The role of the M-band myomesin proteins in muscle integrity and cardiac disease.

Transversal structural elements in cross-striated muscles, such as the M-band or the Z-disc, anchor and mechanically stabilize the contractile apparatus and its minimal unit-the sarcomere. The ability of proteins to target and interact with these structural sarcomeric elements is an inevitable necessity for the correct assembly and functionality of the myofibrillar apparatus. Specifically, the M-band is a well-recognized mechanical and signaling hub dealing with active forces during contraction, while impairment of its function leads to disease and death. Research on the M-band architecture is focusing on the assembly and interactions of the three major filamentous proteins in the region, mainly the three myomesin proteins including their embryonic heart (EH) isoform, titin and obscurin. These proteins form the basic filamentous network of the M-band, interacting with each other as also with additional proteins in the region that are involved in signaling, energetic or mechanosensitive processes. While myomesin-1, titin and obscurin are found in every muscle, the expression levels of myomesin-2 (also known as M-protein) and myomesin-3 are tissue specific: myomesin-2 is mainly expressed in the cardiac and fast skeletal muscles, while myomesin-3 is mainly expressed in intermediate muscles and specific regions of the cardiac muscle. Furthermore, EH-myomesin apart from its role during embryonic stages, is present in adults with specific cardiac diseases. The current work in structural, molecular, and cellular biology as well as in animal models, provides important details about the assembly of myomesin-1, obscurin and titin, the information however about the myomesin-2 and -3, such as their interactions, localization and structural details remain very limited. Remarkably, an increasing number of reports is linking all three myomesin proteins and particularly myomesin-2 to serious cardiovascular diseases suggesting that this protein family could be more important than originally thought. In this review we will focus on the myomesin protein family, the myomesin interactions and structural differences between isoforms and we will provide the most recent evidence why the structurally and biophysically unexplored myomesin-2 and myomesin-3 are emerging as hot targets for understanding muscle function and disease.

Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.

Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.

Inter-Regulation of Kv4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies.

BACKGROUND: Genetic variants in voltage-gated sodium channels (Nav) encoded by SCNXA genes, responsible for INa, and Kv4.3 channels encoded by KCND3, responsible for the transient outward current (Ito), contribute to the manifestation of both Brugada syndrome (BrS) and spinocerebellar ataxia (SCA19/22). We examined the hypothesis that Kv4.3 and Nav variants regulate each other's function, thus modulating INa/Ito balance in cardiomyocytes and INa/I(A) balance in neurons. METHODS: Bicistronic and other constructs were used to express WT or variant Nav1.5 and Kv4.3 channels in HEK293 cells. INa and Ito were recorded. RESULTS: SCN5A variants associated with BrS reduced INa, but increased Ito. Moreover, BrS and SCA19/22 KCND3 variants associated with a gain of function of Ito, significantly reduced INa, whereas the SCA19/22 KCND3 variants associated with a loss of function (LOF) of Ito significantly increased INa. Auxiliary subunits Navß1, MiRP3 and KChIP2 also modulated INa/Ito balance. Co-immunoprecipitation and Duolink studies suggested that the two channels interact within the intracellular compartments and biotinylation showed that LOF SCN5A variants can increase Kv4.3 cell-surface expression. CONCLUSION: Nav and Kv4.3 channels modulate each other's function via trafficking and gating mechanisms, which have important implications for improved understanding of these allelic cardiac and neuronal syndromes.

Impaired excitation-contraction coupling in muscle fibres from the dynamin2R465W mouse model of centronuclear myopathy.

