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Single-nucleus RNA/ATAC-seq in early-stage hypertrophic-cardiomyopathy models predicts SWI/SNF-activation in mutant-myocytes and allele-specific differences in fibroblasts

ABSTRACT: Hypertrophic cardiomyopathy (HCM) is associated with phenotypic variability. To gain insights into transcriptional regulation of cardiac phenotype, single-nucleus linked RNA-/ATAC-seq was performed in 5-week-old control mouse-hearts (WT) and two HCM-models (R92W-TnT, R403Q-MyHC) that exhibit differences in heart size/function and fibrosis; mutant data was compared to WT. Analysis of 23,304 nuclei from mutant hearts, and 17,669 nuclei from WT, revealed similar dysregulation of gene expression, activation of AP-1 TFs (FOS, JUN) and the SWI/SNF complex in both mutant ventricular-myocytes. In contrast, marked differences were observed between mutants, for gene expression/TF enrichment, in fibroblasts, macrophages, endothelial cells. Cellchat predicted activation of pro-hypertrophic IGF-signaling in both mutant ventricular-myocytes, and profibrotic TGFβ-signaling only in mutant-TnT fibroblasts. In summary, our bioinformatics analyses suggest that activation of IGF-signaling, AP-1 TFs and the SWI/SNF chromatin remodeler complex promotes myocyte hypertrophy in early-stage HCM. Selective activation of TGFβ-signaling in mutant-TnT fibroblasts contributes to genotype-specific differences in cardiac fibrosis.

ORGANISM(S): Mus musculus

PROVIDER: GSE265808 | GEO | 2025/10/20

REPOSITORIES: GEO

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Project description:Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ~1:200-500 and predisposes individuals to sudden death and heart failure. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and Results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured primary cardiac non-myocytes and analyzed the conditioned media by proteomics, we found that several ECM proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially expressed. We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac non-myocytes drives upregulation of ECM genes including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac non-myocytes. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac non-myocytes. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

Project description:Growth differentiating factor (GDF)15 is a TGFβ superfamily cytokine and a reported biomarker of heart failure. Myocardial expression of GDF15 is increased in heart failure. Yet, the mechanisms that control synthesis and release of GDF15 as well as the autocrine/paracrine functions of GDF15 on fibroblast function is lacking. Thus, the aim of this study was to investigate signaling pathways and functions of GDF15 in cardiac fibroblasts. Cardiac fibroblasts and cardiac myocytes were isolated from adult C57/BL6 mice, maintained in primary culture and stimulated with recombinant (r)GDF15 or recombinant (r)CCN2. Short-term stimulation (30 minutes) of cardiac fibroblasts demonstrated a GDF15-induced activation of several intracellular signaling pathways including a concentration-dependent increase of phospho-Smad3(Ser423/425) (p<0.05, n=3), phospho-AKT(Ser473) (p<0.05, n=3) and phospho-IκBα(Ser32/36) (p<0.05, n=3) levels. However, rGDF15 did not phosphorylate Smad3(Ser423/425) or IκBα(Ser32/36) in cardiac myocyte. Cardiac fibroblasts exposed to rGDF15 for 48 hours displayed differentiation towards myofibroblasts reflected by increased levels of the differentiation marker α-smooth muscle actin (SMA) similar to cardiac fibroblasts stimulated with TGFβ. The effect of GDF15 on α-SMA was dose-dependent ranging from 500 nM - 20 nM rGDF15 (p<0.05, n=3). Differentiation towards a myofibroblast phenotype in the presence of GDF15 was also supported by higher matrix metalloproteinase (MMP) enzyme activity in the cell culture medium (6±1 fold increase, n=3, p<0.05) and increased expression and release of MMP-3, 9 and 13. Immunoreactive GDF15 was predominantly found in cardiac myocytes. Recombinant CCN2 substantially induced GDF15 expression in cardiac myocytes, but not in cardiac fibroblasts. In conclusion, our data demonstrate that GDF15 is a paracrine factor in myocardial tissue and specifically regulated by CCN2 in cardiac myocytes. GDF15 has similar effects as TGFβ on fibroblasts by activation of intracellular signaling pathways and differentiation to a myofibroblasts phenotype.

Project description:Rationale Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and is associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with findings that microtubule detyrosination (dTyr-MTs) is markedly elevated in heart failure. Acute reduction of dTyr-MTs by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, presenting a new perspective for HCM treatment. Objective In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model (Mybpc3-knock-in; KI), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents, in 3-week-old KI mice, and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show: i) TTL for 6 weeks dose-dependently reduced dTyr-MTs and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes. ii) TTL for 12 weeks reduced the abundance of dTyr-MTs in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in KI mice. iii) TTL for 10 days normalized cell area in HCM hiPSC-cardiomyocytes. iv) TTL overexpression activates transcription of several tubulin isoforms, and omics GO analysis revealed enrichment of components of the cytoskeleton, sarcomere Z-disc, mitochondria, and intercalated disc in KI mice. v) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

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Single-nucleus RNA/ATAC-seq in early-stage hypertrophic-cardiomyopathy models predicts SWI/SNF-activation in mutant-myocytes and allele-specific differences in fibroblasts

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