Action Potential Duration Research Articles

ER stress modulates Kv1.5 channels via PERK branch in HL-1 atrial myocytes: Relevance to atrial arrhythmogenesis and the effect of tetramethylpyrazine

Endoplasmic reticulum (ER) stress is implicated in cardiac arrhythmia whereas the associated mechanisms remain inadequately understood. Kv1.5 channels are essential for atrial repolarization. Whether ER stress affects Kv1.5 channels is unknown. This study aimed to elucidate the response of Kv1.5 channels to ER stress by clarifying the unfolded protein response (UPR) branch responsible for the channel modulation. In addition, the effect of tetramethylpyrazine (TMP) on Kv1.5 channels was studied. Patch clamp and western-blot results revealed that exposure of HL-1 atrial myocytes to ER stress inducer tunicamycin upregulates Kv1.5 expression, increases Kv1.5 channel current (IKur) (14.91 ± 1.11 vs. 6.11 ± 1.31 pA/pF, P < 0.001), and shortened action potential duration (APD) (APD90: 82.79 ± 5.25 vs.121.11 ± 6.72 ms, P < 0.01), which could be reverted by ER stress inhibitors. Blockade of the PERK branch while not IRE1 and ATF6 branches of UPR downregulated Kv1.5 expression, accompanied by a decreased IKur (9.03 ± 0.99 pA/pF) and a prolonged APD90 (113.69 ± 4.41 ms) (P < 0.01). PERK-mediated increases of Kv1.5 expression and IKur were also observed in HL-1 cells incubated with thapsigargin. TMP suppressed the enhancement of IKur (10.52 ± 0.97 vs. 17.52 ± 2.25 pA/pF, P < 0.05), prevented the shortening of APD (APD90: 110.16 ± 5.36 vs. 84.84 ± 4.58 ms, P < 0.05), and inhibited the upregulation of Kv1.5 triggered by ER stress. Our study suggests that ER stress induces upregulation and activation of Kv1.5 channels in atrial myocytes through the PERK branch of UPR. TMP prevents Kv1.5 upregulation/activation and the resultant APD shortening by inhibiting ER stress. These results may shed light on the mechanisms of atrial arrhythmogenesis and the antiarrhythmic effect of the traditional Chinese herb TMP.

Arrhythmias in thyroid dysfunction

Thyroid dysfunction is associated with characteristic changes in heart rate and arrhythmias. Thyroid hormones act through genomic and non-genomic effects on myocytes and influence contractility, relaxation and action potential duration through avariety of mechanisms. Atrial fibrillation is the most common arrhythmia associated with thyroid dysfunction, it occurs in both euthyroidism and hyperthyroidism in clear association with T4 levels. Mechanistically, in the hyperthyroid state, increased automaticity and triggered activity, together with ashortened refractory period and slowing of the conduction speed, lead to the initiation and maintenance of multiple intraatrial reentry circuits. Influences from the autonomic nervous system and hemodynamics controlled by thyroid hormones act as modulators for arrhythmias, which are promoted by acorresponding substrate (significant impact of comorbidities). Concerning therapy, in addition to treating hyperthyroidism, the initial therapeutic focus is on adequate rate control and anticoagulation in patients with ahigh risk of thromboembolism. Ablation of atrial fibrillation can be considered later on, although there is an increased likelihood of recurrence compared to patients without hyperthyroidism.Prolongation of the QT interval and increase in QT dispersion are involved in the formation of ventricular arrhythmias. Epidemiological data suggest an association of elevated T4 levels with ventricular arrhythmias and sudden cardiac death. However, this seems to be mainly relevant for patients with underlying cardiac disease (e.g. ICD users).

Sympathetic structural and electrophysiological remodeling in a rabbit model of reperfused myocardial infarction.

Chondroitin sulfate proteoglycans (CSPGs) inhibit sympathetic reinnervation in rodent hearts post-myocardial infarction (MI), causing regional hypoinnervation that is associated with supersensitivity of β-adrenergic receptors and increased arrhythmia susceptibility. To investigate the role of CSPGs and hypoinnervation in the heart of larger mammals, we used a rabbit model of reperfused MI and tested electrophysiological responses to sympathetic nerve stimulation (SNS). Innervated hearts from MI and sham rabbits were optically mapped using voltage and Ca2+-sensitive dyes. SNS was performed with electrical stimulation of the spinal cord, and β-adrenergic responsiveness was tested using isoproterenol. Sympathetic nerve density and CSPG expression were evaluated using immunohistochemistry. CSPGs were robustly expressed in the infarct region of all MI hearts, and the presence of CSPGs was associated with reduced sympathetic nerve density in the infarct versus remote region. Action potential duration (APD) dispersion and tendency for induction of ventricular tachycardia/fibrillation (VT/VF) were increased with SNS in MI but not sham hearts. SNS decreased APD at 80% repolarization (APD80) in MI but not sham hearts, whereas isoproterenol decreased APD80 in both groups. Isoproterenol also shortened Ca2+ transient duration at 80% repolarization in both groups but to a greater extent in MI hearts. Our data suggest that sympathetic remodeling post-MI is similar between rodents and rabbits, with CSPGs associated with sympathetic hypoinnervation. Despite a reduction in sympathetic nerve density, the infarct region of MI hearts remained responsive to both physiological SNS and isoproterenol, potentially through preserved or elevated β-adrenergic responsiveness, which may underlie increased APD dispersion and tendency for VT/VF.NEW & NOTEWORTHY Here, we show that CSPGs are present in the infarcts of rabbit hearts with reperfused MI, where they are associated with reduced sympathetic nerve density. Despite hypoinnervation, sympathetic responsiveness is maintained or enhanced in MI rabbit hearts, which also demonstrate increased APD dispersion and tendency for arrhythmias following sympathetic modulation. Together, this study indicates that the mechanisms of sympathetic remodeling post-MI are similar between rodents and rabbits, with hypoinnervation likely associated with enhanced β-adrenergic sensitivity.

Abstract P383: Increased renal afferent nerve activity and peak response to pro-inflammatory cytokine interleukin 6 in isolated dorsal root ganglion neurons from hypertensive rats

Renal afferent nerves are reported to mediate the progression of hypertension in preclinical models, putatively via an aberrant increase in activation. The underlying source of this nerve activation is unknown but we hypothesized renal inflammatory cytokines are a primary driver. We previously reported interleukin 6 (IL-6) is increased in the kidneys of hypertensive rats, and intrarenal administration of IL-6 can activate renal afferent nerves. Though these data are promising, the direct effects of IL-6 on afferent renal nerves remain unclear. We hypothesized that isolated renal afferent neurons from hypertensive rats will have increased activity and responsiveness to inflammatory cytokine IL-6 compared to normotensive controls. To test our hypothesis, we tested the direct effects of interleukin-6 (IL-6) on renal afferent nerve action potentials (AP) generated in freshly isolated dorsal root ganglion (DRG) neurons. To model salt-sensitive hypertension, uninephrectomized rats (n=6) were administered deoxycorticosterone acetate (DOCA; 100mg, sc) and 0.9% saline for drinking water for 21 days. Control rats (n=6) were uninephrectomized. To label renal afferent nerves, neuronal tracer DiI was introduced by intrarenal injection 6 days prior to DRG collection. DRGs from T10-L1 were isolated, enzymatically digested and dissociated, and cultured overnight for whole-cell patch-clamp the following day. DiI-positive neurons from both control and DOCA rats generated AP during the delivery of 100, 200, and 300 pA current steps. Data were analyzed by two-way, repeat-measures ANOVA (α=.05). At baseline, DOCA neurons had a higher (p&lt;0.05) mean AP frequency compared to controls (no difference in AP mean peak amplitude or AP duration). In both groups, increasing concentrations of IL-6 (0. 4, 4, and 40 ng/ml) elicited a dose-dependent increase in AP peak amplitude compared to baseline. No difference was detected in IL-6 responses between DOCA and controls. Overall, these data supported our original hypothesis that pro-inflammatory cytokine IL-6 would increase renal afferent nerve activity, and that said activity is increased in hypertensive DOCA-salt rats. IL-6 accumulation triggered by end organ damage due to hypertension might lead to increased afferent renal nerve activity, which exacerbates the hypertensive phenotype through a sympatho-excitatory response. Additional measurements of isolated DRG responses to pro-inflammatory mediators are underway for direct comparison.

C-Src Is Responsible for Mitochondria-Mediated Arrhythmic Risk in Ischemic Cardiomyopathy.

