SUMMARY This proposal explores the hypothesis that very long chain ceramides serve as a causal mediator in the structural and functional changes which underlie diastolic heart failure (HF). Moreover, this suggests a rare therapeutic opportunity for diseases such as heart failure with preserved ejection fraction (HFpEF), which have few evidence- based therapies. The idea is predicated upon data presented herein showing that the genetic deletion of a ceramide synthesis enzyme, dihydroceramide desaturase 1 (Des1), prevents hypertrophy and impairments in diastolic function in 2 distinct mouse models of diastolic HF. The theory is further supported by untargeted lipidomics from HFpEF patients, which demonstrate ceramides as the most upregulated lipids in serum. Moreover, biopsies obtained from hearts with diastolic failure show elevated ceramides compared to control. Cardiac-specific ablation of acid ceramidase, is sufficient to drive ceramide accumulation and diastolic dysfunction without overt changes in systolic function. Administration of a pharmacological inhibitor of ceramide biosynthesis to rodents preserves diastolic function and prevents fibrosis and hypertrophy, suggesting therapeutic potential if safe, effective ceramide synthesis inhibitors are identified. We will determine if ceramides are necessary and sufficient mediators of diastolic heart failure that can be targeted for therapeutic intervention. We will evaluate our hypotheses through the following Aims: • First, we will determine the effects of genetic induction or reduction of ceramides selectively in cardiomyocytes in mouse models of diastolic HF. • Second, we will determine the efficacy of Des1 antagonism or ablation as a therapeutic approach for the treatment or prevention of diastolic HF. • Third, we will elucidate the mechanisms linking ceramides to cardiac fibrosis and mitochondrial dysfunction using cells, mice, and human tissues. Findings obtained from these studies could uncover new nutrient-sensing regulatory mechanisms that modulate mitochondrial function, cardiomyocyte survival, fibrosis, and hypertrophy. Moreover, the translational component of this work could propel the development of promising therapeutics for preventing or treating diastolic HF.