Study On The Effect Of Cytochrome C Release And Its Mediation Mechanism Of Mitochondrial Apoptosis Activation On Yak Meat Tenderness During Postmortem Aging

Tenderness is an important quality index for evaluating yak meat quality and affecting consumers'desire to purchase.Postmortem aging process is an important way to improve the meat tenderness.In the last decades,Apoptotic process has been found that can significantly improve meat tenderness and has been continuously studied and applied to explain the tenderization mechanism during meat postmortem aging.At present,numerous studies on the effects of apoptosis on meat tenderness mainly focus on the degradation of myofibrillar protein by caspases,but there were few related studies on the activation mechanism of mitochondrial apoptotic pathway of yak meat during postmortem aging.In this paper,ten yak cattles were selected as the experimental objects,longissimus dorsi were selected as the test samples,and several specific inhibitors and inducers were used to treat postmortem yak meat to study the possible pathways and influence factors for the release of mitochondrial Cytochrome C?Cyt-c?.The main pathways and influencing factors of Cyt-c release and the effect of Cyt-c oxidative status on the mitochondrial apoptotic cascade activation were determined to reveal the influence mechanism of these factors on the release of Cyt-c,and ultimately clarify the mitochondrial Cyt-c release and mediate the activation mechanism of mitochondrial apoptotic pathway and its effect on yak meat tenderness during postmortem aging period.These works can help to further improve the theory of apoptotic tenderization of yak meat after slaughter,provide data support and theoretical basis for improving yak meat tenderness and fully utilizing yak resources.The main findings are as follows:1.Possible pathways for mitochondrial Cyt-c release and changes in apoptosome composition were studied.During early postmortem aging?0-72 h?,the pH and ATP content decreased significantly,MPTP presented irreversibly opening,and the mitochondrial membrane potential also showed a significant decrease tendency.Changes in these factors can provide favorable conditions for the release of Cyt-c.At the early postmortem aging period,cytosolic Cyt-c expression level increased significantly,and the expression of Apaf-1 also increased significantly,then decreased significantly?P<0.05?.During postmortem aging,the expression of procaspase-9 decreased significantly;6-72 h,the expression of caspase-3 was also significantly decreased?P<0.05?.Thses results indicated that the opening of MPTP may mediate the release of mitochondrial Cyt-c and the mitochondrial membrane potential,the pH value and ATP of intramuscular environmental factors may play a significant role in promoting the release of Cyt-c.Cyt-c released from the mitochondria to the cytoplasm in early aging period forming apoptosome by combining with Apaf-1 and procaspase-9,which further mediated the downstream caspase-3 activation,and finally initiated the mitochondrial apoptotic cascade reaction.2.It is clear that oxidized Cyt-c mediates the activation of the mitochondrial apoptotic pathway and influences muscle tenderness.At the early postmortem aging stage?6-24 h?,Cyt-c expression level in the cytoplasm of the control group was significantly or extremely significantly higher than that of the TMPD?apoptotic body inhibitor?treatment group,and the expression level of caspase-3 in the control group was significantly lower than that in the TMPD treatment group?P<0.05?.TMPD significantly or extremely significantly increased the Cyt-c reduction level,inhibited caspase-9/3 activities,apoptotic rate,and meat tenderization during postmortem aging period,as a whole,oxidized Cyt-c mediated the formation of apoptosome and activated downstream caspase-9/3,which further inducing the mitochondrial apoptotic cascade reaction and improving the yak meat tenderness.3.MPTP opening mediates the release of mitochondrial Cyt-c and activates the mitochondrial apoptotic cascade reaction.CsA promoted the decrease of pH value at the late aging period?72-168 h?and inhibited the decrease of ATP content.During postmortem aging,compared with the control group,CsA as a whole significantly inhibited the opening of MPTP and the decrease of the motichonadrial membrane potential,and increased the Cyt-c expression level of mitochondria,and decreased the Cyt-c expression level of the cytoplasm,caspase-9/3 activities,apoptotic rate and inhibited the expression of Hsp27 and the degree of meat tenderization.Above-mentioned results demonstrated that MPTP mediated the release of Cyt-c and the activation of downstream caspase-9/3 further promoted the mitochondrial apoptotic pathway and its degradation of myofibrillar skeleton proteins;CsA may also regulate the above process by affecting the Hsp27 expression level and intramuscular environmental factors.Ca²⁺and ROS may be the main