Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease, frequently accompanied by sudden cardiac death and terminal heart failure. Genotyping of ARVC patients might be used for palliative treatment of the affected family. We genotyped a cohort of 22 ARVC patients referred to molecular genetic screening in our heart center for mutations in the desmosomal candidate genes JUP, DSG2, DSC2, DSP and PKP2 known to be associated with ARVC. In 43% of the cohort, we found disease-associated sequence variants. In addition, we screened for desmin mutations and found a novel desmin-mutation p.N116S in a patient with ARVC and terminal heart failure, which is located in segment 1A of the desmin rod domain. The mutation leads to the aggresome formation in cardiac and skeletal muscle without signs of an overt clinical myopathy. Cardiac aggresomes appear to be prominent, especially in the right ventricle of the heart. Viscosimetry and atomic force microscopy of the desmin wild-type and N116S mutant isolated from recombinant Escherichia coli revealed severe impairment of the filament formation, which was supported by transfections in SW13 cells. Thus, the gene coding for desmin appears to be a novel ARVC gene, which should be included in molecular genetic screening of ARVC patients.

Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

Chronic conditions like diabetes mellitus (DM) leading to altered metabolism might cause cardiac dysfunction. Hyperglycemia plays an important role in the pathogenesis of diabetic complications including accumulation of methylglyoxal (MG), a highly reactive alpha-dicarbonyl metabolite of glucose degradation pathways and increased generation of advanced glycation endproducts (AGEs). The aim of this investigation was to study the extent of the MG-modification argpyrimidine in human diabetic heart and in rat cardiomyoblasts grown under hyperglycemic conditions. Left ventricular myocardial samples from explanted hearts of patients with cardiomyopathy with (n=8) or without DM (n=8) as well as nonfailing donor organs (n=6), and rat cardiac myoblasts H9c2 treated with glucose were screened for the MG-modification argpyrimidine. The small heat shock protein 27 (Hsp27) revealed to be the major argpyrimidine containing protein in cardiac tissue. Additionally, the modification of arginine leading to argpyrimidine and the phosphorylation of Hsp27 are increased in the myocardium of patients with DM. In H9c2 cells hyperglycemia leads to a decrease of the Hsp27-expression and an increase in argpyrimidine content and phosphorylation of Hsp27, which was accompanied by the induction of oxidative stress and apoptosis. This study shows an association between diabetes and increased argpyrimidine-modification of myocardial Hsp27, a protein which is involved in apoptosis, oxidative stress, and cytoskeleton stabilization.

BackgroundAlthough numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate.Methodology/Principal FindingsTo test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation.Conclusions/SignificanceThe ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study.

Cardiomyocytes derived from pluripotent embryonic stem cells (ESC) have the advantage of providing a source for standardized cell cultures. However, little is known on the regulation of the genome during differentiation of ESC to cardiomyocytes. Here, we characterize the transcriptome of the mouse ESC line CM7/1 during differentiation into beating cardiomyocytes and compare the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium. We observe that the cardiac gene expression pattern of fully differentiated CM7/1-ESC is highly similar to adult primary cardiomyocytes and murine myocardium, respectively. This finding is underlined by demonstrating pharmacological effects of catecholamines and endothelin-1 on ESC-derived cardiomyocytes. Furthermore, we monitor the temporal changes in gene expression pattern during ESC differentiation with a special focus on transcription factors involved in cardiomyocyte differentiation. Thus, CM7/1-ESC-derived cardiomyocytes are a promising new tool for functional studies of cardiomyocytes in vitro and for the analysis of the transcription factor network regulating pluripotency and differentiation to cardiomyocytes.

Diabetic cardiomyopathy is a myocardial disease caused by diabetes mellitus unrelated to vascular and valvular pathology or systemic arterial hypertension. Clinical and experimental studies have shown that diabetes mellitus causes myocardial hypertrophy, apoptosis and necrosis, and increases interstitial tissue. The pathophysiology of diabetic cardiomyopathy is incompletely understood and several mechanistical approaches are under debate. Metabolic impairments like hyperglycemia, hyperlipidemia, hyperinsulinemia, and alterations in the cardiac metabolism lead to structural and functional changes which show cellular effects leading to increased oxidative stress, interstitial fibrosis, myocyte death, and disturbances in ion transport and homeostasis. Diastolic dysfunction which consecutively results in systolic dysfunction with increased left ventricular volume and reduced ejection fraction is an early diagnostic parameter. Treatment of diabetic cardiomyopathy does not differ from myocardiopathies of other etiologies and therefore has to follow the appropriate guidelines. Early intervention to reverse metabolic toxicity is the most effective method of prevention.

Hyperglycemia plays an important role in the pathogenesis of diabetic complications including accumulation of methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways. MG reacts in a non-enzymatic reaction with arginine residues of proteins which results in the formation of argpyrimidine. In several cell lines Hsp27 was found to be the most argpyrimidine containing protein. Whether Hsp27, which is high expressed in cardiac tissue, is also modified in heart, remains unclear. The aim of this investigation was to study the extent of Hsp27-modifications in heart failure and diabetes mellitus. Transmural myocardial samples from the left ventricle of explanted terminal failing hearts (diabetic: n=8; non-diabetic: n=8) and non-failing myocardium (NF) (n=6) without apparent signs of cardiac failure were collected. Cardiac tissue samples and rat cardiomyoblast cells H9c2, which were grown under normal and hyperglycaemic conditions, were investigated for the Hsp27-expression and modification by real-time PCR, western blotting and immunohistology. Hsp27 was significantly decreased (p<0.05) in CM/DM (cardiomyopathy and diabetes mellitus) compared to NF. Relating to Hsp27-expression the MG-modification in diabetic hearts was significantly increased 3-fold (p<0.05) compared to CM (cardiomyopathy) and 9-fold (p<0.05) compared to NF. Consistent with the results in human heart samples, high glucose levels induced a down-regulation of Hsp27 in H9c2-cells in a dose-dependent manner. We also found an increased MG-modification of Hsp27 with higher concentrations of glucose. Noteworthy, we detected Hsp27 in the myocardial intercalated disc and in the M-line of the sarcomer in human cardiac tissue by immunohistology. In summary, we observed an increased argpyrimidine-modification of Hsp27 in the myocardium of patients with terminal heart failure and diabetes in comparison to non-failing donor hearts and explanted hearts from patients with cardiomyopathy. The argpyrimidine-modification of Hsp27 probably results from the diabetic myocardial metabolism and might be of impact for the progress of a cardiomyopathy in patients with diabetes mellitus.

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