Sex Differences in Electrophysiology and Calcium Handling in Atrial Health and Fibrillation.

The importance of biological sex on disease etiology and outcomes has long been underinvestigated. While recent focus on characterizing sex differences in cardiac pathophysiology has led to improved inclusion of both sexes in scientific studies and clinical trials, much is still unknown about underlying differences in normal cardiac physiology. This is particularly true for the atria, where the most common arrhythmia, atrial fibrillation (AF), occurs. AF is associated with adverse structural, electrophysiological, and calcium handling remodeling that leads to patient morbidity and mortality. Differences in the onset, prevalence, presentation, and prognosis of AF are known to differ between males and females, yet the sex-specific baseline phenotypes from which AF arises are not well characterized. This review examines what is currently known about sex differences in atrial physiology, the alterations that occur in AF, potential mechanisms underlying sex divergence, and the need for sex-targeted therapeutic strategies.

Enhanced Ca2+-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation.

BACKGROUND: Small-conductance Ca2+-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 (ISK) 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: ISK 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 IK1 and ISK as major regulators of repolarization. Increased ISK 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 ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK 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 ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. CONCLUSIONS: ISK 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 ISK, 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.

Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes.

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) 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 If during the cardiac cycle in mouse SAMs. We found that If 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, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that ß-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If 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.

Unveiling the intricacies of intracellular Ca2+ regulation in the heart.

Recent studies have provided valuable insight into the key mechanisms contributing to the spatiotemporal regulation of intracellular Ca2+ release and Ca2+ signaling in the heart. In this research highlight, we focus on the latest findings published in Biophysical Journal examining the structural organization of Ca2+ handling proteins and assessing the functional aspects of intracellular Ca2+ regulation in health and the detrimental consequences of Ca2+ dysregulation in disease. These important studies pave the way for future mechanistic investigations and multiscale understanding of Ca2+ signaling in the heart.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: II. Ca2+ -handling protein variation.

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 Ca2+ -handling proteins. Together this ultrastructural and ionic remodelling is associated with aberrant Ca2+ cycling and electrophysiological instabilities that underlie arrhythmic activity. However, due to the concurrent changes in TATs and Ca2+ -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 Ca2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca2+ -handling proteins and variable TATS density on Ca2+ -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) Ca2+ buffer calsequestrin have varying pro- and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca2+ leak-load and Ca2+ -voltage relationships. Interestingly, the impact of protein remodelling on Ca2+ -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 Ca2+ -handling protein remodelling that underlies dysfunctional Ca2+ cycling and electrophysiological instability in disease. KEY POINTS: In our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca2+ 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 Ca2+ -handling proteins known to be remodelled in disease on Ca2+ 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 Ca2+ handling protein remodelling are profound in intermediately tubulated cells. Our work shows the interaction between TATS and Ca2+ -handling protein remodelling that underlies the Ca2+ -driven proarrhythmic behaviour observed in atrial fibrillation and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodelling.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: I. Transverse-axial tubule variation.

Intracellular calcium (Ca2+ ) 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 Ca2+ -handling proteins such as the L-type Ca2+ channel and Na+ -Ca2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca2+ 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 Ca2+ -handling and Ca2+ -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 Ca2+ -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 Ca2+ release. We found that varying TATS density and thus the localization of key Ca2+ -handling proteins has profound effects on Ca2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca2+ concentration through decreased Ca2+ extrusion. This elevated Ca2+ increases RyR open probability causing spontaneous Ca2+ 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 Ca2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca2+ and voltage instabilities through reduced Na+ -Ca2+ exchanger-mediated Ca2+ removal, cleft Ca2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca2+ 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 Ca2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

Dual effects of the small-conductance Ca2+-activated K+ current on human atrial electrophysiology and Ca2+-driven arrhythmogenesis: an in silico study.

