To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca2+ transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val49 → Gly mutant (2-fold), which is a superinhibitor of SR Ca2+-ATPase affinity for Ca2+, were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC50 level of SR Ca2+ uptake for Ca2+ (0.67 ± 0.09 μM) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca2+ signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired β-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca 2+ cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.