Increased mitochondrial Ca2+ uptake has been implicated in the QT prolongation and lethal arrhythmias associated with nonischemic cardiomyopathy. We attempted to define the role of mitochondria in ischemic arrhythmic risk and to identify upstream regulators. Myocardial infarction (MI) was induced in wild-type FVB/NJ mice by ligation of the left anterior descending coronary artery. Western blot, immunoprecipitation, ECG telemetry, and patch-clamp techniques were used. After MI, c-Src (proto-oncogene tyrosine-protein kinase Src) and its active form (p-Src Y416) were increased. The activation of c-Src was associated with increased diastolic Ca2+ sparks, action potential duration prolongation, and arrhythmia in MI mice. c-Src upregulation and arrhythmia could be reversed by treatment of mice with the Src inhibitor PP1 but not with the inactive analogue PP3. Tyrosine phosphorylated mitochondrial Ca2+ uniporter (MCU) was upregulated in the heart tissues of MI mice and patients with ischemic cardiomyopathy. In a heterologous expression system, c-Src could bind MCU and phosphorylate MCU tyrosines. Overexpression of wild-type c-Src significantly increased the mitochondrial Ca2+ transient while overexpression of dominant-negative c-Src significantly decreased the mitochondrial Ca2+ transient. c-Src inhibition by PP1, MCU inhibition by Ru360, or MCU knockdown could reduce the action potential duration, Ca2+ sparks, and arrhythmia after MI. The human heart tissue showed that patients with ischemic cardiomyopathy had significantly increased c-Src active form associated with increased MCU tyrosine phosphorylation and ventricular arrhythmia. MI leads to increased c-Src active form that results in MCU tyrosine phosphorylation, increased mitochondrial Ca2+ uptake, QT prolongation, and arrhythmia, suggesting c-Src or MCU may represent novel antiarrhythmic targets.

Developmental Changes in the Excitation-Contraction Mechanisms of the Ventricular Myocardium and Their Sympathetic Regulation in Small Experimental Animals.

The developmental changes in the excitation-contraction mechanisms of the ventricular myocardium of small animals (guinea pig, rat, mouse) and their sympathetic regulation will be summarized. The action potential duration monotonically decreases during pre- and postnatal development in the rat and mouse, while in the guinea pig it decreases during the fetal stage but turns into an increase just before birth. Such changes can be attributed to changes in the repolarizing potassium currents. The T-tubule and the sarcoplasmic reticulum are scarcely present in the fetal cardiomyocyte, but increase during postnatal development. This causes a developmental shift in the Ca2+ handling from a sarcolemma-dependent mechanism to a sarcoplasmic reticulum-dependent mechanism. The sensitivity for beta-adrenoceptor-mediated positive inotropy decreases during early postnatal development, which parallels the increase in sympathetic nerve innervation. The alpha-adrenoceptor-mediated inotropy in the mouse changes from positive in the neonate to negative in the adult. This can be explained by the change in the excitation-contraction mechanism mentioned above. The shortening of the action potential duration enhances trans-sarcolemmal Ca2+ extrusion by the Na+-Ca2+ exchanger. The sarcoplasmic reticulum-dependent mechanism of contraction in the adult allows Na+-Ca2+ exchanger activity to cause negative inotropy, a mechanism not observed in neonatal myocardium. Such developmental studies would provide clues towards a more comprehensive understanding of cardiac function.

The Kv4 potassium channel modulator NS5806 attenuates cardiac hypertrophy in vivo and in vitro

The compound NS5806 is a Kv4 channel modulator. This study investigated the chronic effects of NS5806 on cardiac hypertrophy induced by transverse aortic constriction (TAC) in mice in vivo and on neonatal rat ventricular cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) in vitro. Four weeks after TAC, NS5806 was administered by gavage for 4 weeks. Echocardiograms revealed pronounced left ventricular (LV) hypertrophy in TAC-treated mice compared with sham mice. NS5806 attenuated LV hypertrophy, as manifested by the restoration of LV wall thickness and weight and the reversal of contractile dysfunction in TAC-treated mice. NS5806 also blunted the TAC-induced increases in the expression of cardiac hypertrophic and fibrotic genes, including ANP, BNP and TGF-β. Electrophysiological recordings revealed a significant prolongation of action potential duration and QT intervals, accompanied by an increase in susceptibility to ventricular arrhythmias in mice with cardiac hypertrophy. However, NS5806 restored these alterations in electrical parameters and thus reduced the incidence of mouse sudden death. Furthermore, NS5806 abrogated the downregulation of the Kv4 protein in the hypertrophic myocardium but did not influence the reduction in Kv4 mRNA expression. In addition, NS5806 suppressed in vitro cardiomyocyte hypertrophy. The results provide novel insight for further ion channel modulator development as a potential treatment option for cardiac hypertrophy.

