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Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies—an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9—to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM. Two approaches using an adenine base editor and a Cas9 nuclease prevented the development of hypertrophic cardiomyopathy in mice carrying a pathogenic mutation on the Myh6 gene, highlighting the potential of single-dose genetic therapies for the treatment of cardiac disease.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.

Comparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of MICU2, a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if MICU2 expression was cardioprotective, we produced and characterized Micu2 −/− mice. Mutant mice had left atrial enlargement and Micu2 −/− cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction. RNA sequencing (RNA-seq) of Micu2 −/− ventricular tissues revealed markedly reduced transcripts encoding the apelin receptor (Micu2 −/− vs. wild type, P = 7.8 × 10−40), which suppresses angiotensin II receptor signaling via allosteric transinhibition. We found that Micu2 −/− and wild-type mice had comparable basal blood pressures and elevated responses to angiotensin II infusion, but that Micu2 −/− mice exhibited systolic dysfunction and 30% lethality from abdominal aortic rupture. Aneurysms and rupture did not occur with norepinephrine-induced hypertension. Aortic tissue from Micu2 −/− mice had increased expression of extracellular matrix remodeling genes, while single-cell RNA-seq analyses showed increased expression of genes related to reactive oxygen species, inflammation, and proliferation in fibroblast and smooth muscle cells. We concluded that Micu2 −/− mice recapitulate features of diastolic heart disease and define previously unappreciated roles for Micu2 in regulating angiotensin II-mediated hypertensive responses that are critical in protecting the abdominal aorta from injury.

Loss of Bcl2-associated athanogene 3 (BAG3) is associated with dilated cardiomyopathy (DCM). BAG3 regulates sarcomere protein turnover in cardiomyocytes; however, the function of BAG3 in other cardiac cell types is understudied. In this study, we used an isogenic pair of BAG3-knockout and wild-type human induced pluripotent stem cells (hiPSCs) to interrogate the role of BAG3 in hiPSC-derived cardiac fibroblasts (CFs). Analysis of cell type-specific conditional knockout engineered heart tissues revealed an essential contribution of CF BAG3 to contractility and cardiac fibrosis, recapitulating the phenotype of DCM. In BAG3-/- CFs, we observed an increased sensitivity to TGF-β signaling and activation of a fibrogenic response when cultured at physiological stiffness (8 kPa). Mechanistically, we showed that loss of BAG3 increased transforming growth factor-β receptor 2 (TGFBR2) levels by directly binding TGFBR2 and mediating its ubiquitination and proteasomal degradation. To further validate these results, we performed single-nucleus RNA sequencing of cardiac tissue from DCM patients carrying pathogenic BAG3 variants. BAG3 pathogenic variants increased fibrotic gene expression in CFs. Together, these results extend our understanding of the roles of BAG3 in heart disease beyond the cardiomyocyte-centric view and highlight the ability of tissue-engineered hiPSC models to elucidate cell type-specific aspects of cardiac disease.

Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte (CM) TTN expression, we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human CMs from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, whereas heterozygous mice showed an allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted binding sites of NK2 homeobox 5 (NKX2-5) and myocyte enhancer factor 2 (MEF2) within E1 abolished its transcriptional activity. In embryonic mice expressing E1 reporter gene constructs, we validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5- and MEF2-binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2-binding motif that was identified in a patient with unexplained dilated cardiomyopathy (DCM) showed reduced TTN expression. Together, these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.

Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4 , FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

Deep nucleotide sequencing enables the unbiased, broad-spectrum detection of viruses in clinical samples without requiring an a priori hypothesis for the source of infection. However, its use in clinical research applications is limited by low cost-effectiveness given that most of the sequencing information from clinical samples is related to the human genome, which renders the analysis of viral genomes challenging. To overcome this limitation we developed ViroFind, an in-solution target-enrichment platform for virus detection and discovery in clinical samples. ViroFind comprises 165,433 viral probes that cover the genomes of 535 selected DNA and RNA viruses that infect humans or could cause zoonosis. The ViroFind probes are used in a hybridization reaction to enrich viral sequences and therefore enhance the detection of viral genomes via deep sequencing. We used ViroFind to detect and analyze all viral populations in the brain of 5 patients with progressive multifocal leukoencephalopathy (PML) and of 18 control subjects with no known neurological disease. Compared to direct deep sequencing, by using ViroFind we enriched viral sequences present in the clinical samples up to 127-fold. We discovered highly complex polyoma virus JC populations in the PML brain samples with a remarkable degree of genetic divergence among the JC virus variants of each PML brain sample. Specifically for the viral capsid protein VP1 gene, we identified 24 single nucleotide substitutions, 12 of which were associated with amino acid changes. The most frequent (4 of 5 samples, 80%) amino acid change was D66H, which is associated with enhanced tissue tropism, and hence likely a viral fitness advantage, compared to other variants. Lastly, we also detected sparse JC virus sequences in 10 of 18 (55.5%) of control samples and sparse human herpes virus 6B (HHV6B) sequences in the brain of 11 of 18 (61.1%) control subjects. In sum, ViroFind enabled the in-depth analysis of all viral genomes in PML and control brain samples and allowed us to demonstrate a high degree of JC virus genetic divergence in vivo that has been previously underappreciated. ViroFind can be used to investigate the structure of the virome with unprecedented depth in health and disease state.

