Cardiac Transverse Tubules in Physiology and Heart Failure.

In mammalian cardiac myocytes, the plasma membrane includes the surface sarcolemma but also a network of membrane invaginations called transverse (t-) tubules. These structures carry the action potential deep into the cell interior, allowing efficient triggering of Ca2+ release and initiation of contraction. Once thought to serve as rather static enablers of excitation-contraction coupling, recent work has provided a newfound appreciation of the plasticity of the t-tubule network's structure and function. Indeed, t-tubules are now understood to support dynamic regulation of the heartbeat across a range of timescales, during all stages of life, in both health and disease. This review article aims to summarize these concepts, with consideration given to emerging t-tubule regulators and their targeting in future therapies.

Junctophilin-2 is a double-stranded RNA-binding protein that regulates cardiomyocyte-autonomous innate immune response.

Junctophilin-2 (JPH2) is traditionally recognized as a cardiomyocyte-enriched structural protein that anchors the junction between the plasma membrane and the endo/sarcoplasmic reticulum, facilitating excitation-induced cardiac contraction. In this study, we uncover a novel function of JPH2 as a double-stranded RNA (dsRNA)-binding protein, which forms complexes with dsRNA both in vitro and in cells. Stimulation by cytosolic dsRNA enhances the interaction of JPH2 with the dsRNA sensor MDA5. Notably, JPH2 inhibits MDA5's binding to its dsRNA ligand, likely by sequestering the dsRNA. Silencing JPH2 in cardiomyocytes increased the interaction between MDA5 and its dsRNA ligands, activated the MAVS/TBK1 signaling, and triggered spontaneous interferon-beta (IFNb1) production in the absence of foreign pathogen. Mouse hearts deficient in JPH2 exhibited upregulation of innate immune signaling cascade. Collectively, these findings identify JPH2 as a regulator of dsRNA sensing and highlight its role in suppressing the automatic activation of innate immune responses in cardiomyocytes, suggesting the cytosolic surface of the endo/sarcoplasmic reticulum as a hub for dsRNA sequestration.

RBFox2-miR-34a-Jph2 axis contributes to cardiac decompensation during heart failure.

Heart performance relies on highly coordinated excitation-contraction (EC) coupling, and defects in this critical process may be exacerbated by additional genetic defects and/or environmental insults to cause eventual heart failure. Here we report a regulatory pathway consisting of the RNA binding protein RBFox2, a stress-induced microRNA miR-34a, and the essential EC coupler JPH2. In this pathway, initial cardiac defects diminish RBFox2 expression, which induces transcriptional repression of miR-34a, and elevated miR-34a targets Jph2 to impair EC coupling, which further manifests heart dysfunction, leading to progressive heart failure. The key contribution of miR-34a to this process is further established by administrating its mimic, which is sufficient to induce cardiac defects, and by using its antagomir to alleviate RBFox2 depletion-induced heart dysfunction. These findings elucidate a potential feed-forward mechanism to account for a critical transition to cardiac decompensation and suggest a potential therapeutic avenue against heart failure.

One gene, two modes of inheritance, four diseases: A systematic review of the cardiac manifestation of pathogenic variants in JPH2-encoded junctophilin-2.

Rare variants in JPH2 have been associated with a range of cardiac disease, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmias, and sudden cardiac death (SCD); however, our understanding of how variants in JPH2 correspond to specific modes of inheritance and correlate clinical phenotypes has not been comprehensively explored. In this systematic review, we assess current case reports and series that describe patients with JPH2 variants and cardiac disease. We identified a total of 61 variant-positive individuals, approximately 80% of whom had some form of cardiac disease, including 47% HCM, 18% DCM, and 14% arrhythmia/SCD. In analyzing the 24 probands described in the studies, we found that autosomal recessive, loss-of-function variants are associated with severe, early onset DCM, while autosomal dominant missense variants are associated with a wider range of cardiac disease, including HCM, arrhythmia, SCD, and cardiac conduction disease.

Junctional Ectopic Tachycardia Caused by Junctophilin-2 Expression Silencing Is Selectively Sensitive to Ryanodine Receptor Blockade.

Junctional ectopic tachycardia (JET) is a potentially fatal cardiac arrhythmia. Hcn4:shJph2 mice serve as a model of nodal arrhythmias driven by ryanodine type 2 receptor (RyR2)-mediated Ca2+ leak. EL20 is a small molecule that blocks RyR2 Ca2+ leak. In a novel in vivo model of JET, Hcn4:shJph2 mice demonstrated rapid conversion of JET to sinus rhythm with infusion of EL20. Primary atrioventricular nodal cells demonstrated increased Ca2+ transient oscillation frequency and increased RyR2-mediated stored Ca2+ leak which was normalized by EL20. EL20 was found to be rapidly degraded in mouse and human plasma, making it a potential novel therapy for JET.

The Role of METTL3-Mediated N6-Methyladenosine (m6A) of JPH2 mRNA in Cyclophosphamide-Induced Cardiotoxicity.

