Abstract 4143368: Glycosphingolipid-Mediated Peripheral Immune Cell Inflammatory Activation Impairs Cardiomyocyte Function in Hypoplastic Left Heart Syndrome

Introduction: Hypoplastic Left Heart Syndrome (HLHS) is the leading indication for heart transplant and the most common cause of cardiovascular death in infants, yet our understanding of the immunologic alterations in HLHS is inadequate. In this study we aimed to (1) assess whether peripheral blood immune cells (PBMCs) from HLHS patients display a pro-inflammatory phenotype, (2) identify specific factors associated with inflammation in HLHS, and (3) investigate the interaction between HLHS PBMCs and cardiomyocytes. Methods: PBMCs were isolated from whole blood by density gradient centrifugation and gene expression changes were assessed using next-generation RNA-sequencing. Mass-spectrometry was used to quantify PBMC lipid species. Healthy PBMCs were cultured with liposomes containing 10μM lactosylceramide (LacCer) and conditioned media (CM) was collected. Cytokine arrays were used to quantify LacCer-mediated inflammatory changes. Cardiomyocytes were treated with CM and mitochondrial bioenergetics were assessed using the Seahorse Bioanalyzer. Results: PBMCs from HLHS patients display significant enrichment of pathways associated with immune cell activation, and cytokine signaling that are suggestive of a pro-inflammatory phenotype. Glycosphingolipids (GSLs) are one of the most abundant glycolipids in humans and play an important role in immune cell signaling and function. We identified that the GSL, LacCer, is significantly enriched in HLHS PBMCs and that exogenous LacCer treatment of healthy PBMCs induces secretion of proinflammatory mediators (e.g., VCAM1, IL-8, CD14). Additionally, in vitro assays determined that CM from healthy PBMCs treated with LacCer is sufficient to promote mitochondrial dysfunction in primary cardiomyocytes. Conclusions: These data suggest that immune cells in HLHS display a pro-inflammatory phenotype that is mediated, at least in part, by increased LacCer. Further, these data provide insight into LacCer’s role in paracrine-mediated crosstalk between immune cells and cardiomyocytes in the setting of HLHS. This suggests a novel role for LacCer-rich PBMCs to potentiate progressive cardiac dysfunction in HLHS and provide an avenue for therapeutic intervention in this vulnerable population.