Targeting heterozygous dominant negative variant of KCNA2 using Gapmer ASO for the treatment of drug-resistant epilepsy

A missense mutation c.1220C>G of KCN2A gene was recently identified in an infant with epilepsy. KCNA2 encodes Kv1.2 subunits that form voltage-gated potassium channels (VGKC) via tetrameric assembly. The mutation results in amino acid change P407R at the highly conserved PVP motif. Functional characterization revealed that mutant Kv1.2_P407R subunits formed loss-of-function channels and suppressed both Kv1.2 and Kv1.1 channel activities. Hetero-tetrameric assembly of the Kv1.2_P407R subunits with other neuronal voltage-gated potassium channels of Shaker subfamily could lead to general deficit of repolarising potassium current and potentially underlie the enhanced seizure susceptibility. Indeed, expression of human Kv1.2_P407R in early postnatal rat cortical neurons or genetically engineered hESC-derived neurons disclosed broadening of action potential duration and early afterdepolarization (EAD), associating with reduced potassium current. We hypothesize that Gapmer antisense oligonucleotides (ASO) targeted to c.1220C>G mutation will selectively degrade the mutant mRNA while allowing the remaining WT subunits to form functional channels. As a proof of principle, delivery of Gapmer packaged in lipid nanoparticle into cortical neurons selectively suppressed Kv1.2_P407R over the WT protein expression, reversing the broadening of action potential duration, abrogating the EAD and leading to overall increase in potassium current.

14-3-3 promotes sarcolemmal expression of cardiac Ca V 1.2 and nucleates isoproterenol-triggered channel super-clustering.

The L-type Ca 2+ channel, Ca V 1.2, plays an essential role in excitation-contraction coupling in the heart and in part regulates the overall strength of contraction during basal and fight- or-flight β -adrenergic signaling conditions. Proteins that modulate the trafficking and/or activity of Ca V 1.2 are interesting both from a physiological and pathological perspective, since alterations in Ca V 1.2 can impact action potential duration and cause arrythmias. A small protein called 14-3-3 regulates other ion channels in the heart and other Ca 2+ channels, but how it may interact with Ca V 1.2 in the heart has never been studied. Examining factors that affect Ca V 1.2 at rest and during β -adrenergic stimulation is crucial for our ability to understand and treat disease and aging conditions where these pathways are altered.

Modulation of NOX2 causes obesity-mediated atrial fibrillation.

Obesity is linked to an increased risk of atrial fibrillation (AF) via increased oxidative stress. While NADPH oxidase 2 (NOX2), a major source of oxidative stress and reactive oxygen species (ROS) in the heart, predisposes to AF, the underlying mechanisms remain unclear. Here, we studied NOX2-mediated ROS production in obesity-mediated AF using Nox2-knockout mice and mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs). Diet-induced obesity (DIO) mice and hiPSC-aCMs treated with palmitic acid (PA) were infused with a NOX blocker (apocynin) and a NOX2-specific inhibitor, respectively. We showed that NOX2 inhibition normalized atrial action potential duration and abrogated obesity-mediated ion channel remodeling with reduced AF burden. Unbiased transcriptomics analysis revealed that NOX2 mediates atrial remodeling in obesity-mediated AF in DIO mice, PA-treated hiPSC-aCMs, and human atrial tissue from obese individuals by upregulation of paired-like homeodomain transcription factor 2 (PITX2). Furthermore, hiPSC-aCMs treated with hydrogen peroxide, a NOX2 surrogate, displayed increased PITX2 expression, establishing a mechanistic link between increased NOX2-mediated ROS production and modulation of PITX2. Our findings offer insights into possible mechanisms through which obesity triggers AF and support NOX2 inhibition as a potential novel prophylactic or adjunctive therapy for patients with obesity-mediated AF.

Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

PITX2 deficiency leads to atrial mitochondrial dysfunction.