By sensing changes in intracellular Ca2+, small-conductance Ca2+-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 (ISK) 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 ISK 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 ISK in human atrial cardiomyocyte electrophysiology. We simulate several protocols to quantify how ISK modulation affects the regulation of AP duration (APD), Ca2+ transient, refractoriness, and occurrence of alternans and delayed afterdepolarizations (DADs). Our simulations show that ISK activation shortens the APD and atrial effective refractory period, limits Ca2+ cycling, and slightly increases the propensity for alternans in both nSR and cAF conditions. We also show that increasing ISK counteracts DAD development by enhancing the repolarization force that opposes the Ca2+-dependent depolarization. Taken together, our results suggest that increasing ISK in human atrial cardiomyocytes could promote reentry while protecting against triggered activity. Depending on the leading arrhythmogenic mechanism, ISK 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 Ca2+-activated K+ current (ISK) in the regulation of cell function and the development of Ca2+-driven arrhythmias. We found that ISK 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.

Biocatalytic Cascade Synthesis of Enantioenriched Epoxides and Triols from Biomass-Derived Synthons Driven by Specifically Designed Enzymes.

Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.

Enzymatic Oxy- and Amino-Functionalization in Biocatalytic Cascade Synthesis: Recent Advances and Future Perspectives.

Biocatalytic cascades are a powerful tool for building complex molecules containing oxygen and nitrogen functionalities. Moreover, the combination of multiple enzymes in one pot offers the possibility to minimize downstream processing and waste production. In this review, we illustrate various recent efforts in the development of multi-step syntheses involving C-O and C-N bond-forming enzymes to produce high value-added compounds, such as pharmaceuticals and polymer precursors. Both in vitro and in vivo examples are discussed, revealing the respective advantages and drawbacks. The use of engineered enzymes to boost the cascades outcome is also addressed and current co-substrate and cofactor recycling strategies are presented, highlighting the importance of atom economy. Finally, tools to overcome current challenges for multi-enzymatic oxy- and amino-functionalization reactions are discussed, including flow systems with immobilized biocatalysts and cascades in confined nanomaterials.

Enhancing the Peroxygenase Activity of a Cofactor-Independent Peroxyzyme by Directed Evolution Enabling Gram-Scale Epoxide Synthesis.

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H2 O2 with water being the sole by-product. These biocatalysts can provide 'green' routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4-oxalocrotonate tautomerase (4-OT), catalysing the epoxidation of a variety of α,ß-unsaturated aldehydes with H2 O2 , is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,ß-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4-OT. Herein we report the directed evolution of a tandem-fused 4-OT variant, which showed an ∼150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,ß-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-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 ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-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 ß1AR 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 ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR 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.

Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes.

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 Ca2+ overload and increased generation of both spontaneous Ca2+ waves and delayed afterdepolarizations. However, similar Ca2+-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+-Ca2+ exchanger proteins within these structures, as reduction in Na+ pump activity locally inhibited Ca2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca2+ 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 (IKur); 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.

Intracellular Na+ Modulates Pacemaking Activity in Murine Sinoatrial Node Myocytes: An In Silico Analysis.

Background: The mechanisms underlying dysfunction in the sinoatrial node (SAN), the heart's primary pacemaker, are incompletely understood. Electrical and Ca2+-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+/Ca2+ 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 Ca2+ and membrane potential clocks underlying SAN firing. Conclusions: Our study generates new testable predictions and insight linking Na+ homeostasis to Ca2+ handling and membrane potential dynamics in SAN myocytes that may advance our understanding of SAN (dys)function.

Engineered C-N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners.

Aspartic acid derivatives with branched N-alkyl or N-arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N-substituted aspartic acids, including N-(3,3-dimethylbutyl)-l-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine-N,N'-disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C-N lyase (mutant D290M/Y320M) displayed a remarkable 1140-fold increase in activity for the selective hydroamination of fumarate compared to that of the wild-type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives.

