The proteostasis interactomes of trafficking-deficient K V 11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue.

Introduction: The voltage gated potassium ion channel K V 11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which can be rescued by pharmacological chaperones like E-4031. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery, comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants, and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. Methods: We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes in human embryonic kidney (HEK293) cells expressing wild-type (WT) K V 11.1 or trafficking-deficient channel variants in the presence or absence of E-4031. Resultsa: We identified 573 core K V 11.1 protein interactors. Both variants K V 11.1-G601S and K V 11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We found that proteasomal degradation is a key component for K V 11.1 degradation and that the K V 11.1-G601S-G965* variant was more responsive to E-4031 treatment. This suggests a role in the C-terminal domain and the ER retention motif of K V 11.1 in regulating trafficking. Conclusion: Our report characterizes the proteostasis network of K V 11.1, two trafficking deficient K V 11.1 variants, and variants treated with a pharmacological chaperone. The identified protein interactions could be targeted therapeutically to improve K V 11.1 trafficking and treat Long QT Syndrome.

Hypertrophic cardiomyopathy dysfunction mimicked in human engineered heart tissue and improved by sodium-glucose cotransporter 2 inhibitors.

AIMS: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, often caused by pathogenic sarcomere mutations. Early characteristics of HCM are diastolic dysfunction and hypercontractility. Treatment to prevent mutation-induced cardiac dysfunction is lacking. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a group of antidiabetic drugs that recently showed beneficial cardiovascular outcomes in patients with acquired forms of heart failure. We here studied if SGLT2i represent a potential therapy to correct cardiomyocyte dysfunction induced by an HCM sarcomere mutation. METHODS AND RESULTS: Contractility was measured of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) harbouring an HCM mutation cultured in 2D and in 3D engineered heart tissue (EHT). Mutations in the gene encoding ß-myosin heavy chain (MYH7-R403Q) or cardiac troponin T (TNNT2-R92Q) were investigated. In 2D, intracellular [Ca2+], action potential and ion currents were determined. HCM mutations in hiPSC-CMs impaired relaxation or increased force, mimicking early features observed in human HCM. SGLT2i enhance the relaxation of hiPSC-CMs, to a larger extent in HCM compared to control hiPSC-CMs. Moreover, SGLT2i-effects on relaxation in R403Q EHT increased with culture duration, i.e. hiPSC-CMs maturation. Canagliflozin's effects on relaxation were more pronounced than empagliflozin and dapagliflozin. SGLT2i acutely altered Ca2+ handling in HCM hiPSC-CMs. Analyses of SGLT2i-mediated mechanisms that may underlie enhanced relaxation in mutant hiPSC-CMs excluded SGLT2, Na+/H+ exchanger, peak and late Nav1.5 currents, and L-type Ca2+ current, but indicate an important role for the Na+/Ca2+ exchanger. Indeed, electrophysiological measurements in mutant hiPSC-CM indicate that SGLT2i altered Na+/Ca2+ exchange current. CONCLUSION: SGLT2i (canagliflozin > dapagliflozin > empagliflozin) acutely enhance relaxation in human EHT, especially in HCM and upon prolonged culture. SGLT2i may represent a potential therapy to correct early cardiac dysfunction in HCM.

ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

Eicosanoid-Regulated Myeloid ENaC and Isolevuglandin Formation in Human Salt-Sensitive Hypertension.

BACKGROUND: The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel-dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. METHODS: Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography-mass spectrometry. RESULTS: Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt-induced increase in isolevuglandin+ APC. CONCLUSIONS: Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading.

Ryanodine receptor 2 inhibition reduces dispersion of cardiac repolarization, improves contractile function, and prevents sudden arrhythmic death in failing hearts.

Sudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) is a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)-mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. We tested the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. We used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. DS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient ß-adrenergic challenge, and improved heart rate variability and cardiac function. Inhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca2+ leak-induced increases in diastolic Ca2+ and ROS-mediated RyR2 oxidation, thereby reducing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.

