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.