Search

Key points Intracellular [Na+] ([Na+]i) is elevated in heart failure (HF) and causes arrhythmogenic cellular [Ca2+]i loading., In HF, hyperactivity of Ca2+-calmodulin-dependent protein kinase II (CaMKII), a key mediator of electrical and mechanical dysfunction in myocytes, causes elevated [Na+]i., We developed a computational model of mouse ventricular myocyte electrophysiology including Ca2+ and CaMKII signalling and quantitatively confirmed evidence suggesting that not only does CaMKII cause elevated [Na+]i, but this additional [Na+]i also promotes further CaMKII activation by increasing [Ca2+]i., We found that a 3-4 m m gain in [Na+]i (similar to that reported in HF) perturbs Ca2+ and membrane potential homeostasis in part via CaMKII activation. This disrupted Ca2+ homeostasis is exacerbated by CaMKII overexpression, and strongly relies upon CaMKII-Na+-Ca2+-CaMKII feedback., CaMKII inhibition in HF may be beneficial, in part by inhibiting [Na+]i loading, and thereby normalizing Ca2+ and membrane potential dynamics without disrupting systolic function., Abstract Ca2+-calmodulin-dependent protein kinase II (CaMKII) hyperactivity in heart failure causes intracellular Na+ ([Na+]i) loading (at least in part by enhancing the late Na+ current). This [Na+]i gain promotes intracellular Ca2+ ([Ca2+]i) overload by altering the equilibrium of the Na+-Ca2+ exchanger to impair forward-mode (Ca2+ extrusion), and favour reverse-mode (Ca2+ influx) exchange. In turn, this Ca2+ overload would be expected to further activate CaMKII and thereby form a pathological positive feedback loop of ever-increasing CaMKII activity, [Na+]i, and [Ca2+]i. We developed an ionic model of the mouse ventricular myocyte to interrogate this potentially arrhythmogenic positive feedback in both control conditions and when CaMKIIδC is overexpressed as in genetically engineered mice. In control conditions, simulation of increased [Na+]i causes the expected increases in [Ca2+]i, CaMKII activity, and target phosphorylation, which degenerate into unstable Ca2+ handling and electrophysiology at high [Na+]i gain. Notably, clamping CaMKII activity to basal levels ameliorates but does not completely offset this outcome, suggesting that the increase in [Ca2+]i per se plays an important role. The effect of this CaMKII-Na+-Ca2+-CaMKII feedback is more striking in CaMKIIδC overexpression, where high [Na+]i causes delayed afterdepolarizations, which can be prevented by imposing low [Na+]i, or clamping CaMKII phosphorylation of L-type Ca2+ channels, ryanodine receptors and phospholamban to basal levels. In this setting, Na+ loading fuels a vicious loop whereby increased CaMKII activation perturbs Ca2+ and membrane potential homeostasis. High [Na+]i is also required to produce instability when CaMKII is further activated by increased Ca2+ loading due to β-adrenergic activation. Our results support recent experimental findings of a synergistic interaction between perturbed Na+ fluxes and CaMKII, and suggest that pharmacological inhibition of intracellular Na+ loading can contribute to normalizing Ca2+ and membrane potential dynamics in heart failure. [ABSTRACT FROM AUTHOR]

Disruption of the transverse-axial tubule system (TATS) in diseases such as heart failure and atrial fibrillation occurs in combination with changes in the expression and distribution of key Ca 2+ -handling proteins. Together this ultrastructural and ionic remodelling is associated with aberrant Ca 2+ cycling and electrophysiological instabilities that underlie arrhythmic activity. However, due to the concurrent changes in TATs and Ca 2+ -handling protein expression and localization that occur in disease it is difficult to distinguish their individual contributions to the arrhythmogenic state. To investigate this, we applied our novel 3D human atrial myocyte model with spatially detailed Ca 2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca 2+ -handling proteins and variable TATS density on Ca 2+ -handling abnormality driven membrane instabilities. We show that modulating the expression and distribution of the sodium-calcium exchanger, ryanodine receptors and the sarcoplasmic reticulum (SR) Ca 2+ buffer calsequestrin have varying pro- and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca 2+ leak-load and Ca 2+ -voltage relationships. Interestingly, the impact of protein remodelling on Ca 2+ -driven proarrhythmic behaviour varied dramatically depending on TATS density, with intermediately tubulated cells being more severely affected compared to detubulated and densely tubulated myocytes. This work provides novel mechanistic insight into the distinct and interactive consequences of TATS and Ca 2+ -handling protein remodelling that underlies dysfunctional Ca 2+ cycling and electrophysiological instability in disease. KEY POINTS: In our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca 