Mechanisms of stretch-induced electro-anatomical remodeling and atrial arrhythmogenesis.

Atrial fibrillation (AF) is the most common cardiac rhythm disorder, often occurring in the setting of atrial distension and elevated myocardialstretch. While various mechano-electrochemical signal transduction pathways have been linked to AF development and progression, the underlying molecular mechanisms remain poorly understood, hampering AF therapies. In this review, we describe different aspects of stretch-induced electro-anatomical remodeling as seen in animal models and in patients with AF. Specifically, we focus on cellular and molecular mechanisms that are responsible for mechano-electrochemical signal transduction and the development of ectopic beats triggering AF from pulmonary veins, the most common source of paroxysmal AF. Furthermore, we describe structural changes caused by stretch occurring before and shortly after the onset of AF as well as during AF progression, contributing to longstanding forms of AF. We also propose mechanical stretch as a new dimension to the concept "AF begets AF", in addition to underlying diseases. Finally, we discuss the mechanisms of these electro-anatomical alterations in a search for potential therapeutic strategies and the development of novel antiarrhythmic drugs targeted at the components of mechano-electrochemical signal transduction not only in cardiac myocytes, but also in cardiac non-myocyte cells.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Neutral sphingomyelinase regulates mechanotransduction in human engineered cardiac tissues and mouse hearts.

Cardiovascular disease is the leading cause of death in the USA and is known to be exacerbated by elevated mechanical stress from hypertension. Caveolae are plasma membrane structures that buffer mechanical stress but have been found to be reduced in pathological conditions associated with chronically stretched myocardium. To explore the physiological implications of the loss of caveolae, we used human engineered cardiac tissue (ECT) constructs, composed of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived cardiac fibroblasts, to develop a long-term cyclic stretch protocol that recapitulates the effects of hypertension on caveolae expression, membrane tension, and the ß-adrenergic response. Leveraging this new stretch protocol, we identified neutral sphingomyelinases (nSMase) as mechanoregulated mediators of caveolae loss, ceramide production and the blunted ß-adrenergic response in this human cardiac model. Specifically, in our ECT model, nSMase inhibition via GW4869 prevented stretch-induced loss of caveolae-like structures, mitigated nSMase-dependent ceramide production, and maintained the ECT contractile kinetic response to isoprenaline. These findings are correlated with a blood lipidomic analysis in middle-aged and older adults, which revealed an increase of the circulating levels of ceramides in adults with hypertension. Furthermore, we found that conduction slowing from increased pressure loading in mouse left ventricle was abolished in the context of nSMase inhibition. Collectively, these findings identify nSMase as a potent drug target for mitigating stretch-induced effects on cardiac function. KEY POINTS: We have developed a new stretch protocol for human engineered cardiac tissue that recapitulates changes in plasma membrane morphology observed in animal models of pressure/volume overload. Stretch of engineered cardiac tissue induces activation of neutral sphingomyelinase (nSMase), generation of ceramide, and disassembly of caveolae. Activation of nSMase blunts cardiac ß-adrenergic contractile kinetics and mediates stretch-induced slowing of conduction and upstroke velocity. Circulating ceramides are increased in adults with hypertension, highlighting the clinical relevance of stretch-induced nSMase activity.

Caveolin-3 prevents swelling-induced membrane damage via regulation of ICl,swell activity.

