Reduced Ejection Fraction in Elite Endurance Athletes: Clinical and Genetic Overlap With Dilated Cardiomyopathy.

BACKGROUND: Exercise-induced cardiac remodeling can be profound, resulting in clinical overlap with dilated cardiomyopathy, yet the significance of reduced ejection fraction (EF) in athletes is unclear. The aim is to assess the prevalence, clinical consequences, and genetic predisposition of reduced EF in athletes. METHODS: Young endurance athletes were recruited from elite training programs and underwent comprehensive cardiac phenotyping and genetic testing. Those with reduced EF using cardiac magnetic resonance imaging (defined as left ventricular EF <50%, or right ventricular EF <45%, or both) were compared with athletes with normal EF. A validated polygenic risk score for indexed left ventricular end-systolic volume (LVESVi-PRS), previously associated with dilated cardiomyopathy, was assessed. Clinical events were recorded over a mean of 4.4 years. RESULTS: Of the 281 elite endurance athletes (22±8 years, 79.7% male) undergoing comprehensive assessment, 44 of 281 (15.7%) had reduced left ventricular EF (N=12; 4.3%), right ventricular EF (N=14; 5.0%), or both (N=18; 6.4%). Reduced EF was associated with a higher burden of ventricular premature beats (13.6% versus 3.8% with >100 ventricular premature beats/24 h; P=0.008) and lower left ventricular global longitudinal strain (-17%±2% versus -19%±2%; P<0.001). Athletes with reduced EF had a higher mean LVESVi-PRS (0.57±0.13 versus 0.51±0.14; P=0.009) with athletes in the top decile of LVESVi-PRS having an 11-fold increase in the likelihood of reduced EF compared with those in the bottom decile (P=0.034). Male sex and higher LVESVi-PRS were the only significant predictors of reduced EF in a multivariate analysis that included age and fitness. During follow-up, no athletes developed symptomatic heart failure or arrhythmias. Two athletes died, 1 from trauma and 1 from sudden cardiac death, the latter having a reduced right ventricular EF and a LVESVi-PRS >95%. CONCLUSIONS: Reduced EF occurs in approximately 1 in 6 elite endurance athletes and is related to genetic predisposition in addition to exercise training. Genetic and imaging markers may help identify endurance athletes in whom scrutiny about long-term clinical outcomes may be appropriate. REGISTRATION: URL: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=374976&isReview=true; Unique identifier: ACTRN12618000716268.

DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

BACKGROUND: Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. METHODS: Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. RESULTS: Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. CONCLUSIONS: Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Irreversible triggers for hypertrophic cardiomyopathy are established in the early postnatal period.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein genes, and left ventricular hypertrophy (LVH) develops as an adaptive response to sarcomere dysfunction. It remains unclear whether persistent expression of the mutant gene is required for LVH or whether early gene expression acts as an immutable inductive trigger. OBJECTIVES: The aim of this study was to use a regulatable murine model of HCM to study the reversibility of pathological LVH. METHODS: The authors generated a double-transgenic mouse model, tTAxαMHCR403Q, in which expression of the HCM-causing Arg403Gln mutation in the α-myosin heavy chain (MHC) gene is inhibited by doxycycline administration. Cardiac structure and function were evaluated in groups of mice that received doxycycline for varying periods from 0 to 40 weeks of age. RESULTS: Untreated tTAxαMHCR403Q mice showed increased left ventricular (LV) mass, contractile dysfunction, myofibrillar disarray, and fibrosis. In contrast, mice treated with doxycycline from conception to 6 weeks had markedly less LVH and fibrosis at 40 weeks. Transgene inhibition from 6 weeks reduced fibrosis but did not prevent LVH or functional changes. There were no differences in LV parameters at 40 weeks between mice with transgene inhibition from 20 weeks and mice with continuous transgene expression. CONCLUSIONS: These findings highlight the critical role of the early postnatal period in HCM pathogenesis and suggest that mutant sarcomeres manifest irreversible cardiomyocyte defects that induce LVH. In HCM, mutation-silencing therapies are likely to be ineffective for hypertrophy regression and would have to be administered very early in life to prevent hypertrophy development.

