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Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function 1 , 2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts 3 , 4 , but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFβ1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.

Atrial fibrillation (AF) is the most common sustained arrhythmia in humans, yet the molecular basis of AF remains incompletely understood. To determine the cell type-specific transcriptional changes underlying AF, we perform single-nucleus RNA-seq (snRNA-seq) on left atrial (LA) samples from patients with AF and controls. From more than 175,000 nuclei we find that only cardiomyocytes (CMs) and macrophages (MΦs) have a significant number of differentially expressed genes in patients with AF. Attractin Like 1 (ATRNL1) was overexpressed in CMs among patients with AF and localized to the intercalated disks. Further, in both knockdown and overexpression experiments we identify a potent role for ATRNL1 in cell stress response, and in the modulation of the cardiac action potential. Finally, we detect an unexpected expression pattern for a leading AF candidate gene, KCNN3. In sum, we uncover a role for ATRNL1 which may serve as potential therapeutic target for this common arrhythmia. Characterizing atrial fibrillation (AF) at the single cell level is challenging. Here, the authors perform snRNA-seq on 18 patients with AF to investigate the cell composition, and gene expression shifts associated with this common arrhythmia.

In cardiovascular tissues, changes in the mechanical properties of the extracellular matrix are associated with cellular de-differentiation and with subsequent functional declines. However, the underlying mechanoreceptive mechanisms are largely unclear. Here, by generating high-resolution, full-field strain maps of cardiomyocyte nuclei during contraction in vitro, complemented with evidence from tissues from patients with cardiomyopathy and from mice with reduced cardiac performance, we show that cardiomyocytes establish a distinct nuclear organization during maturation, characterized by the reorganization of H3K9me3-marked chromatin towards the nuclear border. Specifically, we show that intranuclear tension is spatially correlated with H3K9me3-marked chromatin, that reductions in nuclear deformation (through environmental stiffening or through the disruption of complexes of the linker of nucleoskeleton and cytoskeleton) abrogate chromatin reorganization and lead to the dissociation of H3K9me3-marked chromatin from the nuclear periphery, and that the suppression of H3K9 methylation induces chromatin reorganization and reduces the expression of cardiac developmental genes. Overall, our findings indicate that, by integrating environmental mechanical cues, the nuclei of cardiomyocytes guide and stabilize the fate of cells through the reorganization of epigenetically marked chromatin. Strain maps of cardiomyocyte nuclei during contraction indicate that, by integrating environmental mechanical cues, the nuclei of cardiomyocytes stabilize the fate of cells through the reorganization of epigenetically marked chromatin.

Heart failure (HF) is a leading cause of mortality. Failing hearts undergo profound metabolic changes, but a comprehensive evaluation in humans is lacking. We integrate plasma and cardiac tissue metabolomics of 678 metabolites, genome-wide RNA-sequencing, and proteomic studies to examine metabolic status in 87 explanted human hearts from 39 patients with end-stage HF compared with 48 nonfailing donors. We confirm bioenergetic defects in human HF and reveal selective depletion of adenylate purines required for maintaining ATP levels. We observe substantial reductions in fatty acids and acylcarnitines in failing tissue, despite plasma elevations, suggesting defective import of fatty acids into cardiomyocytes. Glucose levels, in contrast, are elevated. Pyruvate dehydrogenase, which gates carbohydrate oxidation, is de-repressed, allowing increased lactate and pyruvate burning. Tricarboxylic acid cycle intermediates are significantly reduced. Finally, bioactive lipids are profoundly reprogrammed, with marked reductions in ceramides and elevations in lysoglycerophospholipids. These data unveil profound metabolic abnormalities in human failing hearts.

Differentiation of cardiac fibroblasts to myofibroblasts is necessary for matrix remodeling and fibrosis in heart failure. We previously reported that mitochondrial calcium signaling drives α-ketoglutarate-dependent histone demethylation, promoting myofibroblast formation. Here we investigate the role of ATP-citrate lyase (ACLY), a key enzyme for acetyl-CoA biosynthesis, in histone acetylation regulating myofibroblast fate and persistence in cardiac fibrosis. We show that inactivation of ACLY prevents myofibroblast differentiation and reverses myofibroblasts towards quiescence. Genetic deletion of Acly in post-activated myofibroblasts prevents fibrosis and preserves cardiac function in pressure-overload heart failure. TGFβ stimulation enhances ACLY nuclear localization and ACLY–SMAD2/3 interaction, and increases H3K27ac at fibrotic gene loci. Pharmacological inhibition of ACLY or forced nuclear expression of a dominant-negative ACLY mutant prevents myofibroblast formation and H3K27ac. Our data indicate that nuclear ACLY activity is necessary for myofibroblast differentiation and persistence by maintaining histone acetylation at TGFβ-induced myofibroblast genes. These findings provide targets to prevent and reverse pathological fibrosis.

