丙酮酸激酶M2异构体调控心肌细胞中线粒体生物发生及能量代谢

Pyruvate kinase M2 isoform regulates mitochondrial biogenesis and energy metabolism in cardiomyocytes

目的探讨丙酮酸激酶M2异构体(PKM2)在缺氧条件下心肌细胞中线粒体生物发生、线粒体动力学及能量代谢中的作用。方法给予大鼠心肌细胞系H9C2不同浓度梯度的氯化钴(CoCl_(2))刺激,模拟体内的缺氧环境,通过反转录聚合酶链反应和western blot方法检测PKM2和线粒体生物发生及能量代谢相关分子的表达量;分离H9C2心肌细胞中的胞浆和胞核蛋白,western blot检测PKM2在H9C2心肌细胞中的定位,同时免疫荧光染色检测PKM2在H9C2心肌细胞中的定位;使用三磷酸腺苷(ATP)检测试剂盒检测PKM2对缺氧条件下H9C2心肌细胞中ATP生成的影响;使用PKM2质粒和PKM2 siRNA转染H9C2心肌细胞,并给予CoCl_(2)(300μmol/L)刺激24 h,western blot检测PKM2对于H9C2心肌细胞中线粒体生物发生和线粒体融合的影响。结果PKM2在缺氧的H9C2心肌细胞中表达逐渐减少,与线粒体生物发生和功能相关分子的表达量密切相关。PKM2主要表达于心肌细胞的胞浆中,与线粒体标记分子热休克蛋白60(HSP60)共定位。PKM2过表达能促进缺氧心肌细胞中线粒体的生物发生、线粒体融合和ATP生成;反之,PKM2敲低能抑制缺氧心肌细胞中线粒体的生物发生、线粒体融合和ATP生成。结论PKM2对于缺氧条件下心肌细胞中的线粒体生物发生和线粒体动力学至关重要,从而控制心肌细胞的能量代谢。

Objective To investigate the role of pyruvate kinase M2 isomer(PKM2)in mitochondrial biogenesis,mitochondrial dynamics and energy metabolism in cardiomyocytes under hypoxia.Methods The rats cardiomyocytes were stimulated with cobalt chloride(CoCl_(2))at different concentration gradients of H9C2 to simulate hypoxia environment in vivo.The expression levels of PKM2,mitochondrial biogenesis and energy metabolism related molecules were detected by reverse transcription polymerase chain reaction and western blot.The cytoplasmic and cytonuclear proteins in H9C2 cardiomyocytes were isolated,and the location of PKM2 in H9C2 cardiomyocytes was detected by western blot,while the location of PKM2 in H9C2 cardiomyocytes was detected by immunofluorescence staining.Adenosine triphosphate(ATP)assay kit was used to detect the effect of PKM2 on ATP production in H9C2 cardiomyocytes under hypoxia.H9C2 cardiomyocytes were transfected with PKM2 plasmid and PKM2 siRNA and stimulated with CoCl_(2)(300μmol/L)for 24 hours.Western blot analysis was performed to determine the effects of PKM2 on mitochondrial biogenesis and mitochondrial fusion in H9C2 cardiomyocytes.Results The expression of PKM2 decreased gradually in hypoxic H9C2 cardiomyocytes,which was closely related to the expression of molecules related to mitochondrial biogenesis and function.PKM2 was mainly expressed in the cytoplasm of cardiomyocytes and colocates with the mitochondrial marker molecule heat shock protein 60(HSP60).PKM2 overexpression could promote mitochondrial biogenesis,mitochondrial fusion and ATP production in hypoxic cardiomyocytes.Conversely,PKM2 knockdown inhibited mitochondrial biogenesis,mitochondrial fusion and ATP production in hypoxic cardiomyocytes.Conclusion PKM2 is essential for mitochondrial biogenesis and dynamics in H9C2 cardiomyocytes under hypoxia conditions,thus controlling energy metabolism in H9C2 cardiomyocytes.