The Role Of Effector Caspases During Postmortem Aging Of Beef Muscle

Tenderness has been considered as the most important eating quality characteristic of meat. Aging is one of the most effective methods improving postmortem tenderness of meat. It has been widely demonstrated that myofibrils limited proteolysis by calpain is a major determinant of postmortem tenderization. However, individual calpains could not univocally explain all the changes observed during postmortem aging of meat. For example, in the presence of some calpain inhibitors, myofibrillar proteins are still cleaved. Therefore, it is possible that, meat aging is a multi-enyzmatic process, and in addition to well-studied calpain system, caspases are also likely to be involved in postmortem aging of muscle. Recent researches showed that postmortem muscle cells die through apoptosis and caspase-3could also be activated during the early postmortem period. This provide theoretic foundation for further investigation the contribution of caspases to postmortem aging of muscle. The present paper was designed to investigate the degradation of myofibrillar proteins from beef muscle by caspase-3, the interaction between caspase and calpain during postmortem aging, the expression of apoptotic factors and pathways to apoptosis in postmortem beef muscle, and the influences of Ca2x and Zn2+on the expression of caspases and apoptotic factors. The aim was to consummate the mechanism of meat aging by introducing the role of caspase in the conversation muscle to meat. The detailed contents and results are shown as follows.1The effects of caspase-3inhibitor DEVD and calpain inhibitor MDL on the degradation of myofibrillar proteins in postmortem beef muscle.In this study, the degradation of myofibrillar proteins in postmortem beef muscle treated with caspase-3selective inhibitor DEVD and calpain inhibitor MDL was investigated by use of SDS-PAGE and immunoblotting. The results showed that the proteolysis of titin, nebulin, desmin and the appearance of28-30kDa degradation fragments from troponin-T were inhibited significantly by calpain inhibitor MDL during14-days postmortem storage; Inhibitive effect of DEVD on these proteins was significant during the early postmortem period. The activities of calpains were inhibited noticeably by MDL, which reveals that inhibitive action of MDL on myofibrils degradation mainly results from calpain; DEVD had little effect on calpain activities, which eliminates the possibility of the effect of DEVD on myofibrils degradation resulting from calpain activities. Therefore, it can be concluded that calpain is a major contributor to postmortem tenderization, and caspase-3may be also involved in the conversion of muscle into meat.2In vitro proteolysis of myofibrillar proteins from beef skeletal muscle by caspase-3and caspase-6The objective of this study was to investigate the contribution of effector caspases to meat postmortem tenderization by examining in vitro degradation of myofibrils from beef muscle by caspase-3or-6. Results showed that caspase-3and-6reproduced the degradation patterns of titin and nebulin observed during normal postmortem (PM) aging; however, they only reproduced the28kDa fragment derived from troponin-T. Caspase-3induced only minor degradation of desmin. However, caspase-6caused increasing degradation of desmin with extended incubation time and produced three degradation fragments (45,29, and27kDa) of which only the45kDa fragment has been reported in aged beef. Therefore, caspase-3or-6could only reproduce a part of myofibrillar protein degradation or degradation fragments observed in naturally aged meat and may be involved in PM proteolysis of muscle proteins together with other endogenous proteases.3Cleavage of the calpain inhibitor, calpastatin, during postmortem aging of beef skeletal muscleThe objective of this study was to investigate the interaction between caspase and calpain by examining the contribution of caspase and calpain to the degradation of calpastatin during postmortem storage of beef muscle. Result showed that calpain inhibitor MDL inhibited calpastatin degradation significantly during the first3d postmortem; caspase-3inhibitor DEVD only retarded the calpastatin degradation within the first1d postmortem.μ-calpain, in vitro, reproduced the degradation of calpastatin and its breakdown products in postmortem beef muscle; caspase-3degraded calpastatin into two degradation fragments (100, and90kDa) of which only the90kDa fragment has been reported in aged beef; caspase-6also induced the degradation of calpastatin, but it’s production was not similar with that occurred in naturally aged beef muscle. The activity of caspase-3function within the first1-3d postmortem, and the inactivity of caspase-3maybe resulted from the proteolysis of it by calpain in beef muscle. Therefore, calpain is a main contributor to calpastatin postmortem degradation, and caspase-3could be involved in the proteolysis of calpastatin during the early period postmortem. Calpastatin proteolysis by caspase-3possibly enhanced the activation of calpain, and calpain maybe lead to inactivation of caspases during postmortem tenderization of beef muscle.4Changes in the expression of caspase and apoptotic factors during the postmortem tenderization of beef muscleThe objective of the study was to investigate the pathways to apoptosis (effector caspase activation) in beef muscle cells by examining the expression of apoptotic factors during postmortem storage of beef muscle. Results showed that caspase-3could be activated in postmortem beef muscle. The expression of cytochrome c in cytosolic fraction increased and that in mitochondrial fraction decreased significantly during the early postmortem period, which demonstrated that cytochrome c was released from mitochondrion to cytoplasm. The expression of anti-apoptotic factor Bcl showed no visible change and that of pro-apoptotic factor Bax increased during postmortem storage. A significant decrease in the expression of Hsp27occurred with the postmortem time extended. These results demonstrated that mitochondrial pathway is one of the most important apoptosis pathway in postmortem beef muscle and, Bax and Hsp27were two key apoptotic factors in the process of apoptosis in postmortem beef muscle.5Influences of Ca2+and Zn2+on the expression of caspase-3and apoptotic factors in postmortem beef muscleThe study was designed to investigate whether influences of Ca2+and Zn2+on postmortem tenderness are conducted through another endogenous enzyme caspase, except calpain, by examining the expression of caspase and apoptotic factors in postmortem beef muscle treated with Ca2+and Zn2+. Results showed that Ca2+accelerate the activation of caspase-3in postmortem beef muscle, whereas Zn2+retarded it. Ca+increased the expression of cytochrome c, whereas Zn2+had no significant influence on the expression of it. This results demonstrated that the effect of Ca2+on caspase-3was performed indirectly by regulating some apoptotic factors in upstream pathway, whereas Zn2+had direct influence on caspase-3. It was therefore concluded that caspase-3, besides calpain, may be also an contributor to the influences of Ca2+and Zn2+on postmortem tenderness.