Insights into regulatory mechanisms of dilated cardiomyopathy in children by single RNA nuclei sequencing

More than half of childhood cardiomyopathies are diagnosed as dilated cardiomyopathy with a 5-year rate of death or transplantation of ≈30% to 50%. The underlying etiology of DCM includes myocarditis and sarcomeric mutations or is unknown. The limited knowledge regarding the cellular contributions and the mechanisms that lead to cardiac dysfunction in pediatric HF are challenging the development of effective therapeutic strategies in children, in whom large cohort studies are particularly difficult to perform.

In the last years new techniques have been developed with the aim to personalize the treatment of these children. One of these promising technologies is single cell/single-nuclei sequencing providing high throughput data of individual cells that can be used to generate detailed knowledge of cellular signatures and functional states. Specifically, droplet sequencing of isolated nuclei instead of cells was used to elucidate transcriptomes of cardiomyocytes. Nuclei sequencing covers all types of heart cells including fibroblasts, endothelial cells, and inflammatory cells allowing insights into the pathophysiological adaptive and maladaptive responses of individual cell types in the heart, which may otherwise be overlooked in bulk RNA or proteomic approaches.

In order to obtain new insights into the molecular processes of DCM in pediatric patients, Nicin et al. analyzed the nuclear transcriptomes of explanted hearts of pediatric patients with DCM by using single nuclei RNA sequencing (snRNA-seq). In particular, ventricular tissue of 6 children with an age of 0.5, 0.75, 5, 6, 12, and 13 years who required heart transplantation for ultimate palliation of end-stage DCM was evaluated. Following snRNA-seq, data of all cells were pooled, and unsupervised clustering was performed with a total of 18 211 nuclei to annotate the respective cell populations. Here, 14 distinct clusters were found with 6 major cell types, including cardiomyocytes (3 clusters), fibroblasts (1 cluster), endothelial cells (3 clusters), leukocytes (1 cluster), pericytes (1 cluster), smooth muscle cells (1 cluster), and inflammatory cells (1 cluster).

Interestingly, an age-dependent increase in the relative representation of cells in the fibroblast clusters was found, whereas the percentage of number of nuclei in the cardiomyocyte cluster dropped with increasing age of the pediatric patients’ DCM samples. These finding were confirmed by histological analysis and by cardiac magnetic resonance imaging revealing an age-related increase in cardiac fibrosis. Fibroblasts of patients with DCM >6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms, and signatures of fibroblast activation. Moreover, a part of the cardiomyocytes revealed a high proregenerative profile in infant patients with DCM but was absent in children >6 years of age. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes, and alterations in ß-adrenergic signaling genes.

From the results it can be concluded that infants with a predominantly regenerative cardiomyocyte profile may preferentially receive treatment strategies to support cardiac regeneration, whereas patients with a pattern associable with cardiac fibrosis will benefit from an early antifibrotic therapy to avoid diastolic dysfunction. Despite impracticality of large cohort studies in pediatric DCM, tailored pharmacological treatment is possibly nevertheless realistic, for example, based on the expression of ß-adrenergic signaling genes.