Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell‐derived CFBs, epicardial (EpiC‐FB), and second heart field (SHF‐FB) impacts transcriptional and functional properties. Both EpiC‐FBs and SHF‐FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell‐derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC‐FBs exhibiting higher stress‐induced activation potential akin to myofibroblasts and SHF‐FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC‐FBs have utility in modeling fibrotic diseases while SHF‐FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.

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More than 60% of hypertrophic cardiomyopathy (HCM)-causing mutations are found in the gene loci encoding cardiac myosin-associated proteins including myosin heavy chain (MHC) and myosin binding protein C (MyBP-C). Moreover, patients with more than one independent HCM mutation may be at increased risk for more severe disease expression and adverse outcomes. However detailed mechanistic understanding, especially at early stages of disease progression, is limited. To identify early-stage HCM triggers, we generated single (MYH7 c.2167C > T [R723C] with a known pathogenic significance in the MHC converter domain) and double (MYH7 c.2167C > T [R723C]; MYH6 c.2173C > T [R725C] with unknown significance) myosin gene mutations in human induced pluripotent stem cells (hiPSCs) using a base-editing strategy. Cardiomyocytes (CMs) derived from hiPSCs with either single or double mutation exhibited phenotypic characteristics consistent with later-stage HCM including hypertrophy, multinucleation, altered calcium handling, metabolism, and arrhythmia. We then probed mutant CMs at time points prior to the detection of known HCM characteristics. We found MYH7/MYH6 dual mutation dysregulated extracellular matrix (ECM) remodeling, altered integrin expression, and interrupted cell-ECM adhesion by limiting the formation of focal adhesions. These results point to a new phenotypic feature of early-stage HCM and reveal novel therapeutic avenues aimed to delay or prohibit disease onset.

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Introduction: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valve interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct.Methods: To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays.Results: Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs.Conclusion: These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valve interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts.