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Ca(2+)-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 Ca(2+) ([Ca(2+)]i) overload by altering the equilibrium of the Na(+)-Ca(2+) exchanger to impair forward-mode (Ca(2+) extrusion), and favour reverse-mode (Ca(2+) influx) exchange. In turn, this Ca(2+) overload would be expected to further activate CaMKII and thereby form a pathological positive feedback loop of ever-increasing CaMKII activity, [Na(+)]i, and [Ca(2+)]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 [Ca(2+)]i, CaMKII activity, and target phosphorylation, which degenerate into unstable Ca(2+) 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 [Ca(2+)]i per se plays an important role. The effect of this CaMKII-Na(+)-Ca(2+)-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 Ca(2+) channels, ryanodine receptors and phospholamban to basal levels. In this setting, Na(+) loading fuels a vicious loop whereby increased CaMKII activation perturbs Ca(2+) and membrane potential homeostasis. High [Na(+)]i is also required to produce instability when CaMKII is further activated by increased Ca(2+) 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 Ca(2+) and membrane potential dynamics in heart failure.

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]

Background: Increased vascular Ca V 1.2 channel function causes enhanced arterial tone during hypertension. This is mediated by elevations in angiotensin II/protein kinase C signaling. Yet, the mechanisms underlying these changes are unclear. We hypothesize that α1 C phosphorylation at serine 1928 (S1928) is a key event mediating increased Ca V 1.2 channel function and vascular reactivity during angiotensin II signaling and hypertension., Methods and Results: The hypothesis was examined in freshly isolated mesenteric arteries and arterial myocytes from control and angiotensin II-infused mice. Specific techniques include superresolution imaging, proximity ligation assay, patch-clamp electrophysiology, Ca 2+ imaging, pressure myography, laser speckle imaging, and blood pressure telemetry. Hierarchical "nested" and appropriate parametric or nonparametric t test and ANOVAs were used to assess statistical differences. We found that angiotensin II redistributed the Ca V 1.2 pore-forming α1 C subunit into larger clusters. This was correlated with elevated Ca V 1.2 channel activity and cooperativity, global intracellular Ca 2+ and contraction of arterial myocytes, enhanced myogenic tone, and altered blood flow in wild-type mice. These angiotensin II-induced changes were prevented/ameliorated in cells/arteries from S1928 mutated to alanine knockin mice, which contain a negative modulation of the α1 C S1928 phosphorylation site. In angiotensin II-induced hypertension, increased α1 C clustering, Ca V 1.2 activity and cooperativity, myogenic tone, and blood pressure in wild-type cells/tissue/mice were averted/reduced in S1928 mutated to alanine samples., Conclusions: Results suggest an essential role for α1 C S1928 phosphorylation in regulating channel distribution, activity and gating modality, and vascular function during angiotensin II signaling and hypertension. Phosphorylation of this single vascular α1 C amino acid could be a risk factor for hypertension that may be targeted for therapeutic intervention.

