| Objectives:Exposure to chronic hypoxia of myocardium is a main patho-physiological feature of various heart diseases.Mitochondrial biogenesis is one of the generally accepted regulatory mechanisms in the heart under chronic hypoxia.The precise quantity and quality control of mitochondria is critical for the survival and function of cardiomyocytes.Mitochondrial autophagy,also called mitophagy,which selectively eliminates dysfunctional and unwanted mitochondria,is the most important type of mitochondrial quality control.AMPK is Ser/Thr kinase in eukaryotic cells,it is well known playing a important role in maintaining intracellular homoeostasis.AMPK is activated under chronic hypoxia,furthermore,it has been demonstrated that AMPK regulates autophagy via numerous downstream pathways.So,we speculate that AMPK activation might paly a protective role to promote cellular survival via modulating mitophagy in the heart under chronic hypoxia.In this study,we observe the mitochondrial biogenesis and function of the cardiomyocyte,detect the AMPK activation and the influence on mitophagy and mitochondrial quality control of cardiomyocyte under chronic hypoxia.We want to investigate the regulatory mechanisms responsible for the mitophagy of cardiomyocyte under chronic hypoxia,find a new therapeutic strategy to improve the clinical treatment of heart disease under hypoxia.Methods:1.Patients with acyanotic(n = 10)or cyanotic(n = 10)cardiac disease were enrolled.Samples were taken from the right ventricular outflow tract in surgical operation.We used the qRT-PCR analysis to detect the mtDNA copies.The protein level of AMPK and p-AMPK were evaluated by western blot.2.H9c2 cells were devided into normoxic group,hypoxic group,AMPK activation group and AMPK inhibition group.The cells in the hypoxic group were placed in a cultivator containing a gaseous mixture of 94% N2,5% CO2 and 1% O2 at 37 °C for 48 h.In addition,the AMPK activation group was treated with AICAR,while the inhibition group was exposed to Compound C as the AMPK inhibitor.Cells in normoxic group were incubated under the same conditions except for 21% O2 concentration.The mtDNA copy of the cells in nomorxic group and hopoxic group is analyzed by qRT-PCR,The expression level of AMPK and p-AMPK were evaluated by western blot,and the mitophagy was detected by immunofluorescence.Fluorescence microscopy was performed in order to detect ??m in the cell cultivated under both hypoxia and normoxia condition staining by rhodamine-123 and mitotracker.We used the flow cytometry to evaluated the apoptosis and the mitochondrial function of H9c2 cellsResults:1.When compared the cyanotic patients with the acyanotic patients,we found the relative mtDNA / nDNA ratio was higher in the tissue of right ventricular outflow tract from the cyanotic patients[acyanotic patients(1.17 ± 0.26);cyanotic patients(1.97 ± 0.39),p < 0.05].Meanwhile,the ratio of p-AMPK / AMPK was higher in cyanotic patients[acyanotic patients(1);cyanotic patients(1.83 ± 0.21),p < 0.05].2.When compared with H9c2 cells in the normoxic group,the relative mtDNA / nDNA ratio was higher in the hypoxic group[normoxic group(1);hypoxic group(3.43 ± 0.65),p < 0.05],and the ratio of p-AMPK / AMPK was higher[normoxic group(1);hypoxic group(1.72 ± 0.16),p < 0.05].The percentage of the mitochondrial with intact mitochondrial membrane potential(??m)was decreased [normoxic group(89.00 ± 2.94)%;hypoxic group(67.75 ± 8.66)%,p < 0.05] and the ratio of the cells with decreased ??m was increased [normoxic group(25.90 ± 2.72)%;hypoxic group(32.73 ± 2.15)%,p < 0.05] in the hypoxic group.Furthermore,mitophagosomes were increased in the H9c2 cells cultured under hypoxia [treat without chloroquine,normoxic group(1.4 ± 1.14),hypoxic group(10.6 ± 3.05),p < 0.05;treat with chloroquine,normoxic group(4.2 ± 1.92),hypoxic group(21.6 ± 6.73),p < 0.05],and the percentage of apoptotic cells was higher in the hypoxic group[normoxic group [(1.69 ± 0.66)%;hypoxic group(5.01 ± 2.18)%,p < 0.05].3.When compared with the H9c2 cells in the hypoxic group,mitophagosomes were increased [treat without chloroquine,hypoxic group(10.6 ± 3.05),AMPK activation group(28.3 ± 3.96),p < 0.01;treat with chloroquine,hypoxic group(21.6 ± 6.73),AMPK activation group(42.4 ± 5.73),p < 0.01] and the percentage of the mitochondrial with intact ??m was increased in the AMPK activation group [ hypoxic group(67.75 ± 8.66)%,AMPK activation group(87.25 ± 2.75)%,p < 0.05],the ratio of H9c2 cells with the decreased ??m was decreased [ hypoxic group(32.73 ± 2.15)%;AMPK activation group(24.60 ± 2.01)%,p < 0.01] and the percentage of apoptotic cells was reduced [ hypoxic group(5.01 ± 2.18)%;AMPK activation group(2.10 ± 0.94)%,p < 0.05] in the AMPK activation group.In contrast,mitophagosomes were decreased [treat without chloroquine,hypoxic group(10.6 ± 3.05),AMPK inhibition group(2.6 ± 2.07),p < 0.01;treat with chloroquine,hypoxic group(21.6 ± 6.73),AMPK inhibition group(4.80 ± 2.39),p < 0.01],the percentage of the mitochondrial with intact ??m was decreased.[ hypoxic group(67.75 ± 8.66)%,AMPK inhibition group(44.19 ± 3.66)%,p < 0.05],the ratio of H9c2 cells with the reduced ??m was increased [ hypoxic group(32.73 ± 2.15)%;AMPK inhibition group(41.10 ± 1.16)%,p < 0.01],and the percentage of apoptotic cells was increased in the AMPK inhibition group[ hypoxic group(5.01 ± 2.18)%;AMPK inhibition group(7.06 ± 2.23)%,p < 0.05].Conclusions:1.Chronic hypoxia promoted the mitochondrial biogenesis,but induced mitochondrial damage.Mitophagy was facilitated under chronic hypoxia condition.2.Also,hypoxia induced the activation of AMPK.AMPK activation played a critical role in mitochondria quality control and promoting the cellular survival via modulating mitophagy in the heart under chronic hypoxia. |
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