{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/136934"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/136934","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Postmortem energy metabolisms' role in calpain activation, inhibition, and subsequent function","abstract":"The calpain system is the most extensively studied group of proteases involved in postmortem beef tenderization. Tenderness is a critical quality characteristic that significantly influences consumer satisfaction and repeat purchases. Among the various proteolytic systems active in muscle after exsanguination, the calpain system, particularly calpain-1, is considered central to the tenderization process. However, the specific biochemical conditions that govern calpain-1 activation and its ability to degrade myofibrillar proteins are not yet fully understood. These studies aim to investigate the temporal, biochemical, and muscle-specific factors that regulate calpain-1-mediated proteolysis during early postmortem aging in beef. To accomplish this, a novel in vitro digestion assay, an in vitro system simulating postmortem glycolytic metabolism, and in vivo carcass interventions, specifically electrical stimulation (ES), were used to study the calpain system in muscle postmortem. First, an in vitro digestion assay was developed to evaluate protease activity from the longissimus thoracis et lumborum (LTL) and extensor carpi radialis (ER) muscles at 0, 1, 2, 7, and 14 d postmortem. Purified myofibrils served as substrates to monitor degradation of desmin and troponin-T, two surrogates of muscle proteolysis postmortem. Minimal proteolysis occurred with early postmortem samples (0 and 1 d), while proteolytic activity increased notably by day 2 in LTL. ER samples exhibited limited proteolysis across all timepoints studied. Inhibition of in vitro proteolysis by EGTA, cysteine protease inhibitor, and calpastatin strongly suggested the proteolysis observed in our system was indeed calpain-dependent. Second, we explored the relationship between calcium availability, calpastatin abundance, and calpain-1 autolysis in the aforementioned system. In LTL, calcium addition significantly enhanced calpain-1 autolysis and desmin degradation in 1-day samples suggesting that calcium availability is rate-limiting to muscle proteolysis in 24 hr aged beef carcasses. However, in the 0-day LTL samples, an increase in calpain-1 autolysis was also observed with added calcium, yet no increase in proteolysis was detected. These data argue that high levels of calpastatin in the 0-day samples prevented proteolysis, whereas 1-day samples lacked the inhibitory effects of calpastatin but had limited calpain-1 autolysis. Calpastatin abundance decreased earlier in LTL than in ER, where its persistence contributed to lower proteolytic activity in the ER. These results demonstrate muscle-specific regulation of calpain-1 activity based on calcium availability and calpastatin presence. Next, we examined the influence of pH decline on the calpain-1 system using an in vitro glycolytic simulation system. Treatments altering glycogen and creatine concentrations, and ATPase levels generated distinct pH declines in our in vitro digestions. Faster pH declines accelerated calpain-1 autolysis and accumulation of the active 76 kDa subunit in both LTL and ER. Despite enhanced autolysis, desmin degradation remained minimal, however, suggesting that calpain-1 activation alone does not ensure effective proteolysis and implicating additional regulatory mechanisms. Lastly, electrical stimulation (ES) of beef carcasses was used to evaluate the impact of rapid pH decline on the calpain system in vivo. ES accelerated pH decline and calpain-1 autolysis, particularly in the LTL and semitendinosus (ST) muscles, and increased calpastatin degradation in ST. However, ES did not enhance myofibrillar protein degradation compared to non-stimulated controls. These findings suggest that although ES promotes calpain-1 autolysis, it does not necessarily translate to increased proteolytic breakdown of structural proteins. Collectively, results of these studies demonstrate that the regulation of calpain-1-mediated proteolysis is multifactorial, involving the interplay of pH dynamics, calcium availability, calpastatin inhibition, and in a muscle-specific manner. The in vitro assay developed herein provides a novel and sensitive tool for monitoring protease activity in samples during postmortem and studying the regulation of proteases in muscle tissues during the maturation of beef. Moreover, carcass interventions offer insight into the mechanisms that result from ES. Together, these studies add to our understanding of postmortem muscle biology and beef tenderization.","abstract_html":"The calpain system is the most extensively studied group of proteases involved in postmortem beef tenderization. Tenderness is a critical quality characteristic that significantly influences consumer satisfaction and repeat purchases. Among the various proteolytic systems active in muscle after exsanguination, the calpain system, particularly calpain-1, is considered central to the tenderization process. However, the specific biochemical conditions that govern calpain-1 