Endurance Training Provokes Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Heterozygous Desmoglein-2 Mutants: Alleviation by Preload Reduction.

Desmoglein-2 mutations are detected in 5-10% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Endurance training accelerates the development of the ARVC phenotype, leading to earlier arrhythmic events. Homozygous Dsg2 mutant mice develop a severe ARVC-like phenotype. The phenotype of heterozygous mutant (Dsg2mt/wt) or haploinsufficient (Dsg20/wt) mice is still not well understood. To assess the effects of age and endurance swim training, we studied cardiac morphology and function in sedentary one-year-old Dsg2mt/wt and Dsg20/wt mice and in young Dsg2mt/wt mice exposed to endurance swim training. Cardiac structure was only occasionally affected in aged Dsg20/wt and Dsg2mt/wt mice manifesting as small fibrotic foci and displacement of Connexin 43. Endurance swim training increased the right ventricular (RV) diameter and decreased RV function in Dsg2mt/wt mice but not in wild types. Dsg2mt/wt hearts showed increased ventricular activation times and pacing-induced ventricular arrhythmia without obvious fibrosis or inflammation. Preload-reducing therapy during training prevented RV enlargement and alleviated the electrophysiological phenotype. Taken together, endurance swim training induced features of ARVC in young adult Dsg2mt/wt mice. Prolonged ventricular activation times in the hearts of trained Dsg2mt/wt mice are therefore a potential mechanism for increased arrhythmia risk. Preload-reducing therapy prevented training-induced ARVC phenotype pointing to beneficial treatment options in human patients.

Reduced plakoglobin increases the risk of sodium current defects and atrial conduction abnormalities in response to androgenic anabolic steroid abuse.

Androgenic anabolic steroids (AAS) are commonly abused by young men. Male sex and increased AAS levels are associated with earlier and more severe manifestation of common cardiac conditions, such as atrial fibrillation, and rare ones, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical observations suggest a potential atrial involvement in ARVC. Arrhythmogenic right ventricular cardiomyopathy is caused by desmosomal gene defects, including reduced plakoglobin expression. Here, we analysed clinical records from 146 ARVC patients to identify that ARVC is more common in males than females. Patients with ARVC also had an increased incidence of atrial arrhythmias and P wave changes. To study desmosomal vulnerability and the effects of AAS on the atria, young adult male mice, heterozygously deficient for plakoglobin (Plako+/- ), and wild type (WT) littermates were chronically exposed to 5α-dihydrotestosterone (DHT) or placebo. The DHT increased atrial expression of pro-hypertrophic, fibrotic and inflammatory transcripts. In mice with reduced plakoglobin, DHT exaggerated P wave abnormalities, atrial conduction slowing, sodium current depletion, action potential amplitude reduction and the fall in action potential depolarization rate. Super-resolution microscopy revealed a decrease in NaV 1.5 membrane clustering in Plako+/- atrial cardiomyocytes after DHT exposure. In summary, AAS combined with plakoglobin deficiency cause pathological atrial electrical remodelling in young male hearts. Male sex is likely to increase the risk of atrial arrhythmia, particularly in those with desmosomal gene variants. This risk is likely to be exaggerated further by AAS use. KEY POINTS: Androgenic male sex hormones, such as testosterone, might increase the risk of atrial fibrillation in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), which is often caused by desmosomal gene defects (e.g. reduced plakoglobin expression). In this study, we observed a significantly higher proportion of males who had ARVC compared with females, and atrial arrhythmias and P wave changes represented a common observation in advanced ARVC stages. In mice with reduced plakoglobin expression, chronic administration of 5α-dihydrotestosterone led to P wave abnormalities, atrial conduction slowing, sodium current depletion and a decrease in membrane-localized NaV 1.5 clusters. 5α-Dihydrotestosterone, therefore, represents a stimulus aggravating the pro-arrhythmic phenotype in carriers of desmosomal mutations and can affect atrial electrical function.

First report on an inotropic peptide activating tetrodotoxin-sensitive, "neuronal" sodium currents in the heart.

BACKGROUND: New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels. METHODS AND RESULTS: All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue. CONCLUSIONS: Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents.

Overexpression of cAMP-response element modulator causes abnormal growth and development of the atrial myocardium resulting in a substrate for sustained atrial fibrillation in mice.

BACKGROUND AND METHODS: Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. The substrate of AF is composed of a complex interplay between structural and functional changes of the atrial myocardium often preceding the occurrence of persistent AF. However, there are only few animal models reproducing the slow progression of the AF substrate to the spontaneous occurrence of the arrhythmia. Transgenic mice (TG) with cardiomyocyte-directed expression of CREM-IbΔC-X, an isoform of transcription factor CREM, develop atrial dilatation and spontaneous-onset AF. Here we tested the hypothesis that TG mice develop an arrhythmogenic substrate preceding AF using physiological and biochemical techniques. RESULTS: Overexpression of CREM-IbΔC-X in young TG mice (<8weeks) led to atrial dilatation combined with distension of myocardium, elongated myocytes, little fibrosis, down-regulation of connexin 40, loss of excitability with a number of depolarized myocytes, atrial ectopies and inducibility of AF. These abnormalities continuously progressed with age resulting in interatrial conduction block, increased atrial conduction heterogeneity, leaky sarcoplasmic reticulum calcium stores and the spontaneous occurrence of paroxysmal and later persistent AF. This distinct atrial remodelling was associated with a pattern of non-regulated and up-regulated marker genes of myocardial hypertrophy and fibrosis. CONCLUSIONS: Expression of CREM-IbΔC-X in TG hearts evokes abnormal growth and development of the atria preceding conduction abnormalities and altered calcium homeostasis and the development of spontaneous and persistent AF. We conclude that transcription factor CREM is an important regulator of atrial growth implicated in the development of an arrhythmogenic substrate in TG mice.

