Abstract Body (Do not enter title and authors here): Introduction: Metabolic symbiosis has been discovered in several contexts between cells with a high energy demand and supporting cells. Recently, reports have highlighted the existing lactate shuttle between cardiomyocytes (CMs) and cardiac fibroblasts. Epicardium is the embryonic source of cardiac fibroblasts and an active player in ischemic heart repair, however, its contribution to heart metabolism is unstudied. TFEB is a known regulator of energy metabolism in different cell types. Previously, we demonstrated that in mouse embryonic epicardium TFEB modulates TGFβ-driven epithelial-to-mesenchymal transition.
Methods and Results: In adult mice with epicardium-specific Tfeb knock-out, no morphological defects were observed in the hearts. However, echocardiography demonstrated impaired heart contractility, specifically, reduced fractional shortening. The analysis of lysates of mutant hearts revealed reduced activity of enzymes involved in glycolysis, oxidative phosphorylation, and ATP production. We developed in vitro co-cultures between CMs differentiated from H9C2 cells and mouse epicardial cells (MEC), silenced for Tfeb or control. Tfeb knockdown in MEC led to decreased glucose metabolism, as assessed by biochemical assays and Seahorse analysis, resulting in reduced lactate secretion. By measuring the activity of lactate dehydrogenase and C14 tracing, we showed that CMs consumed lactate produced by MEC utilizing it to fuel their metabolism. Knockdown of lactate transporters Mct1 and Mct4 in MEC led to the same inhibitory effect on the metabolism of CMs as Tfeb silencing, while the addition of lactate to the medium rescued CMs’ metabolic parameters. RNAseq and ChIPseq analyses in MEC pointed to TFEB targets, DUSP4 and DUSP6 phosphatases. They were upregulated in MEC siTfeb, leading to dephosphorylation of AKT and ERK kinases, possibly deregulating glucose metabolism. To investigate whether the metabolic deficiency in CMs could cause functional impairment, we co-cultured primary rat CMs with MEC siTfeb and analysed contraction dynamics in microscopy videos. CMs co-cultured with MEC siCtrl but not with MEC siTfeb performed better in terms of contraction amplitude and frequency than CMs cultured alone, and the addition of lactate to the medium rescued the deficit.
Conclusions: These data suggest a novel mechanism of lactate shuttle between epicardial cells and CMs with a potential role for TFEB in regulation of glucose metabolism in epicardial cells.