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Aims: Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable improvement in managing the progression of heart disease, developing effective therapies for heart failure (HF) remains a challenge. A better understanding of molecular mechanisms that maintain proper Ca2+ levels and contractility in the injured heart could be of therapeutic value., Methods and Results: Here, we report that transcription factor zinc finger E-box-binding homeobox 2 (ZEB2) is induced by hypoxia-inducible factor 1-alpha (HIF1α) in hypoxic cardiomyocytes and regulates a network of genes involved in Ca2+ handling and contractility during ischaemic heart disease. Gain- and loss-of-function studies in genetic mouse models revealed that ZEB2 expression in cardiomyocytes is necessary and sufficient to protect the heart against ischaemia-induced diastolic dysfunction and structural remodelling. Moreover, RNA sequencing of ZEB2-overexpressing (Zeb2 cTg) hearts post-injury implicated ZEB2 in regulating numerous Ca2+-handling and contractility-related genes. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban at both serine-16 and threonine-17, implying enhanced activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), thereby augmenting SR Ca2+ uptake and contractility. Furthermore, we observed a decrease in the activity of Ca2+-dependent calcineurin/NFAT signalling in Zeb2 cTg hearts, which is the main driver of pathological cardiac remodelling. On a post-transcriptional level, we showed that ZEB2 expression can be regulated by the cardiomyocyte-specific microRNA-208a (miR-208a). Blocking the function of miR-208a with anti-miR-208a increased ZEB2 expression in the heart and effectively protected from the development of pathological cardiac hypertrophy., Conclusion: Together, we present ZEB2 as a central regulator of contractility and Ca2+-handling components in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in regulating Ca2+ homeostasis in cardiomyocytes is an essential step towards the development of improved therapies for HF., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background and Purposes: Reduced inward rectifier potassium channel (K ir 2.1) functioning is associated with heart failure and may cause Andersen-Tawil Syndrome, among others characterized by ventricular arrhythmias. Most heart failure or Andersen-Tawil Syndrome patients are treated with β-adrenoceptor antagonists (β-blockers) or sodium channel blockers; however, these do not specifically address the inward rectifier current (I K1 ) nor aim to improve resting membrane potential stability. Consequently, additional pharmacotherapy for heart failure and Andersen-Tawil Syndrome treatment would be highly desirable. Acute propafenone treatment at low concentrations enhances I K1 current, but it also exerts many off-target effects. Therefore, discovering and exploring new I K1 -channel openers is necessary., Experimental Approach: Effects of propafenone and 10 additional propafenone analogues were analysed. Currents were measured by single-cell patch-clamp electrophysiology. K ir 2.1 protein expression levels were determined by western blot analysis and action potential characteristics were further validated in human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMCs). Molecular docking was performed to obtain detailed information on drug-channel interactions., Key Results: Analogues GPV0019, GPV0057 and GPV0576 strongly increased the outward component of I K1 while not affecting the K ir 2.1 channel expression levels. GPV0057 did not block I Kr at concentrations below 0.5 μmol L -1 nor Na V 1.5 current below 1 μmol L -1 . Moreover, hiPSC-CMC action potential duration was also not affected by GPV0057 at 0.5 and 1 μmol L -1 . Structure analysis indicates a mechanism by which GPV0057 might enhance K ir 2.1 channel activation., Conclusion and Implications: GPV0057 has a strong efficiency towards increasing I K1 , which makes it a good candidate to address I K1 deficiency-associated diseases., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The development of the normal human heart, ranging from gestational age to the mature adult heart, relies on a very delicate and timely orchestrated order of processes. One of the most striking alterations in time is the gradual extinction of the ability for cardiomyocytes to proliferate. Once passing this event, cardiomyocytes grow and increase in contractile strength by means of physiological hypertrophy. This process, importantly, seems to depend on an adequate development of electromechanical coupling that is achieved by the appropriate formation of the intercellular junction named the intercalated disc (ICD). In this report, we describe two sudden death cases of young and apparently healthy-born individuals without external abnormalities compared to an age-matched control. Histological examination, including the comparison with the age-matched and histology-matched controls, showed a disturbed formation of the protein machinery composing the electromechanical junctions at the ICD and an increased nuclei count for both patients. As a cause or consequence, cardiomyocytes in both sudden death cases showed signs of a delayed developmental stage, presumably resulting in an exaggerated degree of hyperplasia.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 ( PKP2 ). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell-derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive disease characterized by electrophysiological and structural remodeling of the ventricles. However, the disease-causing molecular pathways, as a consequence of desmosomal mutations, are poorly understood. Here, we identified a novel missense mutation within desmoplakin in a patient clinically diagnosed with ACM. Using CRISPR-Cas9, we corrected this mutation in patient-derived human induced pluripotent stem cells (hiPSCs) and generated an independent knockin hiPSC line carrying the same mutation. Mutant cardiomyocytes displayed a decline in connexin 43, NaV1.5, and desmosomal proteins, which was accompanied by a prolonged action potential duration. Interestingly, paired-like homeodomain 2 (PITX2), a transcription factor that acts a repressor of connexin 43, NaV1.5, and desmoplakin, was induced in mutant cardiomyocytes. We validated these results in control cardiomyocytes in which PITX2 was either depleted or overexpressed. Importantly, knockdown of PITX2 in patient-derived cardiomyocytes is sufficient to restore the levels of desmoplakin, connexin 43, and NaV1.5., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End-stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are among the CKD-related factors that have been linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients are cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible