Adenoviral gene transfer of SERCA2a to hypertrophic heart in vivo is consistently reported to lead to enhanced myocardial contractility. It is unknown if the faster skeletal muscle isoform, SERCA1, expressed in whole heart in early failure leads to similar improvements, and whether metabolic requirements are maintained during an adrenergic challenge. In this study, Ad.cmv.SERCA1 was delivered in vivo to aortic-banded and sham operated Sprague Dawley rat hearts. Total SERCA content increased 34%. At 48-72 hrs post transfer, echocardiograms were acquired, hearts were excised, retrograded perfused, and hemodynamics were measured parallel to NMR measures of PCr/ATP and energy substrate selection at basal and high workloads (isoproterenol). In the Langendorff mode, RPP was enhanced 27% with SERCA1 in hypertrophic hearts and 10% in shams. The adrenergic response to isoproterenol was significantly potentiated in both groups with SERCA1. ³¹P NMR analysis of PCr/ATP revealed the ratio remained low in the hypertrophic group with SERCA1 overexpression, and was not further compromised with adrenergic challenge. ¹³C NMR analysis revealed fat and carbohydrate oxidation were unaffected at basal with SERCA1 expression, however there was a shift from fats to carbohydrates at higher workloads with SERCA1 in both groups. Transport of NADH reducing equivalents into the mitochondrial via the KG-malate transporter was not affected by either SERCA1 overexpression or adrenergic challenge in both groups. Echocardiograms revealed an important distinction between in vivo versus ex vivo data. In contrast to earlier SERCA2a studies, the echocardiogram data revealed SERCA1 expression compromised function (fractional shortening) in the hypertrophic group. Shams were unaffected. While our ex vivo findings support much of the earlier cardiomyocyte and transgenic data, the in vivo data challenge previous reports of improved cardiac function in heart failure models after SERCA intervention.