The contractile activity of cardiomyocytes (CMs) critical to heart function emerges from the collective shortening of sarcomeres. However, how these sarcomeric forces are transmitted within CMs during this process remains poorly understood. Traction force microscopy has been used to measure overall forces exerted by CMs, but it falls short in providing insights into which specific proteins within sarcomeres transmit and whether cell shape influences forces within each sarcomere. Here, we aimed to characterize force generation on α-actinin-2, a z-disk protein that crosslinks anti-parallel actin filaments from adjacent sarcomeres and transmits force within a cell. By incorporating a Förster resonance energy transfer (FRET)-based molecular tension sensor in α-actinin-2, we measured contraction-induced deformation of the α-actinin-2 sensor in human-induced pluripotent stem cell-derived cardiomyocytes cultured on rectangular and circular adhesive patterns. We observed α-actinin-2 localized within sarcomeres, and actinin-2 loading correlated with sarcomere maturation and organization. α-actinin-2 tension increased in contracting rectangular-shaped cells, but not in circular cells. Moreover, the increase in tension was only observed in rectangular cardiomyocytes that were cultured for 5 days, and not after 24 h. Interestingly, the spread of FRET index values was increased in both rectangular and circular cells after 5 days in culture, compared to cells that were kept for 24 h in culture. Together, these data suggest that cell shape and maturation modulates tension on a load-bearing sarcomeric protein, α-actinin-2, and highlights the importance of characterizing tension across sarcomeric structures to understand cardiomyocyte contractile activity.

BACKGROUND:Atrial septal defects (ASDs) are a prevalent type of congenital heart disease. Previous GWAS (Genome-Wide Association Studies) have identified common variants associated with ASDs, though their mechanisms remain unknown. We aimed to expand insights into the architecture of common variants associated with ASD risk and elucidate functional mechanisms.METHODS:We conducted a GWAS using isolated ASD cases and healthy controls and replicated findings in an independent cohort. We examined epigenetic marks within this ASD locus in human induced pluripotent stem cell-derived cardiomyocytes and fetal human hearts. We characterized the consequences of deletions introduced by CRISPR-Cas9 mutagenesis of human induced pluripotent stem cells to assess the effect on downstream gene expression. In addition, we investigated the 3-dimensional genome architecture of the locus using chromosome conformation capture sequencing.RESULTS:We identified a novel ASD locus on chromosome 3p12.3 encompassing the ROBO2 gene, which encodes the Roundabout guidance receptor 2 for Slit ligands. This locus includes 15 common single nucleotide polymorphisms, an enhancer, and a CCCTC-binding factor (CTCF)-binding site. Deletions of varying lengths within the ASD-associated locus in human induced pluripotent stem cell-derived cardiomyocytes reduced ROBO2 expression and dysregulated the expression of extracellular matrix genes. Chromosome conformation capture sequencing indicated that this region physically interacts with the ROBO2 promoter and demonstrates that the CTCF-binding site is essential for this contact.CONCLUSIONS:Novel common single nucleotide polymorphisms in regulatory elements controlling ROBO2 transcription contribute to risk for ASDs. These data infer key roles for the Roundabout guidance receptor 2 and Slit ligands in embryogenic development and maturation of the atrial septa.

Pulmonary arterial hypertension (PAH) is a progressive vascular syndrome characterized by aberrant signaling, severe pulmonary artery remodeling, and right ventricular (RV) failure, a major driver of morbidity and mortality. Dysregulation of the apelinergic pathway has been implicated in pulmonary vascular remodeling in PAH. Using a sugen-hypoxia rat model of PAH, we assessed the ability of a potentially novel apelin analog, resistant to native peptidase degradation, to reverse the pathological hallmarks of PAH and RV dysfunction. Apelin analog therapy corrected the vascular lesions in the lungs and nearly normalized pulmonary arterial pressures. Early cardiorenal syndrome, RV dilation, and dysfunction, as well as RV cardiomyocyte and fibroblast activation induced by pressure overload, were also reversed by apelin analog treatment. Single-nucleus RNA-seq of the lungs and RV revealed apelin-analog treatment activated several protective pathways, including rebalancing protective bone morphogenetic protein receptor type 2 (BMPR2) signaling to counteract excessive pathogenic TGF-β receptor 2 (TGFBR2) activity in PAH. These findings highlight the therapeutic potential of exogenous apelin in reversing pulmonary vascular and cardiac pathologies in PAH and support further investigation to evaluate the clinical benefits of apelin analog treatment in patients with PAH and RV failure.

Hypertrophic cardiomyopathy (HCM) is driven by sarcomeric mutations that cause energetic failure and secondary inflammation. This study demonstrates that targeting this metabolic-inflammatory axis with pioglitazone or its peroxisome proliferator-activated receptor gamma inactive enantiomer, R-pioglitazone, reverses disease progression in a murine HCM model. Both agents restored mitochondrial function (including Mitochondrial Pyruvate Carrier 1 [MPC1] levels) and resolved inflammation. Notably, R-pioglitazone showed superior efficacy, reducing interstitial fibrosis by >95% and hypertrophy by 33% without affecting healthy control hearts. These findings identify R-pioglitazone as a promising, mechanism-based candidate for disease-modifying therapy in HCM.

Hypertrophic cardiomyopathy (HCM) variants in genes encoding the myosin heavy chain (MHC) ( MYH7 ), myosin light chains ( MYL2 and MYL3 ), and cardiac myosin binding protein-C (cMyBP-C, MYBPC3 ) lead to cardiac hypertrophy, with abnormal contractility, relaxation, and energy consumption. Here, we defined the structural consequences of pathogenic and benign missense variants in these genes by mapping 233 variants ( MYH7 , n = 175; MYBPC3 , n = 41; MYL2 , n = 12; MYL3 , n = 5) onto a cryo-EM-based atomic model of the human cardiac thick filament. We identified HCM variants residing in 30 molecular interfaces of the complex thick filament interactome, including the two main interfaces of the myosin interacting-heads motif (IHM), and interfaces involving the MHC, essential and regulatory light chains, and cMyBP-C. None of the 21 variants classified as benign were within interfaces. We demonstrated earlier disease onset and adverse outcomes in HCM patients with pathogenic variants within vs. outside of molecular interfaces, emphasizing their importance in normal thick filament function and improving risk stratification of patients.

BACKGROUND: Cardiac sarcoidosis (CS) is an enigmatic disorder characterized by unexplained patchy, sterile granulomas intermixed with preserved myocardium and fibrotic regions without granuloma. CS causes arrhythmias, sudden cardiac death, and heart failure. The mechanisms producing this remarkable histopathology and disease progression remain unexplained.METHODS: Using comprehensive single-cell and spatial transcriptomic analyses, we characterized the cellular composition and gene expression in preserved, granulomatous, and fibrotic regions of human CS hearts. From unexpectedly identified clonally expanded cardiac B cells with rearranged immunoglobulin sequences, we reconstructed antibodies and screened libraries comprising the human peptidome or microbial and allergen peptides to define reactive epitopes in CS hearts.RESULTS: Cellular composition and gene expression differed substantially in CS tissues with preserved, granulomatous, or fibrotic histopathology. Cardiomyocytes upregulated arrhythmogenic and inflammasome transcripts associated with pyroptosis. Cardiomyocytes and fibroblasts activated chemoattractant cytokines that sustained myeloid and lymphoid infiltration. Granulomas contained abundant macrophages expressing modulators of cell-cell fusion, along with Th17-skewed T cells that upregulated B-cell-activating factor, thereby promoting antibody production. Fibrotic regions, without active granulomas, exhibited tertiary lymphoid structures, with clonal expansion of mature B and plasma cells. Reconstructed antibodies derived from expanded B-cell clones were inert to microbial and allergen peptides, but reacted to PPL (periplakin), a desmosome protein, and other peptides expressed on cardiac cells.CONCLUSIONS: Progressive inflammatory signals in CS are mediated by chemoattractant genes in cardiomyocytes and fibroblasts within preserved myocardium, cell-cell fusion modulators in activated macrophages within granulomatous regions, and tertiary lymphoid structures in fibrotic regions that produce patient-specific autoimmune antibodies. Identification of PPL as a CS autoantigen may account for shared clinical manifestations in CS and arrhythmic desmosomal cardiomyopathies. CS autoantigens may underlie enigmatic histopathologic findings, perpetuate disease, and contribute to adverse outcomes. Uncovering an intracardiac humoral autoimmune axis in CS provides specific therapeutic opportunities to limit granuloma formation and B-cell activation, which may reduce arrhythmogenicity and progressive dysfunction. Parallel analytic strategies have potential to define autoantigens in other enigmatic cardiac immune disorders.

