The Mechanism Of Effects Of Mitochondrial Pathway Signaling Mediated Apoptosis On Bovine Muscle Tenderization During Postmortem Aging

Tenderness is one of the most important indexes to determine beef quality and affects purchase decision of consumers.Postmortem aging is a crucial way to improve meat tenderness.Recently,as a new potential contributor,the mechanism of mitochondrial cytochrome c mediated caspase activation is closely related to the muscle tenderization during postmortem aging.It has been suggested that among intrinsic mitochondrial pathways,the broad-spectrum inhibitor of caspase could not completely prevent cytochrome c mediated caspase pathway apoptosis in certain models.Besides,lysosome could involve in apoptosis through the classic mitochondrial apoptosis pathway.However,the mechanism of AIF and lysosome involvement in postmortem muscle tenderization remains unknown.In this study,the longissimus dorsi?LD?,triceps brachii?TB?and semitendinosus?ST?from Simmental crossbreeding local yellow cattle were selected to study the mechanism of mitochondrial cytochrome c release mediated caspase apoptosis,the effects of changes of cytochrome c structure on apoptosis in postmortem bovine LD;to analysis the mechanism of mitochondrial AIF release mediated apoptosis and the relationship between AIF release and cytochrome c mediated caspase activation;to clarify the effects of lysosomal-mitochondrial apoptosis pathway and lysosomal Fe mediated apoptosis on muscle protein degradation and meat tenderness of LD muscle during postmortem aging;to determine the influence of mitochondrial apoptosis in different muscle types?LD,TB and ST?on muscle protein degradation and tenderization.It provides a new perspective for fully elucidating mitochondrial apoptosis pathway and the mechanism of meat tenderization in different muscle types during aging.The results are as follows:1.Effects of cytochrome c mediated apoptosis on meat tenderness.The abundance of cytochrome c in cytosolic was increased at 6¹2 h postmortem?P<0.05?.The caspase-9 and caspase-3 reached its highest activity at 12 h and 24 h postmortem,respectively.The apoptotic nucleus was increased from 24 h postmortem?P<0.05?.The results indicate that cytochrome c release from mitochondria initiated upstream caspase-9 activation and then activates caspase-3,thus leading to apoptosis during early postmortem aging.pH was decreased at 0⁷2 h and mitochondrial membrane permeability was increased at 6²4 h?P<0.05?.The process is the period of cytochrome c release and caspase activation.Low pH and increased mitochondria membrane permeability are necessary conditions for cytochrome c release in postmortem muscle.In addition,cytochrome c mediated apoptosis was significantly related to Raman Spectrum Vibrational Assignment of Fe ligands of cytochrome c.The Raman Spectrum Vibrational Frequency of Fe ligands was red shifted with the extension of aging time indicated that cytochrome c binds more closely to mitochondria and mediated apoptosis.Moreover,shear force was increased and then decreased at 0¹68 h postmortem,and caspase-3 activity was significantly related with shear force?r=0.762,P<0.05?,suggesting that postmortem aging could improve meat tenderness and caspase-3 was involved in the mechanism of meat tenderization during aging.2.Effects of mitochondrial AIF mediated apoptosis?the relationship between AIF and caspase?on meat tenderness.The abundance of AIF in mitochondria was decreased at 6⁷2 h postmortem?P<0.05?while it was increased in nucleus at 12¹20 h postmortem?P<0.05?.The volume of cell nucleus was gradually increased and some nuclei were dissolved and broken during bovine postmortem aging.There was no significant change in the abundance of AIF in mitochondria and nuclear morphology with the caspase inhibitor treatment.The results indicate that AIF release from mitochondria to nucleus mediated caspase-dependent apoptosis from 6 h postmortem.Furthermore,mitochondrial swelling,ROS content,Ca²⁺concentration were increased and calpain and cathepsin were activated with the AIF release from mitochondria?P<0.05?.Caspase inhibitor significantly inhibited AIF release and apoptosis through inhibiting the increases in mitochondrial swelling,ROS content,Ca²⁺concentration,and calpain and cathepsin activity,indicating that mitochondrial swelling,ROS content and Ca²⁺concentration,as well as activated calpain I and cathepsins,are necessary for AIF release during postmortem aging.In addition,caspase inhibitor significantly increased shear force by inhibiting AIF release,suggesting that AIF mediated apoptosis plays a crucial role in postmortem bovine muscle tenderization.3.Effects of the mechanism of lysosomal?cathepsin?-mitochondrial apoptosis on meat tenderness.It can be seen from 2 that cathepsin is one of the factors affecting the release of mitochondrial apoptotic protein.The generation of ROS targets lysosome and destroys lysosomal membrane,and then the cathepsin B and cathepsin D are released from unstable lysosomal membrane.ROS destroys lysosomal membrane stability is due to the accumulation of Fe in lysosome.Pepstatin A,a cathepsin D specific inhibitor,could significantly inhibit the abundance of Bid and Bax,the mitochondrial membrane permeabilization,and caspase activity?P<0.05?,indicating that cathepsin D induces mitochondrial membrane permeabilization by activating Bid and Bax,thus leading to apoptosis.Lysosomal membrane instability is an upstream event of mitochondrial membrane permeabilization.Furthermore,cathepsin D,not cathepsin B,involvement in mitochondrial apoptosis improves meat tenderness.4.Lysosomal Fe mediated mitochondrial apoptosis contributes to muscle protein degradation during postmortem aging.It can be seen from 3 that lysosomal Fe could be involved in mitochondrial apoptosis.DFO significantly decreased mitochondrial membrane permeability,Ca²⁺concentration and cytochrome c oxidation level,and increased mitochondrial membrane potential?P<0.05?,suggesting that lysosomal Fe induced apoptosis through damaging to mitochondrial membrane,inducing Ca²⁺accumulation and increasing cytochrome c oxidation level.Besides,the abundance of cytochrome c and Bid was significantly increased in DFO group?P<0.05?,however,there was no change in caspase-9 and caspase-3 activity,indicating that lysosomal Fe could increase the abundance of cytochrome c and Bid and activate caspase-9 and caspase-3,thus leading to apoptosis.Moreover,desmin and troponin-T intact in the DFO group was higher than that in the control group.Consistently,the desmin and troponin-T degradation in the DFO group was lower than that in the control group?P<0.05?.The results indicate that lysosomal Fe involvement in mitochondrial apoptotic protein mediated caspase cascade would improve meat tenderness.5.Muscle protein degradation and meat tenderization mediated by mitochondrial apoptosis could be muscle specific.The apoptosis existed and led to tenderization in three postmortem yak muscles?TB,LD and ST?,regardless of muscle type,as evidenced by the increases in mitochondrial membrane permeability,lipid peroxidation,Ca²⁺concentration,cytochrome c oxidation,the decreases in cytochrome c abundance as well as the activation of caspase-9 and caspase-3,and the degradation of desmin and troponin-T at late postmortem aging?P<0.05?,suggesting that the changes in mitochondrial apoptosis result in subsequent increases in muscle protein degradation and meat tenderization.TB exhibited the higher apoptotic ability than LD and ST,however,LD showed the more degradation of myofibrillar proteins.TB showed the higher mitochondrial apoptotic ability but exhibited the less degradation of myofibrillar proteins,indicating that muscle protein degradation and meat tenderization mediated by mitochondrial apoptosis could be muscle specific.