Human iPSCs for Elucidating Stress-mediated Paracrine Signaling in Dilated Cardiomyopathy

点击翻译按钮获取中文摘要

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is among the most common forms of inherited heart disease, characterized by systolic dysfunction and ventricular chamber enlargement. Although DCM is often associated with mutations in myocyte-specific genes that impair contractile function, pathological hallmarks also include non-myocyte dysfunction, including cardiac fibrosis and endotheliopathy. Fibrosis in particular correlates with the extent of DCM progression and is an important indicator of adverse patient outcomes (e.g., heart failure), suggesting that cardiomyocyte (CM) dysfunction and aberrant activation of fibroblasts could be causally coupled. Potential pathological crosstalk signaling between the two cell types seems increasingly plausible given that diseased or stressed CMs have been shown to produce remarkably distinct secretory profiles compared to control CMs. However, precise mechanisms of intercellular communication in the heart remain unclear, in part because the human cardiac secretome to date has been poorly defined, hampered by the difficulty of distinguishing proteins secreted by the heart versus other organs in patient plasma. Here, I will leverage iPSC-derived engineered heart tissue (iPSC-EHT), genome-editing technology, and cutting- edge proteomics to test the hypothesis that stress-induced CM secretome signaling promotes fibroblast activation and fibrosis in DCM pathogenesis. To achieve this, I will first generate iPSC-derived cardiomyocytes (iPSC-CMs) from DCM patients that carry mutations in three common sarcomeric genes, along with genome- edited isogenic lines. The iPSC-CMs will be used to create 3D iPSC-EHTs, which will enable enhanced CM maturation as well as examination of cellular responses to electrical stimulation and/or increased mechanical load. The secreted proteins and exosomes from healthy versus diseased iPSC-EHTs will then be comprehensively profiled under defined conditions using high-throughput proteomics platforms. To elucidate mechanisms and downstream effects of potential crosstalk signaling, activation of iPSC-derived cardiac fibroblasts (iPSC-CFs) will be examined by treatment with conditioned media and by co-culture assays. Successful completion of the proposed studies will lead to new mechanistic insights into DCM pathogenesis, and help identify novel therapeutic targets that can disrupt pathological signaling in DCM.