activation and its ability to degrade myofibrillar proteins are not yet fully understood. These studies aim to investigate the temporal, biochemical, and muscle-specific factors that regulate calpain-1-mediated proteolysis during early postmortem aging in beef. To accomplish this, a novel in vitro digestion assay, an in vitro system simulating postmortem glycolytic metabolism, and in vivo carcass interventions, specifically electrical stimulation (ES), were used to study the calpain system in muscle postmortem. First, an in vitro digestion assay was developed to evaluate protease activity from the longissimus thoracis et lumborum (LTL) and extensor carpi radialis (ER) muscles at 0, 1, 2, 7, and 14 d postmortem. Purified myofibrils served as substrates to monitor degradation of desmin and troponin-T, two surrogates of muscle proteolysis postmortem. Minimal proteolysis occurred with early postmortem samples (0 and 1 d), while proteolytic activity increased notably by day 2 in LTL. ER samples exhibited limited proteolysis across all timepoints studied. Inhibition of in vitro proteolysis by EGTA, cysteine protease inhibitor, and calpastatin strongly suggested the proteolysis observed in our system was indeed calpain-dependent. Second, we explored the relationship between calcium availability, calpastatin abundance, and calpain-1 autolysis in the aforementioned system. In LTL, calcium addition significantly enhanced calpain-1 autolysis and desmin degradation in 1-day samples suggesting that calcium availability is rate-limiting to muscle proteolysis in 24 hr aged beef carcasses. However, in the 0-day LTL samples, an increase in calpain-1 autolysis was also observed with added calcium, yet no increase in proteolysis was detected. These data argue that high levels of calpastatin in the 0-day samples prevented proteolysis, whereas 1-day samples lacked the inhibitory effects of calpastatin but had limited calpain-1 autolysis. Calpastatin abundance decreased earlier in LTL than in ER, where its persistence contributed to lower proteolytic activity in the ER. These results demonstrate muscle-specific regulation of calpain-1 activity based on calcium availability and calpastatin presence. Next, we examined the influence of pH decline on the calpain-1 system using an in vitro glycolytic simulation system. Treatments altering glycogen and creatine concentrations, and ATPase levels generated distinct pH declines in our in vitro digestions. Faster pH declines accelerated calpain-1 autolysis and accumulation of the active 76 kDa subunit in both LTL and ER. Despite enhanced autolysis, desmin degradation remained minimal, however, suggesting that calpain-1 activation alone does not ensure effective proteolysis and implicating additional regulatory mechanisms. Lastly, electrical stimulation (ES) of beef carcasses was used to evaluate the impact of rapid pH decline on the calpain system in vivo. ES accelerated pH decline and calpain-1 autolysis, particularly in the LTL and semitendinosus (ST) muscles, and increased calpastatin degradation in ST. However, ES did not enhance myofibrillar protein degradation compared to non-stimulated controls. These findings suggest that although ES promotes calpain-1 autolysis, it does not necessarily translate to increased proteolytic breakdown of structural proteins. Collectively, results of these studies demonstrate that the regulation of calpain-1-mediated proteolysis is multifactorial, involving the interplay of pH dynamics, calcium availability, calpastatin inhibition, and in a muscle-specific manner. The in vitro assay developed herein provides a novel and sensitive tool for monitoring protease activity in samples during postmortem and studying the regulation of proteases in muscle tissues during the maturation of beef. Moreover, carcass interventions offer insight into the mechanisms that result from ES. Together, these studies add to our understanding of postmortem muscle biology and beef tenderization.","abstract_has_math":false,"creators":["Bodmer, Jocelyn Sandra"],"institution":"Virginia Tech","degree_name":"Doctor of Philosophy","degree_level":"doctoral","degree_discipline":"Animal and Poultry Sciences","degree_department":"Animal and Poultry Sciences","school":null,"contributors":[],"advisors":[],"committee_chairs":["Gerrard, David E."],"committee_members":["Silva, Saulo","Shi, Tim Hao","Koohmaraie, Mohammad"],"year":2025,"date_issued":"2025-07-31","date_published":"2025-07-31","updated_at":"2026-07-22T22:18:46Z","subjects":["calpain system","proteolysis","postmortem energy metabolism","myofibrils","pH"],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44431"],"render_values":[{"text":"vt_gsexam:44431","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/10919/136934","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Gerrard, David E."