Reactivation of the Epicardium at the Origin of Myocardial Fibro-Fatty Infiltration During the Atrial Cardiomyopathy.

RATIONALE: Fibro-fatty infiltration of subepicardial layers of the atrial wall has been shown to contribute to the substrate of atrial fibrillation. OBJECTIVE: Here, we examined if the epicardium that contains multipotent cells is involved in this remodeling process. METHODS AND RESULTS: One hundred nine human surgical right atrial specimens were evaluated. There was a relatively greater extent of epicardial thickening and dense fibro-fatty infiltrates in atrial tissue sections from patients aged over 70 years who had mitral valve disease or atrial fibrillation when compared with patients aged less than 70 years with ischemic cardiomyopathy as indicated using logistic regression adjusted for age and gender. Cells coexpressing markers of epicardial progenitors and fibroblasts were detected in fibro-fatty infiltrates. Such epicardial remodeling was reproduced in an experimental model of atrial cardiomyopathy in rat and in Wilms tumor 1 (WT1)CreERT2/+;ROSA-tdT+/- mice. In the latter, genetic lineage tracing demonstrated the epicardial origin of fibroblasts within fibro-fatty infiltrates. A subpopulation of human adult epicardial-derived cells expressing PDGFR (platelet-derived growth factor receptor)-α were isolated and differentiated into myofibroblasts in the presence of Ang II (angiotensin II). Furthermore, single-cell RNA-sequencing analysis identified several clusters of adult epicardial-derived cells and revealed their specification from adipogenic to fibrogenic cells in the rat model of atrial cardiomyopathy. CONCLUSIONS: Epicardium is reactivated during the formation of the atrial cardiomyopathy. Subsets of adult epicardial-derived cells, preprogrammed towards a specific cell fate, contribute to fibro-fatty infiltration of subepicardium of diseased atria. Our study reveals the biological basis for chronic atrial myocardial remodeling that paves the way of atrial fibrillation.

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel: Microtubule and clathrin control of KV1.5 channel.

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusterization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule- dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.

Atrial natriuretic peptide regulates adipose tissue accumulation in adult atria.

The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial-mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1CreERT2+/-RosatdT+/- mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/ß-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.

Lateral Membrane-Specific MAGUK CASK Down-Regulates NaV1.5 Channel in Cardiac Myocytes.

RATIONALE: Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, NaV1.5, carries the sodium current (INa) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, NaV1.5 is present in different membrane domains in myocytes and interacts with several partners. OBJECTIVE: To test the hypothesis that the MAGUK (membrane-associated guanylate kinase) protein CASK (calcium/calmodulin-dependent serine protein kinase) interacts with and regulates NaV1.5 in cardiac myocytes. METHODS AND RESULTS: Immunostaining experiments showed that CASK localizes at lateral membranes of cardiac myocytes, in association with dystrophin. Whole-cell patch clamp showed that CASK-silencing increases INa in vitro. In vivo CASK knockdown similarly increased INa recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of NaV1.5. CASK silencing reduces syntrophin expression without affecting NaV1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of NaV1.5 without changing mRNA levels. Quantification of NaV1.5 expression at the lateral membrane and intercalated disc revealed that the lateral membrane pool only was increased upon CASK silencing. The protein transport inhibitor brefeldin-A prevented INa increase in CASK-silenced myocytes. During atrial dilation/remodeling, CASK expression was reduced but its localization remained unchanged. CONCLUSION: This study constitutes the first description of an unconventional MAGUK protein, CASK, which directly interacts with NaV1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.

Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.

A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is a major cause of heart failure with a high familial recurrence risk. So far, the genetics of DCM remains largely unresolved. We conducted the first genome-wide association study (GWAS) to identify loci contributing to sporadic DCM. METHODS AND RESULTS: One thousand one hundred and seventy-nine DCM patients and 1108 controls contributed to the discovery phase. Pools of DNA stratified on disease status, population, age, and gender were constituted and used for testing association of DCM with 517 382 single nucleotide polymorphisms (SNPs). Three DCM-associated SNPs were confirmed by individual genotyping (P < 5.0 10(-7)), and two of them, rs10927875 and rs2234962, were replicated in independent samples (1165 DCM patients and 1302 controls), with P-values of 0.002 and 0.009, respectively. rs10927875 maps to a region on chromosome 1p36.13 which encompasses several genes among which HSPB7 has been formerly suggested to be implicated in DCM. The second identified locus involves rs2234962, a non-synonymous SNP (c.T757C, p. C151R) located within the sequence of BAG3 on chromosome 10q26. To assess whether coding mutations of BAG3 might cause monogenic forms of the disease, we sequenced BAG3 exons in 168 independent index cases diagnosed with familial DCM and identified four truncating and two missense mutations. Each mutation was heterozygous, present in all genotyped relatives affected by the disease and absent in a control group of 347 healthy individuals, strongly suggesting that these mutations are causing the disease. CONCLUSION: This GWAS identified two loci involved in sporadic DCM, one of them probably implicates BAG3. Our results show that rare mutations in BAG3 contribute to monogenic forms of the disease, while common variant(s) in the same gene are implicated in sporadic DCM.

