissues, perimysium organization is particularly important for background toughness as evidenced by significant correlation between the amount of perimysium and tenderness in both chicken and beef (Strandine,

Koonz and Ramsbottom, 1949). Properties of connective tissues can be affected by species, age and locomotion. Usually, animal muscles that are largely involved in locomotion have greater cross-linkage in connective tissues resulting in a coarse texture. The amount of connective tissues can contribute to the difference in shear force among different muscles

(Ramsbottom and Strandine, 1948). Pork contains less levels connective tissue compared to beef as determined by hydroxyproline content which is the index for the amount of collagen.

Amount of connective tissues is diluted during the rapid growth period as the fiber size increases (Aberle et al., 2001). However, 30 months old beef steers tend to be tougher than market weight beef due to lower solubility and greater amounts of cross-linking in connective tissues.

During conversion of muscle to meat, depletion of ATP makes it difficult to break up cross-bridges between actin and myosin filaments. Postmortem actomyosin and shortened sarcomere length are related to increased meat toughness. Wheeler and Koohmaraie (1994)

reported that sarcomere length of lamb longissimus thoracis et lumborum was reduced from

2.24 µM at 0 hour to 1.69 µM after 24 hour postmortem aging. At the same period, shear

force was significantly increased from 5.07 kg at slaughter to 8.66 kg after 24 hour of

postmortem storage. In the study of Marshall and Leet (1966), tender muscles had sarcomere

lengths of 2.0-2.5 µM, while sarcomere lengths were in the range of 1.7-2.0 µM in tougher

muscles. Consistent with this, strong correlations have been found between meat toughness

and sarcomere length when sarcomere length was less than 2 µM (Bouton, Harris, Shorthose

and Baxter, 1973; Herring, Cassens, Suess, Brungardt and Briskey, 1967). Several methods

have been reported to prevent sarcomere shortening induced meat toughening. Pelvic

suspension by hanging carcass from obturator foramen was reported to prevent shortening of

some muscles and thus inhibit toughening process (Hoestler, Landmann, Link and Fitzhugh,

1970). This suspension was proved to be able to increase sarcomere length compared to

normally shortened samples (Moller, Kirkegaard and Vestergaard, 1987) and improve

tenderness of beef and pork (Eikelenboom, Barnier, Hoving-Bolink, Smulders, and Culioli,

1998; Moller and Vestergaard, 1986; Taylor et al., 1995). When researchers used clamps to

prevent shortening of lamb longissimus, shear force values did not increase during rigor

development in the first 24 hour after slaughter (Koohmaraie, Doumit and Wheeler, 1996).

Claus (1994) developed a new method called tendercut process by stretching or restraining

prerigor muscles to improve meat tenderness. Carcasses were cut between 12th and 13th thoracic vertebrae region in longissimus muscle and the round/loin junction in round and sirloin muscles. This tendercut process could increase sarcomere length and improve meat tenderness in pork and beef (Wang, Claus and Marriott, 1994, 1996; Claus, Wang and

Marriott, 1997). These studies indirectly support the hypothesis that sarcomere shortening is

responsible for meat toughening during rigor development. Two abnormal shortening

phenomenon known as cold shortening and thaw rigor can cause extreme toughness. Thaw

rigor is caused by thaw of frozen prerigor muscle and can lead to 80% of physical shortening

of original length. Cold shortening is developed when prerigor muscles are chilled below 15

oC to 16 oC. Relatively, cold shortening induced muscle contraction is less severe compared to that of thaw rigor (Aberle et al., 2001). Both of these shortening events occur in the presence of ATP and are driven by sudden release of calcium into sarcoplasm or by failure of calcium uptake into sarcoplasmic reticulum (Aberle et al., 2001). Increased accumulation of calcium in sarcoplasm of muscle cells causes severe muscle contraction and the formation of permanent actomyosin to be associated with tough meat. In industry, electrical stimulation is applied to prevent cold shortening by depleting ATP and hastening of rigor mortis in beef and lamb (Carse, 1973; Chrystall, Devine, Ellery and Wade, 1984; Davey, Gilbert and Carse,

1976).

It is well recognized that postmortem proteolysis of myofibrillar and other structural proteins is responsible for the improvement of meat tenderness during aging. An initial study found that Ca2+ containing solutions degraded Z-disk structure of muscle strips (Busch,

Stromer, Goll and Suzuki, 1972). Higher activity of calcium-activated factor was associated

with increased tenderness (Goll, Stromer, Olson, Dayton, Suzuki and Robson, 1974) and this

calcium-activated factor was identified as m-calpain in later studies. In industry, injection of

CaCl2 was applied to improve meat tenderness through enhancing calpain induced

proteolysis (Koohmaraie, Whipple and Crouse, 1990). Calpain induced proteolysis can cause

the disruption of muscle structure including degradation of Z-disks and myofibril rupture at the Z-disk and I band junction. These protein degradation leads to the production of myofibril fragmentation and myofibril fragmentation index is an effective indicator of meat tenderness (Davey and Gilbert, 1969; Olson, Parrish, Dayton and Goll, 1977). The proteins that can be degraded during postmortem aging include nebulin, titin, integrin, dystrophin, troponin, desmin, vinculin, talin and filamin as reviewed by Robson et al. (1997). The degradation patterns of these proteins were similar to the proteolysis patterns seen when proteins were incubated with µ-calpains in vitro (Huff-Lonergan et al., 1996). Degradation of intermediate filament proteins like desmin and filamin could disrupt interconnections between adjacent filaments and the periphery layer of myofibrils to the sarcolemma.

Costameres were largely degraded during 24 h of postmortem storage leading to detachment of myofibril from the sarcolemma (Ouali, 1990; Taylor et al., 1995). Proteolysis of costameric proteins including vinculin and dystrophin may destroy attachments between the myofibrils and the cell sarcolemma in Z-disk and M-line areas (Taylor et al., 1995). The integral structure of muscle could be disrupted by the degradation of cytoskeletal proteins of titin and nebulin and this can lead to more myofibril fragmentation and improved meat tenderness (Huff-Lonergan, Parrish and Robson, 1995; Taylor et al., 1995).

Muscle fiber types and meat quality

Many classifications to differentiate muscle fiber types have been developed based on the structural and functional properties of the muscle fibers. One popular method is to use myosin heavy chain (MHC) isoform to delineate fiber types. According to the major MHC of

adult mammalian skeletal muscle, fiber types are categorized as type I (MHCIβ), type IIA

(MHCIIα), type IIX (MCHIIx) and type IIB (MHCIIb) (Pette and Staron, 2000). Based on

the speed of muscle contraction to stimulation, muscle fibers are also characterized as slow

and fast fibers. Type I fibers are considered slow contraction fibers, while type II are fast

contraction fibers. The muscle fibers are also classified as red (type I and type IIA) and white muscles (type II B and type IIX) based on color intensity (Aberle et al., 2001). Type I and type IIB are also known as slow-oxidative and fast-glycolytic fibers respectively, while type

IIA and IIX are intermediate, fast oxidative-glycolytic fibers (Klont, Brocks and

Eikelenboom, 1998). In postnatal skeletal muscle, MHC isoforms can be reversibly transitioned with a fixed order of MHCIβ, MHCIIα, MCHIIx and MHCIIb by neuromuscular activity, mechanical loading or hormones (Schiaffino and Reggiani, 1996). Muscle fibers have different expression of structural proteins and metabolic enzymes resulting in different chemical and physical properties. Compared with white muscle fibers, red fibers have a higher content of myoglobin, greater proportion of oxidative enzymes, smaller fiber size and more numerous mitochondria (Aberle et al., 2001). Christensen, Kok and Ertbjerg (2006) reported differences in mechanical properties between type I and type II B of single porcine muscle fibers. After 1 day postmortem storage, type II fibers had higher breaking loads for fracture than type I fibers and breaking loads of both type I and II fibers were decreased during 8 day storage. For type I fibers, breaking loads from vastus intermedius muscles was higher than that from longissimus dorsi (Christensen et al., 2006).

Muscle fiber types have been reported to be largely variable among large muscles of the chuck and round muscles in bovine (Hunt and Hedrick, 1977; Kircheofer, Calkins and

Gwartney, 2002). The chemical and physical differences of muscle fibers may affect postmortem metabolism during the conversion of muscle to meat. Fiber types have been found to be associated with different patterns of protein expression and postmortem proteolysis (Christensen, Henckel and Purslow, 2004). The red porcine iliocostalis muscle showed a slower decline in the intensity of desmin and vinculin than the while porcine longissimus muscle (Morrison, Mielche and Purslow, 1998). Longissimus dorsi muscle of pork has been shown to have a higher percentage of MHC IIB and a lower percentage of

MHC IA than semimembranosus. This was associated with slower rate of pH decline and more degradation of desmin and troponin-T (Melody et al., 2004).

Ashmore (1974) reported that improving the quantity of muscle by changing the fast oxidative-glycolytic fibers to fast-glycolytic could have detrimental effects on meat quality.

Consistent with this, the fast oxidative-glycolytic MHC IIA and IIX fibers were found to be associated with favorable meat quality including intense color, higher ultimate pH, better water holding capacity and higher tenderness values in pigs (Chang et al., 2003). The proportion of type IIB fiber of longissimus dorsi muscle in pig was negatively correlated with early postmortem pH and shear force and positively related to drip loss (Karlsson et al.,

1993; Ryu and Kim, 2005). Lonergan, Huff-Lonergan, Rowe, Kuhlers and Jungst (2001) suggested that selection for lean growth efficiency of Duroc pigs was associated with poorer water holding capacity and higher shear force values. This is likely due to the fact that this selection for lean growth tended to increase the percentage of type IIB muscle fibers (Essén-

Gustavsson, 1993). The relationship between muscle fibers and meat quality has also been reported in beef (Calkins et al., 1981; Ozawa et al., 2000). Negative correlations were

detected between large fast glycolytic fibers and meat tenderness and marbling scores, while slow oxidative fibers were found to be positively correlated with these meat attributes

(Calkins et al., 1981; Crouse, Koohmaraie and Seidemann, 1991). In several beef round muscles, MHC type I was found to be positively correlated with juiciness and negatively correlated with chewiness (Anderson, Steadham, Fedler, Prusa, Lonergan and Huff-

Lonergan, 2008). Therefore, it seems that muscle fiber difference may contribute to the variation of fresh meat quality among different muscles.

Calpain family

Calpains

Meat tenderization during postmortem aging is mainly determined by postmortem proteolysis within muscle fibers. Several proteases and their inhibitors that are possibly involved in meat tenderization as reviewed by Sentandreu, Coulis and Ouali (2002), but the calpain system is believed to be the major protease responsible for meat tenderness during postmortem aging (Huff-Lonergan et al., 1996; Koohmaraie, 1994; Taylor et al., 1995).

Calpains are a family of cysteine proteases with 14 known members requiring reducing conditions for their activation (Goll et al., 2003). The calpain superfamily can be divided into three categories based on their tissue distribution and domain localization: ubiquitous calpains (µ- and m-calpain), tissue specific calpains (such as calpain 3 or p94) and atypical calpains (Ono, Sorimachi, and Suzuki, 1998; Sorimachi, Ishiura and Suzuki, 1997). In skeletal muscle, four calpains have been reported to be expressed including ubiquitous µ- and m-calpain, muscle specific isoform calpain 3/p94 and calpain 10.

The calpain system was originally known to be comprised of two ubiquitous proteases

µ- and m-calpain and their specific inhibitor calpastatin. Among all members of calpains, µ-

and m-calpains are the most characterized isoforms and are composed of heterodimers. The

heterodimers are two subunits with molecular weight of 80 kDa and 28 kDa. The large

catalytic 80 kDa subunits of µ- and m-calpain share 50-60% sequence homology, while the

small regulatory 28 kDa subunits are identical in both µ- and m-calpain encoded by a single

gene on chromosome 19 in humans (Carafoli and Molinari, 1998; Ohno et al., 1990). The

typical structure of large subunit is composed of four domains: Domain I: N-terminal domain

or autolytic domain; Domain II: cysteine containing catalytic domain (containing domain IIa

and IIb); Domain III: switch domain and Domain IV: calcium binding or calmodulin-like

domain. The small subunit is composed of Domain V (N-terminal domain of the small

subunit) and Domain VI (calcium binding or calmodulin-like domain (Goll et al., 2003). The

µ- and m-calpain need different calcium concentrations for their proteolytic activation as

their names indicate. m-Calpain requires 200-1000 µM while µ-calpain needs 3-50 µM

2+ calcium for their half maximal proteolytic activity ([Ca ]0.5) in vitro studies (Cong, Goll,

Peterson and Kapprell, 1989). In addition to binding to calmodulin-like domains IV and VI

of calpains, calcium was found to bind negatively charged loops of domain III to form a

catalytic center and mediate interactions with membrane (Strobl et al., 2000). Subdomain IIa

and IIb each can bind one atom of calcium in a peptide loop (Moldoveanu, Hosfield, Lim,

Elce, Jia and Davies, 2002). The calcium binding to multiple sites of both calpains reduces

the distance of Cysteine 105 in domain IIa to Histidine 262 and Asparagine 286 in domain

IIb from 10.5 to 3.7 Å for the formation of a catalytic triad and thus activates the calpains

(Goll et al., 2003).

During postmortem storage, the calpain system is generally believed to be the major

factor for protein degradation and meat tenderization. The earliest study to suggest this

hypothesis was that myofibrils incubated with Ca2+containing solutions had rapid loss of Z- disk structures and this was associated with improved tenderness (Busch et al., 1972). This factor was called Ca2+-activated factor and muscles with more Ca2+-activated factor activity showed higher value of meat tenderness (Goll et al., 1974). This Ca2+-activated factor is

identified as calpains in later studies.

It is known that calpains autolyze once they are activated and extensive autolysis causes

calpains to lose their proteolytic activity (Nagainis, Sathe, Goll and Edmunds, 1983). The

activity of m-calpain has been shown to be relatively stable during postmortem aging. For

example, m-calpain of bovine muscle has been shown to only lose 37% of its initial activity

after 7 days of storage (Boehm, Kendall, Thompson and Goll, 1998). Therefore, m-calpain is

not believed to play major roles in postmortem protein degradation. In addition, the

concentration of calcium ions in sarcoplasmic fluid is only approximately 100 nM in relaxed

muscle. In postmortem muscle, the concentration of free calcium increased to 60-100 µM

immediately after slaughter (Ji and Takahashi, 2006; Nakamura, 1973). m-Calpain needs

much higher [Ca2+] to be activated compared to the concentration of sarcoplasmic calcium.

These considerations have led to the conclusion that µ-calpain instead of m-calpain was

largely responsible for postmortem proteolysis and tenderization (Koohmaraie and Geesink,

2006; Koohmaraie et al., 1987). In the study by Huff-Lonergan et al. (1996), degradation

patterns of muscle proteins including titin, nebulin, filamin, desmin and troponin-T in

postmortem bovine muscle were similar to the proteolysis of myofibrils incubated with µ-

calpain in vitro. The postmortem degradation of desmin, nebulin, dystrophin, vinculin,

metavinculin and troponin-T was largely inhibited in µ-calpain knockout mice (Geesink,

Kuchay, Chishti and Koohmaraie, 2006). This study provided direct evidence for the major roles of µ-calpain in postmortem proteolysis. The degradation of these myofibrillar proteins and associated proteins influence fresh meat quality including water holding capacity and meat tenderness as discussed in the desmin and water holding capacity sections of this literature review.

Calpain 3/p94 is a single 94 kDa polypeptide expressed specifically in skeletal muscle.

Calpain 3 polypeptide contains 821 amino acids which have 54% and 51% of sequence homology to catalytic subunit of µ- and m-calpain (Sorimachi et al., 1989). The additional

20-30 amino acids in domain I, an insertion sequence with 62 amino acids in domain II and an insertion sequence with 77 amino acids in domain III of calpain 3 make calpain 3 polypeptide have greater mass than 80 kDa subunit of µ- and m-calpain (Sorimachi et al.,

1989). Calpain 3 localizes at several regions of the sarcomere by binding to titin at the N2 line and M-line (Kinbara et al., 1998; Sorimachi et al., 1995; Spencer et al., 2002). The N2 line region is the region of titin which is very susceptible to proteolysis. Degradation of this region is linked to meat tenderization during early postmortem aging (Taylor et al., 1995).

This leads to the hypothesis that calpain 3 may be involved in the regulation of fresh meat quality. However, extraction of calpain 3 is very difficult from skeletal muscles due to its tight binding to myofibrils. In addition, calpain 3 can autolyze rapidly even at the

physiological levels of calcium without being affected by calpain inhibitors (Kinbara et al.,

1998). The autolysis can completely digest the enzyme and thus make it impossible to be

purified in its active form (Sorimachi and Suzuki, 1992). These difficulties in purifying and

characterizing calpain 3 lead to controversial reports about possible roles of calpain 3 in

postmortem meat tenderization. In a study using calpain 3 knockout mice, no significant

differences were found about structural changes of postmortem muscle and the degradation

of desmin, nebulin, dystrophin, vinculin and troponin-T between the calpain 3 knockout and

control mice during postmortem aging (Geesink, Taylor and Koohmaraie, 2005). These

results were in agreement with the conclusion from the study of Parr et al. (1999) who did

not find significant correlations between variations in shear force and immunoreactivity and

stability of calpain 3 in longissimus dorsi of pork. The authors concluded that the variability

of meat tenderness during postmortem conditioning could not be explained by the variability

of calpain 3. However, the series of publications from Ilian and coworkers presented

different evidence for the roles of calpain 3 in fresh meat quality. Ilian et al. (2001) reported

significant and strong relationships between mRNA levels of calpain 3 and the relative rate

of tenderization in both bovine (longissimus thoracis et lumborum and psoas major muscles)

and ovine muscles (longissimus thoracis et lumborum, psoas major, semimembranosus and

semitendinosus muscles). However, the expression levels of mRNA do not directly parallel the amounts of protein expression and the activity of this enzyme in skeletal muscles. In another study (Ilian, Bickerstaffe and Greaser, 2003), the intact calpain 3 was reduced by

20% and 90% after 1 and 2 days postmortem storage respectively and no intact calpain 3 was detected at 3 days postmortem determined by immunofluorescence microscopy. The decline

in intensity of intact calpain 3 at the Z- and M-line paralleled the degradation of titin and nebulin during postmortem storage. In two other studies, the kinetics of calpain 3 autolysis was found to be strongly correlated with meat tenderization (r=0.930) and the degradation of nebulin (r=0.930) and desmin (r=0.654) in longissimus thoracis et lumborum of ovine (Ilian,

Bekhit and Bickerstaffe, 2004b; Ilian et al., 2004). These studies led this group to conclude that both calpain 3 and calpain 1 may be involved in meat tenderization by degrading structural proteins during postmortem aging. In conclusion, the roles of calpain 3 in postmortem protein degradation and meat tenderization are a controversial topic with currently published studies. More studies need to be conducted to explore its possible effects on fresh meat quality.

The initial study of calpain 10 is in the area of type 2 diabetes in humans (Horikawa et al., 2000). The calpain 10 gene is expressed as eight different isoforms named from 10a to

10 h (Horikawa et al., 2000). Calpain 10 has different domain architecture from ubiquitous m- and µ-calpain. The T domain of calpain 10 replaces the calmodulin-like calcium binding domain IV of m- and µ-calpain. T-domain does not have EF-hand structures indicating a different calcium activation mechanism of this calpain (Dear, Matena, Vingron and Boehm,

1997). The 74.9 kDa calpain 10a is similar to the domains I-III of the catalytic 80 kDa subunit of m- and µ-calpain. The lower molecular weight of the calpain 10 is due to the lesser amounts of amino acids in this subunit. Compared to m- and µ-calpain, calpain 10 has higher pI value due to smaller percentage of acidic residues and similar percentage of basic residues

(Ilian, Bekhit and Bickerstaffe, 2004a). The only paper about the possible involvement of calpain 10 in fresh meat quality was published by Ilian et al. (2004a). A calpain 10 antibody

detected two different proteins with molecular weight of 70.7 and 73.7 kDa in ovine longissimus thoracis et lumborum. During postmortem storage, both of these protein levels declined in sarcoplasmic fraction and showed a concomitant increase in the myofibrillar fraction. For example, the amount of 73.6 kDa protein declined by 92% in sarcoplasmic fraction and increased by 183% in myofibrillar fraction after 3 days postmortem storage.

However, it is difficult to draw the conclusion that calpain 10 contributes to meat tenderization during postmortem aging based on these very limited studies.

Calpain activity assay

Since calpains play critical roles for fresh meat quality during postmortem aging, methods that are consistent and have high sensitivity are needed to purify calpains and determine their activity. Many methods have been developed in past thirty years and different assays have both their advantages and disadvantages.

Among these methods, the traditional assay is to use casein as substrate to measure the generation of trichloroacetic acid soluble peptides digested from casein by calpain at the wavelength of 278 nm (Dayton, Goll, Zeece, Robson and Reville, 1976; Waxman, 1981).

This assay is less sensitive and unsuitable for continuous detection of calpain activity. To improve the sensitivity of the traditional assay, casein has been labeled with different fluorescent compounds such as fluorescamine (Melloni, Pontreomoli, Salamino, Sparatore,

Michetti and Horecker, 1984), ninhydrin (Doi, Shibata and Matoba, 1981), fluorescein isothiocyanate (FITC) (Lonergan, Johnson and Calkins, 1995; Twining, 1984; Wolfe, Sathe,

Goll, Kleese, Edmunds and Duperret, 1989), and 4,4-difloro-5,7-dimethyl-4-bora-3a,4a–

diaza-s-indacene-3-propionic acid (BODIPY-FL) (Kent, Veiseth, Therkildsen and

Koohmaraie, 2005; Thompson, Saldana, Cong and Goll, 2000). Radiolabeled caseins with 14-

C-methylated (Delgado, Geesink, Marchello, Goll and Koohmaraie, 2001) and [3-H]

(Coolican, Haiech and Hathaway, 1986) have been utilized via measuring the radioactivity of degraded peptides. All these methods with labeled compounds have been reported to be much more sensitive than the standard assay. An assay that uses coomassie brilliant blue G-

250 to bind substrates instead of proteolysis products was developed by Buroker-Kilogore and Wang (1993). Casein zymography can detect calpain activities of crude samples without the previous separation of m- and µ-calpains and their inhibitor calpastatin (Raser, Posner and Wang, 1995). In addition to casein, other substrates have been developed for calpain assay. These substrates include some dipeptides such as Z-Leu-Arg-(7-methoxynaphthy)- amide (Z-Leu-Arg-MNA) and Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA)

(Harris, Gregory and Maycock, 1995; Mallya, Meyer, Bozyczko-Coyne, Siman and Ator,

1998) and cytoskeletal proteins such as microtubule-associated protein 2 (MAP2) (Tompa,

Schad, Baki, Alexa, Batke and Friedrich, 1995). Recently, an assay with capillary electrophoresis has been developed by Gu, Whipple-VanPatter, O’Dwyer and Zeece (2001).

