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Determination of exponential rate constants for calpain-mediated degradation of myofibrillar complexed… Albisser, Tracie 1996

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DETERMINATION OF EXPONENTIAL RATE CONSTANTS FOR CALPAIN-MEDIATED DEGRADATION OF MYOFIBRILLAR COMPLEXED PROTEINS b y TRACIE ALBISSER A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S School of Human Kinetics We accept this thesis as conforming to the required standard U N I V E R S I T Y O F BRITISH C O L U M B I A December, 1996 © Tracie Albisser, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of. British Columbia, I agree that the Library shall make it freely, available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood, that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The purpose of this study was to test the hypothesis that an orderly sequence or time course of calpain action on purified myofibrillar/cytoskeletal complexes from cardiac and fast muscle tissue exists. To test the response of individual substrate proteins to degradation (i.e. susceptibility), exogenous calpain (1.5U/ml) was incubated with 40 ug of highly purified rat myofibrillar complexes from cardiac and gastrocnemius muscles (0 to 60 min), in vitro. Apparent molecular weights (SDS-PAGE) were used to compare and identify individual myofibril proteins. Myofibrillar yields for cardiac (n=9) and fast skeletal (n=9) muscles were 62.93 ± 6.58 and 98.42 ± 9.36 mg/g (p<0.05). Following 30 minutes of calpain treatment, the amount of cardiac desmin, C-protein, a-actinin and troponin-T remaining in complex were 0%, 19%, 23% and 68%, compared to controls (p<0.05). For fast skeletal muscle, the remaining desmin, C-protein, a-actinin and tropomyosin after 30 minutes of calpain digestion was 0%, 38%, 51% and 17%, respectively, compared to control (p<0.05). The estimated exponential rate constants (k) for degradation/loss for each protein (in both tissues) had the following order: desmin>C-protein>a -actinin (p<0.05). The results of this study support the hypothesis that there are selective degradation rates and an ordered sequence of degradation for myofibrillar complexed proteins. Factors contributing to the heterogeneity of k values for myofibril proteins, and therefore their susceptibility to calpain degradation, may be their spatial arrangement (peripheral -> central) within the myofibril and/or their primary amino acid residue composition. ii T A B L E O F C O N T E N T S Abstract ii Table of Contents iii List of Tables v List of Figures vi Introduction 1 Specific Objectives . . 5 Methods ... • 6 Results 11 Discussion 26 References . 36 Appendices 40 Appendix A: Literature Review 41 Mechanisms of Protein Degradation 42 Characteristics of Lysosomal Proteases 42 Chemistry 42 Regulation 45 Physiological Implications. 47 Characteristics of Non-lysosomal Proteases... 48 Chemistry. 48 Regulation 50 Physiological Implications 53 Physiological Stimulation of Altered Protein Degradation.54 Activity . .. 54 Long-Term. 55 Short-Term. 58 Exercise-Induced Muscle Damage 60 Nutrition 61 Starvation 64 Targeting and Susceptibility 68 Covalent Modification 69 Phosphorylation 69 Ubiquitin Conjugation..... 71 Oxidation and Formation of Disulphides....71 iii Substrate Characteristics 73 PEST sequences 74 Conclusion 75 References 78 Appendix B: Methodology 92 Appendix C: Troubleshooting 101 Appendix D: Tables and Statistical Outputs 112 Appendix E: Statistical Outputs 122 iv LIST O F T A B L E S TABLE 1: PERCENTAGE OF MYOFIBRILLAR C O M P L E X E D PROTEIN REMAINING FOLLOWING A 30 MINUTE CALPAIN-DIGESTTON IN FAST M U S C L E TISSUE (individual raw data) 113 TABLE 2: PERCENTAGE OF MYOFIBRILLAR C O M P L E X E D PROTEIN REMAINING FOLLOWING A 30 MINUTE CALPAIN-DIGESTION IN CARDIAC M U S C L E TISSUE (individual raw data) 113 TABLE 3: R A T E OF LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS OVER A 30 MINUTE CALPAIN DIGESTION IN FAST M U S C L E TISSUE, ANDIVIDUAL raw data) 114 TABLE 4: R A T E OF LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS OVER A 30 MINUTE CALPAIN DIGESTION IN CARDIAC M U S C L E TISSUE. (Individual raw data) 114 TABLE 5: PERCENTAGE OF MYOFIBRILLAR C O M P L E X E D PROTEINS REMAINING A T SPECIFIC TIME POINTS OVER 30 MINUTES OF CALPAIN DIGESTION IN FAST M U S C L E TISSUE 115 TABLE 6: PERCENTAGE OF MYOFIBRILLAR C O M P L E X E D PROTEINS REMAINING A T SPECIFIC TIME POINTS OVER 30 MINUTES OF CALPAIN DIGESTION IN CARDIAC M U S C L E TISSUE , 117 TABLE 7: PROTEIN YIELD OF MYOFIBRILLAR ISOLATIONS F R O M CARDIAC A N D FAST M U S C L E TISSUE (individual raw data) .119 TABLE 8: S U M M A R Y OF STATISTICAL ANALYSES 120 LIST O F FIGURES FIGURE 1: SDS-PAGE (5-15% gradient gels) OF FAST M U S C L E MYOFIBRILLAR COMPLEXES DIGESTED B Y 1.5U m-CALPAIN. Purified myofibrils are digested at 37°C and stopped at various time points (given in minutes at bottom) with leupeptin (200ug/ml). Molecular mass standards are indicated on the left of panel in kDa. The 0 minute lane represents control myofibrils 13 FIGURE 2: SDS-PAGE (5-15% gradient gels) OF CARDIAC M U S C L E MYOFIBRILLAR COMPLEXES DIGESTED B Y 1.5U m-CALPAIN. Purified myofibrils are digested at 37°C and stopped at various time points (given in minutes at bottom) with leupeptin (200ug/ml). Molecular mass standards are indicated on the left of panel in kDa. The 0 minute lane represents control myofibrils 15 FIGURE 3:TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS FOLLOWING 30 MINUTES OF CALPAIN DIGESTION, in vitro. Samples of 40ug of purified myofibrils were digested for 30 minutes with 1.5U m-calpain and quenched by leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-P A G E gradient gels and percent remaining at 30 minutes quantified by density analysis using IPLab gel software 17 FIGURE 4: FAST M U S C L E TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS OVER A 30 MINUTE CALPAIN DIGEST, in vitro. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and percent remaining at specific time points quantified by density analysis using IPLab gel software. The density value for each protein of interst was measured as a ratio against the density value of the actin present in that lane. The ratios from the 0.5-30 minute lanes calculated as a percentage of the time 0 lane and fitted into a single exponential decay equation and the rate constant, k, calculated as a best fit value. 20 FIGURE 5: CARDIAC TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS OVER A 30 MINUTE CALPAIN DIGEST, in vitro. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and percent remaining at specific time points quantified by density analysis using IPLab gel software. The density value for each protein of interst was measured as a ratio against the density value of the actin present in that lane. The ratios from the 0.5-30 minute lanes calculated as a percentage of the time 0 lane and fitted into a single exponential decay equation and the rate constant, k, calculated as a best fit value 22 VI FIGURE 6: TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D DESMTN OVER A 30 MINUTE CALPAIN DIGEST, in vitro 24 FIGURE 7: TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D a-ACITNIN OVER A 30 MINUTE CALPAIN DIGEST, in vitro 24 FIGURE 8: TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D C-PROTEIN OVER A MINUTE CALPAIN DIGEST, in vitro 24 FIGURE 9: STRUCTURE OF T H E SARCOMERE. The thick and thin fiaments are arranged around one another in a helical fashion to allow cross bridge formation in the A-band region. C-protein is thought to play a structural role in maintaining this compact internal organization. The boundries of the sarcomere are marked by the Z-line which acts to connect the ends of the thin filaments within the sarcomere, a-actinin and desmin are involved in the structure of the Z-line while tropomyosin and troponin-T are part of the complex that runs the entire length of the thin filament Generally desmin and a-actinin are considered peripheral proteins and C-protein, tropomyosin and troponin-T are considered internalized. If a preferential loss of periferal proteins was to occur first, it could loosen the sarcomeric structure followed by a complte unravelling with the loss of the internal proteins . 30 FIGURELO: TYPICAL DEGRADATION PATTERN OF FAST MYOFIBRILLAR COMPLEXES B Y 1.5U m-CALPAIN. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue. Molecular mass standards are run in the far left lane and lane A depicts 1.5U m-calpain 104 FIGURELL: RESULTS OF A DIGEST PERFORMED WITH CONTAMINATED m-CALPAIN. myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue. Molecular mass standards are run in the far left lane and lane A depicts 1.5U m-calpain 106 FIGURE 12: 60 ug OF PURIFIED FAST M U S C L E MYOFIBRIL COMPLEXES COMBINED WITH COMPONENTS OF CALPAIN DIGESTION BUFFER. Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue 108 FIGURE 13: 60 ug PURIFIED FAST MYOFIBRIL COMPLEXES VISUALIZED A T SUCCESSIVE STAGES OF DIGEST PROCEDURE A T TWO DIFFERENT L E V E L S OF FREE C A 2 + . Individual proteins visualized by 5-15% SDS-and PAGE gradient gels and stained with Coomassie Brilliant Blue 110 vii INTRODUCTION Proteins in skeletal muscle undergo a continuous process of degradation and resynthesis. In comparison with protein synthesis, protein degradation is a much more ambiguous process and as a result is poorly understood. There are a number of stages between the removal of a functioning protein and the release of a free amino acid that may be unique to an individual protein or class of proteins resulting in a wide array of protein half-lives. Despite the compact structure of the myofibril, myofibrillar components turnover with quite different and distinct rates (48). Furthermore, the rates of protein turnover in different skeletal muscles varies according to fiber type and composition (16). This heterogeneity in the process as a whole has resulted in an inadequate explanation of the sequence of events involved in the turnover of striated muscle proteins. A major problem is to account for the removal, degradation, and resynthesis of individual protein subunits within the myofibrillar matrix while maintaining sufficient structural integrity to allow generation and transmission of contractile force (27). Further, a crucial question in the study of protein metabolism is the physiological significance of different protein turnover rates for functionally related myofibrillar proteins (40). Currently, most studies monitoring protein degradation fail to distinguish the selective breakdown of myofibrillar versus non-myofibrillar proteins and the overall contribution of each proteolytic system Furthermore, the mechanisms responsible for selective degradation are poorly understood, thus isolation of specific degradative pathways may provide a more thorough understanding of the mechanisms responsible for protein breakdown. l It is evident that degradation of the myofibrillar complex of striated muscle is a dynamic and precisely controlled process that involves both lysosomal and non-lysosomal processes. Generally, the overall coordination and contribution of these systems to myofibrillar disassembly and degradation are unclear. It has been stated that lysosomes are rarely found in the muscle fibers of normal animals therefore the role they play in striated muscle breakdown is uncertain (6,18,22). Lysosomes are apparent in other cell types present within muscles such as macrophages, mast cells, leukocytes, and endothelial cells (27). If lysosomal enzymes are found extracellulary it is usually a pathological situation (8) therefore it is difficult to be certain how much physiologically relevant information can be gained from characterizing the actions of lysosomal proteases on normal striated muscle proteins. Although lysosomal protease do show action against myofibrillar proteins such as myosin and actin, this activity is increased when acting on the soluble form of these proteins (7). This indicates that optimally the phagocytic action of lysosomal proteases follow a disruption of the myofibril to release large polypeptide fragments leaving them more susceptible. It has been suggested that lysosomal protease activity is preceded by an initial deterioration of myofibrillar structure by non-lysosomal proteases (15). The calcium activated neutral proteases (CANP) (EC 3.4.22.17), or calpains (calcium-dependent papain like), are a group of non-lysosomal cysteine endopeptidases found in both invertebrates and vertebrates. These enzymes function optimally at neutral pH and are in direct contact with the cellular environment. The two isozymes found in striated muscle, u-calpain and m-calpain , have similar biochemical properties except for the calcium (Ca2 +) concentration required for half-maximal activity in vitro(39). The 2 calpains are normally found in the cytosol, with 7-30% associated with membrane structures (24). This distribution is not uniform but localized to membrane components such as membrane phospholipids, structural proteins, ion transport systems, and receptors (24). Calpain has also been found to be associated with the I-band and Z-bands of the sarcomere (24). It is possible that calpain distribution within the cell may indicate substrate specificity and biological function. The activation of calpain in striated muscle is dependent on four factors: 1) the Ca2+ concentration, 2) the autolyzed/unautolyzed state of the enzyme, 3) the concentration of the endogenous inhibitor, Calpastatin, and 4) the availability of digestible substrates (21). Its ubiquitous distribution among tissue types suggests that calpain activation may influence cellular metabolism and protein turnover. Generally, calpain does not digest its substrates in vivo but modifies them producing restricted fragments (8,28). Limited proteolysis of proteins by calpain may cause destabilization of structural rigidity rendering it more susceptible to other cellular proteases (10,2.1). Moreover, calpain proteolysis may be an initial step in the degradation of proteins destined for lysosomal degradation. This protease has been shown to selectively disassemble myofibrillar, cytoskeletal, and membrane proteins in skeletal muscle (9,24). It has been demonstrated that digestion of isolated myofibrils with calpain in the presence of C a 2 + results in the removal of the Z-line by promoting the release of alpha-actinin (34). The Z-line is involved in anchoring the thick and thin filaments together to maintain the three-dimensional architecture of the myofibrils. Reports showing calpain mediated degradation of purified proteins associated with the Z-line region (i.e. desmin, filamin), coincident with a loss of 3 function, supports their role in influencing myofibrillar disassembly (29,30,44). Since desmin may link myofibrils together at the Z-line and anchor alpha-actinin (23), there may be a coordinated pattern to calpain's action in myofibrillar disassembly and degradation. No report of this coordinated removal of selected proteins from a purified myofibrillar/cytoskeletal complex is available in the literature. The purpose of this study was to test the hypothesis that an orderly sequence or time course of calpain action on purified myofibrillar/cytoskeletal complexes from cardiac and fast muscle tissue exists. Because any differences in the selective protein degradation rates of intact tissues/organs may indicate either increases of substrate susceptibility to proteolysis, or calpain activity, or both, an in vitro model of protein degradation was chosen for these experiments . In this way, the addition of exogenous, standardized, calpain activity maximizes the responsiveness of individual proteins to degradation (i.e. a measure of their susceptibility to proteolysis) 4 SPECIFIC O B J E C T I V E S 1) Prepare purified myofibrillar complexes from rat hearts and gastrocnemius muscles and obtain a highly purified preparation of the calcium activated neutral protease, calpain. 2) Identify the proportion of individual proteins (by apparent molecular weight) remaining with the myofibrillar complexes of cardiac and fast muscle tissue after a 30 minute digest with 1.5U of m-calpain activity using SDS-PAGE. 3) Determine the rate constant, k, for exogenous calpain-mediated degradation/loss of identifiable individual substrate proteins of the myofibrillar complexes of cardiac and fast muscle tissue. 