Cellular Composition of Right Atrial and Right Ventricular Samples from Pediatric Patients with Congenital Heart Disease

Background: The molecular nature of congenital heart disease (CHD) is still not well understood. Single nuclei RNA sequencing (SN-RNAseq) is a state-of-the-art technique to analyze the molecular complexity of tissues. We sought to compare the cellular composition of right atrial and right ventricular cardiac biopsies from pediatric CHD patients at the single cell level.

Method: Cardiac biopsies from patients with transposition of the great arteries (TGA), hypoplastic left heart syndrome (HLHS), ventricular septal defects (VSD), or tetralogy of Fallot (TOF) were analyzed. Nine right atrial (TGA n = 2; HLHS n = 3; VSD n = 3; TOF n = 1) and six right ventricular samples (HLHS n = 3; TOF n = 3) were included.

Nuclei were isolated from snap frozen cardiac tissue by homogenization followed by density gradient centrifugation. SN were sorted by flow cytometry and further processed by a droplet-based microfluidic single cell controller. After sequencing, SN transcriptomes were aligned to the human genome. Cell clusters were identified by principal component analysis and unsupervised clustering followed by canonical marker analysis.

Results: More than 135,000 nuclei were analyzed and ~1,400 genes were detected per nucleus (median). All major subtypes of cardiac cells were detected in pediatric CHD samples: cardiomyocytes (CM), cardiac fibroblasts (CF), endothelial cells (EC), smooth muscle cells/pericytes (SMC/PC), macrophages (MP), and neuronal cells (NC). The main cell type in atrial and ventricular samples was CM (30–50%), followed by CF (15–30%) in the atria. In the ventricles, CF and EC were equally abundant (5–20%). Interestingly, atrial samples revealed age-dependent differences. Neonatal atrial biopsies (TGA, HLHS; 5–12 days) showed significantly more CM than infant atrial biopsies (VSD, TOF; 4–11 months), while EC and PC were higher abundant in infant atrial biopsies (p < 0.05). Neonatal (HLHS) and infant (TOF) ventricular samples had a similar cell distribution. However, ventricular samples from HLHS and TOF might be affected by the respective disease and be different from “normal” ventricles. In one HLHS ventricular sample, a specific cell cluster was detected showing markers of fibrosis that might be related to a documented endocardial fibroelastosis for this patient.

Conclusion: SN-RNAseq allows deep insights into the molecular nature of different locations of the pediatric human heart, including variations based on patient age and pathophysiological conditions.

No conflict of interest has been declared by the author(s).

Publication History

Article published online:
28 January 2023