Action Potential Upstroke Velocity Research Articles

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation.

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of INa density, a 69.5% reduction of NaV1.5 expression, and the impaired localization of NaV1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The Ito in BrS-CMs was significantly augmented, and the ICaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of Ito in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

Abstract 477: Modeling the Effect of TBX5 Downregulation on Human Atrial Conduction Using Induced Pluripotent Stem Cell-derived Cardiomyocytes

Atrial fibrillation (AF) poses a notable healthcare burden due to a high incidence in the increasing population over age 65 and limitations of current treatment approaches. One challenge to effectively treat AF is patient-to-patient heterogeneity in the underlying mechanisms of disease. Therefore, a better understanding of AF pathogenesis and more personalized approaches to therapy could reduce risk of side effects and improve therapeutic efficacy. Genome wide association studies (GWAS) have revealed several candidate genes for AF including TBX5 , which encodes for a transcription factor involved in heart development. While work in animal models suggests that loss of TBX5 promotes atrial arrythmias, experimental evidence in human cells is lacking. We created an in vitro model of human atrial conduction using day 60+ induced pluripotent stem cell-derived atrial-like cardiomyocytes (iPSC-aCMs) differentiated from three established healthy iPSC lines. Over 90% atrial-like purity (out of 350+ alpha-actinin positive cardiomyocytes) could be achieved based on MLC2v-/MLC2a+ immunofluorescent staining. TBX5 knockdown via esiRNA resulted in downregulation of genes related to conduction velocity ( GJA5 and SCN5A ), consistent with an enhanced risk of AF. Single cell optical electrophysiology demonstrated slightly reduced action potential amplitude and upstroke velocity for TBX5 knockdown cells versus GFP esiRNA controls, suggesting a functional effect of SCN5A downregulation. Additionally, microelectrode array studies have revealed a trend towards slowed conduction velocity with TBX5 knockdown compared to GFP esiRNA controls (13.1±3.0 cm/s vs 17.0±3.8 cm/s respectively). By further investigating the functional effects of modulating transcription factors such as TBX5 in iPSC-aCMs, our results provide enhanced insight into the regulation of atrial conduction and identify potential AF-related pathways for therapeutic targeting.

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes from Brugada Syndrome Patients with a Loss-of-Function SCN5A Mutation

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell-derived cardiomyocytes from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to cardiomyocytes derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of INa density, a 69.5% reduction of NaV1.5 expression, and the impaired localization of NaV1.5 and connexin 43 at the cell surface. BrS-CMs exhibited reduced action potential upstroke velocity and conduction slowing. The Ito in BrS-CMs was significantly augmented, and the ICaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of Ito in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

Effect of Carvacrol, TRP Channels Modulator, on Cardiac Electrical Activity.

Despite the wide application of carvacrol (CAR) in medicines, dietary supplements, and foods, there is still insufficient electrophysiological data on the mechanisms of action of CAR, particularly with regard to heart function. Therefore, in this study, we attempted to elucidate whether CAR, whose inhibitory effect on both cardiac and vascular TRPM7 and L-type Ca2+ currents has been demonstrated previously, could modify cardiac electrical activity. We used a combination of optical mapping and microelectrode techniques to track the action potentials (APs) and the spread of electrical activity in a Langendorff-perfused rabbit heart model during atrial/endo/epicardial pacing. Simultaneously, ECG recordings were acquired. Because human trials on CAR are still lacking, we tested the action of CAR on human ventricular preparations obtained from explanted hearts. Activation time (AT), AP duration (APD), and conduction velocity maps were constructed. We demonstrated that at a low concentration (10 μM) of CAR, only marginal changes in the AP parameters were observed. At higher concentrations (≥100 μM), a decrease in AP upstroke velocity (dV/dtmax), suggesting inhibition of Na+ current, and APD (at 50 and 90% repolarization) was detected; also slowing in the spread of electrical signals via the atrioventricular node was observed, suggesting impaired functioning of Ca2+ channels. In addition, a decrease in the T-wave amplitude was seen on the ECG, suggesting an impaired repolarization process. Nevertheless, those changes occurred without a significant impact on the resting membrane potential and were reversible. We suggest that CAR might play a role in modulating cardiac electrical activity at high concentrations.