KEY POINTS: Dynamin 2 is a ubiquitously expressed protein involved in membrane trafficking processes. Mutations in the gene encoding dynamin 2 are responsible for a congenital myopathy associated with centrally located nuclei in the muscle fibres. Using muscle fibres from a mouse model of the most common mutation responsible for this disease in humans, we tested whether altered Ca2+ signalling and excitation-contraction coupling contribute to muscle weakness. The plasma membrane network that carries the electrical excitation is moderately perturbed in the diseased muscle fibres. The excitation-activated Ca2+ input fluxes across both the plasma membrane and the membrane of the sarcoplasmic reticulum are defective in the diseased fibres, which probably contributes to muscle weakness in patients. ABSTRACT: Mutations in the gene encoding dynamin 2 (DNM2) are responsible for autosomal dominant centronuclear myopathy (AD-CNM). We studied the functional properties of Ca2+ signalling and excitation-contraction (EC) coupling in muscle fibres isolated from a knock-in (KI) mouse model of the disease, using confocal imaging and the voltage clamp technique. The transverse-tubule network organization appeared to be unaltered in the diseased fibres, although its density was reduced by ∼10% compared to that in control fibres. The density of Ca2+ current through CaV1.1 channels and the rate of voltage-activated sarcoplasmic reticulum Ca2+ release were reduced by ∼60% and 30%, respectively, in KI vs. control fibres. In addition, Ca2+ release in the KI fibres reached its peak value 10-50 ms later than in control ones. Activation of Ca2+ transients along the longitudinal axis of the fibres was more heterogeneous in the KI than in the control fibres, with the difference being exacerbated at intermediate membrane voltages. KI fibres exhibited spontaneous Ca2+ release events that were almost absent from control fibres. Overall, the results of the present study demonstrate that Ca2+ signalling and EC coupling exhibit a number of dysfunctions likely contributing to muscle weakness in DNM2-related AD-CNM.

The genetics underlying acquired long QT syndrome: impact for genetic screening.

AIMS: Acquired long QT syndrome (aLQTS) exhibits QT prolongation and Torsades de Pointes ventricular tachycardia triggered by drugs, hypokalaemia, or bradycardia. Sometimes, QTc remains prolonged despite elimination of triggers, suggesting the presence of an underlying genetic substrate. In aLQTS subjects, we assessed the prevalence of mutations in major LQTS genes and their probability of being carriers of a disease-causing genetic variant based on clinical factors. METHODS AND RESULTS: We screened for the five major LQTS genes among 188 aLQTS probands (55 ± 20 years, 140 females) from Japan, France, and Italy. Based on control QTc (without triggers), subjects were designated 'true aLQTS' (QTc within normal limits) or 'unmasked cLQTS' (all others) and compared for QTc and genetics with 2379 members of 1010 genotyped congenital long QT syndrome (cLQTS) families. Cardiac symptoms were present in 86% of aLQTS subjects. Control QTc of aLQTS was 453 ± 39 ms, shorter than in cLQTS (478 ± 46 ms, P < 0.001) and longer than in non-carriers (406 ± 26 ms, P < 0.001). In 53 (28%) aLQTS subjects, 47 disease-causing mutations were identified. Compared with cLQTS, in 'true aLQTS', KCNQ1 mutations were much less frequent than KCNH2 (20% [95% CI 7-41%] vs. 64% [95% CI 43-82%], P < 0.01). A clinical score based on control QTc, age, and symptoms allowed identification of patients more likely to carry LQTS mutations. CONCLUSION: A third of aLQTS patients carry cLQTS mutations, those on KCNH2 being more common. The probability of being a carrier of cLQTS disease-causing mutations can be predicted by simple clinical parameters, thus allowing possibly cost-effective genetic testing leading to cascade screening for identification of additional at-risk family members.

Endogenous glucuronyltransferase activity of LARGE or LARGE2 required for functional modification of α-dystroglycan in cells and tissues.

Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAß1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.

Whole-body muscle magnetic resonance imaging in SEPN1-related myopathy shows a homogeneous and recognizable pattern.