5 M E T H O D S ANIMALS; Male Wistar rats (350g) were sacrificed by a lethal intraperatoneal injection of euthanol (pentobarbital sodium) (n=9). The gastrocnemius muscle and heart ventricles were dissected, partitioned into samples, trimmed of all visible fat and connective tissue, and then placed in liquid nitrogen. All samples were stored at -70°C. MYOFIBRIL ISOLATION: Muscle samples were homogenized in a borate-KCl buffer containing 39mM sodium borate, 25 mM KC1, 1 mM phenylmethylsulfonyl floride and 5mM E G T A (1). The homogenate was then centrifuged at 13, 000 rpm for 15 minutes (Hermle 360Z, rotor VO2805) and the supernatant discarded. Pelleted material was washed (twice) with a 1% Triton X-100, 0.1 M KC1, 50 mM Tris (pH 7.0), and 1 mM DTT. The myofibrillar pellet was then resuspended in an Low Salt Buffer containing 0.1 M KC1, 2 mM MgCl 2 , 2 mM E G T A , 10 mM Tris-maleate (pH 7.0), and 1 mM DTT. All procedures were performed at 4°C. Purity of the preparations were confirmed by a lack of succinate dehydrogenase activity and by the calcium uptake capacity. The average Ca 2 + -Mg^-ATPase activity in the cardiac samples was 150 mmole Pi /mg/min. MEASUREMENT OF PROTEIN CONCENTRATION: The protein concentration of the myofibril isolations was established using the method of Lowry et al. (26). All protein concentrations were expressed relative to wet weight muscle. CALPAIN ACTIVITY: Purified (>95% by protein, data not shown) high calcium requiring (m-) isoforms of calpain were assayed for activity by assessing the proteolysis of caesin at 37°C in the presence of 2mg/ml caesin, ImM DTT, 750mM free Ca 2 + , and 50mM Tris (pH 7.4). After 30 minutes the caesinolysis was quenched by the addition of 500ul of ice cold 5% 6 trichloracetic acid (TCA). A unit of calpain activity was defined as the amount of T C A soluble product resulting in an increase of 0.1 optical density units at a wavelength of 280nm. Assays of calpain activity were performed in duplicates. PROTEOLYTIC DIGESTION: Myofibril preparations were digested over 0 to 30 minutes at a ratio of 40 ug myofibrillar complexes to 1.5U calpain activity. Proteolytic digestions were conducted in 5mM CaCl 2 , lOmM DTT, and 20mM Tris buffer (pH 7.4) at 37°C and terminated at selected time points with leupeptin (200ug/ml). Following centrifugation (12 500 rpm for 15 minutes in a Hermle Z360K centrifuge), the supernatant was aspirated and the samples resuspended in 5mM Tris buffer (pH 8.0), centrifuged and once again the supernatant aspirated. To visualize individual proteins, digested samples were mixed with an equal volume of a sample buffer containing : 0.062 M Tris, 4% SDS, 20% glycerol. 0.02% bromophenol blue, 1 mM D T T and incubated for 20 min at 50°G and run on SDS-polyacrylamide slab gel electrophoresis using the Laemmh buffer system Aliquots (40ug/well) were electrophoresed on a 5-15% linear gel at 20mA for approximately 8 hours. The gels were stained for one hour in 0.025% Coomassie Blue, 9% acetic acid, and 45% methanol, followed by destaining in 20% methanol and 5% acetic acid. Molecular weights were estimated using molecular weight standards (Sigma). QUANTIFICATION OF SDS P A G E GELS: Gels were air dried, scanned, and the images then quantified via IPLab Gel™ software. The proteins of interest were identified by determining molecular mass and the gel image was subsequently divided into regions for analysis of individual protein bands. Once all the segments were defined, density analysis was performed on all lanes of each gel image. The density of various segments was 7 calculated as the volume of pixel values within each segment (see appendix for detailed explanation). Sarcomeric actin, which is not degraded by calpain activity, was chosen as the internal standard for each lane of interest (19). The density value for each protein of interest was then measured as a ratio against the density value of the actin present in that lane. The ratio value in the time 0 lane was set as 100% protein for each gel. The ratios from the following lanes (30sec-30min) were then calculated as a percentage of the time 0 ratio. The percentage values from each gel were taken and fitted to a single exponential decay equation via non-linear regression. Once the date points were fitted to the single exponential curve, the rate constant, k, and overall slope were calculated as best-fit values. This procedure is depicted in the following schematic. Ao *=( ln[Ao]/[A])/f [A] Jfc=rate constant Ao=percent protein at time zero [A]=percent protein remaining Time (/) * k calculated along length of entire curve 8 The time points chosen for the linear regression were 0. 0.5, 1, 5, 15, 30 minutes as seen in the following schematic. These points described the shape of the curve well and had the lowest associated standard error after linear regression. It was found that increasing the number of time points between 0 and 30 minutes did not have a noticeable effect on the k value and since the procedure used to achieve the time points was both labor intensive and costly, analysis of additional time points seemed unrealistic. Extending the time course beyond 30 minutes also did not have a significant effect on the k value as the reaction seemed to plateau at 30 minutes. It was thought that this might be due to autolysis of calpain but "spiking" the reaction with more calpain activity at 30 minutes did not change the plateau effect. Therefore, the time points chosen for the single exponential decay equation seemed to be the key points in maintaining the validity of the k value. TIME (min) Statistical Analysis: Statistical comparisons were based on a 2-way factorial design for randomized groups analysis of variance (2-way A N O V A test). It assessed a 2 (muscle type) X 4 (rate for each protein) A N O V A design, with a level of significance set at an 9 alpha (p) level of 0.05. Significant main effects and interactions were then analyzed by 1-way analysis of variance or Students t-tests for independent samples. Individual mean comparisons were accomplished with a Student Newman-Keuls test with an a-level of 0.05. 10 R E S U L T S MYOFIBRILLAR YIELD AND COMPOSITION: The yield of purified myofibrillar complexes was 62.93 ±6.58 mg/g from cardiac muscle tissue and 98.42 ± 9.36 mg/g from fast muscle tissue. Previous reports (4) have shown that the sarcomeric arrangement of the proteins within the myofibrillar complexes is maintained by this isolation and purification procedure. The SDS-PAGE of fast and cardiac myofibrillar complexes exhibit similar patterns of staining and migration as previously reported (Figures 1 and 2) (3,4). Al l major contractile proteins are present and the percentage of each resembles former findings (4,13); myosin heavy chain(55%), actin (20%), a-actinin (1.5%), desmin (1.8%). In addition, each substrate protein was identified by its correct apparent molecular weight: C-protein (140 kDa), a-actinin (95 kDa), desmin (55 kDa), cardiac troponin-T (37 kDa), fast tropomyosin (32 kDa). PERCENTAGE OF MYOFIBRILLAR COMPLEXED PROTEINS REMAINING IN FAST TWITCH AND CARDIAC TISSUE FOLLOWING A 30 MINUTE CALPAIN DIGESTION: When 40ug myofibrillar complexed proteins from fast and cardiac muscle tissue were subjected to a 30 minute proteolytic digestion with 1.5U/ml m-calpain, specific identifiable proteins were degraded to distinct endpoints. Quantification of the endpoints for degradation of individual proteins of the myofibrillar complex in fast and cardiac muscle tissue is presented in Figure 3. C-protein and a-actinin were degraded or lost to a greater extent in cardiac compared to fast muscle tissue (p<0.05). Desmin was completely removed from the myofibrillar complexes in both types of tissue. Within cardiac tissue, a greater percentage of C-protein and a-actinin is degraded than troponin-T (p<0.05). There was no difference between the final percentage n of C-protein and A-actinin remaining in the myofibrillar complexes of cardiac muscle tissue (p>0.05). In fast tissue a 30 minute calpain digest resulted in less tropomyosin remaining in the myofibril than both C-protein and A-actinin (p<0.05). Furthermore, more C-protein was removed than A-actinin (p<0.05). SDS-PAGE OF CALPAIN-digested CARDIAC AND FAST twitch MYOFIBRILLAR COMPLEXED PROTEINS: The effects of l .SU of calpain activity on purified cardiac and skeletal myofibrillar complexes over 30 minutes are shown in Figures 1 and 2. In both cardiac and fast muscle myofibrils, the staining intensity of a 55 kDa band (desmin) is dramatically decreased by 30 seconds and has completely disappeared by 5 minutes. In addition, both the fast muscle C-protein and tropomyosin bands begin to diminish by 1 minute and are markedly faded by 30 minutes (Figure 1). The A-actinin band of fast muscle myofibrils does not begin to decrease in intensity until at least 5 minutes and fades more gradually to the end of the digest. The fading of the A-actinin band is delayed in the cardiac digest until 15 minutes indicating that the release of A-actinin from the Z-line occurs later in cardiac tissue. Cardiac C-protein staining intensity dirninishes more dramatically at 30 minutes than the fast twitch form. Finally the cardiac troporiin-T band displays the least amount of fading by the end of the digest 12 FIGURE 1: SDS-PAGE (5-15% GRADIENT GELS) OF FAST MUSCLE MYOFIBRILLAR COMPLEXES DIGESTED B Y 1.5U M-CALPAIN. PURIFIED MYOFIBRILS ARE DIGESTED AT 37°C AND STOPPED AT VARIOUS TIME POINTS (GIVEN IN MINUTES AT BOTTOM) WITH LEUPEPTIN (200UG/ML). MOLECULAR MASS STANDARDS ARE INDICATED ON THE LEFT OF PANEL IN KDA. THE 0 MINUTE LANE REPRESENTS CONTROL MYOFIBRILS. 13 -myosin heavy chain rf FIGURE 2: SDS-PAGE (5-15% GRADIENT GELS) OF CARDIAC MUSCLE MYOFIBRILLAR COMPLEXES DIGESTED BY 1.5U M-CALPAIN. PURIFIED MYOFIBRILS ARE DIGESTED AT 37°C AND STOPPED AT VARIOUS TIME POINTS (GIVEN IN MINUTES AT BOTTOM) WITH LEUPEPTIN (200UG/ML). MOLECULAR MASS STANDARDS ARE INDICATED ON THE LEFT OF PANEL IN KDA. THE 0 MINUTE LANE REPRESENTS CONTROL MYOFIBRILS. 15 MrX10: 200-11 6-97-• H t m * -myosin heavy chain -c-protein -alpha-actinin 66-•desmin -actin -troponin-T 31 Time 0 0.5 1 5 15 30 min lie FIGURE 3:TISSUE SPECIFIC LOSS OF MYOFIBRILLAR COMPLEXED PROTEINS FOLLOWING 30 MINUTES OF CALPAIN DIGESTION, IN VITRO. SAMPLES OF 40UG OF PURIFIED MYOFIBRILS WERE DIGESTED FOR 30 MINUTES WITH L5U M-CALPAIN AND QUENCHED BY LEUPEPTIN (200UG/ML). INDIVIDUAL PROTEINS VISUALIZED BY 5-15% SDS-PAGE GRADIENT GELS AND PERCENT REMAINING AT 30 MINUTES QUANTIFIED BY DENSITY ANALYSIS USING IPLAB GEL SOFTWARE. 17 100 OPROTEN A-ACT1MN DESMIN a=p<0.05 card ac vs fast b= p<0.05 card ac tn-t vs. c-protein and a-actinin c= p<0.05 fast tm vs. c-protein and a-actinin d= p<a05 fast c-protein vs. a-actinin 18 Rate of loss of myofibrillar complexed proteins from fast twitch and cardiac tissue over a 30 minute calpain digest: PURIFIED MYOFIBRILLAR COMPLEXES FROM FAST AND CARDIAC MUSCLE TISSUE TREATED WITH 1.5U/ML M-CALPAIN SHOWED DEFINITE PATTERNS OF MYOFIBRILLAR DEGRADATION/LOSS. QUANTIFICATION OF INDIVIDUAL DEGRADATION RATE CONSTANTS (k) FOR IDENTIFIABLE SUBSTRATE PROTEINS OF THE MYOFIBRILLAR COMPLEXES IS PRESENTED IN FIGURES 4 AND 5. IN BOTH FAST AND CARDIAC TISSUE, THE EXPONENTIAL RATE CONSTANT (k) FOR DESMIN WAS EXTREMELY HIGH RELATIVE TO BOTH C-PROTEIN AND a-ACTININ, INDICATING THAT THE DEGRADATION OF DESMIN WAS MUCH MORE RAPID THAN THE OTHER TWO SUBSTRATES (P<0.05). FAST TROPOMYOSIN AND CARDIAC TROPONIN-T WERE BOTH DEGRADED MORE SLOWLY THAN THE DESMIN PRESENT IN THAT PARTICULAR TISSUE (P<0.05). WHEN THE REMAINING TISSUES SPECIFIC PROTEINS WERE COMPARED, FURTHER DIFFERENCES WERE REVEALED. C-PROTEIN AND TROPOMYOSIN WERE BOTH DEGRADED MORE RAPIDLY THAN a-ACTININ IN FAST TWITCH MYOFIBRILS (P<0.05). MINIMAL DIFFERENCES WERE NOTED BETWEEN THE RATES OF DEGRADATION FOR FAST C-PROTEIN AND TROPOMYOSIN (P>0.05). IN CARDIAC MYOFIBRILLAR COMPLEXES, C-PROTEIN WAS DEGRADED MORE RAPIDLY THAN a-ACTININ AND TROPONIN-T. (P<0.05). THE RATE OF LOSS OF CARDIAC TROPONIN-T WAS NOT DIFFERENT FROM THAT OF a-ACTININ (P>0.05). WHEN THE TISSUE SPECIFIC DEGRADATION RATES FOR DESMIN WERE COMPARED, NO DIFFERENCES WERE FOUND BETWEEN CARDIAC AND FAST MUSCLE (P>0i05, FIGURE 6). THE FAST ISOFORMS OF a-ACTININ AND C-PROTEIN BOTH HAD HIGHER RATES OF DEGRADATION/LOSS THAN FOUND FOR THE CARDIAC ISOFORMS (P<0.05, FIGURES 7 AND 8). THESE PATTERNS AND RATES OF DEGRADATION AND/OR LOSS CAN BE DIRECTLY ATTRIBUTED TO CALPAIN BECAUSE DIGESTS FOR ALL SAMPLES CONTAINING LEUPEPTIN (TIME 0) DID NOT SHOW ANY EVIDENCE OF PROTEIN DEGRADATION. 19 Figure 4: FAST M U S C L E TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS OVER A 30 MINUTE CALPAIN DIGEST, in vitro. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and percent remaining at specific time points quantified by density analysis using IPLab gel software. The density value for each protein of interst was measured as a ratio against the density value of the actin present in that lane. The ratios from the 0.5-30 minute lanes calculated as a percentage of the time 0 lane and fitted into a single exponential decay equation and the rate constant, k, calculated as a best fit value. 20 R A T E CONSTANT (K) P<0.05 A-ACTININ (N=7) 0.35 VS. C-PROTEIN; TROPOMYOSIN C-PROTEIN (N=3) 0.97 TROPOMYOSIN (N=4) 1.14 DESMIN (N=5) 4.30 VS. A-ACTININ; TROPOMYOSIN; C-PROTEIN desmin 4 1 i i ^ i i i i I i i i i l i i i i I i i i i I i i i i 0 5 10 15 20 25 30 TINE (min) 21 Figure 5: CARDIAC TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D PROTEINS OVER A 30 MINUTE CALPAIN DIGEST, in vitro. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and percent remaining at specific time points quantified by density analysis using IPLab gel software. The density value for each protein of interst was measured as a ratio against the density value of the actin present in that lane. The ratios from the 0.5-30 minute lanes calculated as a percentage of the time 0 lane and fitted into a single exponential decay equation and the rate constant, k, calculated as a best fit value. 22 R A T E C O N S T A N T (k) p<0.05 a-actinin (n=5) 0.07 troponin-T (n=5) 0.09 C-protein (n=5) 0.23 vs. a-actinin; tn-T desmin (n=5) 4.60 vs. a-actinin; tn-T; C-protein 0 5 10 15 20 25 30 TIME (min) 23 FIGURE 6: TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D DESMIN OVER A 30 MINUTE CALPAIN DIGEST, in vitro. FIGURE 7: TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D a-ACTININ OVER A 30 MINUTE CALPAIN DIGEST, in vitro. FIGURE 8: TISSUE SPECIFIC LOSS OF MYOFIBRILLAR C O M P L E X E D C-PROTEIN OVER A 30 MINUTE CALPAIN DIGEST, in vitro. 24 o z 70 60 50 40 H 0 FASrTWTCH(r»=S) • CWOAC(n=5) 100 00 80 — 70 1 80 h G [S SO -Jl 40 -fi 30 20 h J I L. J 1 1 i^J 1 2 3 FIGURE 6 5 30 10 H o F*sr™rrcH(c*=7) J07 i i i i I i i . i i I i i i i I i i i i I i i i i I i i i i I 10 15 20 mE(irin) FIGURE 7 25 X O FASTTWTCH(ite3) • CJWMC(n=5) FIGURE 8 25 DISCUSSION The calpam-mediated digestion of myofibril complexes observed in this study confirmed that desmin, C-protein, a-actinin, tropomyosin, and troponin- are substrate proteins for calpain proteolysis. Interestingly, the degree/extent to which each protein was digested varied. Using these proteins as substrates, the results of this study support the hypothesis that there are selective degradation rates and an ordered sequence of degradation for proteins of the myofibrillar complex from striated muscle* From the data set it is clear that the degradation rate constants (it) of three common substrate proteins occur in the same order in both muscle tissue types (desmin>C-protein>a -actinin), albeit with different k values between muscles. Comparison of cardiac and fast muscle myofibrils showed k values for C-protein and a-actinin to be greater for fast muscle, while minimal differences were reported for desmin. The identification that desmin, C-protein, a-actinin, tropomyosin and troponin-T are substrates for calpain, while other major sarcomeric proteins, myosin and actin, are not agrees with previous reports (35). One of the novel aspects arising from this study is that each substrate protein was degraded/lost to varying degrees, even in the presence of a standardized calpain activity and calcium concentration. This suggests that a preferred degradation of some myofibrillar proteins occurs over others (i.e. susceptibility). For example, myofibrillar complexed desmin is completely degraded, whereas C-protein and a-actinin were on average (combined cardiac and skeletal muscle) only partially degraded (-30% and 40% remaining, respectively). Even with a longer incubation and/or addition of more exogenous calpain, no further degradation of these proteins was observed (2,3). 2 6 This variation in the extent of degradation noted for individual myofibrillar complexed proteins in this study may underlie the earlier reports of relatively different degradation rates for desmin, troponin-I, tropomyosin, C-protein and a-actinin (35). There is a considerable degree of polymorphism among individual contractile proteins, however nothing is known about the extent to which these differences influence the susceptibility of the proteins to proteolysis. Although, some structural features of soluble proteins are closely associated with their rates of degradation such as large or acidic proteins degrade faster than small or basic proteins (11,12), it is difficult to perceive whether this applies to proteins linked within the myofibrillar complex. If dissociation from the myofibrillar complex is considered a rate limiting step in the disassembly or degradative process, then those particular proteins that interact more weakly with the other proteins in the complex might be more susceptible to proteolytic attack (27). Furthermore, the presence of C a 2 + ions has been shown to affect the interaction of myofibrillar proteins, particularly the tropomyosin-troponin complex (14). Unfortunately, the lack of published apparent k values estimated for these calpain substrate proteins, makes any systematic comparison between tissues and/or proteins difficult. Moreover, without k values it is difficult to assess other factors which may underlie the variation for individual proteolytic rates and/or the order/sequence of degradation among protein substrates. These other contributing factors may be the susceptibility of the substrate proteins due to the spatial arrangement of the proteins within the myofibrillar complex and/or their compositional characteristics. Spatial Arrangement: The concept of susceptibility to proteolysis being a consequence of accessibility of proteins grouped in a complex, e.g. myofibrils, has never been fully 27 demonstrated. Although Van der Westhuyzen et al., (45) showed that a pool of myofilaments known as easily releasable myofilaments (ERM), which lack desmin and cc-actinin, are derived from a peripheral pool of myofilaments, no test of their susceptibility to proteolysis was attempted. More recent studies by Belcastro et al. (4) have shown that the degradation of troponin-T occurs more rapidly in the ERM's as opposed to that of intact myofibrils. Although this difference between ERM's and intact myofibrils is suggestive of a decreased spatial hindrance of calpain action due to a reduction in the number of proteins present within the myofibrillar structure, the use of end-point degradation rates without appropriate k values may have confounded the interpretation of these results. Although the precise physical arrangement between cytoskeletal and contractile proteins is only partially understood a number of identifying factors are available in the literature (43,46). Generally desmin and a-actinin may be considered peripheral proteins with respect to the myofibrillar complex, whereas tropomyosin, C-protein and troponin-T may be considered internalized proteins (38,46). If spatial alignment was a factor influencing susceptibility of complexed proteins to degradation, then it could be predicted that desmin and a-actinin would have the greatest k values relative to the internalized tropomyosin, C-protein and troponin-T. If correct, the theory would propose that a preferential loss/degradation of peripheral proteins would occur first to possibly loosen the sarcomere, followed by complete unraveling with the loss of internal proteins. The results of this study provides partial support for this theory, in that desmin degradation consistently shows the highest k value (by 2 orders of magnitude), while C-protein, 2 8 tropomyosin, and troponin-T show appreciably lower k values for either cardiac or skeletal muscle. Whether this theory of a peripheral to central disassembly of the myofibrillar complex occurs in vivo is unknown and requires further investigation. 