Regional and Temporal Changes in Atrial Electrophysiology Contribute to Atrial Fibrillation in Angiotensin II Induced Hypertension

Atrial fibrillation (AF) is highly prevalent in hypertensive heart disease and is associated with altered angiotensin II (Ang II) signaling. We have recently demonstrated that chronic hypertension (3 weeks of Ang II infusion) in mice increases AF susceptibility in association with distinct patterns of structural and electrical remodelling of the right and left atrium. However, the progressive effects of Ang II infusion on atrial electrophysiology have not been investigated. Accordingly, the goal of this study was to characterize the effects of Ang II infusion on mouse atrial electrophysiology at three different timepoints (3, 10, 21 days). AF susceptibility was increased to 35.7% following 10 days and 53.8% after 21 days of Ang II infusion. This occurred in association with progressive increases in P wave duration and AERP measurements at 3, 10, and 21 days of Ang II infusion. In isolated right atrial myocytes, action potential (AP) morphology and potassium currents (IK) were not altered after 10 days of Ang II infusion whereas AP duration was prolonged and potassium currents (IK) were reduced following 21 days of Ang II infusion. In contrast, AP morphology was differentially altered in the left atrium at all timepoints investigated. Specifically, AP upstroke velocity was reduced after 10 and 21 days of Ang II infusion with a corresponding reduction in sodium current at these timepoints. In addition, AP duration was similarly prolonged following 3, 10, and 21 days of Ang II infusion and IK was reduced to a similar extent at each of these timepoints. Collectively, these data demonstrate that Ang II infusion is associated with distinct regional and temporal alterations in atrial electrophysiology. These alterations provide insight into the changes in atrial electrophysiology that create a substrate for AF.

Biophysical mechanisms for QRS- and QTc-interval prolongation in mice with cardiac expression of expanded CUG-repeat RNA

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, results from the expression of toxic gain-of-function transcripts containing expanded CUG-repeats. DM1 patients experience cardiac electrophysiological defects, including prolonged PR-, QRS-, and QT-intervals, that increase susceptibility to sudden cardiac death (SCD). However, the specific biophysical and molecular mechanisms that underlie the electrocardiograph (ECG) abnormalities and SCD in DM1 are unclear. Here, we addressed this issue using a novel transgenic mouse model that exhibits robust cardiac expression of expanded CUG-repeat RNA (LC15 mice). ECG measurements in conscious LC15 mice revealed significantly prolonged QRS- and corrected QT-intervals, but a normal PR-interval. Although spontaneous arrhythmias were not observed in conscious LC15 mice under nonchallenged conditions, acute administration of the sodium channel blocker flecainide prolonged the QRS-interval and unveiled an increased susceptibility to lethal ventricular arrhythmias. Current clamp measurements in ventricular myocytes from LC15 mice revealed significantly reduced action potential upstroke velocity at physiological pacing (9 Hz) and prolonged action potential duration at all stimulation rates (1-9 Hz). Voltage clamp experiments revealed significant rightward shifts in the voltage dependence of sodium channel activation and steady-state inactivation, as well as a marked reduction in outward potassium current density. Together, these findings indicate that expression of expanded CUG-repeat RNA in the murine heart results in reduced sodium and potassium channel activity that results in QRS- and QT-interval prolongation, respectively.

Absence of Functional Nav1.8 Channels in Non-diseased Atrial and Ventricular Cardiomyocytes

PurposeSeveral studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species.MethodsThe effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs).ResultsA-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs.ConclusionWe here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs.