Background: Substantial sex-based differences have been reported in atrial fibrillation (AF), but the underlying mechanisms are poorly understood., Objectives: This study sought to gain a mechanistic understanding of Ca 2+ -handling disturbances and Ca 2+ -driven arrhythmogenic events in male vs female atrial cardiomyocytes and establish their responses to Ca 2+ -targeted interventions., Methods: We integrated reported sex differences and AF-associated changes (ie, expression and phosphorylation of Ca 2+ -handling proteins, cardiomyocyte ultrastructural characteristics, and dimensions) into our human atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca 2+ -handling processes. Sex-specific responses of atrial cardiomyocytes to arrhythmia-provoking protocols and Ca 2+ -targeted interventions were evaluated., Results: Simulated quiescent cardiomyocytes showed increased incidence of Ca 2+ sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca 2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca 2+ transients compared with the male model. Sensitivity analysis uncovered distinct arrhythmogenic contributions of each component involved in sex and/or AF alterations. Specifically, increased ryanodine receptor phosphorylation emerged as the major SCR contributor in female AF cardiomyocytes, whereas reduced L-type Ca 2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulated Ca 2+ -targeted interventions identified potential strategies (eg, t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca 2 ⁺-ATPase) to attenuate Ca 2+ -driven arrhythmogenic events in women, and revealed enhanced efficacy when applied in combination., Conclusions: Sex-specific modeling uncovers increased Ca 2+ -driven arrhythmogenic events in female vs male atria in AF, and suggests combined Ca 2+ -targeted interventions are promising therapeutic approaches in women., Competing Interests: Funding Support and Author Disclosures This work was supported by American Heart Association Career Development Award 24CDA1258695 (to Dr Ni), Postdoctoral Fellowships 20POST35120462 (to Dr Ni), 24POST1195229 (to Dr Wu), and Predoctoral Fellowship 20PRE35120465 (to Dr Zhang); National Institutes of Health/National Heart, Lung, and Blood Institute grants R01HL131517 (to Drs Grandi, Louch, and Dobrev), R01HL170521 (to Drs Grandi and Ni), R01HL141214 and P01HL141084 (to Dr Grandi), R01HL136389 (to Dr Dobrev), R01HL089598 (to Dr Dobrev), R01HL163277 (to Dr Dobrev), R01HL160992 (to Dr Dobrev), R01HL165704 (to Dr Dobrev), and R00HL138160 (to Dr Morotti); National Institutes of Health Stimulating Peripheral Activity to Relieve Conditions Grant 1OT2OD026580-01 (to Dr Grandi); the Research Council of Norway (Grant No. 287395 to Dr Louch); UC Davis School of Medicine Dean’s Fellow Award (to Dr Grandi); the European Union (large-scale integrative project MAESTRIA, No. 965286, to Dr Dobrev); and the Burroughs Wellcome Fund–Doris Duke Charitable Foundation "COVID-19 Fund to Retain Clinical Scientists" Award (to Dr Morotti). All authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca 2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND)., Methods: Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca 2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking., Results: In both mouse and human SANs, caveolae compartmentalized HCN4, Ca v 1.2, Ca v 1.3, Ca v 3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca 2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca 2+ current, a negative shift of the HCN current ( I f ) activation curve, and a 40% reduction of Na + /Ca 2+ -exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND., Conclusions: SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

Despite evidence that women are at higher risk of drug-induced torsade de pointes and sudden cardiac death, female sex is vastly underrepresented in cardiovascular research, thus limiting our fundamental understanding of sex-specific arrhythmia mechanisms and our ability to predict arrhythmia propensity. To address this urgent clinical and preclinical need, we developed a quantitative tool that predicts the electrophysiological response to drug administration in female cardiomyocytes starting from data collected in males. We demonstrate the suitability of our translator for sex-specific cardiac safety assessment and include proof-of-concept application of our translator to in vitro and in vivo data., Competing Interests: Funding Support and Author Disclosures This work was funded by National Institutes of Health grants R01-HL131517, 1OT2OD026580-01, R01HL141214, and P01HL141084 to Dr Grandi and grant R00HL138160 to Dr Morotti; Burroughs Wellcome Fund (Doris Duke Charitable Foundation “COVID-19 Fund to Retain Clinical Scientists” award) to Dr Morotti; American Heart Association Postdoctoral Fellowship 20POST35120462 to Dr Ni. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Atrial fibrillation (AF), the most prevalent clinical arrhythmia, is associated with atrial remodelling manifesting as acute and chronic alterations in expression, function, and regulation of atrial electrophysiological and Ca2+-handling processes. These AF-induced modifications crosstalk and propagate across spatial scales creating a complex pathophysiological network, which renders AF resistant to existing pharmacotherapies that predominantly target transmembrane ion channels. Developing innovative therapeutic strategies requires a systems approach to disentangle quantitatively the pro-arrhythmic contributions of individual AF-induced alterations., Methods and Results: Here, we built a novel computational framework for simulating electrophysiology and Ca2+-handling in human atrial cardiomyocytes and tissues, and their regulation by key upstream signalling pathways [i.e. protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)] involved in AF-pathogenesis. Populations of atrial cardiomyocyte models were constructed to determine the influence of subcellular ionic processes, signalling components, and regulatory networks on atrial arrhythmogenesis. Our results reveal a novel synergistic crosstalk between PKA and CaMKII that promotes atrial cardiomyocyte electrical instability and arrhythmogenic triggered activity. Simulations of heterogeneous tissue demonstrate that this cellular triggered activity is further amplified by CaMKII- and PKA-dependent alterations of tissue properties, further exacerbating atrial arrhythmogenesis., Conclusions: Our analysis reveals potential mechanisms by which the stress-associated adaptive changes turn into maladaptive pro-arrhythmic triggers at the cellular and tissue levels and identifies potential anti-AF targets. Collectively, our integrative approach is powerful and instrumental to assemble and reconcile existing knowledge into a systems network for identifying novel anti-AF targets and innovative approaches moving beyond the traditional ion channel-based strategy., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

