Zhang Xiao-Hua, Wei Hua, Šarić Tomo, Hescheler Jürgen, Cleemann Lars, Morad Martin
Cardiac Signaling Center of USC, MUSC, & Clemson University, Charleston, SC, USA.
Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
Cell Calcium. 2015 May;57(5-6):321-36. doi: 10.1016/j.ceca.2015.02.003. Epub 2015 Feb 18.
The quintessential property of developing cardiomyocytes is their ability to beat spontaneously. The mechanisms underlying spontaneous beating in developing cardiomyocytes are thought to resemble those of adult heart, but have not been directly tested. Contributions of sarcoplasmic and mitochondrial Ca(2+)-signaling vs. If-channel in initiating spontaneous beating were tested in human induced Pluripotent Stem cell-derived cardiomyocytes (hiPS-CM) and rat Neonatal cardiomyocytes (rN-CM). Whole-cell and perforated-patch voltage-clamping and 2-D confocal imaging showed: (1) both cell types beat spontaneously (60-140/min, at 24°C); (2) holding potentials between -70 and 0mV had no significant effects on spontaneous pacing, but suppressed action potential formation; (3) spontaneous pacing at -50mV activated cytosolic Ca(2+)-transients, accompanied by in-phase inward current oscillations that were suppressed by Na(+)-Ca(2+)-exchanger (NCX)- and ryanodine receptor (RyR2)-blockers, but not by Ca(2+)- and If-channels blockers; (4) spreading fluorescence images of cytosolic Ca(2+)-transients emanated repeatedly from preferred central cellular locations during spontaneous beating; (5) mitochondrial un-coupler, FCCP at non-depolarizing concentrations (∼50nM), reversibly suppressed spontaneous pacing; (6) genetically encoded mitochondrial Ca(2+)-biosensor (mitycam-E31Q) detected regionally diverse, and FCCP-sensitive mitochondrial Ca(2+)-uptake and release signals activating during INCX oscillations; (7) If-channel was absent in rN-CM, but activated only negative to -80mV in hiPS-CM; nevertheless blockers of If-channel failed to alter spontaneous pacing.
发育中的心肌细胞的典型特性是其自发搏动的能力。发育中心肌细胞自发搏动的机制被认为与成年心脏相似，但尚未经过直接验证。在人诱导多能干细胞衍生的心肌细胞（hiPS-CM）和大鼠新生心肌细胞（rN-CM）中，测试了肌浆网和线粒体Ca(2+)信号与If通道在引发自发搏动中的作用。全细胞和穿孔膜片钳电压钳制以及二维共聚焦成像显示：（1）两种细胞类型均能自发搏动（在24°C时为60 - 140次/分钟）；（2）-70至0mV之间的钳制电位对自发起搏无显著影响，但会抑制动作电位的形成；（3）在-50mV时的自发起搏激活了胞质Ca(2+)瞬变，同时伴有同相内向电流振荡，该振荡被钠钙交换体（NCX）和兰尼碱受体（RyR2）阻滞剂抑制，但未被Ca(2+)和If通道阻滞剂抑制；（4）在自发搏动期间，胞质Ca(2+)瞬变的扩散荧光图像反复从细胞内的特定中心位置发出；（5）线粒体解偶联剂FCCP在非去极化浓度（约50nM）下可逆地抑制自发起搏；（6）基因编码的线粒体Ca(2+)生物传感器（mitycam-E31Q）检测到在INCX振荡期间区域不同且对FCCP敏感的线粒体Ca(2+)摄取和释放信号被激活；（7）rN-CM中不存在If通道，而在hiPS-CM中仅在负于-80mV时被激活；然而，If通道阻滞剂未能改变自发起搏。