2 9 S t r u c t u r e of the S a r c o m e r e C-protein Z-LIN E (desm in, alpha-actinin) thin filament thick filam ent Tropom yosln, Troponln-T C r o s s s e c t i o n at A - b a n d Z- | fpe Figure 9: STRUCTURE OF T H E SARCOMERE. The thick and thin filaments are arranged around one another in a helical fashion to allow cross bridge formation in the A-band region. C-protein is thought to play a structural role in maintaining this compact internal organization. The boundaries of the sarcomere are marked by the Z-line which acts to connect the ends of the thin filaments within the sarcomere, a-actinin and desmin are involved in the structure of the Z-line while tropomyosin and troponin-T are part of the complex that runs the entire length of the thin filament. Generally desmin and a-actinin are considered peripheral proteins and C-protein, tropomyosin and troponin-T are considered internalized If a preferential loss of peripheral proteins was to occur first, it could loosen the sarcomeric structure followed by a complete unraveling with the loss of the internal proteins. 3 0 The k values for the Z-line structural protein, a-actinin, do not support the concept of a peripheral to central disassembly process for striated muscle myofibrillar complexes. The results of this study showed that the k values of 0.07 and 0.35, for cardiac and fast skeletal muscle, respectively, were significantly lower compared to those for desmin, tropomyosin, C-protein and troponin-T. Considering the spatial arrangement of this protein within the myofibrillar complex, it seemed reasonable to predict that a-actinin should have k values approximating those of desmin a peripherally located protein in relation to the Z-line (38). Although the factors) contributing to a lower k value for a-actinin is uncertain, the report that a-actinin is not a true substrate for calpain proteolysis may lend partial support for its unexpected deviation from the pattern of spatial arrangements relative to the other proteins (20). Moreover the observation by Goll et al. (20) that when a-actinin is modified by calpain's action, no difference in its ability to bind polymerized F-actin results, thereby indicating that the structural role of these two proteins may not be a primary target for calpain action, but rather a secondary event related to the degradation of the other proteins. Another possibility for the a-actinin k values not supporting the peripheral to central theory of disassembly may be related to the suggestion that the constituent proteins of the sarcomere are assembled in chronological order (32), with titin and a-actinin addition being the first events in sarcomeric assembly followed by the association of tropomyosin, myosin, nebulin, and C-protein. If this theory of assembly is correct then it could be conceivable that disassembly would follow in a reverse order with a-actinin being one of the last proteins to leave the myofibrillar complex (i.e. low k value). Although speculative, there appears to be sufficient evidence 31 provided by Dr. Goll's laboratory (and others; 47) that a-actinin is not a typical protein substrate of calpain and any variation of its k value from the peripheral to central disassembly process should not detract from this theory. Compositional Characteristics: There is considerable evidence that isolated purified proteins may be targeted for degradation by selective proteases, including calpain, due to a number of factors, including their primary structure (i.e. sequence of amino acid residues), phosphorylation status and or reduction-oxidation (redox) status (36). For example, proteins containing regions rich in proline (P), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T) (called "PEST' sequences) are a unique feature of many calpain protein substrates (37,47). The strength of these PEST sequences can be evaluated with a scoring system that evaluates the sequence bases on the number of residues present in the PEST enriched region and the degree of hydrophobicity (37). Although these "PEST" sequences seem to confer calpain susceptibility, they are not an absolute prerequisite for degradation of all calpain protein substrates (47). In theory, it has been suggested that these regions not only attract calpain, but could also support the increased binding of Ca 2 + , thereby increasing [Ca 2 +]i to levels sufficient to activate the localized calpain (37). Whether or not this PEST factor has a role in protein degradation/disassembly of organelle structures, such as the myofibrils used in these experiments, is unknown. However, a thorough examination of the primary sequences of desmin, C-protein, tropomyosin, troponin-T and a-actinin revealed that only C-protein and a-actinin contained PEST sequences (47, and PEST-FIND program in PCGENE software). For example, cardiac C-protein contains 1 'good' (£ 5) and 16 'poor' (<5) PEST sequences, with the 'good' 32 sequence (score = 10.45) occurring between amino acid residues 114 to 143 and consisting of: E V P A P A T E L E E S V S S P E G S V S V T Q L X J S A A E . In contrast, desmin, tropomyosin and troponin-T do not contain any P E S T related sequences (17,31,41). If P E S T related sequences were primary targets for the grouped proteins of the myofibrillar complex then it would be predicted that C-protein and cc-actinin would have the highest k values. Clearly this is not the case, therefore it may be concluded from the results of this study that primary amino acid sequences ( P E S T score) are not primary factors regulating the degradation and disassembly characteristics of complexed myofibril proteins. Because phosphorylation and redox status confer different susceptibility patterns of calpain protein substrates (39), which are more prominent with pathological conditions (3,36), these experiments were conducted on myofibrils prepared from resting animals, and incubated without protein kinases (to minimize their phosphorylation), and 1 mM D T T to stabilize their redox status. These experimental conditions were carefully controlled so that representative k values for each digestible protein in the myofibrillar complex could be investigated with minimal interference from agents known to increase protein turnover in the cell (27,36). Whether or not these agents alter individual k values from myofibrillar complexes prepared from pathological conditions warrants further investigation. Physiological Implications of Calpain Action on Myofibrillar Complexes: A feature of the results of this study is the dramatic k value noted for desmin. This implies that desmin 33 loss may be a rapid event in vivo. That this effect would be more pronounced during periods of increased contractile activity and/or different pathological conditions has been suggested where distinct patterns of myofibrillar disorientation, particularly at the level of the Z-line, have been observed (5,33). It is possible that mechanical tension from myofibrillar contractile proteins is transmitted via intermediate filaments associated with the Z-line (desmin, vimentin) to the extracellular matrix (25). A loss of these connections may result in a decreased efficiency in the transmission of myofibrillar contractile tension. Although the precise mechanism by which skeletal disruption could result in altered muscle mechanical properties is not known, earlier reports have implicated cytoskeletal proteins in transmitting force along the muscle fiber (42) Calpain mediated degradation may have further functional implications with regards to maintenance of the myofibrillar conformation needed for cross-bridge formation and regulation of cross-bridge cycling. In striated muscle, the Ca2+-sensitive control mechanism for contraction and relaxation is mediated through the interactions of the tropomyosin-troponin complex located on the thin filament (14). The peripheral to central theory of disassembly proposed in this study would imply that the unraveling of the thin filament resulting from calpain proteolysis of tropomyosin and troponin would disrupt the conformational regulation of actomyosin interaction. 34 S U M M A R Y • The results of this investigation confirmed that desmin, C-protein, a-actinin, tropomyosin, and troponin-T are substrate proteins for calpain proteolysis. • Using these proteins as substrate, these finding support the hypothesis that there are selective degradation rates and an ordered sequence of degradation and/or disassembly for proteins of the myofibrillar complex. • The contributing factors to the varied rates of degradation for the individual substrate proteins may be their susceptibility based on the spatial arrangement of the proteins within the myofibrillar complex and/or their compositional characteristics. F U T U R E DIRECTIONS • A complete examination of the amino acid composition of the myofibrillar substrate proteins to identify meaningful characteristics such as potential phosphorylation sites. • Analysis of the fragments produced as a result of calpain action in order to specify sites of cleavage and possible functional implications. • Examination of the order of degradation and disassembly of the myofibrillar substrate proteins in conditions of altered metabolic status. 35 R E F E R E N C E S 1. Belcastro, A. N. Skeletal muscle calcium-activated neutral protease (calpain) with exercise. / . Appl. Physiol. 74: 1381-1386,1993.(Abstract) 2. Belcastro, A . N. , J. S. Gilchrist, and J. Scrubb. Function of skeletal muscle sarcoplasmic reticulum vesicles with exercise. / . Appl. Physiol. 75:2412-2418,1993.(Abstract) 3. Belcastro, A. N., J. S. Gilchrist, J. A. Scrubb, and G . Arthur. 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Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticuluum protein structure and calcium release. / . Biol. Chem. 267: 20857-20865, 1992. 20. Goll, D. E . , W. R. Dayton, I. Singh, and R. M . Robson. Studies of the alpha-actinin/actin interaction in the z-disk by using calpain. / . Biol. Chem. 266: 8501-8510, 1991.(Abstract) 21. Goll, D. E . , V . F. Thompson, R. G. Taylor, and T. Zalewska. Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin. BioEssays 14: 549-556,1992.(Abstract) 22. Guarnieri, G. , G. Toigo, R. Situlin, M . A. Del Bianco, and L. Crapesi. Cathepsin B and D activity in human skeletal muscle in health and disease. In: Proteases II: Potential Role in Health and Disease, edited by W. H. Horl and A. Heidland. New York: Plenum Press, 1988, p. 243-256. 23. Janmey, P. A. Mechanical properties of cytoskeletal polymers. Curr Opin Cell Biol 3: 4-11,1991. 24. Johnson, P. Calpain: structure-activity relationship and involvement in normal and abnormal cellular metabolism. Int. J. Biochem. 263: 823-828, 1990.(Abstract) 25. Lieber, R. L . , L . Thornell, and J. Friden. Muscle cytoskeletal disruption occurs within the first 15 minutes of cyclic eccentric contraction. J. Appl. Physiol. 80: 278-284, 1996. 37 26. Lowry, O. H. , A. J. Rosebrough, A. J. Fair, and A. J. Randall. Protein measurement with the folin phenol reagent. / . Biol. Chem. 193: 265-275, 1951. 27. Millward, D., P. C. Bates, J. G. Brown, S. R. Rosochacki, and M . J. Rennie. Protein degradation and the regulation of protein balance in muscle. In: Protein Degradation in Health and Disease. Netherlands: Ciba Foundation, 1980, p. 307-329. 28. Murachi, T. Intracellular regulatory system involving calpain and calpastatin. Biochemistry International 18:263-294,1989.(Abstract) 29. Nelson, W. J. and P. Traub. 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Stringent requirement for Ca2+ in the removal of z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase. Biochem. J. 209: 635-641, 1983. 35. Reddy, P. A., T. E . Anandavalli, and M . P. Anandaraj. Calcium activated neutral protease (u- and m-CANP) and endogenous CANP inhibitor of muscle in Duchenne muscular dystrophy (DMD). Clin ChimActa 160: 281-288,1986.(Abstract) 36. Rivett, A . J. Regulation of intracellular protein turnover: covalent modification as a mechanism of marking proteins for degradation. Curr. Topics Cell. Regul. 28:291-337, 1986. 37. Rogers, S. W. and M . C. Rechsteiner. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 234: 364-368, 1986. 38 38. Saetersdal, T., H . Dalan, and J. Roli. Immunoflorescence and immunogold electron microscopy of desmin in isolated adult heart myocytes of the rat. Histochem. 92:467-473, 1989. 39. Saido, T. C , H . Sorimachi, and K. Suzuki. Calpain: new perspective in molecular diversity and physiological-pathological involvement FASEB J. 8: 814-822, 1994.(Abstract) 40. Seene, T. Turnover of skeletal muscle contractile proteins in glucocorticoid myopathy. /. Steroid Biochem. Molec. Biol. 50: 1-4,1994. 41. Stone, D. and L. B. Smillie. The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terrninal half and complete sequence. / . Biol. Chem. 253: 1137-1148,1978. 42. Street, S. F. Lateral transmission of tension in frog myofibers. A myofibrillar network and transverse cytoskeletal connections are possible transmitters. / . Cell Physiol. 114: 346-364,1983. 43. Thornell, L . and M . Price. The cytoskeleton in muscle cells in relation to function. Biochem. Soc. Trans. 19: 1116-1120, 1991. 44. Traub, P. and W. J. Nelson. Occurrence in various mammalian cells and tissues of the Ca2-t~ activated protease specific for the intermediate-sized filament proteins vimentin and desmin. Eur. J. Cell Biol. 26: 61-67,1981. 45. Van der Westhuyzen, D. R., K. Matsumoto, and J. D. Etlinger. Easily releasable myofilaments from skeletal and cardiac muscles maintained in vivo. / . Biol. Chem. 256: 11791-11797,1981.(Abstract) 46. Wang, K. Sarcomere-associated cytoskeletal lattices in striated muscle. / . Cell Muscle Motility 6: 315-369, 1985. 47. Wang, K. K. W., A. Villalobo, and B. D. Roufogalis. Calmodulin-binding proteins as calpain substrates. Biochem. J. 262: 693-706,1989.(Abstract) 48. Waterlow, J. C , P. J. Garlick, and D. J. Millward. Protein turnover in mammalian tissue and in whole body. In: North Amsterdam, Holland: North-Holland Pub. Co. 1978, 39 APPENDICES 40 APPENDIX A : L I T E R A T U R E R E V I E W 41 M E C H A N I S M S O F P R O T E I N D E G R A D A T I O N Proteins in eukaryotic cells undergo continuous turnover which occurs under precise control mechanisms. In a steady state condition a basal level of intracellular proteolysis acts to: a) selectively degrade proteins with abnormal conformations or sequences, b) post-translationally process newly synthesized proteins, and c) breakdown inactivated proteins that have lost their function (33,85). In addition to this basal level of activity an accelerated rate of proteolysis occurs with different pathological conditions (muscular dystrophy, diabetes, etc.), myopathies (glucocorticoid myopathy, cardiomyopathy etc.), adverse dietary conditions, and hormonal or amino acid deficiency (6,62). These intracellular proteolytic events in skeletal muscle cells are controlled by multiple pathways with distinct characteristics. Although the exact mechanism of degradation of cellular proteins during normal and accelerated protein turnover remain to be elucidated, generally the two types of protease systems involved in skeletal muscle protein degradation are lysosomal and non-lysosomal. Characteristics of Lysosomal Proteases- Cathepsins Chemistry Lysosomes and/or lysosomal proteases are present in all mammalian cell types except enucleated red blood cells. The concentration of lysosomes varies in different tissue types and is particularly high in the liver, spleen , kidney, and macrophages. The properties of lysosomal cathepsins in different species and cell types, however, are very similar. In general, lysosomal proteases are small monomeric units ranging from 20 000 to 42 40 000 (19). These proteases are optimally active at acidic pH values and are unstable at neutral and alkaline pH. As a result they are compartmentalized and not in contact with the cellular environment (19). Most lysosomal enzymes are glycoproteins and are active against a wide range of small peptides and large peptide substrates (19). The best characterized lysosomal enzymes are the cathepsins. Cathepsin B, L , and H are cysteine proteases which presumably require a highly reducing environment to function properly (19). Cathepsin D is an aspartic protease that resolves into several forms of similar molecular weight and different isoelectric points upon purification (19). Cathepsin L is the most powerful lysosomal cysteine protease against protein substrates, degrading proteins at least ten times faster than other cellular cysteine proteases (18,19). Cathepsin L has been found in many species such as human, pig, and rat and in many organs including liver, kidney and muscle. This endopeptidase degrades proteins in the pH range of 3 to 7 in vitro. The specificity for bonds cleaved seem to be broad in the acidic range, but becomes narrow at pH 7 (86). Cathepsin B is one of major proteinases in the lysosomal pathway and appears to be ubiquitous in rnammals (18,74). This lysosomal cysteine proteases has been purified from various tissue such as human (7) and rat (135,146) liver, as well as rat and monkey skeletal muscle (71,74,135). It operates optimally at a pH of 5.4 as a peptidyldipeptidase. Cathepsins B and H are unique in that they display both endopeptidase and exopeptidase activity depending on the substrate (19).The exact specificity of its' endopeptidase activity is still somewhat unclear and its' activity against protein substrates is low in comparison with Cathepsin L (18). 