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and Y.H. sphingosine-1-phosphate (S1P). We hypothesized that Acid Ceramidase (AC) overexpression Mouse monoclonal to DKK3 would counteract the negative effects of elevated ceramide and promote cell survival, therefore providing cardioprotection after MI. Methods: We performed transcriptomic, sphingolipid and protein analyses to evaluate sphingolipid rate of metabolism and signaling post MI. We investigated the effect of altering ceramide rate of metabolism through a loss (chemical inhibitors) or gain (altered mRNA (modRNA)) of AC function post hypoxia or MI. Results: We found that several genes involved in ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1 and C24) levels had significantly improved 24 hours after MI. AC inhibition post hypoxia or MI resulted in reduced AC activity and improved cell death; by contrast, enhancing AC activity via AC modRNA treatment improved cell survival post hypoxia or MI. AC modRNA-treated mice experienced significantly better heart function, longer survival and smaller scar size than control mice 28 days post MI. We attributed Naproxen the improvement in heart function post MI following AC modRNA delivery to decreased ceramide levels, lower cell death rates and changes in the composition of the immune cell populace in the LV manifested by lowered large quantity of pro-inflammatory detrimental neutrophils. Conclusions: Our findings suggest that transiently altering sphingolipid rate of metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post MI, therefore highlighting the restorative potential of AC modRNA in ischemic heart disease. ceramide synthesis in rodents can improve heart function post MI.8, 9 Ceramidases hydrolyze ceramide to generate free fatty acids and sphingosine, which is then phosphorylated by sphingosine kinase (Sphk) to produce sphingosine 1-phosphate (S1P), a pro-survival lipid mediator with both intra- and extracellular functions.11, 12 More specifically, acid ceramidase (AC) is encoded from the gene and catalyzes ceramide hydrolysis to free fatty acids and sphingosine, which is then phosphorylated by Sphk (1 and 2) to generate S1P.13 gene mutations lead to ceramidase deficiency and cause Farber lipogranulomatosis, a lysosomal storage disease.13 AC is essential for embryogenesis, and activity in Naproxen acidic conditions,14 and belongs to the N-terminal nucleophile hydrolase family. The autoproteolytic cleavage of AC produces two active subunits: the subunit having a Naproxen molecular excess weight of ~14 kDa and the subunit having a molecular excess weight of ~43 kDa.15 The autoproteolytic cleavage of the precursor triggers a conformational change that reveals the active site and activates the enzyme toward sphingolipid metabolism.16 It has been suggested that interfering with the signal transduction pathways mediated by sphingolipids could prevent cell death post MI. Recent studies have suggested that S1P could be used like a restorative target in individuals with heart failure17 and MI,18 to prolong cardiac cell survival and consequently improve heart function. While S1P lyase inhibition causes improved cardiac S1P levels and bradycardia in rats,19 S1P receptor agonist, FTY720, boosts myocardial salvage and enhances heart function inside a porcine model of ischemia/reperfusion injury.20 Inhibiting ceramide synthesis has also been suggested as a strategy for reducing the pro-apoptotic effect of ceramide post MI.9 Indeed, inhibiting acid sphingomyelinase, Naproxen which hydrolyzes spingomyelin to generate ceramide, limits ceramide accumulation in post-ischemic hearts.10 Moreover, adiponectin seems to exert its anti-apoptotic effect on CMs through adiponectin receptor-mediated ceramidase activity.21 We investigated using AC and/or Sphk enzymes to inhibit cell death and initiate cell survival through ceramide hydrolysis and S1P production. Delivering AC or Sphk proteins is definitely safe and controlled, but their effects are limited by these proteins half-lives. Conversely, using DNA or viruses (DNA or RNA viruses) is not controlled and may elicit an immune response that could compromise genome integrity. We consequently delivered AC and Sphk via synthetic altered mRNA (modRNA), a nucleic acid delivery tool, to transiently alter sphingolipid rate of metabolism. Our group.