Alpha Protein Kinase 3 Gene Therapy Restores Heart Function in Mouse and Human Models of Cardiomyopathy
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE328808 | GEO | 2026/06/18
REPOSITORIES: GEO
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Project description: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 TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, while heterozygous mice demonstrated allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive the 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 NKX2-5- and MEF2-binding sites within E1 abolished its transcriptional activity. Embryonic mice expressing E1 reporter gene constructs 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 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.
2024-12-14 | GSE283866 | GEO
Project description: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 TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, while heterozygous mice demonstrated allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive the 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 NKX2-5- and MEF2-binding sites within E1 abolished its transcriptional activity. Embryonic mice expressing E1 reporter gene constructs 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 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.
2024-11-27 | GSE282693 | GEO
Project description: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 TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, while heterozygous mice demonstrated allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive the 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 NKX2-5- and MEF2-binding sites within E1 abolished its transcriptional activity. Embryonic mice expressing E1 reporter gene constructs 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 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.
2024-11-27 | GSE282670 | GEO
Project description:Dilated cardiomyopathy (DCM), defined by left ventricular (LV) enlargement associated with impaired cardiac performance, is a major cause of heart failure (HF). This results in a dilated, thin-walled left ventricle that fails to supply sufficient blood to the body. Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory DCM. The mechanisms for how TTNtv lead to cardiac dilation are unclear. Here, we show that reduction of Ttn expression by shRNA (Ttn shRNA) generated DCM in both mouse and rat. Ttn shRNA transduced mice developed typical DCM manifestations including impaired cardiac performance, enlarged LV and reduced LV wall thickness. Gene profiling indicates cardiac metabolism, cell proliferation and survival related genes are significantly dysregulated in Ttn shRNA-induced DCM. TUNEL assay showed Ttn shRNA induced a significant increase of cardiac cell apoptosis. A screen of 15 dysregulated downstream genes identified candidates, including Esrra, Esrrb and Yy1 significantly suppressed Ttn shRNA-induced cardiac dilation and/or DCM. Ttn shRNA induced cardiac cell apoptosis was ameliorated by Yy1. Importantly, by inducing D-type cyclin, Yy1 initiated cardiomyocyte cell cycle reentry facilitating the restoration of cardiac performance. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically enhancing cardiac cell proliferation and survival.
2020-03-26 | GSE112319 | GEO
Project description:RBM20 is a cardiac splicing factor responsible for splicing of several cardiac genes such as TTN, TRDN, RyR2, PDLIM1, and CAMK2D. Mutations in RBM20 are a major cause of familial dilated cardiomyopathy (DCM), and lead to missplicing of RBM20 target genes. Here, we describe a novel pathogenic truncating mutation, RBM20 c.1222dupC, identified in a patient with mitral valve prolapse and late onset familial DCM. This mutation introduces a premature termination codon and generates a truncated protein of ~55 kDa in vitro. Splicing assays demonstrated complete loss of activity and no dominant-negative effect on WT RBM20. Overexpression in NRCMs revealed that the truncated protein localized to both cytoplasm and nucleus, partially co-localizing with WT RBM20, despite lacking the RS and RRM domains. To model the patient’s condition, we generated heterozygous c.1222dupC iPSC-derived cardiomyocytes. Western blot analysis of endogenous RBM20 revealed a strong reduction in RBM20 protein level. RT-PCR revealed splicing defects in canonical RBM20 targets, and RNA-sequencing identified widespread splicing abnormalities, including in established RBM20 targets (TTN, RyR2, CAMK2D, and CACNA1G). Together, these findings establish RBM20 c.1222dupC as a pathogenic truncating variant that causes DCM primarily through haploinsufficiency.
2026-03-18 | GSE308500 | GEO
Project description:N6-methyladenosine (m6A) is the most common internal modification in eukaryotic messenger RNAs (mRNAs) and plays essential roles in mammals. The function of this chemical modification is deciphered by m6A-specific binding proteins of the YTH family. Recent studies indicated that m6A methyltransferase METTL3 ('writer') and demethylase FTO ('eraser') play critical roles in cardiovascular diseases. However, function of m6A 'reader' proteins in the heart is still largely unknown. Here, we report that cardiac-specific ablation of Ythdc1 in postnatal heart exhibits progressive dilated cardiomyopathy, heart failure and dramatically increases the incidence of postnatal lethality. Mechanically, the transcript of Titin-the major DCM and heart failure related gene-is decorated by m6A modification and directly recognized by YTHDC1, deficiency of Ythdc1 in heart results in abnormal splicing of TTN and disarray of sarcomeric structures in the cardiomyocytes subsequently. Collectively, we demonstrated that YTHDC1-dependent TTN splicing is crucial for the postnatal heart development and cardiac function, which probably provides a potential target for treating DCM through tuning m6A modification of TTN mRNA.