Caveola membrane structures harbor mechanosensitive chloride channels (MCCs; including chloride channel 2, chloride channel 3, and SWELL1, also known as LRRC8A) that form a swelling-activated chloride current (ICl,swell) and play an important role in cell volume regulation and mechanoelectrical signal transduction. However, the role of the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCC expression, activity, and contribution to membrane integrity in response to mechanical stress remains unclear. Here we showed that Cav3-transfected (Cav3-positive) HEK293 cells were significantly resistant to extreme (<20 milliosmole) hypotonic swelling compared with native (Cav3-negative) HEK293 cells; the percentage of cells with membrane damage decreased from 45% in Cav3-negative cells to 17% in Cav3-positive cells (p < 0.05). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the ICl,swell-selective inhibitor 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (10 µM). These results were recapitulated in isolated mouse ventricular myocytes, where the percentage of cardiomyocytes with membrane damage increased from 47% in control cells to 78% in 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid-treated cells (p < 0.05). A higher resistance to hypotonic swelling in Cav3-positive HEK293 cells was accompanied by a significant twofold increase of ICl,swell current density and SWELL1 protein expression, whereas ClC-2/3 protein levels remained unchanged. Förster resonance energy transfer analysis showed a less than 10-nm membrane and intracellular association between Cav3 and SWELL1. Cav3/SWELL1 membrane Förster resonance energy transfer efficiency was halved in mild (220 milliosmole) hypotonic solution as well as after disruption of caveola structures via cholesterol depletion by 1-h treatment with 10 mM methyl-ß-cyclodextrin. A close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. Our findings indicate that, in the MCCs tested, SWELL1 abundance and activity are regulated by Cav3 and that their association relies on membrane tension and caveola integrity. This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity.

Region-specific distribution of transversal-axial tubule system organization underlies heterogeneity of calcium dynamics in the right atrium.

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Caveolin-3 is required for regulation of transient outward potassium current by angiotensin II in mouse atrial myocytes.

Angiotensin II (AngII) is a key mediator of the renin-angiotensin system and plays an important role in the regulation of cardiac electrophysiology by affecting various cardiac ion currents, including transient outward potassium current, Ito. AngII receptors and molecular components of Ito, Kv4.2 and Kv4.3 channels, have been linked to caveolae structures. However, their functional interaction and the importance of such proximity within 50- to 100-nm caveolar nanodomains remain unknown. To address this, we studied the mechanisms of Ito regulation by AngII in atrial myocytes of wild-type (WT) and cardiac-specific caveolin-3 (Cav3) conditional knockout (Cav3KO) mice. We showed that in WT atrial myocytes, a short-term (2 h) treatment with AngII (5 µM) significantly reduced Ito density. This effect was prevented 1) by a 30-min pretreatment with a selective antagonist of AngII receptor 1 (Ang1R) losartan (2 µM) or 2) by a selective inhibition of protein kinase C (PKC) by BIM1 (10 µM). The effect of AngII on Ito was completely abolished in Cav3-KO mice, with no change in a baseline Ito current density. In WT atria, Ang1Rs co-localized with Cav3, and the expression of Ang1Rs was significantly decreased in Cav3KO in comparison with WT mice, whereas no change in Kv4.2 and Kv4.3 protein expression was observed. Overall, our findings demonstrate that Cav3 is involved in the regulation of Ang1R expression and is required for the modulation of Ito by AngII in mouse atrial myocytes.NEW & NOTEWORTHY Angiotensin II receptor 1 is associated with caveolae and caveolar scaffolding protein caveolin-3 in mouse atrial myocytes that is required for the regulation of Ito by angiotensin II. Downregulation of caveolae/caveolin-3 disrupts this regulation and may be implicated in pathophysiological atrial remodeling.

Human iPSC-engineered cardiac tissue platform faithfully models important cardiac physiology.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and the intact human myocardium. Fulfilling this potential is hampered by their relative immaturity, leading to poor physiological responsiveness. hiPSC-CMs grown in traditional two-dimensional (2D) culture lack a t-tubular system, have only rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as an energy substrate. Culturing hiPSC-CM in a variety of three-dimensional (3D) environments and the addition of nutritional, pharmacological, and electromechanical stimuli have proven, to various degrees, to be beneficial for maturation. We present a detailed assessment of a novel model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are cocultured in a 3D fibrin matrix to form engineered cardiac tissue constructs (hiPSC-ECTs). The hiPSC-ECTs are responsive to physiological stimuli, including stretch, frequency, and ß-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Furthermore, transcript levels of various genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a relatively short period of time in culture (6 wk vs. 2 wk in hiPSC-ECTs). A comparison of the hiPSC-ECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to aid selection of the most appropriate platform for the research question at hand. Important and noteworthy aspects of this human cardiac model system are its reliance on "off-the-shelf" equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological, and electromechanical stimuli that may elicit unintended effects on function.NEW & NOTEWORTHY This study seeks to provide an in-depth assessment of contractile performance of human iPSC-derived cardiomyocytes cultured together with fibroblasts in a 3-dimensional-engineered tissue and compares performance both over time as cells mature, and with corresponding measures found in the literature using alternative 3D culture configurations. The suitability of 3D-engineered human cardiac tissues to model cardiac function is emphasized, and data provided to assist in the selection of the most appropriate configuration based on the target application.