Hypertrophic cardiomyopathy (HCM), a genetic heart disease defined by unexplained cardiac wall thickening, is a leading cause of sudden death worldwide. However, the three-dimensional organization of cardiac tissue underlying left ventricular hypertrophy remains poorly understood. We developed CaMVIA-3D, a deep-learning volumetric imaging and analysis pipeline to characterize cardiac microarchitecture. Analysis of tissues from HCM hearts revealed genotype-specific differences in cardiomyocyte volume, morphology, and extracellular volume, with pathogenic variants exhibiting greater concentric cellular hypertrophy and disarray and variant-negative cases showing predominant fibrosis. Longitudinal profiling of a pig HCM model revealed early-onset fibrosis preceding cardiomyocyte hypertrophy. Integrating transcriptomic and morphologic changes, we identified genes associated with cellular and extracellular remodeling. These findings define genotype-specific microstructural differences in HCM, offering insights to improve diagnostics and targeted therapies.

Somatically acquired mitochondrial DNA (mtDNA) mutations accumulate with age, but the mechanisms and consequences of this accumulation are poorly understood. Here we show that transient injuries induce a burst of persistent mtDNA mutations that impair resilience to future injuries. mtDNA mutations suppressed energy-intensive nucleotide metabolism. Repletion of adenosine, but not other nucleotides, restored adenosine triphosphate generation, which required a nuclear-encoded purine biosynthetic enzyme, adenylate kinase 4 (AK4). Analysis of 369,912 UK Biobank participants revealed a graded association between mutation burden and chronic kidney disease severity as well as an independent increase in the risk of future acute kidney injury events (P < 10-7). Heteroplasmic mtDNA mutations may therefore reflect the cumulative effect of acute injuries to metabolically active cells, impairing major functions in a fashion amenable to nuclear-controlled purine biosynthesis.

Background SMAD2 is a coregulator that binds a variety of transcription factors in human development. Heterozygous SMAD2 loss‐of‐function and missense variants are identified in patients with congenital heart disease (CHD) or arterial aneurysms. Mechanisms that cause distinct cardiovascular phenotypes remain unknown. We aimed to define transcriptional and epigenetic effects of SMAD2 variants and their role in CHD. We also assessed the function of SMAD2 missense variants of uncertain significance. Methods and Results Rare SMAD2 variants (minor allele frequency ≤10−5) were identified in exome sequencing of 11 336 participants with CHD. We constructed isogenic induced pluripotent stem cells with heterozygous or homozygous loss‐of‐function and missense SMAD2 variants identified in CHD probands. Wild‐type and mutant induced pluripotent stem cells were analyzed using bulk RNA sequencing, chromatin accessibility (Assay for Transposase‐Accessible Chromatin With Sequencing), and integrated with published SMAD2/3 chromatin immunoprecipitation data. Cardiomyocyte differentiation and contractility were evaluated. Thirty participants with CHD had heterozygous loss‐of‐function or missense SMAD2 variants. SMAD2 haploinsufficiency altered chromatin accessibility at promoters and dysregulated expression of 385 SMAD regulated genes, including 10 CHD‐associated genes. Motifs enriched in differential Assay for Transposase‐Accessible Chromatin peaks predicted that SMAD2 haploinsufficiency disrupts interactions with transcription factors NANOG (homeobox protein NANOG), ETS, TEAD3/4 (transcriptional enhanced associate domain 3/4), CREB1 (cAMP response element binding protein 1), and AP1 (activator protein 1). Compared with SMAD2‐haploinsufficient cells, induced pluripotent stem cells with R114C or W274C variants exhibited distinct and shared chromatin accessibility and transcription factor binding changes. Conclusions SMAD2 haploinsufficiency disrupts transcription factor binding and chromatin interactions critical for cardiovascular development. Differences between the molecular consequences of loss‐of‐function and missense variants likely contribute to phenotypic heterogeneity. These findings indicate opportunities for molecular analyses to improve reclassification of SMAD2 variants of uncertain clinical significance.