2+ handling with subcellular spatial details governed by the transverse-axial tubule system (TATS). Here we utilize this model to mechanistically examine the impact of TATS loss and changes in the expression and distribution of key Ca 2+ -handling proteins known to be remodelled in disease on Ca 2+ homeostasis and electrophysiological stability. We demonstrate that varying the expression and localization of these proteins has variable pro- and anti-arrhythmic effects with outcomes displaying dependence on TATS density. Whereas detubulated myocytes typically appear unaffected and densely tubulated cells seem protected, the arrhythmogenic effects of Ca 2+ handling protein remodelling are profound in intermediately tubulated cells. Our work shows the interaction between TATS and Ca 2+ -handling protein remodelling that underlies the Ca 2+ -driven proarrhythmic behaviour observed in atrial fibrillation and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodelling., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Intracellular calcium (Ca 2+ ) cycling is tightly regulated in the healthy heart ensuring effective contraction. This is achieved by transverse (t)-tubule membrane invaginations that facilitate close coupling of key Ca 2+ -handling proteins such as the L-type Ca 2+ channel and Na + -Ca 2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca 2+ store. Although less abundant and regular than in the ventricle, t-tubules also exist in atrial myocytes as a network of transverse invaginations with axial extensions known as the transverse-axial tubule system (TATS). In heart failure and atrial fibrillation, there is TATS remodelling that is associated with aberrant Ca 2+ -handling and Ca 2+ -induced arrhythmic activity; however, the mechanism underlying this is not fully understood. To address this, we developed a novel 3D human atrial myocyte model that couples electrophysiology and Ca 2+ -handling with variable TATS organization and density. We extensively parameterized and validated our model against experimental data to build a robust tool examining TATS regulation of subcellular Ca 2+ release. We found that varying TATS density and thus the localization of key Ca 2+ -handling proteins has profound effects on Ca 2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca 2+ concentration through decreased Ca 2+ extrusion. This elevated Ca 2+ increases RyR open probability causing spontaneous Ca 2+ releases and the promotion of arrhythmogenic waves (especially in the cell interior) leading to voltage instabilities through delayed afterdepolarizations. In summary, the present study demonstrates a mechanistic link between TATS remodelling and Ca 2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca 2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca 2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca 2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca 2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca 2+ and voltage instabilities through reduced Na + -Ca 2+ exchanger-mediated Ca 2+ removal, cleft Ca 2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca 2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca 2+ releases are larger and more frequent in the cell interior than at the periphery. Our work provides mechanistic insight into how atrial TATS remodelling can lead to Ca 2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Key Points: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) capture patient-specific genotype-phenotype relationships, as well as cell-to-cell variability of cardiac electrical activity Computational modelling and simulation provide a high throughput approach to reconcile multiple datasets describing physiological variability, and also identify vulnerable parameter regimes We have developed a whole-cell model of iPSC-CMs, composed of single exponential voltage-dependent gating variable rate constants, parameterized to fit experimental iPSC-CM outputs We have utilized experimental data across multiple laboratories to model experimental variability and investigate subcellular phenotypic mechanisms in iPSC-CMs This framework links molecular mechanisms to cellular-level outputs by revealing unique subsets of model parameters linked to known iPSC-CM phenotypes ABSTRACT: There is a profound need to develop a strategy for predicting patient-to-patient vulnerability in the emergence of cardiac arrhythmia. A promising in vitro method to address patient-specific proclivity to cardiac disease utilizes induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). A major strength of this approach is that iPSC-CMs contain donor genetic information and therefore capture patient-specific genotype-phenotype relationships. A cited detriment of iPSC-CMs is the cell-to-cell variability observed in electrical activity. We postulated, however, that cell-to-cell