Ginsenoside Rb1 exerts antiarrhythmic effects by inhibiting INa and ICaL in rabbit ventricular myocytes

Ginsenoside Rb1 exerts its pharmacological action by regulating sodium, potassium and calcium ion channels in the membranes of nerve cells. These ion channels are also present in cardiomyocytes, but no studies have been reported to date regarding the effects of Rb1 on cardiac sodium currents (INa), L-type calcium currents (ICaL) and action potentials (APs). Additionally, the antiarrhythmic potential of Rb1 has not been assessed. In this study, we used a whole-cell patch clamp technique to assess the effect of Rb1 on these ion channels. The results showed that Rb1 inhibited INa and ICaL, reduced the action potential amplitude (APA) and maximum upstroke velocity (Vmax), and shortened the action potential duration (APD) in a concentration-dependent manner but had no effect on the inward rectifier potassium current (IK1), delayed rectifier potassium current (IK) or resting membrane potential (RMP). We also designed a pathological model at the cellular and organ level to verify the role of Rb1. The results showed that Rb1 abolished high calcium-induced delayed afterdepolarizations (DADs), depressed the increase in intracellular calcium ([Ca2+]i), relieved calcium overload and protected cardiomyocytes. Rb1 can also reduce the occurrence of ventricular premature beats (VPBs) and ventricular tachycardia (VT) in ischemia-reperfusion (I-R) injury.

Studying Brugada Syndrome With an SCN1B Variants in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BackgroundAmong rare channelopathies BrS patients are at high risk of sudden cardiac death (SCD). SCN5A mutations are found in a quarter of patients. Other rare gene mutations including SCN1B have been implicated to BrS. Studying the human cellular phenotype of BrS associated with rare gene mutation remains lacking.ObjectivesWe sought to study the cellular phenotype of BrS with the SCN1B gene variants using human-induced pluripotent stem cell (hiPSCs)–derived cardiomyocytes (hiPSC-CMs).Methods and ResultsA BrS patient suffering from recurrent syncope harboring a two variants (c.629T > C and c.637C > A) in SCN1B, which encodes the function-modifying sodium channel beta1 subunit, and three independent healthy subjects were recruited and their skin biopsies were used to generate hiPSCs, which were differentiated into cardiomyocytes (hiPSC-CMs) for studying the cellular electrophysiology. A significantly reduced peak and late sodium channel current (INa) and a shift of activation curve to more positive potential as well as a shift of inactivation curve to more negative potential were detected in hiPSC-CMs of the BrS patient, indicating that the SCN1B variants impact the function of sodium channels in cardiomyocytes. The reduced INa led to a reduction of amplitude (APA) and upstroke velocity (Vmax) of action potentials. Ajmaline, a sodium channel blocker, showed a stronger effect on APA and Vmax in BrS cells as compared to cells from healthy donors. Furthermore, carbachol was able to increase arrhythmia events and the beating frequency in BrS.ConclusionOur hiPSC-CMs from a BrS-patient with two variants in SCN1B recapitulated some key phenotypic features of BrS and can provide a platform for studies on BrS with SCN1B variants.

Rat caval vein myocardium undergoes changes in conduction characteristics during postnatal ontogenesis.

The electrophysiological properties of the superior vena cava (SVC) myocardium, which is considered a minor source of atrial arrhythmias, were studied in this study during postnatal development. Conduction properties were investigated in spontaneously active and electrically paced SVC preparations obtained from 7-60-day-old male Wistar rats using optical mapping and microelectrode techniques. The presence of high-conductance connexin 43 (Cx43) was evaluated in SVC cross-sections using immunofluorescence. It was found that SVC myocardium is excitable, electrically coupled with the atrial tissue, and conducts excitation waves at all stages of postnatal development. However, the conduction velocity (CV) of excitation and action potential (AP) upstroke velocity in SVC were significantly lower in neonatal than in adult animals and increased with postnatal maturation. Connexins Cx43 were identified in both neonatal and adult rat SVC myocardium; however, the abundance of Cx43 was significantly less in neonates. The gap junction uncoupler octanol affected conduction more profound in the neonatal than in adult SVC. We demonstrated for the first time that the conduction characteristics of SVC myocardium change from a slow-conduction (nodal) to a high-conduction (working) phenotype during postnatal ontogenesis. An age-related CV increase may occur due to changes of AP characteristics, electrical coupling, and Cx43 presence in SVC cardiomyocyte membranes. Observed changes may contribute to the low proarrhythmicity of adult caval vein cardiac tissue, while pre- or postnatal developmental abnormalities that delay the establishment of the working conduction phenotype may facilitate SVC proarrhythmia.