By sensing changes in intracellular Ca 2+ , small-conductance Ca 2+ -activated K + (SK) channels dynamically regulate the dynamics of the cardiac action potential (AP) on a beat-to-beat basis. Given their predominance in atria versus ventricles, SK channels are considered a promising atrial-selective pharmacological target against atrial fibrillation (AF), the most common cardiac arrhythmia. However, the precise contribution of SK current ( I SK ) to atrial arrhythmogenesis is poorly understood, and may potentially involve different mechanisms that depend on species, heart rates, and degree of AF-induced atrial remodeling. Both reduced and enhanced I SK have been linked to AF. Similarly, both SK channel up- and downregulation have been reported in chronic AF (cAF) versus normal sinus rhythm (nSR) patient samples. Here, we use our multiscale modeling framework to obtain mechanistic insights into the contribution of I SK in human atrial cardiomyocyte electrophysiology. We simulate several protocols to quantify how I SK modulation affects the regulation of AP duration (APD), Ca 2+ transient, refractoriness, and occurrence of alternans and delayed afterdepolarizations (DADs). Our simulations show that I SK activation shortens the APD and atrial effective refractory period, limits Ca 2+ cycling, and slightly increases the propensity for alternans in both nSR and cAF conditions. We also show that increasing I SK counteracts DAD development by enhancing the repolarization force that opposes the Ca 2+ -dependent depolarization. Taken together, our results suggest that increasing I SK in human atrial cardiomyocytes could promote reentry while protecting against triggered activity. Depending on the leading arrhythmogenic mechanism, I SK inhibition may thus be a beneficial or detrimental anti-AF strategy. NEW & NOTEWORTHY Using our established framework for human atrial myocyte simulations, we investigated the role of the small-conductance Ca 2+ -activated K + current ( I SK ) in the regulation of cell function and the development of Ca 2+ -driven arrhythmias. We found that I SK inhibition, a promising atrial-selective pharmacological strategy against atrial fibrillation, counteracts the reentry-promoting abbreviation of atrial refractoriness, but renders human atrial myocytes more vulnerable to delayed afterdepolarizations, thus potentially increasing the propensity for ectopic (triggered) activity.

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.)

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca 2+ /calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca 2+ sparks, contraction and L-type Ca 2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca 2+ load was assessed by rapid caffeine (10 mM) induced Ca 2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca 2+ spark frequency, SR Ca 2+ load, L-type Ca 2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca 2+ ] i decline, lower Ca 2+ spark rate and lower RyR phosphorylation, but with similar SR Ca 2+ load, L-type Ca 2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca 2+ uptake and RyR sensitivity, but not altering L-type Ca 2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca 2+ handling., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier Ltd.)

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.)

Background: Small-conductance Ca 2+ -activated K + (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study., Methods: Apamin-sensitive SK-channel current (I SK ) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF)., Results: I SK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified I K1 and I SK as major regulators of repolarization. Increased I SK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and I SK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced I SK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater I SK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased I SK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced I SK -upregulation., Conclusions: I SK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in I SK , which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating β adrenergic receptors (βARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight increase in AP firing rate. We found that while βAR stimulation significantly altered nearly all AP waveform parameters, the increase in firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the βAR-sensitive component of If showed that it accounts for ∼41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate., (© 2022 Peters et al.)

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish ( Danio rerio ) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411 , in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells., Competing Interests: YD, DL, JY, HB, HL, KJ, JY, HN, SM, TL, KC, JP, HC, YZ, JW, EG, ZL, YS, YL, AG, XX No competing interests declared, SE Reviewing editor, eLife, (© 2022, Ding et al.)