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Silva, Saulo","Shi, Tim Hao","Koohmaraie, Mohammad"]},{"key":"dc:contributor.department","label":"Department","values":["Animal and Poultry Sciences"]},{"key":"dc:creator","label":"Author","values":["Bodmer, Jocelyn Sandra"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-08-01T08:00:31Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-08-01T08:00:31Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-07-31"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Animal and Poultry Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["calpain system","proteolysis","postmortem energy metabolism","myofibrils","pH"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44431"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10919/136934"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The calpain system is the most extensively studied group of proteases involved in postmortem beef tenderization. Tenderness is a critical quality characteristic that significantly influences consumer satisfaction and repeat purchases. Among the various proteolytic systems active in muscle after exsanguination, the calpain system, particularly calpain-1, is considered central to the tenderization process. However, the specific biochemical conditions that govern calpain-1 activation and its ability to degrade myofibrillar proteins are not yet fully understood. These studies aim to investigate the temporal, biochemical, and muscle-specific factors that regulate calpain-1-mediated proteolysis during early postmortem aging in beef. To accomplish this, a novel in vitro digestion assay, an in vitro system simulating postmortem glycolytic metabolism, and in vivo carcass interventions, specifically electrical stimulation (ES), were used to study the calpain system in muscle postmortem. First, an in vitro digestion assay was developed to evaluate protease activity from the longissimus thoracis et lumborum (LTL) and extensor carpi radialis (ER) muscles at 0, 1, 2, 7, and 14 d postmortem. Purified myofibrils served as substrates to monitor degradation of desmin and troponin-T, two surrogates of muscle proteolysis postmortem. Minimal proteolysis occurred with early postmortem samples (0 and 1 d), while proteolytic activity increased notably by day 2 in LTL. ER samples exhibited limited proteolysis across all timepoints studied. Inhibition of in vitro proteolysis by EGTA, cysteine protease inhibitor, and calpastatin strongly suggested the proteolysis observed in our system was indeed calpain-dependent. Second, we explored the relationship between calcium availability, calpastatin abundance, and calpain-1 autolysis in the aforementioned system. In LTL, calcium addition significantly enhanced calpain-1 autolysis and desmin degradation in 1-day samples suggesting that calcium availability is rate-limiting to muscle proteolysis in 24 hr aged beef carcasses. However, in the 0-day LTL samples, an increase in calpain-1 autolysis was also observed with added calcium, yet no increase in proteolysis was detected. These data argue that high levels of calpastatin in the 0-day samples prevented proteolysis, whereas 1-day samples lacked the inhibitory effects of calpastatin but had limited calpain-1 autolysis. Calpastatin abundance decreased earlier in LTL than in ER, where its persistence contributed to lower proteolytic activity in the ER. These results demonstrate muscle-specific regulation of calpain-1 activity based on calcium availability and calpastatin presence. Next, we examined the influence of pH decline on the calpain-1 system using an in vitro glycolytic simulation system. Treatments altering glycogen and creatine concentrations, and ATPase levels generated distinct pH declines in our in vitro digestions. Faster pH declines accelerated calpain-1 autolysis and accumulation of the active 76 kDa subunit in both LTL and ER. Despite enhanced autolysis, desmin degradation remained minimal, however, suggesting that calpain-1 activation alone does not ensure effective proteolysis and implicating additional regulatory mechanisms. Lastly, electrical stimulation (ES) of beef carcasses was used to evaluate the impact of rapid pH decline on the calpain system in vivo. ES accelerated pH decline and calpain-1 autolysis, particularly in the LTL and semitendinosus (ST) muscles, and increased calpastatin degradation in ST. However, ES did not enhance myofibrillar protein degradation compared to non-stimulated controls. These findings suggest that although ES promotes calpain-1 autolysis, it does not necessarily translate to increased proteolytic breakdown of structural proteins. Collectively, results of these studies demonstrate that the regulation of calpain-1-mediated proteolysis is multifactorial, involving the interplay of pH dynamics, calcium availability, calpastatin inhibition, and in a muscle-specific manner. The in vitro assay developed herein provides a novel and sensitive tool for monitoring protease activity in samples during postmortem and studying the regulation of proteases in muscle tissues during the maturation of beef. Moreover, carcass interventions offer insight into the mechanisms that result from ES. Together, these studies add to our understanding of postmortem muscle biology and beef tenderization."]