Examination of the Effects of Conduction Slowing on the Upstroke of Optically Recorded Action Potentials.

IntroductionThe upstroke of optical action potentials (APs) recorded from intact hearts are generally recognized to be slower than those recorded with microelectrodes. This is thought to reflect spatial signal averaging within the volume of tissue that makes up the optical signal. However, to date, there has been no direct experimental study on the relationship between conduction velocity (CV) and optical AP upstroke morphology in the intact heart. Notably, it is known that sodium channel block and gap junction inhibition, which both slow CV, exert differential effects on the upstroke velocity of microelectrode-recorded APs. Whether such differences are evident in optical APs is not known. The present study sought to determine the relationship between tissue CV and optical AP upstroke velocity in intact mouse hearts.Materials and MethodsIsolated, perfused mouse hearts were stained with the potentiometric dye Rh-237. Fluorescent signals were recorded from across the anterior surface of the left and right ventricles during constant pacing. Maximum rate of change in fluorescence (dF/dtmax) and tissue CV were assessed in control conditions, during an acute period of low-flow ischemia, and following perfusion of flecainide (1–3 μmol/L), a sodium channel blocker, or carbenoxolone (10–50 μmol/L), a gap junction inhibitor.ResultsDuring epicardial pacing, an anisotropic pattern was observed in both activation and dF/dtmax maps, with more rapid optical AP upstroke velocities orientated along the fastest conduction paths (and vice versa). Low-flow ischemia resulted in a time-dependent slowing of ventricular CV, which was accompanied by a concomitant reduction in optical AP upstroke velocity. All values returned to baseline on tissue reperfusion. Both flecainide and carbenoxolone were associated with a concentration-dependent reduction in CV and decrease in optical AP upstroke velocity, despite distinct mechanisms of action. Similar responses to carbenoxolone were observed for low- (156 μm pixel with) and high- (20 μm pixel width) magnification recordings. Comparison of data from all interventions revealed a linear relationship between CV and upstroke dF/dt.ConclusionIn intact mouse hearts, slowing of optical AP upstroke velocity is directly proportional to the change in CV associated with low-flow ischemia, sodium channel block, and gap junction inhibition.

Fatty Acids Enhance the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells.

SummaryAlthough human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a novel platform for heart regeneration, disease modeling, and drug screening, their immaturity significantly hinders their application. A hallmark of postnatal cardiomyocyte maturation is the metabolic substrate switch from glucose to fatty acids. We hypothesized that fatty acid supplementation would enhance hPSC-CM maturation. Fatty acid treatment induces cardiomyocyte hypertrophy and significantly increases cardiomyocyte force production. The improvement in force generation is accompanied by enhanced calcium transient peak height and kinetics, and by increased action potential upstroke velocity and membrane capacitance. Fatty acids also enhance mitochondrial respiratory reserve capacity. RNA sequencing showed that fatty acid treatment upregulates genes involved in fatty acid β-oxidation and downregulates genes in lipid synthesis. Signal pathway analyses reveal that fatty acid treatment results in phosphorylation and activation of multiple intracellular kinases. Thus, fatty acids increase human cardiomyocyte hypertrophy, force generation, calcium dynamics, action potential upstroke velocity, and oxidative capacity. This enhanced maturation should facilitate hPSC-CM usage for cell therapy, disease modeling, and drug/toxicity screens.