Animal experimentation is key in the evaluation of cardiac efficacy and safety of novel therapeutic compounds. However, interspecies differences in the mechanisms regulating excitation-contraction coupling can limit the translation of experimental findings from animal models to human physiology and undermine the assessment of drugs’ efficacy and safety. Here, we built a suite of translators for quantitatively mapping electrophysiological responses in ventricular myocytes across species. We trained these statistical operators using a broad dataset obtained by simulating populations of our biophysically detailed computational models of action potential and Ca 2+ transient in mouse, rabbit, and human. We then tested our translators against experimental data describing the response to stimuli, such as ion channel block, change in beating rate, and β-adrenergic challenge. We demonstrate that this approach is well suited to predicting the effects of perturbations across different species or experimental conditions and suggest its integration into mechanistic studies and drug development pipelines.

Torsade de Pointes (TdP), a rare but lethal ventricular arrhythmia, is a toxic side effect of many drugs. To assess TdP risk, safety regulatory guidelines require quantification of hERG channel block in vitro and QT interval prolongation in vivo for all new therapeutic compounds. Unfortunately, these have proven to be poor predictors of torsadogenic risk, and are likely to have prevented safe compounds from reaching clinical phases. Although this has stimulated numerous efforts to define new paradigms for cardiac safety, none of the recently developed strategies accounts for patient conditions. In particular, despite being a well-established independent risk factor for TdP, female sex is vastly under-represented in both basic research and clinical studies, and thus current TdP metrics are likely biased toward the male sex. Here, we apply statistical learning to synthetic data, generated by simulating drug effects on cardiac myocyte models capturing male and female electrophysiology, to develop new sex-specific classification frameworks for TdP risk. We show that (i) TdP classifiers require different features in females vs. males; (ii) male-based classifiers perform more poorly when applied to female data; and (iii) female-based classifier performance is largely unaffected by acute effects of hormones (i.e., during various phases of the menstrual cycle). Notably, when predicting TdP risk of intermediate drugs on female simulated data, male-biased predictive models consistently underestimate TdP risk in women. Therefore, we conclude that pipelines for preclinical cardiotoxicity risk assessment should consider sex as a key variable to avoid potentially life-threatening consequences for the female population., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (I f ) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify I f during the cardiac cycle in mouse SAMs. We found that I f is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, I f carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by I f , consistent with a contribution of I f to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of I f Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of I f during the sinoatrial AP. These results establish a new conceptual framework for the role of I f in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions., Competing Interests: The authors declare no competing interest.

Background : The mechanisms underlying dysfunction in the sinoatrial node (SAN), the heart's primary pacemaker, are incompletely understood. Electrical and Ca 2+ -handling remodeling have been implicated in SAN dysfunction associated with heart failure, aging, and diabetes. Cardiomyocyte [Na + ] i is also elevated in these diseases, where it contributes to arrhythmogenesis. Here, we sought to investigate the largely unexplored role of Na + homeostasis in SAN pacemaking and test whether [Na + ] i dysregulation may contribute to SAN dysfunction. Methods : We developed a dataset-specific computational model of the murine SAN myocyte and simulated alterations in the major processes of Na + entry (Na + /Ca 2+ exchanger, NCX) and removal (Na + /K + ATPase, NKA). Results : We found that changes in intracellular Na + homeostatic processes dynamically regulate SAN electrophysiology. Mild reductions in NKA and NCX function increase myocyte firing rate, whereas a stronger reduction causes bursting activity and loss of automaticity. These pathologic phenotypes mimic those observed experimentally in NCX- and ankyrin-B-deficient mice due to altered feedback between the Ca 2+ and membrane potential clocks underlying SAN firing. Conclusions : Our study generates new testable predictions and insight linking Na + homeostasis to Ca 2+ handling and membrane potential dynamics in SAN myocytes that may advance our understanding of SAN (dys)function.

Rationale: CaMKII (Ca 2+ -Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca 2+ handling and CaMKII activation in hypertrophy and HF., Objective: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice., Methods and Results: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca 2+ content, Ca 2+ transient amplitude, and [Ca 2+ ] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca 2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca 2+ ] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions., Conclusions: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca 2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca 2+ -CaMKIIδC axis contributes to eccentric hypertrophy and HF.