INTRODUCTION: The aim of this study was to delineate the spectrum of muscle involvement in patients with a myopathy due to mutations in SEPN1 (SEPN1-RM). METHODS: Whole-body magnetic resonance imaging (WBMRI) was used in 9 patients using T1-weighted turbo spin-echo (T1-TSE) sequences and short tau inversion recovery (STIR) in 5 patients. RESULTS: Analysis of signal and volume abnormalities by T1-TSE sequences in 109 muscles showed a homogeneous pattern characterized by a recognizable combination of atrophy and signal abnormalities in selected muscles of the neck, trunk, pelvic girdle, and lower limbs. Severe wasting of sternocleidomastoid muscle and atrophy of semimembranosus were detected. Selective paraspinal, gluteus maximus, and thigh muscle involvement was also observed. The lower leg was less constantly affected. CONCLUSIONS: WBMRI scoring of altered signal and atrophy in muscle can be represented by heatmaps and is associated with a homogeneous, recognizable pattern in SEPN1-RM, distinct from other genetic muscle diseases.

A Common Mutation of Long QT Syndrome Type 1 in Japan.

BACKGROUND: Previous studies of long QT syndrome (LQTS) have revealed the presence of country-specific hot spots in KCNQ1 mutations, and the purpose of this study was to evaluate the influence of a common mutation on clinical phenotypes in Japanese LQT1 patients. METHODS AND RESULTS: We retrospectively studied the frequency of each mutation in 190 LQT1 Japanese probands and evaluated the clinical severity of LQT1 among carriers with a common mutation. We also compared it with that of carriers with other mutations. In the Japanese cohort, the most common mutation was p. A344spl (c.1032 G>A), comprising a substitution of a guanine for an adenine at the last base of exon 7, and it was found in 17 probands (8.9%). Regarding the clinical characteristics of A344spl carriers, the mean age-of-onset was 10±4 years, >40% were symptomatic, and the mean corrected QT interval was 461±30 ms. The prognosis for carriers of the A344spl mutation (n=31) was intermediate between that for the A341V mutation reported to be associated with severe phenotypes (n=24) and other mutations (n=290). CONCLUSIONS: The A344spl mutation was a frequent LQTS genotype in Japan, which indicates that the influence of country-specific hot spots should be considered when studying LQT1 clinical phenotypes.

A centronuclear myopathy--dynamin 2 mutation impairs autophagy in mice.

Dynamin 2 (Dnm2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. Heterozygous (HTZ) mutations in the DNM2 gene cause dominant centronuclear myopathy or Charcot-Marie-Tooth neuropathy. We generated a knock-in Dnm2R465W mouse model expressing the most frequent human mutation and recently reported that HTZ mice progressively developed a myopathy. We investigated here the cause of neonatal lethality occurring in homozygous (HMZ) mice. We show that HMZ mice present at birth with a reduced body weight, hypoglycemia, increased liver glycogen content and hepatomegaly, in agreement with a defect in neonatal autophagy. In vitro studies performed in HMZ embryonic fibroblasts point out to a decrease in the autophagy flux prior to degradation at the autolysosome. We show that starved HMZ cells have a higher number of immature autophagy-related structures probably due to a defect of acidification. Our results highlight the role of Dnm2 in the cross talk between endosomal and autophagic pathways and evidence a new role of Dnm2-dependent membrane trafficking in autophagy which may be relevant in DNM2-related human diseases.

Absence of triadin, a protein of the calcium release complex, is responsible for cardiac arrhythmia with sudden death in human.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant triadin in triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of triadin for the normal function of the cardiac calcium release complex in humans.

Complex Brugada syndrome inheritance in a family harbouring compound SCN5A and CACNA1C mutations.

Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads and is associated with increased risk of sudden cardiac death. We have recently reported families with BrS and SCN5A mutations where some affected members do not carry the familial mutation. We evaluated the involvement of additional genetic determinants for BrS in an affected family. We identified three distinct gene variants within a family presenting BrS (5 individuals), cardiac conduction defects (CCD, 3 individuals) and shortened QT interval (4 individuals). The first mutation is nonsense, p.Q1695*, lying within the SCN5A gene, which encodes for NaV1.5, the α-subunit of the cardiac Na(+) channel. The second mutation is missense, p.N300D, and alters the CACNA1C gene, which encodes the α-subunit CaV1.2 of the L-type cardiac Ca(2+) channel. The SCN5A mutation strictly segregates with CCD. Four out of the 5 BrS patients carry the CACNA1C variant, and three of them present shortened QT interval. One of the BrS patients carries none of these mutations but a rare variant located in the ABCC9 gene as well as his asymptomatic mother. Patch-clamp studies identified a loss-of-function of the mutated CaV1.2 channel. Western-blot experiments showed a global expression defect while increased mobility of CaV1.2 channels on cell surface was revealed by FRAP experiments. Finally, computer simulations of the two mutations recapitulated patient phenotypes. We report a rare CACNA1C mutation as causing BrS and/or shortened QT interval in a family also carrying a SCN5A stop mutation, but which does not segregate with BrS. This study underlies the complexity of BrS inheritance and its pre-symptomatic genetic screening interpretation.