Load-reducing therapy prevents development of arrhythmogenic right ventricular cardiomyopathy in plakoglobin-deficient mice.

OBJECTIVES: We used a murine model of arrhythmogenic right ventricular cardiomyopathy (ARVC) to test whether reducing ventricular load prevents or slows development of this cardiomyopathy. BACKGROUND: At present, no therapy exists to slow progression of ARVC. Genetically conferred dysfunction of the mechanical cell-cell connections, often associated with reduced expression of plakoglobin, is thought to cause ARVC. METHODS: Littermate pairs of heterozygous plakoglobin-deficient mice (plako(+/-)) and wild-type (WT) littermates underwent 7 weeks of endurance training (daily swimming). Mice were randomized to blinded load-reducing therapy (furosemide and nitrates) or placebo. RESULTS: Therapy prevented training-induced right ventricular (RV) enlargement in plako(+/-) mice (RV volume: untreated plako(+/-) 136 ± 5 µl; treated plako(+/-) 78 ± 5 µl; WT 81 ± 5 µl; p < 0.01 for untreated vs. WT and untreated vs. treated; mean ± SEM). In isolated, Langendorff-perfused hearts, ventricular tachycardias (VTs) were more often induced in untreated plako(+/-) hearts (15 of 25), than in treated plako(+/-) hearts (5 of 19) or in WT hearts (6 of 21, both p < 0.05). Epicardial mapping of the RV identified macro-re-entry as the mechanism of ventricular tachycardia. The RV longitudinal conduction velocity was reduced in untreated but not in treated plako(+/-) mice (p < 0.01 for untreated vs. WT and untreated vs. treated). Myocardial concentration of phosphorylated connexin43 was lower in plako(+/-) hearts with VTs compared with hearts without VTs and was reduced in untreated plako(+/-) compared with WT (both p < 0.05). Plako(+/-) hearts showed reduced myocardial plakoglobin concentration, whereas ß-catenin and N-cadherin concentration was not changed. CONCLUSIONS: Load-reducing therapy prevents training-induced development of ARVC in plako(+/-) mice.

PITX2c is expressed in the adult left atrium, and reducing Pitx2c expression promotes atrial fibrillation inducibility and complex changes in gene expression.

BACKGROUND: Intergenic variations on chromosome 4q25, close to the PITX2 transcription factor gene, are associated with atrial fibrillation (AF). We therefore tested whether adult hearts express PITX2 and whether variation in expression affects cardiac function. METHODS AND RESULTS: mRNA for PITX2 isoform c was expressed in left atria of human and mouse, with levels in right atrium and left and right ventricles being 100-fold lower. In mice heterozygous for Pitx2c (Pitx2c(+/-)), left atrial Pitx2c expression was 60% of wild-type and cardiac morphology and function were not altered, except for slightly elevated pulmonary flow velocity. Isolated Pitx2c(+/-) hearts were susceptible to AF during programmed stimulation. At short paced cycle lengths, atrial action potential durations were shorter in Pitx2c(+/-) than in wild-type. Perfusion with the ß-receptor agonist orciprenaline abolished inducibility of AF and reduced the effect on action potential duration. Spontaneous heart rates, atrial conduction velocities, and activation patterns were not affected in Pitx2c(+/-) hearts, suggesting that action potential duration shortening caused wave length reduction and inducibility of AF. Expression array analyses comparing Pitx2c(+/-) with wild-type, for left atrial and right atrial tissue separately, identified genes related to calcium ion binding, gap and tight junctions, ion channels, and melanogenesis as being affected by the reduced expression of Pitx2c. CONCLUSIONS: These findings demonstrate a physiological role for PITX2 in the adult heart and support the hypothesis that dysregulation of PITX2 expression can be responsible for susceptibility to AF.

Regional, age-dependent, and genotype-dependent differences in ventricular action potential duration and activation time in 410 Langendorff-perfused mouse hearts.

Although numerous studies have reported the effects of genetic alterations on murine electrophysiology, the range of normal values for ventricular activation, repolarization, and arrhythmias in mouse hearts is not known. We analyzed right ventricular (RV), left ventricular (LV), and septal activation times, monophasic action potential durations (APD), and right ventricular effective refractory periods during spontaneous rhythm, induced AV nodal block, right ventricular pacing (100-300 ms paced cycle length), and programmed stimulation in 410 beating, Langendorff-perfused, wild-type mouse hearts of CD1, DBAC3H, FVBN, C57/Bl6, and hybrid backgrounds (age 203 +/- 132 days). Action potential duration was longer at longer cycle lengths. LV-APD prolonged more than RV-APD, resulting in an increased heterogeneity of APD at longer pacing cycle lengths. Higher heart weight/body weight ratio and DBAC3H and FVB/N backgrounds were associated with long APD, C57Bl/6 background was associated with short APD. Activation times were longer in older hearts. There were no clear-cut sex-dependent APD differences. Sustained spontaneous arrhythmias occurred in 1% of hearts, non-sustained arrhythmias in 18%. Induction of AV block and C57Bl/6 genetic background were associated with spontaneous arrhythmias. Programmed stimulation induced arrhythmias in 51% of hearts. Inducible arrhythmias were associated with advanced age and shorter refractory periods. Ventricular APD in beating mouse hearts show rate- and site-dependent changes comparable to man and large animals. Bradycardia provokes spontaneous arrhythmias in mouse heart, while age-dependent conduction slowing and short refractory periods predispose to induced arrhythmias. Genetic background influences repolarization and arrhythmogenesis. These findings provide systematic data for the design and interpretation of arrhythmia studies in murine disease models.