Abstract Body (Do not enter title and authors here): INTRODUCTION: Hypoplastic left heart syndrome (HLHS) is a cardiovascular malformation that is a leading cause of cardiovascular related infant mortality. The cause of HLHS and the progression of heart failure (HF) in this population remains unclear. Additionally, there are no current medical therapies for the treatment of prevention of HF in HLHS. Our data suggests that the glycosphingolipid lactosylceramide (LacCer) is elevated in the heart of HLHS infants when compared to healthy age-matched controls. Based on this we hypothesize that elevated levels of LacCer contributes to cardiomyocyte dysfunction in HLHS.

METHODS: To test the effect of LacCer on myocytes directly, neonatal rat ventricular myocytes (NRVMs) were isolated and treated with liposomes containing 3µM LacCer (C18:0 and C24:0) or vehicle. Global cardiomyocyte proteomics changes were analyzed via mass spectrometry (n=11 for vehicle, n=13 for LacCer). The mitochondrial uptake of long chain fatty acids was examined using a stable-isotope based carnitine palmitoyltransferase (CPT) activity assay (n=4 for vehicle, n=3 for LacCer). Mitochondrial bioenergetics, including fatty acid oxidation +/- etomoxir (CPT inhibitor), were assessed using an Agilent Seahorse Bioanalyzer (n=5 for each treatment).

RESULTS: Our proteomics data identified 188 significantly differentially regulated proteins in response to LacCer treatment, with a significant number of proteins involved in glycolysis, oxidative phosphorylation, the tricarboxylic acid cycle, and muscle contraction (Fig A). Additionally, Lacer treatment was also sufficient to impair mitochondrial CPT activity, which is required for the uptake of long-chain fatty acids. Correspondingly, LacCer treatment significantly impaired fatty acid oxidation, illustrated by significantly decreased maximal mitochondrial respiration and energetic reserve capacity (Fig B -C).

CONCLUSIONS: Together, these data demonstrate the potential role of LacCer in the pathophysiological remodeling of HLHS cardiomyocyte metabolic function and substrate utilization. Moreover, it also indicates LacCer as a potential target of therapy to halt or delay heart failure progression in HLHS patients.