43 Cathepsin H has been detected in all organs and tissues of the rat and has a pH optima between 6 and 7 (18). This protease shows limited action against proteins (less than 5% compared to that of Cathepsin L) (18). Aside from it functioning as both an endo- and exopeptidase, Cathepsin H is unusual in that it is the only lysosomal aminopeptidase to be characterized as yet (18). Therefore, it possible that Cathepsin H functions mainly as an aminopeptidase on peptides released by cathepsin L and in some more specialized cells, by Cathepsin D (18). Cathepsin D cleaves preferentially between hydrophobic amino acid residues at a pH optima ranging from 3 to 5 depending on the substrate (18). This lysosomal carboxyl protease may be primarily located in non-muscle elements (especially the liver) and therefore its importance in muscle proteolysis is uncertain (144). As a rule, lysosomal proteolysis is initiated by endopeptidases, which are the rate-limiting proteases. This event is then immediately followed by exopeptidase activity and diffusion of the free amino acids and some dipeptides through the lysosomal membrane. Cytosolic exopeptidase activity rapidly cleaves the remaining dipeptides and the resulting free amino acids can be utilized as substrates for synthesis of new proteins or catabolized for energy production (18). Early investigations into the action of cathepsins B and D on myofibrils found that they displayed limited proteolysis of native myosin and other myofibrillar proteins (16,135). Bird et al. (16) found that these proteases did act on myofibrillar proteins and that soluble denatured myosin was degraded more extensively than insoluble native myosin. Hirao et al. (74) reported that cathepsin B prepared from monkey skeletal muscle 44 degraded skeletal and cardiac muscle myosin and actin (74). A later study examined the action of rabbit skeletal muscle cathepsin B towards myofibrils. This enzyme degraded the myosin heavy chain, actin ,and troponin-T, but not a-actinin, tropomyosin, troponin-I and troponin C (100). In accordance with this, actin and different troponins are also susceptible to proteolysis by cathepsin L (18). Purified cathepsins H and L demonstrate a greater specificity of action against myosin than cathepsin B (5 and 10 times respectively). Cathepsin L broke down myosin to peptides less than 5000D or to individual amino acids (16). It has been stated that muscle lysosomes are rarely found in skeletal muscle fibers of normal animals (15). When lysosomal enzymes are found extracellulary it is usually a pathological situation. For example, some malignant tissues secrete a "cathepsin-B-like' cysteine protease that is thought to be involved in the destruction of the extracellular matrix. In addition, lysosomal enzymes from macrophages are released at sites of irdlarnmation and may be damaging to the normal tissue and structural proteins in the area (19). Since cathepsins have been purified from a variety of skeletal muscles in different species, it is interesting to consider the source of these proteases. These lysosomal proteases may arrive at the muscle cell from sources other than myogenic ones (i.e. extracellular origins). It is therefore difficult to be certain of how much physiologically relevant information can be gained from characterizing the actions of lysosomal proteases on skeletal muscle proteins. Regulation 45 The proteolytic activity in cells must be highly regulated to accomplish the aims of the many processes in which proteases are involved and to prevent inappropriate and uncontrolled degradation of proteins. Regulation of cellular protease activity is achieved in many ways (19). Although some factors have been distinguished, generally the signals that regulate the rates of protein turnover remain vague. Compartmentalization of cathepsin proteases in lysosomes provides an acidic environment for activity and stabilization while restricting their action to proteins that enter the compartment. Leakage from the lysosome results in inactivation of the cathepsins by the higher pH of the cytosol (19). Endogenous inhibitors found intracellularly also may function to prevent inappropriate proteolysis in the cytosol by lysosomal enzymes. Cystatins and stefins are two intracellular agents that inhibit lysosomal cathepsins. It is possible that intracellular inhibitors are important in the control of cellular proteases and that fluctuations in proteases activity are due to changes in inhibitors rather than proteinase concentrations (19). Hormones may also act as mediators in the metabolism of skeletal muscle proteins. For example, Millward et al. (107) looked at the effects of dietary protein deficiency and stress on lysosomal proteases and suggested that these could be mediated by thyroid hormones. Insulin, an important regulator of energy substrates storage and release, has a direct action on protein utilization by increasing amino acid uptake and increase muscle protein synthesis. It has been demonstrated that protein synthesis is extremely sensitive to small doses of insulin and its effects are maximal at physiological levels (80). Other studies have shown that synthesis is halted in insulin-deficient states, but the exact 46 mechanisms by which insulin promotes protein synthesis are not fully determined (38,81,156,157). In terms of protein breakdown, some authors believe that insulin acts mainly to regulate the lysosomal component of degradation (162). However, whether insulin is involved in regulation of muscle protein breakdown still a matter of debate (34). Intracellular nucleotide concentrations, especially of ATP, may affect the energy-dependent proton pumps that maintain the optimal acidity of the lysosomes in vivo (18,104). When the effects of ATP on cathepsin L activity was investigated, it was found that ATP interacted with the substrate (aldolase) rather than the protease itself. This interaction resulted in the substrate becoming more susceptible to proteolysis by cathepsin L (103). •Physiological Implications Skeletal muscle contains few lysosomes which has been suggested to limit the rate of catabolism of muscle proteins (63,69). Some authors have reported that lysosomes do not contribute significantly to overall protein breakdown in muscles incubated under optimal conditions and therefore are not involved in degradation of myofibrillar proteins (60,98). The activity of several cathepsin enzymes has been shown to increase during different conditions of enhanced proteolysis such as starvation/fasting (13,112) and muscle wasting diseases (60). However, under these conditions the lysosomal pathway may rally be responsible for increased protein breakdown of non-myofibrillar proteins (98). Furthermore, although lysosomal proteases do show action against myosin and actin, the activity is increased when acting on soluble forms of the proteins (16). This indicates that optimally the phagocytic action of lysosomal proteases would be preceded by an initial 47 deterioration of the myofibrillar structure. Such a disruption would act to release large polypeptide fragments and leave them more susceptible to lysosomal proteolysis. Therefore, it is possible that the two degradative pathways within skeletal muscle operate synergistically. After cytoplasmic initiation of proteolysis by limited and specific non-lysosomal protein degradation, a "cascade effect " may follow in lysosomes (69). The sequestered polypeptide fragments would be more extensively degraded by a non-selective bulk process within the lysosome (18). Characteristics of Non-lysosomal proteases- Calpain Chemistry The best characterized non-lysosomal protease, calpain, was defined as a calcium activated neutral protease cysteine endopeptidase (EC. 3.4.22.17) (110,129). This enzyme was found to be ubiquitously distributed in a variety of vertebrate tissue types including human skeletal muscle, erythrocytes and platelets (17,121,141,155). Recent molecular biological studies have now established that calpain actually constitutes a large family of distinct isozymes differing in structure and distribution. Basically , this family can be divided into two groups based on distribution: ubiquitous and tissue specific (129). The ubiquitous calpains consist of a u- and a m-calpain species. Each isozyme is heterodimer made up of a large (80kDa) and a small (28kDa) subunit (129). The 28kDa subunits are identical in both species and are encoded by a single gene. The 80kDa subunits are different gene products but share 50% sequence homology (67). Both isozymes have similar biochemical properties except for the calcium (Ca2 +) concentration required for half maximal activity in vitro. The u-calpain is activated in the presence of uM 48 levels of C a 2 + (3-50uM) and binds four C a 2 + atoms per molecule, while m-calpain is activated by mM levels of C a 2 + (200-1000uM) and binds five to six C a 2 + atoms per molecule (67,129). Since intracellular C a 2 + concentrations normally fluctuate just below uM levels, it is more likely the u-calpain functions in the cells under physiological conditions. There may be unknown in vivo mechanisms, involving for example nuclear materials, that are responsible for m-calpain activation. The novel tissue specific calpain isozymes, n-calpains, have similar but distinct structures and exhibit strict tissue specificity in comparison to the ubiquitous forms. For example, n-calpain 1 is expressed exclusively in skeletal muscle (129). This group of calpains have not yet been fully characterized but may have a 94kDa and a 74-76kDa species both of which have a very high C a 2 + requirement (3-4 mM)(67). These isozymes display about 50% sequence homology with the human 80kDa subunits of both u- and m-calpain and have no small subunits (67). The calpains are normally intracellular enzymes found mainly in the cytosol with between 7-30% of calpain being associated with membrane structures (82). Within the membrane it seems likely that the distribution of calpain is not uniform but associated with various membrane components such as membrane phospholipids, structural proteins, ion transport systems and receptors (82). Within the sarcomere, calpain has been found to be associated with the I-band and Z-band regions (82). As a result of being in direct contact with the cellular environment, the activity of these proteases is under the direct influence of intracellular events (19). 49 In general, all non-lysosomal proteases including calpain, are not digestive but more regulatory in function, as they produce restricted fragments from their specific protein substrates in vivo (19,110). This action usually results in alteration of a given substrate. For example, protein kinase C isozymes are converted by both u- and m-calpain to their active forms (129). Furthermore, limited proteolysis of a given protein by calpain is likely to cause destabilization of its structural rigidity as well as making it more sensitive to attacks by various cellular proteases. It has been suggested that calpain activation may be the initial step in the degradation of skeletal muscle proteins. Its' action may be to release peptide fragments from the a compact myofibrillar structure for further degradation by lysosomal proteases (9,67). The calpains selectively degrade myofibrillar proteins (21), cytoskeletal proteins, membrane proteins (82). Cytoskeletal substrates include; a-actinin, tropomyosin, lamins, vimentin, neurofilament protein, tubulin, microtubule-associated protein, C-protein, and desmin (82). Proteins indirectly involved in myofibril cross-bridge formation are also substrates such as myosin light chain, tropomyosin, troponin-T-like protein (in smooth muscle), caldesmon, calponin, and a-actinin(82). Calpain does not cleave undenatured actin or myosin (66). Regulation In addition to its limited proteolysis and strict substrate specificity, calpain resembles other processing proteases in that its activity is under tight control (129). Calpain activity in striated muscle is generally controlled by 1) the C a 2 + concentration as previously discussed, 2) autolyzed/unautolyzed state of the enzyme, 3) the level of inhibitor (calpastatin), and 4) the availability of digestible substrates (47,66,142). 50 Calpain undergoes autolytic activation to become fully active which may be due to its' role as a regulatory or processing protease in response to increased intracellular calcium concentrations. The actual role of the autolytic reaction is not very clear . It is possible that autolysis removes the ammo-terminal of the propeptide portion that apparently suppresses the autolytic activity. Or it may cause a conformational change in the molecule leading to the enzyme activation. Conversely, autolysis may arise as a consequence of the conformational changes induced by C a 2 + levels (129).Suzuki et al. (143) described that chick skeletal muscle calpain undergoes autolysis quickly in the presence of sufficient calcium and that autolysis lowers the calcium concentration required for its proteolytic activity. Brief autolysis decreases the C a 2 + concentration required for half maximal activity of u-calpain from 3-50uM to 0.6-0.8uM and of m-calpain from 200-1000uM to 50-150uM (67). This event occurs without affecting the specific activity of either isozyme. The C a 2 + concentration required to initiate autolysis is just slightly higher than that required to initiate proteolytic activity (67). Examination of the kinetics of this process with rriammalian u-calpain indicates that the autolysis of the catalytic large subunit and then of the small subunit precedes proteolysis of the substrate (128). Subunit autolysis seems to be an important early event in the intramolecular activation process of this protease (129). Calpastatin is a naturally occurring specific inhibitor of u-calpain and m-calpain and is equally effective in inhibiting both isoforms of the enzyme (19). The inhibitor binds to the larger subunit of calpain in the presence of C a 2 + and is not cleaved by the proteinase (19). At least two kinds of calpastatin exist in vertebrates. The most prominent species 51 which exists in all tissues except erythrocytes has a molecular weight of 73kDa in humans, while a smaller form is found only in erythrocytes and has a molecular weight of 48kDa. The large calpastatin can inhibit up to four u- or m-calpain molecules and the smaller form can act on three u- or m-calpain molecules. Calpastatin relies on C a 2 + for binding to the calpains as well as the inhibition of activity (84). Therefore, the calpain protease system exists in a Ca2+-regulated paradox where C a 2 + is required for both proteolytic activity and inhibition. This illustrates why there is still alot of uncertainty regarding the in vivo regulation of these proteases A long standing question regarding the in vivo calpain activation mechanism concerns the discrepancy between the in vitro C a 2 + requirement of calpain and the physiological intracellular calcium concentrations. Even the u-calpain requires greater than 10"6 M C a 2 + concentrations in vitro whereas intracellular C a 2 + usually fluctuates between 10"8 and 10"6 M . Coolican and Hathway's (31) finding that phosphatidylserine and phosphatidylinositol decreases C a 2 + concentrations required for calpain autolysis led to the emergence of the membrane-activation hypothesis. This suggested that calpain is activated intracellularly by attachment of the small subunit to a phospholipid site on the plasma membrane. Possibly, subunit attachment to the phospholipid decreases C a 2 + requirement for autolysis. Once autolysis occurs, the enzyme is released into the cell cytoplasm where C a 2 + concentration is now high enough for activation. Although this hypothesis is appealing and has gained popularity, recent findings indicate that it may require modification or that an alternative mechanism may exist (67). The intracellular C a 2 + concentrations are still not high enough to initiate autolysis of most