Effects Of CaCl₂ Treatment On AMPK Activity,Energy Metabolism And Meat Quality During Postmortem Maturation Of Beef At Different Altitudes

To study the changes of CaMKKβ-dependent AMPK activation pathway,energy metabolism and intramuscular environment during the postmortem maturation of muscle at different altitudes.In this experiment,CaCl₂ was applied to beef of different altitudes.By measuring the adaptability to hypoxia of three altitude cattles,the control of CaMKKβ-dependent AMPK activation pathway,key products of muscle energy metabolism and the products of muscle energy metabolism during the maturation,the changes in rate-limiting enzyme activity,myofibrillar protein（MP）characteristics,mitochondria,PGC-1α,and microstructure of muscle cells of three altitude muscles in control and CaCl₂ group were analyzed,which provide a theoretical reference for the in-depth study of the relationship between AMPK and postmortem muscle energy metabolism,intramuscular environment and quality,so as to realize the regulation of postmortem muscle quality.The research results are as follows:1.The hypoxia adaptability of cattle is related to altitude.The mRNA expression of postmortem muscle HIF-1,PRKAA2,GLUT4 and the content of myoglobin increased with the elevation（P<0.05）.No significant difference in HIF-1 gene mRNA levels between Gannan yak and Yushu yak was found.The mRNA expression of PRKAA1 in Yushu yak was lower than that of Gannan yak（P<0.05）.The protein expression of PRKAA1 in Yushu yak is not significantly different from that of Gannan yak.At the same time,the protein expression of PRKAA2 in the longissimus doris at three altitudes is higher than PRKAA1,which may be a unique metabolic mechanism for yak to adapt to hypoxia and achieve energy supply and demand balance.2.The energy metabolism of the longissimus doris of yak at high altitude was higher than that of Local cattle at low altitude during postmortem maturation,and the energy metabolism of postmortem muscle was significantly accelerated by CaCl₂ treatment.Adenosine monophosphate-activated protein kinase（AMPK）activity,CaMKKβactivity,lactic acid,Ca²⁺-ATPase,Na/K⁺-ATPase,and brightness（L*）rise first and then decrease;glycogen,pH,lactic acid Dehydrogenase（LDH）and redness（a*）decreased overall;myofibrillation index（MFI）increased overall.At 12 h,the AMPK activity of the longissimus doris of CaCl₂ group in three altitudes was 150.96±4.89,171.05±4.41,and 196.98±4.02U/L,and the CaMKKβactivity was 116.37±3.74,121.66±3.78,and 147.32±4.01 U/L.After treated with CaCl₂,the metabolic rate increased and the enzyme activity level was higher in the early postmortem period.3.With the increase of altitude,the thermal stability of myofibrillar of the longissimus doris in three altitude bovines gradually decreased,and CaCl₂ treatment significantly changed the characteristics of myofibrillar of the longissimus doris.The thermal degeneration temperature（Tg）of myofibrillar protein in the longissimus doris of control group in three altitudes increased and then decreased.The thermal denaturation temperature of the CaCl₂group of Locla cattle and Gannan yak gradually decreased,and the Tg of Yushu yak in CaCl₂group increased first and then decreased.There are differences in the Tg of bovine in different altitudes.As the altitude increases,the thermal stability of the myofibrillar protein of the longissimus doris at three altitudes gradually decreases.At 72 h,the random curl content of the Local cattle in both control and CaCl₂ groups reached the maximum,and the CaCl₂ group was higher than that in control group（P<0.05）.Compared with other two types of longissimus doris,Yushu yak group has a higher protein content.CaCl₂ treatment makes the muscle protein of Yushu yak more fully decomposed.At 72-120 h,theα-helix andβ-sheet contents of the control and the CaCl₂ groups in Yushu yak were the lowest,and theβ-turn angle and random curl content were higher,indicating that the mature rate of Yushu yak muscle was faster during the later stages of maturation and the protein secondary structure is more damaged.4.With the increase of altitude,the fluorescence signals of muscle mitochondria and PGC-1αincreased gradually,and there was a significant difference in the active status of mitochondria and PGC-1αin CaCl₂ group of three altitude bovines.Compared with the other two groups,the mitochondrial fluorescence signal of the Yushu Yak group was stronger,and the distribution was mostly concentrated around the cell membrane,and the fluorescence signal inside the cells was stronger.Consistent with mitochondrial imaging results,the expression of PGC-1αwas higher in the Yushu Yak group.The degree of damage to the muscle fiber structure in the CaCl₂ group was greater than that in the control group.CaCl₂may increase the degree of the damage in the muscle fiber structure by increasing the postmortem glycolysis process,and then accelerate the postmortem muscle maturation process.In summary,AMPK activity is related to the hypoxia adaptability of bovine at different altitudes.At the same time,postmortem CaCl₂ treatment can activate AMPK through the CaMKKβcascade,thereby regulating the glycolytic metabolic pathway downstream of muscle AMPK.The possible mechanism for AMPK to regulate meat quality is that after activation of AMPK,it changes the muscle glycogen content,lactic acid accumulation,pH value,intramuscular environment,myofibrillar protein characteristics and microstructure,changes the degree and speed of glycolysis,and affects the maturation process,the beef enters rigidity and completes maturity earlier.Therefore,AMPK and its cascade effect are important targets for improving meat quality.In the future,AMPK activity can be adjusted to improve meat quality.