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.

A compartmentalized mathematical model of mouse atrial myocytes.

Various experimental mouse models are extensively used to research human diseases, including atrial fibrillation, the most common cardiac rhythm disorder. Despite this, there are no comprehensive mathematical models that describe the complex behavior of the action potential and [Ca2+]i transients in mouse atrial myocytes. Here, we develop a novel compartmentalized mathematical model of mouse atrial myocytes that combines the action potential, [Ca2+]i dynamics, and ß-adrenergic signaling cascade for a subpopulation of right atrial myocytes with developed transverse-axial tubule system. The model consists of three compartments related to ß-adrenergic signaling (caveolae, extracaveolae, and cytosol) and employs local control of Ca2+ release. It also simulates ionic mechanisms of action potential generation and describes atrial-specific Ca2+ handling as well as frequency dependences of the action potential and [Ca2+]i transients. The model showed that the T-type Ca2+ current significantly affects the later stage of the action potential, with little effect on [Ca2+]i transients. The block of the small-conductance Ca2+-activated K+ current leads to a prolongation of the action potential at high intracellular Ca2+. Simulation results obtained from the atrial model cells were compared with those from ventricular myocytes. The developed model represents a useful tool to study complex electrical properties in the mouse atria and could be applied to enhance the understanding of atrial physiology and arrhythmogenesis.NEW & NOTEWORTHY A new compartmentalized mathematical model of mouse right atrial myocytes was developed. The model simulated action potential and Ca2+ dynamics at baseline and after stimulation of the ß-adrenergic signaling system. Simulations showed that the T-type Ca2+ current markedly prolonged the later stage of atrial action potential repolarization, with a minor effect on [Ca2+]i transients. The small-conductance Ca2+-activated K+ current block resulted in prolongation of the action potential only at the relatively high intracellular Ca2+.

Epigenetic Priming of Human Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Accelerates Cardiomyocyte Maturation.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.

Long QT syndrome caveolin-3 mutations differentially modulate Kv 4 and Cav 1.2 channels to contribute to action potential prolongation.

KEY POINTS: Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 channels responsible for Ito . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating ICa,L but, for Cav3-S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches. ABSTRACT: Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Cav 1.2+Cav ß2cN4 channels, as well as Kv 4.2 and Kv 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca2+ -dependent inactivation of ICa,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+ -dependent inactivation of ICa,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future.

Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure.

RATIONALE: Disruption in subcellular targeting of Ca(2+) signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. OBJECTIVE: To explore microdomain-targeted remodeling of ventricular L-type Ca(2+) channels (LTCCs) in HF. METHODS AND RESULTS: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium-calmodulin kinase II-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias. CONCLUSIONS: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease.

Reduced response to IKr blockade and altered hERG1a/1b stoichiometry in human heart failure.

