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

Background: Heart rhythm relies on complex interactions between the electrogenic membrane proteins and intracellular Ca 2+ signaling in sinoatrial node (SAN) myocytes; however, the mechanisms underlying the functional organization of the 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, however 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: In vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca 2+ imaging, immunofluorescent and electron microscopy analysis 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 40% reduction of Na + /Ca 2+ -exchanger function. 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 representing a new dimension of SAN remodeling and providing a newly recognized target for therapy.

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.

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.

A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene.

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.

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.

Electrophysiological and Molecular Mechanisms of Sinoatrial Node Mechanosensitivity.

The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.

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.

Cellular and Molecular Mechanisms of Functional Hierarchy of Pacemaker Clusters in the Sinoatrial Node: New Insights into Sick Sinus Syndrome.

The sinoatrial node (SAN), the primary pacemaker of the heart, consists of a heterogeneous population of specialized cardiac myocytes that can spontaneously produce action potentials, generating the rhythm of the heart and coordinating heart contractions. Spontaneous beating can be observed from very early embryonic stage and under a series of genetic programing, the complex heterogeneous SAN cells are formed with specific biomarker proteins and generate robust automaticity. The SAN is capable to adjust its pacemaking rate in response to environmental and autonomic changes to regulate the heart's performance and maintain physiological needs of the body. Importantly, the origin of the action potential in the SAN is not static, but rather dynamically changes according to the prevailing conditions. Changes in the heart rate are associated with a shift of the leading pacemaker location within the SAN and accompanied by alterations in P wave morphology and PQ interval on ECG. Pacemaker shift occurs in response to different interventions: neurohormonal modulation, cardiac glycosides, pharmacological agents, mechanical stretch, a change in temperature, and a change in extracellular electrolyte concentrations. It was linked with the presence of distinct anatomically and functionally defined intranodal pacemaker clusters that are responsible for the generation of the heart rhythm at different rates. Recent studies indicate that on the cellular level, different pacemaker clusters rely on a complex interplay between the calcium (referred to local subsarcolemmal Ca2+ releases generated by the sarcoplasmic reticulum via ryanodine receptors) and voltage (referred to sarcolemmal electrogenic proteins) components of so-called "coupled clock pacemaker system" that is used to describe a complex mechanism of SAN pacemaking. In this review, we examine the structural, functional, and molecular evidence for hierarchical pacemaker clustering within the SAN. We also demonstrate the unique molecular signatures of intranodal pacemaker clusters, highlighting their importance for physiological rhythm regulation as well as their role in the development of SAN dysfunction, also known as sick sinus syndrome.

Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats.

Right ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.

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.

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.

Genetic Loss of IK1 Causes Adrenergic-Induced Phase 3 Early Afterdepolariz ations and Polymorphic and Bidirectional Ventricular Tachycardia.

BACKGROUND: Arrhythmia syndromes associated with KCNJ2 mutations have been described clinically; however, little is known of the underlying arrhythmia mechanism. We create the first patient inspired KCNJ2 transgenic mouse and study effects of this mutation on cardiac function, IK1, and Ca2+ handling, to determine the underlying cellular arrhythmic pathogenesis. METHODS: A cardiac-specific KCNJ2-R67Q mouse was generated and bred for heterozygosity (R67Q+/-). Echocardiography was performed at rest, under anesthesia. In vivo ECG recording and whole heart optical mapping of intact hearts was performed before and after adrenergic stimulation in wild-type (WT) littermate controls and R67Q+/- mice. IK1 measurements, action potential characterization, and intracellular Ca2+ imaging from isolated ventricular myocytes at baseline and after adrenergic stimulation were performed in WT and R67Q+/- mice. RESULTS: R67Q+/- mice (n=17) showed normal cardiac function, structure, and baseline electrical activity compared with WT (n=10). Following epinephrine and caffeine, only the R67Q+/- mice had bidirectional ventricular tachycardia, ventricular tachycardia, frequent ventricular ectopy, and/or bigeminy and optical mapping demonstrated high prevalence of spontaneous and sustained ventricular arrhythmia. Both R67Q+/- (n=8) and WT myocytes (n=9) demonstrated typical n-shaped IK1IV relationship; however, following isoproterenol, max outward IK1 increased by ≈20% in WT but decreased by ≈24% in R67Q+/- (P<0.01). R67Q+/- myocytes (n=5) demonstrated prolonged action potential duration at 90% repolarization and after 10 nmol/L isoproterenol compared with WT (n=7; P<0.05). Ca2+ transient amplitude, 50% decay rate, and sarcoplasmic reticulum Ca2+ content were not different between WT (n=18) and R67Q+/- (n=16) myocytes. R67Q+/- myocytes (n=10) under adrenergic stimulation showed frequent spontaneous development of early afterdepolarizations that occurred at phase 3 of action potential repolarization. CONCLUSIONS: KCNJ2 mutation R67Q+/- causes adrenergic-dependent loss of IK1 during terminal repolarization and vulnerability to phase 3 early afterdepolarizations. This model clarifies a heretofore unknown arrhythmia mechanism and extends our understanding of treatment implications for patients with KCNJ2 mutation.