Background and Purpose: Pharmacotherapy of atrial fibrillation (AF), the most common cardiac arrhythmia, remains unsatisfactory due to low efficacy and safety concerns. New therapeutic strategies target atrial-predominant ion-channels and involve multichannel block (poly)therapy. As AF is characterized by rapid and irregular atrial activations, compounds displaying potent antiarrhythmic effects at fast and minimal effects at slow rates are desirable. We present a novel systems pharmacology framework to quantitatively evaluate synergistic anti-AF effects of combined block of multiple atrial-predominant K + currents (ultra-rapid delayed rectifier K + current, I Kur , small conductance Ca 2+ -activated K + current, I KCa , K 2P 3.1 2-pore-domain K + current, I K2P ) in AF., Experimental Approach: We constructed experimentally calibrated populations of virtual atrial myocyte models in normal sinus rhythm and AF-remodelled conditions using two distinct, well-established atrial models. Sensitivity analyses on our atrial populations was used to investigate the rate dependence of action potential duration (APD) changes due to blocking I Kur , I K2P or I KCa and interactions caused by blocking of these currents in modulating APD. Block was simulated in both single myocytes and one-dimensional tissue strands to confirm insights from the sensitivity analyses and examine anti-arrhythmic effects of multi-atrial-predominant K + current block in single cells and coupled tissue., Key Results: In both virtual atrial myocytes and tissues, multiple atrial-predominant K + -current block promoted favourable positive rate-dependent APD prolongation and displayed positive rate-dependent synergy, that is, increasing synergistic antiarrhythmic effects at fast pacing versus slow rates., Conclusion and Implications: Simultaneous block of multiple atrial-predominant K + currents may be a valuable antiarrhythmic pharmacotherapeutic strategy for AF., (© 2020 The British Pharmacological Society.)

Rationale: Cardiotoxic β 1 adrenergic receptor (β 1 AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β 1 AR and organizes a receptor signalosome., Objective: We aim to elucidate the dynamics of β 1 AR-SAP97 signalosome and its potential role in chronic cardiotoxic β 1 AR-CaMKII signaling that contributes to development of heart failure., Methods and Results: The integrity of cardiac β 1 AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β 1 AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β 1 AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β 1 AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β 1 AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β 1 AR-SAP97 complex and increases in CaMKII activity in hearts., Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β 1 AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Rationale: Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes., Objective: To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes., Methods and Results: Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K + ] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca 2+ overload and increased generation of both spontaneous Ca 2+ waves and delayed afterdepolarizations. However, similar Ca 2+ -dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na + -K + ATPase and Na + -Ca 2+ exchanger proteins within these structures, as reduction in Na + pump activity locally inhibited Ca 2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca 2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca 2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na + current, as they were rapidly blocked by tetrodotoxin. Na + current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K + current (I Kur ); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes., Conclusions: Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.

This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration-sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model-based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm., (© 2019 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

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.)

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

Quantitative systems modeling aims to integrate knowledge in different research areas with models describing biological mechanisms and dynamics to gain a better understanding of complex clinical syndromes. Heart failure (HF) is a chronic complex cardiac disease that results from structural or functional disorders impairing the ability of the ventricle to fill with or eject blood. Highly interactive and dynamic changes in mechanical, structural, neurohumoral, metabolic, and electrophysiological properties collectively predispose the failing heart to cardiac arrhythmias, which are responsible for about a half of HF deaths. Multiscale cardiac modeling and simulation integrate structural and functional data from HF experimental models and patients to improve our mechanistic understanding of this complex arrhythmia syndrome. In particular, they allow investigating how disease-induced remodeling alters the coupling of electrophysiology, Ca 2+ and Na + handling, contraction, and energetics that lead to rhythm derangements. The Ca 2+ /calmodulin-dependent protein kinase II, which expression and activity are enhanced in HF, emerges as a critical hub that modulates the feedbacks between these various subsystems and promotes arrhythmogenesis. This article is categorized under: Physiology > Mammalian Physiology in Health and Disease Models of Systems Properties and Processes > Mechanistic Models Models of Systems Properties and Processes > Cellular Models Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models., (© 2018 Wiley Periodicals, Inc.)