remain poorly understood. The current study aimed to assess the vulnerability of action potential repolarization caused by exposure to pre-identified UTs at clinically relevant concentrations. We exposed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and HEK293 chronically (48 h) to the UTs indoxyl sulfate, kynurenine, or kynurenic acid. We used optical and manual electrophysiological techniques to assess action potential duration (APD) in the hiPSC-CMs and recorded I Kr currents in stably transfected HEK293 cells (HEK-hERG). Molecular analysis of K V 11.1, the ion channel responsible for I Kr , was performed to further understand the potential mechanism underlying the effects of the UTs. Chronic exposure to the UTs resulted in significant APD prolongation. Subsequent assessment of the repolarization current I Kr , often most sensitive and responsible for APD alterations, showed decreased current densities after chronic exposure to the UTs. This outcome was supported by lowered protein levels of K V 11.1. Finally, treatment with an activator of the I Kr current, LUF7244, could reverse the APD prolongation, indicating the potential modulation of electrophysiological effects caused by these UTs. This study highlights the pro-arrhythmogenic potential of UTs and reveals a mode of action by which they affect cardiac repolarization.

Chronic kidney disease (CKD) and cardiovascular disease (CVD) have an estimated 700-800 and 523 million cases worldwide, respectively, with CVD being the leading cause of death in CKD patients. The pathophysiological interplay between the heart and kidneys is defined as the cardiorenal syndrome (CRS), in which worsening of kidney function is represented by increased plasma concentrations of uremic toxins (UTs), culminating in dialysis patients. As there is a high incidence of CVD in CKD patients, accompanied by arrhythmias and sudden cardiac death, knowledge on electrophysiological remodeling would be instrumental for understanding the CRS. While the interplay between both organs is clearly of importance in CRS, the involvement of UTs in pro-arrhythmic remodeling is only poorly investigated, especially regarding the mechanistic background. Currently, the clinical approach against potential arrhythmic events is mainly restricted to symptom treatment, stressing the need for fundamental research on UT in relation to electrophysiology. This review addresses the existing knowledge of UTs and cardiac electrophysiology, and the experimental research gap between fundamental research and clinical research of the CRS. Clinically, mainly absorbents like ibuprofen and AST-120 are studied, which show limited safe and efficient usability. Experimental research shows disturbances in cardiac electrical activation and conduction after inducing CKD or exposure to UTs, but are scarcely present or focus solely on already well-investigated UTs. Based on UTs data derived from CKD patient cohort studies, a clinically relevant overview of physiological and pathological UTs concentrations is created. Using this, future experimental research is stimulated to involve electrophysiologically translatable animals, such as rabbits, or in vitro engineered heart tissues., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

At least one-half of the growing heart failure population consists of heart failure with preserved ejection fraction (HFpEF). The limited therapeutic options, the complexity of the syndrome, and many related comorbidities emphasize the need for adequate experimental animal models to study the etiology of HFpEF, as well as its comorbidities and pathophysiological changes. The strengths and weaknesses of available animal models have been reviewed extensively with the general consensus that a "1-size-fits-all" model does not exist, because no uniform HFpEF patient exists. In fact, HFpEF patients have been categorized into HFpEF phenogroups based on comorbidities and symptoms. In this review, we therefore study which animal model is best suited to study the different phenogroups-to improve model selection and refinement of animal research. Based on the published data, we extrapolated human HFpEF phenogroups into 3 animal phenogroups (containing small and large animals) based on reports and definitions of the authors: animal models with high (cardiac) age (phenogroup aging); animal models focusing on hypertension and kidney dysfunction (phenogroup hypertension/kidney failure); and models with hypertension, obesity, and type 2 diabetes mellitus (phenogroup cardiometabolic syndrome). We subsequently evaluated characteristics of HFpEF, such as left ventricular diastolic dysfunction parameters, systemic inflammation, cardiac fibrosis, and sex-specificity in the different models. Finally, we scored these parameters concluded how to best apply these models. Based on our findings, we propose an easy-to-use classification for future animal research based on clinical phenogroups of interest., Competing Interests: This work was supported by Netherlands Cardiovascular Research Initiative, with the support of the Dutch Heart Foundation, the Netherlands (CVON2018-30 PREDICT2 to Drs van Ham and van Veen, Senior Clinical Scientist grant 2020T058 to Dr Handoko; EARLY-HFpEF Young Talent Grant 2015-10 to Dr Kessler, RECONNEXT 2020B008 and IMPRESS 2020B004 to Drs Kessler, Handoko, den Ruijter, and de Jager). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Alteration of the inward rectifier current I K 1 , carried by K IR 2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired K IR 2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in K IR 2.1 trafficking processes is unknown. We aimed to quantify the dependence of K IR 2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged K IR 2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new K IR 2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted K IR 2.1. Patch clamp electrophysiology was used to quantify I KIR 2 . 1 . Biochemical methods were used for cytoskeleton isolation and detection of K IR 2.1-cytoskeleton interactions. Cytochalasin B (20 μM), Nocodazole (30 μM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 μM, 24 h) was used to impair K IR 2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of K IR 2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (* p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (* p < 0.05 vs. control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited I KIR 2 . 1 . Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of K IR 2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of K IR 2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits K IR 2.1 currents. Internalized K IR 2.1 channels become recruited to the cytoskeleton, presumably in lysosomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Loen, van Ham, Kool, van der Heyden and Takanari.)

