PROJECT SUMMARY: With advancements in operative techniques and perioperative management, there is an increasing number of patients with Hypoplastic Left Heart Syndrome (HLHS) that are surviving into childhood and beyond. Due to the chronic pressure and volume load placed on the single systemic ventricle, these patients remain at constant risk for the development and progression of cardiac failure. Unfortunately, very little is known about how the failing HLHS heart differs from the failing adult biventricular heart, but the extrapolation of proven adult heart failure medications to the HLHS population has been unsuccessful, suggesting focused study is necessary to better understand the mechanisms underlying maladaptive remodeling and eventual heart failure in HLHS. Our preliminary data suggest glycosphingolipids (GSLs) play a role in modulating cardiomyocyte and immune cell function in HLHS, via both autocrine and paracrine signaling. Specifically, we show accumulation of LacCer in the myocardium of HLHS patients. To test the effect of LacCer on cardiac myocytes directly, we treated primary cardiomyocytes with liposomes carrying LacCer and determined that LacCer is sufficient to decrease mitochondrial OXPHOS, increase oxidative stress, and upregulate pro-inflammatory gene expression. Similarly, in vivo administration of LacCer induced right ventricular hypertrophy and decreased cardiac mitochondrial respiratory capacity. Further, we determined that PBMCs from HLHS patients display increased levels of LacCer, upregulated pro-inflammatory gene expression, and decreased mitochondrial OXPHOS. Similarly, treatment of healthy PBMCs with LacCer promotes decreased mitochondrial OXPHOS. Additionally, conditioned media from healthy PBMCs treated with LacCer is sufficient to promote mitochondrial dysfunction in primary cardiomyocytes, suggesting LacCer-induced crosstalk via paracrine mediated effects. Therefore, our central hypothesis is that aberrant glycosphingolipids drive maladaptive cardiac and immune cell responses and predispose HLHS patients to life-limiting complications including cardiac dysfunction. Aim 1 will investigate whether LacCer accumulation elicits a proinflammatory and metabolically dysfunctional phenotype in peripheral blood immune cells (PBMCs), and Aim 2 will elucidate the mechanisms by which GSL accumulation alters cardiac myocyte function. Our long-term goal is to ensure that HLHS patients benefit from rigorous pre-clinical studies in order to provide the necessary framework to identify novel therapies, influence clinical care, and improve outcomes. Successful completion of these aims will establish key mechanisms and modulators of heart failure progression in HLHS and will elucidate a potential therapeutic pathway to mitigate progressive cardiac dysfunction in this vulnerable population.