Reduced left atrial PITX2 is associated with atrial cardiomyopathy and atrial fibrillation. PITX2 is restricted to left atrial cardiomyocytes in the adult heart. The links between PITX2 deficiency, atrial cardiomyopathy and atrial fibrillation are not fully understood. To identify mechanisms linking PITX2 deficiency to atrial fibrillation, we generated and characterized PITX2-deficient human atrial cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) and their controls. PITX2-deficient hiPSC-derived atrial cardiomyocytes showed shorter and disorganised sarcomeres and increased mononucleation. Electron microscopy found an increased number of smaller mitochondria compared to the control. Mitochondrial protein expression was altered in PITX2-deficient hiPSC-derived atrial cardiomyocytes. Single-nuclear RNA-sequencing found differences in cellular respiration pathways and differentially expressed mitochondrial and ion channel genes in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2 repression in hiPSC-derived atrial cardiomyocytes replicated dysregulation of cellular respiration. Mitochondrial respiration was shifted to increased glycolysis in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2-deficient human hiPSC-derived atrial cardiomyocytes showed higher spontaneous beating rates. Action potential duration was more variable with an overall prolongation of early repolarization, consistent with metabolic defects. Gene expression analyses confirmed changes in mitochondrial genes in left atria from 42 patients with atrial fibrillation compared to 43 patients in sinus rhythm. Dysregulation of left atrial mitochondrial (COX7C) and metabolic (FOXO1) genes was associated with PITX2 expression in human left atria. In summary, PITX2 deficiency causes mitochondrial dysfunction and a metabolic shift to glycolysis in human atrial cardiomyocytes. PITX2-dependent metabolic changes can contribute to the structural and functional defects found in PITX2-deficient atria.

KCNQ1 suppression-replacement gene therapy in transgenic rabbits with type 1 long QT syndrome.

Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

Abstract Mo021: Role of Estrogen and Testosterone in promoting Sex Dimorphism in Doxorubicin Cardiotoxicity.

Background: Doxorubicin (DOX), a cancer chemotherapeutic agent, causes sex-specific cardiotoxicity. Lower cardiotoxicity in females compared to males is typically attributed to the cardioprotective effects of estrogen (E2). However, the role of the sex hormones, E2 and testosterone (T), in modulating DOX induced cardiac dysfunction and its underlying mechanisms are not known. Methods: C57BL/6J mice were divided into four groups, 1) Control, 2) DOX (5 mg/kg/week for 6 weeks), 3) Reverse hormone profile (RevH, castrated males+E2 and ovariectomized females+T), and 4) RevH + DOX. Mouse morbidity was tracked daily. Total body weight and cardiac function was assessed biweekly for 6 weeks by echocardiography, ECG and optical mapping. Dependence on fatty acid versus glucose for metabolism was measured by seahorse analysis. Results: DOX treatment reduced total body weight (11%) in males, and increased morbidity in males and females. This was associated with reduced mechanical function (15% decrease in stroke volume) in males and electrical remodeling (29% action potential duration prolongation) in females. In RevH mice, no changes in morbidity or cardiac function were observed compared to Control. In RevH+DOX males, the onset of morbidity was delayed by two weeks compared to DOX males. This cardioprotective effect was associated with preserved mechanical and electrical function. On the other hand, in RevH+DOX females, morbidity was increased, and this correlated with mechanical (20% reduction in cardiac output) and electrical remodeling (22% increase in action potential duration). Comparing RevH+DOX males to females, males had greater fatty acid dependency while females had greater glucose dependency for metabolism. Conclusions: These results suggest that sex hormones play a significant role in modulating specific aspects of DOX cardiotoxicity. While E2 has a critical role in preserving cardiac mechanics during DOX therapy, high T levels were associated with mechanical dysfunction in both males and RevH females. Switching of metabolic substrates with greater preference for fatty acid metabolism in mice with higher E2 could underlie cardiac mechanics preservation. Finally, DOX induced electrical remodeling was not altered by sex hormones.

Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an In Vitro Model to Evaluate the Efficacy of a Gene Replacement Therapy for Plakophilin-2- Associated Arrhythmogenic Right Ventricular Cardiomyopathy

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G&gt;C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.

