Introduction: The molecular mechanisms underlying heart failure (HF) in the single ventricle (SV) population are poorly understood, and further investigation is limited by the lack of a model system to study SV. We previously demonstrated that treatment of neonatal rat ventricular myocytes (NRVMs) with SV patient serum induces pathologic gene expression changes. The purpose of this study was to characterize changes in the transcriptome of SV myocardium and compare them to SV serum-treated NRVMs using next generation sequencing.
Methods: RNAseq (2x150 total RNAseq, Illumina HiSEQ 4000) was performed on age-matched explanted RV myocardium from non-failing (NF) controls (n=4) and SV HF (n=9 systolic HF) patients. Additionally, RNAseq (1X150 directional mRNAseq) was performed on NRVMs treated with NF (n=3) and SV HF (n=3) sera for 72 hours. Aligned reads were normalized and annotated using the edgeR pipeline. Significant changes in gene expression were calculated using a non-parametric t-test (p<0.05). Differentially expressed genes were clustered hierarchically using R. Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, and Gene Ontology categorization using PANTHER were performed to investigate biological processes and toxicity functions associated with differentially regulated genes.
Results: RNAseq identified more than 4,000 differentially expressed transcripts between NF and SV RV, and unsupervised hierarchical clustering (HC) separated NF and SV samples. In NRVMs treated with NF vs SV serum there are 1,136 transcripts differentially expressed and HC separated NF and SV serum-treated cells. Transcriptome pathway analysis demonstrated multiple pathways that are similarly dysregulated in SV RV and SV-serum treated NRVMs, including those related to cardiac failure, necrosis/cell death and arrhythmia.
Conclusions: Compared to control, SV RV and SV serum-treated NRVMs are characterized by: (1) a unique gene expression pattern and (2) similar dysregulation of cardiac-specific pathways. While confirmation of these findings is necessary, SV serum-treated NRVMs have potential as a novel model of SV, and further exploration of SV-specific transcriptional changes may provide insight into novel mechanisms of HF in this population.