This method could improve the sensitivity and the speed of calpain assay. Two to three ng of calpain activity could be detected using Oregon Green labeled β-casein as substrate. The detailed procedures of these assays and their advantages and disadvantages are discussed in the paper section of this thesis.

Calpain autolysis

The autolysis of m-calpain in the presence of Ca2+ was discovered by Suzuki and

coworkers (1981a, 1981b). The autolysis of 80 kDa subunit to 78 kDa in the presence of 1

2+ 2+ 2+ mM free Ca reduced Ca concentration required for [Ca ]0.5 of m-calpain in chicken skeletal muscle. The following studies suggested that µ-calpain also underwent autolysis in

2+ 2+ the presence of 0.1-1 mM Ca and this autolysis decreased [Ca ]0.5 from 50 to 0.5-2 µM

depending on the experimental conditions (Dayton, 1982; Mellgren, Repetti, Muck and Easly,

1982). Now, it is well known that autolysis reduces the mass of 80 kDa subunit of µ-calpain

to a 76 kDa product through a 78 kDa intermediate and that of m-calpain to a 78 kDa product.

For small 28 kDa subunits, both m- and µ-calpain have the same autolysis pattern and both of

them are reduced to 18 kDa fragments. Autolysis of calpains occurs in the domain I of large

catalytic subunit and domain V of small regulatory subunit. Several steps are involved in the

autolysis of calpains by deleting different amounts of amino acids. Three sequential

fragments could be produced from the autolysis of the small subunit with molecular weights

of 26-27 kDa (29 amino acids removed from N-terminal), 22-23 kDa (37 amino acids

removed) and 18 kDa (28 amino acids removed) (McClelland, Lash and Hathawaw, 1989).

For the large subunit of µ-calpain, removal of 14 amino acids from the N-terminal produces a

78 kDa intermediate and the final 76 kDa fragment is formed after the removal of another

additional 12 amino acids (Zimmerman and Schlaepfer, 1991). The sequential removal of 9

amino acids and then 10 amino acids separately from N-terminus produces 78 kDa fragments

from the 80 kDa subunit of m-calpain (Brown and Crawford, 1993). The physiological

significance of calpain autolysis is still unclear and has been a controversial subject for many

years in vivo (Goll et al., 2003).

In postmortem muscle, one of the significant roles of calpain autolysis is to reduce their

2+ calcium requirement for their activation. The value of [Ca ]0.5 of m-calpain was reduced from 400-800 to 50-150 µM and that of µ-calpain from 3-50 to 0.5-2.0 µM (Goll, Thompson,

Taylor, Edmunds and Cong, 1995). This is significant for µ-calpain because this decline of

2+ 2+ calcium requirement may bring [Ca ]0.5 to be closer with free Ca concentration (0.300-1.2

µM) in the sarcoplasm of skeletal muscle cells. However, the Ca2+ requirement for calpain autolysis is much higher than the actual Ca2+ concentrations in living cells. Some studies

suggest that the interaction with other components of muscle cells may decrease Ca2+

requirement for autolysis (Collican and Hathaway, 1984). Autolysis of calpains can be

regulated in vitro by the association with some plasma membrane lipids such as

phosphatidylinositol. The Ca2+ concentration required for half-maximal autolysis of m-

calpain and µ-calpain was reduced by 49% (from 740-780 to 370-400 µM) and 30% (from

190-210 to 140-150 µM) respectively in the presence of 80 µM phosphatidylinositol (Cong,

et al., 1989). In addition, the calpain concentration itself may influence the rate of autolysis

as reported by Cottin Thompson, Sathe, Szpacenko and Goll (2001). The rate of autolysis of

m-calpain from bovine skeletal muscle was decreased by decreasing calpain concentration at

Ca2+ concentration of 350 µM or less indicating autolysis was intermolecular process at this

condition. However, calpain concentration and casein presence did not show significant

effects on the autolytic rate of m-calpain when calpain was 1000 µM or higher. At pH 6.5, µ-

calpain showed slower rate of autolysis than at pH 7.5 after incubation with 100 µM CaCl2

for 60 minutes. This slower autolysis resulted in greater maximum activity at pH 6.5

independent of ionic strength (Maddock, Huff-Lonergan, Rowe and Lonergan, 2005). Elce et

al. (1997) reported that both small and large subunits at Ala9-Lys10 of m-calpain autolyzed

2+ within 1 minute. Only the autolysis of 80 kDa subunit influenced [Ca ]0.5 value and stability of m-calpain. The authors concluded that the roles of autolysis may generate active and instable forms of calpain to limit the duration of its activity.

Several papers have indicated that the autolysis of both m- (DeMartino et al., 1986) and

µ-calpain (Saido et al., 1994) proceeded with their proteolytic activities. Therefore, autolysis can be used as an index for proteolytic activation of calpain especially in postmortem muscle.

The relative intensity of 80 kDa µ-calpain at 24 hour postmortem was positively correlated with the intensity of intact desmin after 1 and 5 days of storage, while the 76 kDa fragments at day 1 showed negative correlation with the intensity of intact desmin at the same time points (Bee et al., 2007; Gardner et al., 2005; Zhang et al., 2006). This indicates autolysis of

µ-calpain is associated with increased desmin degradation in later postmortem storage.

Earlier autolysis during postmortem aging may be an indicator that the calpains are activated earlier resulting in protein degradation at an early stage. This was supported by Melody et al.

(2004) which showed that psoas major had earlier autolysis of µ-calpain than longissimus dorsi which was associated with faster rate of degradation of desmin. In the same study, psoas major was shown to have earlier appearance of myofibril-bound µ-calpain indicating that rate of autolysis may be associated the re-localization of µ-calpain within muscle cells.

Therefore, the extent of µ-calpain autolysis during postmortem aging could be an effective indicator of the postmortem degradation of proteins and variation the fresh meat quality.

Posttranslational modifications of calpain including oxidation and nitrosylation can regulate its activation and autolysis of calpain. Thus these modifications may be involved with regulations of fresh meat quality. How these modifications influence calpain activity and fresh meat quality will be discussed in protein oxidation and nitrosylation sections.

Calpastatin

The specific inhibitor of µ- and m-calpain is calpastatin. Calpastatin is a polymorphic protein with four repeated domains of 140 amino acids each and an N-terminal domain without inhibitory activity. Each of four repeated domains has inhibitory activity composed of three subdomains A, B and C (Goll, Thompson, Taylor, Ouali and Chou, 1999).

Subdomain B of calpastatin plays central role in calpain inhibition (Wendt, Thompson and

Goll, 2004). Subdomain B can be associated with both sides of the substrate-binding active site cleft of calpains to obstruct the orientation for substrate binding (Moldoveanu, Gehring and Green, 2008). Subdomain A binds to domain IV and subdomain C interacts with domain

VI at the penta-EF-hand in domain IV and VI and this binding can increase the overall interaction between calpain and calpastatin (Hanna, Campbell and Davies, 2008). The four domains have different inhibitory abilities for m-calpain with the order of domain I > domain

IV > domain III > domain II (Clarke, 1991; Kawasaki, Emori, Imajoh-Ohmi, Minami and

Suzuki, 1989). Calcium is required for the interaction between calpains and calpastatin. The calcium concentration required for the binding of calpastatin to calpain was lower than calcium concentration for half maximal activity of both intact and autolyzed m-calpain. For

µ-calpain, the calcium concentration for half maximal activity for intact enzyme was higher

and was close for autolyzed products compared with the calcium concentration for the interaction of calpastatin with intact µ-calpain (Kapprell and Goll, 1989; Otsuka and Goll,

1987). Interestingly, calpastatin is a substrate of the calpains (Mellgren, Mericle, and Lane,

1986). The intact 130 kDa calpastatin was found to be almost completely degraded by calpain after 72 hours of postmortem refrigerated storage. The molecular weights of degradation products from calpastatin include 100, 80, 65, 54, 32 and 29 kDa fragments in postmortem muscle (Doumit and Koohmaraie, 1999). However, the degradation products of calpastatin could still retain some inhibitory activity for calpains (DeMartino et al., 1988).

The rate and extent of meat tenderization are variable among species. Several studies have shown that calpastatin activity at 24 hours postmortem is a good indicator for subsequent extent of proteolysis and meat tenderness. Pork and lamb muscles need 4-6 days for optimum tenderness while bovine muscles need 10-15 days for better tenderness (Monin and Ouali, 1991). The variation in calpastatin activity could explain the variability of the extent of protein degradation and the rate of tenderization between different species and between different breeds (Koohmaraie et al., 1991; Whipple, Koohmaraie, Dikeman, Crouse,

Hunt and Klemm, 1990). Animals with the callipyge genotype have greatly exaggerated muscling, higher calpastatin activity and compromised tenderness compared to animals without the callipyge genotype (Koohmaraie, Shackelford, Wheeler, Lonergan and Doumit,

1995). The calpastatin activity at 24 hours postmortem was found to be negatively correlated with meat tenderization. Calpastatin activity was the best variable to account for tenderness variation (Shackelford, Koohmaraie, Cundiff, Gregory, Rohrer and Savell, 1994). In the study of Kent, Spencer and Koohmaraie (2004), calpastatin was overexpressed in transgenic

mice with a human calpastatin gene. The degradation of desmin and troponin T were

inhibited in calpastatin overexpressed groups compared to their degradation in control

groups. Lamb with the callipyge genotype is known to have higher calpastatin activities.

Biceps femoris from callipyge lambs showed less postmortem proteolysis including titin,

nebulin, vinculin, desmin and troponin T than biceps femoris from normal lamb during

extended postmortem storage (Geesink and Koohmaraie, 1999). Both of these studies further

confirm the critical roles of calpastatin in protein degradation and meat tenderization during

postmortem aging. The polymorphism of the calpastatin gene showed major effects on the

drip loss of longissimus lumborum muscle at 48 hours and 96 hours postmortem in pigs

(Krzecio, Kurl, Kocwin-Podsiadla and Monin, 2005). In the same study, significant

interactions were found between genotypes of calpastatin and the ryanodine receptors (also named calcium release channel which regulates the calcium release from sarcoplasmic reticulum to cytoplasm of muscle cells) for 48 hour drip loss and 24 hour pH. All these studies support the hypothesis that calpastatin is related to postmortem protein turnover and fresh meat quality (Goll, Thompson, Taylor and Ouali, 1998).

Protein modification and meat quality

Protein oxidation

Oxidation is known as one of the major causes for quality deterioration during

processing and storage of food products. Protein oxidation is defined as the covalent modification of a protein induced either by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress (Shacter, 2000). The main oxidative of

modifications of the side chains of amino acids are thiol oxidation (reaction between active

sulfhydryl groups and oxygen to produce disulfide bonds), aromatic hydroxylation (hydrogen

abstraction and deamination of tyrosine and phenylalanine groups) and formation of carbonyl

groups (Stadtman, 1990). Among all amino acid side chains, cysteine and methionine are the

most prone to be oxidized because both of them contain reactive sulfur atoms (Shacter,

2000). Sulfur anion is the most powerful nucleophile and is rich with electrons which can be

removed one or two at a time to cause oxidation. Reactive oxygen species include free

• •- • • radicals OH, O2 , RS and ROO , nonradical species H2O2 and ROOH and reactive aldehydes and ketones (Butterfield and Stadtman, 1997). Oxidation can directly attack the backbone of protein side-chains to cause fragmentation and conformational changes of secondary and tertiary structure of proteins. Disulfide and dityrosine and other intermolecular bridges induced by oxidation can lead to protein aggregation and polymerization to change the proteolytic properties of these proteins (Martinaud, Mercier, Marinova, Tassy, Gatellier and Renerre, 1997; Morzel, Gatellier, Sayd, Renerre and Laville, 2006). This is mainly due to oxidation induced unfolding process and increases of the surface hydrophobicity of the oxidized protein during unfolding (Davies and Delsignore, 1987). These alterations can influence the physical and chemical properties of proteins including solubility, hydrophobicity, water holding capacity, meat tenderness, gelation functions and even the nutritional value (Liu and Xiong, 2000; Rowe, Maddock, Lonergan, and Huff-Lonergan,

2004a, 2004b; Srinicasan and Hultin, 1997; Srinivasan and Xiong, 1996).

In postmortem muscle, protein oxidation has been become gradually recognized as one important factor for meat quality. During postmortem storage, muscle has a decreased ability

to maintain its antioxidant defense system and this can cause the increased accumulation of reactive oxygen and nitrogen species (Renerre et al., 1996). Using fluorescence microscopy to detect carbonyl content of muscle cells, Astruc, Marinova, Labas, Gatellier and Santé-

Lhoutellier determined the localization of protein oxidation in bovine rectus abdominis with

2+ three different treatments (chemical oxidation reagents of Fe /H2O2, refrigerated storage at 4 oC and cooking at 100 oC). All these treatments induced protein oxidation at both the periphery and the inside of muscle cells. Of particular interest, the amount of protein carbonyl was increased by 4 times from 0 to 10 days of refrigerated storage. In addition, the levels of protein oxidation at day 10 after refrigerated storage was similar to that induced by

1 mM of oxidant reagents with the mixture of FeSO4 and H2O2. The authors concluded that protein oxidation was initiated from the muscle membrane to the inside of muscle cells.

Lipophilic antioxidants such as vitamin E may be powerful reagents to limit the deterioration of protein oxidation (Astruc et al., 2007; Rowe et al., 2004a).

Rowe et al (2004a and 2004b) studied the influence of protein oxidation on fresh meat attributes including color and tenderness. They also examined its influence on calpain system. In this study, steers were fed with 1,000 IU vitamin E per steer daily in finishing diet and irradiation of the beef steaks (longissimus dorsi) was utilized as the tool to accelerate protein oxidation. Irradiation-induced oxidation could cause the discoloration of steaks to decrease their L*, a* and b* values. Carbonyl contents of myofibrillar and sarcoplasmic proteins were found to be negatively correlated with meat tenderness at day 1, 3 and 7.

Increased shear force associated with protein oxidation was suggested by the author due to the aggregation of myofibrillar proteins or to the inhibition of proteolytic enzyme activity

(Rowe et al., 2004a). Vitamin E was found to be effective to decrease the amount and the

extent of oxidation of sarcoplasmic proteins. In parallel study by Rowe et al. (2004b), effects

of oxidation on protein degradation and the calpain system were further studied. In

nonirradiated samples, steaks from vitamin E supplemented animals had earlier degradation

of troponin-T than steaks from animals that were not supplemented with vitamin E.

Irradiation was found to decrease the extent of desmin degradation. This decreased

proteolysis was likely due to the inhibition of µ-calpain activity by irradiation (Rowe et al.,

2004b). All of these studies supported that oxidizing conditions may have negative effects on

fresh meat quality through inhibiting protein degradation and inactivating of µ-calpain.

Carlin et al. (2006) further determined the effects of oxidation on the activity of purified

m- and µ-calpain and the interaction between µ-calpain and calpastatin. Proteolytic activity

of µ-calpain and m-calpain was shown to be inhibited by hydrogen peroxide (H2O2) induced oxidation at pH 6.0, 6.5 and 7.5. Interestingly, H2O2 could decrease the percentage of

inhibition of µ-calpain activity by calpastatin at pH 6.5 and 7.5. Addition of H2O2 could allow µ-calpain to degrade desmin in the presence of calpastatin compared to only H2O2

addition (no calpastatin) treatment (Carlin et al., 2006). They reported oxidation decreased

the rate of conversion of 80 kDa subunit to 76 kDa without changing the extent of autolysis

of large subunit. Both intact 80 kDa and autolyzed 76 kDa products of large subunit of µ-

calpain were sensitive to oxidative environment. Inactivation of intact µ-calpain could be

reversed by a reducing agent such as dithiothreitol (Guttmann, Elce, Bell, Isbell and Johnson,

1997). In another study, the activity inhibition of µ-calpain by oxidation stress was reported

to be associated with the formation of a disulfide bond between Cys115 and Cys108 within

active site by LC-MS/MS analysis (Lametsch, Lonergan and Huff-Lonergan, 2008).

Antioxidant supplementation may be an effective method to limit protein oxidation

induced negative effects on meat quality. For example, vitamin E supplementation has been

known to limit both protein and lipid oxidation resulting in improved meat quality (Arnold,

Arp, Scheller, William and Schaefer, 1993; Chea et al., 1995; den Hertog-Meischke, et al.,

1997; Liu, Scheller, Arp, Schaefer and Williams, 1996; Mercier, Gatellier, Viau, Remignon

and Renerre, 1998; Rowe et al., 2004a). In a study about the effects of oxidation on beef

tenderization (Rowe et al., 2004b), dietary vitamin E supplementation could cause faster degradation of troponin-T at 2 days postmortem in beef steaks through lowing protein oxidation. In conclusion, protein oxidation is involved with the modifications of side chain amino acid groups and the alteration of the physical and chemical properties of proteins.

These oxidative changes can influence fresh meat quality during postmortem aging and

subsequent functionality of meat products. Addition of antioxidant in animal diets such as

vitamin E could be a possible method to limit deteriorative effects of protein oxidation on

fresh meat quality.

Protein nitrosylation

Nitric oxide is a molecular messenger that plays important roles in physiological and

pathophysiological conditions. S-Nitrosylation is referred as the attachment of a nitric oxide

group on the sulfur atom of one or more cysteine residues (Hess, Matsumoto, Kim, Marshall

and Stamler, 2005). For example, cysteines of proteins can react with nitric oxide and its

secondary products to produce disulfides and sulfinic acids. In recent years, protein

nitrosylation has become recognized as another significant mechanism for posttranslational

regulation of most classes of proteins much like phosphorylation. There is some synergy

between phosphorylation and nitrosylation in regulating the redox status of biological

systems. The biogenesis of nitric oxide in muscle systems is catalyzed by a family of enzyme

nitric oxide synthases (NOS) during the conversion of L-arginine to L-citrulline. This

reaction requires cofactors including oxygen, NADPH, tetrahydrobiopterin, flavin aenine

mono- and dinucleotides (Brannan and Decker, 2002). Three isoforms of NOS have been

identified and named with the cells or systems where they are initially discovered. These

isoforms are neuronal NOS (NOS1 or nNOS), macrophage (immune)/calcium calmodulin-

independent or inducible NOS (NOS2 or iNOS) and endothelial NOS (eNOS or NOS)

(Stamler and Meissner, 2001). Although all three isoforms can be expressed in muscle cells,

nNOS is the muscle specific splice variant and the most expressed isoform within skeletal

muscles. nNOS is specially targeted at the membrane surface by binding a dystrophin-

associated protein α1-syntrophin (Miyagoe-Suzuki and Takeda, 2001). The NOS expression has been reported in many species including human, rats, hamsters, chicken, gold fish and rabbit. The distribution of nNOS is species and muscle fiber dependent. For example, greater amounts of NOS are expressed in slow-twitch than fast-twitch muscle fibers in humans, while rat muscles have opposite expression patterns (Higaki, Hirshman, Fujii and Goodyear,

2001; McConell and Kingwell, 2006).

Nitric oxide can react with superoxide to cause the formation of peroxynitrite which can initiate lipid oxidation through different pathways. Peroxynitrite can increase prooxidant

activity of metmyoglobin, cause the production lipid hydroperoxides of membrane lipids and

activate metal-independent prooxidant (Brannan and Decker, 2001; Rubbo et al., 1994). In

postmortem muscle, NOS activity was determined in several species including chicken,

turkey, pork and trout by Brannan and Decker (2001). They reported that all of these fresh

muscle foods showed NOS activity and their activity was gradually decreased during

refrigerated storage. As for pork, most of its original activity remained after 24 h of

refrigerated storage and no activity was shown at 48 h of storage in this study. No significant difference in NOS activity was reported in the range of pH from 4.5 to 7.4. This provides the possibility for the involvement of nitric oxide with the regulation of fresh meat quality since pH decreased from approximately 7.4 in living muscle to ultimate pH of 5.3-5.7 in postmortem muscle.

Andrade, Reid, Allen and Westerblad (1998) investigated the effects of nitric oxide on the contractile function of single mouse skeletal muscle fibers. Nitric oxide donors S-nitroso-

N-acetylcysteine decreased the myofibrillar sensitivity to calcium due to impaired calcium activation of the actin filament. S-nitroso-N-acetylcysteine inhibited the activity of myosin

ATPase to result in the decline of maximal contraction force and maximal shortening velocity (Galler, Hilber and Gobesberger, 1997). Injection of NOS inhibitor N-nitro-L- arginine methyl ester was found to increase force production during slow-twitch contraction,

while S-nitroso-N-acetylcysteine attenuated force production of both fast twitch and tetanic

contractions (King-Vanvlack, Curtis, Mewburn, Cain and Chapler, 1995; Murrant, Frisbee

and Barclay, 1997). In the study of Kobzik et al. (1994), nitric oxide can both promote

muscle relaxation by regulating cGMP and increased the contraction through the interaction

with reactive oxygen intermediates. NOS activity can partly explain the difference in force

generated by type I and type II fibers such as soleus, diaphragm and extensor digitorum

longus. The cytoskeleton and focal contacts were stabilized by nitric oxide. This stabilization

was due to the increase of protein accumulation of talin and vinculin and the decrease of talin

proteolysis (Zhang, Kraus and Truskey, 2004). These studies indicate that nitric oxide

potentially influences the properties of proteins and the excitation-contraction coupling of

skeletal muscles.

Several papers have been published during past 10 years about the effects of nitric oxide on fresh meat quality during ante- and postmortem storage. In a study by Cook, Scott and

Devine (1998), bull longissimus lumborum, obtained within 2 hours of slaughter, was soaked with three different solutions including control (L-arginine), NOS inhibitor (N-nitro-L- arginine and N-nitro-L-arginine methyl ester hydrochloride) and nitric oxide enhancer

(diethylenetriamine/nitric oxide adduct and S-nitroso-N-acetylpenicillamine) containing groups to study the influence of nitric oxide on meat tenderization. Nitric oxide concentration in nitric enhancer group was higher than NOS inhibitor samples. Nitric oxide enhancer treatment showed lower values of shear force at day 3 than control and NOS inhibitor groups during postmortem aging. At day 6, the NOS inhibitor group had greater shear force values than the other two groups. However, all three groups exhibited similar tenderness values after

8 days of refrigerated storage. The author suggested that the faster rate of tenderization induced by nitric oxide may be related to the mediation of free radicals and the activation of calpain.