Impacts of a high-fat diet on the metabolic profile and the phenotype of atrial myocardium in mice.

AIMS: Obesity, diabetes, and metabolic syndromes are risk factors of atrial fibrillation (AF). We tested the hypothesis that metabolic disorders have a direct impact on the atria favouring the formation of the substrate of AF. METHODS AND RESULTS: Untargeted metabolomic and lipidomic analysis was used to investigate the consequences of a prolonged high-fat diet (HFD) on mouse atria. Atrial properties were characterized by measuring mitochondria respiration in saponin-permeabilized trabeculae, by recording action potential (AP) with glass microelectrodes in trabeculae and ionic currents in myocytes using the perforated configuration of patch clamp technique and by several immuno-histological and biochemical approaches. After 16 weeks of HFD, obesogenic mice showed a vulnerability to AF. The atrial myocardium acquired an adipogenic and inflammatory phenotypes. Metabolomic and lipidomic analysis revealed a profound transformation of atrial energy metabolism with a predominance of long-chain lipid accumulation and beta-oxidation activation in the obese mice. Mitochondria respiration showed an increased use of palmitoyl-CoA as energy substrate. APs were short duration and sensitive to the K-ATP-dependent channel inhibitor, whereas K-ATP current was enhanced in isolated atrial myocytes of obese mouse. CONCLUSION: HFD transforms energy metabolism, causes fat accumulation, and induces electrical remodelling of the atrial myocardium of mice that become vulnerable to AF.

Kv4 potassium channels form a tripartite complex with the anchoring protein SAP97 and CaMKII in cardiac myocytes.

Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, I(to), in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited I(to) in cardiac myocytes, whereas its overexpression by using an adenovirus increased I(to). Kv4.3 channels without the SAL sequence were no longer regulated by Ca2+/calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels.

Distinct calcium/calmodulin-dependent serine protein kinase domains control cardiac sodium channel membrane expression and focal adhesion anchoring.

BACKGROUND: Membrane-associated guanylate kinase proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein calcium/calmodulin-dependent serine protein kinase (CASK) negatively regulates the main cardiac sodium channel NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex and, like syntrophin, binds to the C-terminal domain of the channel. OBJECTIVE: The purpose of this study was to unravel the mechanisms of CASK-mediated negative INa regulation and interaction with the dystrophin-glycoprotein complex in cardiac myocytes. METHODS: CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASKΔCAMKII, CASKΔL27A, CASKΔL27B, CASKΔPDZ, CASKΔSH3, CASKΔHOOK, and CASKΔGUK. A combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy, and biochemistry experiments was conducted in cardiac myocytes to study the functional consequences of domain deletions. RESULTS: We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. CONCLUSION: This study demonstrates that the multimodular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocytes.

Mutations in the Z-band protein myopalladin gene and idiopathic dilated cardiomyopathy.

AIMS: Idiopathic dilated cardiomyopathy (DCM) is a cardiac disorder characterized by left ventricular dilatation and impaired systolic contraction. It is a major cause of heart failure and heart transplantation. DCM is of genetic origin in approximately 30% of cases and genetically heterogeneous with the identification of numerous disease genes. However, many new disease genes remain to be discovered. Focusing on gene products located in the sarcomere of cardiomyocytes as disease-causing candidates, we screened the gene encoding the sarcomeric Z-band protein myopalladin (MYPN, OMIM 608517) for mutation. METHODS AND RESULTS: We sequenced the coding region in 114 (65 familial and 49 sporadic cases) independent DCM patients' DNA and functionally analysed the identified mutations. We identified four independent heterozygous mutations in two families (R1088H and I83fsX105) and two sporadic cases (V1195M, P1112L). For the three missense mutations, the substituted amino acids were conserved among species. All mutations were absent from 400 control subjects. Specific immunolabelling of heart tissue from a proband carrying the R1088H mutation showed a decreased localization of myopalladin at the Z-band area of left ventricular cardiac myofibrils. Analysis of the effects of the mutations after transfection in rat neonate cardiomyocytes indicated sarcomere disorganization and premature cell death associated with the V1195M and P1112L myopalladin expression. Allele-specific expression analysis of mRNA from a patient harbouring the I83fsX105 mutation indicated the absence of the mutated transcript, suggesting a haploinsufficiency mechanism. CONCLUSION: Based on genetic, histological, and functional evidence, we identified a new gene associated with DCM and observed mutations in 3-4% of cases in a population of European descent.

A novel genetic variant in the transcription factor Islet-1 exerts gain of function on myocyte enhancer factor 2C promoter activity.

