Multi-Omic and Functional Metabolic Analysis Identified Dysregulated Lipid and Mitochondrial Metabolism in the Pediatric Failing Single Ventricle Heart

Anastacia (Tasha) Garcia, PhD

University of Colorado Anschutz Medical Campus, Children’s Hospital Colorado, Assistant Professor

Abstract: Congenital heart disease with single ventricle physiology (SV) encompasses a group of severe abnormalities in cardiac structure where improper development of the fetal heart results in only one functional pumping chamber. From a molecular standpoint, it is not well understood how the SV myocardium adapts to the chronic altered hemodynamic conditions of SV physiology, and cardiac dysfunction and ultimately heart failure (HF) are a common complication in the SV population. The purpose of this study was to characterize the transcriptomic, metabolomic, and lipidomic profiles in SV myocardium from both failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we conducted high-resolution respirometry (Oroboros Oxygraph system) to assess myocardial mitochondrial respiratory function in each of these populations. Lastly, we measured carnitine palmitoyltransferase (CPT) activity, a mitochondrial enzyme that allows the uptake of long-chain fatty acids for their subsequent oxidation. Multi-omics pathway analysis demonstrated multiple pathways that are similarly dysregulated in SVNF and SVHF, while pathways involved in mitochondrial and lipid metabolism were significantly dysregulated specifically in the SVHF population. Moreover, functional mitochondrial oxygen flux and CPT activity were significantly decreased in SVHF relative to BVNF controls. Therefore, these results provide new insights into SVHF by identifying unique gene, metabolite and lipid changes, including those related to mitochondrial metabolic function, which may serve as potential therapeutic targets for the treatment or prevention of HF in the SV population. These data are corroborated by the significant decrease seen in functional assessments of mitochondrial oxygen flux and CPT activity.  Based on these findings, we have also recently begun to explore circulating peripheral mononuclear cells a potential biomarkers of myocardial mitochondrial function in the SV population. We propose that peripheral blood mononuclear cells (PBMCs) isolated from children with SV could serve as surrogate circulating molecular biomarkers for myocardial mitochondrial alterations and provide predictive prognostic value to this unique patient population. Our preliminary data demonstrates PBMCs from SVHF patients display significant changes in mitochondrial oxygen flux manifesting in decreased respiratory capacity, ATP production, and coupling efficiency, and increased reactive oxygen species relative to controls. Therefore, we hypothesize that myocardial mitochondrial metabolism represents a biomarker of disease progression in SV patients, and that patient-derived PBMCs serve as a proxy for myocardial cellular respiration.

Predicting Outcomes in Single Ventricle Heart Disease Through Circulating miRNAs

Stephanie Nakano, MD

Assistant Professor, Pediatric Cardiology; Children’s Hospital Colorado

Abstract: Single ventricle congenital heart disease (SV) is universally fatal without intervention and is the leading cause of cardiovascular death in infancy. Currently, the most common management strategy for SV is a series of palliative surgeries. While these surgeries represent significant advances in medical care, mortality remains high, with 30% of patients dying in the first year of life. Importantly, the ability to predict which infants will do well with surgical palliation is lacking.

Circulating miRNAs are increasingly recognized as effective biomarkers in a broad range of medical disciplines, aiding in both diagnosis and prognosis. Our hypothesis was that circulating miRNA profiles prior to surgical palliation may correlate with SV survival at one year and assist with risk-stratification in this population. Serum samples from subjects with SV (of right ventricular morphology) were obtained at the following time points: Pre-Norwood (n=71), Pre-Glenn (n=46) and Pre-Fontan (n=25). Outcomes were classified as alive versus death or heart transplant listing by one year of age. Serum was subject to three freeze/heat cycles to maximize miRNA release, then miRNAs were reverse transcribed using a pool of primers specific for each miRNA. Real-time PCR was performed in 384-well plates containing sequence-specific primers and TaqMan probes in the ABI7900HT. Analysis of Pre-Norwood samples demonstrated downregulation of miR-15b, -192, and 193b in patients who died or required heart transplant listing (n=22) compared to those who were alive (n=49) at one year of age. Additionally, miR-let-7b, -26a, and -454 were differentially expressed between the Pre-Norwood, Pre-Glenn, and Pre-Fontan groups, suggesting an association with surgical stage and/or age. Circulating miRNA profiles are distinct in pediatric SV patients at each surgical stage, and demonstrate promise as prognostic biomarkers of 1-year outcome in the Pre-Norwood SV population.