Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome-wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A). Mass-spectrometric analysis and bisulphite-sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13. In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure.

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein ␤␥ dimer (G␤␥) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in G␤ 1␥2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating G␣ s and G␣i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish G␤ 1 orthologs, G␤1 and G␤1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of G␤ 1/G␤1like is associated with a down-regulation in caveolins, AC-regulating G␣-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with G␤␥ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.cAMP ͉ cardiac contractility ͉ G proteins ͉ NDPK ͉ zebrafish S ignaling through the activation of G proteins represents the most widely used signaling pathway in mammalian biology (1). A variety of G protein-coupled receptors (GPCRs) mediate extracellular signals via heterotrimeric G proteins, which are composed of a guanine nucleotide binding ␣-subunit (G␣), as well as a ␤-subunit (G␤) and a ␥-subunit (G␥). Upon GPCR activation, the bound GDP in G␣ is exchanged for GTP and both the GTP-liganded G␣ and the stable dimer G␤␥ regulate downstream effectors (2).Nucleoside diphosphate kinases (NDPKs), which catalyze the transfer of ␥-phosphate between NTPs and NDPs, represent a family of multifunctional proteins encoded by nine human nm23 genes. The two major isoforms, NDPK A and B (17-21 kDa), play crucial roles in a wide array of cellular processes [for review see (3)]. Despite their high sequence homology and the well known formation of heterohexamers to perform their house keeping enzyme activity (4), NDPK A and B have distinct cellular functions, which are based on the possibility of both isoforms contributing to multimeric protein complexes like the SET complex (5) and a complex formed with Ca 2ϩ -activated potassium channel KCa3.1 (6). In such complexes, NDPK not only supplies NTPs but also acts as protein kinase (6). We have previously shown that NDPK B, but not NDPK A, forms a complex with G␤␥ (7, 8) and acts as a histidine kinase for G␤. The high energetic phosphate on G␤ can be specifically transferred to GDP and the GTP that is formed, induces G protei...