Hypertrophic cardiomyopathy (HCM) caused by autosomal-dominant mutations in genes coding for structural sarcomeric proteins, is the most common inherited heart disease. HCM is associated with myocardial hypertrophy, fibrosis and ventricular dysfunction. Hypoxia-inducible transcription factor-1α (Hif-1α) is the central master regulators of cellular hypoxia response and associated with HCM. Yet its exact role remains to be elucidated. Therefore, the effect of a cardiomyocyte-specific Hif-1a knockout (cHif1aKO) was studied in an established α-MHC719/+ HCM mouse model that exhibits the classical features of human HCM. The results show that Hif-1α protein and HIF targets were upregulated in left ventricular tissue of α-MHC719/+ mice. Cardiomyocyte-specific abolishment of Hif-1a blunted the disease phenotype, as evidenced by decreased left ventricular wall thickness, reduced myocardial fibrosis, disordered SRX/DRX state and ROS production. cHif1aKO induced normalization of pro-hypertrophic and pro-fibrotic left ventricular remodeling signaling evidenced on whole transcriptome and proteomics analysis in α-MHC719/+ mice. Proteomics of serum samples from patients with early onset HCM revealed significant modulation of HIF. These results demonstrate that HIF signaling is involved in mouse and human HCM pathogenesis. Cardiomyocyte-specific knockout of Hif-1a attenuates disease phenotype in the mouse model. Targeting Hif-1α might serve as a therapeutic option to mitigate HCM disease progression.

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.

While exome and whole genome sequencing have transformed medicine by elucidating the genetic underpinnings of both rare and common complex disorders, its utility to predict clinical outcomes remains understudied. Here, we use artificial intelligence (AI) technologies to explore the predictive value of whole exome sequencing in forecasting clinical outcomes following surgery for congenital heart defects (CHD). We report results for a prospective observational cohort study of 2,253 CHD patients from the Pediatric Cardiac Genomics Consortium with a broad range of complex heart defects, pre- and post-operative clinical variables and exome sequencing. Damaging genotypes in chromatin-modifying and cilia-related genes are associated with an elevated risk of adverse post-operative outcomes, including mortality, cardiac arrest and prolonged mechanical ventilation. The impact of damaging genotypes is further amplified in the context of specific CHD phenotypes, surgical complexity and extra-cardiac anomalies. The absence of a damaging genotype in chromatin-modifying and cilia-related genes is also informative, reducing the risk for some adverse postoperative outcomes. Thus, genome sequencing enriches the ability to forecast outcomes following congenital cardiac surgery.

