Single nuclei sequencing reveals novel insights into cardiac cell signatures in human pediatric dilated cardiopathy

Abstract

The mechanism underlying dilated cardiomyopathy (DCM) in children without a known genetic disorder are unclear. In contrast to adult DCM patients, there is an unmet need for therapeutic options that improve survival in pediatric DCM. Therefore, we performed single nuclei RNA sequencing (snRNA-seq) from heart tissue obtained from children undergoing heart transplantation due to severe heart failure. We processed heart tissue from 6 children with DCM (EF: 18.67±2.11%) of an age of 0.5, 0.75, 5, 6, 12 and 13 years (y). After snRNA-seq, unsupervised clustering was performed identifying 8 major cell types, including cardiomyocytes (CM), fibroblasts (FB), endothelial cells, leukocytes, pericytes, smooth muscle cells, neuronal-like cells and an endothelial-fibroblast-like cluster. Relative numbers of FB clusters correlated with increasing age of the children which was clinically validated by measuring late enhancement (LE) with cardiac magnetic resonance imaging in 68 pediatric DCM patients. The mean age of patients with LE was 5.86±0.53y vs. 2.36±0.53y in patients without LE (p<0.05). Further analysis of unique highly expressed genes (DEGs) between the 3 age groups identified a profound alteration of gene expression in FB clusters. FBs of explants of <1y old patients showed high expression of anti-fibrotic, development and remodeling associated genes. In contrast, FBs of 12-13y old children highly expressed pro-fibrotic and FB activation associated genes as transforming growth factor beta binding protein (6.63 fold), cytochrome P450 1B1 (3.79 fold), and periostin (7.67 fold) (all p<0.05). Moreover, we observed a switch in collagen expression patterns and in thrombospondin isoforms (from THBS1 to THBS4). Furthermore, our analysis revealed most profound transcriptional changes in CMs. We identified a cluster of CMs with a pro-regenerative profile in <1y old patients, which could not be detected during adolescence. This CM cluster showed high expression of genes associated with proliferation (e.g. cyclin D2), glycolytic metabolism and anti-oxidant markers. Increased cyclin D2 was confirmed by immunostaining (1.43 fold higher in <1y vs. 12-13y). Since all of these gene expression patterns might be affected by the underlying disease of the pediatric heart recipients, we explored their expression in FBs and CMs of postnatal vs. adult mice. Importantly, we could recapitulate the vast majority of the findings from humans in the mice experiments. Together, these data demonstrate an age-dependent decrease in CM numbers concomitant with increased FBs in pediatric DCM. FBs of <1y old pediatric patients revealed a distinct collagen expression profile and showed lower levels of pro-fibrotic genes. CMs of <1y old donors where characterized with a regeneration enabling gene expression profile, which include pro-proliferative genes. The expression patterns of the CMs indicates, that regeneration might also occur in humans during the firs year of life.