In contrast to Cook’s study, most of the studies support nitric oxide can inhibit calpain

activity and have negative effects on meat tenderness. The inhibitory effects of NO-donors

on cysteine proteases in different living organisms including caspase, calpain and cathepsin

of mammalian system have been reviewed by Ascenzi, Salvati, Bolognesi, Colasanti,

Polticelli and Venturini (2001). Five S-nitroso compounds were reported to have inhibitory

effects on the enzyme papain through the formation of mixed disulfide species with the

cysteine in the active site (Xian, Chen, Liu, Wang and Wang, 2000). In transnitrosylation,

cysteines of the proteins could directly attack the S-NO bonds resulting in the formation of a

mixed disulfide bond. In this condition, cysteine needs to be activated and forms a strong

nucleophile with a nearby histidine as described by Mohr, Hallak, de Bioitte, Lapetina and

Brüne (1999). The NO generating agent sodium nitroprusside decreased the proteolytic activity of m-calpain by 90% and of µ-calpain by 20% respectively at pH 7.5 (Michetti et al.,

1995). The inhibition of m-calpain activity was recovered by 82% after incubation with dithiothreitol for 12 hours indicating that the inactivation of m-calpain may be associated with the formation of disulfide bonds. In the same study, the decrease of calpain activity was shown to be pH dependent for both µ- and m-calpain in different ways. The inhibition of activity was sharply decreased as pH was shifted to acidic conditions for m-calpain.

Inactivation of µ-calpain was gradually decreased as the pH was shifted to neutral conditions from acidic environment. In intact neutrophils, cells pre-incubated with 2 mM SNP under light completely prevented the autolysis of 80 kDa µ-calpain in the presence of calcium.

A recent study determined the influence of pre-slaughter injection of NOS inhibitor N- nitro-L-arginine methyl ester on the meat tenderness and glycolysis of ovine muscles

(Cottrell et al., 2008). They reported injection of 30 mg/kg L-NAME at 135 minutes pre- slaughter significantly decreased Warner-Bratzler shear force value of longissimus thoracis et lumborum independent of pH, temperature and sarcomere length at day 3 of postmortem aging. These decreases of Warner-Bratzler shear force was not seen in the semimembranosus. The authors suggested this may be due to the different composition of the two muscles as semimembranosus had a higher percentage of connective tissues. In contrast, exercise increased Warner-Bratzler shear force of both longissimus thoracis et lumborum and semimembranosus after 3 days of postmortem aging. N-nitro-L-arginine methyl ester injection was shown to increase the rate of postmortem glycolysis which was evidenced by reduced glycogen content of both muscles and increased lactate concentration of semimembranosus in pre-slaughtered exercised lambs. The possible mechanism is that nitric oxide may stimulate the activation of soluble guanylate cyclase to increase the production of cyclic guanosine momophosphate (cGMP) and improve glucose uptake in skeletal muscles

(McConell and Kingwell, 2006; Young, Radda and Leighton, 1997).

Nitric oxide may be involved in the metabolism of postmortem muscle by regulating the concentration of sarcoplasmic calcium. Both sarcoplasmic/endoplasmic reticulum calcium

ATPase (SERCA) and RyR are rich in cysteine residues which are known to be sensitive to nitric oxide induced nitrosylation and to oxidation (Stamler, Lamas and Fang, 2001). Intense exercise has been shown to cause increased nitrosylation of RyR1 and reduce the binding affinity of calstabin to RyR1 to cause calcium leak from sarcoplasmic reticulum (Bellinger et al., 2008; Bellinger, Mongillo and Marks, 2008). Since the binding of calstabin to RyR1 stabilizes the closed state of calcium release channel, exhausting exercise could cause the

leakage of calcium from sarcoplasmic reticulum to sarcoplasm resulting in muscle damage, impaired contractility and even malignant hyperthermia (Stamler, Sun and Hess, 2008). In contrast, nitric oxide induced nitrosylation showed inhibitory effects on the calcium uptake channels of SERCA during excitation-contraction coupling of cardiac muscles (Hare, 2003).

The consequence of this regulation of RyR and SERCA by nitrosylation is to increase cytosolic calcium concentration. Since calcium plays critical roles in the early postmortem period for homeostasis and rate of muscle metabolism, extensive nitrosylation may have some roles in fresh meat quality by regulating the sarcoplasmic calcium concentration.

However, no studies in this specific area have been conducted to the best of our knowledge.

Summary

This literature review addressed the topics of skeletal muscle structure, calpains and calpastatin, water holding capacity, drip loss formation and meat tenderness, two muscle proteins of integrin and desmin, protein oxidation and nitrosylation, and relationship between muscle fiber types and meat quality. Biochemical processes during postmortem refrigerated storage are largely responsible for the variations of meat quality. Skeletal muscles have a well organized structure to exert their functions. Degradation of myofibrillar and myofibrillar-associated proteins including integrin and desmin in postmortem storage disrupts their integral structure. This proteolysis can contribute to fresh meat quality including water holding capacity and tenderness. Decreased ability to regulate the redox of postmortem muscles leads to the accumulation of reactive oxygen and nitrogen species.

These reactive species can regulate protein functions through protein oxidation and S-

nitrosylation. Muscle fiber composition can explain variations of meat quality among different muscles and species. A complete understanding of these biochemical reactions and their relationships to postmortem muscle metabolism can help us to deliver consistent meat to our consumers.

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Methods to determine the activity of calpain protease

A paper to be submitted to Meat Science

Wan-Gang Zhang, Steven M. Lonergan, and Elisabeth Huff-Lonergan

Abstract

The calpain proteolytic system is generally thought to play major roles in protein degradation and meat tenderization during postmortem refrigerated storage. Therefore, exploring some sensitive and continuous methods to monitor the status of calpain system is critical for controlling the variation of meat quality. This review is to track the development of calpain protease assays and discuss both advantages and disadvantages of these assays especially focusing on the field of meat science. We also briefly discuss appropriate conditions that these different assays can be applied for. In this review, these methods are divided into different categories including standard assay, labeled-casein based assays, casein zymography, colorimetric assays, capillary electrophoretic analysis and assays using other compounds instead of casein as substrates.

Introduction

Calpains are involved in regulation of many cellular processes including signal transduction, cell mobility, and apoptosis. In meat science area, it has been repeatedly reported that calpain system plays critical roles in governing meat quality during postmortem

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aging. Many studies have shown that calpains are involved in the proteolysis of myofibrillar

and cytoskeletal proteins of skeletal muscles (Geesink, Kuchay, Chishti and Koohmaraie

2006; Huff-Lonergan et al., 1996; Koohmaraie, 1992). This degradation is generally believed

to be associated with meat tenderness and water-holding capacity during postmortem aging

(Melody, Lonergan, Rowe, Huiatt, Mayes and Huff-Lonergan, 2004; Rowe, Huff-Lonergan

and Lonergan, 2001; Swatland and Belfry, 1985; Zhang, Lonergan, Gardner and Huff-

Lonergan, 2006). Although much information has been obtained about the calpain system

since it was purified in 1976, their physiological function in vivo is still poorly understood.

For example, how calpains are activated in physiological conditions is not completely

understood especially with the consideration of the lower Ca2+ concentration in vivo. A critical step to better understand calpain system is to purify and analyze its activity accurately. Hence, it is of particular importance to explore some continuous and consistent methods to track properties of this enzyme system.

During past thirty years, many methods have been developed to determine the activity of calpain proteases. Among these methods, casein has been the most widely used substrate.

The most commonly used assay works by detecting the amount of trichloroacetic acid (TCA) soluble peptides hydrolyzed from casein at the wavelength of 278 nm (Dayton, Goll, Zeece,

Robson and Reville, 1976; Waxman, 1981). This method is typically thought of as standard assay. The low sensitivity of standard assay has driven scientists to explore some compounds to label casein to improve its sensitivity. Fluorescent compound–labeled substrates have been developed by several labs. These methods work by measuring the fluorescence of labeled fragments of casein. These fluorescent compounds include fluorescamine (Melloni,

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Pontreomoli, Salamino, Sparatore, Michetti and Horecker, 1984), ninhydrin (Doi, Shibata and Matoba, 1981), fluorescein isothiocyanate (FITC) (Lonergan, Johnson and Calkins,

1995; Twining, 1984; Wolfe, Sathe, Goll, Kleese, Edmunds and Duperret, 1989), and 4,4- difloro-5,7-dimethyl-4-bora-3a,4a–diaza-s-indacene-3-propionic acid (BODIPY-FL) to label casein in different assays (Kent, Veiseth, Therkildsen and Koohmaraie, 2005; Thompson,

Saldana, Cong and Goll, 2000). Radiolabeled substrates such as 14C-methylated (Delgado,

Geesink, Marchello, Goll, and Koohmaraie, 2001; Koohmaraie, 1992) and [3H] (Coolican,

Haiech and Hathaway, 1986) are alternative compounds to label casein for calpain assay via measuring the radioactivity of degraded peptides.

One of the main disadvantages of standard assay is that it is required to separate undigested substrates from peptides or amino acids derived from proteolysis. This disadvantage makes standard assay unsuitable for continuous detection. Raser, Posner and

Wang (1995) developed a method to measure calpain activity using casein zymography. This assay can detect calpain activity of crude extracts even in the presence of its inhibitor and other enzymes. Buroker-Kilogore and Wang (1993) used coomassie brilliant blue G-250 to bind substrates instead of peptide fragments and measured the absorbance at the wavelength of 595 nm. Both these assays can eliminate the requirement of standard assay to precipitate undigested substrates to measure extent of proteolysis.

Several studies have reported to use other calpain substrates instead of casein to measure its activity. Harris, Gregory and Maycock (1995) have detected calpain activity by using dipeptide Z-Leu-Arg-(7-methoxynaphthy)-amide (Z-Leu-Arg-MNA) as substrate.

They found that the hydrolysis rate of this substrate was linear with the increase of enzyme

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concentration. Similarly, another dipeptide Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-

LY-MNA) has been used as a continuous recording fluorogenic assay for calpain activity

(Mallya, Meyer, Bozyczko-Coyne, Siman and Ator, 1998). This method could be sensitive

enough to detect 10 picomolar calpain. Tompa, Schad, Baki, Alexa, Batke and Friedrich

(1995) have described that a natural substrate of calpain dichlorotriazinylamino-fluorescein

(DTAF)-labeled microtubule-associated protein 2 (MAP2) could be used to detect calpain

activity in the pictogram range. In addition, a high sensitive technique using electrophoretic

analysis for calpain assay has been presented recently by Gu, Whipple-VanPatter, O’Dwyer

and Zeece (2001). The main modification of this new assay is that capillary electrophoresis is

used to analyze the TCA-soluble peptides derived from Oregon Green-labeled casein. The

high sensitivity of this technique makes it suitable for detecting 2-3 ng of calpain activity.

This review is mainly to describe the development of these assays and compare their

advantages and disadvantages under different situations.

Assays and discussions

Standard assay

Casein is well known as a good substrate for calpain proteases. It can be digested very

rapidly at several sites to produce short peptide fragments or amino acids (Dayton et al.,

1976). The general procedures for this assay include incubating calpains with casein buffers

at 25 oC for 1 hour. After incubation, trichloroacetic acid (TCA) is added into the solutions to stop reaction and dissolve peptide products. Undigested caseins are separated by precipitation. The final step is to measure the absorbance of the supernatant fluid at the

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wavelength of 278 nm. Koohmaraie (1990) found that both the caseinolytic activity of µ-

calpain and m-calpain were linear with time within 1 h at 25 oC during incubation. One unit of activity of calpains is calculated as one increase of one absorbance unit at 278 nm.

The main advantage of this assay is economic and straightforward because casein is a ecnomical commercial product. The similar hydrolysis model of casein with natural proteolytic proteins of calpain also contributes to its wide acceptance as a substrate for calpain assay. Compared with other assay methods, standard assay is relatively simple. It just needs three-step procedure around 90 minutes to complete all analysis. The limitations of this standard method have been discussed in detail by Tompa et al. (1995). The main disadvantage of the assay is that it is not sensitive enough under common experimental conditions. Usually, microgram quantities of calpain are required to determine it activities.

This method is relatively tedious because of the required procedure to separate final products of peptides from casein substrate. Due to this limitation, the standard assay is not suitable for continuous measurements and mechanistic studies. Although standard assay has its own limitations, it is still widely used for activity assay of purified calpains especially in the area of meat science because of affordability and availability of casein.

Labeled-casein based assays

In subsequent studies, overcoming low sensitivity of standard assay is the major goal for improving calpain assays. Using some special compounds to label casein can significantly improve the sensitivity of standard assay. Wolfe et al. (1989) reported fluorescence assay was ten to twenty times more sensitive than the standard assay with measuring the absorbance of fluorescent peptides at 278 nm. Based on the fact that fluorescein

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isothiocyanate (FITC) is a sensitive fluorescent label for casein, several authors have provided modified methods to improve the sensitivity of the standard assay using FITC labeled casein (Lonergan et al., 1995; Twing, 1984; Wolfe et al., 1989). FITC-casein is an inexpensive and stable substrate for many proteases including trypsin, chymotrypsin, elastase and thermolysin cleaved in a linear time-dependent model (Twing, 1984). The basic reaction is FITC can react with amino groups of casein to produce high fluorescence compound of fluorescein thiocarbamoyl derivative. In the study of Lonergan et al. (1995), final fluorescence of labeled peptides digested from calpain was measured with 490 nm as excitation wavelength and 513 nm as emission wavelength. Fluorometric results were highly

correlated with standard assay (r=0.985) and activity titration assay (r=0.997). FITC-labeled

assay was able to increase the sensitivity at least by five times of the standard assay which

makes it good enough for calpain assay of cell extracts and biopsy-sized samples. The

sensitivity of this assay can be further improved by the increase of FITC concentration. The

author mentioned several factors that could cause inconsistent results about this fluorescent

assay. For example, readings of blank fluorescence were relatively high if the unbound FITC

was not completely removed. Lower calpain activity could be achieved if procedure failed to

completely neutralize TCA supernatant (Lonergan et al., 1995). A large number of assay

additives could cause possible interference of this assay (Buroker-Kilgore et al., 1993)

Using DTAF labeled-casein as substrate to detect calpain activity was found to result in

high background fluorescence in microtiter plate. To overcome this disadvantage, BODIPY-

FL labeled casein was introduced as a calpain substrate in autoquenching assays (Thompson,

Saldnana, Cong and Goll, 2000). This new technique could increase fluorescence by five to

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ten times compared to the FITC-casein assay. It was sensitive enough to detect 10 ng of

calpain and it is one of the most sensitive assay methods thus far. Many limitations of other

assays could be overcome by this new modified technique. Compared to standard assay and

other methods, it is time-saving because this technique does not require step to precipitate

and remove undigested substrates from the peptides fragments. The whole process could be

completed within 15 minutes plus the incubation time for assaying 96 samples (Thompson, et

al., 2000). The spectra of BIODIPY- FL are not sensitive to pH and polarity of other solvents

because there are no ionic groups in this BIODIPY- FL molecule. Based on these advantages

of this assay, the author recommended that this method was advanced to monitor any

proteases that cleave caseins in a microtiter plate format. Especially, it can be easily applied

to continuous measurement of casein proteolysis and screening for a large amount of

samples.

Using radiolabeled substrates is alternative method to increase sensitivity of standard

assay for calpain activity. Coolican et al. (1986) reported that calpain activity could be

determined by [3H]α-labeled casein. Radioactivity was counted from the TCA soluble

peptides derived from [3H]α-casein. In the study of Delgado et al. (2001), 14C-methylate radiolabeled casein was effective to detect activity of myofibril-bound calpain. Casein was labeled by reductive alkylation with 14C-formaldehyde and sodium cyanoborohydride

(Dottavio-Martin and Raven, 1978). Calpain activity was calculated as the TCA-soluble

radioactivity released from 14C-labeled peptides. The paper showed that 100 ng of enzyme could be detected by this technique. The linearity between the release of peptides and incubation time could be as long as 48 h (Delgodo, et al., 2001). Although this method could

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improve sensitivity of standard assay, it is not widely accepted because of its several

disadvantages. Radiolabeled assay was quite expensive due to the high price of materials and

the disposal of radioactive waste. In addition, these radiolabeled wastes were considered to

be hazardous products for the environment (Thompson et al., 2000).

Casein zymography

Gelatin zymography was initially used to measure the activity of matrix metalloprotease

in tissues and biological fluids. This method could distinguish different enzymes based on

their different mobility properties upon eletrophoresis (Heussen and Dowdle, 1980; Kleiner

and Stetler-Stevenson, 1994; Paech, Christianson and Maurer, 1993). Raser et al. (1995)

further developed this assay to measure activity of µ- and m-calpain for tissue samples or

cultured cells in vitro studies. This assay is advanced in determining the enzyme activity

without the separation of m- and µ-calpain and their endogenous inhibitor calpastatin. This method used the nondenaturing casein as the substrate for calpains in polyacrylamide gels.

DTT and EGTA were added into calpain buffers to stabilize calpains. After electrophoresis, gels were incubated overnight with a buffer containing CaCl2 to activate embedded calpain

bands. Coomassie blue G250 was used to stain casein gels. m-Calpain and µ-calpain were

shown as different bands in the gels because of their different motilities upon electrophoresis.

The brightness of clearing bands in gels was proportional to the amount and the activity of

loaded calpains.

Measuring activity of calpains separately without chromarographic separation of m-

calpain, µ-calpain and calpastatin from each other is one of the major advancements of this

method. This is mainly based on different mobility of these two calpain isoforms and their

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inhibitor during electrophoresis. m-Calpain has higher negative charge compared with µ- calpain. Calpain activity even could be measured in the presence of an excessive amount of its inhibitor. This is due to the chelator in samples which could prevent interaction between calpain and calpastatin (Kapprell and Goll, 1989). Therefore, this method could be used to

analyze crude homogenate of cultured cells or tissues. It is hard to detect activities of other

cellular proteases under experimental conditions described by Raser et al. (1995). Compared

to standard assay, this method does not need centrifugation step to separate undigested

substrates from reaction products. This method is also advanced as a sensitive assay which

can detect as many as 20 ng of m-calpain and µ-calpain (Raser et al., 1995). The major

disadvantage is that it needs much time to complete all the procedures. Therefore, it is not

suitable for measuring a large amount of samples. Just like as Raser et al. (1995) described, it

needs to spend 24 to 28 hours to complete electrophoresis, incubation and final staining.

Colorimetric assay

Buroker-Kilgore and Wang (1993) developed this two-step colorimetric assay using

coomassie brilliant blue G-250 to bind calpain substrate. This method could eliminate

procedure of separating small peptides from protein substrate of the standard assay.

Coomassie brilliant blue G-250 is chosen as the reagent because this dye reagent only to bind

proteins. In addition, it does not have interaction with reaction products such as peptide

fragments or amino acids digested from casein (Buroker-Kolgore and Wang, 1993). This

assay was more sensitive than other colorimetric assays for calpains by using azo-casein

(Moss, Gutierrez, Perez and Kobayashi, 1991) and azo-collagen (Compton and Jones, 1985).

Calpains are incubated with casein buffers with adding appropriate amount of CaCl2 to start

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proteolysis at 25 oC for 30 min. After incubation, dye reagent is added into the aliquot to bind

left caseins. Absorbance value is measured at the wavelength of 595 nm. The calpain activity

is calculated based on difference of absorbance between with CaCl2 and without CaCl2

groups.

This modified assay is advanced in its simplicity. It just needs to incubate casein with

enzyme and dye reagent separately for around 40 minutes. Eliminating required step of

precipitating of undigested substrate is another advantage to make it much faster compared to

the standard method. However, real-time recording of the calpain activity may be not

detected very well because of the high background from dye molecules interference (Tompa

et al., 1995). This modified caseinolysis assay uses casein as proteolytic substrate. It is able

to detect 5-10 µg of calpain which is less sensitive than the fluorescence and the radiolabeled

assays. High background of this method (1.6-2.0 OD) is another disadvantage for accurate assay of calpain protease.

Assays using other substrates instead of casein

Some other substrates have been proposed to be effective on measuring calpain activity.

MAP2 is well known as one of the cytoskeletal proteins that are sensitive to calpains in vitro

(Goll, Thompson, Li, Wei and Cong, 2003) and in vivo (Johnson and Foley, 1993). Tompa et al. (1995) reported that DTAF-labeled MAP2 could be used as a substrate to detect calpain activity. The fluorescence intensity of DTAF-labeled MAP2 could increase sensitivity by three folds of magnitude. After incubation with DTAF, labeled MAP2 was separated from fluorescent dye by gel filtration through special column calibrated with calpains. The final fluorescence was measured at excitation wavelength of 420 nm and emission wavelength of

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520 nm. Again, this method skips procedure to separate reactants and substrates. This permits it to be suitable for continuous detection which is significant for enzyme kinetic study and screening biological samples. Since MAP2 is quite sensitive than widely used casein, the sensitivity of this assay is much higher than the standard assay. Two hundred pictograms of calpains were capable of being detected by this substrate as reported by Tompa et al. (1995).

The main limitation of this method is the inhomogeneity of MAP2 because MAP2 could be labeled and cleaved at multiple sites. The competition from other endogenous proteases may result in significant background in fluorescence and interfere the analysis for crude samples.

Some peptides have been known to be sensitive hydrolyzed substrates for calpains

(Croall and Demartino, 1991; Sasaki, Yumoto, Yoshimura and Murachi, 1984). Using Z-

Leu-Arg-MNA as substrate, Harris et al. (1995) developed a continuous and fluorogenic assay for µ-calpain. They found that the rate of hydrolysis was linear with the concentration of calpain. Mallya et al. (1998) described another dipeptide Suc-LY-MNA to be an excellent substrate for calpain assay. This assay was shown to be one of the most sensitive methods thus far. Ten picomolar of µ-calpain could be detected efficiently by this assay in the presence of 0.1% CHAPS (3-[(3-Cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate) which interacted with substrates to improve fluorescence (Mallya et al., 1998). This method has the advantage in the continuity. The rate of hydrolysis of Suc-LY-MNA was linear with a wide range of calpain concentration (Tompa et al., 1995). It could be easily applied in kinetic study in screening a large number of samples. Using labeled dipeptides to determine calpain assay is more convenient and rapid than using proteins as substrates. One of the limitations using synthetic peptides for calpain assay is that most of these peptides are inherently

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different with the degradation model of endogenous substrates of calpain. Therefore, this

method may not provide accurate activity as in physiological conditions. When it is used in

tissue samples, the contaminating protease is possible to cause background drift. In addition,

other substrates may compete with MAP2 to cause underestimated calpain activity.