Each year, more than 300,000 people experience cardiac arrest or sudden cardiac death. Sudden cardiac death is caused by irregular heartbeats known as ventricular tachycardia or ventricular fibrillation, which prevent the heart from pumping blood. During a regular heart rhythm, the heart muscles contract and relax, regulated by a coordinated rise and fall of calcium ions within heart cells. In the cells of diseased hearts, on the other hand, calcium leaks out of a compartment known as the sarcoplasmic reticulum in an uncontrolled manner. This happens because an ion channel in the membrane of the sarcoplasmic reticulum known as ryanodine receptor 2 becomes hyperactive and releases calcium in an uncontrolled manner. This abnormal calcium release leads to irregular calcium waves, which can make the heart's electrical properties unstable, causing ventricular tachycardia, ventricular fibrillation and sudden cardiac death. Joshi et al. tested whether dantrolene, a molecule that blocks ryanodine receptor 2, can stop calcium leaks from the sarcoplasmic reticulum and prevent lethal arrhythmias and sudden cardiac death in failing hearts. To investigate this, Joshi et al. induced heart failure in guinea pigs that have abnormal heart calcium signalling similar to human heart failure, and then treated the animals with either dantrolene or a placebo. The results indicate that blocking ryanodine receptor 2 hyperactivity with dantrolene prevents lethal arrhythmias and sudden cardiac death by blocking calcium leaks and by preventing the instability of the electrical properties of the heart. Additionally, Joshi et al. found that dantrolene also improved the diseased heart's ability to pump adequate amounts of blood, allowing failing hearts to meet increased cardiovascular demands, and thereby improving the heart's overall function. The proposed studies come from a strong clinical need to improve bad outcomes in people who keep having fatal heart rhythm episodes despite getting the best medical care. Many heart failure patients are plagued by recurrent defibrillator shocks to abort sudden cardiac death from relentless lethal heart rhythms. These shocks are painful, injure the heart, and worsen the quality of life. Unfortunately, management options are extremely limited for these patients. The findings of Joshi et al. indicate that dantrolene may be a potential treatment for people with fatal heart rhythms who are at risk of sudden cardiac death and could have a positive impact on these people's quality of life. However, before this can happen, dantrolene will first have to be thoroughly tested to ensure effectivity and safety in humans. In any case, Joshi et al. have opened a new avenue in the search for medications to treat deadly arrhythmias and sudden cardiac death.

Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3-4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin's interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin's interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in the development of cardiac disease.

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

Ryanodine receptor 2 inhibition reduces dispersion of cardiac repolarization, improves contractile function and prevents sudden arrhythmic death in failing hearts.

Introduction: Sudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) are a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca 2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)- mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. Objective: To test the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca 2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. Methods: We used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca 2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. Results: DS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient ß-adrenergic challenge, and improved heart rate variability and cardiac function. Conclusion: Inhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca 2+ leak-induced increases in diastolic Ca 2+ and ROS-mediated RyR2 oxidation, thereby increasing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca 2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.

Ryanodine receptor inhibition with acute dantrolene treatment reduces arrhythmia susceptibility in human hearts.

Ryanodine receptor 2 (RyR2) hyperactivity is observed in structural heart diseases that are a result of ischemia or heart failure. It causes abnormal calcium handling and calcium leaks that cause metabolic, electrical, and mechanical dysfunction, which can trigger arrhythmias. Here, we tested the antiarrhythmic potential of dantrolene (RyR inhibitor) in human hearts. Human hearts not used in transplantation were obtained, and right ventricular outflow tract (RVOT) wedges and left ventricular (LV) slices were prepared. Pseudo-ECGs were recorded to determine premature ventricular contraction (PVC) incidences. Optical mapping was performed to determine arrhythmogenic substrates. After baseline optical recordings, tissues were treated with 1) isoproterenol (250 nM), 2) caffeine (200 mM), and 3) dantrolene (2 or 10 mM). Optical recordings were obtained after each treatment. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Isoproterenol shortened action potential duration (APD) in the RV, RVOT, and LV regions and shortened calcium transient duration (CaTD) in the LV. Caffeine further shortened APD in the RV, did not modulate APD in the RVOT, and prolonged APD in the LV. In addition, in the LV, CaTD prolongation was also observed. More importantly, adding dantrolene did not alter APD in the RV or RVOT regions but produced a trend toward APD prolongation and significant CaTD prolongation in the LV, restoring these parameters to baseline values. In conclusions, dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates in the human heart and could be a novel antiarrhythmic therapy in patients with structural heart disease.NEW & NOTEWORTHY Ryanodine receptor 2 hyperactivity is observed in structural heart diseases caused by ischemia or heart failure. It causes abnormal calcium leaks, which can trigger arrhythmias. To prevent arrhythmias, we applied dantrolene in human hearts ex vivo. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates and could be a novel antiarrhythmic therapy in patients with structural heart disease.

ent -Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice.