Voltage-gated potassium 11.1 (K v 11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated K v 11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal K v 11.1 forward trafficking and thus reduce functional K v 11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a K v 11.1 allosteric modulator/activator, to rescue K v 11.1 trafficking and produce functional K v 11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-K v 11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT K v 11.1 levels was rescued by 10 μM dofetilide or 10 μM dofetilide + 5 μM LUF7244. In trafficking defective G601S-K v 11.1 cells, dofetilide (10 μM) or dofetilide + LUF7244 (10 + 5 μM) also restored K v 11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased I Kv 11.1 despite the presence of dofetilide (1 μM) in WT K v 11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 μM) and dofetilide (1 μM) increased I Kv11.1 compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues K v 11.1 trafficking and produces functional I Kv11.1 Thus, combined administration of LUF7244 and an I Kv11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced K v 11.1 trafficking defects. SIGNIFICANCE STATEMENT: Decreased levels of functional K v 11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of K v 11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired K v 11.1 trafficking defects, resulting in functional K v 11.1 current., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Purpose: K v 11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of K v 11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model., Experimental Approach: LUF7244 was tested in vitro for (a) increasing human I Kv11.1 and canine I Kr and (b) decreasing dofetilide-induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block., Key Results: LUF7244 (0.5-10 μM) concentration dependently increased I Kv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on I KIR2.1 , I Nav1.5 , I Ca-L , and I Ks , doubled I Kr , shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide-induced early afterdepolarizations. LUF7244 (2.5 mg·kg -1 ·15 min -1 ) in dogs with sinus rhythm was not proarrhythmic and shortened, non-significantly, repolarization parameters (QTc: -6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide-induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block., Conclusions and Implications: LUF7244 counteracted dofetilide-induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of K v 11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The ether-à-go-go (Eag) K v superfamily comprises closely related K v 10, K v 11, and K v 12 subunits. K v 11.1 (termed hERG in humans) gained much attention, as drug-induced inhibition of these channels is a frequent cause of sudden death in humans. The exclusive drug sensitivity of K v 11.1 can be explained by central drug-binding pockets that are absent in most other channels. Currently, it is unknown whether K v 12 channels are equipped with an analogous drug-binding pocket and whether drug-binding properties are conserved in all Eag superfamily members., Experimental Approach: We analysed sensitivity of recombinant K v 12.1 channels to quinine, a substituted quinoline that blocks K v 10.1 and K v 11.1 at low micromolar concentrations., Key Results: Quinine inhibited K v 12.1, but its affinity was 10-fold lower than for K v 11.1. Contrary to K v 11.1, quinine inhibited K v 12.1 in a largely voltage-independent manner and induced channel opening at more depolarised potentials. Low sensitivity of K v 12.1 and characteristics of quinine-dependent inhibition were determined by histidine 462, as site-directed mutagenesis of this residue into the homologous tyrosine of K v 11.1 conferred K v 11.1-like quinine block to K v 12.1(H462Y). Molecular modelling demonstrated that the low affinity of K v 12.1 was determined by only weak interactions of residues in the central cavity with quinine. In contrast, more favourable interactions can explain the higher quinine sensitivity of K v 12.1(H462Y) and K v 11.1 channels., Conclusions and Implications: The quinoline-binding "motif" is not conserved within the Eag superfamily, although the overall architecture of these channels is apparently similar. Our findings highlight functional and pharmacological diversity in this group of evolutionary-conserved channels., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)