Melloni et al. (1984) used denatured globin as substrate and measured free amino

groups with fluorescamine. The enzyme activity was defined as the amount to release of one

µM of free amino groups in 10 min reaction period. Other cytoskeletal proteins such as

spectrin and tau are possible substrate for calpain assay (Goll et al., 2003). However, these

proteins have not been used a lot compared with casein because of their availability,

solubility and sensitivity to calpains.

Capillary electrophoresis

Several labs have shown that capillary electrophoresis is a rapid and reproductive

technique to analyze peptides produced from degradation of phospholipase and cathepsin D

(Choi, Lee, Na and Yoo, 1999; Chu, O’Dwyer and Zeece, 1997). Gu et al. (2001) recently

used this capillary eletrophoretic technique to measure degradation products of Oregon

Green labeled αs-casein and β-casein due to calpains. Sodium dodecyl sulfate (SDS) instead of TCA was chosen to stop reaction because SDS was considered as one highly efficient surfactant under this experimental condition for micellar separation. Products were separated using capillary electrophoresis in micellar electrokinetic capillary chromatography mode.

Laser-induced fluorescence detection was used to detect separated peptides at excitation wavelength of 488 nm and emission wavelength of 520 nm. This method found that β-casein was more sensitive than αs-casein. This technique may be the most sensitive method for

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calpain assay thus far. One to two ng calpain was enough to determine all of the points in the

curve. Compared with the TCA-soluble based methods, this assay only needs 100 times less

enzymes for analysis (Gu et al., 2001).

Summary

Developing a sensitive, simple and continuous assay for calpain is critical for meat

scientists to monitor the changes of calpain system. This could be helpful to predict the

subsequent meat quality based on the status of calpain system. Many different assays have

been developed during past thirty years for different conditions. Standard assay is using

casein as substrate to monitor the amount of TCA-soluble peptides. It is usually applied when

tissue samples or calpains are abundant because of the low sensitivity of this assay. Using

labeled-casein as substrates for calpains is developed to overcome the disadvantage of low sensitivity of the standard assay. These label compounds include FITC, BODIPY-FL, and radiolabeled compounds 14-C-methylated and [3-H]. The methods mentioned above are

tedious to separate final peptide fragments from undigested substrates. Casein zymography

and colorimetric assays could eliminate the step of precipitating left substrates. Hence, they

are effective to be used for continuous recording of calpain activity. Other substrates instead

of casein have been mentioned including natural proteolytic substrate MAP2 and some other

dipeptides Z-Leu-Arg-MNA and Suc-LY-MNA. Recently, a high sensitive assay method has

been described using capillary electrophoresis to separate the peptides. This method is

suitable for monitoring low concentration of calpain analysis. It is noteworthy that different

methods really can achieve different activities. For example, the specific activity of m-

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calpain was two fold more than µ-calpain when measured with a FITC-casein. However,

these two calpains show almost identical specific activities in a fluorescent microplate assay

(Thompson et al., 2000). Even the separation methods for three components of calpain

system can influence detected activity (Geesink and Koohmaraie, 1999). In future, more dependable and accurate methods need to be further developed to monitor the status of calpain system especially during pre-slaughter and postmortem period. This can help us further understand how this amazing enzyme works in different conditions and thus produce high quality of fresh meat.

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Zhang, W. G., Lonergan, S. M., Gardner, M. A., and Huff-Lonergan, E. (2006). Contribution of postmortem changes of integrin, desmin and µ-calpain to variation in water holding capacity of pork. Meat Science, 74:578-585.

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Contribution of postmortem changes of integrin, desmin and µ-calpain to variation in

water holding capacity of pork1

A paper published in Meat Science

Wangang Zhang2, Steven M. Lonergan, Matt A. Gardner and Elisabeth Huff-Lonergan3

Abstract

The purpose of this study was to examine the relationship between integrin, desmin, µ-

calpain and water-holding capacity in fresh pork. High levels of intact integrin at one day

postmortem were negatively correlated with day 1 (P < 0.05) and days 1-5 (cumulative) drip

loss (P < 0.05). High levels of intact integrin at five days postmortem were negatively

correlated with days 1-7 (cumulative) purge loss (P < 0.05). Intensity of intact desmin at one

day postmortem was positively correlated with days 1-7 purge loss (P < 0.01). There were

positive correlations between intensity of intact desmin at day 7 and day 1 (P < 0.01), days 1-

5 drip loss (P < 0.01) and days 1-7 purge loss (P < 0.05). Autolysis of µ-calpain was

associated with the degradation of desmin and drip or purge loss postmortem. Our results

indicate that low levels of degradation of integrin and high levels of desmin degradation were associated with low drip loss values in fresh pork.

1Reprinted with permission of Meat Science, 2006, 74,578-585.

2Graduate student, Department of Animal Science, Iowa State University.

3Author for correspondence: 2378 Kildee Hall; Tel: +1 515 294 9125; fax: +1 515 294 9143; E-mail address: [email protected]

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Introduction

The ability of meat to retain moisture during postmortem storage is referred to as its

water holding capacity (WHC). Low WHC is associated with high drip or purge loss and

subsequently reduces profitability in meat production. Numerous and complex factors

contribute to the considerable variability in WHC of meat. Genotype (Hamilton, Ellis, Miller,

McKeith and Parrett, 2000), different stunning methods (Stoier, Aaslyng, Olsen and Henckel,

2001), aging (Kristensen and Purslow, 2001), chilling rate (Maribo, Olsen, Barton-Gade,

Moller and Karlsson, 1998) and even protein oxidation (Rowe, Maddock, Lonergan and

Huff-Lonergan, 2004) may all affect the WHC of meat. Recently, several authors have

shown that degradation of cytoskeletal and other structural proteins plays an important role in

drip loss (Huff-Lonergan and Lonergan, 2005; Kristensen and Purslow, 2001; Melody,

Lonergan, Rowe, Huiatt, Mayes and Huff-Lonergan, 2004; Schafer, Rosenvold, Purslow,

Andersen and Henckel, 2002) and may work in concert with biophysical forces to influence

WHC (for a review, see Huff-Lonergan and Lonergan, 2005; Offer and Trinick, 1983).

Integrin, the heterodimeric cell adhesion molecule that links the extracellular matrix to

the cytoskeleton, is important in controlling many steps in cell membrane-cytoskeleton

attachments and in signaling pathways (Hynes, 1992). In integrins, 18 α- and 8 β-subunits

have been identified (Plow, Haas, Zhang, Loftus and Smith, 2000; van der Flier and

Sonnenberg, 2001). β-Chain integrin is responsible for the attachment of cell membrane to

the cell cytoskeleton (van der Flier and Sonnenberg, 2001). The degradation of β1 integrin has been suggested to increase the drip channel formation between the cell and cell

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membrane. The resulting drip channel contributes to drip loss postmortem in pork (Lawson,

2004).

One of the protease systems that is commonly thought to play a role in the degradation

of several muscle cell proteins is the calpain system (Huff-Lonergan and Lonergan, 2005).

Two of the more well studied isoforms are µ- and m-calpain. Under hypoxic conditions, µ-

calpain can be activated and cleaves the β3 integrin domains in human endometrial cells

(Aoyama et al., 2004). m-Calpain co-localizes with β1 chain integrin containing focal

adhesions. The inhibition of calpain activity has been shown to reduce both integrin

degradation and drip channel formation in postmortem muscle (Lawson, 2004).

Degradation of cytoskeletal proteins also plays an important role in determining

tenderness and WHC of meat during postmortem storage (Melody et al., 2004; Morrison,

Mielche and Purslow, 1998). It has been documented that myofibrils experience shrinkage

during postmortem storage and this process can cause increased drip loss (Johnson, Jeremiah,

and Robertson, 1993; Offer and Trinick, 1983; Swatland and Belfry, 1985). Desmin is one of

the intermediate filament proteins that ties the myofibrils to the cell membrane and connects

adjacent myofibrils (Robson et al., 1997). Desmin is a known substrate of µ-calpain (Huff-

Lonergan et al., 1996). The rate and extent of degradation of desmin affects WHC during postmortem storage (Melody et al., 2004). Limited degradation of desmin maintains the connections between the membrane and the myofibrils and increases the transfer of the shrinkage of myofibrils into the shrinkage of the whole cells increasing drip or purge loss postmortem (Kristensen and Purslow, 2001).

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µ-Calpain is known to be involved in the proteolysis of several myofibrillar and other cytoskeletal proteins in bovine skeletal muscle during postmortem storage (Huff-Lonergan et al., 1996). µ-Calpain is composed of an 80 kDa and a 28 kDa subunit and both of them autolyze in the presence of calcium (Cong, Goll, Peterson and Kapprell, 1989). The large 80 kDa subunit of µ-calpain is reduced to 76 kDa through a 78 kDa intermediate form (Cottin,

Thompson, Sathe, Szpacenko and Goll, 2001). Although both unautolyzed and autolyzed forms of µ-calpain have proteolytic activity, it has been suggested that autolysis has significance in early postmortem muscle (Melody et al., 2004). The autolyzed form has a reduced calcium concentration requirement for activity (Goll, Thompson, Taylor, Edmunds and Cong, 1995) and it binds its substrates more tightly than the unautolyzed form (Boehm,

Kendall, Thompson and Goll, 1998). Additionally, autolysis of µ-calpain has been suggested to be indicative of proteolytic activation in living muscle and in postmortem muscle or meat

(Geesink and Koohmaraie, 1999; Goll, Thompson, Li, Wei and Cong, 2003; Melody et al.,

2004). Greater autolysis of µ-calpain in postmortem muscle is associated with degradation of some proteins in pork like desmin. The early postmortem degradation of these proteins may influence WHC and tenderness (Melody, et al., 2004; Rowe, Huff-Lonergan and Lonergan,

2001).

Because of the different function and location of integrin and desmin in postmortem muscle, we hypothesized that these proteins differentially influence water-holding capacity.

Therefore, the aim of this study was to determine the relationship between degradation of integrin and desmin, µ-calpain autolysis and WHC in pork during postmortem storage.

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Materials and methods

Animals

Sixty-four halothane negative Duroc x Yorkshire gilts (n = 32) and barrows (n = 32) weighing approximately 113 kg were slaughtered using humane practices at the Iowa State

University Meat Laboratory. All pigs originated from the same farm, and were held in lairage without feed overnight.

Sample collection

At one day postmortem, loin samples were excised from the left sides of the carcasses.

These samples were used for drip loss measurement, µ-calpain, integrin and desmin analysis.

The posterior ends of the boneless loins (sirloin end, approximately 1.2 kg) were used for purge analysis. Unless otherwise noted, the samples were stored at 4 °C.

Water-holding capacity

Drip loss was measured in duplicate using 2.54-cm thick loin samples. At day 1 postmortem, samples were placed in plastic bags under atmospheric conditions at 4 °C.

Immediately prior to being placed in bags, chops were towel dried and the initial weight of the chops was recorded. After one day of storage, samples were removed from their individual bags and were towel dried and weighed again. The chops were then placed in new bags and stored for an additional four days. Following five days of storage, chops were again towel dried and weighed. Drip loss after one day or five days of storage was calculated as a difference between final and initial weight expressed as a percentage of the initial weight.

Purge loss was measured on the sirloin portion after 7 days of storage at 1 ºC in a vacuum bag. Prior to storage, samples were towel dried to remove excess surface moisture,

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weighed to determine initial weight, and vacuum packaged. After storage, samples were

removed from their packaging, towel dried, and weighed again. Purge loss was calculated as

a percentage of the final weight from each sample compared to the initial weight.

SDS-PAGE Sample Preparation

Whole-muscle extracts of the longissimus dorsi were prepared using the methods

described by Lonergan, Huff-Lonergan, Rowe, Kuhlers and Jungst (2001). The

concentration of protein in the extract was determined for each sample using methods

described by Lowry, Rosebrough, Farr and Randall (1951) for each sample using prepared

reagents (DC Protein Assay, Bio-Rad, Hercules, CA). Protein concentration was then

adjusted to a concentration of 6.4 mg/mL by diluting with deionized water. One volume of

each diluted sample was combined with 0.5 volumes of tracking dye solution (3 mM EDTA,

3%[wt/vol] SDS, 30%[vol/vol] glycerol, 0.001% [wt/vol] pyronin Y, and 30 mM Tris-HCl,

pH 8.0) and 0.1 volume of ß-mercaptoethanol for a final protein concentration of 4

mg/mL. The gel samples were heated for 20 min at 50 ºC and frozen at -80 ºC for

storage. These whole-muscle samples were used to analyze µ-calpain, integrin, and desmin using SDS-PAGE and western blotting techniques.

SDS-PAGE

A 10% polyacrylamide separating gel (acrylamide:N,N'-bis-methylene acrylamide =

100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.05% [vol/vol] TEMED, 0.05% [wt/vol] APS, and 0.5 M

Tris•HCl, pH 8.8) was used for determination of µ-calpain autolysis, integrin and desmin

(whole-muscle samples). A 5% polyacrylamide gel (acrylamide:N,N'-bis-methelyene

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acrylamide = 100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.125% [vol/vol] TEMED, 0.075% [wt/vol]

APS, and 0.125 M Tris•HCl, pH 6.8) was used for the stacking gel.

Running Conditions

Gels (10 cm wide x 8 cm tall x 1.5mm thick) were run on SE 260 Hoefer Mighty Small

II electrophoresis units (Hoefer Scientific Instruments). The running buffer contained 25 mM

Tris, 192 mM glycine, 2 mM EDTA, and 0.1% [wt/vol] SDS. Gels were loaded with 80 µg per lane of total proteins for integrin (whole-muscle extracts), 30 µg per lane of total proteins

for desmin (whole-muscle extracts) and 60 µg per lane of total proteins for µ-calpain and run

at a constant voltage of 120 V.

Transfer Conditions

Gels for integrin, desmin and µ-calpain were transferred to polyvinylidene difluoride

(PVDF) membranes (Schleicher and Schuell, Inc., Keene, NH) using a TE22 Mighty Small

Transphor electrophoresis unit (Hoefer Scientific Instruments) at a constant voltage of 90 V for 1.5 h. The transfer buffer consisted of 25 mM Tris, 192 mM glycine, 2 mM EDTA, and

15% (vol/vol) methanol. The temperature of the transfer buffer was maintained using a refrigerating circulating water bath set at -1.0 °C (Ecoline RE106; Lauda Brinkmann,

Westbury, NY).

Western Blotting

After transfer, membranes were blocked in a solution composed of 80 mM disodium

hydrogen orthophosphate, 20 mM sodium dihydrogen orthophosphate, 100 mM sodium

chloride, 0.1% (vol/vol) polyoxyethylene sorbitan monolaurate (Tween-20)(PBS-Tween),

and 5% nonfat dry milk (wt/vol) for 1 h at room temperature. After blocking, membranes

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were placed in their respective primary antibody diluted in PBS-Tween. The integrin primary

antibody (mouse anti-integrin β 1D monoclonal antibody, MAB1900; Chemicon

International, Temecula, CA) and µ-calpain primary antibody (monoclonal anti-µ-calpain

antibody, MA3-940; Affinity Bioreagents, Inc., Golden, CO) were diluted 1:10,000 in PBS-

Tween and were incubated with the blots overnight at 4°C. The desmin primary antibody

(polycolonal rabbit anti-desmin antibody, V2022; Biomedia, Foster City, CA) was used at a

dilution of 1:10,000 in PBS-Tween for 2 h at room temperature. After primary antibody

incubations were complete, integrin membranes were washed four times (10 min for first

three washes and 5 min for the fourth wash) using PBS-Tween at room temperature. Desmin

and µ-calpain membranes were washed three times (10 min/wash) at room temperature using

PBS-Tween before incubation with the secondary antibody. Integrin, desmin and µ -calpain blots were incubated 1 h at room temperature with the secondary antibody diluted 1:5,000 in

PBS-Tween (goat anti-mouse conjugated horseradish peroxidase for integrin and µ-calpain, catalog No. A2554; goat anti-rabbit conjugated with horseradish peroxidase for desmin, catalog No. A9169, Sigma Chemical Co.). After completion of the secondary antibody incubation, integrin membranes were washed four times (10 min for first three washes and 5 min for the fourth wash) and desmin and µ-calpain membranes were washed three times (10 min/wash) using PBS-Tween at room temperature. Detection was initiated using premixed reagents (ECL Plus Western Blotting Detection Reagents, RPN2132, Amersham Bioscience

UK Limited, Little Chalfont Buckinghamshire, England). Chemiluminescence was detected using a 16-bit mega-pixel CCD camera FluorChem 8800 (Alpha Innotech Corp., San

Leandro, CA) and FluorChem IS-800 software (Alpha Innotech Corp.). Densitometric

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measurements were completed using the AlphaEaseFC software (FluorChem 8800, Alpha

Innotech Corp.).

The western blots of intact integrin are displayed in Fig. 1. Data for integrin degradation was calculated as the ratio of the intensity of the intact integrin band in each sample over intensity of the intact integrin band in the internal designated densitometry standard

(Reference sample-Lane 5 in Fig. 1). Desmin degradation was determined and recorded as a ratio of each sample to the intact desmin in a reference sample that was run on all gels

(Melody et al., 2004).

Autolysis of µ-calpain was evaluated on immunoblots of whole-muscle extracts of samples aged 1 d postmortem. Data were reported as percentage of the large subunit present as 80, 78 or 76 kDa polypeptide.

Statistical analysis

All data were analyzed using SAS Version 9.1 (Cary, NC) and significance was reported at the P < 0.05 and P < 0.01 level. Pearson correlation coefficients were calculated to determine the relationship between different parameters.

Results and discussion

Correlations between intact desmin and drip or purge loss

The intensity of intact desmin was positively correlated with the drip and purge loss during postmortem storage (Table 1). At one day postmortem, there were significant positive correlations between intensity of the intact desmin and sirloin purge loss after day 7

(P<0.01). The cumulative drip loss after five days tended to be related to intensity of the

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intact desmin at day 1 (P=0.054). At day 7, there was a significant positive correlation between the intensity of the intact desmin and day 1 drip loss (P<0.01), total drip loss after day 5 (P<0.01), and sirloin purge loss after day 7 (P<0.05). These results show that a greater amount of intact desmin is associated with increased drip or purge loss during postmortem storage.

Skeletal muscle has a very complex structure and cytoskeletal proteins, such as desmin, play important roles in maintaining the integrity of that structure at the cellular level. As one of the major intermediate filament proteins, the protein desmin plays an important role in maintaining the lateral alignment of adjacent myofibrils and also links the peripheral layer of myofibrils to the cell membrane skeleton. Thus, the position of desmin allows it to transmit the transverse shrinkage of the myofibrils to the entire cell and thus influence drip or purge loss postmortem. Desmin has been shown to be highly sensitive to postmortem proteolysis.

Degradation of desmin and other cytoskeletal and intermediate filament proteins does contribute to the development of meat tenderness during postmortem storage (Huff-Lonergan et al., 1996; Taylor, Geesink, Thompson, Koohmaraie and Goll, 1995). Several authors have shown that a high level of desmin degradation is correlated with high WHC during postmortem storage (Kristensen and Purslow, 2001; Melody, et al., 2004; Rowe et al., 2001).

Thus, continued study of this protein may yield important insights into the mechanism underlying drip production in product with normal or near normal ultimate pH. In our current study, intensity of intact desmin at one day and seven days was positively correlated with drip loss after five days of storage and sirloin purge loss after seven days of storage. These results provide evidence to support the hypothesis that lower level of desmin degradation

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result in the shrinkage of the muscle cells that translates into high drip loss or purge loss during postmortem storage (Huff-Lonergan and Lonergan, 2005, Kristensen and Purslow,

2001). Offer and Cousins (1992) provided a hypothesis that shrinkage of the myofilament lattice could be transferred to the shrinkage muscle fibers, which ultimately contributes to formation of gaps between muscle cells and between muscle bundles. Our results are in agreement with Kristensen and Purslow (2001), who described that the degradation of the cytoskeleton was responsible for the WHC increases during aging. However, Schafer et al.

(2002) reported that variations in the degradation of desmin only accounted for 13% of the variation in drip loss using the PLS1 (Partial Least Squares) models and concluded that the degree of degradation of desmin was not correlated with drip loss. But it is noteworthy that

Schafer et al. (2002) only measured the early structural changes of cytoskeletal proteins from

3 to 24 h, while the best relationships were noted after 24 hours in current study and in

Kristensen and Purslow (2001).

Purge loss is a significant industry concern because many water-soluble proteins are lost from the muscle with purge loss and this purge loss has a negative impact on the nutritional value of meat (Savage, Warriss and Jolley, 1990). In addition, purge also represents a significant loss of weight and thus is of economic concern (Wright et al., 2005). Our results showed that purge loss was highly correlated with the intensity of the intact desmin at both one and seven days after storage. Davis, Sebranek, Huff-Lonergan and Lonergan (2004) showed that the samples with the highest purge loss had least desmin degradation at day 1 and day 4 postmortem. All of these results clearly indicate that a high level of intact desmin

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in postmortem muscles could not only increase the drip loss, but its degradation may also

improve purge loss during postmortem storage.

Correlations between calpain µ-autolysis and drip or purge loss

Several authors have hypothesized that the degradation of cytoskeletal proteins is

associated with the tenderness and water holding capacity of meat (Kristensen and Purslow,

2001; Melody et al., 2004;; Morrison et al., 1998). Degradation of there proteins, including

desmin, is thought to be mainly due to µ-calpain in postmortem muscle (Huff-Lonergan et al.,

1996; Koohmaraie, 1992; Rowe et al., 2004). In postmortem muscle, autolysis of µ-calpain is associated with its activation (Geesink and Koohmaraie, 1999). Therefore, autolysis of µ- calpain at 24 h postmortem was examined in our current study to determine the relationship between µ-calpain autolysis (and presumed activation) and drip or purge loss (Table 2). The

78 kDa autolysis product at day 1, the intermediate product of µ-calpain large subunit autolysis, was positively correlated with the drip loss or purge loss (P<0.05). However, the

76 kDa subunit was negatively correlated with drip loss or purge loss postmortem (P<0.05).