Congenital heart disease (CHD) is a leading cause of infant mortality. We analyzed de novo mutations (DNMs) and very rare transmitted/unphased damaging variants in 248 prespecified genes in 11,555 CHD probands. The results identified 60 genes with a significant burden of heterozygous damaging variants. Variants in these genes accounted for CHD in 10.1% of probands with similar contributions from de novo and transmitted variants in parent–offspring trios that showed incomplete penetrance. DNMs in these genes accounted for 58% of the signal from DNMs. Thirty-three genes were linked to a single CHD subtype while 12 genes were associated with 2 to 4 subtypes. Seven genes were only associated with isolated CHD, while 37 were associated with 1 or more extracardiac abnormalities. Genes selectively expressed in the cardiomyocyte lineage were associated with isolated CHD, while those widely expressed in the brain were also associated with neurodevelopmental delay (NDD). Missense variants introducing or removing cysteines in epidermal growth factor (EGF)-like domains of NOTCH1 were enriched in tetralogy of Fallot and conotruncal defects, unlike the broader CHD spectrum seen with loss of function variants. Transmitted damaging missense variants in MYH6 were enriched in multiple CHD phenotypes and account for ~1% of all probands. Probands with characteristic mutations causing syndromic CHD were frequently not diagnosed clinically, often due to missing cardinal phenotypes. CHD genes that were positively or negatively associated with development of NDD suggest clinical value of genetic testing. These findings expand the understanding of CHD genetics and support the use of molecular diagnostics in CHD.

BACKGROUND AND AIMS:Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disorder characterized by left ventricular hypertrophy (LVH), diastolic dysfunction, and impaired metabolic efficiency. This study investigates the therapeutic potential of the sodium-glucose cotransporter 2 inhibitor (SGLT2i) empagliflozin (EMPA) in ameliorating these pathological features in a mouse model carrying the myosin R403Q mutation. METHODS:Male mice harbouring the R403Q mutation were treated with EMPA for 16 weeks. Multi-nuclear magnetic resonance spectroscopy (31P, 13C, and 23Na MRS), echocardiography, transcriptomic, proteomic, and phosphoproteomic profiling were utilized to assess metabolic, structural, and functional changes. RESULTS:Empagliflozin facilitated the coupling of glycolysis with glucose oxidation and normalized elevated intracellular sodium levels. Treatment resulted in a significant reduction in LVH and myocardial fibrosis as evidenced by echocardiography and histopathology. These structural improvements correlated with enhancements in mitochondrial adenosine triphosphate (ATP) synthesis, fatty acid oxidation, and branched-chain amino acid catabolism. Furthermore, EMPA improved left ventricular diastolic function and contractile reserve, underscored by improved ATP production and reduced energy cost of contraction. Notably, these benefits were linked to down-regulation of the mammalian target of rapamycin signalling pathway and normalization of myocardial substrate metabolic fluxes. CONCLUSIONS:Empagliflozin significantly mitigates structural and metabolic dysfunctions in a mouse model of HCM, underscoring its potential as a therapeutic agent for managing this condition. These findings suggest broader applicability of SGLT2i in cardiovascular diseases, including those due to myocardial-specific mutations, warranting further clinical investigation.

Human females and males differ in cardiac physiology and pathology, even after controlling for sex differences in anthropometrics, lifestyle, and environment. For example, females and males differ in cardiac stroke volume and ventricular thickness, and they exhibit different rates and symptoms of cardiovascular disease. Less is understood about molecular differences in female and male hearts, such as sex differences in gene expression. Here we present an integrative framework utilizing bulk and single-nucleus RNA-sequencing data to study sex differences in the cardiac transcriptome. We show that genes of the fatty acid oxidation (FAO) pathway, the primary source of energy in the heart, are expressed more highly in healthy female than in healthy male hearts. We demonstrate that this sex difference is due to cardiomyocyte-specific, female-biased expression of FAO genes and cannot be explained by sex differences in cardiac cellular composition or number of mitochondria, where FAO takes place. Finally, we observe increased cardiac flux and energetic utilization of free fatty acids in female compared to male hearts. Overall, our results demonstrate that male and female human hearts exhibit fundamental differences in metabolism that likely contribute to sex differences in cardiac physiology and pathology. ### Competing Interest Statement The authors have declared no competing interest.