of the calpain species 52 under normal conditions. Available evidence indicates that calpain and calpastatin are not preferentially located near plasma membranes (87,89,161). And recent studies show that calpain binds to proteins and not phospholipids in membrane vesicles (78,79,88). Further, the membrane-hypothesis does not include any role for calpastatin which completely inhibits both autolyzed and unautolyzed forms of u- and m-calpain and does so at C a 2 + concentrations lower than those required to initiate either proteolytic activity or autolysis. Immunolocalization studies have also shown that calpain and calpastatin are colocalized within the cell (89). Therefore, the cell must possess a mechanism that decreases that C a 2 + concentration requirement for proteolytic activity of the calpains, but also to regulate the binding of calpastatin. Evidence has indicated that regulation involves specific responses to C a 2 + binding at one or more of the Ca2+-binding sites on the calpain molecule. Calpain has four C a 2 + properties (in order of increased C a 2 + concentration requirements): 1) binding to subcellular organelles/plasma membrane; 2) binding to calpastatin; 3) proteolytic activity; 4) autolysis. Therefore, regulation may involve a "cascade" of events that occur in order of increasing C a 2 + concentrations (67). Physiological Implications Although much is known about the structural and enzymatic properties of u- and m-calpains, information regarding their physiological roles are quite limited The ubiquitous presence of those isozymes and the lack of a cell permeable and completely specific inhibitory reagents make it difficult to analyze their in vivo functions (129). Some of the substrates of calpain activity may be indicative of its' in vivo functions. For example, a-actinin is a protein in the Z-line to which the f-actin molecule of the thin 53 filament attach (112) and tropomyosin is a regulatory protein that acts to block the myosin binding site on each actin molecule (12). Thus calpain may be involved in the maintenance of the myofibrillar conformation needed for cross-bridge formation and regulation of cross-bridge cycling. Limited proteolysis of a given protein by calpain is likely to cause destabilization of its structural rigidity as well as making it more sensitive to attacks by various other cellular proteases. In vitro, calpain is responsible for complete removal of the Z-line proteins (a-actinin) and important cytoskeletal proteins (i.e. desmin, vimentin) which are involved in the maintenance of the sarcomeric structure (25,123). Degradation of the z-disks and C-protein would cause disordering of the three dimensional array of thick and thin filaments (39) and degradation of tropomyosin and troponin would initiate disassembly of their filaments to actin monomers or dimers (16). Thus, it has been suggested that calpains' role in muscle protein degradation is directed exclusively at the myofibrillar or cytoskeletal proteins where its effects result in disassembly of the myofibril and release of large polypeptide fragments, but not in the liberation of free amino acids. Therefore, calpain is an ideal enzyme for initiating disassembly of myofibrils (66) and has been shown to be activated in a variety of states including diabetes (9,22), starvation/fasting (23,113), and with exercise (8). P H Y S I O L O G I C A L STIMULI O F A L T E R E D P R O T E I N D E G R A D A T I O N Activity Exercise, whether it be long or short term, induces changes to peripheral fuel sources and promotes a metabolic environment which is conducive to muscle protein 54 degradation. Glucose stored as glycogen and fatty acids stored as triglycerides (TG's) are the major quantitatively important energy sources during exercise. Of these, glycogen is the more important fuel for strenuous exercise that requires 65-100% V02max (153). Conversely, during prolonged exercise (requiring less than 65% V02max), free fatty acids serve as an important fuel source. This results in a sparing of glycogen and prolonged exercise time before glycogen depletion occurs. Hepatic glycogen stores are generally depleted within 90-120 minutes of moderately intense exercise (160). Once this occurs, proteins may provide some additional energy requirements. Although all changes in protein metabolism are not identified, it is known that during endurance exercise rates of amino acid oxidation and conversion of amino acids to glucose are increased and that these changes may be physiologically important. Long-Term AEROBIC/ENDURANCE It has been found that there are significant increases in blood urea concentrations occurring at 60 to 70 rninutes into an exercise bout (37). Once the portion of the increased concentration due to reduced kidney function was considered, it was surmised that the remaining increase was due to elevated amino acid oxidation. An increase in protein catabolism at this time of exercise is probably related to a decrease in carbohydrate availability. Similar results were found by Dohm et al. (45) in both rat and human running protocols. Salminen and Vinko (131) found that prolonged subrnaximal exercise caused an increase in cathepsin D activity and exhaustive exercise increased the activity of several acid hydrolases in skeletal muscle (151). Later research showed that exhaustive exertion 55 induced an increase in both cathepsin C and D activity but no change for cathepsin B (130). Heavy exercise has also been found to cause necrotic lesions in mouse skeletal muscle and stimulates the lysosomal system of the muscle fibers adjacent to the necrotic foci (132,151). Evidence from other research has shown that myofibrillar degradation/fiber necrotic lesions are completed by intrinsic lysosomal proteases and may be preceded by non-lysosomal protease activity (23,39,149,162). Belcastro (8) studied the relationship between prolonged running and the activation of calpain. It was concluded that calpain activity was increased as a result of enhanced Ga 2 + sensitivity of the protease and increased susceptibility of myofibrillar substrate proteins. Thus it appears that muscle protein damage and amino acid catabolism increase with longer periods of exercise. In a large variety of cell types C a 2 + is recognized as a mediator of stimulus-response coupling. In muscle, the role of C a 2 + release from the sarcoplasmic reticuluum (SR) is to regulate contractility following excitation. Vollestad and Sejersted (152) suggested that an inability of the SR to reaccumulate C a 2 + would contribute to a decrease in the degree of actin-myosin interaction. Chronic presence of C a 2 + within the muscle cell may progressively decrease C a 2 + sensitivity of troponin-C which contributes to altered contractile mechanics (68). A significant decrease in C a 2 + sensitivity of myofibrils may contribute to a manifestation of exercise induced muscle fatigue by progressively decreasing contractile tension which can be produced at a given level of intracellular C a 2 + (158). Therefore, an exercise induced loss of intracellular C a 2 + homeostasis may contribute to peripheral fatigue by contributing to dysfunction within the contractile mechanism. Furthermore, the changes in intracellular C a 2 + may be promoted by both 56 lysosomal and non lysosomal proteolytic pathways (11). Haelberle et al. (70) showed that mechanical coordination of muscle contraction and proper contractile function was lost in vitro following calpain specific removal of intermediate filament proteins. Many of the intracellular factors which alter C a 2 + homeostasis and/or calpain protease activity under in vitro conditions may also be influential in promoting an increase in calpain proteolytic activity during in vivo exercise states. In which case, it appears that there is an association between exercise stress and possible intracellular changes which contribute to a loss of C a 2 + homeostasis and subsequent activation of calpain. Exercise stress may affect intracellular C a 2 + homeostasis by causing damage to the muscle cell membrane. The inability of muscle cell to regulate influx of extracellular C a 2 + may increase susceptibility to calpain activity. Moreover, this effect may be enhanced by intracellular A T P depletion (161). A decrease of ATP resynthesis may be affected by efflux of creatine kinase (CK) from intracellular compartment as a result of exercise induced damage to sarcolemmal membrane (3). Skeletal muscle C K has been shown to be to be a substrate for m-calpain activity in vitro (4) In addition, elevation of intracellular C a 2 + can alter ATP production by inhibiting mitochondrial oxidative phosphorylation (11). Exercise stress also may contribute to increase in intracellular C a 2 + by altering SR. Byrd et al. (27) found that exercise stress caused decrease in SR C a 2 + - uptake capacity which was associated with decreased activity of C a 2 + transport enzyme, Ca2 +-ATPase. The physiological stress of prolonged exercise also causes increases in bom SR and mitochondrial surface area (101) and dilation of SR from exposed skeletal muscle fibers (12). 57 One might expect that evidence of calpain- induced myofibrillar damage would accompany exercise-induced ultrastructural changes to subcellular organelles (SR and mitochondrial), since both processes would be influenced by changes in intracellular Ca 2 + . Myofibrils from exercised muscle tissue display a pattern of myofibrillar degradation which is characteristic of calpain protease activity (i.e. absence of Z-lines/ Z-line materials). For example, Belcastro et al. (12) described specific removal of Z-line structure from skeletal muscle myofibrils exposed to exercise stress was observed to be associated with a decrease in content of 95 and 58kDa proteins from myofibrillar structure. The size of these proteins closely corresponds to molecular weight of the Z-line proteins, a-actinin, and the exosarcomeric protein, desmin. In vitro, the fate of both these proteins is influenced by the proteolytic activity of calpain which readily degrades desmin, while promoting the releases of a-actinin (10). Therefore, calpain proteolysis contributes to disruption of myofibrillar structure during periods of acute prolonged exercise but the effect may vary according to the intensity of exercise. Booker et al. (20) revealed that calpain activity was increased with increasing intensity of exercise in the plantaris muscle and this intensity effect was not seen when exercise duration was low. Fiber type may have also been a factor since the effect was only seen in plantaris muscle (consisting mainly of fast twitch fibers) and not when it was assessed in soleus muscle (consisting mainly of slow twitch fibers). Short Term ANAEROBIC/ECCENTRIC EXERCISE 58 The sarcolemrnal membrane of skeletal muscle fibers is quite susceptible to high levels of myofibrillar contractile tension developed during intense anaerobic /eccentric exercise (3). When muscle cells are exposed to high levels of mechanical stress, it will disrupt the cell membrane structure and permit influx of Ca 2 + . Sarcolemrnal damage from high levels of muscle tension developed during eccentric muscle contractions can be evaluated by efflux of creatine kinase (CK) levels (57). Friden et al. (57) found a 36% increase in blood C K levels after acute bout eccentric muscle contractions. A sirnilar change in plasma C K was reported following intense anaerobic exercise of 15-60 sec (119). It is possible that is mechanical stress to sarcolemrnal membrane which permits a CK-efflux may promote a Ca2+-influx. Prolonged eccentric exercise yielded an increase in C K efflux which was paralleled by significant increase in mitochondrial calcium concentration (46). An increase in intracellular C a 2 + may be promoted by 1) C a 2 + influx through stretch-activated channels in sarcolemrnal membrane which allows C a 2 + ions to move in muscle cell down the C a 2 + concentration gradient and 2) C a 2 + -influx through ruptures/lesions in sarcolemrnal membrane produced by high levels of muscle tension (46). But since intense eccentric muscle contractions are often associated with increased CK-efflux the latter mechanism of Ca2+-influx may be more plausible. Therefore anaerobic and eccentric exercise promotes damage to muscle cell membranes causing loss of C a 2 + homeostasis due to increased influx of C a 2 + but evidence of increased calpain activity is somewhat altered. Extracellular matrix proteins which surround muscle fibers seem to be quite susceptible to myofibrillar tension developed during eccentric contractions (139). 59 Regardless of the mechanism by which C a z + enters muscle cell during eccentric contractions, tissue damage as indicated by SR vacuolization and Z-line disruption is present. The characteristic pattern of complete Z-line removal which normally follows calpain activity is replaced by extreme Z-line smearing/blurring which indicates severe myofibrillar structural disruption (56,59). But the specificity of the pattern of disruption coupled with selective damage to certain exosarcomeric matrix proteins (e.g. desmin) suggests calpain activity is increased (56). The change in the pattern of calpain degradation seems to be a function of proteolytic activity which is found during periods of excessive contractile force. When comparing the pattern of Z-line streammg/blurring from eccentric exercise versus Z-line removal from prolonged exercise, calpain proteolytic activity may be mediated by different intracellular factors which are a function of exercise demands (56,72). The Z-line structure is a weak link between series of sarcomeres which constitutes a myofibril and bears the bulk of the contractile tension, and therefore it is subject to exercise induced mechanical damage (56). The extent of Z-line damage within the myofibril would be determined by the balance between metabolic and mechanical factors which are developed during different types of exercise induced stress. Increases in intracellular C a 2 + which accompanies eccentric exercise may be quite transient which accounts for variance in Z-line damage in aerobic versus anaerobic/eccentric exercise (2). Exercise-Induced Muscle Damage Skeletal muscle damage has been shown to occur as a result of heavy exercise of short duration or prolonged exercise of moderate intensity in both human (75) and animal 60 species (3,8). With this type of damage, there appears to be a disruption of both the contractile and connective tissue (28,51) characterized by mitochondrial and SR vacuolization (3,58) and sarcomeric Z-line disruption (28,30,51). The underlying mechanism of these morphological observations is not known but may involve the activation of calpain. In vitro investigations have shown that this enzyme produces changes identical to those reported in vivo after exercise (47,66). Furthermore, these changes may be due to an enhanced sensitivity of the protease and an enhanced susceptibility of the myofibrillar substrate proteins as a result of the metabolic changes that occur during exercise (8) Nutrition The quantity and source of macronutrients that constitute dietary intake can influence the type of substrate utilized and substrate oxidation rates in muscle. McGarger et al. (102) found that a larger amount of fat and less carbohydrate was oxidized in subjects fed a high fat diet. The different trends in substrate oxidation was measured by the Respiratory Quotient (RQ) which was higher for subjects fed a high carbohydrate diet. Thomas et al. (145) reported similar results and further suggested that fat oxidation was significantly and directly related to fat intake levels but only for lean subjects. Sauer et al. (133,134) looked at substrate utilization of infants intravenously fed high fat formula and found increased fat oxidation as indicated by a decrease in RQ. Furthermore, fat combustion was found to be much higher during exercise after a high fat diet as compared to a high carbohydrate or normal diet (96). It is possible that these alterations in substrate oxidation influence the breakdown of body tissue proteins, particularly in skeletal muscle. 61 Increases in fat oxidation induced by diabetes enhance calpain activity and calpain degradation rates in the Z-line area of myofibrils (9,85). There is also mdirect evidence that the Z-line region is selectively affected when fat oxidation is increased as a result of fasting (35) and exercise (8). It is not known whether non-lysosomal proteases are responsible for this event The efficiency of protein utilization also depends upon the composition of the diet as a whole. Protein and non-protein energy may stimulate protein synthesis and/or degradation through different mechanisms and therefore may have an additive effect on protein turnover (102). Dietary carbohydrates and fats serve as the major sources of dietary energy for support of body protein metabolism. Carbohydrates lower the body nitrogen output in fasted subjects whereas fat does not (1). Munro et al.(109) reported that when normal subjects receiving a mixed diet were fed a diet where carbohydrates were isocalorically replaced by fat, an initial negative nitrogen balance occurred. Since amino acids represent supplemental fuel sources, it is logical that there would be an arnino acid sparing effect with increased glucose availability. Davies et al. (37) found leucine oxidation during exercise was approximately three times that during rest, but glucose ingestion rapidly reduces the oxidation of leucine. Based on urea and sweat losses, Lemon and Mullen (91) reported that during prolonged exercise by human subjects, amino acids provide about 5-10% of fuel utilized. Whereas in glycogen-depleted subjects, arnino acids and protein utilization increased and accounted for a greater percentage of total fuel supplied. 