A computational model of induced pluripotent stem-cell derived cardiomyocytes incorporating experimental variability from multiple data sources.

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.

Different paths, same destination: divergent action potential responses produce conserved cardiac fight-or-flight response in mouse and rabbit hearts.

KEY POINTS: Cardiac electrophysiology and Ca2+ 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 Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ -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 ß-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.

Transient outward K+ current can strongly modulate action potential duration and initiate alternans in the human atrium.

Efforts to identify the mechanisms for the initiation and maintenance of human atrial fibrillation (AF) often focus on changes in specific elements of the atrial "substrate," i.e., its electrophysiological properties and/or structural components. We used experimentally validated mathematical models of the human atrial myocyte action potential (AP), both at baseline in sinus rhythm (SR) and in the setting of chronic AF, to identify significant contributions of the Ca2+-independent transient outward K+ current ( Ito) to electrophysiological instability and arrhythmia initiation. First, we explored whether changes in the recovery or restitution of the AP duration (APD) and/or its dynamic stability (alternans) can be modulated by Ito. Recent reports have identified disease-dependent spatial differences in expression levels of the specific K+ channel α-subunits that underlie Ito in the left atrium. Therefore, we studied the functional consequences of this by deletion of 50% of native Ito (Kv4.3) and its replacement with Kv1.4. Interestingly, significant changes in the short-term stability of the human atrial AP waveform were revealed. Specifically, this K+ channel isoform switch produced discontinuities in the initial slope of the APD restitution curve and appearance of APD alternans. This pattern of in silico results resembles some of the changes observed in high-resolution clinical electrophysiological recordings. Important insights into mechanisms for these changes emerged from known biophysical properties (reactivation kinetics) of Kv1.4 versus those of Kv4.3. These results suggest new approaches for pharmacological management of AF, based on molecular properties of specific K+ isoforms and their changed expression during progressive disease. NEW & NOTEWORTHY Clinical studies identify oscillations (alternans) in action potential (AP) duration as a predictor for atrial fibrillation (AF). The abbreviated AP in AF also involves changes in K+ currents and early repolarization of the AP. Our simulations illustrate how substitution of Kv1.4 for the native current, Kv4.3, alters the AP waveform and enhances alternans. Knowledge of this "isoform switch" and related dynamics in the AF substrate may guide new approaches for detection and management of AF.

Antiarrhythmic mechanisms of beta blocker therapy.

Sympathetic activity plays an important role in modulation of cardiac rhythm. Indeed, while exerting positive tropic effects in response to physiologic and pathologic stressors, ß-adrenergic stimulation influences cardiac electrophysiology and can lead to disturbances of the heart rhythm and potentially lethal arrhythmias, particularly in pathological settings. For this reason, ß-blockers are widely utilized clinically as antiarrhythmics. In this review, the molecular mechanisms of ß-adrenergic action in the heart, the cellular and tissue level cardiac responses to ß-adrenergic stimulation, and the clinical use of ß-blockers as antiarrhythmic agents are reviewed. We emphasize the complex interaction between cardiomyocyte signaling, contraction, and electrophysiology occurring over multiple time- and spatial-scales during pathophysiological responses to ß-adrenergic stimulation. An integrated understanding of this complex system is essential for optimizing therapies aimed at preventing arrhythmias.

Non-ion channel therapeutics for heart failure and atrial fibrillation: Are CaMKII inhibitors ready for clinical use?

The Ca2+-calmodulin dependent protein kinase II (CaMKII) is an established central mediator of electrophysiological and contractile responses to cardiac stress, and its hyper-activation in cardiac diseases has been linked to heart failure (HF) and arrhythmia. Here we summarize the evidence supporting the role of CaMKII as a critical nodal point for therapeutic intervention against HF and atrial and ventricular tachyarrhythmias. Targeting of CaMKII in heart with inhibitors possessing appropriate selectivity might represent a novel therapeutic approach for HF and arrhythmias.