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Shear Fluid‐induced Ca2+ Release and the Role of Mitochondria in Rat Cardiac Myocytes

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 (∼20 ms) application of shear fluid forces (∼25 dynes/cm2) to rat cardiac myocytes triggers slowly (∼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 (ΔΨm) 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 μM digitonin and perfused with an “intracellular” solution containing 10 μM 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 ΔΨm, while application of a pressurized solution produced a transient ΔΨm 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.