Ca 2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca 2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent- (+)-verticilide ( ent -1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product ( nat -(-)-verticilide). Here, we examined its 18-membered ring-size oligomer ( ent -verticilide B1; " ent -B1") in single RyR2 channel assays, [ 3 H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent -B1 inhibited RyR2 single-channels and [ 3 H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca 2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent -B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent -B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent -B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent -B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statement: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

RyR2 inhibition with dantrolene is antiarrhythmic, prevents further pathological remodeling, and improves cardiac function in chronic ischemic heart disease.

Diastolic Ca2+ leak due to cardiac ryanodine receptor (RyR2) hyperactivity has been widely documented in chronic ischemic heart disease (CIHD) and may contribute to ventricular tachycardia (VT) risk and progressive left-ventricular (LV) remodeling. Here we test the hypothesis that targeting RyR2 hyperactivity can suppress VT inducibility and progressive heart failure in CIHD by the RyR2 inhibitor dantrolene. METHODS AND RESULTS: CIHD was induced in C57BL/6 J mice by left coronary artery ligation. Four weeks later, mice were randomized to either acute or chronic (6 weeks via implanted osmotic pump) treatment with dantrolene or vehicle. VT inducibility was assessed by programmed stimulation in vivo and in isolated hearts. Electrical substrate remodeling was assessed by optical mapping. Ca2+ sparks and spontaneous Ca2+ releases were measured in isolated cardiomyocytes. Cardiac remodeling was quantified by histology and qRT-PCR. Cardiac function and contractility were measured using echocardiography. Compared to vehicle, acute dantrolene treatment reduced VT inducibility. Optical mapping demonstrated reentrant VT prevention by dantrolene, which normalized the shortened refractory period (VERP) and prolonged action potential duration (APD), preventing APD alternans. In single CIHD cardiomyocytes, dantrolene normalized RyR2 hyperactivity and prevented spontaneous intracellular Ca2+ release. Chronic dantrolene treatment not only reduced VT inducibility but also reduced peri-infarct fibrosis and prevented further progression of LV dysfunction in CIHD mice. CONCLUSIONS: RyR2 hyperactivity plays a mechanistic role for VT risk, post-infarct remodeling, and contractile dysfunction in CIHD mice. Our data provide proof of concept for the anti-arrhythmic and anti-remodeling efficacy of dantrolene in CIHD.

Cellular and electrophysiological characterization of triadin knockout syndrome using induced pluripotent stem cell-derived cardiomyocytes.

Triadin knockout syndrome (TKOS) is a malignant arrhythmia disorder caused by recessive null variants in TRDN-encoded cardiac triadin. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two unrelated TKOS patients and an unrelated control. CRISPR-Cas9 gene editing was used to insert homozygous TRDN-p.D18fs∗13 into a control line to generate a TKOS model (TRDN-/-). Western blot confirmed total knockout of triadin in patient-specific and TRDN-/- iPSC-CMs. iPSC-CMs from both patients revealed a prolonged action potential duration (APD) at 90% repolarization, and this was normalized by protein replacement of triadin. APD prolongation was confirmed in TRDN-/- iPSC-CMs. TRDN-/- iPSC-CMs revealed that loss of triadin underlies decreased expression and co-localization of key calcium handling proteins, slow and decreased calcium release from the sarcoplasmic reticulum, and slow inactivation of the L-type calcium channel leading to frequent cellular arrhythmias, including early and delayed afterdepolarizations and APD alternans.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/-) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/- mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/- mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/- mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.

In Vivo Pharmacokinetic and Pharmacodynamic Properties of the Antiarrhythmic Molecule ent-Verticilide.

The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat- and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within 5 minutes, whereas ent-verticilide showed <1% degradation over 6 hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak C max and area under the plasma-concentration time curve (AUC) scaled proportionally to dose, and the half-life was 6.9 hours for the 3-mg/kg dose and 6.4 hours for the 30-mg/kg dose. Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 minutes after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 minutes after administration in a concentration-dependent manner, with an estimated potency (IC50) of 266 ng/ml (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the US Food and Drug Administration-approved pan-RyR blocker dantrolene, the RyR2-selective blocker ent-verticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development. SIGNIFICANCE STATEMENT: ent-Verticilide has therapeutic potential to treat cardiac arrhythmias, but little is known about its pharmacological profile in vivo. The primary purpose of this study is to determine the systemic exposure and pharmacokinetics of ent-verticilide in mice and estimate its efficacy and potency in vivo. The current work suggests ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.