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Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes.

ABSTRACT: Cardiac mitochondria can take up Ca(2+), competing with Ca(2+) transporters like the sarcoplasmic reticulum (SR) Ca(2+)-ATPase. Rapid mitochondrial [Ca(2+)] transients have been reported to be synchronized with normal cytosolic [Ca(2+)](i) transients. However, most intra-mitochondrial free [Ca(2+)] ([Ca(2+)](mito)) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca(2+)](mito) in single rat myocytes using the ratiometric Ca(2+) indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca(2+)](mito) dependence on [Ca(2+)](i) (with 5 mM EGTA) was sigmoid with [Ca(2+)](mito)<[Ca(2+)](i) for [Ca(2+)](i) below 475 nM. With low [EGTA] (50 microM) and 150 nM [Ca(2+)](i) (+/-15 mM Na(+)) cyclical spontaneous SR Ca(2+) release occurred (5-15/min). Changes in [Ca(2+)](mito) during individual [Ca(2+)](i) transients were small ( approximately 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca(2+) handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca(2+)](mito) and spontaneous Ca(2+) transients (confirming that SR Ca(2+) releases caused the [Ca(2+)](mito) rise). Confocal imaging of local [Ca(2+)](mito) (using rhod-2) showed that [Ca(2+)](mito) rose rapidly with a delay after SR Ca(2+) release (with amplitude up to 10 nM), but declined much more slowly than [Ca(2+)](i) (time constant 2.8+/-0.7 s vs. 0.19+/-0.06 s). Total Ca(2+) uptake for larger [Ca(2+)](mito) transients was approximately 0.5 micromol/L cytosol (assuming 100:1 mitochondrial Ca(2+) buffering), consistent with prior indirect estimates from [Ca(2+)](i) measurements, and corresponds to approximately 1% of the SR Ca(2+) uptake during a normal Ca(2+) transient. Thus small phasic [Ca(2+)](mito) transients and gradually integrating [Ca(2+)](mito) signals occur during repeating [Ca(2+)](i) transients.

SUBMITTER: Andrienko TN

PROVIDER: S-EPMC2683203 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis.

Project description:Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca²+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca²+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca²+ dynamics: 1) the biphasic increment during the upstroke of the Ca²+ transient resulting from the delay between the peripheral and central SR Ca²+ release, and 2) the relative contribution of SL Ca²+ current and SR Ca²+ release to the Ca²+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca²+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca²+ release sites define the interface between Ca²+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca²+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca²+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca²+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes.

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Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes.

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