Cyclophosphamide (CYP)-induced cardiotoxicity is a common side effect of cancer treatment. Although it has received significant attention, the related mechanisms of CYP-induced cardiotoxicity remain largely unknown. In this study, we used cell and animal models to investigate the effect of CYP on cardiomyocytes. Our data demonstrated that CYP-induced a prolonged cardiac QT interval and electromechanical coupling time courses accompanied by JPH2 downregulation. Moreover, N6-methyladenosine (m6A) methylation sequencing and RNA sequencing suggested that CYP induced cardiotoxicity by dysregulating calcium signaling. Importantly, our results demonstrated that CYP induced an increase in the m6A level of JPH2 mRNA by upregulating methyltransferases METTL3, leading to the reduction of JPH2 expression levels, as well as increased field potential duration and action potential duration in cardiomyocytes. Our results revealed a novel mechanism for m6A methylation-dependent regulation of JPH2, which provides new strategies for the treatment and prevention of CYP-induced cardiotoxicity.

SPEG: a key regulator of cardiac calcium homeostasis.

Proper cardiac Ca2+ homeostasis is essential for normal excitation-contraction coupling. Perturbations in cardiac Ca2+ handling through altered kinase activity has been implicated in altered cardiac contractility and arrhythmogenesis. Thus, a better understanding of cardiac Ca2+ handling regulation is vital for a better understanding of various human disease processes. 'Striated muscle preferentially expressed protein kinase' (SPEG) is a member of the myosin light chain kinase family that is key for normal cardiac function. Work within the last 5 years has revealed that SPEG has a crucial role in maintaining normal cardiac Ca2+ handling through maintenance of transverse tubule formation and phosphorylation of junctional membrane complex proteins. Additionally, SPEG has been causally impacted in human genetic diseases such as centronuclear myopathy and dilated cardiomyopathy as well as in common acquired cardiovascular disease such as heart failure and atrial fibrillation. Given the rapidly emerging role of SPEG as a key cardiac Ca2+ regulator, we here present this review in order to summarize recent findings regarding the mechanisms of SPEG regulation of cardiac excitation-contraction coupling in both physiology and human disease. A better understanding of the roles of SPEG will be important for a more complete comprehension of cardiac Ca2+ regulation in physiology and disease.

Use of Proximity Labeling in Cardiovascular Research.

Protein-protein interactions are of paramount importance in regulating normal cardiac physiology. Methodologies to elucidate these interactions in vivo have been limited. Recently, proximity-dependent biotinylation, with the use of BioID, TurboID, and ascorbate peroxidase, has been developed to uncover cellular neighborhoods and novel protein-protein interactions. These cutting-edge techniques have enabled the identification of subcellular localizations of specific proteins and the neighbors or interacting proteins within these subcellular regions. In contrast to classic methods such as affinity purification and subcellular fractionation, these techniques add covalently bound tags in living cells, such that spatial relationships and interaction networks are not disrupted. Recently, these methodologies have been used to identify novel protein-protein interactions relevant to the cardiovascular system. In this review, we discuss the development and current use of proximity biotin-labeling for cardiovascular research.

Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization.

OBJECTIVES: This study sought to study the role of junctophilin-2 (JPH2) in atrial fibrillation (AF). BACKGROUND: JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca(2+) handling and modulation of ryanodine receptor Ca(2+) channels (RyR2). Whereas defective RyR2-mediated Ca(2+) release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias. METHODS: Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudoknock-in (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation. RESULTS: PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF than wild type (WT)-PKI mice, whereas A399S-PKI mice expressing a hypertrophic cardiomyopathy-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca(2+) release events. These changes were attributed to reduced binding of E169K-JPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca(2+) spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients and an increased frequency of spontaneous Ca(2+) release events. CONCLUSIONS: Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2/RyR2 ratios can promote SR Ca(2+) leak and atrial arrhythmias, representing a potential novel therapeutic target for AF.

FGF12 is a candidate Brugada syndrome locus.

BACKGROUND: Less than 30% of the cases of Brugada syndrome (BrS) have an identified genetic cause. Of the known BrS-susceptibility genes, loss-of-function mutations in SCN5A or CACNA1C and their auxiliary subunits are most common. On the basis of the recent demonstration that fibroblast growth factor (FGF) homologous factors (FHFs; FGF11-FGF14) regulate cardiac Na(+) and Ca(2+) channel currents, we hypothesized that FHFs are candidate BrS loci. OBJECTIVE: The goal of this study was to test whether FGF12 is a candidate BrS locus. METHODS: We used quantitative polymerase chain reaction to identify the major FHF expressed in the human ventricle and then queried a phenotype-positive, genotype-negative BrS biorepository for FHF mutations associated with BrS. We queried the effects of an identified mutant with biochemical analyses combined with electrophysiological assessment. We designed a novel rat ventricular cardiomyocyte system in which we swapped the endogenous FHF with the identified mutant and defined its effects on multiple ionic currents in their native milieu and on the cardiac action potential. RESULTS: We identified FGF12 as the major FHF expressed in the human ventricle. In 102 individuals in the biorepository, we identified a single missense mutation in FGF12-B (Q7R-FGF12). The mutant reduced binding to the NaV1.5 C terminus, but not to junctophilin-2. In adult rat cardiac myocytes, Q7R-FGF12, but not wild-type FGF12, reduced Na(+) channel current density and availability without affecting Ca(2+) channel function. Furthermore, the mutant, but not wild-type FGF12, reduced action potential amplitude, which is consistent with a mutant-induced loss of Na(+) channel function. CONCLUSIONS: These multilevel investigations strongly suggest that Q7R-FGF12 is a disease-associated BrS mutation. Moreover, these data suggest for the first time that FHF effects on Na(+) and Ca(2+) channels are separable. Most significantly, this study establishes a new method to analyze effects of human arrhythmogenic mutations on cardiac ionic currents.