Background Congenital heart disease (CHD) is an important cause of childhood mortality as well as morbidity in children and adults. While genetic risk contributes to the majority of CHD, most individuals with CHD do not have an identified genetic diagnosis. Short tandem repeat (TR) elements are composed of repeated base pair motifs for 2–6 basepairs that are highly polymorphic in length between individuals. These regions had been difficult to study with short read sequencing, and they have not been studied at a large scale in the context of CHD. New software and sequencing platforms have allowed for more accurate TR element genotyping. Therefore, we aimed to identify TR element variants that could impact the expression of known CHD genes. Results We identified de novo and inherited TR element variants near known CHD genes in participants with CHD ( n  = 1,899) in the Pediatric Cardiac Genomics Consortium cohort as well as unaffected participants ( n  = 1,932) from the Simons Foundation Autism Research Initiative using short-read sequencing followed by variant calling with the gangSTR pipeline. Comparison with long-read sequencing confirmed proband genotypes for 75% (91/120) of the TR element variants identified using short read sequencing. 114 TR element regions had 3 or more de novo TR element variants, compared to an expectation of 74 TR element regions (1.54-fold enrichment, p  < 1.5E-5). CHD genes CACNA1C and EVC2 had the strongest enrichment of TR element variants in the CHD cohort, determined by a higher frequency of nearby de novo TR length variants in the CHD cohort compared to the non-CHD cohort. Within CHD trios, there was over-transmission of a TR element variant near Tab  2 . Conclusions In a targeted analysis of de novo and transmitted TR element variants in a large cohort of CHD probands, each individual had ~ 1 de novo TR element variant near a CHD gene, and participants with CHD demonstrate clustering of variants within TR element regions. Long-read sequencing confirmed the majority of TR element variants identified using the gangSTR pipeline. D e novo variants in known CHD genes were enriched in participants with CHD, with specific enrichment in TR elements near CACNA1C , EVC2 , and Tab  2 in the CHD cohort. Many individual TR element variants were in known regulatory regions, but further work is needed to determine their functional impact.

Hypertrophic cardiomyopathy is usually caused by mutations in sarcomere proteins, but in some patients such mutations are not found. This study identified mutations in genes encoding enzymes involved in glycogen metabolism as causes of hypertrophic cardiomyopathy. Thus, glycogen storage diseases may sometimes present as hypertrophic cardiomyopathy, owing to accumulation of glycogen-filled vacuoles in myocytes. This study identified mutations in genes encoding enzymes involved in glycogen metabolism as causes of hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy, an autosomal dominant disorder associated with increased morbidity and premature mortality, is traditionally diagnosed on the basis of increased cardiac mass with histopathological findings of myocyte enlargement, myocyte disarray, and cardiac fibrosis. 1 – 3 However, given the availability of sophisticated noninvasive imaging techniques, an echocardiographic demonstration of unexplained left ventricular hypertrophy constitutes the current basis for a diagnosis of hypertrophic cardiomyopathy. 3 Echocardiography has shown that there is considerable diversity in the manifestations of hypertrophic cardiomyopathy, including variable age at onset, from early childhood to late adulthood, and severity of left ventricular hypertrophy. Left ventricular wall thickness in hypertrophic cardiomyopathy . . .

Congenital heart disease (CHD) occurs in ∼1% of newborns. CHD arises from many distinct etiologies, ranging from genetic or genomic variation to exposure to teratogens, which elicit diverse cell and molecular responses during cardiac development. To systematically explore the relationships between CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation and environmental exposures that modulate critical biological systems during heart development.