Permeabilized Rat Cardiomyocyte Response Demonstrates Intracellular Origin of Diffusion Obstacles
Permeabilized Rat Cardiomyocyte Response Demonstrates Intracellular Origin of Diffusion Obstacles
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DOI：
10.1016/j.bpj.2011.09.025
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发表时间：
2011-11-02
影响因子：
3.4
通讯作者：
Vendelin, Marko
中科院分区：
文献类型：
--
作者：
Jepihhina, Natalja;Beraud, Nathalie;Vendelin, Marko
细胞内的ADP和其他分子的扩散限制已被预测较早的基础上渗透纤维或心肌细胞的实验。然而，由于纤维制剂中细胞的聚集和细胞的不完全分离，有效扩散距离可能大于细胞尺寸。这项工作的目的是检查是否存在扩散限制内大鼠心肌细胞或大的有效扩散距离所造成的。为此，我们确定了氧化磷酸化（OxPhos）的反应，外源性ADP和ATP刺激透化大鼠心肌细胞使用荧光显微镜。通过NADH和黄素蛋白自发荧光监测OxPhos的状态。通过改变流动室中ADP或ATP的浓度，我们确定OxPhos在心肌细胞中具有低亲和力。在荧光显微镜下观察到ADP诱导的心肌细胞自发荧光变化。ATP对OxPhos的刺激作用在荧光仪中比在显微镜下更强，这归因于ADP在荧光仪中的积累。通过计算周围的细胞在显微镜室中的流动轮廓和比较模型的解决方案，测量数据，我们证明，细胞内结构施加显着的扩散障碍，在大鼠心肌细胞。
Intracellular diffusion restrictions for ADP and other molecules have been predicted earlier based on experiments on permeabilized fibers or cardiomyocytes. However, it is possible that the effective diffusion distance is larger than the cell dimensions due to clumping of cells and incomplete separation of cells in fiber preparations. The aim of this work was to check whether diffusion restrictions exist inside rat cardiomyocytes or are caused by large effective diffusion distance. For that, we determined the response of oxidative phosphorylation (OxPhos) to exogenous ADP and ATP stimulation in permeabilized rat cardiomyocytes using fluorescence microscopy. The state of OxPhos was monitored via NADH and flavoprotein autofluorescence. By varying the ADP or ATP concentration in flow chamber, we determined that OxPhos has a low affinity in cardiomyocytes. The experiments were repeated in a fluoronneter on cardiomyocyte suspensions leading to similar autofluorescence changes induced by ADP as recorded under the microscope. ATP stimulated OxPhos more in a fluorometer than under the microscope, which was attributed to accumulation of ADP in fluorometer chamber. By calculating the flow profile around the cell in the microscope chamber and comparing model solutions to measured data, we demonstrate that intracellular structures impose significant diffusion obstacles in rat cardiomyocytes.