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 ( 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.

Multimodality of Ca2+ signaling in rat atrial myocytes.

It has been suggested that the multiplicity of Ca(2+) signaling pathways in atrial myocytes may contribute to the variability of its function. This article reports on a novel Ca(2+) signaling cascade initiated by mechanical forces induced by "puffing" of solution onto the myocytes. Ca(i) transients were measured in fura-2 acetoxymethyl (AM) loaded cells using alternating 340- and 410-nm excitation waves at 1.2 kHz. Pressurized puffs of bathing solutions, applied by an electronically controlled micro-barrel system, activated slowly (approximately 300 ms) developing Ca(i) transients that lasted 1,693 +/- 68 ms at room temperature. Subsequent second and third puffs, applied at approximately 20 s intervals activated significantly smaller or no Ca(i) transients. Puff-triggered Ca(i) transients could be reactivated once again following caffeine (10 mM)-induced release of Ca(2+) from sarcoplasmic reticulum (SR). Puff-triggered Ca(i) transients were independent of [Ca(2+)](o), and activation of voltage-gated Ca(2+) or cationic stretch channels or influx of Ca(2+) on Na(+)/Ca(2+)exchanger, because puffing solution containing no Ca(2+), 10 microM diltiazem, 1 mM Cd(2+), 5 mM Ni(2+), or 100 microM Gd(3+) failed to suppress them. Puff-triggered Ca(i) transients were enhanced in paced compared to quiescent myocytes. Electrically activated Ca(i) transients triggered during the time course of puff-induced transients were unaltered, suggesting functionally separate Ca(2+) pools. Contribution of inositol 1,4,5-triphosphate (IP(3))-gated or mitochondrial Ca(2+) pools or modulation of SR stores by nitric oxide/nitric oxide synthase (NO/NOS) signaling were evaluated using 0.5 to 500 microM 2-aminoethoxydiphenyl borate (2-APB) and 0.1 to 1 microM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 1 mM Nomega-Nitro-L-arginine methyl ester (L-NAME) and 7-nitroindizole, respectively. Only FCCP appeared to significantly suppress the puff-triggered Ca(i) transients. It was concluded that neither Ca(2+) influx nor depolarization was required for activation of this signaling pathway. These studies suggest that pressurized puffs of solutions activate a mechanically sensitive receptor, which signals in turn the release of Ca(2+) from a limited Ca(2+) store of mitochondria. How mechanical forces are sensed and transmitted to mitochondria to induce Ca(2+) release and what role such a Ca(2+) signaling pathway plays in the physiology or pathophysiology of the heart remain to be worked out.