Maastricht antiarrhythmic drug evaluator (MANTA): A computational tool for better understanding of antiarrhythmic drugs

Cardiac arrhythmias are a global health burden, contributing significantly to morbidity and mortality worldwide. Despite technological advances in catheter ablation therapy, antiarrhythmic drugs (AADs) remain a cornerstone for the management of cardiac arrhythmias. Experimental and translational studies have shown that commonly used AADs exert multiple effects in the heart, the manifestation of which strongly depends on the exact experimental or clinical conditions. This diversity makes the optimal clinical application of AADs challenging.Here, we present a novel computational tool designed to facilitate a better understanding of the complex mechanisms of action of AADs (the Maastricht Antiarrhythmic Drug Evaluator, MANTA). In this tool, we integrated published computational cardiomyocyte models from different species (mouse, guinea pig, rabbit, dog, and human), regions (atrial, ventricular, and Purkinje cells) and disease conditions (atrial fibrillation- and heart failure-related remodeling). Subsequently, we investigated the effects of clinically available AADs (Vaughan-Williams Classes I, III, IV and multi-channel blockers) on action potential (AP) properties and the occurrence of proarrhythmic effects such as early afterdepolarizations. Steady-state drug effects were simulated based on a newly compiled overview of published IC50 values for each cardiac ion channel and by integrating state-dependent block of the cardiac Na+-current by Class I AADs using a Markov-model approach. Using MANTA, we demonstrated and characterized important species-, rate-, cell-type-, and disease-state-specific AAD effects, including 1) a stronger effect of Class III AADs in large mammals than in rodents; 2) a rate-dependent decrease in upstroke velocity with Class I AADs and reverse rate-dependent effects of Class III AADs on action potential duration; 3) ventricular-predominant effects of pure IKr blockers; 4) preferential reduction in atrial AP upstroke velocity with vernakalant; and 5) excessive AP prolongation with Class III AADs other than amiodarone under heart failure conditions.In conclusion, the effects of AADs are highly complex and strongly dependent on the experimental or clinical conditions. MANTA is a powerful and freely available tool reproducing a wide range of AAD characteristics that enables analyses of the underlying ionic mechanisms. Use of MANTA is expected to improve our understanding of AAD effects on cellular electrophysiology under a wide range of conditions, which may provide clinically-relevant information on the safety and efficacy of AAD treatment.

A cellular model of Brugada syndrome with SCN10A variants using human-induced pluripotent stem cell-derived cardiomyocytes.

Brugada syndrome (BrS) is associated with a pronounced risk to develop sudden cardiac death (SCD). Up to 21% of patients are related to mutations in SCN5A. Studies identified SCN10A as a contributor of BrS. However, the investigation of the human cellular phenotype of BrS in the presence of SCN10A mutations remains lacking. The objective of this study was to establish a cellular model of BrS in presence of SCN10A mutations using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Dermal fibroblasts obtained from a BrS patient suffering from SCD harbouring the SCN10A double variants (c.3803G>A and c.3749G>A) and three independent healthy control subjects were reprogrammed to hiPSCs. Human-induced pluripotent stem cells were differentiated into cardiomyocytes (hiPSC-CMs).The hiPSC-CMs from the BrS patient showed a significantly reduced peak sodium channel current (INa) and a significantly reduced ATX II (sea anemone toxin, an enhancer of late INa) sensitive as well as A-887826 (a blocker of SCN10A channel) sensitive late sodium channel current (INa) when compared with the healthy control hiPSC-CMs, indicating loss-of-function of sodium channels. Consistent with reduced INa the action potential amplitude and upstroke velocity (Vmax) were significantly reduced, which may contribute to arrhythmogenesis of BrS. Moreover, Ajmaline effects on action potentials were stronger in BrS-hiPSC-CMs than in healthy control cells. This is in agreement with the higher susceptibility of patients to sodium channel blocking drugs in unmasking BrS. Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of BrS with SCN10A mutations and may provide novel opportunities to further elucidate the cellular disease mechanism.

