Belmonte Steve, Morad Martin
Department of Pharmacology, Georgetown University, 3900 Reservoir Road, Washington, DC 20007, USA.
Ann N Y Acad Sci. 2008 Mar;1123:58-63. doi: 10.1196/annals.1420.007.
Cardiac myocyte contraction occurs when Ca2+ influx through voltage-gated L-type Ca2+ channels causes Ca2+ release from ryanodine receptors of the sarcoplasmic reticulum (SR). Although mitochondria occupy about 35% of the cell volume in rat cardiac myocytes, and are thought to be located <300 nm from the junctional SR, their role in the beat-to-beat regulation of cardiac Ca2+ signaling remains unclear. We have recently shown that rapid ( approximately 20 ms) application of shear fluid forces ( approximately 25 dynes/cm2) to rat cardiac myocytes triggers slowly ( approximately 300 ms) developing Cai transients that were independent of activation of all transmembrane Ca2+ transporting pathways, but were suppressed by FCCP, CCCP, and Ru360, all of which are known to disrupt mitochondrial function. We have here used rapid 2-D confocal microscopy to monitor fluctuations in mitochondrial Ca2+ levels ([Ca2+]m) and mitochondrial membrane potential (Delta Psi m) in rat cardiac myocytes loaded either with rhod-2 AM or tetramethylrhodamine methyl ester (TMRM), respectively. Freshly isolated intact rat cardiac myocytes were plated on glass coverslips and incubated in 5 mM Ca2+ containing Tyrode's solution and 40 mM 2,3-butanedione monoxime (BDM) to inhibit cell contraction. Alternatively, myocytes were permeabilized with 10 microM digitonin and perfused with an "intracellular" solution containing 10 microM free [Ca2+], 5 mM EGTA, and 15 mM BDM. Direct [Ca2+]m measurements showed transient mitochondrial Ca2+ accumulation after exposure to 10 mM caffeine, as revealed by a 66% increase in the rhod-2 fluorescence intensity. Shear fluid forces, however, produced a 12% decrease in signal, suggesting that application of a mechanical force releases Ca2+ from the mitochondria. In addition, caffeine and CCCP or FCCP strongly reduced Delta Psi m, while application of a pressurized solution produced a transient Delta Psi m hyperpolarization in intact ventricular myocytes loaded with TMRM. The close proximity of mitochondria to ryanodine receptors and large [Ca2+] that develop in microdomains following calcium release are likely to play a critical role in regulating cytosolic Ca2+ signaling. We suggest that mitochondria may accumulate and release Ca2+ in response to mechanical forces generated by blood flow, independent of surface membrane-regulated CICR. The extent to which such a signaling mechanism contributes to stretch-induced increase in myocardial force and pathogenesis of arrhythmias remains to be assessed.
当钙离子通过电压门控L型钙离子通道内流，导致肌浆网（SR）的兰尼碱受体释放钙离子时，心肌细胞发生收缩。尽管在大鼠心肌细胞中，线粒体占据细胞体积的约35%，并且被认为位于距离连接肌浆网小于300纳米处，但其在心脏钙离子信号逐搏调节中的作用仍不清楚。我们最近发现，对大鼠心肌细胞快速（约20毫秒）施加剪切流动力（约25达因/平方厘米）会触发缓慢（约300毫秒）发展的胞内钙离子瞬变，该瞬变独立于所有跨膜钙离子转运途径的激活，但被FCCP、CCCP和Ru360抑制，所有这些物质都已知会破坏线粒体功能。我们在此使用快速二维共聚焦显微镜来监测分别用罗丹明-2 AM或四甲基罗丹明甲酯（TMRM）加载的大鼠心肌细胞中线粒体钙离子水平（[Ca2+]m）和线粒体膜电位（ΔΨm）的波动。将新鲜分离的完整大鼠心肌细胞铺在玻璃盖玻片上，在含有5 mM钙离子的台氏液和40 mM 2,3 - 丁二酮单肟（BDM）中孵育以抑制细胞收缩。或者，用10 μM洋地黄皂苷使心肌细胞通透化，并用含有10 μM游离[Ca2+]、5 mM乙二醇双四乙酸（EGTA）和15 mM BDM的“细胞内”溶液灌注。直接[Ca2+]m测量显示，暴露于10 mM咖啡因后线粒体钙离子出现短暂积累，罗丹明-2荧光强度增加66%表明了这一点。然而，剪切流动力使信号降低了12%，这表明施加机械力会使钙离子从线粒体中释放出来。此外，咖啡因和CCCP或FCCP会强烈降低ΔΨm，而施加加压溶液会使加载TMRM的完整心室肌细胞中ΔΨm出现短暂超极化。线粒体与兰尼碱受体的紧密接近以及钙释放后微区中产生的大量[Ca2+]可能在调节胞质钙离子信号中起关键作用。我们认为，线粒体可能响应血流产生的机械力而积累和释放钙离子，独立于表面膜调节的钙诱导钙释放。这种信号机制在多大程度上导致牵张诱导的心肌力增加和心律失常的发病机制仍有待评估。
