In failing hearts, deficiency in sarcoplasmic reticulum (SR) calcium (Ca²⁺)-ATPase (SERCA2a) results in abnormal Ca²⁺ handling and diminished contraction. In addition, a decrease in phosphorylation of phospholamban (PLB) has been reported. Gene transfer of antisense PLB (asPLB) can improve the contractile function in failing human myocardium. Gene transfer of SERCA2a improves survival and the energy potential in failing hearts. The aim of present study was to evaluate whether enhancement of SERCA2a function prevents acute Ca²⁺ overload-induced left ventricle (LV) dysfunction in rat hearts. We ablated PLB using adenoviral gene transfer of asPLB by a new and less invasive gene delivery method, which involved a percutaneous technique. Experiments were performed on 13 excised cross-circulated rat hearts; five rats underwent sham operations, 4 rats underwent gene transfer of the reporter gene, -galactosidase (Ad. gal) and 4 rats underwent gene transfer of the asPLB (Ad.asPLB). After clearance of high Ca²⁺ infused into the coronary, there was an LV contractile dysfunction associated with decreased myocardial O₂ consumption per beat (VO₂) intercept (=decreased VO₂ for Ca²⁺ handling in excitation-contraction coupling) of VO₂ - systolic pressure-volume area (PVA: a total mechanical energy per beat) linear relation in the hearts underwent sham operation and infected with Ad. gal. The hearts infected with Ad.asPLB, were rescued from LV contractile dysfunction associated with unchanged VO₂ intercept of VO₂ -PVA linear relation. We conclude that SERCA2a function enhanced by adenoviral gene transfer of asPLB prevents Ca²⁺ overload-induced LV contractile dysfunction in terms of mechanical work and especially energetics.