The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca2+ and Mg2+ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.

Correct detection of membrane currents under whole-cell patch–clamp conditions is limited by the transfer function of a recording system. The low-pass output filter of a recording amplifier alters the time course of membrane current and causes errors in relevant descriptors. To solve these problems, we developed a practical procedure for reconstruction of the time course of membrane currents based on deconvolution of recorded currents in frequency domain. The procedure was tested on membrane capacitance estimates from current responses to step voltage pulses. The reconstructed current responses, in contrast to original current records, could be described exactly by an adequate impedance model of a recorded cell. The reconstruction allowed to increase the accuracy and reliability of membrane capacitance measurements in wide range of cell sizes and to suppress the cross-talk errors well below the noise. Moreover, it allowed resolving the instabilities in recording conditions arising from parasitic capacitance and seal resistance variation. Complex tests on hardware models, on simulated data sets, and on living cells confirmed robustness and reliability of the deconvolution procedure. The aptitude of the method was demonstrated in isolated rat cardiac myocytes by recording of spontaneous vesicular events, by discerning the formation of a fusion pore, and by revealing artefacts due to unstable seal resistance.

Recent data on structure of dyads in cardiac myocytes indicate variable clustering of RyR calcium release channels. The question arises as to how geometric factors of RyR arrangement translate to their role in formation of calcium release events (CRE). Since this question is not experimentally testable in situ, we performed in silico experiments on a large set of calcium release site (CRS) models. The models covered the range of RyR spatial distributions observed in dyads, and included gating of RyRs with open probability dependent on Ca2+ and Mg2+ concentration. The RyR single-channel calcium current, varied in the range of previously reported values, was set constant in the course of CRE simulations. Other known features of dyads were omitted in the model formulation for clarity. CRE simulations initiated by a single random opening of one of the RyRs in a CRS produced spark-like responses with characteristics that varied with RyR vicinity, a newly defined parameter quantifying spatial distribution of RyRs in the CRSs, and with the RyR single-channel calcium current. The CRE characteristics followed the law of mass action with respect to a CRS state variable, defined as a weighed product of RyR vicinity and RyR single-channel calcium current. The results explained the structure-function relations among determinants of cardiac dyads on synergy principles and thus allowed to evolve the concept of CRS as a dynamic unit of cardiac dyad.