Effects Of HIF-1α On Postmortem Metabolic Pathway And Tenderness Of Yak Meat

Tenderness is a feature highly valued by consumers.Postmortem metabolism is an important way to affect the tenderness of yak meat at different altitudes.In recent years,with postmortem blood loss in animals and the resulting hypoxia,activation of Hypoxia inducible factor-1α（HIF-1α）in cells through glycolysis increases lactic acid and H⁺accumulation in muscle affecting meat quality.Therefore,understanding the factors that control the rate and extent of postmortem metabolism can affect the likelihood of producing high quality products.At present,postmortem metabolism speed and degree have important effects on the formation of meat quality traits,but how it is regulated by postmortem hypoxia is still unclear,and the influencing factors of quality differences of yaks at different altitudes have not been reported.Taking longissimus dorsi muscle of yaks as samples,this paper studied the difference of meat quality and the relationship between HIF-1αand meat quality of yaks at different altitudes.To investigate the effects of HIF-1αon glycolysis pathway and mitochondrial function in post-slaughter maturation of yak meat.To investigate the effects of HIF-1αon Ca²⁺release,apoptosis,Ca²⁺/calmodulin-dependent protein kinase kinaseβ（CaMKKβ）and AMP-activated protein kinase（AMPK）activity in endoplasmic reticulum.By inhibiting PI3K/Akt signaling pathway,the changes of HIF-1αexpression,glycolysis pathway and mitochondrial metabolic pathway were investigated.The effects of hypoxia-modified packaging on the apoptosis pathway and the postmortem tenderness of yak meat were investigated.This study provides new ideas and approaches for further revealing postmortem metabolism of yak,enriching and expanding postmortem metabolism theory,improving and controlling meat quality during storage and processing,and reducing loss of inferior meat.The main research results are as follows:1.The quality of yak meat at different altitudes was significantly different and highly correlated with HIF-1αexpression.HIF-1αexpression in all groups was firstly increased and then decreased with the extension of post-slaughter maturation time（P<0.05）,the HIF-1αprotein expression and HIF-1αm RNA expression of all groups increased with the increase of altitude 0-120 h postmortem（P<0.05）;HIF-1αshowed the highest VIP by PLS-DA analysis,which indicated that HIF-1αwas the main biomarker.The above changes indicated that HIF-1αwas the main protein marker reflecting the difference of meat quality at different altitudes and was positively correlated with HIF-1αexpression and altitude.Glycolytic potential（GP）,a*,p H and shear force were significantly increased with elevation（P<0.05）,while b*,L*,water retention and MFI were significantly decreased（P<0.05）,the expression of HIF-1αprotein was highly correlated with glycolysis potential,p H value,meat color,tenderness and water retention,suggesting that HIF-1α-induced glycolysis pathway may be the key to affect the quality of postmortem yak meat.2.HIF-1αincreased the glycolysis rate,maintained mitochondrial function,and decreased the tenderness of yak meat.GLUT-1,LDH and HK increase first and then decrease with the extension of maturation time（P<0.01）,all the groups treated with HIF-1αinhibitor YC-1 had decreased lactic acid content and GLUT-1 expression（P<0.01）,LDH and HK decreased,indicating that postmortem hypoxia environment provided the necessary conditions for HIF-1αexpression,and HIF-1αaccelerated glycolysis rate by upregulating glucose GLUT-1 and glycolytic enzymes（LDH and HK）during maturation.After YC-1 treatment,the expression of PDK-1 increased significantly within 12 h and decreased after 24-120 h（P<0.05）,the activity of F0F1-ATP decreased,while the mitochondrial morphology did not change significantly,indicating that HIF-1αdid not change the integrity of mitochondria,upregulated the expression of PDK-1 protein to maintain mitochondrial function,and increased the activity of F0F1-ATP（P<0.05）,reduces the flow of pyruvate into the mitochondria,thereby increasing lactic acid production and prolonging the glycolytic pathway.In addition,the shear force of the control group was significantly higher than that of the treatment group（P<0.01）,indicating that high expression of HIF-1αwould increase the glycolysis rate and negatively affect meat tenderization during postmortem maturation.3.HIF-1αpromoted the release of calcium in endoplasmic reticulum,inhibited the occurrence of apoptosis,and resulted in the decrease of tenderness of yak meat.The ryanodine receptor（Ry R）in the endoplasmic reticulum increased with the increase of HIF-1αexpression during the maturation of yaks after slaughter,and the expression of RYR increased first and then decreased with the extension of maturation time after