},{"key":"dc:description.abstractgeneral","label":"General Abstract","values":["Tenderness is one of the most important qualities consumers look for when selecting beef products. While color may influence the initial purchase, tenderness is often what brings consumers back. A group of enzymes called proteases, naturally found in muscle, plays a vital role in improving tenderness during postmortem aging. Among these, calpain-1 is particularly important. However, the precise conditions that control how calpain-1 becomes active and 'breaks down' muscle proteins remain unclear. Our research investigated the key factors that regulate this protease and beef tenderization using a combination of laboratory (in vitro) experiments and carcass treatments, such as electrical stimulation (ES). First, we developed a new digestion assay to simulate postmortem muscle aging and identify when and for how long proteases are active. This method showed protein breakdown begins after 24 hrs postmortem, while very little occurs during the first 24 hrs. Additionally, muscles with higher oxidative capacity showed minimal proteolysis even after 14 d of aging. By using a calcium chelator, a protease inhibitor, and a natural inhibitor of the protease (calpain-1), we confirmed that it was the protease responsible for the digestions observed in our assays. Next, we explored why proteolysis is limited early postmortem. Calpain-1 requires some calcium to become active through self-cleavage. We found that muscle samples from 0 and 1 day postmortem had limited calcium availability and higher inhibitor, which together suppressed proteolysis. Additionally, we used a system that simulates postmortem energy metabolism in a tube to demonstrate that the rate at which pH declines, as is normal during meat aging, affects how quickly proteases activate. However, even when the protease activates, it does not lead to significant breakdown of the proteins, suggesting activation alone isn't the sole factor determining the extent of proteolysis. Finally, we used electrical stimulation (ES), a common practice used in the beef industry to improve tenderness, to study proteolysis. While ES activated protease and increased pH decline, it didn't lead to a change in protein breakdown compared to untreated carcasses. Altogether, these studies show that beef tenderization is influenced by a number of interrelated factors, including type of muscle, calcium levels, pH changes, and the amount of protease inhibitor. Our findings expand our how muscle matures during the aging process to produce tender meat."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["ETD"]},{"key":"dc:title","label":"Title","values":["Postmortem energy metabolisms' role in calpain activation, inhibition, and subsequent function"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Gerrard, David E."],"dc:contributor.committeemember":["Silva, Saulo","Shi, Tim Hao","Koohmaraie, Mohammad"],"dc:contributor.department":["Animal and Poultry Sciences"],"dc:creator":["Bodmer, Jocelyn Sandra"],"dc:date.accessioned":["2025-08-01T08:00:31Z"],"dc:date.available":["2025-08-01T08:00:31Z"],"dc:date.issued":["2025-07-31"],"dc:description.abstract":["The calpain system is the most extensively studied group of proteases involved in postmortem beef tenderization. Tenderness is a critical quality characteristic that significantly influences consumer satisfaction and repeat purchases. Among the various proteolytic systems active in muscle after exsanguination, the calpain system, particularly calpain-1, is considered central to the tenderization process. However, the specific biochemical conditions that govern calpain-1 activation and its ability to degrade myofibrillar proteins are not yet fully understood. These studies aim to investigate the temporal, biochemical, and muscle-specific factors that regulate calpain-1-mediated proteolysis during early postmortem aging in beef. To accomplish this, a novel in vitro digestion assay, an in vitro system simulating postmortem glycolytic metabolism, and in vivo carcass interventions, specifically electrical stimulation (ES), were used to study the calpain system in muscle postmortem. First, an in vitro digestion assay was developed to evaluate protease activity from the longissimus thoracis et lumborum (LTL) and extensor carpi radialis (ER) muscles at 0, 1, 2, 7, and 14 d postmortem. Purified myofibrils served as substrates to monitor degradation of desmin and troponin-T, two surrogates of muscle proteolysis postmortem. Minimal proteolysis occurred with early postmortem samples (0 and 1 d), while proteolytic activity increased notably by day 2 in LTL. ER samples exhibited limited proteolysis across all timepoints studied. Inhibition of in vitro proteolysis by EGTA, cysteine protease inhibitor, and calpastatin strongly suggested the proteolysis observed in our system was indeed calpain-dependent. Second, we explored the relationship between calcium availability, calpastatin abundance, and calpain-1 autolysis in the aforementioned system. In LTL, calcium addition significantly enhanced calpain-1 autolysis and desmin degradation in 1-day samples suggesting that calcium availability is rate-limiting to muscle proteolysis in 24 hr aged beef carcasses. However, in the 0-day LTL samples, an increase in calpain-1 autolysis was also observed with added calcium, yet no increase in proteolysis was detected. These data argue that high levels of calpastatin in the 0-day samples prevented proteolysis, whereas 1-day samples