62 Many studies of the effect of nonprotein energy on nitrogen balance are in conflict Nitrogen balance studies in humans (32,120) looked at altered lipid and carbohydrate energy sources and found no difference between carbohydrates and fats in amount of nitrogen retained per kcal of added energy. However in normal subjects when carbohydrate was isocalorically exchanged for fat, a transient increase in nitrogen excretion developed and then returned to normal after a few days (80). Richardson (124) found significant improvements in nitrogen utilization (13%) when carbohydrates were the major source of dietary energy intake. The magnitude of protein sparing was greatest when the total energy intake was less than 50 kcal/kg/day. Therefore, at lower caloric intakes, carbohydrates significantly increased nitrogen retention as compared to fats. Much of the research regarding the effects of dietary composition has included hospitalized and depleted patients. Use of such subjects confounds the results with disease related variables. Inadequate dietary protein intake stimulates adaptations in order to conserve proteins and/or amino acids and maintain homeostasis. In rat skeletal muscle the capacity for intralysosomal proteolysis decreased by 55-75% and extracts of muscles showed 30-70 % lower activity of many lysosomal proteases including cathepsins B, H , and C as well as carboxypeptidases A and C (144). These findings coincide with Millward et al.(106) who found cathepsin B activity was reduced with dietary protein deficiency. In contrast, the capacity for Ca2+-induced activity increased by 66% as did the activity of the calpains (150-250%) (144). Goicoechea et al.(65) looked at the effects of dietary levels of protein on the activity of various enzymes in the liver that exhibit similar properties in skeletal 63 muscle. This study was performed on livers from normal, 5-days protein depleted, and 16 hours refed animals, representing basal, increased, and decreased levels of protein degradation respectively. The depleted livers contained an average of 5.5 to 3.1 times less calpastatin when compared to normal and refed livers. Calpain activity per whole liver did not change with depletion and refeeding. However, on the basis of enzyme /substrate ratio's (i.e. activity/mg liver soluble protein) the activity increased with depletion and decreased with refeeding. Since total calpain content decreased with depletion, these results were mainly explained by the large decrease in calpastatin. Thus, it seems that the major degradative processes in skeletal muscle respond in unique ways to protein deficiency which indicate the distinct roles each may serve in the turnover of muscle proteins. This discrepancy may be due to a dynamic metabolic environment that results in alterations of regulators and substrate selectivity. Starvation Prolonged starvation is characterized by mobilization of triglyceride stores and synthesis of glucose de novo coupled with suppressed glucose oxidation (140). This process acts to conserve glucose for obligatory glucose users such as the central nervous system To achieve glucose homeostasis, an early response is the mobilization of hepatic glycogen as a result of a decrease in circulating insulin/glucagon concentration ratio. Then increased lipolysis generates glycerol and non-esterified fatty acids which provides substrates for hepatic beta oxidation . The increase in fat oxidation stimulates hepatic gluconeogenesis and the released glycerol acts as a gluconeogenic precursor. Thus , the 64 oxidation of lipid fuels can be seen to exert a major role both on glucose oxidation and conservation (146). O'Donnel et al. (115) found that a prolonged fast promotes increases in the uptake of free fatty acids (FFA's) by skeletal muscle which serve as a source of peripheral fuel. A trademark of increased FFA uptake by skeletal muscle is the increased content of lipid deposits located within muscle cell (49). Enhanced FFA mobilization also occurs in response to increased presence of catecholamines, glucagon, and the glucocorticoid steroid hormones (137). The increase of FFA uptake into the muscle cell may potentially contribute to a loss of Ca2+ homeostasis. Messineo et al. (105) suggested that certain long chain fatty acids (LCFA's) prevent the uptake of C a 2 + by the SR when ATP availability is restricted. The presence of LCFA's promotes disruption within the SR membrane which decreases C a 2 + uptake potential and increases membrane permeability to C a 2 + efflux. Bindoli et al. (14) found similar SR disruptions. It is possible these types of perturbations to the SR may be physiologically relevant during conditions which are associated with an increase in FFA's. An extrapolation of these findings link changes in C a 2 + homeostasis with altered protein metabolism and increased calpain protease activity. Starvation also leads to mobilization of muscle proteins resulting from a decrease in protein synthesis and an increases in non-myofibrillar and myofibrillar protein breakdown (92,93,98,108). During periods of increased protein catabolism, it has been reported that myofibrillar proteins are affected more so than some non-myofibrillar proteins (94). The release of 3-methylhistidine (3MH) and urea nitrogen derived from degradation of myofibrillar proteins and muscle amino acids has been shown to be directly related to 65 hepatic gluconeogenesis found in the fasted state (64). Thus when animals and humans are subjected to adverse dietary conditions the result is a negative nitrogen (N) balance originating from striated muscle (62,97). In vivo, the evidence from isotopic studies and the measurement of excretion rates of 3MH indicates that during starvation, muscle protein is not degraded but progressively conserved until later stages of starvation (108). However, in vitro studies show that during the early stages of starvation, muscles from rats experience protein degradation (36). For example, fasted rat skeletal muscle isolated in situ showed increased protein breakdown and refeeding caused a decrease as measured by plasma tyrosine concentrations (138). Vasquez et. al.(150) demonstrated that a 7-day starvation period significantly increased the rate of leucine oxidation in obese female subjects. The oxidation of leucine and other branched chain amino acids (BCAA), which occurs primarily in skeletal muscles, has repetitively been shown to increase in both humans and animals with starvation (1,26). The rate of B C A A oxidation is equal to their rate of alpha-decarboxylation catalyzed by branched-chain alpha-ketoacid dehydrogenase (BCKA-DH). This enzyme exists in an inactive (phosphorylated) form and an active (dephosphorylated) form and is the rate limiting enzyme in the catabolic pathway of these amino acids (90). Studies in rats show that starvation results in an increase in the conversion of B C K A - D H from the inactive to the active form (118) which partially accounts for the increased B C A A oxidation seen in this condition. These findings correspond with changes that occur during the exercise state which show that the activity of B C K A - D H and the relative oxidation rates of leucine increase with increasing metabolic rates (90,159). This seems reasonable as 6 6 periods of starvation/fasting would expose skeletal muscle to many of the same metabolic and hormonal changes which occur in the exercise condition. These changes included altered glucose and lipid metabolism, disturbed C a 2 + homeostasis, insulin deficiency, and progressive proteolysis of skeletal muscle tissue. Presently the rate limiting event underlying increased myofibrillar protein degradation is unknown. Until the controlling factor(s) have been identified, determination of a critical protease is not possible. Studies investigating the effects of fasting on mRNA concentrations encoding u- and m-calpains, and Cathepsin D showed an increase in all protease mRNAs (76,77). These data implicated both lysosomal and non-lysosomal proteolytic systems in the increase of myofibrillar degradation with fasting. This coincides with other studies that have found increases in calpain activity with starvation and fasting (23,113) as well as that of cathepsin D (113). It is possible that degradation of specific myofibrillar proteins initiated by calpain increases the susceptibility of the remaining proteins of the contractile apparatus to lysosomal proteases which are capable of extensive myofibrillar degradation. (23). As a result of the dynamic metabolic environment found in vivo, it is difficult to identify all of the regulating factors involved in controlling myofibrillar degradation. If calpain proteolysis is the initiating event in the breakdown of myofibrillar proteins, then it is important to understand the elements that determine substrate selection and enhance the proteolysis of these substrates. The responses of different muscles to dietary deprivation may be partially detennined by the fiber type makeup of that particular muscle. Frayn and Maycock (55) investigated protein degradation with fasting and found that the extensor digitorum longus 67 muscle of fasted rats had a significantly higher rate of protein degradation (3.36 +/- .21 umol tyrosine released /gram protein ) compared to control rats (2.54 +/- .1 umol tyrosine released/gram protein) as measured by [3H]tyrosine. These observations were dependent on fiber type as there was significantly higher protein degradation exhibited in the extensor digitorum longus muscle (primarily fast twitch fibers) as compared to the soleus muscle (primarily slow twitch fibers). Similarly, Hian and Forsberg (77) reported that fasting caused a two fold increase in the calpain mRNA concentrations of the vastus lateralis muscle (containing 1% slow twitch fibers), whereas in the biceps femoris (containing 10% slow twitch fibers) the effect was much larger (76). It was suggested that the proteases gene expression during fasting could differ temporally among individual muscles. T A R G E T I N G AND SUSCEPTIBILITY Different proteins turnover with distinctive half-lives and the rate of degradation of a given protein can vary with changes in the metabolic state of the cell (125). A particular metabolic environment may promote protein breakdown, for example, ATP depletion has been shown to stimulate calpain dependent proteolysis of chick skeletal muscle proteins (50). Moreover, degradation of intracellular proteins to amino acids and small peptides is at least somewhat determined by the structural characteristics of protein substrates. Therefore, regulation of protein turnover may occur through modulation of the protein structure by intrinsic and extrinsic factors (19). The heterogeneity in the half lives of different proteins in a cell indicates that some proteins are more susceptible to degradation than others. Abnormal protein structures are degraded more rapidly than 68 normal proteins indicating the importance of structure as a determinant for proteolysis. One way in which highly selective protein degradation could be achieved within a cell is by selection of individual proteins for degradation by some kind of marking reaction (125). Covalent Modification Ctovalent-rnarking reactions could provide the mechanism for metabolic control of degradation and therefore account for the heterogeneity of protein half-lives. Covalent modification of proteins may mark them for degradation by rendering them more susceptible to proteolytic attack (125). A native protein may be relatively resistant to proteolytic attack and therefore protected from ^discriminate degradation. Whereas a modified protein may be rapidly degraded. Therefore degradation could occur in a two-step mechanism in which the protein is first modified in some way that allows recognition by intracellular proteases (125). There are several types of covalent modification including phosphorylation, oxidation and formation disulfides, and ubiquitin conjugation. Phosphorylation Reversible phosphorylation of serine and threonine residues is an important mechanism for controlling activity of enzymes in eukaryotic cells and can cause conformational changes which may also affect the susceptibility of a protein to proteolytic attack (125). In vivo inactivation of various enzymes by way of phosphorylation has been shown to be followed by degradation. Hemmings (73) established that phosphorylation was involved in the degradation of yeast NAD-dependent glutamate D H . The phosphorylated form of 3-hydroxy-3-methglutaryl-CoA from rat liver is preferentially 69 degraded by calpain, a Ca2+-dependent protease (117). Toyo-Oka (147) also demonstrated that phosphorylation of the troponins, regulatory proteins in striated muscle myofibrils, by cyclic AMP-dependent protein kinase (PKA) increases their degradation by this protease. In contrast, Di Lisa et al. (40) showed that PKA phosphorylation decreased the rate of troponin proteolysis by calpain but the rate was enhanced when phosphorylation was catalyzed by protein kinase C. Litersky and Johnson (95) examined the effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on degradation of the microtubule associated protein, tau, by calpain. Tau is a major antigenic component of paired helical filaments (PHF's) which are present in neurofibrillary tangles (NFT's) found in abundance in the brains of patients with Alzheimer's disease. It is thought that abnormally phosphorylated species of tau in susceptible neurons precede the formation of NFT's. When purified bovine brain tau was phosphorylated by cAMP-PK its proteolysis by calpain was significantly inhibited compared to untreated tau. Therefore, it is possible that phosphorylation plays an important role in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease. Actin-binding protein (ABP) has been shown to undergo hydrolysis by calpain during platelet aggregation (54) In vitro this cleavage causes irreversible loss in the ability of ABP to cross-link actin filaments into cytoskeletal networks (148). Phosphorylation of ABP by cAMP dependent protein kinase has been shown in to increase its resistance to proteolysis by calpain both in vitro (163) and in situ (29). Pant (116) found that 70 degradation of dephosphorylated neurofilament proteins (NFP's) by calpain occurred at a higher rate and to a greater extent than that of the phosphorylated NFP's. Thus it appears that dephosphorylation of NFP's can increase their sensitivity to calpain degradation Ubiquitin Conjugation Ubiquitin is a protein found in eukaryotic cells either free or covalently joined to a variety of cytoplasmic and nuclear proteins (52). It is thought that ubiquitin acts as a signal for attack by proteases and ubiquitin-protein conjugates are intermediates in ubiquitin and ATP-dependent protein degradation. Ubiquitin conjugation represents a unique form of marking specific for denatured or abnormal substrates and may have an important physiological function in the degradation of abnormal proteins which can accumulate during stress (125). Studied show that heat shock perturbs the ubiquitin system and mactivation/overloading of the ubiquitin system plays a role in triggering aspects of the stress response. In fact the ubiquitin dependent proteolytic pathway and the heat shock response may be complementary systems designed to prevent the cellular damage that abnormal proteins could inflict (52). The ubiquitin dependent pathway is non-lysosomal as is most of the highly selective turnover of intracellular proteins under normal conditions. Biological and genetic evidence indicates that conjugation of ubiquitin to short-lived proteins is essential for their selective degradation in vivo (125). Oxidation and Formation of Disulphides Oxidation of thiol groups and formation of mixed disulphides may mark a number of different proteins for degradation. In muscle, the susceptibility of aldolase is increased with oxidation possibly due to formation of mixed disulphides which decreases its 71 conformational stability (103). Murphy and Kehrer (111) examined the pectoralis major muscle from genetically dystrophic chickens and found a higher content of protein carbonyl groups and loss or oxidation of protein thiol groups. These changes were thought to be physiologically relevant estimates of oxidative stress and may reflect significant damage to cellular proteins in this disease. Nagy and Samaha (114) demonstrated a 10 fold increase calpain activity within dystrophic tissue. Furthermore, digestion of SR membrane proteins by calpain causes alterations in C a 2 + ion transfer which is considered to promote the progressive stages of degradation. In the diabetic state, the more oxidized cardiac fibers exhibit increased calpain degradation rates. These fibers have a lower sulfhydryl group reactivity and content thus making them more vulnerable substrates for calpain (10). Another study showed that physiologically induced modifications of myofibrillar proteins due to exercise made them more susceptible to calpain cleavage (8). It was suggested that the observed accelerated degradation rates were the result of protein substrate modifications that occurred with exercise such as altered oxidation/reduction status. Widespread interest in the regulation of E.coli glutamine synthetase (GS) activity has resulted in proposal that mixed function oxidation of proteins may be a mechanism for marking GS for degradation. Oxidized GS is degraded more rapidly than the native enzyme and is preferentially degraded by several mammalian proteases from rat and mouse liver including calpain I and II, and cathepsin D. In addition, mixed function oxidation of rabbit muscle enolase, pyruvate kinase, creatine kinase, and phosphoglycerate kinase renders them more susceptible to degradation (125). 