Greater presence of the 76 kDa autolysis product at one day postmortem indicates that µ- calpain may have been activated earlier and could degrade its substrates (including desmin) earlier postmortem. A high level of degradation of desmin is associated with lower drip or purge loss during postmortem storage. It was notable that the 76 kDa form of µ-calpain at day 1 had a negative correlation with purge loss after seven days (P<0.01). These results indicate that greater autolysis of µ-calpain at day 1 is predictive of lower drip loss or purge loss postmortem in pork likely through enhancing proteolysis of specific key muscle cell proteins. Melody et al. (2004) reported that the early postmortem biochemical factors, such

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as µ-calpain autolysis and protein degradation, might be involved with subsequent meat tenderness and water holding capacity. Based on the significant correlations of autolysis of

µ-calpain and drip or purge loss, autolysis of µ-calpain at one day postmortem may prove to be a useful tool to predict the drip or purge loss postmortem and may begin to allow us to decipher the mechanism underlying differences in WHC.

Correlations between intact integrin and drip or purge loss

Table 3 shows the Pearson correlation coefficients between intensity of the intact integrin band at day 1 and day 5 of storage and drip or purge loss measurements. Overall, all the coefficients were negative for the relationships between intact integrin and drip or purge loss. Negative correlations between intact integrin at day 1 of storage and day 1 drip loss

(P<0.05) and total drip loss (over the entire five days of storage) (P<0.05) were detected. At day 5 of storage, intensity of intact integrin was negatively correlated with sirloin purge loss after day 7 of storage (P<0.05). These results show that a greater amount of detectable intact integrin is associated with decreased drip loss or purge loss during postmortem storage in pork.

Integrin-containing adhesions (Hynes, 1999) and the dystrophin/dystroglycan

(Zubrzycka-Gaarn et al., 1991) complex of proteins are major factors that attach the cell membrane to the cell cytoskeleton. Integrins are a family of adhesion receptors that mediate interactions between the extracellular matrix and the cytoskeleton (van der Flier and

Sonnenberg, 2001). α- and β-subunits are the two chains of integrin that form many different combinations. Each of these combinations has different biological functions. The β chains of integrin play the major role for the adhesion of cell cytoskeleton to the cell membrane

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(Cukierman, Pankov and Yamada, 2002). Although several β-subunits have been described,

the β1-subunit is the best studied and it is most common in muscle tissue (de Melker and

Sonnenberg, 1999).

Lawson (2004) showed that the degradation of β1-integrin in pork was associated with formation of drip channels. These gaps were shown to exist in all samples by 24 h of postmortem storage in pork (Lawson, 2004). Drip channels appeared as early as 3 h postmortem in products with high drip loss, while the channels took as long as 9 h to form in products with relatively low drip loss (Lawson, 2004). Detectable space between single cells has been shown as early as 3 h in pork postmortem and this space has been estimated to account for 47% of the variation in drip loss (Schafer et al., 2002). These results support the observations in the current study, which show significant negative correlations between intact integrin and drip or purge loss during postmortem storage. The degradation of β1-subunit integrin of integrin weakens the interaction between the cell/membrane attachment. This may mean that degradation of membrane associated proteins could allow drip channel formation and may be another important factor that influences drip loss. These results indicate that intact integrin may play a role to block the drip formation between cell membranes and cell cytoskeleton and between muscle cells and thus a higher level of intact integrin could decrease drip or purge loss during postmortem storage.

Correlations between intact integrin, desmin and µ-calpain autolysis

The proteases µ- and m-calpain are the two most thoroughly studied calpains among several isoforms. Both of them autolyze when incubated with free Ca2+. The biological significance of autolysis has not been completely understood. The 80 kDa subunit of µ-

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calpain is first to be reduced to the 78 kDa fragment and then to the 76 kDa fragment

(Zimmerman and Schlaepfer, 1991). Several authors have reported that the autolysis of µ- calpain reduced the Ca2+ concentration required for its proteolytic activity from 3-50 µM to

0.5-2.0 µM (Cong et al., 1989; Goll et al., 1995). This autolysis could have important

physiological significance because it decreases the Ca2+ requirement for proteolytic activity

making it closer to the Ca2+ concentration in vivo. Baki, Tompa, Molnar and Friedrich (1996)

and Cottin, Poussard, Desmazes, Georgescauld and Ducastaing (1991) hypothesized that the

autolysis of µ-calpain preceded its proteolytic activity. Hence, it is generally accepted that

the autolysis of µ-calpain can be used as an indicator for its proteolytic activation in

postmortem muscle (Geesink and Koohmaraie, 1999; Rowe et al., 2004). The current study

examined the extent to which autolysis of µ-calpain is associated with proteolytic changes in

integrin and desmin. µ-Calpain autolysis was determined by the decrease of 80 kDa subunits and the increase of 78 kDa and 76 kDa autolysis products.

Table 4 and Table 5 illustrate correlation coefficients between µ-calpain autolysis at day

1 and the intensity of intact integrin and desmin postmortem. There were no significant correlations between intensity of the intact integrin and different stages of autolysis of µ- calpain at day 1 or day 5 (Table 4). However, there was a significant, positive correlation between the intensity of intact desmin at day 7, the 80 kDa subunit (P<0.05) and the 78 kDa autolysis product (P<0.05). Intensity of the intact desmin was negatively correlated with the

76 kDa autolysis product (P<0.01) of µ-calpain at day 7. At day 1, intensity of intact desmin was not significantly correlated to µ-calpain autolysis.

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Du et al. (1995) showed that calpain was the enzyme that cleaved the integrin β3 subunit of the cytoplasmic domain. Pfaff, Du and Ginsberg (1999) subsequently found that calpain was also responsible for the cleavage of other integrin β cytoplasmic domains in vitro. In human endometrial cells, the β3 integrin domain was cleaved by µ-calpain under hypoxic conditions (Aoyama et al., 2004). However, few studies have focused on the relationship between integrin degradation and µ-calpain in postmortem muscle tissues. Lawson (2004) reported that calpains were present at the cell surface where the β1-integrin subunits localize

in postmortem muscle tissue. The cysteine protease inhibitor E-64 reduced the integrin

degradation and decreased the drip channel formation. Lawson (2004) showed that m-calpain

co-localizes with β1-integrin on the muscle cell surface, while µ-calpain was spread throughout the muscle cells. The relationship between calpain and integrin degradation in postmortem muscle appears to be complex and needs more detailed investigation in the future.

Our current results showed negative correlations between autolysis of µ-calpain at day 1 and intact desmin intensity at day 7 indicating that there is a possible relationship between µ- calpain autolysis and desmin degradation. Melody et al. (2004) also found that the autolysis of µ-calpain was associated with degradation of desmin in postmortem porcine muscle. The positive correlations between intensity of the intact desmin and 80 kDa subunits of µ-calpain provides evidence that slow or impeded autolysis of the 80 kDa intact catalytic subunit to 76 kDa autolysis product at day 1 (and thus slower activation of µ-calpain) is related to low levels of desmin degradation at day 7.

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Conclusions

Integrin and desmin may play different roles in water holding capacity postmortem in

pork. The degradation of integrin could contribute to the formation of drip channels between the cell membrane and cell body and subsequently increases the drip loss. Intact desmin helps to transfer the shrinkage of myofibrils to whole cell level and force the water out of the myofibrils. Autolysis of µ-calpain is associated with the degradation of desmin and autolysis of µ-calpain at 24 h is a potent indicator to predict the degree of desmin degradation and drip or purge loss postmortem.

Acknowledgements

Appreciation is extended to the National Pork Board for funding a portion of this research. The authors are grateful for the significant efforts of Mr. Randy Petersohn and the

ISU Meat Laboratory for assistance in harvesting and fabrication of pork carcass.

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Table 1 Correlations between intensity of the intact desmin and drip or purge lossa

Intact Intact Drip loss Total drip Sirloin desmin desmin (day 1) loss purge loss (day 1) (day 7) (days 1-5) (days 1-7)

Intact desminb 1.0000 0.3712 0.1359 0.2462 0.4800 (day 1) (P=0.003) (P=0.292) (P=0.054) (P<0.001)

Intact desminb 1.0000 0.3631 0.3938 0.2789 (day 7) (P=0.004) (P=0.002) (P=0.031)

Drip loss 1.0000 0.8949 0.4321 (day 1) (P<0.001) (P=0.001)

Drip loss 1.0000 0.5961 (days 1-5) (P<0.001)

Sirloin purge 1.0000 loss (days 1-7) aPearson correlations were determined to be significant at P<0.05 and P<0.01 levels. bRatios were calculated as intensity of the intact integrin band in each sample over intensity of the intact integrin band in the internal designated densitometry standard.

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Table 2 Correlations between intact µ-calpain autolysis and drip or purge lossa

µ-Calpain µ-Calpain µ-Calpain Drip loss Total drip Sirloin (80 kDa) (78 kDa) (76 kDa) (day 1) loss purge loss (days 1-5) (days 1-7)

µ-Calpainb 1.0000 0.2791 -0.8087 0.1315 0.2048 0.3817 (80 kDa) (P=0.041) (P<0.001) (P=0.343) (P=0.138) (P=0.004)

µ-Calpainb 1.0000 -0.7906 0.2713 0.3272 0.4066 (78 kDa) (P<0.001) (P=0.047) (P=0.016) (P=0.002)

µ-Calpainb 1.0000 -0.2497 -0.3308 -0.4927 (76 kDa) (P=0.069) (P=0.015) (P<0.001)

Drip loss 1.0000 0.8949 0.4321 (day 1) (P<0.001) (P=0.001)

Total drip 1.0000 0.5961 loss (P<0.001) (days 1-5) 1.0000 Sirloin purge loss (days 1-7) aPearson correlations were determined to be significant at P<0.05 and P<0.01 levels. bRatios were calculated as the percentage of intensity of different subunit of µ-calpain over total intensity of three different subunits.

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Table 3 Correlations between intensity of the intact integrin and drip or purge lossa

Intact integrin Intact integrin Drip loss Total drip loss Sirloin purge (day 1) (day 5) (day 1) (days 1-5) loss (days 1-7)

Intact integrinb 1.0000 0.3090 -0.3022 -0.2864 -0.0720 (day 1) (P=0.018) (P=0.020) (P=0.028) (P=0.588)

Intact integrin 1.0000 -0.0554 -0.1617 -0.2866 b (P=0.672) (P=0.213) (P=0.025) (day 5)

Drip loss 1.0000 0.8949 0.4321 (day 1) (P<0.001) (P=0.001)

Total drip loss 1.0000 0.5961 (days 1-5) (P<0.001)

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Table 4 Correlations between intensity of the intact integrin and µ-calpain autolysisa

Intact integrin Intact integrin µ-Calpain µ-Calpain µ-Calpain (day 1) (day 5) (80 kDa) (78 kDa) (76 kDa)

Intact integrinb 1.0000 0.3090 0.2094 -0.1499 -0.0452 (day 1) (P=0.018) (P=0.136) (P=0.289) (P=0.750)

Intact integrinb 1.0000 0.1185 -0.1664 0.0263 (day 5) (P=0.393) (P=0.229) (P=0.850)

µ-Calpainc 1.0000 0.2791 -0.8087 (80 kDa) (P=0.041) (P<0.001)

µ-Calpainc 1.0000 -0.7906 (78 kDa) (P<0.001)

µ-Calpainc 1.0000 (76 kDa) aPearson correlations were determined to be significant at P<0.05 and P<0.01 levels. bRatios were calculated as intensity of the intact integrin band in each sample over intensity of the intact integrin band in the internal designated densitometry standard. cRatios were calculated as the percentage of intensity of different subunit of µ-calpain over total intensity of three different subunits of µ-calpain.

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Table 5 Correlations between intensity of the intact desmin and µ-calpain autolysisa

Intact Intact µ-Calpain µ-Calpain µ-Calpain desmin desmin (80 kDa) (78 kDa) (76 kDa) (day 1) (day 7)

Intact desminb 1.0000 0.3712 0.2043 0.1346 -0.2129 (day 1) (P=0.004) (P=0.138) (P=0.332) (P=0.122)

Intact desminb 1.0000 0.3464 0.2863 -0.3957 (day 7) (P=0.011) (P=0.038) (P=0.003)

µ-Calpainc 1.0000 0.2791 -0.8087 (80 kDa) (P=0.041) (P<0.001)

µ-Calpainc 1.0000 -0.7906 (78 kDa) (P<0.001)

µ-Calpainc 1.0000 (76 kDa) aPearson correlations were determined to be significant at P<0.05 and P<0.01 levels.

bRatios were calculated as intensity of the intact integrin band in each sample over intensity of the intact

integrin band in the internal designated densitometry standard.

cRatios were calculated as the percentage of intensity of different subunit of µ-calpain over total intensity

of three different subunits of µ-calpain .

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1 2 3 4 5* 6 7 8

Intact integrin

0.402 0.688 0.890 0.618 1.000 0.278 0.207 1.068 Ratios of the intensity of intact integrin

1.44 1.35 1.84 1.58 1.46 2.50 2.25 1.22 Drip loss

Fig.1. Western blots of intact integrin (top dark bands) in purified myofibrils at 24 h. All lanes were loaded with 80 µg per lane of total proteins. Ratios were calculated as intensity of the intact integrin band in each sample over intensity of the intact integrin band in the internal designated densitometry standard (lane 5).

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S-nitrosoglutathione affects the activity and autolysis of m- and µ-calpain

A paper to be submitted to Journal of Animal Science

Wan-gang Zhang, Steven M. Lonergan and Elisabeth Huff-Lonergan

Abstract

Effects of S-nitrosoglutathione (GSNO) on nitrosylation, activity and autolysis of

calpains in the presence and absence of calcium at pH 6.5 and 7.5 were determined. The

hypothesis was that nitric oxide can regulate the function of m- and µ-calpain through the S- nitrosylation. The objective was to determine the effects of a nitric oxide donor on the activity, nitrosylation and autolysis of m- or µ-calpain. Porcine skeletal muscle m- and µ- calpain were nitrosylated with GSNO. Nitrosylation of µ-calpain was identified using a commercial S-nitrosylated Protein Detection Assay Kit. Calpain activity and autolysis were measured to determine the extent to which nitrosylation influence these property of calpain.

The five treatments for calpains (either m- or µ-calpain) were (in order of addition): 1): control: calpain; 2): GSNO: calpain+GSNO; 3): CaCl2: calpain+CaCl2; 4): CaCl2+GSNO: calpain+ CaCl2+GSNO; 5): GSNO+CaCl2: calpain+GSNO+CaCl2. One additional (DTT)

treatment for µ-calpain nitrosylation was added in which µ-calpain was pre-incubated with dithiothreitol for 12 hours. m-Calpain activity was lower in treatments with combination of

GSNO and calcium at both pH 6.5 and 7.5 regardless of the addition order of calcium and

GSNO (P<0.05). The decrease of m-calpain activity due to GSNO was greater at pH 6.5 than

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at pH 7.5. Activity of µ-calpain was higher when it was exposed to GSNO before calcium

compared to only exposed to calcium (P<0.05). GSNO did not affect the activity of calpains at either pH in the absence of calcium (P>0.05). Autolysis of the 80 kDa subunit of µ-calpain was slowed when it was exposed to GSNO before calcium at both pH compared to calcium only group. Nitrosylation detection showed endogenous purified µ-calpain was nitrosylated and part of this nitrosylation could be reversed by DTT incubation. GSNO could further cause µ-calpain nitrosylation in both the absence and the presence of calcium at pH 6.5. In both pH 6.5 and 7.5, very little nitrosylation was detected in groups in which µ-calpain was only incubated with calcium. Our results indicate that GSNO induced protein nitrosylation may play a role in regulating proteolytic activity of m- and µ-calpain and mediating autolysis of µ-calpain.

Keywords: autolysis, calpain, nitric oxide, nitrosylation, S-nitrosoglutathione

Introduction

The calpain system is generally believed to be responsible for the degradation of muscle proteins in postmortem muscle. This proteolysis is known to be associated with development of meat quality including meat tenderness and water holding capacity during postmortem storage (Huff-Lonergan et al., 1996; Koohmaraie, 1992a). Three members of the calpain system are the proteases m- and µ-calpain and their inhibitor calpastatin (Goll et al., 2003).

Calcium, pH, temperature, oxidation and ionic strength have been reported to influence calpain activity (Carlin et al., 2006; Goll et al., 1992; Koohmaraie, 1992b; Rowe et al.,

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2004). Calpains autolyze in the presence of calcium and this autolysis is indicative of their

proteolytic activation in postmortem muscles (Geesink and Koohmaraie, 1999).

Nitric oxide (NO) is a free radical that can be released in many tissues in vivo including

muscle. Endogenous nitric oxide can react with protein residues resulting in protein S- nitrosylation. S-Nitrosylation is the covalent attachment of a nitrogen monoxide group to thiol side chain of cysteine (Hess et al., 2005). The formation of S-nitrosothiols and mixed disulfides through S-nitrosylation can influence catalytic activity of cysteine protease in vivo and vitro (Ascenzi et al., 2001; Michetti et al., 1995). Nitrosylation may be involved in

regulation of meat quality during pre- or post-slaughter periods through influencing protein

function and enzyme activity (Cottrell et al., 2008; Warner et al., 2005; Bellinger et al.,

2008).

S-Nitrosothiols play an important role in NO level as a potential storage and transport

vehicle of NO in vivo (Stamler et al., 1992). GSNO is one of these S-nitrosothiol species

which spontaneously releases NO under physical conditions. Since m- and µ-calpain are

cysteine proteases, this study was designed to test the hypothesis that NO induced

nitrosylation may influence the properties of µ- and m-calpain at different pH conditions. To

achieve this objective, GSNO was used as a NO donor in experiment of highly purified µ-

and m-calpain.

Materials and methods

Purification of µ- and m-calpain

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Purification of µ- and m-calpain was according to the procedures described by

Thompson and Goll (2000) with minor modifications from Maddock et al. (2005). The

muscles were ground and homogenized in six volumes of 10 mM EDTA, 0.1% (vol/vol) β-

mercaptoethanol (MCE) and 100 mM Tris-HCl, pH 8.3, containing 2.5 µM trans-

epoxysuccnyl-L-leucylamido-[4-guanidino]butane (E-64), 0.1 mg/ml ovomucoid trypsin

inhibitor and 0.2 mM phenylmethy-lsulfonylfluoride (PMSF). The homogenate was

centrifuged at 10,000 x g for 30 min at 4 oC. After filtration of the supernatant, proteins were salted out between 0 and 45% ammonium sulfate saturation. Proteins were pelleted at 10,000 x g at 4 oC for 30 min and resuspended in 1 mM EDTA, 0.1% (vol/vol) MCE, and 40 mM

Tris-HCl (TEM, pH=7.4). The solutions were stirred overnight at 4 oC and dialyzed against

TEM. The supernatant was loaded onto a 5 cm x 50 cm QA-52 (GE Healthcare, Piscataway,

NJ) anion-exchange column previously equilibrated in TEM. The column was eluted with a

linear gradient of 0 to 500 mM KCl in TEM (pH=7.5) after removing unbound proteins with

TEM. Crude µ-calpain eluted between 160 and 190 mM KCl and m-calpain eluted between

250 and 280 mM KCl. Fractions from the Q-sepharose column containing µ-calpain activity were pooled. Pooled µ-calpain was purified using successive chromatography over a Phenyl

Sepharose 6 Fast Flow (GE Healthcare, Piscataway, NJ), Butyl Sepharose 4 Fast Flow (GE

Healthcare, Piscataway, NJ), EMD TMAE 650 S (EM Science, Gibbstown, NJ), and DEAE-

TSK Toyopearl (Supelco, Bellefonte, PA). Fractions from the Q-sepharose column

containing m-calpain activity were pooled and purified using successive chromatography over a Phenyl Sepharose 6 Fast Flow (GE Healthcare, Piscataway, NJ), Reactive Red 120

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(Sigma, St. Louis, MO) and DEAE-TSK Toyopearl (Supleco, Bellefonte, PA). The purified

µ-calpain and m-calpain were stored in TEM with the addition of 1 mM sodium azide at 4°C.

Incubations of µ-Calpain and m-calpain

µ-Calpain and m-calpain were incubated in 50 mM HEPES. S-nitrosoglutathione

(GSNO, Sigma, US) was added at a ratio of 2:1 (w:w) GSNO to calpain in corresponding treatments. Anhydrous calcium chloride (J. T. Baker, Phillipsburg, NJ) was used to make calcium solutions. The concentrations of calcium chloride were either 0 or 1 mM in different treatments for activity assay and nitrosylation detection. The specific activity and concentration for m-calpain were 86 unit/mg and 0.194 mg/ml for pH 6.5 and 130 unit/mg and 0.457 mg/ml for pH 7.5. The specific activity and concentration for µ-calpain were 73 unit/mg and 0.413 mg/ml for pH 6.5 and 33 unit/mg and 0.410 mg/ml for pH 7.5.

The five treatments for µ-calpain were (in order of addition): 1): control: µ-calpain; 2):

GSNO: µ-calpain+GSNO; 3): CaCl2: µ-calpain+CaCl2; 4): CaCl2+GSNO: µ-calpain+CaCl2+

GSNO; 5): GSNO +CaCl2: µ-calpain+GSNO+CaCl2. For detection of µ-calpain nitrosylation, one more group was added in which purified µ-calpain was incubated with 5 mM dithiothreitol (DTT) for 12 hours before nitrosylation detection. The amount of µ- calpain was 29 µg in each treatment. The five treatments for m-calpain were (in order of addition): 1): control: m-calpain; 2): GSNO: m-calpain+GSNO; 3): CaCl2: m-calpain+CaCl2;

4): CaCl2+GSNO: m-calpain+CaCl2+GSNO; 5): GSN+CaCl2: m-calpain+ GSNO+CaCl2.

The amount of m-calpain was 27 µg at pH 6.5 and 32 µg at pH 7.5 in each treatment.

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All reactions were done for 60 minutes on ice in cold room. After incubation, protein

concentration of solutions was measured for each treatment using the Braford assay (Bio-Rad

Protein Assay, BioRad, Hercules, CA).

Calpain activity assay

Activity of calpain was measured by standard assay through measuring the release of trichloroacetic acid (TCA)-soluble polypeptides as described by Koohmaraie (1990). Two µg of m-calpain and 1.5 µg of µ-calpain based on the results of protein concentration assay were used in each assay (Bio-Rad Protein Assay, BioRad, Hercules, CA). The assay buffer for measuring calpain activity was made up of 1 volume of TE (40 mM Tris and 0.5 mM EDTA, pH=7.35), 1 volume of assay media (7 mg/ml casein in 100 mM Tris-acetate, pH=7.50) and

0.1 volume of 100 mM CaCl2. To determine whether nitrosylation of calpain could be

reversed by reducing conditions, assay buffers were made with mercaptoethanol (MCE) and

without MCE. The solutions were incubated with corresponding assay buffer at 25 oC for 60 minutes in water bath. After incubation, 5% TCA was added to stop reactions. Reactions were centrifuged at 2,000×g at 20 oC for 20 minutes to precipitate undigested casein from

TCA-soluble peptides. Absorbance value of the supernatant was measured using UV/

Spectrophotometer Ultrospec 3000 (Pharmacia, Peapack, NJ). The final activities of µ- and

m-calpain were calculated as specific activity (U/mg). One unit (U) of activity is defined as

one increase of one absorbance unit at 278 nm after 60 min incubation at 25 oC.

