Abstract Body: Although heart failure (HF) is extensively studied, the mechanisms underlying cardiac metabolism in HF patients remain incomplete. Creatine is essential for maintaining proper cardiac function, as the creatine-phosphocreatine buffer allows for sufficient energy storage and heart contraction. In the endogenous pathway, L-arginine:glycine amidinotransferase (AGAT) catalyzes the formation of Guanidinoacetate (GAA) from arginine and glycine. Guanidinoacetate N-methyltransferase (GAMT) then converts GAA to Creatine. From previous studies within the lab, reduced creatine levels and increased GAA levels are observed in HF patients. Transporters Crt1 and Crt2 facilitate the translocation of creatine across the plasma membrane, and decreased transporter expression may cause reduced creatine levels in heart failure. Previously we have shown that Methionine adenosyltransferase 2A cardiac knockout (Mat2a cKO) mice exhibit heart failure with decreased creatine and an accumulation of GAA. In this study, we aimed to rescue heart failure symptoms in Mat2a cKO mice by increasing creatine in cardiomyocytes via overexpression of Crt1 or Crt2. We designed a cardiac specific myofibril-AAV (myo-AAV) to overexpress Crt1 or Crt2, with western blots and qPCR confirming the myo-AAV’s specificity and potency. Surprisingly, compared to the control, Crt1 OE did not alter survival or creatine levels. However, Crt2 overexpression mice exhibit increased survival and ejection fraction. Interestingly, metabolomics results showed decreased creatine levels in both WT Crt2 OE and KO Crt2 OE mice, indicating a creatine independent survival mechanism. Additionally, given Crt2 has a GAA efflux role in the kidney and in human heart failure samples GAA levels are increased, we hypothesized the cardioprotective mechanism of Crt2 overexpression is due to a release of GAA buildup. Under fed conditions at night when the heart is active, we indeed observed decreased GAA levels in Mat2a cKO Crt2 OE mice. Overall, these findings show cardiac specific Crt2 overexpression can mitigate heart failure, and our future studies aim to elucidate the mechanism behind this cardioprotection. Our current results show Crt2 OE does not alter GAA under fasted conditions, yet in fed conditions decreases GAA levels in Mat2a cKO mice and increases GAA in WT mice. Therefore, we believe Crt2’s role is altered based on creatine and overall nutrient availability, and we plan to test this using heavy isotope tracing.