Models of the cardiac L-type calcium current: a quantitative comparison

The L-type calcium current (ICaL) plays a critical role in cardiac electrophysiology, and models of ICaL are vital tools to predict arrhythmogenicity of drugs and mutations. Five decades of measuring and modelling ICaL have resulted in several competing theories (encoded in mathematical equations). However, the introduction of new models has not typically been accompanied by a data-driven critical comparison with previous work, so that it is unclear where predictions overlap or conflict, or which model is best suited for any particular application. We gathered 71 mammalian ICaL models, compared their structure, and reproduced simulated experiments to show that there is a large variability in their predictions, which was not substantially diminished when grouping by species or other categories. By highlighting the differences in these competing theories, listing major data sources, and providing simulation code, we have laid strong foundations for the development of a consensus model of ICaL.

L-type channel inactivation balances the increased peak calcium current due to absence of Rad in cardiomyocytes

The L-type Ca2+ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (ICa,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates ICa,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of ICa,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak ICa,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in ICa,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell ICa,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of ICa,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β1,β2-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β1,β2-AR still yielded modulated ICa,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca2+ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β1,β2-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.

Inward calcium current through the polycystin-2-dependent channels of renal primary cilia

In 15% of cases, autosomal dominant polycystic kidney disease (ADPKD) arises from defects in polycystin-2 (PC2). PC2 is a member of the TRPP subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a pro-cystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca2+ have yielded widely varying results. Here we report the mean macroscopic Ca2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from murine inner medullary collecting duct. Under conditions designed to isolate inward Ca2+ currents, a small inward Ca2+ current was detected in cilia with active PC2 channels, but not in cilia lacking those channels. The current was activated by addition of 10 µM internal Ca2+, which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was −1.8 pA at −190 mV; its conductance from −50 to −200 mV averaged 20 pS. Thus the native PC2 channels of renal primary cilium are able to conduct a small but detectable Ca2+ influx under the conditions tested. The possible consequences of this influx are discussed.

Characterization and modelling of the abnormal electrophysiological response under beta-adrenergic stimulation in hypertrophic cardiomyopathy

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Introduction Hypertrophic Cardiomyopathy (HCM) is the most common inheritable heart pathology and the main cause of sudden cardiac death in young adults. HCM patients often present an enhanced arrhythmogenicity that can lead to lethal arrhythmias, especially during exercise. Recent studies have shown an abnormal response of HCM myocytes to β-adrenergic stimulation (β-ARS), with prolongation of their action potential duration (APD). The mechanisms underlying this aberrant response to sympathetic stimulation remain unknown. Purpose To investigate the key ionic mechanisms underlying the HCM abnormal response to β-ARS using human-based experimental and computational methodologies. Methods Experimental ionic currents, action potential and calcium transient were recorded in human adult cardiomyocytes from control and HCM patients. Isoproterenol (10-7 mol/L) was used to elicit β-ARS. Whole-cell ruptured patch voltage clamp experiments were conducted to characterise L-type calcium and potassium currents, with recordings performed before and after 3 min of drug exposure. The latest models of human ventricular electrophysiology and beta-adrenergic receptor signalling were integrated and calibrated using the human measured data. Simulations under isoproterenol were performed to quantify the effects of β-ARS on the action potential and calcium transient. The role of the main ion currents affected by β-ARS and by HCM remodelling was evaluated. Results In vitro, isoproterenol shortened APD (-16 ± 3%) in control, while prolonging APD in HCM myocytes (+23 ± 8%). Analysis of the measured data indicated two possible mechanisms contributing to APD prolongation in HCM myocytes. Firstly, a protracted L-type calcium current, presenting slower inactivation kinetics in HCM compared to control. The relative increase of potassium currents under β-ARS was also lower in HCM myocytes. The developed in silico models of β-ARS replicated the behaviour observed in the experimental data, based on slower L-type calcium current inactivation kinetics and a smaller increase of potassium currents in HCM. In absence of β-ARS, simulated HCM cardiomyocytes exhibited prolonged APD compared to control (525 ± 88 vs 281 ± 56 ms, p < 0.001). Under β-ARS, APD in control was reduced (-16.46%), whereas APD was prolonged in HCM (+11.63%). Further analysis showed that the reduction of the potassium currents increment under β-ARS was the main cause of the APD prolongation in HCM myocytes, with L-type calcium inactivation minimally contributing to APD prolongation. Conclusions In this study we assessed the effects of β-ARS on ion currents and APD in control and HCM myocytes. Our modelling results suggest that the increase of potassium repolarising currents under β-ARS is greatly reduced in HCM cardiomyocytes, being the main mechanism underlying their APD prolongation. This APD prolongation may have severe consequences in HCM patients, increasing the risk of exercise-induced arrhythmias. Abstract Figure.

In silico simulations reveal that RYR distribution affects the dynamics of calcium release in cardiac myocytes

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.

Long-QT founder variant T309I-Kv7.1 with dominant negative pattern may predispose delayed afterdepolarizations under β-adrenergic stimulation

The variant c.926C > T (p.T309I) in KCNQ1 gene was identified in 10 putatively unrelated Czech families with long QT syndrome (LQTS). Mutation carriers (24 heterozygous individuals) were more symptomatic compared to their non-affected relatives (17 individuals). The carriers showed a mild LQTS phenotype including a longer QTc interval at rest (466 ± 24 ms vs. 418 ± 20 ms) and after exercise (508 ± 32 ms vs. 417 ± 24 ms), 4 syncopes and 2 aborted cardiac arrests. The same haplotype associated with the c.926C > T variant was identified in all probands. Using the whole cell patch clamp technique and confocal microscopy, a complete loss of channel function was revealed in the homozygous setting, caused by an impaired channel trafficking. Dominant negativity with preserved reactivity to β-adrenergic stimulation was apparent in the heterozygous setting. In simulations on a human ventricular cell model, the dysfunction resulted in delayed afterdepolarizations (DADs) and premature action potentials under β-adrenergic stimulation that could be prevented by a slight inhibition of calcium current. We conclude that the KCNQ1 variant c.926C > T is the first identified LQTS-related founder mutation in Central Europe. The dominant negative channel dysfunction may lead to DADs under β-adrenergic stimulation. Inhibition of calcium current could be possible therapeutic strategy in LQTS1 patients refractory to β-blocker therapy.

AMPK mediates regulation of voltage-gated calcium channels by leptin in isolated neurons from arcuate nucleus

Our results readily support the hypothesis that AMPK is responsible for the maintenance of the calcium current and mediates the fine-tuning modulation of the leptin response. The novelty of these results strengthens the critical role of AMPK in the general energy balance and homeostasis.

Nociceptin/orphanin FQ peptide receptor mediates inhibition of N-type calcium currents in vestibular afferent neurons of the rat

Our results show that in primary vestibular afferent neurons, activation of the nociceptin/orphanin FQ peptide receptor inhibits the N-type calcium current by a mechanism mediated by G proteins. We propose that calcium current inhibition modulates neurotransmitter release from vestibular afferents, producing a presynaptic modulation of vestibular input to vestibular nuclei, thus contributing to gain control in the vestibular afferent input.