Abstract
Ischaemic heart failure is due to the irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could regenerate infarcted hearts and improve cardiac function. However, these hPSC-derived cardiomyocytes remained immature. Epicardial-myocardial crosstalk underpins key events during cardiac development and maturation. Recently we showed that incorporating hPSC-derived epicardial cells improved cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). However, the key signaling pathways underpinning epicardial-myocardial crosstalk remain unclear.We found that Fibronectin (FN1) was uniquely upregulated in 3D-EHTs co-cultured with hESC-epicardium, compared to other stromal cells such as mesenchymal stem cells. Thus, we posited that epicardially-secreted fibronectin (FN1) is a key mediator of epicardial-cardiomyocyte crosstalk driving cardiomyocyte maturation.To test this hypothesis, we performed a series of loss of FN function experiments with peptide inhibition (pUR4), CRISPR/Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs. We found that the loss of epicardial-FN, at both protein and gene levels, resulted in immature hPSC-derived cardiomyocytes with decreased contractile function, inefficient Ca2+ handling, and disorganized sarcomeric apparatus. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Furthermore, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs.
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