A heterozygous variant in the human cardiac miR-133 gene, MIR133A2, alters miRNA duplex processing and strand abundance.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Sequential cleavage of miRNA precursors results in a ~22 nucleotide duplex of which one strand, the mature miRNA, is typically loaded into the RNA-induced silencing complex (RISC) while the passenger strand is degraded. Very little is known about how genetic variation might affect miRNA biogenesis and function. RESULTS: We re-sequenced the MIR1-1, MIR1-2, MIR133A1, MIR133A2, and MIR133B genes, that encode the cardiac-enriched miRNAs, miR-1 and miR-133, in 120 individuals with familial atrial fibrillation and identified 10 variants, including a novel 79T > C MIR133A2 substitution. This variant lies within the duplex at the 3' end of the mature strand, miR-133a-3p, and is predicted to prevent base-pairing and weaken thermostability at this site, favoring incorporation of the passenger strand, miR-133a-5p, into RISC. Genomic DNA fragments containing miR-133a-2 precursor sequences with 79T and 79C alleles were transfected into HeLa cells. On Northern blotting the 79T allele showed strong expression of miR-133a-3p with weak expression of miR-133a-5p. In contrast, the 79C allele had no effect on miR-133a-3p but there was a significant increase (mean 3.6-fold) in miR-133a-5p levels. Deep sequencing of small RNA libraries prepared from normal human and murine atria confirmed that nearly all the mature miR-133a was comprised of miR-133a-3p and that levels of miR-133a-5p were very low. A number of isomiRs with variations at 5' and 3' ends were identified for both miR-133a-3p and miR-133a-5p, with 2 predominant miR-133a-3p isomiRs and one predominant miR-133a-5p isomiR. Bioinformatics analyses indicate that the major miR-133a-3p and 5p isomiRs have numerous predicted target mRNAs, only a few of which are in common. CONCLUSIONS: Multiple miR-133a isomiRs with potential different mRNA target profiles are present in the atrium in humans and mice. We identified a human 79T > C MIR133A2 variant that alters miRNA processing and results in accumulation of the miR-133a-5p strand that is usually degraded.

R222Q SCN5A mutation is associated with reversible ventricular ectopy and dilated cardiomyopathy.

OBJECTIVES: The goal of this study was to characterize a variant in the SCN5A gene that encodes the alpha-subunit of the cardiac sodium channel, Nav1.5, which was identified in 1 large kindred with dilated cardiomyopathy (DCM) and multiple arrhythmias, including premature ventricular complexes (PVCs). BACKGROUND: Treatment guidelines for familial DCM are based on conventional heart failure therapies, and no gene-based interventions have been established. METHODS: Family members underwent clinical evaluation and screening of the SCN5A and LMNA genes. Cellular electrophysiology and computational modeling were used to determine the functional consequences of the mutant Nav1.5 protein. RESULTS: An R222Q missense variant located in a Nav1.5 voltage-sensing domain was identified in affected family members. Patch-clamp studies showed that R222Q Nav1.5 did not alter sodium channel current density, but did left shift steady-state parameters of activation and inactivation. Using a voltage ramp protocol, normalized current responses of R222Q channels were of earlier onset and greater magnitude than wild-type channels. Action potential modeling using Purkinje fiber and ventricular cell models suggested that rate-dependent ectopy of Purkinje fiber origin is the predominant ventricular effect of the R222Q variant and a potential cause of DCM. In R222Q carriers, there were only modest responses to heart failure therapies, but PVCs and DCM were substantially reduced by amiodarone or flecainide, which are drugs that have sodium channel-blocking properties. CONCLUSIONS: The R222Q SCN5A variant has an activating effect on sodium channel function and is associated with reversible ventricular ectopy and DCM. Elucidation of the genetic basis of familial DCM can enable effective gene-targeted therapy to be implemented.

Epistatic effects of potassium channel variation on cardiac repolarization and atrial fibrillation risk.

OBJECTIVES: The aim of this study was to evaluate the role of cardiac K(+) channel gene variants in families with atrial fibrillation (AF). BACKGROUND: The K(+) channels play a major role in atrial repolarization but single mutations in cardiac K(+) channel genes are infrequently present in AF families. The collective effect of background K(+) channel variants of varying prevalence and effect size on the atrial substrate for AF is largely unexplored. METHODS: Genes encoding the major cardiac K(+) channels were resequenced in 80 AF probands. Nonsynonymous coding sequence variants identified in AF probands were evaluated in 240 control subjects. Novel variants were characterized using patch-clamp techniques and in silico modeling was performed using the Courtemanche atrial cell model. RESULTS: Nineteen nonsynonymous variants in 9 genes were found, including 11 rare variants. Rare variants were more frequent in AF probands (18.8% vs. 4.2%, p < 0.001), and the mean number of variants was greater (0.21 vs. 0.04, p < 0.001). The majority of K(+) channel variants individually had modest functional effects. Modeling simulations to evaluate combinations of K(+) channel variants of varying population frequency indicated that simultaneous small perturbations of multiple current densities had nonlinear interactions and could result in substantial (>30 ms) shortening or lengthening of action potential duration as well as increased dispersion of repolarization. CONCLUSIONS: Families with AF show an excess of rare functional K(+) channel gene variants of varying phenotypic effect size that may contribute to an atrial arrhythmogenic substrate. Atrial cell modeling is a useful tool to assess epistatic interactions between multiple variants.