Laboratory of Systems Biology, Institute of Cybernetics, Tallinn University of Technology, Tallinn, Estonia.
Biophys J. 2010 Jun 16;98(12):2785-93. doi: 10.1016/j.bpj.2010.03.025.
Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.
心肌细胞具有细胞内扩散限制，这些限制将 ADP 和 ATP 在空间上分隔开。然而，用于预测扩散限制的模型使用了在大鼠心脏通透纤维中生成的数据集，其中扩散距离可能是不均匀的。通过使用分离的通透心肌细胞可以避免这种情况。本工作的目的是分析大鼠通透心肌细胞中 ATP 和 ADP 的细胞内扩散。为此，我们测量了呼吸速率、周围溶液中的 ATP 酶速率和 ADP 浓度。使用反映细胞不同程度区室化的数学模型对数据进行了分析。与先前的研究一致，我们发现线粒体外膜存在显著的扩散限制，并证实了线粒体与细胞中一部分 ATP 酶之间的功能偶联。此外，我们的实验数据表明，通透化后心肌细胞中仍存在相当数量的内源性丙酮酸激酶 (PK) 的活性。没有这种内源性 PK 的 ATP 反馈，一部分 ATP 酶是无活性的。在分析数据时，我们仅通过将内源性 PK 和 ATP 酶的一部分紧密偶联的数学模型才能重现测量结果。据我们所知，这是首次在通透化的心肌细胞中证明 PK 与 ATP 酶之间存在如此强的偶联。