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. 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

Clinical observations of patients with congenital heart disease carrying SMAD2 genetic variants revealed correlations with multi-organ impairments at the developmental and functional levels. Many patients with congenital heart disease present with glomerulosclerosis, periglomerular fibrosis and albuminuria. It remains largely unknown whether SMAD2 variants associated with congenital heart disease can directly alter kidney cell fate, tissue patterning and organ-level function. Here we investigate the role of pathogenic SMAD2 variants in podocytogenesis, nephrogenic cell lineage specification and glomerular filtration barrier function using a combination of CRISPR-based disease modelling, stem cell and microfluidic organ-on-a-chip technologies. We show that the abrogation of SMAD2 results in altered patterning of the mesoderm and intermediate mesoderm cell lineages, which give rise to nearly all kidney cell types. Following further differentiation of intermediate mesoderm cells, the mutant podocytes failed to develop arborizations and interdigitations. A reconstituted glomerulus-on-a-chip system showed substantial albumin leakage, as observed in glomerulopathies. This study implicates chronic heart disease-associated SMAD2 mutations in kidney tissue malformation that might inform targeted regenerative therapies. This study investigates the role of SMAD2 genetic variants associated with congenital heart disease on renal development using a CRISPR-engineered human induced pluripotent stem cell model on a microphysiological system.

Myocarditis, characterized by inflammatory cell infiltration, can have multiple etiologies, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or, rarely, mRNA-based coronavirus disease 2019 (COVID-19) vaccination. The underlying cellular and molecular mechanisms remain poorly understood. In this study, we performed single-nucleus RNA sequencing on left ventricular endomyocardial biopsies from patients with myocarditis unrelated to COVID-19 (Non-COVID-19), after SARS-CoV-2 infection (Post-COVID-19) and after COVID-19 vaccination (Post-Vaccination). We identified distinct cytokine expression patterns, with interferon-γ playing a key role in Post-COVID-19, and upregulated IL16 and IL18 expression serving as a hallmark of Post-Vaccination myocarditis. Although myeloid responses were similar across all groups, the Post-Vaccination group showed a higher proportion of CD4+ T cells, and the Post-COVID-19 group exhibited an expansion of cytotoxic CD8+ T and natural killer cells. Endothelial cells showed gene expression changes indicative of vascular barrier dysfunction in the Post-COVID-19 group and ongoing angiogenesis across all groups. These findings highlight shared and distinct mechanisms driving myocarditis in patients with and without a history of SARS-CoV-2 infection or vaccination. Maatz, Lindberg et al. identify molecular alterations and immune response changes in endomyocardial biopsies from patients with myocarditis after COVID-19 infection, after anti-COVID-19 vaccination or from non-COVID-related causes.

Hypertrophic cardiomyopathy (HCM) is a common, serious, genetic heart muscle disorder. Although the biophysical mechanisms by which gene variants in sarcomeric proteins disrupt cardiomyocyte function are largely understood, the cellular and molecular pathways leading to the complex, variable, and adverse remodeling of the non-myocyte compartment are unexplained. Here, we report that postmortem and explanted human HCM hearts exhibited chronic focal leukocyte infiltration and prominent activation of immune cells. Gene set enrichment analysis (GSEA) revealed that active immune responses were present in the mid- and late-stage HCM human hearts and in mouse hearts from several HCM mouse models. The alpha cardiac actin 1-E99K (Actc1E99K) HCM mouse model was selected for the study because it closely recapitulates the features of progressive remodeling and fibrosis seen in advanced disease in patients. Genetic depletion of lymphocytes in recombination activating gene 1-knockout (Rag-1KO) mice led to marked exacerbation of adverse cardiac remodeling in the Actc1E99K mice. Detailed characterization of cardiac regulatory T cells (Treg cells) demonstrated a time-dependent increase in Actc1E99K hearts with altered immunosuppressive profiles. Adoptive transfer of splenic Treg cells reduced cardiac fibrosis and improved systolic dysfunction in Actc1E99K mice with or without lymphocytes. In addition, low-dose interleukin-2 (IL-2)/anti-IL-2 complex (IL-2/c), which specifically induced Treg cell expansion in vivo, ameliorated cardiac fibrosis and reduced macrophage infiltration and activation in Actc1E99K mice. These data contribute to our understanding of HCM and support the use of Treg cells as a clinically testable therapeutic strategy for cardiac fibrosis in the HCM heart.

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.