Sample preparation for µ-calpain nitrosylation detection

To detect extent of µ-calpain nitrosylation, µ-calpain was further treated with S-

Nitrosylated Protein Detection Assay Kit (No. 10006518, Cayman Chemical Company, Ann

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Arbor, MI) after incubation on ice at different time points. This detection assay was a

modification named “biotin switch method” from Jaffrey et al. (2001). This assay method

contains three steps to convert nitrosylated cysteins into biotinylated cysteins. These three

steps were: blocking free thiols, reducing nitrosothiols to free thiols and labeling reduced

thiols to N-[6-(biotinamido)hexyl]-3´-(2´-pyridyldithio)propionamide (biotin-HPDP). Biotin-

labeled proteins were further identified by western blotting. Four volumes of ice-cold acetone

were added into tubes after µ-calpain was incubated with different reagents containing

HEPES buffer, GSNO or calcium. Samples were incubated with acetone at -20 oC for 60 min followed by centrifugation to precipitate proteins with mini centrifuge (Fisher Scientific,

Pittsburgh, PA) for 15 min at 4 oC. After decanting supernatants, five volumes of Buffer A

containing S-Nitrosylation Blocking Reagent (No. 10006518, Cayman Chemical Company,

Ann Arbor, MI) were added into microcentrifuge tube to block free thiols of µ-calpain.

Samples were blocked on a rocker with gentle agitation for 45 min in cold room. After blocking, four volumes of ice-cold acetone were used to precipitate µ-calpain as same procedure above. After precipitation, 0.25 mL Buffer B containing Reducing and Labeling

Reagents (No. 10006518, Cayman Chemical Company, Ann Arbor, MI) were added to resuspend the protein pellet for 1 h at room temperature. During this procedure, S- nitrosothiols of µ-calpain were reduced to free thiols and these thiols were labeled with maleimide-biotin. Ice-cold acetone was used to incubate samples at -20 oC for 60 min.

Acetone-containing supernatants were removed after centrifugation with mini centrifuge

(Fisher Scientific, Pittsburgh, PA). Protein was dissolved in 60 µL cold washing buffer (No.

10006518, Cayman Chemical Company, Ann Arbor, MI) and samples were stored at -80 oC

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for subsequent analysis. Biotinylated proteins were detected by S-Nitrosylation Detection

Reagent I (HPR, No. 10006524; Cayman Chemical Company, Ann Arbor, MI, USA) with

western blotting.

µ-Calpain autolysis analysis and SDS-PAGE for nitrosylation detection

An 8% polyacrylamide separating gel (acrylamide:N,N'-bis-methylene acryla-

mide=100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.05% [vol/vol] TEMED, 0.05% [wt/vol] APS, and

0.5 M Tris•HCl, pH 8.8) was used for determination of autolysis and extent of µ-calpain

nitrosylation. A 5% polyacrylamide gel (acrylamide:N,N'- bis-methelyene acrylamide=100:1

[wt/wt], 0.1% [wt/vol] SDS, 0.125% [vol/vol] TEMED, 0.075% [wt/vol] APS, and 0.125 M

Tris•HCl, pH 6.8) was used for the stacking gel.

To determine whether GSNO influenced the autolysis of µ-calpain, samples were removed from reaction solutions at different time points during incubation on ice. Samples were run on SDS-PAGE gels to analyze the autolytic status of 80 kDa larger subunit of µ- calpain. The calcium concentrations used for autolysis study were 0.5 mM in corresponding treatments at pH 6.5 and pH 7.5. Gels (10 cm wide x 8 cm tall x 1.5mm thick) for µ-calpain autolysis and nitrosylation detection were run on SE 260 Hoefer Mighty Small II electrophoresis units (Hoefer Scientific Instruments, Holliston, MA). The running buffer

contained 25 mM Tris, 192 mM glycine, 2 mM EDTA, and 0.1% [wt/vol] SDS. Gels were loaded with 1.5 µg of µ-calpain per lane for autolysis analysis and 0.18 µg of µ-calpain per lane for nitrosylation detection. The gels were run at a constant voltage of 120 V at room temperature.

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After completion of running, gels for µ-calpain autolysis were silver stained

(FASTSilverTM, Bioscience, Bozeman, MT). Protein bands were recorded using a 16-bit

mega-pixel CCD camera FluorChem 8800 (Alpha Innotech Corporation San Leandro, CA)

and FluorChem IS-800 software (Alpha Innotech Corporation San Leandro, CA).

Transfer conditions for nitrosylation detection

Gels for nitrosylation detection of µ-calpain were transferred to polyvinylidene

difluoride (PVDF) membranes (Schleicher and Schuell, Inc., Keene, NH) using a TE22

Mighty Small Transphor electrophoresis unit (Hoefer Scientific Instruments, Grotton, CT) at

a constant voltage of 90 V for 90 min. The transfer buffer consisted of 25 mM Tris, 192 mM

glycine, 2 mM EDTA, and 15% (vol/vol) methanol. The temperature of transfer buffer was

maintained using a refrigerating circulating water bath set at -1.0 °C (Ecoline RE106; Lauda

Brinkmann, Westbury, NY).

Nitrosylation detection

After transfer, membranes were blocked in StartingBlockTM T20 Blocking Buffers (No.

37543, Pierce, Rockford, IL) for 30 min at room temperature. S-Nitrosylation Detection

Reagent I (HPR, No. 10006524, Cayman Chemical Company, Ann Arbor, MI) dissolved in

400 µl of distilled water was used for nitrosylation detection. The concentration of HPR was

1:75 diluted in 80 mM disodium hydrogen orthophosphate, 20 mM sodium dihydrogen orthophosphate, 100 mM sodium chloride and 0.1% polyoxyethylene sorbitan monolaurate

(Tween-20) (PBS-Tween). Blots were placed on a rocker with gentle agitation overnight at 4

°C in cold room. After incubations were completed, membranes were warmed for 30 min at room temperature. Blots were washed three times using PBS-Tween at room temperature and

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each wash time was 10 min. Detection was initiated using premixed reagents (ECL Plus

Western Blotting Detection Reagents, RPN2132, Amersham Bioscience UK Limited, Little

Chalfont Buckinghamshire, England). Chemiluminescence was detected using a 16-bit mega- pixel CCD camera FluorChem 8800 (Alpha Innotech Corporation, San Leandro, CA) and

FluorChem IS-800 software (Alpha Innotech Corp Corporation, San Leandro, CA).

Densitometric measurements were completed using the AlphaEaseFC software (FluorChem

8800, Alpha Innotech Corp Corporation, San Leandro, CA).

Data analysis

All data were analyzed using SAS version 9.1 (NC, US) and significance was reported at the levels of P<0.05 and P<0.01. GLM and analysis of variance (ANOVA) were used to determine significance of the effect of GSNO and calcium on activities of m- and µ-calpain.

Results

Effects of GSNO on activity of m-calpain

At pH 6.5 with MCE in assay buffer, no significant differences of m-calpain activity were found between control and GSNO only group (P>0.05, Table 1). This suggests that

GSNO may not have significant influence on m-calpain activity in the absence of calcium at pH 6.5. The addition of calcium alone for final concentration of 1 mM in control group tended to decrease m-calpain activity (P=0.08). However, m-calpain activity was significantly lowered in groups with combination of GSNO and calcium regardless of the order that GSNO and calcium were added into solutions compared to control groups

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(P<0.05). The m-calpain activity of calcium+GSNO and GSNO+calcium groups was

reduced by 22% and 17% respectively of control group (Table 1).

As expected, without MCE in assay buffer at pH 6.5, m-calpain activities were lower

than corresponding treatments with MCE in assay buffer (Table 1). For instance, m-calpain

activity of control group without MCE in assay buffer was 38 U/mg protein lower than that

of MCE in assay buffer. This difference may be due to that MCE in assay buffer may partly

reverse oxidized m-calpain during standard assay. MCE reduces disulfide bonds of µ-calpain

to thiol groups and thus increases its proteolytic activity compared to treatments without

MCE in assay buffer (Carlin, Huff-Lonergan, Rowe and Lonergan, 2006; Lametsch,

Lonergan and Huff-Lonergan, 2008). Similar to MCE groups, there were no significant

differences among control and GSNO groups (P>0.05). Again, these results indicate that

GSNO may not influence m-calpain activity at pH 6.5 in the absence of calcium. Calcium

only group had tendency to decrease m-calpain activity compared to control groups (P=0.10).

The activity of calcium+GSNO group was 77% of control group and 84% of calcium group

(Table 1). When GSNO was added before calcium into calpain solutions, GSNO showed similar effects on m-calpain activity as calcium was added before GSNO. The activity of

GSNO+calcium group was significantly lower than control and GSNO (P<0.05).

At pH 7.5 with MCE in assay buffer, control and GSNO groups had similar values of m- calpain activity. Addition of calcium with 1 mM concentration in solutions did not significantly influence m-calpain activity at pH 7.5 compared to control group (P=0.12). m-

Calpain activity of calcium+GSNO and GSNO+calcium groups were both significantly lower than control group and GSNO group (P<0.05). It is worthy noting that differences

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between control and calcium+GSNO groups and between control and GSNO+calcium group at pH 7.5 were not as much as shown at pH 6.5. For example, the difference between control and calcium+GSNO groups was 11% at pH 7.5 compared to 22% difference between control and calcium+GSNO groups at pH 6.5. These results suggest that GSNO induced inactivation of m-calpain activity may be pH dependent. Activity of m-calpain tended to be different between calcium group and calcium+GSNO (P=0.08) and between calcium group and

GSNO+calcium groups (P=0.10). Without MCE in assay buffer at pH 7.5, GSNO tended to have lower m-calpain activity in the absence of calcium (P=0.06). Similar with pH 6.5, control group showed higher activity than calcium+GSNO and GSNO+calcium groups

(P<0.05). Both activities of these two groups were significantly lower than GSNO group

(P<0.05). Again differences at pH 7.5 were less than corresponding differences between these groups shown at pH 6.5.

Effects of GSNO on activity of µ-Calpain

At pH 6.5, µ-calpain with MCE in assay buffer had higher activity values than corresponding groups without MCE in assay buffer. With MCE in assay buffer, no significant difference was found between control and GSNO group (P>0.05). Calcium group showed lower µ-calpain activity than both control and GSNO group (P<0.05). However, µ- calpain activity of calcium+GSNO group was lower than control and GSNO group which was not observed at pH 7.5 (P<0.05). There were no significant differences of µ-calpain activity among GSNO+calcium, control and GSNO groups (P>0.05). However, µ-calpain activity in GSNO+calcium group was significantly higher than calcium and calcium+GSNO group as shown at pH 7.5 (P<0.05). Again, these results indicate that reaction between NO

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and µ-calpain before addition of calcium may protect activity loss by calcium induced autolysis. Without MCE in assay buffer at pH 6.5, no significant differences were shown among control, GSNO, calcium and GSNO+calcium groups (P>0.05). The only significant difference at pH 6.5 with MCE in assay buffer was found between calcium+GSNO and control group (P<0.05).

Similar to m-calpain activity, activity of µ-calpain was not influenced by GSNO in the absence of calcium at pH 7.5 (P>0.05, Table 2). With MCE in assay buffer, incubation with

1 mM of calcium lowered µ-calpain activity by 32% compared to control group (P<0.05).

This decrease may be due to rapid autolysis of µ-calpain in the presence of calcium and this autolysis caused loss of some of its proteolytic activity (Dayton, 1982; Suzuki et al., 1981).

However, addition of GSNO in the presence of calcium (calcium+GSNO group) did not influence µ-calpain activity compared with calcium groups (P>0.05). This indicates GSNO could not further influence µ-calpain activity in the presence of calcium as shown for m- calpain. µ-Calpain activity in the GSNO+calcium treatment was significantly higher than calcium only group (P<0.05). There were no significant differences between GSNO+calcium and control group (P=0.16) and GSNO groups (P=0.18).

Without MCE in assay buffer at pH 7.5, calcium group and calcium+GSNO group had lower µ-calpain activity than treatments of control and GSNO (P<0.05). Again, the addition of calcium for final 1 mM concentration significantly decreased µ-calpain activity (P<0.05).

The activity of calcium+GSNO group was not significantly different from calcium group, but it was significantly lower than GSNO and control group (P<0.05). These results suggest that

GSNO when added after calcium did not further decrease µ-calpain activity in the presence

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of calcium as shown for m-calpain at pH 7.5. This may be mainly due to rapid autolysis of µ-

calpain in the presence of 1 mM calcium. GSNO+calcium group had higher activity than

calcium and calcium+GSNO groups as seen with MCE in assay buffer (P<0.05). Again, this

result indicates that GSNO may suppress activity loss of µ-calpain caused by autolysis in

current experimental conditions. These data were consistent with autolysis results which

showed that GSNO tended to slow the autolysis rate of µ-calpain when µ-calpain was

exposed to GSNO at pH 7.5 particular before calcium as discussed in nitrosylation section of

this paper.

Effects of GSNO on autolysis of µ-calpain

The interesting results from activity assay drove us to determine the extent to which

GSNO governs autolysis. It is not surprising to see that 80 kDa subunit of µ-calpain in control and GSNO groups had little autolysis after 5 and 60 min incubation at pH 7.5 (Figure

1). This is due to the fact that calpain autolysis requires the presence of calcium (Cong et al.,

1989; Dayton, 1982). As for other three groups with calcium in solutions at pH 7.5, almost all of 80 kDa subunits of µ-calpain were completely autolyzed to 76 kDa product after 60 minutes incubation in the presence of 0.5 mM calcium. The most interesting results were that calcium+GSNO and GSNO+calcium groups showed slower rate of autolysis after 5 minutes incubation than calcium only group at pH 7.5 (Figure 1). This was evidenced by more 80 kDa and 78 kDa products of µ-calpain in GSNO+calcium group were detected than calcium only group after 5 min incubation. At this time point, a large portion of 80 kDa subunits in calcium only group were autolyzed to 76 kDa products. However, around half of 80 kDa

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subunits remained intact in GSNO+calcium group. The autolysis rate of large subunit of µ- calpain in calcium+GSNO group seemed to be between calcium and GSNO+calcium groups.

Similar to pH 7.5, the autolysis rate of 80 kDa µ-calpain was slowed by GSNO in the presence of calcium at pH 6.5 (Figure 2). After 5 min and 10 min incubation at pH 6.5, a large portion of 80 kDa subunit of µ-calpain was autolyzed quickly to 76 kDa products in calcium only group. However, about half of 80 kDa subunits of µ-calpain in GSNO+calcium group were kept intact and it seemed to be more 78 kDa intermediate products than calcium group after 5 min incubation. The autolysis rate of calcium+GSNO group was intermediate compared with control and GSNO+calcium groups similar with pH 6.5. There was a greater proportion of large subunit present as the intact (unautolyzed) products after 10 min incubation in GSNO+calcium group than calcium group. At this point, lesser 80 kDa µ- calpain in calcium group could be detected while more of 80 kDa subunit of µ-calpain remained in GSNO+calcium group. After 60 min incubation on ice, almost all of the 80 kDa subunit was autolyzed to 76 kDa fragments in all three treatments with calcium at pH 6.5. On the contrast, most 80 kDa subunit of µ-calpain of control group was still kept intact by the end of incubation on ice. These results were in line with another study in intact human neutrophils (Michetti et al., 1995). Autolysis of 80 kDa subunit of calpain was completely inhibited in the presence of 20 and 50 µM calcium when cells were previously exposed to

SNP in the light (Michetti et al., 1995). Along with current study, these results suggest that

NO induced nitrosylation may be able to regulate autolytic process of calpains. However, how µ-calpain nitrosylation induced by GSNO inhibits its autolysis rate in the presence of

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calcium is unknown. Obviously, more studies needs to be conducted to determine the

relationship between the nitrosylation and autolytic procedure of µ-calpain.

Effects of GSNO on nitrosylation of calpain

In order to determine whether µ-calpains were really nitrosylated in this study, we used commercial S-Nitrosylated Protein Detection Assay Kit to identify the extent of µ-calpain nitrosylation in different treatments (Figure 3 and Figure 4).

As Figure 3 showed, we detected nitrosylation of µ-calpain in most of groups at pH 6.5.

Of particular interest, endogenous purified µ-calpains were shown to be nitrosylated in control group. This indicates that some of endogenous µ-calpain may already be nitrosylated.

Compared to control groups, it seemed that more µ-calpain was nitrosylated in the GSNO group as evidenced by the higher intensity of the nitrosylation band at pH 6.5.

Calcium+GSNO and GSNO+calcium showed higher intensity of the nitrosylation than control group and GSNO group. These results suggest that µ-calpain could be further nitrosylated in both the presence and the absence of calcium at pH 6.5. Calcium may improve the extent of µ-calpain nitrosylation and these were consistent with the results of activity assay. However, very little nitrosylation was found in calcium only group compared to other groups. DTT treated group showed less nitrosylation intensity compared with control group indicating that part of nitrosylated µ-calpain could be reversed by DTT during overnight incubation on ice.

At pH 7.5, purified µ-calpain was shown again to be nitrosylated in control group

(Figure 4). DTT incubated µ-calpain showed less nitrosylation intensity compared with control group. Very little µ-calpain nitrosylation was detected in calcium only group which

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was consistent with results at pH 6.5. GSNO further nitrosylated µ-calpain in the absence of calcium evidenced by higher intensity of nitrosylation in GSNO group. Not much nitrosylation of µ-calpain was found in calcium+GSNO group. This may be due to rapid autolysis of µ-calpain at this pH value as shown in other studies (Maddock et al., 2005). In

GSNO+calcium group, the intensity of µ-calpain nitrosylation was 5 times of that calcium only group and two folds of that calcium+GSNO groups. These differences were consistent with the slower rate of µ-calpain autolysis when it was exposed to GSNO before calcium.

Discussion

Effects of nitric oxide on activity of calpains and meat quality

The current study shows that GSNO could not significantly influence the activity of m- and µ-calpain in the absence of calcium at both pH 6.5 and 7.5. GSNO could differently affect the activity of m- and µ-calpain. For m-calpain, lower proteolytic activity was detected after incubation with GSNO and calcium compared to control groups regardless of whether calcium or GSNO were added into the solutions first. There are two possible explanations. It is possible that calcium can cause conformational changes and this can cause the nitrosylation of the cysteine residue in active site of m-calpain resulting in inactivation of m- calpain. From another aspect, GSNO can result in calpain more active in the presence of calcium during incubation prior to activity assay. This greater activation in calcium+GSNO and GSNO+calcium groups caused calpain to lose more of it proteolytic activity during incubation compared to calcium only groups. Influence of GSNO on µ-calpain activity seemed to be more complicated. One mM calcium could significantly lower µ-calpain

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activity compared with control group. This activity decrease may be due to calcium-induced

autolysis (Nagainis et al., 1983). In most conditions, GSNO+calcium groups showed higher

µ-calpain activity than calcium and calcium+GSNO groups (P<0.05). These interesting results suggest that nitrosylation may slow activity loss of µ-calpain resulting from calcium induced autolysis. Consistent with this study, Michetti et al. (1995) reported nitric oxide generating agent SNP could inactivate both calpains. m-Calpain activity was largely inhibited by SNP when pH was near neutral range. These results were in agreement with current study which shows that GSNO could have significant inhibitory effects on m-calpain activity at pH

6.5 and 7.5. The activity of µ-calpain was shown to be decreased by SNP when pH was shifted in acidic range (Michetti et al., 1995). Several studies have shown that NO induced nitrosylation could influence meat quality through manipulating the activity of NOS and the levels of nitric oxide during pre- or post-slaughter periods (Cottrell et al., 2008; Warner et al.,

2005). Through incubating meat with NOS inhibitors or NO enhancers, Cook et al. (1998) reported that NO could change the rate of tenderization of bovine longissimus lumborum.

NO enhancer injection could improve meat tenderness during 2-6 aging days, while NO inhibitors caused tougher meat at same postmortem storage period. In another study, infusing of a NOS inhibitor L-NAME at 3 h preslaughter improved the tenderness of longissimus thoracis et lumborum (LTL) and resulted in tougher meat in semimembranosus (SM) at 3 day postmortem (Cottrell et al., 2002). The recent published study showed that exercise could lead to tougher meat in LTL and SM at day 3 of postmortem storage through increasing muscle NOS activity (Cottrell et al., 2008). These limited studies have shown controversial conclusions about the function of NO on meat tenderness. Possible explanations may be due

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to different agents for NO donor, NOS inhibitor and NO enhancer in different studies. It is

also noteworthy that the production of nitric oxide during postmortem needs the presence of

enzyme NOS and many cofactors, such as NADPH, FMN, FAD, and oxygen (Brannan and

Decker, 2002; Grozdanovic, 2001). Different levels of co-factors may cause the variation of

different studies. The current results may partly explain inconsistent effects of nitric oxide on

meat tenderness. µ-Calpain is generally believed to be the enzyme for postmortem protein

degradation and meat tenderness (Huff-Lonergan et al., 1996; Koohmaraie et al., 1987;

Geesink et al., 2006). After animals are slaughtered, the concentration of cytoplasm calcium

could increase rapidly due to the loss of regulatory ability of sarcoplasmic reticulum. Parrish

et al. (1981) reported that the ultimate concentration of free calcium in the sarcoplasm could

increase to be 970 µM. At the same time, reactive oxygen species and reactive nitrogen

species are accumulated during postmortem storage because of the decreased ability of

antioxidant defense system (Renerre et al., 1996). NOS activity was detected in postmortem

muscle in several species by Brannan and Decker (2002) indicating the possibility of NO

production in aging muscle foods. The order of exposure to NO and calcium may have

different effects on µ-calpain activity. These studies support that earlier nitrosylation of µ-

calpain may contribute to variation of fresh meat quality. In addition to influence calpain

system, NO has been proved to influence protein properties of skeletal muscles. Andrade et

al. (1998) reported that NO induced nitrosylation could decrease myofibrillar Ca2+ sensitivity and thus damage Ca2+ activation of the actin filaments in mouse when using S-nitroso-N- acetylcysteine and SNP as nitric oxide donors.