Heart failure (HF) claims 250,000 lives per year in the US, and nearly half of these deaths are sudden and presumably due to ventricular tachyarrhythmias. QT interval and action potential (AP) prolongation are hallmark proarrhythmic changes in the failing myocardium, which potentially result from alterations in repolarizing potassium currents. Thus, we aimed to examine whether decreased expression of the rapid delayed rectifier potassium current, IKr, contributes to repolarization abnormalities in human HF. To map functional IKr expression across the left ventricle (LV), we optically imaged coronary-perfused LV free wall from donor and end-stage failing human hearts. The LV wedge preparation was used to examine transmural AP durations at 80% repolarization (APD80), and treatment with the IKr-blocking drug, E-4031, was utilized to interrogate functional expression. We assessed the percent change in APD80 post-IKr blockade relative to baseline APD80 (∆APD80) and found that ∆APD80s are reduced in failing versus donor hearts in each transmural region, with 0.35-, 0.43-, and 0.41-fold reductions in endo-, mid-, and epicardium, respectively (p=0.008, 0.037, and 0.022). We then assessed hERG1 isoform gene and protein expression levels using qPCR and Western blot. While we did not observe differences in hERG1a or hERG1b gene expression between donor and failing hearts, we found a shift in the hERG1a:hERG1b isoform stoichiometry at the protein level. Computer simulations were then conducted to assess IKr block under E-4031 influence in failing and nonfailing conditions. Our results confirmed the experimental observations and E-4031-induced relative APD80 prolongation was greater in normal conditions than in failing conditions, provided that the cellular model of HF included a significant downregulation of IKr. In human HF, the response to IKr blockade is reduced, suggesting decreased functional IKr expression. This attenuated functional response is associated with altered hERG1a:hERG1b protein stoichiometry in the failing human LV, and failing cardiomyoctye simulations support the experimental findings. Thus, of IKr protein and functional expression may be important determinants of repolarization remodeling in the failing human LV.

Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes.

BACKGROUND: Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). METHODS AND RESULTS: Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈ 40% greater current. Optical mapping of Ca(2+) transients revealed that rat AMs presented ≈ 3-fold as many spontaneous Ca(2+) release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca(2+) transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-ß-cyclodextrin, with an associated ≈ 30% whole-cell ICa,L reduction. Heart failure (16 weeks post-myocardial infarction) in rats resulted in a T-tubule degradation (by ≈ 40%) and significant elevation of spontaneous Ca(2+) release events. Although heart failure did not affect LTCC occurrence, it led to ≈ 25% decrease in T-tubule LTCC amplitude. CONCLUSIONS: We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1.

AIMS: Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. METHODS AND RESULTS: In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex. CONCLUSIONS: Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients.

Upregulation of adenosine A1 receptors facilitates sinoatrial node dysfunction in chronic canine heart failure by exacerbating nodal conduction abnormalities revealed by novel dual-sided intramural optical mapping.

BACKGROUND: Although sinoatrial node (SAN) dysfunction is a hallmark of human heart failure (HF), the underlying mechanisms remain poorly understood. We aimed to examine the role of adenosine in SAN dysfunction and tachy-brady arrhythmias in chronic HF. METHODS AND RESULTS: We applied multiple approaches to characterize SAN structure, SAN function, and adenosine A1 receptor expression in control (n=17) and 4-month tachypacing-induced chronic HF (n=18) dogs. Novel intramural optical mapping of coronary-perfused right atrial preparations revealed that adenosine (10 µmol/L) markedly prolonged postpacing SAN conduction time in HF by 206 ± 99 milliseconds (versus 66 ± 21 milliseconds in controls; P=0.02). Adenosine induced SAN intranodal conduction block or microreentry in 6 of 8 dogs with HF versus 0 of 7 controls (P=0.007). Adenosine-induced SAN conduction abnormalities and automaticity depression caused postpacing atrial pauses in HF versus control dogs (17.1 ± 28.9 versus 1.5 ± 1.3 seconds; P<0.001). Furthermore, 10 µmol/L adenosine shortened atrial repolarization and led to pacing-induced atrial fibrillation in 6 of 7 HF versus 0 of 7 control dogs (P=0.002). Adenosine-induced SAN dysfunction and atrial fibrillation were abolished or prevented by adenosine A1 receptor antagonists (50 µmol/L theophylline/1 µmol/L 8-cyclopentyl-1,3-dipropylxanthine). Adenosine A1 receptor protein expression was significantly upregulated during HF in the SAN (by 47 ± 19%) and surrounding atrial myocardium (by 90 ± 40%). Interstitial fibrosis was significantly increased within the SAN in HF versus control dogs (38 ± 4% versus 23 ± 4%; P<0.001). CONCLUSIONS: In chronic HF, adenosine A1 receptor upregulation in SAN pacemaker and atrial cardiomyocytes may increase cardiac sensitivity to adenosine. This effect may exacerbate conduction abnormalities in the structurally impaired SAN, leading to SAN dysfunction, and potentiate atrial repolarization shortening, thereby facilitating atrial fibrillation. Atrial fibrillation may further depress SAN function and lead to tachy-brady arrhythmias in HF.