Mutations in dynamin 2 cause dominant centronuclear myopathy.

Autosomal dominant centronuclear myopathy is a rare congenital myopathy characterized by delayed motor milestones and muscular weakness. In 11 families affected by centronuclear myopathy, we identified recurrent and de novo missense mutations in the gene dynamin 2 (DNM2, 19p13.2), which encodes a protein involved in endocytosis and membrane trafficking, actin assembly and centrosome cohesion. The transfected mutants showed reduced labeling in the centrosome, suggesting that DNM2 mutations might cause centronuclear myopathy by interfering with centrosome function.

Satellite cell loss and impaired muscle regeneration in selenoprotein N deficiency.

Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.

A KCNQ1 mutation causes a high penetrance for familial atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and its incidence is expected to grow. A genetic predisposition for AF has long been recognized, but its manifestation in these patients likely involves a combination of rare and common genetic variants. Identifying genetic variants that associate with a high penetrance for AF would represent a significant breakthrough for understanding the mechanisms that associate with disease. METHOD AND RESULTS: Candidate gene sequencing in 5 unrelated families with familial AF identified the KCNQ1 missense mutation p.Arg231His (R231H). In addition to AF, several of the family members have abnormal QTc intervals, syncope or experienced sudden cardiac arrest or death. KCNQ1 encodes the voltage-gated K(+) channel that conducts the slowly activating delayed rectifier K(+) current in the heart. Functional and computational analyses suggested that R231H increases KCNQ1 current (I(KCNQ1)) to shorten the atrial action potential (AP) duration. R231H is predicted to minimally affect ventricular excitability, but it prevented the increase in I(KCNQ1) following PKA activation. The unique properties of R231H appeared to be caused by a loss in voltage-dependent gating. CONCLUSIONS: The R231H variant causes a high penetrance for interfamilial early-onset AF. Our study indicates R231H likely shortens atrial refractoriness to promote a substrate for reentry. Additionally, R231H might cause abnormal ventricular repolarization by disrupting PKA activation of IKCNQ1 . We conclude genetic variants, which increase IKs during the atrial AP, decrease the atrial AP duration, and/or shorten atrial refractoriness, present a high risk for interfamilial AF.

Mutational spectrum in the Ca(2+)--activated cation channel gene TRPM4 in patients with cardiac conductance disturbances.

Very recently, mutations in the TRPM4 gene have been identified in four pedigrees as the cause of an autosomal dominant form of cardiac conduction disease. To determine the role of TRPM4 gene variations, the relative frequency of TRPM4 mutations and associated phenotypes was assessed in a cohort of 160 unrelated patients with various types of inherited cardiac arrhythmic syndromes. In eight probands with atrioventricular block or right bundle branch block--five familial cases and three sporadic cases--a total of six novel and two published TRPM4 mutations were identified. In patients with sinus node dysfunction, Brugada syndrome, or long-QT syndrome, no mutations were found. The novel mutations include six amino acid substitutions and appeared randomly distributed through predicted TRPM4 protein. In addition, eight polymorphic sites including two in-frame deletions were found. Mutations separated from polymorphisms by absence in control individuals and familial cosegregation in some families. In summary, TRPM4 gene mutations appear to play a major role in cardiac conduction disease but not for other related syndromes so far. The phenotypes are variable and clearly suggestive of additional factors modulating the disease phenotype in some patients.