72 Native enzymes which are relatively resistant to proteolytic attack may be made more susceptible to degradation by covalent modification. Different modifications mark different types of proteins for degradation but a given type of covalent modification can make a particular protein susceptible to proteolysis by any one of several different proteases. And each protease may degrade several different kinds of modified proteins. Therefore susceptibility of proteins to degradation seems to be determined by features of the substrate protein structure not by the peptide bond specificity of the intracellular proteases (125). This argument is supported by the fact that any one of several different types of proteases (having different bond specificities) can extensively degrade oxidized GS. Substrate Characteristics The structural features which determine susceptibility of proteins to proteolytic attack still remain largely unknown. Some structural features of soluble proteins are closely associated with their rates of degradation such as large or acidic proteins degrade faster than small or basic proteins (42,43). Some other considerations can be found in correlations between physiochemical properties of soluble mammalian proteins and their half-lives in vivo. Unfortunately the half-life of a given protein varies in different tissues and conditions resulting in conflicting studies with respect to subunit molecular weight, isoelectric point, and hydrophobicity (44,99,126). Since altered proteins degraded faster than native proteins (48), another possibilities would be to look at changes in a protein which occur as a result of marking 73 For example, changes in GS as a result of oxidation include a decrease in subunit interactions and thermal stability. Also oxidized GS molecules tend to aggregate which may be a result of increased surface hydrophobicity. It has been suggested that increases in thermal liability and surface hydrophobicity are related to proteolytic susceptibility but results conflict (125). PEST sequences A unique feature of many calpain substrates is the presence of certain amino acid sequences that seem to make them recognizable to this protease. These amino acid groups are called "PEST" sequences and calpain cleavage seems to occur at or near these regions. Initial studies examined the amino acid sequences of 10 short lived proteins and found that common to each protein was at least one region rich in proline (P), glutamic acid (E), aspartic acid (D)* serine (S) and threonine (T), now known as "PEST" regions. Similar analysis of another group of 35 longer lived proteins showed that only 15 of these contained these so called PEST regions (127). The strength of a PEST region can be evaluated by its PEST score. Basically the two types of PEST regions that exist are "weak" and "strong". It was also revealed that 9 of the 10 short lived proteins contained at least one strong PEST region whereas only 3 of the 35 stable proteins had a strong score. It was suggested that the presence of PEST regions rendered proteins susceptible to rapid degradation. In addition, since the E and D residues in the PEST sequence are both negatively charged and the S and T residues are potentially phosphorylatable, phosphorylation of PEST sequences could provide very negatively charged regions which may bind Ca 2 + . As a consequence of the localized increase in C a 2 + concentration, calpain 74 proteolysis could be activated (127). PEST regions are also very hydrophilic and are thought to form surface loops on the protein making this area easily accessible to calpain (122). Many calmodulin binding proteins, which are known to be substrates for calpain, generally showed the presence of PEST like regions located at or near the site of cleavage by calpain (154). C O N C L U S I O N The two pathways involved in skeletal muscle protein breakdown could serve distinct but complementary functions. The non-lysosomal pathway appears to be responsible for initiation of myofibrillar breakdown via selective and limited fragmentation. More extensive non-selective degradation of the resulting polypeptides may then follow in the lysosomes. The factors that act to enhance muscle protein proteolysis in various conditions have not been fully elucidated but several commonalties are evident. Such similarities suggest a dominant route for catabolism of muscle proteins during periods of accelerated proteolysis. Altered energy metabolism associated with different physiological states has been identified as a major stimulus for altered rates of protein degradation. Prolonged exercise, starvation, and altered dietary composition have all been shown to affect substrate oxidation with a shift from glucose catabolism to increased oxidation of fatty acids. In addition, conditions that exhibit perturbed carbohydrate metabolism are often characterized by insulin deficiency. Prolonged exercise, starvation, and diabetes all display hypomsulinemia with a resultant increase in gluconeogenesis and progressive proteolysis 75 of skeletal muscle tissue (53). Despite the fact that C a 2 + dependent proteolysis is not decreased by the presence of insulin (83), calpain activity is increased in a variety of insulin-deficient states (10,22,24). The mobilization of free fatty acids during exercise is promoted by a significant decrease in circulating insulin levels. This creates a similar hormonal environment as other insulin-deficient conditions. An increased reliance on free fatty acids for metabolic energy production may eventually contribute to a loss of C a 2 + homeostasis (105). It is important to understand role of calcium in regulating protein turnover in muscle since its'effects could be an important link between endocrine factors, nutrition, and activity to alterations in protein metabolism (162). C a 2 + has been shown to stimulate overall proteolysis in skeletal muscle and it is possible that the non-lysosomal proteolytic systems may be responsible (83). Calpain is known to proteolyze myofiteillar proteins and is activated in a variety of conditions that exhibit increased muscle protein breakdown. A crucial problem in the study of protein metabolism is the physiological significance of different protein turnover rates for functionally related myofibrillar proteins (136). Currently, most studies monitoring protein degradation fail to distinguish the selective breakdown of myofibrillar versus non-myofibrillar proteins and the overall contribution of each proteolytic system (5). Furthermore the mechanisms responsible for selective degradation are poorly understood (41) and it is important to clarify the events and conditions necessary to facilitate this process. Since a particular and specific loss of myofibrillar proteins occurs under calpain action, a predictable loss of selected muscle proteins would occur under 76 conditions of altered energy metabolism. In addition, the rates of protein turnover in different skeletal muscles vary according to fiber type composition (61). Therefore, it is reasonable to assume that the response of different muscle types to metabolic alterations would not be homogeneous. 77 REFERENCES 1. Adibi, S. A., E . L . Morse, and P. M . Amin. 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Comm. 151: 355-360,1988(Abstract) 91 APPENDIX B: M E T H O D O L O G Y Animals; Male Wistar rats (350g) were sacrificed by a lethal intraperatoneal injection of euthanol (pentobarbital sodium) (n=9). The gastrocnemius muscle and heart were dissected, partitioned into samples, trimmed of all visible fat and connective tissue, and then placed in liquid nitrogen. Al l samples were stored at -70°C. Myofibril Isolation: Muscle samples were homogenized in a borate-KCl buffer containing 39mM sodium borate, 25 mM KC1, 1 mM phenylmethylsulfonyl floride and 5mM E G T A . The homogenate was then centrifuged at 13, 000 rpm for 15 minutes (Hermle 360Z, rotor VO2805) and the supernatant discarded. Pelleted material was washed twice with a 1% Triton X-100 for removal of membrane-bound proteins and resuspended in a 0.1 M KC1,50 mM Tris (pH 7.0), and 1 mM DTT. The homogenate was recentrifuged between each washing. The myofibrillar pellet was then washed in an LSB containing 0.1 M KG1, 2 mM MgCl 2 , 2 mM E G T A , 10 mM Tris-maleate (pH 7.0), and 1 mM DTT. All procedures were performed at 4°C. Measurement of protein concentration: The protein concentration of the myofibril isolations was established using the method of Lowry et al. (1951). All protein concentrations were expressed relative to wet weight muscle. Proteolytic digestion; Myofibrils preparations were digested over 0 to 30 minutes at a ratio of 40 ug myofibrillar complexes to 1.5U m-calpain activity. AVPreparation of Sigma Calpain (25 U) Final Concentration D T T (200mM) 50ul 2mM Tris-OH (250mM, pH 7.4) 4.9 ml 20mM 93 E G T A (lOOrnM) Total Volume 5M 5ml ImM Made up in test tube and vortexed. Then added directly into Sigma botde and swirled gently to avoid air/liquid disturbance. B) Caseinolysis Calpain Tris (250mM. pH7.4) Ca 2 + (50 irJvf) D T T (200mM) Casein (lOmg/ml) Oul 325 ul 50 ul 25 ul 100 ul 10 ul 315 ul 50 ul 25 ul 100 ul 50 ul 275 ul 50 ul 25 ul 100 ul The calpain was added in the last step. Samples incubated at 37°C for 60 minutes. After 30 minutes, the digest was quenched by the addition of 500ul of ice cold 5% T C A and put on ice for 5-10 minutes. Spin at 14 000 rpm for 10 minutes. Activity was measured spectrophotometrically in photometric mode 280nm (program #7) using 1ml quartz cuvettes. Absorbance changes (i.e. increases) with an increased number of soluble digested fragments. Calculations: calpain (lOulV blank X 1000 = units activity 10 calpain (50 ul)- blank X 1000 = units activity 50 The average of the two values estimates the total activity of the stock calpain. 94 O Calpain Digests In each sample, a solution containing a final concentration of; 1.5 U calpain (added last), 40 ug myofibrillar protein, 5 mM Ca 2 + , 10 mM DTT, 20 mM Tris (pH 7.4) to make up a final volume of 50 ul was prepared. Conditions are designed to result in a 50% loss of a-actinin in a 30 minute digest Digestions were conducted over a time course of 0-30 minutes at 37°C and were terminated at selected time points with lOul of 200ug/ml leupeptin. The digested samples were spun at 12 500 rpm for 15 min in a Hermle Z360K centrifuge. The supernatant was wazzu aspirated and the samples were resuspended in 250 ul of 5mM Tris (pH 8.0), centrifuged and once again the supernatant aspirated. The myofibrillar proteins were solubilised by mixing the digested samples with an equal volume of a sample buffer containing: 0.062 M Tris, 4% SDS, 20% glycerol. 0.02% bromophenol blue, 1 mM D T T and incubated for 20 min at 50°C. To identify individual proteins the samples were run on SDS-polyacrylamide slab gel electrophoresis using the Laemmli buffer system Aliquots (40ug/well) were electrophoresed on a 5-15% linear gel at 20mA for approximately 8 hours. Molecular weights were estimated using molecular weight standards (Sigma). D^ Preparation of 5-15% Gels for SDS PAGE The following 5% and 15% working solutions were 98 prepared from stock solutions and used to assemble the 1.5mm thick gradient gels. | 5% 15% 95 acrylamide monomer (30%) 3.75ml 11.25ml 1.5MTris-OH (pH8.8) 5.63 5.63 10% SDS 225 ul 225 ul ddH 2 0 12.83 n/a glycerol 5.39 n/a amm. persulfate (10%) 67.5 13.5 T E M E D 2A 1A Total Volume 22.5 ml 22.5 ml The following 3% working solution was prepared from stock solutions and used to assemble the stacking gels. 3% acrylamide monomer (30%) 2ml 250mM Tris-OH + 10% SDS 10ml ddH 2 0 8ml amm. persulfate (10%) 500ul T E M E D 5_ul Total Volume 20.505ml Each working solution was degassed for 15 minutes before commencement of gel polymerization. Gel polymerization was initiated by the addition of T E M E D and Ammonium Persulphate to the above solutions and the gels were poured immediately after 96 their addition. The gradient gels were cast between two glass plates in a Bio-Rad gel assembly using a linear gradient maker. After pouring, the gel was overlaid with 200ul H 2 0 saturated isobutanol and allowed to polymerize for at least 6 hours. After polymerization, the stacking gel cavity between the plated was rinsed with H 2 0 and then dried using filter paper to absorb excess moisture. With the well comb in place the stacking gel was poured immediately after polymerization was initiated. The stacking gels were allowed to set for about 3-4 hours. Protein samples were solubilised by the addition of 40 ul of 2X sample buffer (after completion of calpain digests) and incubated for 20 minutes at 50°C. During this time polymerized gels were mounted on the central cooling core of the SDS PAGE unit Once the upper chamber was partially filled with the Tank buffer, the samples were overlaid into each well and electrophoresis was started. Once electrophoresis was complete, the gels were stained in 0.025% Coomassie Blue, 9% acetic acid, and 45% methanol solution overnight. Following this, the gels were put in Destain 1 and Destain 2 in succession until stain removed from background. The gels were then submersed in 70% methanol and mounted between 2 sheets of cellophane clamped within a perspex frame until completely dried. Quantification of SDS P A G E gels A) Pre-image analysis Dried gels were scanned with Adobe Photoshop 2.01 software™ and Abaton Scan 300/color scanner. The images were quantified via IPLab Gel™ 1.5 software. The gel 97 image was divided into regions for analysis by separating the protein bands of interest from the background segments. Once all the protein bands were defined, density analysis was performed on all lanes of each gel image. B) Density Analysis The data for analysis was stored as bytes and picture elements as pixels (1 byte per pixel).The program assigned pixel values between 0-255, higher numbers indicating a higher density. The density of various segments was calculated as the volume of pixel values within each segment. The following formula was used to compute density measurements1; Volume= Z (Ly)- N*B N=number of pixels in region Ixy=pixel values within a region B=background value used O Internal Standard The myofibrillar protein, actin, which is not degraded by calpain activity, was used as the standard for each lane of a gel. The density value for each protein of interest was then measured as a ratio against the density value of the actin present in that lane. The ratio value in the time 0 lane was set as 100% protein for each gel. The ratios from the following lanes (30sec-30min) were then calculated as a percentage of the time 0 ratio. 1 Signal Analytics Corporation. IPLab Gel User's Guide. 1994. 98 D) Reaction Rate Equation and Curve Fitting The percentage values from each protein were taken and fitted to a single exponential decay equation via non-linear regression using Grafit™ 3.0 software. Non-linear regression calculates the best-fit parameter by a series of iterations assessed by a reduced chi-squared value. The raw data was smoothed via a binomial technique2. The smoothed value, JC, , is defined as Xi = Xi.i+2xi+xM 4 Where JC,_I and are the previous and subsequent values in the data column. Once the date points were fitted to the single exponential curve, the rate constant,, and overall slope were calculated as best-fit values as depicted in the following schematic. 2 Leatherbarrow .R.J.. Grafit User's Guide. Erithacus Software Limited. 1992. 99 A standard error value was provided with each k of these parameters as calculated by the matrix inversion method. These estimated standard errors were generally not greater than 10% of the parameter values in order to maintain confidence in the calculated parameters. 100 A P P E N D I X C : T R O U B L E S H O O T I N G 101 During data collection, the migration and digestion pattern of the 5-15% SDS-PAGE gels were as depicted in Figure 9. Upon receiving a new batch of isolated and purified (<95%) m-calpain this typical pattern changed radically and it appeared that all the proteins within the myofibril were either being cleaved or altered in a way that prevented them from entering the gel (Figure 10). Each step of the myobrillar isolation procedure was taken into account and the proteins visualized normally (see Time 0 Figure 9), however the atypical digestion pattern remained as that seen in Figure 10. It was assumed that a component of the digestion procedure was causing the alteration. When the myofibrils were suspended in the components of the digestion buffer the migration and visualization of the myofibrillar proteins was normal (Figurell). It was then suspected that either the components of the buffer in which the calpain was suspended or alterations in the calcium levels of the distilled water produced in the laboratory may be illiciting the results. A water analysis report revealled that based on atomic absorption spectrophotometry, there were no detectable levels of calcium present in the distilled water (<0.01 ppm). Furthermore, the lanes 3,4, and 5 in Figure 12 show normal visualization of the myofibrils in conditions prior to combination with the calpain buffer (lane 3) and subseqent to the addition of the buffer at two different levels of the chelating agent, E G T A (lane 4 and 5). These two results indicated that calcium levels were not a contributing factor to the massive proteolysis witnessed with the addition of the new batch of m-calpain. Lanes 6 and 11 depict the unusual migration and visualization of myofibrils that have been digested with the new batch of m-calpain (1.5U activity) at two different levels of free Ca 2 + . Lanes 7-10 and 12-15 represent the resulting pellets and supematants of succesive washings after the 102 digestion procedures. Since all of the supernatant lanes (8,10,13,15) remained empty, it was assumed that no soluble contaminants were present in the preparations. Furthermore, the migration of the pelleted fractions (Lanes 7, 9,12, and 15) remained distorted indicating the contaminate remained present in the fractions and/or the protein fragments were irreversiblely altered. A result that consistently occured coincident to the altered migration and visualization patterns was the appearance of an unusual band in the lanes in which 1.5U of m-calpain had been loaded (see arrows indicating band in Figure 10 and Figure 12). In addition, prior to receiving the new batch of purified m-calpain the myofbrils had appeared normal. Therfore, it was concluded that there was a contaminate present in the m-calpain that was responsibile for the anomalous migration and visualization patterns of the digested myofbrils. 