Data_Sheet_1_Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.pdf

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

表达于心肌细胞（cardiac myocytes）的毒蕈碱受体（muscarinic receptors），在副交感神经系统（parasympathetic nervous system）调控心脏功能的过程中发挥关键作用。目前尚不明确心肌细胞的结构组织如何影响毒蕈碱受体对细胞内钙处理的调控作用。本研究采用共聚焦钙成像、膜片钳技术与免疫细胞化学方法，探究了正常小鼠心室肌细胞，以及经甲酰胺（formamide）处理后出现横小管（t-tubule）系统急性破坏的心肌细胞中，横小管密度与胆碱能调控细胞内钙的关联。毒蕈碱受体激动剂卡巴胆碱（carbachol, CCh，10 μM）对100 nM异丙肾上腺素（isoproterenol, Iso，β肾上腺素能激动剂）存在时经场刺激（field-stimulation）诱发的钙瞬态（Ca2+ transients）振幅的抑制效应，与心肌细胞脱横小管程度呈正相关。研究以钙敏感性染料fluo-4的荧光增幅最大速率的时间点，将图像像素划分为与肌膜（sarcolemma）钙内流（Ca2+ influx, ICa）功能偶联或非偶联的区域。卡巴胆碱可降低功能偶联区域的占比，并抑制钙信号从肌膜向细胞内部的传播。甲酰胺处理可降低钙电流密度，并减少肌浆网（sarcoplasmic reticulum, SR）内的钙储备。在异丙肾上腺素刺激的对照组与甲酰胺处理组心肌细胞中，卡巴胆碱均未改变肌浆网的钙储备水平。在异丙肾上腺素存在的条件下，卡巴胆碱可使对照组与脱横小管组心肌细胞记录到的峰值钙电流降低约20%。通过将电压阶跃从0 mV调整至-25 mV，将钙电流振幅降低至多40%时，仅在甲酰胺处理组（而非对照组）的异丙肾上腺素刺激心肌细胞中，钙瞬态振幅出现下降。卡巴胆碱可抑制钙/钙调蛋白依赖性蛋白激酶II（CaMKII）的活性，而使用KN93抑制CaMKII，可模拟卡巴胆碱对甲酰胺处理组心肌细胞钙瞬态的调控效应。本研究得出结论：横小管的下调与兴奋-收缩偶联（excitation–contraction coupling）效率降低相结合，会使钙释放与传播过程对毒蕈碱受体介导的钙电流与CaMKII活性抑制的敏感性升高。