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Effects of calcium on GSNO induced nitrosylation of calpains

It is very interesting to find that GSNO only shows significant effects on activity of m-

and µ-calpain with the combination of calcium in our current study. From crystal structure of

m-calpain in the absence of calcium, it is known that the active residue Cys105 locates in

domain IIa (Strobl et al., 2000). The m-calpain activation is believed to require the formation

of catalytic triad among Cys105, His262 and Asn286 (Goll et al., 2003). Calcium binding to

the negatively charged loop of domain III can induce conformational change to reduce the

distance of three residues and then activate m-calpain (Goll et al., 2003). This mechanism

may explain the results of current study that GSNO only show significant effects on calpain

activity in the presence of calcium. For m-calpain, we postulate that conformational changes

caused by calcium binding may expose thiol groups in active sites of domain II. This

exposure may increase the reactions between nitric oxide and thiol groups in the presence of

calcium. The nitrosylation of cysteines in active sites may inactivate m-calpain through the

formation of S-nitrosothios as other papers showed (Ascenzi et al., 2001; Michetti et al.,

1995). Otherwise, GSNO could not play significant effects on the activity of m-calpain in the absence of calcium. This is also true for µ-calpain as evidenced by no significant effects of

GSNO on µ-calpain activity without the presence of calcium. In the presence of calcium,

GSNO induced nitrosylation could decrease activity losses of µ-calpain resulting from calcium induced autolysis. The hypothesis for this result could be that nitrosylation of some thiol groups other than active sites of µ-calpain occurs in the absence of calcium. These nitrosylation events could have protective roles on the autolysis rate when µ-calpain is further exposed to calcium. To our knowledge, this is the first paper to show the key role of

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calcium on nitric oxide induced nitrosylation of calpains. The nitrosylation detection results support the hypothesis since more µ-calpain seemed to be further nitrosylated in the presence of calcium compared with control group. The possibility for these results may be that calcium binding induces conformational changes of calpain structure (Suzuki and Sorimachi,

1998) and this conformation change exposes more free thiols to react with nitric oxide. In another study, Lai et al. (2001) reported similar effects of calcium on the regulation of nitrosylation and activity of transglutaminase. Eight cysteines out of eighteen free cysteines of this enzyme were nitrosylated in the absence of calcium using S-nitrosocysteine as nitric oxide carrier. However, this nitrosylation of free cysteines could not influence enzyme activity in the absence of calcium. In the presence of calcium, as many as fifteen free cysteines of transglutaminase were nitrosylated and the activity of transglutaminase was inhibited with a calcium concentration dependent tendency.

Effects of pH on extent of nitrosylation of calpains

Maddock et al. (2005) found that pH had significant effects on activity of µ-calpain and m-calpain. They reported that µ-calpain had the greatest activity at pH 6.5 due to the slow rate of autolysis compared with pH 7.5 when incubated with calcium. On the other hand, m- calpain showed higher activity at pH 7.5 than pH 6.0 and pH 7.5 (Maddock et al., 2005). The pH could play a regulatory role in the extent of inhibitory effects of µ- and m-calpain activity caused by NO induced nitrosylation. Michetti et al. (1995) described that the highest inhibition of m-calpain activity by nitric oxide at pH 7.5. At this pH, it also showed its maximal catalytic activity. At pH 7.5, m-calpain lost 90% of its activity when it was incubated with 4 mM SNP. The extent of inactivation of m-calpain was decreased when the

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pH was shifted to acidic or basic values. In current study, GSNO tends to decrease m-calpain activity more at pH 6.5 than at pH 7.5 regardless of the presence of MCE in assay buffer. For example, m-calpain activity in calcium+GSNO group was decreased by 39% compared to control group at pH 6.5 with MCE in assay buffer. However, the activity of calcium+GSNO group is 89% of control group and is decreased by 11% at pH 7.5 compared with control group when MCE was added in assay buffer. In the study of Michetti et al. (1995), pH showed different tendency on the inactivation of µ-calpain caused by SNP and inactivation was improved when pH of the incubation solutions was progressively shifted to acidic values. The most inhibition was observed at pH 5.5 at which 80% of its proteolytic activity was inactivated (Michetti et al., 1995). In current study, pH did not show big effects on µ- calpain activity as was shown for m-calpain activity. For example, with the presence of MCE in assay buffer, calcium+GSNO could decrease the catalytic activity by 20% at both pH 7.5 and pH 6.5. If µ-calpain activity could be regulated at pH 7.5, 6.5 and more acidic conditions, this is significant because muscle pH decreases from 7.3 in living muscles to pH

5.7 within 6 to 8 h postmortem (Aberle et al., 2001). Hence, NO induced nitrosylation may play roles in early postmortem metabolism through regulating calpains. In current study, we do not know whether NO donors have different abilities to release NO under different pH conditions or if pH itself could influence the extent of nitrosylation on calpain activity instead of the amount of NO release from nitric oxide donors. Actually, one study described that the pH really could influence the extent of NO release from GSNO (Noble et al., 1999).

If this conclusion is true, we need further to determine whether it occurs in our experimental

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conditions. Before that, it is difficult to simply provide the conclusion that pH influence nitric

oxide induced inactivation of calpains through regulating the extent of nitrosylation.

Effects of nitrosylation on autolysis of µ-calpains

Many factors have been reported to have regulatory effects on µ-calpain autolysis.

Koohmaraie (1992b) reported that rate of autolysis of 80 kDa subunit of µ-calpain was decreased with temperature changing from 25 to 5 oC. After incubation at 5 oC for 120 min at pH 7.0 with the addition of 3.8 mM CaCl2, µ-calpain retained 95.1% of its original activity

(Koohmaraie 1992b). This remaining activity may be due to autolytic products of µ-calpain.

In another study, the 56 kDa polypeptide was found to retain the activity of µ-calpain in the absence of 80 kDa subunit (Nagainis et al., 1988). These results above could partly explain why µ-calpain still retains some of its proteolytic activity after 60 min incubation with 1 mM calcium on ice with its 80 kDa subunit being autolyzed to 76 kDa products. Autolysis of calpain is a natural procedure in the presence of calcium. Extensive autolysis destroys the enzyme structure and causes complete losses of its proteolytic activity (Edmunds et al., 1991;

Goll et al., 2003). Autolysis results in this study showed that the 80 kDa subunit of µ-calpain was detected to be autolyzed to 76 kDa products after 60 min incubation on ice for all treatments in the presence of 0.5 mM calcium.

In this study, we found that the rate of µ-calpain autolysis was slowed when it was first exposed to GSNO before calcium. This was evidenced by more 80 and 78 kDa products and less 76 products were left after 5 minutes incubation on ice at pH 7.5 compared to calcium only group. At pH 6.5, more intact 80 kDa and 78 products could be detected after 5 and 10 minutes incubation in GSNO+calcium group than calcium only group. These results were in

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agreement with the report by Michetti et al. (1995). In that study, they treated intact

neutrophils with SNP in the presence of calcium ionophore and different calcium

concentrations ranged from 0 to 50 µM. In neutrophil cells exposed with SNP previously in

the light, the autolysis of calpain catalytic subunit was completely inhibited at 20 and 50µM

calcium. However, calpain was completely autolyzed in the group without being treated with

SNP in advance at same calcium concentration. Both these studies support the hypothesis that NO may be considered as a regulator for autolytic procedure of calpains. When µ- calpain is first exposed to NO agent, NO induced nitrosylation may arrest the rate of µ- calpain autolysis when it is further exposed to calcium. To the best of our knowledge, how

NO induced nitrosylation influences the rate of µ-calpain autolysis is not clear. We also do not know whether nitrosylation occurs in different thiol groups of calpains in the presence and absence of calcium. Further experiments need to be conducted to explore the behind mechanism about the effect of NO on autolytic procedure of µ-calpain.

Conclusions

GSNO differently influences properties of µ- and m-calpain with the combination of calcium. For m-calpain, groups with combination of GSNO and calcium show lower proteolytic activity at both pH 6.5 and 7.5. The activity differences at pH 6.5 tend to be more significant than at pH 7.5. GSNO may play protective roles on the activity loss of µ-calpain caused by calcium induced autolysis at both pH 7.5 and pH 6.5. GSNO could not further decrease µ-calpain activity after being exposed to 1 mM calcium. Neither activity of µ- nor m-calpain could be influenced by GSNO in the absence of calcium. MCE could not show

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reversible ability on calpain nitrosylation in this study. GSNO could affect the rate of µ-

calpain autolysis when it is exposed to GSNO before calcium at both pH 6.5 and 7.5.

Nitrosylation of calpain proteinases may be an important regulator of calpain mediated

proteolysis. Based on the results of this study, the effects of nitrosylation on the meat quality

deserve our attention, especially on the status of calpains during early postmortem storage.

More experiments need to be conducted to figure out how nitrosylation influences the rate of autolytic procedure of µ-calpain. The basic mechanism behind regulatory roles of calcium

and pH in the extent of calpain nitrosylation also needs to be further studied.

186

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Table 1 Effects of GSNO on the activity of m-calpain at pH 6.5 and 7.5

Control* GSNO** Calcium*** Calcium GSNO +GSNO +Calcium

pH=6.5 with 89.75±3.19a 85.00±4.02a 78.50±4.75ab 69.75±4.40b 74.13±4.47b MCE****

pH=6.5 51.50±2.91a 51.13±2.78a 45.88±2.45ab 39.50±1.81b 43.38±1.70b without MCE

pH=7.5 with 134.80±4.55a 135.10±4.93a 126.70±2.96ab 120.10±2.41b 122.80±1.72b MCE

pH=7.5 98.40±3.49a 90.60±3.34ab 93.60±1.44a 85.30±2.11b 86.00±2.08b without MCE a–b Means in the same row without a common superscript letter differ significantly (P < 0.05).

*Treatment (in order of addition): 1): control: m-calpain; 2): GSNO: m-calpain+GSNO; 3): CaCl2: m- calpain +CaCl2; 4): CaCl2+GSNO: m-calpain+CaCl2+GSNO; 5): GSNO+CaCl2: m-calpain+GSNO

+CaCl2.

** GSNO concentrations:: 1.05 mM.

*** CaCl2 concentration: 1 mM.

****Assay buffer for calpain activity was made with MCE or without MCE to determine whether nitrosylation could be reversed by reducing conditions.

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Table 2 Effects of GSNO on the activity of µ-calpain at pH 7.5 and 6.5

Control* GSNO** Calcium*** Calcium GSNO +GSNO +Calcium pH=6.5 with 75.33±2.18a 73.67±1.21a 58.50±2.63b 60.00±1.80b 68.58±3.54a MCE**** pH=6.5 49.33±1.17a 47.42±1.11ab 47.42±1.98ab 43.08±2.31b 47.42±2.02ab without MCE pH=7.5 32.84±4.66a 32.50±2.88a 22.33±1.40b 26.57±2.92ab 30.23±2.52ab with MCE pH=7.5 22.67±1.15a 23.78±0.22a 18.22±1.56b 16.59±1.93b 22.67±0.78a without MCE a–b Means in the same row without a common superscript letter differ significantly (P < 0.05).

*Treatment (in order of addition): 1): control: m-calpain; 2): GSNO: m-calpain+GSNO; 3): CaCl2: m- calpain +CaCl2; 4): CaCl2+GSNO: m-calpain+CaCl2+GSNO; 5): GSNO+CaCl2: m-calpain+GSNO

+CaCl2.

** GSNO concentrations:: 1.05 mM.

*** CaCl2 concentration: 1 mM.

****Assay buffer for calpain activity was made with MCE or without MCE to determine whether nitrosylation could be reversed by reducing conditions.

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Calpain Ca Ca+GSNO GSNO+Ca 80 kDa 60 5 60 5 60 5 60 5 78 kDa 76kDa 1 2 3 4 5 6 7 8

Figure 1: Autolysis of 80 kDa subunit of µ-calpain after 60 and 5 minutes incubation on ice in the presence of 0.5 mM CaCl2 at pH 7.5.

Treatment (in order of addition): Lane 1 and 2: control after 60 and 5 minutes incubation: µ-calpain; Lane

3 and 4: calcium after 60 and 5 minutes: µ-calpain+calcium; Lane 5 and 6: calcium+GSNO** after 60 and

5 minutes: µ-calpain+calcium+GSNO; Lane 7 and 8: GSNO+calcium after 60 and 5 minutes: µ- calpain+GSNO+calcium.

** GSNO concentrations: 1.15 mM.

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Calpain Ca Ca+GSNO GSNO+Ca

60 5 10 60 5 10 60 5 10 60 80 kDa 78 kDa

76 kDa 1 2 3 4 5 6 7 8 9 10

Figure 2: Autolysis of 80 kDa subunit of µ-calpain after 5, 10 and 60 minutes incubation on ice in the presence of 0.5 mM CaCl2 at pH 6.5.

Treatment (in order of addition): Lane 1: control 60 min: µ-calpain; Lane 2, 3 and 4: calcium after 5, 10 and 60 minutes: µ-calpain+calcium; Lane 5, 6 and 7: calcium+GSNO after 5, 10 and 60 minutes: µ- calpain+calcium+GSNO; Lane 8, 9 and 10: GSNO+calcium after 5, 10 and 60 minutes: µ-calpain+

GSNO+calcium.

** GSNO concentrations: 1.15 mM.

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DTT Control GSNO Calcium Calcium GSNO +GSNO +calcium

1.00 1.58 1.94 0.29 2.51 2.72 Ratios of nitrosylation intensity

Figure 3: Nitrosylation of 80 kDa µ-calpain subunit at pH 6.5 after 60 min incubation

Lane1: µ-calpain incubated with DTT incubation for 12 h; Lane2: control: µ-calpain without DTT treatment; Lane3: GSNO: µ-calpain+GSNO; Lane4: calcium: µ-calpain+calcium; Lane5: calcium+GSNO:

µ-calpain+calcium+GSNO; Lane6: GSNO+calcium: µ-calpain+GSNO+calcium

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DTT Calpain GSNO Calcium Calcium GSNO +GSNO +calcium

1.00 1.18 1.54 0.27 0.62 1.29 Ratios of nitrosylation intensity

Figure 4: Nitrosylation of 80 kDa µ-calpain subunit at pH 7.5 after 2 min incubation

Lane1: DTT: µ-calpain incubated with DTT for 12 h; Lane2: control: µ-calpain without DTT treatment;

Lane3: GSNO: µ-calpain+GSNO; Lane4: calcium: µ-calpain+calcium; Lane5: calcium+GSNO: µ- calpain+calcium+GSNO; Lane6: GSNO+calcium: µ-calpain+GSNO+calcium

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Comparison of protein nitrosylation between postmortem beef longissimus dorsi and

psoas major

A paper to be submitted to Meat Science

Wan-Gang Zhang, Steven M. Lonergan and Elisabeth Huff-Lonergan

Abstract

This study was designed to determine protein nitrosylation in postmortem beef longissimus dorsi (LD) and psoas major (PM) muscles. LD had 73% of type IIA/X and 27% of type I myosin heavy chain isoforms, while PM had 54.85% type IIA/X and 45.15% type I myosin heavy chain isoforms. LD showed higher percentage of type IIA/X and lower percentage of type I isoforms than PM (P<0.01). Several protein bands were detected as being nitrosylated. At day 1 of postmortem storage, LD showed higher intensity of protein nitrosylation than PM (P<0.05). No significant differences were found about protein nitrosylation across aging days (P>0.05). In conclusion, protein nitrosylation can be detected in postmortem beef muscles. Muscle fiber composition may be related to protein nitrosylation between muscles.

Introduction

In recent years, nitric oxide (NO) has been recognized as a critical molecule for many biological processes in skeletal muscle system (Jaffrey, Erdjument-Bromage, Ferris, Tempst,

197

& Snyder, 2001; Stamler, Sun & Hess, 2008). The function of sulfhydryl-containing proteins

can be regulated by NO induced S-nitrosylation. For some cysteine proteases, NO can inhibit

their proteolytic activity by oxidizing thiol groups to S-nitrosothiol and mixed disulfide

(Ascenzi, Salvati, Bolognesi, Colasanti, Polticelli, & Venturini, 2001; Xian, Chen, Liu, Wang,

& Wang, 2000). The proteolytic activity of both m- and µ-calpain has been shown to be

arrested by NO donors at different pHs (Koh & Tidball, 2000; Michetti, Salamino, Melloni,

& Pontremoli, 1995). NO may be involved in regulating muscle glycogen metabolism and

calcium flux between sarcoplasm and sarcoplasmic reticulum of muscle cells (Bellinger et al.,

2008; McConell & Kingwell, 2006; Stamler, Lamas, & Fang, 2001; Young, Radda, &

Leighton, 1997). In addition, Ca2+ induced actin filaments activation was impaired by NO and NO decreased myofibrillar Ca2+ sensitivity (Andrade, Reid, Allen, & Westerblad, 1998).

With these functions, NO may have regulatory roles in the conversion of muscle to meat and thus influence fresh meat quality (Cook, Scott, & Devine, 1998; Cottrell, McDonagh,

Dunshea, & Warner, 2008).

NO is synthesized during the conversion of L-arginine to L-citrulline which is catalyzed by a family of enzymes called nitric oxide synthase (NOS). Three isoforms of NOS have been identified including neuronal NOS (NOS1 or nNOS), macrophage (immune)/calcium calmodulin-independent or inducible NOS (NOS2 or iNOS) and endothelial NOS (eNOS or

NOS) (Stamler & Meissner, 2001). All three isoforms of NOSs are identified in skeletal muscles of mammals, but nNOS is the most predominant expressed isoform (Stamler &

Meissner, 2001). The expression and localization of nNOS in skeletal muscle are dependent on age, innervation, hormone exposure, species and muscle fiber types. For example,

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immunolocalization showed that nNOS was mainly localized with type II fiber cell

membranes in rat skeletal muscles (Kobzik, Reid, Bredt, & Stamler, 1994). The activity of

nNOS was positively correlated with the percentage of type II fibers in different rat skeletal

muscles. NOS activity has been reported in several muscle foods within 8 hours postmortem

including chicken, pork, trout and turkey (Brannan & Decker, 2002). However, no studies

have been published about protein nitrosylation in meat science area to the best of our

knowledge. Therefore, we have two-fold objectives in current study: 1) to determine protein

nitrosylation in postmortem beef; 2) to compare the extent of protein nitrosylation between longissimus dorsi and psoas major of postmortem beef.

Materials and methods

Animals and sample collections

Four market weight beef cattle were used in this study. They were slaughtered using human methods at the Iowa State University Meat Laboratory. Two muscles (LD and PM) were removed from both sides of the carcasses at 24 h after slaughter. Muscles were cut into

2.54-cm thick steaks and steaks were separately vacuum packaged. Steaks were aged at 4 °C for 1 (frozen immediately after packaging), 3 (aging for 2 more days after cut) and 7 days

(aging for 6 more days after cut) postmortem. After aging, steak samples were frozen stored until for nitrosylation analysis.

Whole muscle Sample Preparation

Whole muscle extracts were prepared as described by Lonergan, Huff-Lonergan, Rowe,

Kuhlers and Jungst (2001). The protein concentration in the extracts was determined using

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methods described by Lowry, Rosebrough, Farr and Randall (1951) for each sample using

prepared reagents (DC Protein Assay, Bio-Rad, Hercules, CA). Protein concentration was

then adjusted to a concentration of 2 mg/mL by diluting with deionized water.

Muscle fiber type analysis

Whole muscle samples were diluted with distilled deionized water to the final concentration of 0.256 mg/mL. One volume of this diluted samples were combined with one

volume of tracking dye solution (50% [vol/vol] glycerol, 2% [wt/vol] SDS, 0.1% [wt/vol]

bromophenol blue, and 60 mM Tris•HCl, pH 6.8) and 0.05 vol of MCE. The final protein

concentration of these samples was 0.125 mg/mL and they are stored at -80°C until for

myosin heavy chain analysis.

Myosin heavy chain (MHC) isoforms were determined by a 6% acrylamide separating gel (acrylamide:N,N'-bis-methylene acrylamide=50:1 [wt/wt], 0.4% [wt/vol] SDS, 0.05%

[vol/vol] TEMED, 0.1% [wt/vol] APS, 30% [vol/vol] glycerol, 100 mM glycine, and 200 mM

Tris, pH 8.8) and a 4% acrylamide stacking gel (acrylamide:N,N'-bis-methylene

acrylamide=50:1 [wt/wt], 0.4% [wt/vol] SDS, 0.05% [vol/vol] TEMED, 0.1% [wt/vol] APS,

30% [vol/vol] glycerol, 4 mM EDTA, and 200 mM Tris•HCl, pH 6.7). The running buffer in

the upper chamber contained 200 mM Tris, 300 mM glycine, 0.2% [wt/vol] SDS, and 0.1%

[vol/vol] MCE and in the lower chamber contained 100 mM Tris, 150 mM glycine, 0.1%

[wt/vol] SDS, and 0.1% [vol/vol] MCE (Melody et al., 2004). Each lane was loaded with 2

µg of protein. Gel (18 cm wide x 16 cm tall) for MHC was run on SE 400 Hoefer units

(Hoefer Scientific Instruments) at a constant voltage of 100 V for approximately 66 hours.

After the completion of running, the gel was stained using Silver Stain Plus kit (Catalog No.

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161-0449; Bio-Rad, Hercules, CA). The density of type I and type IIa/IIx MHC bands were quantified by using ChemiImager 5500 (Alpha Innotech, San Leandro, CA) and

AlphaEaseFC (version 2.03, Alpha Innotech, San Leandro, CA). The percentage for each

MHC isoform (type I or type IIa/IIx) was calculated as a percentage of the total MHC isoforms in each lane.

