Soluble adenylyl cyclase: A novel player in cardiac hypertrophy induced by isoprenaline or pressure overload.

AIMS: In contrast to the membrane bound adenylyl cyclases, the soluble adenylyl cyclase (sAC) is activated by bicarbonate and divalent ions including calcium. sAC is located in the cytosol, nuclei and mitochondria of several tissues including cardiac muscle. However, its role in cardiac pathology is poorly understood. Here we investigate whether sAC is involved in hypertrophic growth using two different model systems. METHODS AND RESULTS: In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h ß1-adrenoceptor stimulation using isoprenaline (ISO) and a ß2-adrenoceptor antagonist (ICI118,551). To monitor hypertrophy cell size along with RNA/DNA- and protein/DNA ratios as well as the expression level of α-skeletal actin were analyzed. sAC activity was suppressed either by treatment with its specific inhibitor KH7 or by knockdown. Both pharmacological inhibition and knockdown blunted hypertrophic growth and reduced expression levels of α-skeletal actin in ISO/ICI treated rat cardiomyocytes. To analyze the underlying cellular mechanism expression levels of phosphorylated CREB, B-Raf and Erk1/2 were examined by western blot. The results suggest the involvement of B-Raf, but not of Erk or CREB in the pro-hypertrophic action of sAC. In wild type and sAC knockout mice pressure overload was induced by transverse aortic constriction. Hemodynamics, heart weight and the expression level of the atrial natriuretic peptide were analyzed. In accordance, transverse aortic constriction failed to induce hypertrophy in sAC knockout mice. Mechanistic analysis revealed a potential role of Erk1/2 in TAC-induced hypertrophy. CONCLUSION: Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term ß1-adrenoceptor stimulation or to pressure overload.

A novel desmin (DES) indel mutation causes severe atypical cardiomyopathy in combination with atrioventricular block and skeletal myopathy.

BACKGROUND: DES mutations cause different cardiac and skeletal myopathies. Most of them are missense mutations. METHODS: Using a next-generation sequencing cardiac 174 gene panel, we identified a novel heterozygous in-frame indel mutation (DES-c.493_520del28insGCGT, p.Q165_A174delinsAS) in a Caucasian patient with cardiomyopathy in combination with atrioventricular block and skeletal myopathy. This indel mutation is located in the coding region of the first exon. Family anamnesis revealed a history of sudden cardiac death. We performed cell transfection experiments and in vitro assembly experiments to prove the pathogenicity of this novel DES indel mutation. RESULTS: These experiments revealed a severe filament formation defect of mutant desmin supporting the pathogenicity. In addition, we labeled a skeletal muscle biopsy from the mutation carrier revealing cytoplasmic desmin positive protein aggregates. In summary, we identified and functionally characterized a pathogenic DES indel mutation causing cardiac and skeletal myopathy. CONCLUSION: Our study has relevance for the clinical and genetic interpretation of further DES indel mutations causing cardiac or skeletal myopathies and might be helpful for risk stratification.

Novel Desmin Mutation p.Glu401Asp Impairs Filament Formation, Disrupts Cell Membrane Integrity, and Causes Severe Arrhythmogenic Left Ventricular Cardiomyopathy/Dysplasia.

BACKGROUND: Desmin (DES) mutations cause severe skeletal and cardiac muscle disease with heterogeneous phenotypes. Recently, DES mutations were described in patients with inherited arrhythmogenic right ventricular cardiomyopathy/dysplasia, although their cellular and molecular pathomechanisms are not precisely known. Our aim is to describe clinically and functionally the novel DES-p.Glu401Asp mutation as a cause of inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia. METHODS: We identified the novel DES mutation p.Glu401Asp in a large Spanish family with inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia and a high incidence of adverse cardiac events. A full clinical evaluation was performed on all mutation carriers and noncarriers to establish clinical and genetic cosegregation. In addition, desmin, and intercalar disc-related proteins expression were histologically analyzed in explanted cardiac tissue affected by the DES mutation. Furthermore, mesenchymal stem cells were isolated and cultured from 2 family members with the DES mutation (1 with mild and 1 with severe symptomatology) and a member without the mutation (control) and differentiated ex vivo to cardiomyocytes. Then, important genes related to cardiac differentiation and function were analyzed by real-time quantitative polymerase chain reaction. Finally, the p.Glu401Asp mutated DES gene was transfected into cell lines and analyzed by confocal microscopy. RESULTS: Of the 66 family members screened for the DES-p.Glu401Asp mutation, 23 of them were positive, 6 were obligate carriers, and 2 were likely carriers. One hundred percent of genotype-positive patients presented data consistent with inherited arrhythmogenic cardiomyopathy/dysplasia phenotype with variable disease severity expression, high-incidence of sudden cardiac death, and absence of skeletal myopathy or conduction system disorders. Immunohistochemistry was compatible with inherited arrhythmogenic cardiomyopathy/dysplasia, and the functional study showed an abnormal growth pattern and cellular adhesion, reduced desmin RNA expression, and some other membrane proteins, as well, and desmin aggregates in transfected cells expressing the mutant desmin. CONCLUSIONS: The DES-p.Glu401Asp mutation causes predominant inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia with a high incidence of adverse clinical events in the absence of skeletal myopathy or conduction system disorders. The pathogenic mechanism probably corresponds to an alteration in desmin dimer and oligomer assembly and its connection with membrane proteins within the intercalated disc.

The novel αB-crystallin (CRYAB) mutation p.D109G causes restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare heart disease characterized by diastolic dysfunction and atrial enlargement. The genetic etiology of RCM is not completely known. We identified by a next-generation sequencing panel the novel CRYAB missense mutation c.326A>G, p.D109G in a small family with RCM in combination with skeletal myopathy with an early onset of the disease. CRYAB encodes αB-crystallin, a member of the small heat shock protein family, which is highly expressed in cardiac and skeletal muscle. In addition to in silico prediction analysis, our structural analysis of explanted myocardial tissue of a mutation carrier as well as in vitro cell transfection experiments revealed abnormal protein aggregation of mutant αB-crystallin and desmin, supporting the deleterious effect of this novel mutation. In conclusion, CRYAB appears to be a novel RCM gene, which might have relevance for the molecular diagnosis and the genetic counseling of further affected families in the future.

Calcium Handling Abnormalities as a Target for Atrial Fibrillation Therapeutics: How Close to Clinical Implementation?

Atrial fibrillation (AF) is the most common cardiac arrhythmia with a substantial impact on morbidity and mortality. Antiarrhythmic drugs play a major role in rhythm-control therapy of AF. However, currently available agents exhibit limited efficacy and pronounced adverse effects, notably drug-induced proarrhythmia. Recent experimental studies have identified that Ca handling abnormalities are critical elements in AF pathophysiology with central roles in atrial ectopic activity, reentry, and atrial remodeling suggesting that Ca handling abnormalities could be promising targets for novel AF therapeutics. Here, we summarize key aspects of AF-related Ca-handling abnormalities, describe currently available compounds targeting atrial Ca handling, and highlight potential novel targets and experimental drugs currently under investigation. Finally, we assess how close AF therapeutics based on Ca-handling abnormalities are to clinical implementation.