variability may constitute a strength when appropriately utilized in a computational framework to build cell populations that can be employed to identify phenotypic mechanisms and pinpoint key sensitive parameters. Thus, we have exploited variation in experimental data across multiple laboratories to develop a computational framework for investigating subcellular phenotypic mechanisms. We have developed a whole-cell model of iPSC-CMs composed of simple model components comprising ion channel models with single exponential voltage-dependent gating variable rate constants, parameterized to fit experimental iPSC-CM data for all major ionic currents. By optimizing ionic current model parameters to multiple experimental datasets, we incorporate experimentally-observed variability in the ionic currents. The resulting population of cellular models predicts robust inter-subject variability in iPSC-CMs. This approach links molecular mechanisms to known cellular-level iPSC-CM phenotypes, as shown by comparing immature and mature subpopulations of models to analyse the contributing factors underlying each phenotype. In the future, the presented models can be readily expanded to include genetic mutations and pharmacological interventions for studying the mechanisms of rare events, such as arrhythmia triggers., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: Cardiac electrophysiology and Ca 2+ handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca 2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca 2+ handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca 2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses., Abstract: Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca 2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca 2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca 2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P < 0.05) followed by shortening (46.5 ± 9.1 ms at t = 60 s, P = NS vs. t = 0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by β-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight-or-flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: [Ca 2+ ] i enhanced rabbit ventricular slowly activating delayed rectifier K + current (I Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K + current (I Kr ) amplitude and voltage dependence were unaffected by high [Ca 2+ ] i . When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca 2+ transient or when [Ca 2+ ] i was buffered to 500 nm., Abstract: The slowly activating delayed rectifier K + current (I Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca 2+ ([Ca 2+ ] i ) and β-adrenergic receptor (β-AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca 2+ ] i dependence of I Ks in steady-state conditions and with dynamically changing membrane potential and [Ca 2+ ] i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca 2+ ] i , we found that raising [Ca 2+ ] i from 100 to 600 nm produced similar increases in I Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca 2+ ] i increased maximally activated tail I Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I Ks recorded during a normal physiological Ca 2+ transient was similar to I Ks measured with [Ca 2+ ] i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca 2+ ] i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca 2+ ] i , in the submembrane or junctional cleft space, is not required to maximize [Ca 2+ ] i -dependent I Ks activation during normal Ca 2+ transients., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Sarcoplasmic reticulum (SR) Ca(2+) release mediates excitation–contraction coupling (ECC) in cardiac myocytes. It is triggered upon membrane depolarization by entry of Ca(2+) via L-type Ca(2+) channels (LTCCs), which undergo both voltage- and Ca(2+)-dependent inactivation (VDI and CDI, respectively). We developed improved models of L-type Ca(2+) current and SR Ca(2+) release within the framework of the Shannon-Bers rabbit ventricular action potential (AP) model. The formulation of SR Ca(2+) release was modified to reproduce high ECC gain at negative membrane voltages. An existing LTCC model was extended to reflect more faithfully contributions of CDI and VDI to total inactivation. Ba(2+) current inactivation included an ion-dependent component (albeit small compared with CDI), in addition to pure VDI. Under physiological conditions (during an AP) LTCC inactivates predominantly via CDI, which is controlled mostly by SR Ca(2+) release during the initial AP phase, but by Ca(2+) through LTCCs for the remaining part. Simulations of decreased CDI or K(+) channel block predicted the occurrence of early and delayed after depolarizations. Our model accurately describes ECC and allows dissection of the relative contributions of different Ca(2+) sources to total CDI, and the relative roles of CDI and VDI, during normal and abnormal repolarization.