Nitrosylation detection

To detect protein nitrosylation, 20 µg of whole muscle samples were treated with S-

Nitrosylated Protein Detection Assay Kit (No. 10006518, Cayman Chemical Company, Ann

Arbor, MI, USA). This detection assay was a modification of the “Biotin Switch” method from Jaffrey et al. (2001) which can directly identify S-nitrosylated proteins. The assay contained three steps to convert nitrosylated cysteines into biotimylated cysteines. First, ten

µL of whole muscle extracts were incubated with four volumes of ice-cold acetone at -20 oC

for 60 min. After incubation, samples were precipitated by centrifugation with mini

centrifuge (Fisher Scientific, USA) for 15 min at 4 oC. Acetone-containing supernatants were decanted by pipetting. Five volumes of Buffer A containing S-Nitrosylation Blocking

Reagent (No. 10006518, Cayman Chemical Company, Ann Arbor, MI, USA) was added into microcentrifuge tube to block free thiols of precipitated proteins. Samples were blocked on a rocker with gentle agitation for 45 min at 4 oC. After completion of block, four volume of

ice-cold acetone was used to precipitate proteins again with centrifuge. After proteins were

precipitated and supernatant were decanted, 250 µL of Buffer B containing Reducing and

Labeling Reagents (No. 10006518, Cayman Chemical Company, Ann Arbor, MI, USA) were

used to resuspend protein pellets for 1 h at room temperature. During this step, S-

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nitrosothiols of proteins were reduced to free thiols and these free thiols were labeled with

maleimide-biotin. One mL of ice-cold acetone was added and microcentrifuge tubes were

vortexed before they were incubated at -20 oC for 60 min followed by centrifugation. After

incubation and precipitation, acetone-containing supernatants were removed using pipette

tips. Finally, protein samples were dissolved in 120 µL cold washing buffer (No. 10006518,

Cayman Chemical Company, Ann Arbor, MI, USA) and these samples were kept in frozen

storage for subsequent nitrosylation analysis. Before samples treated with commercial kit

were run on SDS-PAGE, one volume of each sample was combined with 0.5 volumes of

tracking dye solution (3 mM EDTA, 3%[wt/vol] SDS, 30%[vol/vol] glycerol, 0.001%

[wt/vol] pyronin Y, and 30 mM Tris-HCl, pH 8.0) for a final protein concentration of 0.11 mg/mL. The gel samples were heated for 5 min with boiling water and kept on ice for another 5 min before being loaded into gels. The biotin label was covalently bound to

proteins during the heating process. Biotinylated proteins were further detected by S-

Nitrosylation Detection Reagent I (HPR, No. 10006524, Cayman Chemical Company, Ann

Arbor, MI, USA).

SDS-PAGE and running conditions

An 8% polyacrylamide separating gel (acrylamide:N,N'-bis-methylene acryla-

mide=100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.05% [vol/vol] TEMED, 0.05% [wt/vol] APS, and

0.5 M Tris•HCl, pH 8.8) with a 5% polyacrylamide stacking gel (acrylamide:N,N'- bis-

methelyene acrylamide = 100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.125% [vol/vol] TEMED,

0.075% [wt/vol] APS, and 0.125 M Tris•HCl, pH 6.8) was used for the samples.

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Gels (10 cm wide x 8 cm tall x 1.5mm thick) for nitrosylation detection were run on SE

260 Hoefer Mighty Small II electrophoresis units (Hoefer Scientific Instruments). The

running buffer contained 25 mM Tris, 192 mM glycine, 2 mM EDTA, and 0.1% [wt/vol]

SDS. Gels were loaded with 2.2 µg of proteins per lane for nitrosylation detection. The gels

were run at a constant voltage of 120 V.

Transfer conditions

Gels for nitrosylation detection were transferred to polyvinylidene difluoride (PVDF) membranes (Schleicher and Schuell, Inc., Keene, NH) using a TE22 Mighty Small Transphor electrophoresis unit (Hoefer Scientific Instruments) at a constant voltage of 90 V for 1.5 h.

The transfer buffer consisted of 25mM Tris, 192mM glycine, 2mM EDTA, and 15%

(vol/vol) methanol. The transfer buffer was maintained at low temperature using a refrigerating circulating water bath set at -1.0 °C (Ecoline RE106; Lauda Brinkmann,

Westbury, NY).

Protein nitrosylation detection

Membranes were blocked in StartingBlockTM T20 Blocking Buffers (No. 37543, Pierce,

Rockford, IL, USA) for 30 min at room temperature after transfer. S-Nitrosylation Detection

Reagent I (HPR, No. 10006524, Cayman Chemical Company, Ann Arbor, MI, USA) was

dissolved in 400 µl of distilled water to incubate membranes. The concentration of HPR was

further diluted with volume ratio 1:75 in 80 mM disodium hydrogen orthophosphate, 20 mM

sodium dihydrogen orthophosphate, 100 mM sodium chloride and 0.1% polyoxyethylene

sorbitan monolaurate (Tween-20) (PBS-Tween). Blots were placed on a rocker at 4 °C with

gentle agitation in cold room overnight. After incubation, membranes were warmed for 30

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min at room temperature before they were washed with PBS-Tween. Blots were washed

three times at room temperature and each wash time was 10 min. Detection was initiated

using premixed reagents (ECL Plus Western Blotting Detection Reagents, RPN2132,

Amersham Bioscience UK Limited, Little Chalfont Buckinghamshire, England).

Chemiluminescence was detected using a 16-bit mega-pixel CCD camera FluorChem 8800

(Alpha Innotech Corp., San Leandro, CA) and FluorChem IS-800 software (Alpha Innotech

Corp.). Densitometric measurements were completed using the AlphaEaseFC software

(FluorChem 8800, Alpha Innotech Corp.).

Data analysis

All data were analyzed using SAS version 9.1 (NC, US) and significance was reported

at the levels of P<0.05 and P<0.01. GLM and analysis of variance (ANOVA) were used to determine nitrosylation difference between LD and PM and among different aging days.

Results and discussions

Myosin heavy chain analysis

The composition of muscle fiber types in bovine muscles is highly variable (Kirchofer,

Calkins, & Gwartney, 2002). Hunt and Hedrick (1977) studied profile of fiber types of five bovine muscles. The percentages of β-red type in LD and PM were 29.3% and 52.4% respectively, while the percentage of α-white type in LD was 41% higher than that in PM

(Hunt & Hedrick, 1977; Kim, Kim, Lee, & Baik, 2000). The different composition of muscle fiber types could contribute to the variation of fresh meat quality. Compared with LD muscles, PM depleted ATP faster and showed a faster rate of pH decline in early postmortem

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storage in both porcine and bovine muscles (Kim et al., 2000; Melody, Lonergan, Rowe,

Huiatt, Mayes, & Huff-Lonergan, 2004). α-White fiber type was shown to be associated with less favorable meat traits including marbling, tenderness and ultimate pH indicating that muscle fiber type may be related to fresh meat quality (Calkins, Dutson, Smith, Carpenter, &

Davis, 1981; Ozawa et al., 2000).

In current study, significant difference was found about myosin heavy chain composition between LD and PM muscles. In LD, 73.00% of MHC isoform was type IIA/X and 27.00% of MHC isoform was type I. Type II A/X and type I isoforms accounted for

54.85% and 45.15% separately of total MHC isoforms in PM muscle (Figure 1). The percentage of MHC type IIA/X in LD muscle was significantly higher than that in PM muscle (P<0.01). In contrast, LD muscle showed lower percentage of MHC type I fibers than

PM muscles (P<0.01). Within muscles, the percentage of type I fibers was significantly higher in PM and lower in LD than type IIA/X fibers. Consistent with this, Anderson,

Steadham, Fedler, Prusa, Lonergan and Huff-Lonergan (2008) reported that 70% of MHC was type IIA/X in beef LD muscles. The percentage of type IIA/X and type I fiber types were significantly correlated with some beef parameters including juiciness and the levels of troponin-T degradation (Anderson et al., 2008). Muscle fiber type can be associated with the rate of postmortem tenderization due to the different amounts of proteolytic enzymes and proteinases inhibitors (Dransfield, Jones, & MacFie, 1981; Koohmaraie, 1996).

Protein nitrosylation across postmortem aging days

Beef LD and PM were both aged for 1, 3 and 7 days at 4 °C before nitrosylation

analysis. Figure 2 was one of the blots to determine protein nitrosylation for LD and PM at

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different aging days. Several protein bands in each lane were detected by commercial Protein

Nitrosylation Detection Assay Kit. Based on the consistency of the band intensity, we

selected four bands for statistical analysis in this study to compare protein nitrosylation

across aging days.

The nitrosylation intensity across postmortem aging days was shown in Figure 3. No significant differences were found across three aging days for all four selected bands

(P>0.05). Brannan and Decker (2002) reported that fresh muscles less than 8 h postmortem

of several species exhibited NOS activity including chicken, pork, trout and turkey. Pork

muscles retained most of their NOS activity after 24 h refrigerated storage and no NOS

activity was detected after 48 h of postmortem storage. In contrast, chicken and trout lost

their NOS activity measured at 24 h postmortem. In Brannan and Decker’s study, sufficient

levels of cofactors and substrates were utilized for NOS activity analysis in vitro. Thus, NOS

activities were supposed to represent the maximum levels in vivo. Considering the decrease

of cofactors including nicotinamide adenine dinucleotide phosphate (NADPH), flavin

adenine dinucleotides (FAD) and flavin mononucleotides (FMN) for NO production in

postmortem muscle, the authors concluded that NOS catalyzed the formation of NO was

expected to have a short duration for the first 24 hours postmortem. Goreen, Schrammel,

Schmidt and Mayer (1998) found that nNOS structure and function were affected by

different pH conditions. The optimal nNOS activity was between pH 7.0 and 7.5 based on the

formation of L-citrulline. NOS showed decreased activity when pH was shifted to acidic

conditions. No significant differences across aging days in current study support the

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conclusion that NO mediated protein nitrosylation may be a short period event in postmortem

muscles during refrigerated storage (Brannan & Decker, 2002).

NOS activity can be influenced by many preslaughter factors including animal age,

electrical stimulation, exercise and mechanical activity (Balon & Nadler, 1994; Capanni et al.,

1998; Robert, Barnard, Jasman, & Balon, 1999). NO has been shown to be produced with the

rate of 3-5 pM/min/mg muscle in resting rat diaphragm (Reid, Kobzik, Bredt, & Stamler,

1998). In the same study, NO production was increased by almost 6 times in actively

contracting diaphragm under the stimulation of 25 Hz for 1 h. In the study of Cottrell et al.

(2008), pre-slaughter exercise showed negative effects on meat tenderness of ovine SM and

LTL muscles. The author assumed that pre-slaughter exercise may increase NOS activity and

NO production to decrease calpain induced proteolysis. In addition, animals may have large

variation in neurological susceptibility to pre-slaughter stimuli which could be related to the

stress response and immune capacity (Rosenkranz et al., 2003). King et al. (2006) reported

that excitable steers produced carcasses with lower 30 min pH and higher values of WBS

than carcasses harvested from animals with calm temperament. With all of these

considerations, animals may have different levels of NOS activity and protein nitrosylation

under the variation of pre-slaughter treatments and the responsiveness to environmental stress. These variations may cause the inconsistency of fresh meat quality among animals.

Comparison of protein nitrosylation between LD and PM

The distribution and activity of NOS are known to be species and muscle fiber dependent. nNOS was greater expressed in the cytoplasm and at the sarcoplasm of type I fibers than type II fibers inn vastus lateralis muscle of humans (Frandsen, Lopez-Figueroa, &

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Hellsten, 1996). However, type II muscle fibers were shown to be associated with much higher amount of nNOS in other mammals including rat, mice hamsters and guinea pigs

(Grozdanovic & Gossrau, 1998; Grozdanovic, Nakos, Dahrmann, Mayer, & Gossrau, 1995).

In these species, the concentration of nNOS in type IIB fibers seemed to be greater than type

IIA muscle fibers (Grozdanovic & Baumgarten, 1999). In the study of Kobzik et al. (1994),

NOS activity was found to be strongly correlated with the proportion of type II muscle fiber

(r=0.97). For instance, extensor digitorum longus muscle had 98% of type II fibers and its

NOS activity was 5.44 times of that in soleus muscle with 13% type II fibers. LD and PM are known to have different composition of MHC type isoform as measured in this current study.

Therefore, this study further compared the extent of protein nitrosylation between PM and

LD muscles using 1 day aging samples.

One of the blots to compare nitrosylation intensity between LD and PM of day 1 samples was shown in Figure 4. Based on the intensity of bands, LD obviously had greater extent of protein nitrosylation than PM across animals. For all samples, the intensity of protein nitrosylation of LD in selected four bands was greater than that of PM muscle after 1 day of postmortem storage (P<0.05). Higher levels of protein nitrosylation in LD muscle may be indicative of greater NOS activity of this muscle. This supports the hypothesis that muscles with greater proportion of type II fibers to have higher amount of NOS and NO production (Kobzik et al., 1994). NO has been reported to have inhibitory effects on the activity of cysteine protease calpain and calpain induced proteolysis (Koh & Tidball, 2000;

Michetti et al., 1995). This inhibitory effect provides the possibility that NO may be involved in regulating meat tenderization in postmortem muscle (Warner, Dunshea, Ponnampalam, &

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Cottrell, 2005). Cook, Scott and Devine (1998) reported that NO enhancer reagent could

improve the rate of meat tenderization at day 3 and 6 of postmortem beef than NOS inhibitor

groups. However, all groups showed similar values of tenderness after 8 days of storage. In a

recent study (Cottrell et al., 2008), injection of NOS inhibitor pre-slaughter reduced Warner-

Bratzler shear force in the longissimus thoracis et lumborum (LTL) but not

semimembranosous (SM) after 3 days of postmortem storage. This difference is partly due to

the variation of chemical composition including the content and the solubility of collagen

between two muscles. Nitrosylation induced meat toughening could have more inhibitory effects in LTL. The results from current study also support this conclusion since higher levels of protein nitrosylation were detected in LD than PM of beef. In contrast, exercise induced

NO production tended to decrease meat tenderness during aging in both LTL and semimembranosous (Cottrell et al., 2008). Exercise may increase the activity of nNOS in both muscles and thus improve the production of NO. NO induced nitrosylation may inhibit the process of meat tenderization resulting in tougher meat. Based on these studies, variation in the amount and activity of NOS may be contributed to the difference of fresh meat quality among muscles.

Conclusions

Very limited studies have been published about the possible roles of NO and NO induced protein nitrosylation in fresh meat quality during postmortem storage. This study reports that protein nitrosylation can be detected in postmortem muscle. LD shows higher percentage of

type II fiber type and lower percentage of type I fiber types compared to PM. Greater protein

nitrosylation in LD than PM indicates that muscle fiber composition may be associated with

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different NOS activity and NO production. More studies need to be conducted to explore how these protein nitrosylation during early postmortem aging are related to fresh meat quality.

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Table 1 Comparison muscle fiber types between longissimus and psoas major musclesa

Type I Type II P values

LD 27.00±0.94 73.00±0.95 P<0.001

PM 45.15±1.73 54.85±1.73 P=0.007

P values P<0.001 P<0.001 a Percentage of muscle fiber types within longissimus and psoas major muscles were calculated as the percentage of each fiber isoform to divide the total amount of MHC isoforms in each sample.

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Table 2 Intensity of protein nitrosylation across LD and PMa

LD PM P values

Band1 0.97±0.03 0.77±0.04 0.04

Band2 1.00±0.10 0.56±0.11 0.05

Band3 0.82±0.03 0.61±0.06 0.03

Band4 1.07±0.04 0.62±0.11 0.02

aIntensity ratios were calculated as the intensity of the band in each sample over intensity of the band in the internal designated densitometry standard of diaphragm muscle.

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7LD 8LD 9 LD 10 LD 7PM 8 PM 9PM 10PM

Type IIA/X Type I

Figure 1. Coomassie-stained 6% polyacrylamide gel showing differences in myosin heavy-chain isoforms of the LD and PM. The top bands in the figure were the MHC type IIA/X isoform and the lower bands were MHC type I isoform. All lanes were loaded with 2 µg of protein.

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LD PM

D1 D3 D7 D1 D3 D7 Band 1 Band 2

Band 3 Band 4

Figure 2: Protein nitrosylation of LD and PM of animal 7 after 1, 3 and 7 days postmortem storage. All lanes were loaded with 2.22 µg of total proteins in each lane. From left to right, the lanes were: LD d 1;

LD d 3 ; LD d 7; PM d 1; PM d 3; PM d 7.

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Band1: SE=0.11 Band2: SE=0.07 Band3: SE=0.08 Band4: SE=0.09

Figure 3: Nitrosylation intensity of muscle proteins of LD and PM across aging days. Ratios of band intensity were calculated as intensity of the band in each sample over the intensity of the band in the internal designated densitometry standard of diaphragm muscle (P>0.05).

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DP* 7LD 8LD 9LD 10LD 7PM 8PM 9PM 10PM

Band 1

Band 2

Band 3 Band 4

Figure 4: Nitrosylation Intensity of muscle proteins of LD and PM at day 1 postmortem. All lanes were loaded with 2.22 µg per lane of total proteins. Ratios were calculated as intensity of the band in each sample over the intensity of the band in the internal designated densitometry standard (DP: diaphragm, lane 1 from left side).

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GENERAL SUMMARY

Inconsistency in tenderness and water holding capacity are major problems that impede the profitability of meat industry. Many factors can contribute to variations of these two attributes including genotype, animal feeding, preslaughter stress and postmortem treatments.

Skeletal muscle is a well organized system for contraction and relaxation and has the capacity to remain their redox in living animals. In postmortem muscle, biochemical and biophysical processes including proteolysis can change the structure of skeletal muscle.

Protein degradation has been believed to be related to the tenderization of meat for many years. Relatively, less study has been conducted about the effects of protein degradation on water holding capacity during postmortem aging. Decreased ability to retain reducing cellular environment in postmortem skeletal muscle can increase the accumulation of reactive oxygen and nitrogen species. These species may cause protein oxidation and nitrosylation to regulate protein functions including cysteine enzyme activities. The overall hypothesis of this study was that biochemical factors that influence proteolysis during the antemortem or the early postmortem period can contribute to variations in fresh meat quality.

Our first study suggests that integrin and desmin have different ways to regulate water holding capacity of pork during postmortem refrigerated storage. Significant correlations were found between intensity of intact desmin at day 1 and day 7 and drip loss or purge loss during postmortem aging. These correlations indicate that degradation of desmin may be associated with increased water holding capacity. Desmin degradation can decrease the transmission of the shrinkage of the myofibrils to the shrinkage of entire cells. Therefore, degradation of desmin reduces the formation of drip gaps between muscle cells and between

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muscle bundles and thus results in decreased drip loss. Negative correlations between intensity of intact integrin and drip or purge loss indicate that integrin degradation may contribute to decreased water holding capacity. Integrin proteolysis may weaken the interaction between the membrane and the cytoskeleton of skeletal muscle cells and thus contribute to the formation of drip channels between the cell cytoskeleton and the cell membrane. In the same study, intensity of intact desmin at day 7 was positively correlated with 80 kDa and 78 kDa products of µ-calpain while it was negatively correlated with autolyzed 76 kDa fragments of µ-calpain. In the meanwhile, higher amounts of drip loss or purge loss were associated with higher percentage of 78 kDa µ-calpain and lower percentage of 76 kDa µ-calpain. Since autolysis of µ-calpain is associated with its activation in postmortem muscle, these correlations support the hypothesis that µ-calpain may be involved with desmin degradation and drip or purge loss during postmortem aging.

The results from first study further confirm the critical roles of µ-calpain in regulating fresh meat quality as previous studies reported. It is known that cellular environment changes dramatically in postmortem muscles including pH, ionic strength and oxidation conditions.

Therefore, it is very important to understand how these postmortem changes regulate the activities of m- and µ-calpain. Decreased ability of postmortem muscle to retain reducing condition may increase the accumulation of reactive nitrogen species including nitric oxide.

In second study, we determined the effects of nitric oxide on the properties of m- and µ- calpain using a natural compound S-nitrosoglutathione as nitric oxide donor. In the absence of calcium, the activities of m- and µ-calpain were not influenced by S-nitrosoglutathione at both pH 6.5 and 7.5. In the presence of 1 mM calcium chloride, S-nitrosoglutathione could

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regulate m- and µ-calpain differently. S-Nitrosoglutathione combined with calcium significantly changed m-calpain activity regardless of whether S-nitrosoglutathione or calcium was added first at both pHs. For µ-calpain, S-nitrosoglutathione did not further influence its activity in the presence of calcium. However, S-nitrosoglutathione can decrease the autolysis rate of µ-calpain when it was added before calcium resulting in high proteolytic activity measured with standard assay. We found that µ-calpain was endogenously nitrosylated and S-nitrosoglutathione could further nitrosylated µ-calpain in the presence and absence of calcium in current experimental conditions.

In the third study, the same nitrosylation detection kit was used to detect protein nitrosylation and compared the extent of protein nitrosylation in postmortem beef longissimus dorsi and psoas major muscles. Nitric oxide synthase catalyzes the production of nitric oxide in skeletal muscle. The distribution of nitric oxide synthase is muscle fiber type dependent. Muscles with higher percentage of type II fibers have greater amounts of nitric oxide synthase. Longissimus dorsi has a higher percentage of type II fibers and a lower percentage of type I fibers than the psoas major. Several protein bands were detected as being nitrosylated in all samples. A significant difference was found in protein nitrosylation between the two muscles. At day 1 of postmortem storage, the longissimus dorsi showed higher intensity of protein nitrosylation than the psoas major. This study shows that protein nitrosylation can be detected in postmortem beef muscles during aging. Muscle fiber composition could influence the extent of protein nitrosylation between muscles.

These studies show that biochemical processes including desmin and integrin degradation during postmortem storage may change the structures of postmortem muscles

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and thus influence fresh meat quality. Further studies need to be conducted to explore the behind mechanism that how nitric oxide regulates the properties of calpains. Whether and how nitrosylation of calpain and other muscle proteins in postmortem aging are involved in the regulation of meat quality are deserved more studies. For example, porcine skeletal muscles that vary in tenderness and drip loss can be used to determine the correlations between the extent of protein nitrosylation and meat quality parameters.

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ACKNOWLEDGEMENTS

I would like to take this opportunity to express my thanks to those who helped me with various aspects including conducting research and the writing of this dissertation.

First and foremost, I would like to express my deepest gratitude to my major advisor

Dr. Elisabeth Huff-Lonergan for her excellent guidance, patience and caring throughout this research and the writing of this thesis. I have learned not only science from her, but more importantly the right attitude to science. Her insights and encouragement have always inspired me and supported me with excellent atmosphere to complete my graduate education during the past four years.

I would like to thank Dr. Steven Lonergan for his invaluable recommendations and excellent guidance for my whole research. His advice is incredibly important because it always posed my research to the right directions.

I would like to express my great appreciation to my committee member Dr. Joseph

Sebranek. He always encouraged me and communicated with me friendly especially when I had tough time in my life. His advices for both my research and my life will be invaluable throughout my life.

I would like to thank my other committee members, Dr. Patricia Murphy and Dr.

Richard Robson, for their helpful advice for my class selection, research plan and the writing of my dissertation.

I would like to thank my lab members Dr. Ed Steadham, graduate students Mark

Anderson, Rachel Smith, Kasey Maddock and Cory Wagner and undergraduate students.

Without their help and efforts, it can not imagine that I can finish my lab work successfully.

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Finally, I would like to thank my parents Xiaoyun Zhang and Huizhen Luo, my brother Hongjun Zhang and my sisters Hongmei Zhang and Yan Zhang. Although I have a very long distance from them, their strong support and deep love always stay with me and help me overcome any difficulties that I faced in my life.