Abstract We099: Antisense Oligonucleotide Treatment of Calmodulinopathy

Introduction: Antisense Oligonucleotides (ASOs) are an emerging class of drugs that target RNAs to impede their production, stability, and translation. Calmodulinopathies are severe cardiac arrhythmias caused by dominant mutations in CALM1 , CALM2 , or CALM3 . Each of these genes encodes the identical calmodulin (CaM) protein. Leveraging this genetic redundancy, we hypothesized that ASO-mediated knockdown of the affected CALM gene would treat arrhythmia while functional redundancy of the other CALM genes would maintain CaM protein level and prevent toxicity. Methods: Cells from a patient expressing the heterozygous CALM1 - F142L mutation were reprogrammed into induced pluripotent stem cells (iPSCs; P-CALM1 F142L/+ ). Additionally, the variant or a nonsense codon were introduced into the wild-type reference line, WTC11, to yield isogenic disease (E-CALM1 F142L/+ ) and CALM1 null ( CALM1 Δ/Δ ) lines respectively. iPSCs were differentiated into cardiomyocytes (iPSC-CMs) to assess their action potentials and Ca 2+ transients using multielectrode arrays and fluorescent voltage- and calcium-sensitive dyes. Calm1 N98S/+ mice, previously shown to develop catecholamine-induced ventricular tachycardia, were used for in vivo studies. Using these model systems, we tested ASOs that selectively depleted human or murine CALM1 . Results: Both E-CALM1 F142L/+ and P-CALM1 F142L/+ iPSC-CMs exhibited prolonged action potentials, consistent with QT prolongation observed in the proband. CALM1 Δ/Δ iPSC-CMs expressed normal levels of CaM protein and exhibited no change in action potential duration. ASO that selectively depleted CALM1 transcripts by ~50% did not alter CaM protein level but normalized action potential and Ca 2+ transient duration in CALM1 F142L/+ iPSC-CMs. In Calm1 N98S/+ mice, ASO selectively depleted Calm1 transcripts by ~90% and prevented catecholamine-induced ventricular tachycardia. Calm1 ASO did not alter CaM protein level, cardiac function, or treadmill endurance, or cause significant histopathological changes. Conclusion: ASO-based therapy safely and effectively treated arrhythmias caused by dominant CALM1 variants, suggesting that ASOs are a promising potential therapy for calmodulinopathy.

Abstract Tu083: Oxytocin improves cardiovascular outcomes in a rat model of pressure-overload heart failure

Oxytocin (OT) is a peptide made in the hypothalamus and released into circulation by the pituitary gland. Known for its role in childbirth and lactation, recent research suggests that OT has significant cardiovascular benefits, reducing the risk of cardiovascular diseases such as hypertension and atherosclerosis. We tested if OT has beneficial effects on cardiovascular function in a rat model of pressure overload heart failure (HF). Neonatal Sprague Dawley rats underwent transaortic constriction (TAC). Pressure overload and cardiac dysfunction progressed as animals matured. Echocardiography at 5 weeks of age confirmed reduced ejection fraction. Animals were then randomly assigned to receive daily IP injections of either OT (treatment) or saline (control) for two weeks. Echocardiography was performed again before hearts were excised for optical mapping studies to test for electrophysiological differences between groups. Hearts were electromechanically uncoupled using blebbistatin and transmembrane potential (RH237) and intracellular Ca2+ (Rhod2am) were simultaneously mapped during a dynamic restitution protocol. Preliminary results indicate that rats in the treatment (OT, n=3) group had improved cardiac function compared to controls (saline, n=3), with higher ejection fraction (66.71 ± 9.44 vs. 25.83 ± 17.93, p=0.0125), higher cardiac output (62.4 ± 11.45 vs. 20.35 ± 6.12, p=0.0025), and greater stroke volume (180.2 ± 18.19 vs. 73.96 ± 37.29, p=0.0057). Preliminary optical mapping results indicate that the hearts of OT-treated rats (n=3) have lower steady state action potential duration (APD) compared to controls (n=2). APD60 decreased from 109.5 ± 10.09 to 75.79 ± 21.94 (p=.0725) and APD80 decreased from 133.1 ± 10.22 to 101.1 ± 24.5 (p=.09595), indicating preservation of repolarizing currents corresponding to healthier electrophysiological function. In summary, our preliminary results suggest exogenous IP OT administration could have a potent cardioprotective effect on chronic and severe pressure overload hearts. Future work will investigate potential mechanisms of OT mediated cardio-protection, including sites of action.