Cardiomyocyte structural remodeling is reported as a causal contributor to heart failure (HF) development and progression. Growing evidence highlights the role of organelle apposition in cardiomyocyte function and homeostasis. Disruptions in organelle crosstalk, such as that between the sarcoplasmic reticulum (SR) and mitochondria, are thought to impact numerous cellular processes such as calcium handling and cellular bioenergetics; two processes that are disrupted and implicated in cardiac pathophysiology. While the physical distance between organelles is thought to be essential for homeostatic cardiomyocyte function, whether the interactions and coupling of organelles are altered in human heart failure remains unclear. Here, we utilized transmission electron microscopy and careful quantification of ultrastructure to characterize the changes in organelle apposition in cardiomyocytes isolated from the hearts of patients diagnosed with various types of HF. Subsequently we employed molecular approaches to examine the expression of proposed organelle tethers. We demonstrate that cardiomyocytes isolated from dilated cardiomyopathy, hypertrophic cardiomyopathy and ischemic cardiomyopathy hearts display smaller, more rounded mitochondria, as compared to nonfailing controls. Failing cardiomyocytes also exhibited disrupted SR-mitochondria juxtaposition and changes in the expression of proposed molecular tethers. Further analysis revealed alterations in lipid droplet dynamics including decreased lipid droplet content and less lipid droplets in association with mitochondria in failing cardiomyocytes. Here we observed changes in organelle dynamics in cardiomyocytes isolated from heart failure patients diagnosed with differing etiologies. Our results suggest that organelle structure and apposition may be a ubiquitous contributor to human HF progression.

Heart failure is a growing public health concern which encompasses a heterogenous set of clinical features, converging on impaired cardiac contractile function.1,2 Previous work has highlighted changes in both transcription and protein expression in failing hearts,3,4 but may overlook molecular changes in less prevalent cell types. Here, we identify extensive molecular alterations present in failing hearts at single-cell resolution by performing single-nuclei RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 dilated cardiomyopathy, 15 hypertrophic cardiomyopathy and 16 non-failing hearts. Broadly, the transcriptional profiles of dilated and hypertrophic cardiomyopathy patients converged at the tissue and cell type level. Further, a subset of cardiomyopathy patients harbor a unique population of activated fibroblasts nearly entirely absent from non-failing samples. A CRISPR knockout screen was performed in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition. After knocking out genes associated with fibroblast transition in vivo, we observed a reduction in myofibroblast cell-state transition upon TGFβ1 stimulation for a subset of genes. Our results provide novel insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.

No therapies exist to reverse right ventricular failure (RVF), and the molecular mechanisms that drive RVF remain poorly studied. We recently reported that the developmentally restricted noncanonical WNT receptor ROR2 is upregulated in human RVF in proportion to severity of disease. Here we test mechanistic role of ROR2 in RVF pathogenesis. ROR2 was overexpressed or knocked down in neonatal rat ventricular myocytes (NRVMs). ROR2-modified NRVMs were characterized using confocal microscopy, RNAseq, proteomics, proteostatic functional assays, and contractile properties with pacing. The impact of cardiac ROR2 expression was evaluated in mice by AAV9-mediated overexpression and by AAV9-mediated delivery of shRNA to knockdown ROR2 in a pulmonary artery banded pressure overload RVF model. ROR2-modified mice were evaluated by echocardiography, RV protein synthetic rates and proteasome activity. In NRVMs, we find that ROR2 profoundly dysregulates the coordination between protein translation and folding. This imbalance leads to excess protein clearance by the ubiquitin proteasome system (UPS) with dramatic impacts on sarcomere and cytoskeletal structure and function. In mice, forced cardiac ROR2 expression is sufficient to disrupt proteostasis and drive RVF, while conversely ROR2 knockdown partially rescues proteostasis and cardiac function in a pressure overload model of RVF. In sum, ROR2 is a key driver of RVF pathogenesis through proteostatic disruption and, thus, provides a promising target to treat RVF.