A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

Identification of the gene for familial atrial fibrillation will help to elucidate the molecular basis of the disease and provide insights into acquired forms. The strategy of pooling DNA samples for analysis is more time and cost effective than conventional screening and should accelerate the process of gene mapping in the future.

Rationale A delicate balance between protein synthesis and degradation maintains cardiac size and function. TRIM63 encoding Muscle RING Finger 1 (MuRF1) maintains muscle protein homeostasis by tagging the sarcomere proteins with ubiquitin for subsequent degradation by the Ubiquitin-Proteasome System (UPS). Objectives To determine the pathogenic role of TRIM63 in human hypertrophic cardiomyopathy (HCM). Methods and Results Sequencing of TRIM63 gene in 302 HCM probands (250 Caucasians) and 339 controls (262 Caucasians) led to identification of two missense (p.A48V and p.I130M) and a deletion (p.Q247*) variants exclusively in the HCM probands. These three variants were absent in 751 additional controls screened by TaqMan assays. Likewise, rare variants were enriched in the Caucasian HCM population (11/250, 4.4% vs. 3/262, 1.1%, respectively, p=0.024). Expression of the mutant TRIM63 was associated with mislocalization of TRIM63 to sarcomere Z disks, impaired auto-ubiquitination, reduced ubiquitination and UPS-mediated degradation of myosin heavy chain 6, cardiac myosin binding protein C, calcineurin (PPP3CB), and p-MTOR in adult cardiac myocytes. Induced expression of the mutant TRIM63 in the mouse heart was associated with cardiac hypertrophy, activation of the MTOR-S6K and calcineurin pathways and expression of the hypertrophic markers, which were normalized upon turning off expression of the mutant protein. Conclusions TRIM63 mutations, identified in patients with HCM, impart loss-of-function effects on E3 ligase activity and are likely causal mutations in HCM. The findings implicate impaired protein degradation in the pathogenesis of HCM.

Rationale Mutations in desmosome proteins cause arrhythmogenic cardiomyopathy (AC), a disease characterized by excess myocardial fibro-adipocytes. Cellular origin(s) of fibro-adipocytes in AC is unknown. Objective To identify the cellular origin of adipocytes in AC. Methods and Results Human and mouse cardiac cells were depleted from myocytes and flow sorted to isolate cells expressing platelet-derived growth factor receptor A (PDGFRA) and exclude those expressing other lineage and fibroblast markers (CD32, CD11B, CD45, Lys76, Ly−6c and Ly6c, THY1, and DDR2). The PDGFRApos:Linneg:THY1neg:DDR2neg cells were bipotential, as the majority expressed COL1A1, a fibroblast marker, and a subset CEBPA, a major adipogenic transcription factor, and therefore, were referred to as fibro-adipogenic progenitors (FAPs). FAPs expressed desmosome proteins including desmoplakin (DSP), predominantly in the adipogenic but not fibrogenic subsets. Conditional heterozygous deletion of Dsp in mouse using Pdgfra-Cre deleter led to increased fibro-adipogenesis in the heart and mild cardiac dysfunction. Genetic fate mapping tagged 41.4±4.1% of the cardiac adipocytes in the Pdgfra-Cre:Eyfp:DspW/F mice, indicating an origin from FAPs. FAPs isolated from the Pdgfra-Cre:Eyfp:DspW/F mouse hearts showed enhanced differentiation to adipocytes. Mechanistically, deletion of Dsp was associated with suppressed canonical Wnt signaling and enhanced adipogenesis. In contrast, activation of the canonical Wnt signaling rescued adipogenesis in a dose-dependent manner. Conclusions A subset of cardiac FAPs, identified by the PDGFRApos:Linneg:THY1neg:DDR2neg signature, expresses desmosome proteins and differentiates to adipocyte in AC through a Wnt-dependent mechanism. The findings expand the cellular spectrum of AC, commonly recognized as a disease of cardiac myocytes, to include non-myocyte cells in the heart.

Background-Familial dilated cardiomyopathy (FDCM) and hypertrophic cardiomyopathy (FHCM) are the 2 most common forms of primary cardiac muscle diseases. Studies indicate that mutations in sarcomeric proteins are responsible for FHCM and suggest that mutations in cytoskeletal proteins cause FDCM. Evidence is evolving, however, that such conclusions are premature. Methods and Results-A novel missense mutation in the cardiac troponin T gene was identified by direct sequencing and confirmed by endonuclease restriction analysis in a large family with FDCM that we had previously mapped to chromosome 1q32. The mutation substitutes tryptophan for a highly conserved amino acid, arginine, at amino acid residue 141 (Arg141Trp). The mutation occurs within the tropomyosin-binding domain of cardiac troponin T and alters the charge of the residue. This mutation cosegregates with the disease, being present in all 14 living affected individuals. The mutation was not found in 100 normal control subjects. Clinical features were congestive heart failure with premature deaths. The age of onset and severity of the disease are highly variable, with incomplete penetrance. Because 15 mutations in troponin T are known to cause FHCM, 219 probands with FHCM were screened, and none had the mutation. Conclusions-Thus, the novel cardiac troponin T mutation Arg141Trp is responsible for FDCM in our family. Because several mutations in troponin T have already been recognized to be responsible for FHCM, it appears that the phenotype, whether it be hypertrophy or dilatation, is determined by the specific mutation rather than the gene. (Circulation. 2001;