DPP10 is a new regulator of Nav1.5 channels in human heart

BackgroundCardiac accessory β-subunits are part of macromolecular Nav1.5 channel complexes modulating biophysical properties and contributing to arrhythmias. Recent studies demonstrated the structural interaction between β-subunits of Na+ (Nav1.5) and K+ (Kv4.3) channels. Here, we identified the dipeptidyl peptidase-like protein-10 (DPP10), which is known to modulate Kv4.3-current kinetics, as a new regulator of Nav1.5 channels. MethodsWe assessed DPP10 expression in the healthy and diseased human heart and we studied the functional effects of DPP10 on the Na+ current in isolated rat cardiomyocytes expressing DPP10 after adenoviral gene-transfer (DPP10ad). ResultsDPP10 mRNA and proteins were detected in human ventricle, with higher levels in patients with heart failure. In rat cardiomyocytes, DPP10ad significantly reduced upstroke velocity of action potentials indicating reduction in Na+-current density. DPP10 significantly shifted the voltage-dependent Na+ channel activation and inactivation curve to more positive potentials, resulting in greater availability of Na+ channels for activation, along with increasing window Na+ current. In addition, time-to-peak Na+ current was reduced, whereas time course of recovery from inactivation was significantly accelerated by DPP10ad. DPP10 co-immunoprecipitated with Nav1.5 channels in human ventricles, confirming their physical interaction. ConclusionWe provide first evidence that DPP10 interacts with Nav1.5 channels, linking Na+- and K+-channel complexes in the heart. Our data suggest that increased ventricular DPP10 expression in heart failure might promote arrhythmias by decreasing peak Na+ current, while increasing window Na+ current and channel re-openings due to accelerated recovery from inactivation.

NPR-C (Natriuretic Peptide Receptor-C) Modulates the Progression of Angiotensin II-Mediated Atrial Fibrillation and Atrial Remodeling in Mice.

Atrial fibrillation (AF) commonly occurs in hypertension and in association with elevated Ang II (angiotensin II) levels. The specific mechanisms underlying Ang II-mediated AF are unclear, and interventions to prevent the effects of Ang II are lacking. NPs (natriuretic peptides), which elicit their effects through specific NP receptors, including NPR-C (natriuretic peptide receptor-C), are cardioprotective hormones that affect cardiac structure and function. This study used wild-type and NPR-C knockout (NPR-C-/-) mice to investigate the effects of Ang II (3 mg/kg per day for 3 weeks) on AF susceptibility and atrial function using in vivo electrophysiology, high-resolution optical mapping, patch clamping, and molecular biology. In some experiments, wild-type mice were cotreated with Ang II and the NPR-C agonist cANF (0.07-0.14 mg/kg per day) for 3 weeks. In wild-type mice, Ang II increased susceptibility to AF in association with a prolongation of P-wave duration, increased atrial refractory period, and slowed atrial conduction. These effects were exacerbated in Ang II-treated NPR-C-/- mice. Ang II prolonged action potential duration and reduced action potential upstroke velocity (Vmax). These effects were greater in left atrial myocytes from Ang II-treated NPR-C-/- mice. Ang II also increased fibrosis in both atria in wild-type mice, whereas Ang II-treated NPR-C-/- mice exhibited substantially higher fibrosis throughout the atria. Fibrotic responses were associated with changes in expression of profibrotic genes, including TGFβ and TIMP1. Cotreating wild-type mice with Ang II and the NPR-C agonist cANF dose dependently reduced AF inducibility by preventing some of the Ang II-induced changes in atrial myocyte electrophysiology and preventing fibrosis throughout the atria. NPR-C may represent a new target for the prevention of Ang II-induced AF via protective effects on atrial electrical and structural remodeling.