Electrophysiological Characteristics, Rhythm, Disturbances and Conduction Discontinuities Under Autonomic Stimulation in the Rat Pulmonary Vein Myocardium.

INTRODUCTION: Despite the importance of neurogenic initiation of rapid firing from pulmonary veins (PVs), the mechanism of autonomic modulation of electrophysiological properties of the PV myocardium to form a substrate for atrial arrhythmia remains poorly understood. METHODS AND RESULTS: A 2-microelectrode technique was used to characterize electrophysiological properties of rat PV myocardium and to explore PV arrhythmogenesis, at baseline, during electrical stimulation and/or under autonomic modulation. PV myocardium was characterized by prolonged action potential duration (APD), high degree of APD alternans, and spontaneous depolarizations. Autonomic stimulation resulted in significantly enhanced APD dispersion within the PV, which dynamically changed over time and was associated with intra-PV and atria-PV conduction blocks and could lead to spontaneous fibrillation-like high-frequency activity. In the distal part of the PV we found an unexcitable area that was characterized by depolarized resting potential (-50 ± 4 mV vs. -75 ± 2 mV vs. PV mouth, P < 0.01). This region could be activated during autonomic stimulation or fast pacing that led to multiple conduction discontinuities (uni- and bi-directional conduction blocks, Wenckebach periodicity, electrotonic modulation conduction block, echo phenomenon) in 17/23 preparations, including those occurring under norepinephrine superfusion (14/17) and during pacing frequency changes (3/17). PV echoes (unstable reentrant circuits) were found in 8/23 preparations. In some experiments, several types of conduction abnormalities were observed. CONCLUSION: The PV myocardium demonstrates distinct electrophysiological characteristics, which could be considerably exaggerated by electrical stimulation and/or autonomic nervous system to dynamically form a functional substrate to support re-entry as well as focal activity.

WITHDRAWN: Caveolar Compartmentalization is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

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Functional roles of KATP channel subunits in metabolic inhibition.

ATP-sensitive potassium channel (KATP) activation can drastically shorten action potential duration (APD) in metabolically compromised myocytes. We showed previously that SUR1 with Kir6.2 forms the functional channel in mouse atria while Kir6.2 and SUR2A predominate in ventricles. SUR1 is more sensitive to metabolic stress than SUR2A, raising the possibility that KATP in atria and ventricles may respond differently to metabolic stress. Action potential duration (APD) and calcium transient duration (CaTD) were measured simultaneously in both atria and ventricles by optical mapping of the posterior surface of Langendorff-perfused hearts from C57BL wild-type (WT; n=11), Kir6.2(-/-) (n=5), and SUR1(-/-) (n=6) mice during metabolic inhibition (MI, 0mM glucose+2mM sodium cyanide). After variable delay, MI led to significant shortening of APD in WT hearts. On average, atrial APD shortened by 60.5 ± 2.7% at 13.1 ± 2.1 min (n=6, p<0.01) after onset of MI. Ventricular APD shortening (56.4 ± 10.0% shortening at 18.2 ± 1.8 min) followed atrial APD shortening. In SUR1(-/-) hearts (n=6), atrial APD shortening was abolished, but ventricular shortening (65.0 ± 15.4% at 25.33 ± 4.48 min, p<0.01) was unaffected. In Kir6.2(-/-) hearts, two disparate responses to MI were observed; 3 of 5 hearts displayed slight shortening of APD in the ventricles (24 ± 3%, p<0.05) and atria (39.0 ± 1.9%, p<0.05) but this shortening occurred later and to much less extent than in WT (p<0.05). Marked prolongation of ventricular APD was observed in the remaining hearts (327% and 489% prolongation) and was associated with occurrence of ventricular tachyarrhythmias. The results confirm that Kir6.2 contributes to APD shortening in both atria and ventricle during metabolic stress, and that SUR1 is required for atrial APD shortening while SUR2A is required for ventricular APD shortening. Importantly, the results show that the presence of SUR1-dependent KATP in the atria results in the action potential being more susceptible to metabolically driven shortening than the ventricle.