Changes And Mechanism Of Myocardial Energy Metabolism In Broilers With Ascites Syndrome

The broiler ascites syndrome(AS),known as broiler pulmonary hypertension syndrome(PHS),is a nutritional metabolic disease mainly occurring in fast-growing broilers,characterized by accumulation of a certain amount of light yellow liquid in body cavity and right ventricular hypertrophy.It is one of the important common diseases that seriously affect the healthy development of broiler breeding industry.Our previous studies have shown that the content of macrophage migration inhibitory factor(MIF)and the phosphorylation level of AMP-activated protein kinase(AMPK)were significantly higher in the hearts of clinical ascites broilers than that of healthy broilers in the same group,indicating that MIF was involved in the pathogenesis of AS.In this study,the key enzymes of energy metabolism were studied both in heart of clinical ascites broilers and the hypoxic chicken embryonic cardiomyocytes in order to reveal the pathogenesis of AS in broilers.Part 1: Study on the expression of key enzymes of energy metabolism such as MIF and AMPK in myocardium of clinical ascites broilers.The heart of clinical ascites broilers was studied as the research object,and the heart of healthy broilers in the same group was used as the control.Firstly,HE staining was performed to observe the micropathology of right ventricular muscle tissue.Secondly,western blotting was used to detect the expression level of MIF,the phosphorylation level of AMPK,key enzyme of energy level,as well as the expression of key enzymes of glycolysis pathway [hexokinase 2(HK2),phosphofructokinase 1(PFK1),phosphofructokinase 2(PFK2),and M2 pyruvate kinase(PKM2)],tricarboxylic acid cycle pathway[α-ketoglutarate dehydrogenase(OGDH),isocitrate dehydrogenase 2(IDH2),citrate synthase(CS)] and key proteins of fat acid β-oxidation pathway [carnitine acyl transferase-1A(CPT-1A)and phosphorylated acetyl Co A carboxylase(p-ACC)] in myocardial tissue.The results showed that compared with control,the structure of myofibril and myofilament in the cytoplasm of cardiomyocytes of AS broilers were disordered,and a large number of blood cells were deposited in the blood vessels between muscle fibers,indicating that the right heart failure in AS broilers.Compared with healthy broilers of the same group,the levels of MIF and AMPK phosphorylation in myocardium of AS broilers were significantly increased(P < 0.05);the key enzymes of glycolysis pathway and the key transporters of fatty acid β-oxidation pathway were significantly increased,while the key enzymes of tricarboxylic acid cycle were significantly decreased.The results showed that the energy metabolism of myocardial in broilers with ascites syndrome was enhanced by glycolysis and fatty acid oxidation,while the aerobic energy supply of tricarboxylic acid was weakened.Part 2: Construction of hypoxia model of cardiomyocytes in vitro and observation of the changes of energy metabolism(1)Isolation,culture and identification of primary chicken embryonic cardiomyocytesThe hearts of AA broiler embryos(9-12 days old)were used to obtain primary chicken embryonic cardiomyocytes.The hearts were cut into pieces and digested in trypsin and collagenase type Ⅱ,and then purified by differential adherent separation technique.The viability and morphology of the cells were observed under microscope and identified by immunocytochemistry with α-sarcomeric actin.The results showed that the cardiomyocytes obtained by the combined digestion method were adherent well,and the purity after differential separation was more than 90%.The cell viability was higher and beat spontaneously about 80-130 times/minute after 48 hours.(2)Establishment of hypoxia model of chicken embryo cardiomyocytesChicken embryo cardiomyocytes were treated with chemical hypoxia simulator cobalt chloride(Co Cl2),with the concentration of 400 μM,500 μM,600 μM and 700 μM.Hypoxia inducible factor 1α(HIF-1α)was used as a hypoxia marker.The expressions of HIF-1α and MIF were detected by WB technique to determine the optimal concentration of Co Cl2 to establish the hypoxia model.The results showed that compared with the control group,the expression of HIF-1α was significantly increased when the concentration of Co Cl2 was 500 μM,600 μM and 700 μM,and the highest in 600 μM Co Cl2 group.Therefore,600 μM Co Cl2 was added to the culture medium of isolated chicken embryo cardiomyocytes to establish the hypoxia model of cardiomyocytes.(3)Study on the changes of energy metabolism of chicken embryo cardiomyocytes under hypoxiaIn order to confirm the effect of hypoxia stimulation on the secretion of endogenous MIF,activation of AMPK and energy metabolism of cardiomyocytes,different concentrations of ISO-1(10 μM,50 μM,100 μM;the inhibitor of MIF),were added to hypoxia cultured cardiomyocytes.The expression levels of MIF and the phosphorylation levels of AMPK were detected by WB technique to determine the optimal concentration of ISO-1(100 μM).Then WB and q RT-PCR techniques were used to detect the expression of key proteins related to energy metabolism in chicken embryo cardiomyocytes.Three groups were set up in this study,namely as normoxic control group,hypoxia model group(600 μM Co Cl2)and MIF blocking group(600 μM Co Cl2 + 100 μM ISO-1).The results showed that: Compared with the normoxic group,the expression levels of MIF,phosphorylation levels of AMPK,the key enzymes of glycolysis pathway and the key proteins of fat β-oxidation pathway were significantly increased,while the key enzymes of tricarboxylic acid cycle were significantly decreased.The changes of key enzymes of glycolysis pathway(HK2,PFK1,PFK2),fatty acid oxidation pathway key proteins(p-ACC)and tricarboxylic acid cycle key enzymes(OGDH,IDH2,CS)in the MIF blocking group were opposite to those in the hypoxia model group with the significantly differences.indicating the changes of these key enzymes tended to normoxic culture after inhibition of MIF.The same trend was also confirmed by q RT-PCR(except PKM2 and CPT-1A).It can be concluded that hypoxia can promote the secretion of endogenous MIF,activate AMPK,promote glycolysis and fatty acid oxidation and inhibit aerobic oxidation in cardiomyocytes.In conclusion,in terms of myocardial energy metabolism in broilers,hypoxia promotes the secretion of endogenous MIF in myocardial.MIF activates AMPK indirectly through autocrine or paracrine action,and then activated AMPK to activate key enzymes or proteins in glycolysis and fatty acid metabolism pathways,and inhibit aerobic oxidation to provide compensatory energy supply for hypoxic myocardium,which may be conducive to maintain the function of anoxic myocardium.