Abstract 16826: Reversal of TSPO Upregulation in the Diabetic Heart by Chronic TSPO Gene Silencing Causes Metabolic Sink via an Increase in ROMK Expression

Introduction: The mitochondrial translocator protein (TSPO) is an integral component of a macromolecular complex that regulates cell death & survival pathways. Myocardial TSPO levels are markedly increased in diabetes. Objectives: We sought to investigate the electrophysiological effects of TSPO in diabetes using a cardiotropic TSPO gene silencing approach that avoids off-target effects of TSPO ligands. Methods: In a standard model of diabetes mellitus consisting of Zucker Obese rats, AAV9.shTSPO (5E11 gc/mL) was delivered by tail vein injection (N=5). Non-diabetic lean (N=4) and spontaneously hypertensive (N=16) rats served as treatment controls. Untreated diabetic (N=6) and non-diabetic (N=6) hearts were also studied. The TSPO ligand DZP (64μμ) was perfused in additional diabetic hearts to contrast its effects with those of chronic TSPO knockdown. Optical action potential (AP) mapping was performed 2 wks following gene transfer. Hearts were challenged with 12 min ischemia followed by reperfusion. Results: Myocardial TSPO knockdown by AAV9.shTSPO promoted metabolic sink in diabetic hearts. This manifested as rapid and spatially heterogeneous loss of AP amplitude and upstroke velocity in response to ischemia (Figure). Areas of early loss of excitability remained inexcitable upon reperfusion resulting in wide regions of conduction block that supported VT. These findings were specific to diabetes as AAV9.shTSPO did not alter the response of non-diabetic hearts to ischemia. Loss of excitability was not related to irreversible cell death by TUNEL staining. Analysis of the expression of ATP-sensitive channel subunits revealed marked upregulation of ROMK in response to TSPO knockdown exclusively in the diabetic heart. Unlike TSPO knockdown, DZP did not promote metabolic sink but rather prevented post-ischemic VT. Conclusions: Chronic gene silencing of TSPO promotes VT via a metabolic sink mechanism likely arising from compensatory upregulation of ROMK.

Abstract 17323: Polyinosinic-Polycytidylic Acid Primes Cardiac Progenitors From Human Induced Pluripotent Stem Cells for Enhanced Cell Therapy and Cardiomyocyte Maturation

Cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) hold promise for disease modeling, drug discovery, and therapy, but the challenge remains to create mature cardiomyocytes like those found in the adult heart. While groups have increased the maturity of hiPS-CMs in extended culture with electrical, metabolic, and mechanical stimulation, we hypothesized that epigenetic modulation during the formation of cardiac progenitors (hiPS-CPCs) could enhance their capacity to form mature CMs. We found that priming with the innate immune agonist polyinosinic-polycytidylic acid (pIC) decreased cardiac lineage-HDAC expression during the formation of hiPS-CPCs in defined small molecule monolayer differentiation. While both untreated and primed day 5 hiPS-CPCs contained equivalent >80% purity of KDR+PDGRFα+ CPC populations, gene expression studies using RNAseq demonstrated that pIC priming enhanced the early cardiogenic and Notch signaling programs. When both groups were differentiated in basal media, primed hiPS-CPCs gave rise to more mature cardiomyocytes based on larger cell size, increased optical action potential upstroke velocity, greater oxidative metabolism, enhanced sarcomere maturation, and upregulated transcriptional markers of CM maturation including cTnI, cardiac actin, and αMHC. These maturation effects of pIC treatment were blocked by the Notch inhibitor DAPT. Most impressively, primed hiPS-CPCs improved survival as well as myocardial systolic/diastolic function in a mouse model of myocardial infarction (Figure). Conclusion: pIC-primed hiPS-CPCs with the capacity to give rise to hiPS-CMs of enhanced maturation hold promise for improved disease modeling, drug screening and cell therapy.