High-risk long QT syndrome mutations in the Kv7.1 (KCNQ1) pore disrupt the molecular basis for rapid K(+) permeation.

Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the K(+) channel (Kv7.1) that underlies the slowly activating delayed rectifier K(+) current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss of function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confers a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated nonfunctional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamics simulations of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K(+)-K(+) repulsive forces required for rapid K(+) permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K(+) channel selectivity filter.

A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice.

Autosomal dominant centronuclear myopathy (AD-CNM) is due to mutations in the gene encoding dynamin 2 (DNM2) involved in endocytosis and intracellular membrane trafficking. To understand the pathomechanisms resulting from a DNM2 mutation, we generated a knock-in mouse model expressing the most frequent AD-CNM mutation (KI-Dnm2(R465W)). Heterozygous (HTZ) mice developed a myopathy showing a specific spatial and temporal muscle involvement. In the primarily and prominently affected tibialis anterior muscle, impairment of the contractile properties was evidenced at weaning and was progressively associated with atrophy and histopathological abnormalities mainly affecting mitochondria and reticular network. Expression of genes involved in ubiquitin-proteosome and autophagy pathways was up-regulated during DNM2-induced atrophy. In isolated muscle fibers from wild-type and HTZ mice, Dnm2 localized in regions of intense membrane trafficking (I-band and perinuclear region), emphasizing the pathophysiological hypothesis in which DNM2-dependent trafficking would be altered. In addition, HTZ fibers showed an increased calcium concentration as well as an intracellular Dnm2 and dysferlin accumulation. A similar dysferlin retention, never reported so far in congenital myopathies, was also demonstrated in biopsies from DNM2-CNM patients and can be considered as a new marker to orientate direct genetic testing. Homozygous (HMZ) mice died during the first hours of life. Impairment of clathrin-mediated endocytosis, demonstrated in HMZ embryonic fibroblasts, could be the cause of lethality. Overall, this first mouse model of DNM2-related myopathy shows the crucial role of DNM2 in muscle homeostasis and will be a precious tool to study DNM2 functions in muscle, pathomechanisms of DNM2-CNM and developing therapeutic strategies.

The role of stress test for predicting genetic mutations and future cardiac events in asymptomatic relatives of catecholaminergic polymorphic ventricular tachycardia probands.

AIMS: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmic disorder with a highly malignant clinical course. Exercise-stress test is the first-line approach to diagnose suspected individuals. We sought to elucidate the value of exercise-stress test for predicting mutations and future cardiac events in CPVT-family relatives. METHODS AND RESULTS: The present study included 67 asymptomatic relatives (24 ± 15 years) of 17 genetically positive CPVT probands, who underwent exercise-stress test without any medication and genetic testing. Exercise-stress test, which was considered positive with the induction of ventricular tachycardia or premature ventricular contractions consisting of bigeminy or couplets, was positive in 17 relatives (25%). Genetic analysis disclosed mutations in 16 of these 17 relatives (94%) and in 16 of the 50 relatives (32%) with negative exercise-stress test; the sensitivity and specificity for a positive genotype were 50 and 97%, respectively (P< 0.001). Among 32 mutation carriers, cardiac events occurred in 7 of the 16 relatives with positive and 2 of the 16 relatives with negative exercise-stress test during the follow-up period of 9.6 ± 3.8 years, and four with positive and two with negative stress test were not on regular beta-blocker treatment at these events. In the 16 relatives with positive stress test, those on beta-blocker treatment demonstrated a trend of lower cardiac event rate (Log-rank P= 0.054). CONCLUSION: In asymptomatic relatives of CPVT probands, exercise-stress test can be used as a simple diagnostic tool. Nevertheless, because of the low sensitivity for predicting mutations and future cardiac events in those with negative stress test, genetic analysis should be performed to improve patient management.

Trafficking and Gating Cooperation Between Deficient Nav1.5-mutant Channels to Rescue INa.

