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In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.

The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current (ICa) controls the intensity of calcium release is not clear because of the stochastic nature of voltage-dependent gating of calcium channels (DHPRs) and of calcium-dependent gating of ryanodine receptors (RyRs). To disclose the relation between DHPR openings and the probability of calcium release, local calcium release activation by ICa was investigated in rat ventricular myocytes using patch-clamp and confocal microscopy. Calcium spikes were activated by temporally synchronized DHPR calcium current triggers, generated by instantaneous ‘tail’ICa and modulated by prepulse duration, by tail potential, and by the DHPR agonist BayK 8644. The DHPR–RyR coupling fidelity was determined from the temporal distribution of calcium spike latencies using a model based on exponentially distributed DHPR open times. The analysis provided a DHPR mean open time of ∼0.5 ms, RyR activation time constant of ∼0.6 ms, and RyR activation kinetics of the 4th order. The coupling fidelity was low due to the inherent prevalence of very short DHPR openings but was increased when DHPR openings were prolonged by BayK 8644. The probability of calcium release activation was high, despite low coupling fidelity, due to the activation of many DHPRs at individual release sites. We conclude that the control of calcium release intensity by physiological stimuli can be achieved by modulating the number and duration of DHPR openings at low coupling fidelity, thus avoiding the danger of inadvertently triggering calcium release events.

The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s(-1)) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order (n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from -50 to 0 mV commenced with a mean latency of 4.1 +/- 0.2 ms and peaked 6.7 +/- 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 +/- 0.1 ms, and the time constant of termination was 5.5 +/- 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.

NO donors were found to reduce the rate of Ca2+ release from isolated skeletal muscle sarcoplasmic reticulum (SR) and the open probability of single ryanodine receptor Ca2+ release channels (RyRCs) in planar lipid bilayers, and these effects were prevented by the NO quencher hemoglobin and reversed by 2-mercaptoethanol. Ca2+ release assessed in skeletal muscle homogenates was also reduced by NO that was generated in situ from l-arginine by endogenous, nitro-l-arginine methylester-sensitive NO-synthase. The effect of NO on the RyRC might explain NO-induced depression of contractile force in striated muscles and, since both RyRC isoforms and NOS isoenzymes are ubiquitous, may represent a wide-spread feedback mechanism in Ca2+ signaling; i.e. Ca-dependent activation of NO production and NO-evoked reduction of Ca2+ release from intracellular Ca2+ stores.