Abstract Mo068: The role of FGF13 in modulating gap junction protein Connexin-43

Background: Fibroblast growth factor homologous factors (FHFs) are associated with arrhythmias like Brugada syndrome and atrial fibrillation, but their pathological mechanisms remain poorly understood. FHFs are best characterized as regulators of voltage gated sodium channels (VGSCs), however recent studies suggest broader, non-VGSC-related functions, including regulation of microtubules. Whether and how non-VGSC regulatory functions modulate cardiac rhythm is not known. Hypothesis: FGF13 regulates Cx43 trafficking and expression in ventricular cardiomyocytes via destabilizing microtubules. Aims: Building on an unbiased proteomic screen, we investigated if FGF13, the predominant FHF in rodent heart, affects the expression and trafficking of Connexin-43 (Cx43), the predominant gap junction protein in ventricular myocytes. Methods: To identify additional FGF13-dependent functional roles, we employed proximity labeling proteomics in mouse heart with the TurboID biotin ligase fused to FGF13. We used immunoblots, immunohistochemistry, and immunocytochemistry to further characterize the effects of FGF13 ablation on Cx43 pattern distribution and expression. We also leveraged electrophysiology techniques using gap junction inhibitors to detect any Cx43-dependent functional changes in FGF13-ablated cardiomyocytes. Results: The proteomic data revealed that cell junction proteins associated with the intercalated disc (ID) were among the top FGF13 near neighbors, of which Cx43 was heavily enriched, together with microtubule associated proteins (MAPs) known to be critical for cardiomyocyte function and ion channel trafficking. In ventricular cardiomyocytes from constitutive cardiomyocyte-restricted Fgf13 knockout mice, we found markedly perturbed trafficking of Cx43 and reduced colocalization with the ID marker N-Cadherin. Separately, MAP4 abundance was markedly reduced. The addition of colchicine, a microtubule destabilizing drug, to WT cells altered Cx43 distribution pattern similar to that observed in FGF13-ablated cardiomyocytes. Re-expression of FGF13 restored Cx43 trafficking and ID targeting. Electrophysiology experiments using a Cx43 hemichannel inhibitor, Gap19, showed a perturbation of the action potential duration in knockout cardiomyocytes. Isoproterenol-induced action potential prolongation in knockout myocytes was decreased by the addition of Gap19. Conclusions: FGF13 regulates microtubule-based trafficking and targeting of Cx43.

Abstract Mo054: Long Noncoding RNA LINC00667 Downregulation Leads to SCN5A Alternative Splicing and Arrhythmogenesis

Background: The voltage-gated sodium channel SCN5A, which encodes the Nav1.5 protein, is integral to the propagation of electrical signals in cardiac tissue. SCN5A expression is diminished in cardiomyopathy, significantly increasing the susceptibility to arrhythmias. This reduction in channel activity can be attributed to decreased mRNA stability coupled with aberrant mRNA splicing. The underlying mechanisms driving the dysregulation of mRNA splicing under pathophysiological conditions remain poorly understood. Objective: This study explores the role of long noncoding RNA LINC00667 in the abnormal alternative splicing of SCN5A mRNA. Methods: LINC00667 expression was analyzed using public databases, human heart tissue, iPSC-derived cardiomyocytes (iPSCMs), and cell lines. The impact of SCN5A on action potentials and sodium currents was studied in iPSCMs. LINC00667 and SCN5A expressions were measured using qRT-PCR, while Nav1.5 protein levels were assessed through Western blot. Alternative splicing of SCN5A mRNA was examined using PCR with exon-specific primers. Gene ontology and in silico analysis identified RNA binding proteins (RBPs) potentially interacting with LINC00667. RNA immunoprecipitation (RIP) assays confirmed direct binding with key splice factors. Group differences were assessed using 2-tailed Student’s t-tests, with P &lt; 0.05 indicating significance. Results: LINC00667, found at chromosome 18p11.31, is expressed in the human heart and associated cells, including cardiomyocytes. Its levels are inversely related to arrhythmic risk, decreasing in heart failure and ischemic cardiomyopathy as well as under hypoxic conditions. Gene ontology linked RBPs to SCN5A mRNA splicing, with RBM25, RBM5, and TIA1 identified as LINC00667 interactors. LINC00667 reduction affected regions of SCN5A mRNA encompassing Exons 6, 24, and 28, which could potentially be linked to decreased function/expression or increased unfolded protein response (UPR). Abnormal RNA splicing decreased SCN5A-encoded sodium current, reduced action potential upstroke velocity, and shortened AP duration without altering resting membrane potential. Conclusion: Decreased LINC00667 releases splicing factors that result in abnormal SCN5A mRNA splicing and reduced sodium current. Since LINC00667 is reduced in cardiomyopathy, overexpression may be antiarrhythmic by preventing sodium channel downregulation.