Study On The Effects Of Caspase-3 And Its Association With Calpain During Postmortem Ageing Of Chicken Meat

Tenderness is one extremely important character of meat, to which researchers pay more attentions and by which consumers decide whether to purchase. While then, a better understanding of the effects that account for meat tenderness would help meat producers produce consistently good tender meat to meet the majority of consumers’demands. The tenderizing of meat during the ageing process is highly complex and although there still some controversy regarding the actual mechanism, it is generally accepted that major components affected by proteolysis are the key myofibrillar proteins. Several studies suggest that some of the changes occurring in mammalian muscle ultrastructure during ageing are associated with the action of calpains; moreover, there is much evidence that the calpain system plays a primary role in proteolysis. Early studies on meat focused on postmortem tenderization byμ-calpain and m-calpain. These two ubiquitous calpains, are negatively regulated by interaction with their endogenous specific inhibitor, calpastatin. Although there is large amount of data that shows that the calpain system has the major effect during the postmortem ageing on the tenderness of meat, there is evidence that other proteolytic systems are involved in a complex multi-enzyme system. Soon after animal slaughter, the tissues will be exhausted of oxygenated blood leading to hypoxia-ischemia (HI), which will initiate an apoptotic-necrotic response. Recently, it has been proposed that apoptosis should be considered as a step prior to development of rigor mortis. Apoptosis is a common physiological event that occurs in proliferating and regenerating tissues. Caspase-3, a member of the interleukin-1β-converting enzyme family of cysteine proteases, is a trigger for the execution phase of apoptosis. Recently a novel consideration was focused on the potential relationship of apoptosis key protease caspases and its underlying mechanism for meat tenderization.1. Effects of apoptosis inducing on caspase-3 and myofibrillar disruption of chicken during postmortem ageingIn this study, apoptosis inducers, camptothecin, etoposide and Ca2+as well as Low-frequency and high-power ultrasound (40 kHz,1500 W) were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during days of ageing. All treatments resulted in significantly higher caspase-3 activities during storage (p<0.05), with the natural substrates, whereas, western blotting analysis of theα-spectrin cleavage product,120 kDa peptide (SBDP 120), showed that Ca2+ and ultrasound were more effective than either camptothecin or etopside, and all were most active up to day 3 (p<0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product (p<0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected. The findings revealed that the three apoptosis inducers and ultrasound could increase myofibrillar dissociation and proteolysis during chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat.2. Effects of apoptosis inducing on calpain and energy changing of chicken during postmortem ageingWe used apoptosis inducers, camptothecin, etoposide and Ca2+ to treat chicken muscle after slaughter and followed the outcome during 7 days of ageing. According to the results of western blotting, all the three apoptosis inducers treatments enhanced the band intensity of calpain (p<0.05) but reduced calpastatin (p<0.05) during 7 days of meat ageing. We used Low-frequency and high-power ultrasound (40 kHz,1500 W) to treat chicken muscle 30 minutes or 60 minutes after slaughter and followed the outcome during 5 days of ageing. Our results showed that the treatments stimulated calpain activity significantly during storage time (p<0.05), but reduced calpastatin bands (p<0.05) of Western blots. The findings of this investigation revealed that caspase-3 possibly activates calpain via calpastatin degradation. We speculate that, through the calpain/calpastatin system, caspase-3 has the potential to contribute to the tenderisation of meat during the conversion of muscle tissue into meat. We found Schematic representation of the morphological changes of apoptosis during postmortem ageing of chicken meat, such as condensation of nuclear chromatin and mitochondrial swelling. The apoptosis inducers, camptothecin, etoposide and Ca2+ as well as Low-frequency and high-power ultrasound made the phenomena dramaticly. Our results showed that each treatments decreased the amount of ATP, ADP, AMP and increased IMP significantly than control during storage time (p<0.01). At same time, during the first 24 hours the threatments made the activities of Na+K+-ATPase and Ca2+-ATPase higher than control (p<0.01), but lower than control after 24 hours ageing (p<0.01). We therefore suggest that there is a relationship between caspase-3 and appotosis which contributes to the conversion of muscle tissue into meat.3. Effects of the inhibitors of caspase-3 on calpain during postmortem ageing cf chicken meatIn this study, we used inhibitors of caspase-3, DEVD-CHO and Z-VAD-FMK, to treat chicken muscle immediately after slaughter, and followed the changes in calpain activities together with their expression during 5 days of ageing. Inclusion of inhibitors of caspase-3 DEVD-CHO, led to lower calpain activities (p<0.01), and dramatically reduced expression of calpain-1 and calpain-2 (p<0.01). At same time, this DEVD-CHO inhibition resulted in greater calpastatin expression compared with the control (p<0.01). The findings of this investigation show that calpain prevented the activation of caspase-3, while caspase-3 appeared to enhance the calpain activity during postmortem ageing through inhibition of calpastatin. We therefore suggest that there is a relationship between caspase-3 and calpain which contributes to the conversion of muscle tissue into meat. However, Addition of caspase-3 inhibitor Z-VAD-FMK to the system resulted in significantly lower calpain activities (P<0.01) during storage. Western blot analysis of calpain-1 and calpain-2 showed that the addition of Z-VAD-FMK resulted in the formation of higher amounts compared with the control (p<0.01). While, the Z-VAD-FMK threatment led to lower calpastatin expression compared with the control (p<0.05), and this caspase-3 inhibitor nearly had no effects on western blotting analysis of pro-caspase-3. As there are several common factors of meat ageing and apoptosis, our results showed that each treatments stimulated the amount of ATP, ADP, AMP significantly higher than control but decreased IMP during storage time (p<0.01). At same time, the activities of Na+K+-ATPase and Ca2+-ATPase higher than control (p<0.01). We therefore suggest that there is a relationship between caspase-3 and calpain which contributes to the conversion of muscle tissue into meat.4. Effects of the inhibitors of calpain on caspase-3during postmortem ageing of chicken meatCalpain activity is an important factor in the conversion of muscle into tender meat, and recently consideration has focused on the potential of a key protease of apoptosis, caspase-3, as being involved in the underlying mechanism for the postmortem tenderization of meat. In this study, we used inhibitors of calpain, MDL28170 and Calpeptin, to treat chicken muscle immediately after slaughter, and followed the changes in caspase-3 activities together with their expression during 5 days of ageing. Addition of calpain inhibitors to the system resulted in significantly higher caspase-3 activities (p<0.01) during storage. Western blotting analysis of pro-caspase-3 andα-spectrin cleavage of the 120 kDa peptide (SBDP 120), showed that the addition of calpain inhibitors resulted in the formation of higher amounts of the active form of capsase-3 compared with the control (p<0.01). At same time, this inhibition resulted in greater calpastatin expression compared with the control (P<0.01). The findings of this investigation show that calpain prevented the activation of caspase-3, and also calpain inhibition through calpastatin over expression up-regulates caspase-3 activity during postmortem ageing. As there are several common factors of meat ageing and apoptosis, our results showed that each treatments stimulated the amount of ATP, ADP, AMP significantly higher than control but decreased IMP during storage time (p<0.01). At same time, the activities of Na+K+-ATPase and Ca2+-ATPase higher than control (p<0.01). We therefore suggest that there is a relationship between caspase-3 and calpain which contributes to the conversion of muscle tissue into meat.