Givvimani S, Pushpakumar S B, Metreveli N, Veeranki S, Kundu S, Tyagi S C
Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY 40202, United States.
Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY 40202, United States.
Int J Cardiol. 2015;187:325-33. doi: 10.1016/j.ijcard.2015.03.352. Epub 2015 Mar 25.
Mitochondria constitute 30% of cell volume and are engaged in two dynamic processes called fission and fusion, regulated by Drp-1 (dynamin related protein) and mitofusin 2 (Mfn2). Previously, we showed that Drp-1 inhibition attenuates cardiovascular dysfunction following pressure overload in aortic banding model and myocardial infarction. As dynamic organelles, mitochondria are capable of changing their morphology in response to stress. However, whether such changes can alter their function and in turn cellular function is unknown. Further, a direct role of fission and fusion in cardiomyocyte contractility has not yet been studied. In this study, we hypothesize that disrupted fission and fusion balance by increased Drp-1 and decreased Mfn2 expression in cardiomyocytes affects their contractility through alterations in the calcium and potassium concentrations.
To verify this, we used freshly isolated ventricular myocytes from wild type mouse and transfected them with either siRNA to Drp-1 or Mfn2. Myocyte contractility studies were performed by IonOptix using a myopacer. Intracellular calcium and potassium measurements were done using flow cytometry. Immunocytochemistry (ICC) was done to evaluate live cell mitochondria and its membrane potential. Protein expression was done by western blot and immunocytochemistry.
We found that silencing mitochondrial fission increased the myocyte contractility, while fusion inhibition decreased contractility with simultaneous changes in calcium and potassium. Also, we observed that increase in fission prompted decrease in Serca-2a and increase in cytochrome c leakage leading to mitophagy.
Our results suggested that regulating mitochondrial fission and fusion have direct effects on overall cardiomyocyte contractility and thus function.
线粒体占细胞体积的30%，参与称为裂变和融合的两个动态过程，由动力相关蛋白1（Drp-1）和线粒体融合蛋白2（Mfn2）调节。此前，我们发现抑制Drp-1可减轻主动脉缩窄模型压力超负荷和心肌梗死后的心血管功能障碍。作为动态细胞器，线粒体能够响应应激改变其形态。然而，这种变化是否会改变其功能进而影响细胞功能尚不清楚。此外，裂变和融合在心肌细胞收缩性中的直接作用尚未得到研究。在本研究中，我们假设心肌细胞中Drp-1增加和Mfn2表达减少导致的裂变和融合平衡破坏会通过钙和钾浓度的改变影响其收缩性。
为验证这一点，我们使用从野生型小鼠新鲜分离的心室肌细胞，并用针对Drp-1或Mfn2的小干扰RNA（siRNA）转染它们。使用肌动起搏器通过IonOptix进行心肌细胞收缩性研究。使用流式细胞术测量细胞内钙和钾。进行免疫细胞化学（ICC）以评估活细胞线粒体及其膜电位。通过蛋白质印迹和免疫细胞化学检测蛋白质表达。
我们发现沉默线粒体裂变可增加心肌细胞收缩性，而抑制融合则会降低收缩性，同时钙和钾也发生变化。此外，我们观察到裂变增加促使肌浆网钙ATP酶2a（Serca-2a）减少，细胞色素c泄漏增加，导致线粒体自噬。
我们的结果表明，调节线粒体裂变和融合对心肌细胞的整体收缩性及功能有直接影响。
