The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability.

AIMS: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. METHODS AND RESULTS: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. CONCLUSION: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.

Computational analysis of the human sinus node action potential: model development and effects of mutations.

KEY POINTS: We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi-DiFrancesco model of rabbit SAN cells. Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally. Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model. The model shows that the modulatory role of the 'funny current' (If ) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude. The model may prove useful in the design of experiments and the development of heart-rate modulating drugs. ABSTRACT: The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart.  Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells.  We based our model on the recent Severi-DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller 'funny current' (If ) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss-of-function and gain-of-function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If , fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.

An updated computational model of rabbit sinoatrial action potential to investigate the mechanisms of heart rate modulation.

The cellular basis of cardiac pacemaking is still debated. Reliable computational models of the sinoatrial node (SAN) action potential (AP) may help gain a deeper understanding of the phenomenon. Recently, novel models incorporating detailed Ca(2+)-handling dynamics have been proposed, but they fail to reproduce a number of experimental data, and more specifically effects of 'funny' (I(f)) current modifications. We therefore developed a SAN AP model, based on available experimental data, in an attempt to reproduce physiological and pharmacological heart rate modulation. Cell compartmentalization and intracellular Ca(2+)-handling mechanisms were formulated as in the Maltsev-Lakatta model, focusing on Ca(2+)-cycling processes. Membrane current equations were revised on the basis of published experimental data. Modifications of the formulation of currents/pumps/exchangers to simulate I(f) blockers, autonomic modulators and Ca(2+)-dependent mechanisms (ivabradine, caesium, acetylcholine, isoprenaline, BAPTA) were derived from experimental data. The model generates AP waveforms typical of rabbit SAN cells, whose parameters fall within the experimental ranges: 352 ms cycle length, 80 mV AP amplitude, -58 mV maximum diastolic potential (MDP), 108 ms APD(50), and 7.1 Vs(-1) maximum upstroke velocity. Rate modulation by I(f) -blocking drugs agrees with experimental findings: 20% and 22% caesium-induced (5mM) and ivabradine-induced (3 µM) rate reductions, respectively, due to changes in diastolic depolarization (DD) slope, with no changes in either MDP or take-off potential (TOP). The model consistently reproduces the effects of autonomic modulation: 20% rate decrease with 10 nM acetylcholine and 28%increase with 1 µM isoprenaline, again entirely due to increase in the DD slope,with no changes in either MDP or TOP. Model testing of BAPTA effects showed slowing of rate, -26%, without cessation of beating. Our up-to-date model describes satisfactorily experimental data concerning autonomic stimulation, funny-channel blockade and inhibition of the Ca(2+)-related system by BAPTA, making it a useful tool for further investigation. Simulation results suggest that a detailed description of the intracellular Ca(2+) fluxes is fully compatible with the observation that I(f) is a major component of pacemaking and rate modulation.