Follistatin-like 1 promotes proliferation of matured human hypoxic iPSC-cardiomyocytes and is secreted by cardiac fibroblasts.

The human heart has limited regenerative capacity. Therefore, patients often progress to heart failure after ischemic injury, despite advances in reperfusion therapies generally decreasing mortality. Depending on its glycosylation state, Follistatin-like 1 (FSTL1) has been shown to increase cardiomyocyte (CM) proliferation, decrease CM apoptosis, and prevent cardiac rupture in animal models of ischemic heart disease. To explore its therapeutic potential, we used a human in vitro model of cardiac ischemic injury with human induced pluripotent stem cell-derived CMs (iPSC-CMs) and assessed regenerative effects of two differently glycosylated variants of human FSTL1. Furthermore, we investigated the FSTL1-mediated interplay between human cardiac fibroblasts (cFBs) and iPSC-CMs in hypoxia. Both FSTL1 variants increased viability, while only hypo-glycosylated FSTL1 increased CM proliferation post-hypoxia. Human fetal cardiac fibroblasts (fcFBs) expressed and secreted FSTL1 under normoxic conditions, while FSTL1 secretion increased by iPSC-cFBs upon hypoxia but decreased in iPSC-CMs. Co-culture of iPSC-CMs and cFBs increased FSTL1 secretion compared with cFB mono-culture. Taken together, we confirm that FSTL1 induces iPSC-CM proliferation in a human cardiac in vitro hypoxia damage model. Furthermore, we show hypoxia-related FSTL1 secretion by human cFBs and indications for FSTL1-mediated intercellular communication between cardiac cell types in response to hypoxic conditions.

Metabolic Maturation Increases Susceptibility to Hypoxia-induced Damage in Human iPSC-derived Cardiomyocytes.

The development of new cardioprotective approaches using in vivo models of ischemic heart disease remains challenging as differences in cardiac physiology, phenotype, and disease progression between humans and animals influence model validity and prognostic value. Furthermore, economical and ethical considerations have to be taken into account, especially when using large animal models with relevance for conducting preclinical studies. The development of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has opened new opportunities for in vitro studies on cardioprotective compounds. However, the immature cellular phenotype of iPSC-CMs remains a roadblock for disease modeling. Here, we show that metabolic maturation renders the susceptibility of iPSC-CMs to hypoxia further toward a clinically representative phenotype. iPSC-CMs cultured in a conventional medium did not show significant cell death after exposure to hypoxia. In contrast, metabolically matured (MM) iPSC-CMs showed inhibited mitochondrial respiration after exposure to hypoxia and increased cell death upon increased durations of hypoxia. Furthermore, we confirmed the applicability of MM iPSC-CMs for in vitro studies of hypoxic damage by validating the known cardioprotective effect of necroptosis inhibitor necrostatin-1. Our results provide important steps to improving and developing valid and predictive human in vitro models of ischemic heart disease.