The role of Natriuretic Peptide Receptor C in atrial electrophysiological remodelling in hypertensive heart disease

Atrial fibrillation (AF) is prevalent in hypertension in association with alterations in angiotensin II (Ang II) signaling. Natriuretic peptides (NPs) are a family of hormones with potent effects on atrial electrophysiology mediated in part through the NPR-C receptor. The goal of this study was to determine the role of NPR-C in atrial electrophysiological remodelling in Ang II treated mice (3 mg/kg/day for 3 weeks). Ang II treatment increased the susceptibility to AF in WT (P = 0.001) and was more severe in NPR-C-/- (P = 0.03) mice vs. saline controls. Action potential (AP) upstroke velocity (Vmax; P = 0.01) and peak Na+ current (INa; P < 0.001) were reduced in left atrial (LA) myocytes from Ang II treated WT mice, which was attributed to enhanced PKC⍺ signalling. Ang II differentially prolonged the AP in right atrial (RA; APD50; P < 0.001) and LA (P < 0.001) myocytes. In addition, Ang II reduced Ito in RA myocytes (P < 0.001) and to a greater extent in LA myocytes (P = 0.045). In the RA of NPR-C-/‑ mice, Ang II prolonged (P < 0.05) APD50 to a similar extent vs. WT. In contrast, Vmax was reduced and APD50 was prolonged by Ang II to a greater extent (P < 0.05) in the LA of NPR-C-/- vs. WT mice. Ito was similarly reduced (P<0.05) in the RA and LA of Ang II treated WT and NPR-C-/- mice. Co-treatment with cANF (NPR-C agonist) reduced the susceptibility to AF (P = 0.04) in WT mice vs. Ang II alone. Strikingly, APD50 was reduced (P = 0.01) and Ito was increased (P = 0.001) in the RA, but not LA, by cANF co-treatment. Collectively, these data demonstrate that NPR-C plays a critical role in modulating disease progression in the atria elicited by Ang II treatment.

Description of the Human Atrial Action Potential Derived From a Single, Congruent Data Source: Novel Computational Models for Integrated Experimental-Numerical Study of Atrial Arrhythmia Mechanisms.

Introduction: The development of improved diagnosis, management, and treatment strategies for human atrial fibrillation (AF) is a significant and important challenge in order to improve quality of life for millions and reduce the substantial social-economic costs of the condition. As a complex condition demonstrating high variability and relation to other cardiac conditions, the study of AF requires approaches from multiple disciplines including single-cell experimental electrophysiology and computational modeling. Models of human atrial cells are less well parameterized than those of the human ventricle or other mammal species, largely due to the inherent challenges in patch clamping human atrial cells. Such challenges include, frequently, unphysiologically depolarized resting potentials and thus injection of a compensatory hyperpolarizing current, as well as detecting certain ion currents which may be disrupted by the cell isolation process. The aim of this study was to develop a laboratory specific model of human atrial electrophysiology which reproduces exactly the conditions of isolated-cell experiments, including testing of multiple experimental interventions.Methods: Formulations for the primary ion currents characterized by isolated-cell experiments in the Workman laboratory were fit directly to voltage-clamp data; the fast sodium-current was parameterized based on experiments relating resting membrane potential to maximal action potential upstroke velocity; compensatory hyperpolarizing current was included as a constant applied current. These formulations were integrated with three independent human atrial cell models to provide a family of novel models. Extrapolated intact-cell models were developed through removal of the hyperpolarizing current and introduction of terminal repolarization potassium currents.Results: The isolated-cell models quantitatively reproduced experimentally measured properties of excitation in both control and pharmacological and dynamic-clamp interventions. Comparison of isolated and intact-cell models highlighted the importance of reproducing this cellular environment when comparing experimental and simulation data.Conclusion: We have developed a laboratory specific model of the human atrial cell which directly reproduces the experimental isolated-cell conditions and captures human atrial excitation properties. The model may be particularly useful for directly relating model to experiment, and offers a complementary tool to the available set of human atrial cell models with specific advantages resulting from the congruent input data source.