Abstract
Hypertrophic cardiomyopathy (HCM) is characterized by increased left ventricular wall thickness that can lead to devastating conditions such as heart failure and sudden cardiac death. Despite extensive study, the mechanisms mediating many of the associated clinical manifestations remain unknown and human models are required. To address this, human-induced pluripotent stem cell (hiPSC) lines were generated from patients with a HCM-associated mutation (c.ACTC1G301A) and isogenic controls created by correcting the mutation using CRISPR/Cas9 gene editing technology. Cardiomyocytes (hiPSC-CMs) were differentiated from these hiPSCs and analyzed at baseline, and at increased contractile workload (2 Hz electrical stimulation). Released extracellular vesicles (EVs) were isolated and characterized after a 24-h culture period and transcriptomic analysis performed on both hiPSC-CMs and released EVs. Transcriptomic analysis of cellular mRNA showed the HCM mutation caused differential splicing within known HCM pathways, and disrupted metabolic pathways. Analysis at increasing contraction frequency showed further disruption of metabolic gene expression, with an additive effect in the HCM background. Intriguingly, we observed differences in snoRNA cargo within HCM released EVs that specifically altered when HCM hiPSC-CMs were subjected to increased workload. These snoRNAs were predicted to have roles in post-translational modifications and alternative splicing, processes differentially regulated in HCM. As such, the snoRNAs identified in this study may unveil mechanistic insight into unexplained HCM phenotypes and offer potential future use as HCM biomarkers or as targets in future RNA-targeting therapies.
Highlights
Hypertrophic cardiomyopathy (HCM) is the most commonly inherited heart disease, with a prevalence considered to be greater than 1 in 500 in the general population [1]
Differentiation of these human-induced pluripotent stem cell (hiPSC) lines to high purity (>90% a-actinin+) cardiomyocytes produced an in vitro model in which the mutant cells displayed characteristic HCM phenotypes when compared with the isogenic control line [26,33]
To determine if the HCM phenotypes seen within the cellular model could be analyzed at an expression level, transcriptomic analysis was performed on the cellular mRNA
Summary
Hypertrophic cardiomyopathy (HCM) is the most commonly inherited heart disease, with a prevalence considered to be greater than 1 in 500 in the general population [1]. The hearts of HCM patients consist of enlarged cardiomyocytes, with alignment disarray, and the presence of interstitial fibrosis [3]. HCM is an autosomal-dominant disorder mainly caused by mutations in genes encoding for contractile and structural proteins of the cardiac muscle sarcomere apparatus [4]. The penetrance of the HCM phenotype is variable, with mutation carriers presenting with symptoms that range from fully asymptomatic to fatal cardiac dysfunction [5]. The mechanisms leading to secondary HCM phenotypes, such as myocyte hypertrophy, myocardial disarray, and interstitial fibrosis, are not fully explained. It remains unclear how mutations in genes encoding sarcomeric proteins only expressed in cardiomyocytes, give rise to phenotypes that encompass multiple cardiac cell populations
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