Alterations in myocardial triacylglycerol content have been associated with poor left ventricular function, suggesting that enzymes involved in myocardial triacylglycerol metabolism play an important role in regulating contractile function. Myocardial triacylglycerol catabolism is mediated by adipose triglyceride lipase (ATGL), which is rate limiting for triacylglycerol hydrolysis. To address the influence of triacylglycerol hydrolysis on myocardial energy metabolism and function, we utilized mice with cardiomyocyte-specific ATGL overexpression (MHC-ATGL). Biochemical examination of MHC-ATGL hearts revealed chronically reduced myocardial triacylglycerol content but unchanged levels of long-chain acyl coenzyme A esters, ceramides, and diacylglycerols. Surprisingly, fatty acid oxidation rates were decreased in ex vivo perfused working hearts from MHC-ATGL mice, which was compensated by increased rates of glucose oxidation. Interestingly, reduced myocardial triacylglycerol content was associated with moderately enhanced in vivo systolic function in MHC-ATGL mice and increased isoproterenol-induced cell shortening of isolated primary cardiomyocytes. Most importantly, MHC-ATGL mice were protected from pressure overloadinduced systolic dysfunction and detrimental structural remodeling following transverse aortic constriction. Overall, this study shows that ATGL overexpression is sufficient to alter myocardial energy metabolism and improve cardiac function. © 2012, American Society for Microbiology.