AIMS: The transcription factor Islet-1 (ISL1) is a marker of cardiovascular progenitors and is essential for mammalian cardiogenesis. An ISL1 haplotype has recently been associated with congenital heart disease. In this study we evaluated whether ISL1 variants are associated with hypertrophic (HCM), dilated (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), or with Emery-Dreifuss muscular dystrophy (EDMD). METHODS AND RESULTS: The six exon and intron boundaries of ISL1 were screened for genetic variants in a cohort of 454 index cases. Eleven exonic variants were identified in HCM, DCM, ARVC, and/or EDMD. Out of the five novel variants, two are located in the 5'-untranslated region, two are silent (p.Arg171Arg and p.Asn189Asn), and one is a missense (p.Asn252Ser). The latter was identified in the homozygous state in one DCM patient, and in the heterozygous state in 11 relatives, who did not present with DCM but often with cardiovascular features. This variant was found in one HCM patient also carrying a MYH7 mutation and in 3/96 North-African Caucasian control individuals, but was absent in 138 European Caucasian control individuals. We investigated the effect of the ISL1 wild type and p.Asn252Ser mutant on myocyte enhancer factor 2C (Mef2c) promoter activity, an established ISL1 target. Mef2c promoter activity was ∼4-fold higher in the presence of wild-type and ∼6-fold higher in the presence of mutant ISL1 in both HEK and CHO cells. CONCLUSION: This study describes a new gain-of-function p.Asn252Ser variant in the human ISL1 gene, which could potentially lead to greater activation of downstream targets involved in cardiac development, dilation, and hypertrophy.

Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Na(v)1.5 α-subunits.

AIMS: Brugada syndrome (BrS) is an autosomal-inherited cardiac arrhythmia characterized by an ST-segment elevation in the right precordial leads of the electrocardiogram and an increased risk of syncope and sudden death. SCN5A, encoding the cardiac sodium channel Na(v)1.5, is the main gene involved in BrS. Despite the fact that several mutations have been reported in the N-terminus of Na(v)1.5, the functional role of this region remains unknown. We aimed to characterize two BrS N-terminal mutations, R104W and R121W, a construct where this region was deleted, ΔNter, and a construct where only this region was present, Nter. METHODS AND RESULTS: Patch-clamp recordings in HEK293 cells demonstrated that R104W, R121W, and ΔNter abolished the sodium current I(Na). Moreover, R104W and R121W mutations exerted a strong dominant-negative effect on wild-type (WT) channels. Immunocytochemistry of rat neonatal cardiomyocytes revealed that both mutants were mostly retained in the endoplasmic reticulum and that their co-expression with WT channels led to WT channel retention. Furthermore, co-immunoprecipitation experiments showed that Na(v)1.5-subunits were interacting with each other, even when mutated, deciphering the mutation dominant-negative effect. Both mutants were mostly degraded by the ubiquitin-proteasome system, while ΔNter was addressed to the membrane, and Nter expression induced a two-fold increase in I(Na). In addition, the co-expression of N-terminal mutants with the gating-defective but trafficking-competent R878C-Na(v)1.5 mutant gave rise to a small I(Na). CONCLUSION: This study reports for the first time the critical role of the Na(v)1.5 N-terminal region in channel function and the dominant-negative effect of trafficking-defective channels occurring through α-subunit interaction.

MOG1: a new susceptibility gene for Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is caused mainly by mutations in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Na(v)1.5. However, ≈ 20% of probands have SCN5A mutations, suggesting the implication of other genes. MOG1 recently was described as a new partner of Na(v)1.5, playing a potential role in the regulation of its expression and trafficking. We investigated whether mutations in MOG1 could cause BrS. METHODS AND RESULTS: MOG1 was screened by direct sequencing in patients with BrS and idiopathic ventricular fibrillation. A missense mutation p.Glu83Asp (E83D) was detected in a symptomatic female patient with a type-1 BrS ECG but not in 281 controls. Wild type (WT)- and mutant E83D-MOG1 were expressed in HEK Na(v)1.5 stable cells and studied using patch-clamp assays. Overexpression of WT-MOG1 alone doubled sodium current (I(Na)) density compared to control conditions (P<0.01). In contrast, overexpression of mutant E83D alone or E83D+WT failed to increase I(Na) (P<0.05), demonstrating the dominant-negative effect of the mutant. Microscopy revealed that Na(v)1.5 channels failed to properly traffic to the cell membrane in the presence of the mutant. Silencing endogenous MOG1 demonstrated a 54% decrease in I(Na) density. CONCLUSIONS: Our results support the hypothesis that dominant-negative mutations in MOG1 can impair the trafficking of Na(v)1.5 to the membrane, leading to I(Na) reduction and clinical manifestation of BrS. Moreover, silencing MOG1 reduced I(Na), demonstrating that MOG1 is likely to be important in the surface expression of Na(v)1.5 channels. All together, our data support MOG1 as a new susceptibility gene for BrS.