ADENOVIRAL GENE-TRANSFER OF PHOSPHOLAMBAN IN ISOLATED RAT CARDIOMYOCYTES - RESCUE EFFECTS BY CONCOMITANT GENE-TRANSFER OF SARCOPLASMIC-RETICULUM CA2-ATPASE()

Phospholamban forms an integral part of the cardiac sarcoplasmic retic ulum (SR) and regulates the activity of SR Ca2+-ATPase (SERCA2a). A nu mber of studies have suggested a decrease in SERCA2a relative to phosp holamban in heart failure. To test the hypothesis that changes in the relative abundance of phospholamban to SERCA2a could account for the p athophysiological abnormalities in Ca2+ handling observed in failing m yocardium, we created a recombinant adenovirus designed to overexpress phospholamban (Ad.RSV.PL). In neonatal rat cardiomyocytes, Ad.RSV.PL increased the expression of phospholamban in a concentration-dependent fashion, reaching 280+/-43% at a multiplicity of infection (MOI) of 1 0.0 plaque forming units (pfu)/cell at 48 hours. The relationship betw een Ca2+-ATPase activity and [Ca2+] was shifted rightward in membrane preparations from cardiomyocytes infected with Ad.RSV.PL. Intracellula r Ca2+ transients measured in the neonatal cells infected with Ad.RSV. PL (MOI, 10 pfu/cell) were characterized by (1) a significant prolonga tion of the relaxation phase (344+/-26 versus 710+/-56 milliseconds, P <.01), (2) a decrease in peak [Ca2+](i) (967+/-43 versus 630+/-33 nmol /L, P<.01), and (3) an elevation in resting [Ca2+](i) (143+/-14 versus 213+/-17 nmol/L, P<.05). Similarly, the time course of shortening was prolonged in myocytes infected with Ad.RSV.PL. These effects were par tially restored by simultaneous transduction with an adenovirus carryi ng SERCA2a. Cardiomyocytes infected with Ad.RSV.PL had an abnormal fre quency response: a decrease in peak [Ca2+](i) and an increase in resti ng [Ca2+](i) with increasing frequency. These findings indicate that a denovirus-mediated gene transfer of phospholamban modifies intracellul ar Ca2+ handling and the frequency response in cardiomyocytes. Our res ults suggest that alterations in the ratio of phospholamban to SERCA2a could account for the abnormalities in Ca2+ handling observed in hear t failure and that overexpression of SERCA2a can largely correct these abnormalities.