lacked the inhibitory effects of calpastatin but had limited calpain-1 autolysis. Calpastatin abundance decreased earlier in LTL than in ER, where its persistence contributed to lower proteolytic activity in the ER. These results demonstrate muscle-specific regulation of calpain-1 activity based on calcium availability and calpastatin presence. Next, we examined the influence of pH decline on the calpain-1 system using an in vitro glycolytic simulation system. Treatments altering glycogen and creatine concentrations, and ATPase levels generated distinct pH declines in our in vitro digestions. Faster pH declines accelerated calpain-1 autolysis and accumulation of the active 76 kDa subunit in both LTL and ER. Despite enhanced autolysis, desmin degradation remained minimal, however, suggesting that calpain-1 activation alone does not ensure effective proteolysis and implicating additional regulatory mechanisms. Lastly, electrical stimulation (ES) of beef carcasses was used to evaluate the impact of rapid pH decline on the calpain system in vivo. ES accelerated pH decline and calpain-1 autolysis, particularly in the LTL and semitendinosus (ST) muscles, and increased calpastatin degradation in ST. However, ES did not enhance myofibrillar protein degradation compared to non-stimulated controls. These findings suggest that although ES promotes calpain-1 autolysis, it does not necessarily translate to increased proteolytic breakdown of structural proteins. Collectively, results of these studies demonstrate that the regulation of calpain-1-mediated proteolysis is multifactorial, involving the interplay of pH dynamics, calcium availability, calpastatin inhibition, and in a muscle-specific manner. The in vitro assay developed herein provides a novel and sensitive tool for monitoring protease activity in samples during postmortem and studying the regulation of proteases in muscle tissues during the maturation of beef. Moreover, carcass interventions offer insight into the mechanisms that result from ES. Together, these studies add to our understanding of postmortem muscle biology and beef tenderization."],"dc:description.abstractgeneral":["Tenderness is one of the most important qualities consumers look for when selecting beef products. While color may influence the initial purchase, tenderness is often what brings consumers back. A group of enzymes called proteases, naturally found in muscle, plays a vital role in improving tenderness during postmortem aging. Among these, calpain-1 is particularly important. However, the precise conditions that control how calpain-1 becomes active and 'breaks down' muscle proteins remain unclear. Our research investigated the key factors that regulate this protease and beef tenderization using a combination of laboratory (in vitro) experiments and carcass treatments, such as electrical stimulation (ES). First, we developed a new digestion assay to simulate postmortem muscle aging and identify when and for how long proteases are active. This method showed protein breakdown begins after 24 hrs postmortem, while very little occurs during the first 24 hrs. Additionally, muscles with higher oxidative capacity showed minimal proteolysis even after 14 d of aging. By using a calcium chelator, a protease inhibitor, and a natural inhibitor of the protease (calpain-1), we confirmed that it was the protease responsible for the digestions observed in our assays. Next, we explored why proteolysis is limited early postmortem. Calpain-1 requires some calcium to become active through self-cleavage. We found that muscle samples from 0 and 1 day postmortem had limited calcium availability and higher inhibitor, which together suppressed proteolysis. Additionally, we used a system that simulates postmortem energy metabolism in a tube to demonstrate that the rate at which pH declines, as is normal during meat aging, affects how quickly proteases activate. However, even when the protease activates, it does not lead to significant breakdown of the proteins, suggesting activation alone isn't the sole factor determining the extent of proteolysis. Finally, we used electrical stimulation (ES), a common practice used in the beef industry to improve tenderness, to study proteolysis. While ES activated protease and increased pH decline, it didn't lead to a change in protein breakdown compared to untreated carcasses. Altogether, these studies show that beef tenderization is influenced by a number of interrelated factors, including type of muscle, calcium levels, pH changes, and the amount of protease inhibitor. Our findings expand our how muscle matures during the aging process to produce tender meat."],"dc:description.degree":["Doctor of Philosophy"],"dc:format.medium":["ETD"],"dc:identifier.other":["vt_gsexam:44431"],"dc:identifier.uri":["https://hdl.handle.net/10919/136934"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["calpain system","proteolysis","postmortem energy metabolism","myofibrils","pH"],"dc:title":["Postmortem energy metabolisms' role in calpain activation, inhibition, and subsequent function"],"dc:type":["Dissertation"],"thesis:degree_discipline":["Animal and Poultry Sciences"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:18:46Z"}