BACKGROUND: Pathogenic variants in SCN5A, the gene encoding the cardiac Na+ channel α-subunit Nav1.5, result in life-threatening arrhythmias, e.g., Brugada syndrome, cardiac conduction defects and long QT syndrome. This variety of phenotypes is underlied by the fact that each Nav1.5 mutation has unique consequences on the channel trafficking and gating capabilities. Recently, we established that sodium channel α-subunits Nav1.5, Nav1.1 and Nav1.2 could dimerize, thus, explaining the potency of some Nav1.5 pathogenic variants to exert dominant-negative effect on WT channels, either by trafficking deficiency or coupled gating. OBJECTIVE: The present study sought to examine whether Nav1.5 channels can cooperate, or transcomplement each other, to rescue the Na+ current (INa). Such a mechanism could contribute to explain the genotype-phenotype discordance often observed in family members carrying Na+-channel pathogenic variants. METHODS: Patch-clamp and immunocytochemistry analysis were used to investigate biophysical properties and cellular localization in HEK293 cells and rat neonatal cardiomyocytes transfected respectively with WT and 3 mutant channels chosen for their particular trafficking and/or gating properties. RESULTS: As previously reported, the mutant channels G1743R and R878C expressed alone in HEK293 cells both abolished INa, G1743R through a trafficking deficiency and R878C through a gating deficiency. Here, we showed that coexpression of both G1743R and R878C nonfunctioning channels resulted in a partial rescue of INa, demonstrating a cooperative trafficking of Nav1.5 α-subunits. Surprisingly, we also showed a cooperation mechanism whereby the R878C gating-deficient channel was able to rescue the slowed inactivation kinetics of the C-terminal truncated R1860X (ΔCter) variant, suggesting coupled gating. CONCLUSIONS: Altogether, our results add to the evidence that Nav channels are able to interact and regulate each other's trafficking and gating, a feature that likely contributes to explain the genotype-phenotype discordance often observed between members of a kindred carrying a Na+-channel pathogenic variant.

Novel CALM3 Variant Causing Calmodulinopathy With Variable Expressivity in a 4-Generation Family.

BACKGROUND: CaM (calmodulin), encoded by 3 separate genes (CALM1, CALM2, and CALM3), is a multifunctional Ca2+-binding protein involved in many signal transduction events including ion channel regulation. CaM variants may present with early-onset long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia, or sudden cardiac death. Most reported variants occurred de novo. We identified a novel CALM3 variant, p.Asn138Lys (N138K), in a 4-generation family segregating with LQTS. The aim of this study was to elucidate its pathogenicity and to compare it with that of p.D130G-CaM-a variant associated with a severe LQTS phenotype. METHODS: We performed whole exome sequencing for a large, 4-generation family affected by LQTS. To assess the effect of the detected CALM3 variant, the intrinsic Ca2+-binding affinity was measured by stoichiometric Ca2+ titrations and equilibrium titrations. L-type Ca2+ and slow delayed rectifier potassium currents (ICaL and IKs) were recorded by whole-cell patch-clamp. Cav1.2 and Kv7.1 membrane expression were determined by optical fluorescence assays. RESULTS: We identified 14 p.N138K-CaM carriers in a family where 2 sudden deaths occurred in children. Several members were only mildly affected compared with CaM-LQTS patients to date described in literature. The intrinsic Ca2+-binding affinity of the CaM C-terminal domain was 10-fold lower for p.N138K-CaM compared with wild-type-CaM. ICaL inactivation was slowed in cells expressing p.N138K-CaM but less than in p.D130G-CaM cells. Unexpectedly, a larger IKs current density was observed in cells expressing p.N138K-CaM, but not for p.D130G-CaM, compared with wild-type-CaM. CONCLUSIONS: The p.N138K CALM3 variant impairs Ca2+-binding affinity of CaM and ICaL inactivation but potentiates IKs. The variably expressed phenotype of this variant compared with previously published de novo LQTS-CaM variants is likely explained by a milder impairment of ICaL inactivation combined with IKs augmentation.