2022-08-01 | GSE164353 | GEO
Project description:Mutations in the sarcomeric protein titin are a major cause of human dilated cardiomyopathy. We have developed a knock-in mouse model that imitated a previously identified titin truncation mutation. The heterotygous Ttn-deficient mice develop features of DCM and therefore recapitulate the human phenotype. To investigate the role of microRNAs in titin-based heart failure, we performed a miRNA screen in heterozygous Ttn-deficient mice and their wildtype littermate controls.
2017-01-31 | GSE90972 | GEO
Project description:Background – Hypertrophic cardiomyopathy (HCM) is a common genetic cardiac disorder that is characterized by the presence of left ventricular hypertrophy with histological evidence of myocyte disarray and fibrosis. Pathologic DNA variants in sarcomeric genes are seen in 50–60% of families, but the mechanistic origins of hypertrophy and disarray remain poorly understood. More molecular insights could contribute to a better understanding of these origins and improve therapeutic strategies for HCM. Methods – To identify gene expression patterns relevant to HCM pathology, we quantified intercellular heterogeneity in cardiomyocyte gene expression by performing single-cell RNA sequencing (scRNA-seq) on myectomy tissue from HCM patients. Results – Septal myectomy samples provide a unique opportunity to access diseased human heart tissue for scRNA-seq. We analysed gene expression of individual cardiomyocytes for three HCM patients, who each showed a similar presence of cardiomyocyte subpopulations. This similarity indicated these subpopulations emerge from biological processes, and not from experimental variability. Moreover, our data revealed functional links between genes important to HCM. A subpopulation characterized by elevated titin (TTN) expression showed increased expression of genes involved in muscle contraction and development. A subpopulation of Natriuretic Peptide A (NPPA)-enriched cells showed elevated expression of Natriuretic Peptide B (NPPB). Further analysis of gene co-expression revealed five groups of genes with tightly correlated expression (regulons), one consisting of mostly sarcomeric genes and another containing some well-known stress markers. These correlations were confirmed in 97 additional HCM myectomy samples and allowed us to de novo identify transcription factors related to sarcomeric gene expression and stress. Finally, using forward scatter, we charted functional implications of regulon expression and showed a link between the stress-marker regulon and cell size. Histology samples independently confirmed that regulon member skeletal alpha actin (ACTA1) correlates with cell size. Conclusion – We have shown that single-cell sequencing on myectomy samples from HCM patients identified new functional links between genes, transcription factors, and a functional link between genes and cell size. This highlights the relevance of cardiomyocyte heterogeneity, and additionally provides a wealth of novel insights into molecular events at the base of HCM, which potentially contribute to understanding disease progression.
2022-04-21 | GSE138262 | GEO
Project description:Top-down proteomics for the sarcomeric maturation assessment of hiPSC-CMs cultured on micropatterned PDMS substrate. Dataset includes spectra for hiPSC-CMs (cell count: 50k cells) cultured as monocultured on non-patterned PDMS (Monoculture Monolayer), hiPSC-CMs cultured as co-cultures (with hiPSC-CFs) on non-patterned PDMS (Coculture Monolayer), and hiPSC-CMs co-cultured (with hiSPC-CFs) on patterned PDMS (Micropattern). Additionally, we have included data files from method development using 100k hiPSC-CMs: (A-C) treated with 25% HFIP, (D-F) treated with 25% HFIP and pulse-sonication, (G-I) treated with 90% HFIP, and (J-L) treated with 90% HFIP and pulse sonication.
2025-05-13 | MSV000097864 | MassIVE
Project description:Myosin inhibitor mavacamten is the first targeted treatment available for hypertrophic cardiomyopathy (HCM), a disease caused by hundreds of genetic variants that affect mainly sarcomeric myosin and its negative regulator cardiac myosin-binding protein C (cMyBP-C, encoded by MYBPC3). Here, we have examined whether the reported limited efficacy of mavacamten in a fraction of HCM patients can result from dissimilar HCM pathomechanisms triggered by different genetic variants, a scenario particularly relevant for MYBPC3-associated HCM. To this aim, we have generated knock-in mice including missense pathogenic variant cMyBP-C p.R502W, which, different from patients who carry truncations in the protein, develop progressive pathogenic myocardial remodeling in the absence of alterations of cMyBP-C levels and localization. Mechanistically, mutation R502W reduces the binding affinity of cMyBP-C for myosin, increases Ca2+ sensitivity and promotes the ON structural state of myosin heads without inducing a shift towards more active myosin conformations as observed when cMyBP-C levels are reduced. Despite these differences, we demonstrate that mavacamten blunts myocardial remodeling both in R502W and cMyBP-C-deficient, knock-out hearts, correlating with its ability to normalize the levels of ON myosins in R502W sarcomeres. These beneficial effects are accompanied by improved tolerance to exercise in R502W animals. In human engineered heart tissues carrying R502W, we show that mavacamten opposes hypercontractility induced by the mutation. Hence, in combination our results indicate that myosin inhibition is effective to treat HCM caused by both truncating and missense variants in MYBPC3 regardless of the primary pathomechanisms they elicit.
2026-06-02 | PXD076207 | Pride