The Transcription Factor ETV1 Induces Atrial Remodeling and Arrhythmia.

RATIONALE: Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria. OBJECTIVE: Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes. METHODS AND RESULTS: The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 under control of the myosin heavy chain promoter developed atrial dilatation, fibrosis, thrombosis, and arrhythmia. Cardiac myocyte-specific ablation of ETV1 in mice did not alter cardiac structure and function at baseline. Treatment with Ang II (angiotensin II) for 2 weeks elicited atrial remodeling and fibrosis in control, but not in ETV1-deficient mice. To identify ETV1-regulated genes, cardiac myocytes were isolated and purified from mouse atrial tissue. Active cis-regulatory elements in mouse atrial cardiac myocytes were identified by chromatin accessibility (assay for transposase-accessible chromatin sequencing) and the active chromatin modification H3K27ac (chromatin immunoprecipitation sequencing). One hundred seventy-eight genes regulated by Ang II in an ETV1-dependent manner were associated with active cis-regulatory elements containing ETV1-binding sites. Various genes involved in Ca2+ handling or gap junction formation ( Ryr2, Jph2, Gja5), potassium channels ( Kcnh2, Kcnk3), and genes implicated in atrial fibrillation ( Tbx5) were part of this ETV1-driven gene regulatory network. The atrial ETV1-dependent transcriptome in mice showed a significant overlap with the human atrial proteome of patients with permanent atrial fibrillation. CONCLUSIONS: This study identifies ETV1 as an important component in the pathophysiology of atrial remodeling associated with atrial arrhythmias.

Deciphering the Epigenetic Code of Cardiac Myocyte Transcription.

RATIONALE: Epigenetic mechanisms are crucial for cell identity and transcriptional control. The heart consists of different cell types, including cardiac myocytes, endothelial cells, fibroblasts, and others. Therefore, cell type-specific analysis is needed to gain mechanistic insight into the regulation of gene expression in cardiac myocytes. Although cytosolic mRNA represents steady-state levels, nuclear mRNA more closely reflects transcriptional activity. To unravel epigenetic mechanisms of transcriptional control, cell type-specific analysis of nuclear mRNA and epigenetic modifications is crucial. OBJECTIVE: The aim was to purify cardiac myocyte nuclei from hearts of different species by magnetic- or fluorescent-assisted sorting and to determine the nuclear and cellular RNA expression profiles and epigenetic marks in a cardiac myocyte-specific manner. METHODS AND RESULTS: Frozen cardiac tissue samples were used to isolate cardiac myocyte nuclei. High sorting purity was confirmed for cardiac myocyte nuclei isolated from mice, rats, and humans. Deep sequencing of nuclear RNA revealed a major fraction of nascent, unspliced RNA in contrast to results obtained from purified cardiac myocytes. Cardiac myocyte nuclear and cellular RNA expression profiles showed differences, especially for metabolic genes. Genome-wide maps of the transcriptional elongation mark H3K36me3 were generated by chromatin-immunoprecipitation. Transcriptome and epigenetic data confirmed the high degree of cardiac myocyte-specificity of our protocol. An integrative analysis of nuclear mRNA and histone mark occurrence indicated a major impact of the chromatin state on transcriptional activity in cardiac myocytes. CONCLUSIONS: This study establishes cardiac myocyte-specific sorting of nuclei as a universal method to investigate epigenetic and transcriptional processes in cardiac myocytes of different origins. These data sets provide novel insight into cardiac myocyte transcription.

Short-term safety results from compassionate use of risdiplam in patients with spinal muscular atrophy in Germany.

BACKGROUND: The oral, selective SMN2-splicing modifier risdiplam obtained European approval in March 2021 for the treatment of patients ≥ 2 months old with a clinical diagnosis of 5q-associated spinal muscular atrophy (SMA) 1/2/3 or with 1-4 SMN2 gene copies. For the preceding 12 months, this compassionate use program (CUP) made risdiplam available to patients with SMA1/2 in Germany who could not receive any approved SMA therapy. PATIENTS AND METHODS: Patients with SMA1/2, aged ≥ 2 months at enrollment, could be included if they were not eligible for, no longer responsive to, or not able to tolerate nusinersen or not able to receive onasemnogene abeparvovec. Oral risdiplam dosing ranged from 0.2 mg/kg to 5 mg depending on age and weight. All treatment decisions were made by the attending physicians, who were required to report all adverse events (AEs). RESULTS: Between March 12, 2020 and March 30, 2021, 36 patients with SMA1 and 98 patients with SMA2 were enrolled, with 31 patients and 80 patients receiving ≥ 1 risdiplam dose, respectively. The median (range) age was 10.5 (3-52) years in the SMA1 cohort, and 26.5 (3-60) years in the SMA2 cohort. 22.2% of patients with SMA1 and 48.0% with SMA2 were treatment-naïve. Most patients were not eligible/could not continue to receive nusinersen due to scoliosis/safety risk (SMA1: 75.0%; SMA2: 96.9%), risks associated with sedation (77.8%; 63.3%), or loss of efficacy (30.6%; 12.2%). Safety data were generally in line with the safety profile of risdiplam in ongoing clinical studies. Gastrointestinal disorders were the most common AEs. For patients with SMA1, 30 AEs were reported in 13 cases with 2 serious AEs in 1 patient. For SMA2, 100 AEs were documented in 31 case reports, including 8 serious AEs in 2 patients. CONCLUSIONS: We present the first real-world safety data of risdiplam in patients with SMA in Germany. Our observations indicated no new safety signals under real-world conditions. Real-world SMA1/2 populations comprise considerable numbers of patients who are not eligible for gene therapy and cannot tolerate or have failed nusinersen treatment. This medical need may be addressed by oral risdiplam.

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo.

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

DNA methylation signatures follow preformed chromatin compartments in cardiac myocytes.

Storage of chromatin in restricted nuclear space requires dense packing while ensuring DNA accessibility. Thus, different layers of chromatin organization and epigenetic control mechanisms exist. Genome-wide chromatin interaction maps revealed large interaction domains (TADs) and higher order A and B compartments, reflecting active and inactive chromatin, respectively. The mutual dependencies between chromatin organization and patterns of epigenetic marks, including DNA methylation, remain poorly understood. Here, we demonstrate that establishment of A/B compartments precedes and defines DNA methylation signatures during differentiation and maturation of cardiac myocytes. Remarkably, dynamic CpG and non-CpG methylation in cardiac myocytes is confined to A compartments. Furthermore, genetic ablation or reduction of DNA methylation in embryonic stem cells or cardiac myocytes, respectively, does not alter genome-wide chromatin organization. Thus, DNA methylation appears to be established in preformed chromatin compartments and may be dispensable for the formation of higher order chromatin organization.