Abstract Body: Background
Congenital heart disease (CHD) affects ~1% of infants. Hypoplastic left heart syndrome (HLHS), a severe form of CHD in which the left ventricle is underdeveloped, is associated by a 15% incidence of heart failure by 6 years of age. Only 6% of HLHS patients have a genetic cause identified on exome sequencing, limiting the ability of patients to receive a diagnosis and potentially benefit from targeted treatments.

Hypothesis
Novel HLHS genes are differentially expressed in HLHS cardiac tissues, or in cells with differential abundance in HLHS hearts.

Goals
To use single nucleus RNA sequencing of HLHS patient cardiac tissues to identify candidate HLHS genes.

Methods
Single nucleus RNA sequencing (nucSeq) was performed on paired left and right ventricular tissues (LV, RV) from 9 participants with HLHS. Left ventricular cardiac tissues from children (4) and adults (12) without CHD were used for comparison. Filtering with CellBender and Solo were used to remove low-quality nuclei. Analysis was performed in R using the Seurat package.

Results
HLHS LV tissues had a higher proportion of cardiomyocytes than pediatric or adult control tissues, (78.7% vs 64.1%, p=0.005; 78.7 % vs 47.0%, p=1.8E-08, respectively), and a lower proportion of mural cells (3.1% vs 9.3%, p=3.9E-05; 3.1 % vs 23.5%, p=7.3E-15, respectively). By contrast, there were similar proportions of endothelial cells in the HLHS, pediatric and adult tissues (7.1%, 8.8% and 7.9%, respectively; p=0.56). Within HLHS tissues, the LV had a higher proportion of cardiomyocytes than the RV (78.7% vs 51.2%, p=4.8E-04) while the proportion of endothelial cells were similar in the LV and RV (7.3% and 9.1%, respectively; p=0.33). LV tissues from HLHS participants 2-18 years of age had fewer capillary endothelial cells than pediatric controls (31.2% vs 57.8%, p=0.001), and gene markers of HLHS endothelial cells included PBX1, which is required for Hox D-3 mediated angiogenesis, as well as ARHGAP26 and RORA, inhibitors of VEGF signaling.

Conclusion
HLHS cardiac tissues have a higher proportion of cardiomyocytes in the LV despite a smaller chamber size. Differences in gene expression that accompany the differences in cell proportion could identify novel HLHS genes as well as potential therapeutic targets.