103 Figure 10: TYPICAL DEGRADATION PATTERN OF FAST MYOFIBRILLAR COMPLEXES B Y 1.5U m-CALPAIN. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue. Molecular mass standards are run in the far left lane and lane A depicts 1.5U m-calpain. Note gradual fading of substrate protein bands while myosin heavy chain and actin remain intact. 104 Time (min) A 0 0.5 1 5 15 30 105 Figure 11: RESULTS OF A DIGEST PERFORMED WITH CONTAMINATED m-CALPAIN. Purified myofibrils (40ug) are digested at 37°C with 1.5U m-calpain and stopped at various time points with leupeptin (200ug/ml). Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue. Molecular mass standards are run in the far left lane and lane A depicts 1.5U m-calpain. Note in Lane A the appearance of an unusual band. Over the time course of 0 to 30 minutes all myofibrillar protein bands (including myosin heavy chain and actin) gradually diminish. 106 Time (min) 5 10 1 5 20 25 30 Figure 12: 60 ug OF PURIFIED FAST M U S C L E MYOFIBRIL COMPLEXES COMBINED WITH COMPONENTS OF CALPAIN DIGESTION BUFFER. Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue. Lane 1; 60ug myofibrils + 5mM Ca2+,10mM DTT, 20mM Tris (pH 7.4) Lane 2; 60ug myofibrils + 5mM Ca2+, 20mM Tris (pH 7.4) Lane 3; 60ug myofibrils + lOmM DTT, 20mM Tris (pH 7.4) Lane 4; 60ug myofibrils + 20mM Tris (pH 7.4) Note normal migration pattern and visualization of myofibrillar proteins. 108 Lane 1 2 3 4 Figure 13: 60 ug PURIFIED FAST MYOFIBRIL COMPLEXES VISUALIZED A T SUCCESSIVE STAGES OF DIGEST PROCEDURE A T T W O DIFFERENT L E V E L S OF F R E E C A 2 + . Individual proteins visualized by 5-15% SDS-PAGE gradient gels and stained with Coomassie Brilliant Blue. Lane land 2; 1.5U contaminated m-calpain. Note unusual migration when compared to 60ug fast myofibrils in Low Salt Buffer (LSB). Lane 3; 60ug fast myofibrils in LSB. Lane 4; 60ug fast myofibrils + 5mM E G T A calpain buffer (2mM DTT, 20 mM Tris-O H [pH 7.4], 5mM EGTA). Lane 5; 60ug fast myofibrils + l m M E G T A calpain buffer Lane 6; Suspension of 60ug fast myofibrils with calpain digest components after a 20 minute digest with contaminated m-calpain in l m M E G T A conditions. Lane 7; Pellet of suspension from Lane 6. Lane 8; Supernatant from Lane 6. Lane 9; Pellet from Lane 7 washed with 5mM Tris-HCl (pH 8.0) and recentrifuged. Lane 10; Supernatant from Lane 9. Lane 11; Suspension of 60ug fast myofibrils with calpain digest components after a 20 minute digest with contaminated m-calpain in 5mM E G T A conditions. Lane 12; Pellet of suspension from Lane 11. Lane 13; Supernatant from Lane 12. Lane 14; Pellet from Lane 12 washed with 5mM Tris-HCl (pH 8.0) and recentrifuged. Lane 15; Supernatant from Lane 14. Note: Myofibrils visualized normally until combined with contaminated m-calpain. The unusual migration pattern did not change when more C a 2 + was chelated in the 5mM E G T A conditions (Lane 11-15). 110 Lane 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 m APPENDIX D: T A B L E S T A B L E 1: P E R C E N T A G E O F M Y O F I B R I L L A R C O M P L E X E D P R O T E I N R E M A I N I N G F O L L O W I N G A 30 M I N U T E CALPAIN-DIGESTION IN F A S T M U S C L E TISSUE (individual raw data) C-protein a-actinin desmin tropomvosin sample 1 36.57 52.40 0.00 15.16 2 31.99 48.09 0.00 17.10 3 45.70 56.50 0.00 17.64 4 51.28 0.00 11.77 5 51.28 0.00 6 53.59 7 49.89 mean 38.09 50.92 Owazzu .00 15.42 std dev 6. 3.66 0.00 2.65 T A B L E 2: P E R C E N T A G E O F M Y O F I B R I L L A R C O M P L E X E D P R O T E I N R E M A I N I N G F O L L O W I N G A 30 M I N U T E CALPAIN-DIGESTION IN C A R D I A C M U S C L E TISSUE (individual raw data) C-protein a-actinin desmin troponin-T samDle 1 19.04 23.18 0.00 74.70 2 17.68 26.87 0.00 69.38 3 20.11 23.01 0.00 74.16 4 20.40 27.70 0.00 56.40 5 20.25 15.98 0.00 63.99 mean 19.49 23.34 0.00 67.72 stddev 1.15 4.63 0.00 7.66 113 T A B L E 3: R A T E O F LOSS O F MYOFIBRILLAR C O M P L E X E D PROTEIN O V E R A 30 MINUTE CALPAIN-DIGESTION IN FAST M U S C L E TISSUE (individual raw data) C-protein a-actinin desmin tropomvosin samole 1 0.81 0.20 3.95 1.05 2 1.20 0.14 3.18 1.14 3 0.88 0.45 4.77 1.03 4 0.28 4.06 1.34 5 0.55 5.52 6 0.35 7 0.47 mean 0.96 0.35 4.30 1.14 stddev 0.21 0.15 0.89 0.14 T A B L E 4: R A T E O F LOSS O F MYOFIBRILLAR C O M P L E X E D PROTEIN O V E R A 30 MINUTE CALPAIN-DIGESTION IN CARDIAC M U S C L E TISSUE (individual raw data) C-protein a-actinin desmin troponin-T sample 1 0.19 0.07 4.57 0.09 2 0.26 0.08 3.89 0.08 3 0.30 0.08 4.86 0.06 4 0.17 0.07 4.38 0.13 5 0.22 0.07 5.21 0.11 mean 0.23 .07 4.58 0.09 stddev 0.05 .00 .50 0.03 114 T A B L E 5: P E R C E N T A G E O F MYOFIBRILLAR C O M P L E X E D PROTEIN REMAINING A T SPECIFIC T I M E POINTS O V E R A 30 MINUTE CALPAIN-DIGESTION IN FAST M U S C L E TISSUE (INDIVIDUAL RAW DATA) Time-0 05 I 5 15. 2Q sample 1 95 79 59 45 37 35 2 93 70 47 37 33 30 3 92 75 63 59 53 42 4 26 81 £3. 54 51 44 mean 93.81 76.06 57.94 48.50 43.19 37.69 std dev 1.82 4.98 7.70 9.82 9.83 6.37 a - A L i iwir • Time-0 0 5 1 5 15 2Q sample 1 100 91 88 69 57 51 2 100 94 88 73 55 47 3 100 86 80 65 59 55 4 100 79 71 75 59 44 5 100 97 88 63 54 51 6 100 97 81 53 40 64 7 100 98 87 57 50 51 8 100 29 70 42 44 AA mean 100 90.13 81.63 62.75 52.25 50.87 std dev 0.00 7.90 7.58 9.82 7.05 6.53 DESMIN: Time-0 &5 1 5 sample 1 100 13 5 0 2 100 18 10 0 3 100 9 2 0 4 100 12 6 0 5 IfjQ 6. 3. Q mean 100 11.60 5.20 0.00 std dev 0.00 5.12 3.59 0.00 115 Time-0 (L5_ 1 5. 15. 2Q samole 1 100 72 40 20 15 12 2 100 77 34 31 8 15 3 100 61 50 23. 14 16 4 JCjQ 59. 32 12 12 mean 100 67.25 39.25 24.67 12.25 13.75 std dev 0.00 8.66 7.80 5.69 3.10 2.06 116 T A B L E 6: P E R C E N T A G E O F M Y O F I B R I L L A R C O M P L E X E D P R O T E I N R E M A I N I N G A T SPECIFIC T I M E POINTS O V E R A 30 M I N U T E C A L P A I N -DIGESTION IN C A R D I A C M U S C L E TISSUE (individual raw data) C - P R O T E I N : Time-0 05 I 5 15 30 sample 1 93 76 61 46 29 18 2 95 83 65 42 24 17 3 92 72 56 41 26 17 4 93 75 60 47 32 20 5 92 25 60. 44 29. 20 mean 92.95 76.35 60.00 44.00 27.85 18.30 std dev 1.63 4.48 3.68 2.88 3.69 1.79 a -ACTINTN: Time-0 0.5 1 5 15 30 sample 1 100 93 91 82 47 28 2 100 96 90 79 50 30 3 100 90 84 80 44 26 4 100 88 83 77 53 34 5 1QQ 91 89. & 4Q 21 mean 100 92.80 87.4 80.80 46.80 27.80 std dev 0.00 4.43 3.51 3.87 5.85 5.56 DESMIN: Time-0 1 5 sample 1 100 10 2 0 2 100 14 3 0 3 100 8 5 0 4 100 11 2 0 5 100 2 3 Q mean 100 10.00 3.00 0.00 stddev 0.00 3.16 1.26 0.00 117 TROPONIN-T: Time-0 &5 1 5 15 2Q sample 1 100 100 96 91 84 76 2 100 99 96 90 81 73 3 100 99 96 93 86 78 4 100 98 91 79 66 73 5 91 94 £ 2 £3. mean 99.50 97.55 93.45 87.00 79.25 72.90 stddev 0.78 2.26 3.68 6.45 10.31 5.47 118 T A B L E 7: P R O T E I N Y I E L D O F M Y O F I B R I L L A R ISOLATIONS F R O M C A R D I A C AND F A S T M U S C L E TISSUE (individual raw data) sample F A S T (me/g) C A R D I A C (mg/g) 1 97.2 58.8 2 99.0 50.2 3 104.6 62.4 4 107.8 62.0 5 86.6 52.2 6 93.8 59.6 7 10.1.6 79.0 8 102.4 72.2 9 92.8 70.0 mean 98.42 62.93 std dev 6.58 9.36 119 T A B L E 8: S U M M A R Y O F S T A T I S T I C A L A N A L Y S E S ; 2X3 Anova, 1-way Anovas with Student-Newman-Kuelsd Post Hoc, T-test for independent samples 2X4 Anova: — Effect Dependent Variable protein (p) tissue (t) o X t Rate p=.000 p=.001 p=.004 2X3 Anova: . Effect Dependent Variables protein (p) tissue (t) o X t Offset (% at 30 min) Rate p=.000 p=.000 p=.268 P=.147 p=000 p=.060 1-way Anovas with Student-Newman-Keuls Post Hoc: Offset hv Fast CF proh=.QQffl tropomyosin C-protein a-actinin tropomyosin C-protein a-actinin Offset hv Cardiar-rF prob=000) C-protei in a-actinm *=p<0.05 troponin-T C-protein a-actinin troponin-T *=p<0.05 a-actinin C-protein tropomyosin desmin a-actinin C-protein tropomyosin desmin * * * * Rate hv Cardiac fF proh=.000) 120 a-actinin troponin-T C-protein desmin a-actinin troponin-T C-protein desmin * * *=P<0.05 a-actinin C-protein tropomyosin a-actinin C-protein tropomyosin * * *=P<0.05 Rate by Cardiac CF prob=.0Q0) a-actinin troponin-T C-protein a-actinin troponin-T C-protein *=P<0.05 T-TESTS FOR INDEPENDENT SAMPLES: Desmin RATE fast vs. cardiac p=.232 C-protein OFFSET (% AT 30 MIN) RATE fast vs cardiac p=.023* p=.037* *=p<0.05 a-actinin OFFSET (% AT 30 MIN) RATE fast vs cardiac p=.727 p=.004* *=P<0.05 121 * * * A N A L Y S I S O F V A R I A N C E RATE by PROTEINS TISSUE UNIQUE sums o f squares A l l e f f e c t s e n t e r e d s i m u l t a n e o u s l y Sum o f Mean S i g Source o f V a r i a t i o n Squares DF Square F o f F Main E f f e c t s 121. 199 4 30.300 211 997 .000 PROTEINS 118. 852 3 39.617 277 189 .000 TISSUE 1. 816 1 1.816 12 709 .001 2-Way I n t e r a c t i o n s 2. 378 3 .793 5 547 .004 PROTEINS TISSUE 2. 378 3 .793 5 547 .004 E x p l a i n e d 125. 310 7 17.901 125 251 .000 R e s i d u a l 4 . 431 31 .143 T o t a l 129 . 741 38 3.414 39 cases were p r o c e s s e d . 0 cases (.0 pet) were m i s s i n g . 122 16 Dec 96 SPSS f o r MS WINDOWS Re lease 6.1 Page 3 * * * A N A L Y S I S O F V A R I A N C E RATE by PROTEINS TISSUE UNIQUE sums o f squares A l l e f f e c t s e n t e r e d s i m u l t a n e o u s l y Sum o f Mean S i g Source o f V a r i a t i o n Squares DF Square F o f F Main E f f e c t s 110. 467 3 36.822 202 .344 .000 PROTEINS 109. 965 2 54.982 302 .137 .000 TISSUE • 408 1 .408 2 .241 .147 2-Way I n t e r a c t i o n s 1. 150 2 .575 3 .160 .060 PROTEINS TISSUE 1. 150 2 .575 3 .160 .060 E x p l a i n e d 113. 730 5 22.746 124 .993 .000 R e s i d u a l 4. 367 24 .182 T o t a l 118 . 097 29 4.072 39 cases were p r o c e s s e d . 9 cases (23.1 pet) were m i s s i n g . 123 O N E W A Y V a r i a b l e RATE1 By V a r i a b l e CARDIAC A n a l y s i s o f V a r i a n c e Sum o f Mean F F Source D . F . Squares Squares R a t i o P r o b . Between Groups 3 74.4285 24.8095 393.3426 .0000 W i t h i n Groups 16 1.0092 .0631 T o t a l 19 75.4377 124 16 Dec 96 SPSS f o r MS WINDOWS Re lease 6.1 Page 5 O N E W A Y V a r i a b l e RATE1 By V a r i a b l e CARDIAC M u l t i p l e Range T e s t s : Student-Newman-Keuls t e s t w i t h s i g n i f i c a n c e l e v e l .050 The d i f f e r e n c e between two means i s s i g n i f i c a n t i f MEAN(J)-MEAN(I) >= .1776 * RANGE * SQRT(1/N(I) + 1 /N(J) ) w i t h the f o l l o w i n g v a l u e ( s ) f o r RANGE: Step 2 3 4 RANGE 3.00 3.64 4.04 (*) I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s which are shown i n the lower t r i a n g l e G G G G r r r r P P P P 2 4 1 3 Mean CARDIAC .0731 Grp 2 .0932 Grp 4 .2287 Grp 1 4.5846 Grp 3 * * * Homogeneous Subsets ( h i g h e s t and lowes t means are not s i g n i f i c a n t l y d i f f e r e n t ) Subset 1 Group Grp 2 Grp 4 Grp 1 Mean .0731 .0932 .2287 Subset 2 Group Grp 3 Mean 4.5846 125 O N E W A Y V a r i a b l e RATE2 By V a r i a b l e FASTTW A n a l y s i s o f V a r i a n c e Sum o f Mean F F Source D . F . Squares Squares R a t i o P r o b . Between Groups 3 49.2690 16.4230 71.9990 .0000 W i t h i n Groups 15 3.4215 .2281 T o t a l 18 52.6905 126 16 Dec 96 SPSS f o r MS WINDOWS Re lease 6.1 Page 7 O N E W A Y V a r i a b l e RATE2 By V a r i a b l e FASTTW M u l t i p l e Range T e s t s : Student-Newman-Keuls t e s t w i t h s i g n i f i c a n c e l e v e l .050 The d i f f e r e n c e between two means i s s i g n i f i c a n t i f MEAN(J)-MEAN(I) >= .3377 * RANGE * SQRT(1/N(I) + 1 /N(J) ) w i t h the f o l l o w i n g v a l u e ( s ) f o r RANGE: Step 2 3 4 RANGE 3.02 3.67 4.07 (*) I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s which are shown i n the lower t r i a n g l e G G G G r r r r P P P P 2 1 4 3 Mean FASTTW .3498 Grp 2 .9627 Grp 1 1.1426 Grp 4 4.2953 Grp 3 127 t - t e s t s f o r Independent Samples o f CPROTEIN V a r i a b l e Number o f Cases Mean SD SE o f Mean RATE 3 CPROTEIN c a r d i a c CPROTEIN f a s t t w .2305 .9627 .060 .208 .030 .120 Mean D i f f e r e n c e = - . 7321 Levene ' s T e s t f o r E q u a l i t y o f V a r i a n c e s : F= 7.986 P= .037 t - t e s t f o r E q u a l i t y o f Means V a r i a n c e s t - v a l u e d f 2 - T a i l S i g SE o f D i f f 95% CI f o r D i f f E q u a l Unequal -6.87 -5.91 5 2.25 .001 .021 .107 .124 (-1 .006, ( -1 .213, -.458) -.251) t - t e s t s f o r Independent Samples o f ACTININ V a r i a b l e Number o f Cases Mean SD SE of Mean RATE 4 ACTININ c a r d i a c ACTININ f a s t t w . 0731 .3498 .004 .150 .002 .057 Mean D i f f e r e n c e = - .2767 L e v e n e ' s T e s t f o r E q u a l i t y o f V a r i a n c e s : F= 13.366 P= .004 t - t e s t f o r E q u a l i t y o f Means V a r i a n c e s t - v a l u e d f 2 - T a i l S i g SE o f D i f f 95% CI f o r D i f f E q u a l Unequal -4.07 -4.88 10 6.01 .002 .003 .068 .057 ( - .428, ( - .415 , -.125) -.138) t - t e s t s f o r Independent Samples o f DESMIN V a r i a b l e Number o f Cases Mean SD SE o f Mean RATE 5 DESMIN c a r d i a c DESMIN f a s t t w 4.5846 4.2953 .499 .886 .223 .396 Mean D i f f e r e n c e = .2893 Levene ' s T e s t f o r E q u a l i t y o f V a r i a n c e s : F= 1.672 P= .232 t - t e s t f o r E q u a l i t y o f Means V a r i a n c e s t - v a l u e d f 2 - T a i l S i g SE o f D i f f 95% CI f o r D i f f 128 * * * A N A L Y S I S O F V A R I A N C E * * * OFFSET by PROTEINS TISSUE UNIQUE sums o f squares A l l e f f e c t s e n t e r e d s i m u l t a n e o u s l y Sum o f Mean S i g Source o f V a r i a t i o n Squares DF Square F o f F Main E f f e c t s 686 .408 3 228.803 10 .202 .000 PROTEINS 678 .392 2 339.196 15 .125 .000 TISSUE 28 .858 1 28.858 1 .287 .268 2-Way I n t e r a c t i o n s 9065 .150 2 4532.575 202 .110 .000 PROTEINS TISSUE 9065 .150 2 4532.575 202 .110 .000 E x p l a i n e d 10577 .478 5 2115.496 94 .331 .000 R e s i d u a l 538 .230 24 22 .426 T o t a l 11115 .708 29 383.300 30 cases were p r o c e s s e d . 0 cases (.0 pet) were m i s s i n g . 129 16 Dec 96 SPSS f o r MS WINDOWS Re lease 6.1 Page 12 O N E W A Y V a r i a b l e OFFSET1 By V a r i a b l e FASTTW A n a l y s i s o f V a r i a n c e Source D . F . Sum o f Squares Mean Squares F F R a t i o Prob . Between Groups W i t h i n Groups T o t a l 2 12 14 3362.5312 212.1190 3574.6503 1681.2656 17.6766 95.1126 .0000 130 O N E W A Y V a r i a b l e OFFSET1 By V a r i a b l e FASTTW M u l t i p l e Range T e s t s : Student-Newman-Keuls t e s t w i t h s i g n i f i c a n c e l e v e l .050 The d i f f e r e n c e between two means i s s i g n i f i c a n t i f MEAN(J)-MEAN(I) >= 2.9729 * RANGE * SQRT(1/N(I) + 1 /N(J) ) w i t h the f o l l o w i n g v a l u e ( s ) f o r RANGE: Step 2 3 RANGE 3.08 3.77 (*) I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s which are shown i n the lower t r i a n g l e G G G r r r P P P 3 1 2 Mean FASTTW 15.4178 Grp 3 38.0857 Grp 1 50.9150 Grp 2 O N E W A Y V a r i a b l e By V a r i a b l e OFFSET2 CARDIAC A n a l y s i s o f V a r i a n c e Source Between Groups W i t h i n Groups T o t a l D . F . 2 12 14 Sum o f Squares 7184.0748 326.1107 7510.1855 Mean Squares 3592.0374 27.1759 F F R a t i o P r o b . 132.1773 .0000 131 16 Dec 96 SPSS f o r MS WINDOWS Release 6.1 Page 15 O N E W A Y V a r i a b l e OFFSET2 By V a r i a b l e CARDIAC M u l t i p l e Range T e s t s : Student-Newman-Keuls t e s t w i t h s i g n i f i c a n c e l e v e l .050 The d i f f e r e n c e between two means i s s i g n i f i c a n t i f MEAN(J)-MEAN(I) >= 3.6862 * RANGE * SQRT(1/N(I) + 1 /N(J ) ) w i t h the f o l l o w i n g v a l u e ( s ) f o r RANGE: Step 2 3 RANGE 3.08 3.77 -(*) I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s which are shown i n the lower t r i a n g l e G G G r r r P P P 1 2 3 Mean CARDIAC 19.4946 Grp 1 23.3444 Grp 2 67.7240 Grp 3 * * Homogeneous Subse t s (h ighes t and lowes t means a r e n o t s i g n i f i c a n t l y d i f f e r e n t ) Subset 1 Group Grp 1 Grp 2 Mean 19.4946 23.3444 Subset 2 Group Grp 3 Mean 67.7240 t - t e s t s f o r Independent Samples o f CPROT Number V a r i a b l e o f Cases Mean SD SE o f Mean OFFSET3 CPROT c a r d i a c 5 19.4946 1.147 .513 CPROT f a s t t w 3 38.0857 6.978 4.029 Mean D i f f e r e n c e = -18.5911 Levene ' s T e s t f o r E q u a l i t y o f V a r i a n c e s : F= 9.194 P= .023 t - t e s t f o r E q u a l i t y o f Means 95% V a r i a n c e s t - v a l u e d f 2 - T a i l S i g SE o f D i f f CI f o r D i f f E q u a l - 6 . 1 5 6 .001 3.021 (-25.983, -11.200) Unequal -4 .58 2.07 .042 4.061 ( -35.549, -1 .633) 132 

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