The effects of inotropic agents are blunted in end-stage failing human myocardium. This has been related to a number of subcellular alterations including desensitization of the beta -adrenergic system. However, it is unknown whether alterations in SR Ca(2+)-handling contribute to blunted inotropic responsiveness of failing myocardium. We tested the hypothesis that the reduced effectiveness of Ca(2+)-dependent inotropic interventions results from the inability of the SR to sufficiently increase its Ca(2+)-content in failing human myocardium. Experiments were performed in ventricular muscle preparations from a total of four non-failing and 18 end-stage failing hearts. Isometric twitch force and SR Ca(2+)-content (using rapid cooling contractures; RCCs) were assessed under basal experimental conditions (1 Hz, 37 degrees C, [Ca(2+)](o) 2.5 mmol/l), and at increasing [Ca(2+)](o) (1.25-15 mmol/l), increasing concentrations of the beta -adrenergic agonist isoproterenol (ISO; 0.01-10 micromol/l), or the glycolytic substrate pyruvate (5-15 mmol/l). In addition, paired RCCs were evoked in a subset of experiments to investigate the relative contribution of SR Ca(2+)-uptake v Na(+)/Ca(2+)-exchange to cytosolic Ca(2+)-elimination. In non-failing human myocardium, Ca(2+), ISO, and pyruvate exerted significant positive inotropic effects (increase in twitch force by maximally 396%, 437%, and 82%, respectively). The inotropic effects were associated with increasing RCCs (by 147%, 193%, and 51%, respectively). In failing myocardium, the inotropic effects of Ca(2+) and ISO were significantly less pronounced (with maximal increases in twitch force by 226% and 138%, respectively), associated with blunted effects on RCCs (increase by 33% and 79%, respectively). In contrast, the inotropic effect of pyruvate was unchanged in failing myocardium (increase by 66%), while the corresponding RCCs increased only by 30%. We conclude that the inotropic effects of Ca(2+), ISO, and pyruvate are associated with a significant increase in SR Ca(2+)-content in non-failing human myocardium. In end-stage failing myocardium, the reduced inotropic response to Ca(2+) and ISO is associated with the inability of the SR to appropriately increase its Ca(2+)-content, possibly related to decreased SR Ca(2+)-ATPase and increased Na(+)/Ca(2+)-exchanger expression. In contrast, the maintained inotropic response to pyruvate despite reduced SR Ca(2+)-loading points to additional subcellular effects such as enhanced myofilament Ca(2+)-responsiveness.