slaughter（P<0.05）;SERCA activity was negatively correlated with HIF-1αexpression,and HIF-1αexpression was significantly decreased after YC-1 treatment（P<0.05）;With the increase of HIF-1αexpression,mitochondrial calcium ion concentration,mitochondrial membrane permeability conversion pore（MPTP）and cell apoptosis rate decreased,indicating that HIF-1αactivated the calcium regulatory factor of endoplasmic reticulum,inhibited the release of mitochondrial calcium,and increased cytoplasmic calcium concentration inhibited cell apoptosis.With the increase of HIF-1αexpression,AMPK activity and CaMKKβexpression were significantly increased（P<0.05）and yak shear force increased（P<0.05）,indicating that HIF-1αoverexpression increased the intracellular calcium ion concentration to activate CaMKKβactivity,activate downstream factor AMPK activity,inhibit cytochrome c activity,inhibit the occurrence of cell apoptosis and then lead to the deterioration of tenderness.4.PI3K/Akt signaling pathway up-regulates HIF-1αand increases glycolysis rate,leading to the deterioration of yak tenderness.Hypoxia increased the expression of PI3K,Akt and P-Akt during the maturation of yak meat after slaughter（P<0.05）,inhibition of PI3K/Akt signaling pathway can inhibit HIF-1αexpression（P<0.05）,indicating that hypoxia induced the expression of PI3K,Akt,P-Akt and HIF-1α,and PI3K and Akt were the upstream proteins inducing HIF-1αexpression（P<0.05）,during which Akt phosphorylation is activated and the downstream factor HIF-1αis activated.Inhibition of PI3K/Akt signaling pathway decreased the activities of HK,LDH,AMPK and mitochondrial complexⅤ（P<0.05）,increased the activities of Na⁺/K⁺-ATPase and Ca²⁺-ATPase（P<0.05）,the p H value of the control group was the highest,and the lactic acid content and p H value of the PI3K/Akt inhibited group were the lowest（P<0.05）,which is not conducive to the synthesis of ATP and the activation of glycolysis potential,which slows down the rate of glycogen consumption.During the whole postmortem maturation process,the control group had the highest shear force and minimum MFI,while the PI3K/Akt inhibition group had the lowest shear force and maximum MFI（P<0.05）,indicating that PI3K/Akt signaling pathway up-regulates HIF-1α,maintains mitochondrial energy metabolism,increases muscle postmortem glycolysis potential and down-regulates meat tenderness.5.High oxygen modified packaging down-regulated HIF-1αexpression,maintained calcium flow in endoplasmic reticulum and activated apoptosis pathway,thus improving the tenderness of yak meat.80%O₂-Hi Ox-MAP significantly increased HIF-1α,Ry R expression and SERCA activity in yak（P<0.05）;EDS map showed that the distribution of mitochondrial calcium increased gradually in the group treated with hyperox-modified packaging,indicating that Hi Ox-MAP treatment can reduce the expression of HIF-1α,inhibit the expression of calcium regulatory factors in the endoplasmic reticulum,increase the concentration of mitochondrial calcium ions,and may induce mitochondrial pathway to induce apoptosis.In addition,Hi Ox-MAP treatment increased Caspase-3 activity,apoptosis rate and MFI（P<0.05）,down-regulated the activities of calmodulin protein（CaMKKβ）and AMPK（P<0.05）,indicating that Hi Ox-MAP treatment can improve meat tenderness by decreasing HIF-1αexpression and increasing mitochondrial calcium ion concentration,which can activate Caspase-3 activity and promote apoptosis of muscle cells during postmortem maturation.In conclusion,HIF-1αexpression in yak muscle increases with elevation,and GP,shear force,water retention and a*increase with HIF-1αexpression change.HIF-1αmaintains mitochondrial function by activating GLUT-1 expression and glycolytic enzyme activity,and by activating PDK-1 expression and F0F1-ATPase activity,providing necessary conditions for increasing glycolytic flux.HIF-1αactivates Ca²⁺regulatory factors in the ER and induces a large influx of Ca²⁺into the cytoplasm,thereby inducing increased activities of CaMKKβand AMPK,shutting down MPTP and Ca²⁺into mitochondria,inhibiting the activities of Caspase-9 and downstream Caspase-3 and inhibiting the occurrence of apoptosis.Hypoxia activates PI3K/Akt signaling pathway to induce HIF-1αaccumulation,increase the activities of HK,LDH,AMPK and mitochondrial complex V,decrease the activities of Na⁺/K⁺-ATPase and Ca²⁺-ATPase,and finally lead to the increase of glycolysis potential to affect the changes of meat quality during post-slaughter maturation.Hi Ox-MAP inhibited CaMKKβand AMPK activities by down-regulating HIF-1αexpression and Ry R expression of endoplasmic reticulum,promoted the opening of MPTP and increased mitochondrial calcium ion concentration,activated Caspase-9 and downstream Caspase-3 activities,and led to the occurrence of mitochondrial apoptosis,thus improving the tenderness of yak meat.