factors that promote the opening of MPTP and the decrease of the mitochondrial membrane potential,which thus affect the mitochondrial apoptotic cascade reaction and yak meat tenderization.4.Ca²⁺modulates MPTP opening-mediated Cyt-c release and activates mitochondrial apoptotic pathway through a variety of ways.In the early stage of aging?6-72 h?,the pH,ATP and glycogen content in the Ca²⁺treatment group were significantly or extremely significantly lower than those in the control group,and the lactic acid content was significantly or extremely significantly higher than that in the control group;Ca²⁺treatment group Na⁺/K⁺-The activity of ATPase,Ca²⁺-ATPase,SDH and CS activities were significantly or extremely significantly higher than thoes of the control group?P<0.01;P<0.05?.The mitochondrial ROS level in the Ca²⁺treatment group was significantly higher than that of the control group.The degree of MPTP opening was all stronger than that of the control group,and the mitochondrial membrane potential was significantly lower than that of the control group?P<0.01?.These results suggested that during early aging process,the imbalance of Ca²⁺homeostasis may provide necessary conditions for apoptotic process activation by accelerating the glycolysis process.Meanwhile,the enzymatic activity and the tricarboxylic acid cycle can provide favorable conditions for mitochondrial Ca²⁺overload,and the imbalance of Ca²⁺homeostasis can induce mitochondrial damage by increasing mitochondrial ROS levels,triggering MPTP opening and the membrane potential decline,promoting Cyt-c release and caspase-9/3 activation,and finally accelerating mitochondrial apoptotic process and further improves the yak meat tenderness.5.ROS can cause mitochondrial oxidative damage and promote apoptosis by increasing mitochondrial oxidative stress.During postmortem aging process,the ROS level in the H₂O₂treatment group was significantly higher than that in the control group and the NAC treatment group?P<0.01?,and the activities of SOD,CAT,GSH-PX,and SDH were significantly or extremely significantly lower in the H₂O₂ treatment group from 24 to 168 h.In the control group and NAC treatment group,the MDA content in the H₂O₂ treatment group and the control group was significantly higher than that in the NAC treatment group,and the mitochondrial membrane fluidity was significantly lower than that in the NAC treatment group?P<0.01?.The above results indicated that H₂O₂ induced the ROS production significantly increased the mitochondrial oxidative stress of skeletal muscle cells and reduced the mitochondrial antioxidant capacity,resulting in mitochondrial oxidative damage,which was conducive to postmortem skeletal muscle mitochondrial apoptotic pathway.6.ROS induces MPTP-mediated Cyt-c release by increasing mitochondrial Ca²⁺and Bax/Bcl-2 levels.During postmortem aging period,the mitochondrial Ca²⁺content in the H₂O₂ treatment group was significantly higher than that in the NAC treatment group,and the mitochondrial membrane potential was significantly lower than that in the NAC treatment group.The degree of MPTP opening and Bax content were significantly higher than those in the other two groups;6¹20 h,Bcl-2 content in H₂O₂ treatment group was significantly lower than that in NAC treatment group;Bax/Bcl-2 in H₂O₂ treatment group was significantly higher than that in other two groups?P<0.01?.Meanwhile,ROS mediated oxidative stress by increasing the levels of mitochondrial Ca²⁺and Bax/Bcl-2 resulting in jointly promoting the opening of MPTP and the decrease of membrane potential,further promoting the release of Cyt-c and activating downstream caspase-9/3 and finally inducing the cascade reaction of mitochondrial apoptosis and improving the yak meat tenderness.In summary,Cyt-c released from the mitochondria to the cytoplasm through the opening of MPTP during yak meat postmortem aging,and the mitochondrial membrane potential and the internal environment factors:pH values and ATP may provide favorable conditions for this process.Ca²⁺accelerated the process of glycolysis and provided the necessary conditions for the activation of apoptosis.It can provide favorable conditions for Ca²⁺overload by affecting the activity of cell energy metabolism enzymes and the tricarboxylic acid cycle,and triggered the MPTP opening and the decline of mitochondrial membrane potential membrane by increasing mitochondrial oxidative stress levels,which promoting apoptosis by inducing the release of Cyt-c.ROS-mediated oxidative stress also promoted the opening of MPTP and the decrease of membrane potential and promoted the release of Cyt-c by increasing the levels of mitochondrial Ca²⁺and Bax/Bcl-2.Cyt-c from the mitochondria released into the cytoplasm would by oxidized and then formed apoptosome with Apaf-1 and procaspase-9,further activated downstream caspase-3.Eventually leaded to the mitochondrial apoptotic cascade reaction and improved the tenderness of yak meat.