Deisl Christine, Chung Jay A, Hilgemann Donald W
Department of Physiology, Southwestern Medical Center, Dallas, TX 75235-9040 USA.
Laboratory of Obesity and Aging Research, Cardiovascular Branch, NHLBI, NIH, Bethesda, MD 20892, USA.
bioRxiv. 2023 Jan 3:2023.01.02.522313. doi: 10.1101/2023.01.02.522313.
Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses ∼50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10 cm /s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, ∼20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.
我们运用光学和电学方法记录到，随着分子量从30增加到2000，BL6小鼠心肌细胞胞质中的溶质扩散受限程度增加超过30倍，这大致符合心脏孔蛋白通道尺寸的孔的预期情况。Bodipy-FL ATP类似物在BL6心脏胞质中的扩散速度比在自由水中慢约50倍。从分析的几种荧光团来看，我们对结合荧光团比例的估计范围从2 kD FITC标记的聚乙二醇的0.1到磺基罗丹明的0.93。我们估计未结合荧光团的扩散系数范围为0.5至8×10 cm /s。对钠钾泵和藜芦定修饰的钠通道电流的分析证实，钠的扩散几乎不受限制（与移液管尖端平衡的时间常数约为20秒）。我们使用三种不同方法估计，ATP在BL6心肌细胞胞质中的扩散速度比在自由水中慢8至10倍。为了探究限制是更多由胞质蛋白还是膜网络引起的，我们首先验证了10克%明胶的蛋白凝胶会限制溶质扩散，且对分子量有强烈依赖性。然而，通过吸入大口径移液管尖端在横向受限的膜提取心肌肌丝中的溶质扩散，比在完整心肌细胞中受到的限制要小。值得注意的是，同等提取的骨骼肌（膈肌）肌丝对扩散的限制比心肌肌丝小。用β-七叶皂苷（80 µM）使肌膜通透的心肌细胞中的溶质扩散与完整心肌细胞中的情况类似受限。心脏心肌细胞中的扩散限制具有品系依赖性，BL6心肌细胞中的限制比具有CD1/J6/129svJ背景的心肌细胞大约大两倍。此外，缺乏线粒体孔蛋白Vdac1的CD1/J6/129svJ心肌细胞中的扩散受限程度比野生型CD1/J6/129svJ心肌细胞高2.5倍。我们得出结论，心肌肌丝和线粒体网络都会限制心脏心肌细胞中的扩散。因此，长距离溶质扩散可能优先通过孔蛋白通道和线粒体内膜间隙进行，在这些地方扩散受到的限制比在肌丝间隙中要小。