Arrhythmogenic Interaction Between Sympathetic Tone and Mechanical Stretch in Rat Pulmonary Vein Myocardium.

Rapid firing from pulmonary veins (PVs) frequently initiates atrial fibrillation, which is a common comorbidity associated with hypertension, heart failure, and valvular disease, i.e., conditions that pathologically increase cardiomyocyte stretch. Autonomic tone plays a crucial role in PV arrhythmogenesis, while its interplay with myocardium stretch remains uncertain. Two-microelectrode technique was used to characterize electrophysiological response of Wistar rat PV to adrenaline at baseline and under mild (150 mg of applied weight that corresponds to a pulmonary venous pressure of 1 mmHg) and moderate (10 g, ∼26 mmHg) stretch. Low concentrations of adrenaline (25-100 nmol/L) depolarized the resting membrane potential selectively within distal PV (by 26 ± 2 mV at baseline, by 18 ± 1 mV at 150 mg, P < 0.001, and by 5.9 ± 1.1 mV at 10 g, P < 0.01) suppressing action potential amplitude and resulting in intra-PV conduction dissociation and rare episodes of spontaneous activity (arrhythmia index of 0.4 ± 0.2, NS vs. no activity at baseline). In contrast, 1-10 µmol/L of adrenaline recovered intra-PV propagation. While mild stretch did not affect PV electrophysiology at baseline, moderate stretch depolarized the resting potential within distal PV (-56 ± 2 mV vs. -82 ± 1 mV at baseline, P < 0.01), facilitated the triggering of rapid PV firing by adrenaline (arrhythmia index: 4.4 ± 0.2 vs. 1.3 ± 0.4 in unstretched, P < 0.001, and 1.7 ± 0.8 in mildly stretched preparations, P < 0.005, at 10 µmol/L adrenaline) and induced frequent episodes of potentially arrhythmogenic atrial "echo" extra beats. Our findings demonstrate complex interactions between the sympathetic tone and mechanical stretch in the development of arrhythmogenic activity within PVs that may impact an increased atrial fibrillation vulnerability in patients with elevated blood pressure.

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+.

Induced cardiac progenitor cells repopulate decellularized mouse heart scaffolds and differentiate to generate cardiac tissue.

Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy.

Caveolae-Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis.

Background Atrial fibrillation often occurs in the setting of hypertension and associated atrial dilation with pathologically increased cardiomyocyte stretch. In the setting of atrial dilation, mechanoelectric feedback has been linked to the development of ectopic beats that trigger paroxysmal atrial fibrillation mainly originating from pulmonary veins (PVs). However, the precise mechanisms remain poorly understood. Methods and Results We identify mechanosensitive, swelling-activated chloride ion channels (ICl,swell) as a crucial component of the caveolar mechanosensitive complex in rat and human cardiomyocytes. In vitro optical mapping of rat PV, single rat PV, and human cardiomyocyte patch clamp studies showed that stretch-induced activation of ICl,swell leads to membrane depolarization and decreased action potential amplitude, which trigger conduction discontinuities and both ectopic and reentrant activities within the PV. Reverse transcription quantitative polymerase chain reaction, immunofluorescence, and coimmunoprecipitation studies showed that ICl,swell likely consists of at least 2 components produced by mechanosensitive ClC-3 (chloride channel-3) and SWELL1 (also known as LRRC8A [leucine rich repeat containing protein 8A]) chloride channels, which form a macromolecular complex with caveolar scaffolding protein Cav3 (caveolin 3). Downregulation of Cav3 protein expression and disruption of caveolae structures during chronic hypertension in spontaneously hypertensive rats facilitates activation of ICl,swell and increases PV sensitivity to stretch 10- to 50-fold, promoting the development of atrial fibrillation. Conclusions Our findings identify caveolae-mediated activation of mechanosensitive ICl,swell as a critical cause of PV ectopic beats that can initiate atrial arrhythmias including atrial fibrillation. This mechanism is exacerbated in the setting of chronically elevated blood pressures.