Background: Heart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na + current (I NaL ), L-type Ca 2+ current, and NCX (Na + /Ca 2+ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca 2+ buffers)., Methods: We measured the major inward currents and their Ca 2+ - and β-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control)., Results: AP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of I NaL decreased both parameters to the control level. I NaL was slightly increased in HF versus control even when intracellular Ca 2+ was strongly buffered. But under physiological AP clamp with normal Ca 2+ cycling, I NaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca 2+ /calmodulin-dependent protein kinase II] activity). β-Adrenergic stimulation (often elevated in HF) further enhanced I NaL . L-type Ca 2+ current was decreased in HF when Ca 2+ was buffered, but CaMKII-mediated Ca 2+ -dependent facilitation upregulated physiological L-type Ca 2+ current to the control level. Furthermore, L-type Ca 2+ current response to β-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling., Conclusions: Our results suggest that CaMKII-dependent upregulation of I NaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress.

In cardiac electrophysiology, there exist many sources of inter- and intra-personal variability. These include variability in conditions and environment, and genotypic and molecular diversity, including differences in expression and behavior of ion channels and transporters, which lead to phenotypic diversity (e.g., variable integrated responses at the cell, tissue, and organ levels). These variabilities play an important role in progression of heart disease and arrhythmia syndromes and outcomes of therapeutic interventions. Yet, the traditional in silico framework for investigating cardiac arrhythmias is built upon a parameter/property-averaging approach that typically overlooks the physiological diversity. Inspired by work done in genetics and neuroscience, new modeling frameworks of cardiac electrophysiology have been recently developed that take advantage of modern computational capabilities and approaches, and account for the variance in the biological data they are intended to illuminate. In this review, we outline the recent advances in statistical and computational techniques that take into account physiological variability, and move beyond the traditional cardiac model-building scheme that involves averaging over samples from many individuals in the construction of a highly tuned composite model. We discuss how these advanced methods have harnessed the power of big (simulated) data to study the mechanisms of cardiac arrhythmias, with a special emphasis on atrial fibrillation, and improve the assessment of proarrhythmic risk and drug response. The challenges of using in silico approaches with variability are also addressed and future directions are proposed.

Background: In human cardiac ventricle, I K1 is mainly comprised Kir2.1, but Kir2.2 and Kir2.3 heterotetramers occur and modulate I K1 . Long-QT syndrome-9-associated CAV3 mutations cause decreased Kir2.1 current density, but Kir2.x heterotetramers have not been studied. Here, we determine the effect of long-QT syndrome-9- CAV3 mutation F97C on Kir2.x homo- and heterotetramers and model-associated arrhythmia mechanisms., Methods and Results: Super-resolution microscopy, co-immunoprecipitation, cellular electrophysiology, on-cell Western blotting, and simulation of Purkinje and ventricular myocyte mathematical models were used. Kir2.x isoforms have unique subcellular colocalization in human cardiomyocytes and coimmunoprecipitate with Cav3. F97C-Cav3 decreased peak inward Kir2.2 current density by 50% (-120 mV; P =0.019) and peak outward by 75% (-40 mV; P <0.05) but did not affect Kir2.3 current density. FRET (Förster resonance energy transfer) efficiency for Kir2.2 with Cav3 is high, and on-cell Western blotting demonstrates decreased Kir2.2 membrane expression with F97C-Cav3. Cav3-F97C reduced peak inward and outward current density of Kir2.2/Kir2.1 or Kir2.2/Kir2.3 heterotetramers ( P <0.05). Only Cav3 scaffolding and membrane domains co-immunoprecipitation with Kir2.1 and Kir2.2 and Kir2.x-N-terminal Cav3 binding motifs are required for interaction. Mathematical Purkinje, but not ventricular, myocyte model incorporating simulated current reductions, predicts spontaneous delayed after-depolarization-mediated triggered activity., Conclusions: Kir2.x isoforms have a unique intracellular pattern of distribution in association with specific Cav3 domains and that critically depends on interaction with N-terminal Kir2.x Cav3-binding motifs. Long-QT syndrome-9- CAV3 mutation differentially regulates current density and cell surface expression of Kir2.x homomeric and heteromeric channels. Mathematical Purkinje cell model incorporating experimental findings suggests delayed after-depolarization-type triggered activity as a possible arrhythmia mechanism., (© 2018 American Heart Association, Inc.)

Current pharmacological therapy against atrial fibrillation (AF), the most common cardiac arrhythmia, is limited by moderate efficacy and adverse side effects including ventricular proarrhythmia and organ toxicity. One way to circumvent the former is to target ion channels that are predominantly expressed in atria vs. ventricles, such as K V 1.5, carrying the ultra-rapid delayed-rectifier K + current (I Kur ). Recently, we used an in silico strategy to define optimal K V 1.5-targeting drug characteristics, including kinetics and state-dependent binding, that maximize AF-selectivity in human atrial cardiomyocytes in normal sinus rhythm (nSR). However, because of evidence for I Kur being strongly diminished in long-standing persistent (chronic) AF (cAF), the therapeutic potential of drugs targeting I Kur may be limited in cAF patients. Here, we sought to simulate the efficacy (and safety) of I Kur inhibitors in cAF conditions. To this end, we utilized sensitivity analysis of our human atrial cardiomyocyte model to assess the importance of I Kur for atrial cardiomyocyte electrophysiological properties, simulated hundreds of theoretical drugs to reveal those exhibiting anti-AF selectivity, and compared the results obtained in cAF with those in nSR. We found that despite being downregulated, I Kur contributes more prominently to action potential (AP) and effective refractory period (ERP) duration in cAF vs. nSR, with ideal drugs improving atrial electrophysiology (e.g., ERP prolongation) more in cAF than in nSR. Notably, the trajectory of the AP during cAF is such that more I Kur is available during the more depolarized plateau potential. Furthermore, I Kur block in cAF has less cardiotoxic effects (e.g., AP duration not exceeding nSR values) and can increase Ca 2+ transient amplitude thereby enhancing atrial contractility. We propose that in silico strategies such as that presented here should be combined with in vitro and in vivo assays to validate model predictions and facilitate the ongoing search for novel agents against AF.

The K V 1.5 potassium channel, which underlies the ultra-rapid delayed-rectifier current (I Kur ) and is predominantly expressed in atria vs. ventricles, has emerged as a promising target to treat atrial fibrillation (AF). However, while numerous K V 1.5-selective compounds have been screened, characterized, and tested in various animal models of AF, evidence of antiarrhythmic efficacy in humans is still lacking. Moreover, current guidelines for pre-clinical assessment of candidate drugs heavily rely on steady-state concentration-response curves or IC 50 values, which can overlook adverse cardiotoxic effects. We sought to investigate the effects of kinetics and state-dependent binding of I Kur -targeting drugs on atrial electrophysiology in silico and reveal the ideal properties of I Kur blockers that maximize anti-AF efficacy and minimize pro-arrhythmic risk. To this aim, we developed a new Markov model of I Kur that describes K V 1.5 gating based on experimental voltage-clamp data in atrial myocytes from patient right-atrial samples in normal sinus rhythm. We extended the I Kur formulation to account for state-specificity and kinetics of K V 1.5-drug interactions and incorporated it into our human atrial cell model. We simulated 1- and 3-Hz pacing protocols in drug-free conditions and with a [drug] equal to the IC 50 value. The effects of binding and unbinding kinetics were determined by examining permutations of the forward (k on ) and reverse (k off ) binding rates to the closed, open, and inactivated states of the K V 1.5 channel. We identified a subset of ideal drugs exhibiting anti-AF electrophysiological parameter changes at fast pacing rates (effective refractory period prolongation), while having little effect on normal sinus rhythm (limited action potential prolongation). Our results highlight that accurately accounting for channel interactions with drugs, including kinetics and state-dependent binding, is critical for developing safer and more effective pharmacological anti-AF options.

Enhanced L-type Ca 2+ channel (LTCC) activity in arterial myocytes contributes to vascular dysfunction during diabetes. Modulation of LTCC activity under hyperglycemic conditions could result from membrane potential-dependent and independent mechanisms. We have demonstrated that elevations in extracellular glucose (HG), similar to hyperglycemic conditions during diabetes, stimulate LTCC activity through phosphorylation of Ca V 1.2 at serine 1928. Prior studies have also shown that HG can suppress the activity of K + channels in arterial myocytes, which may contribute to vasoconstriction via membrane depolarization. Here, we used a mathematical model of membrane and Ca 2+ dynamics in arterial myocytes to predict the relative roles of LTCC and K + channel activity in modulating global Ca 2+ in response to HG. Our data revealed that abolishing LTCC potentiation normalizes [Ca 2+ ] i , despite the concomitant reduction in K + currents in response to HG. These results suggest that LTCC stimulation may be the primary mechanism underlying vasoconstriction during hyperglycemia.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

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.)