We used a catheter-based technique to achieve generalized cardiac gene transfer in vivo and to alter cardiac function by overexpressing phospholamban (PL) which regulates the activity of the sarcoplasmic reticulum Ca 2؉ ATPase (SERCA2a). By using this approach, rat hearts were transduced in vivo with 5 ؋ 10 9 pfu of recombinant adenoviral vectors carrying cDNA for either PL, ␤-galactosidase (␤-gal), or modified green f luorescent protein (EGFP). Western blot analysis of ventricles obtained from rats transduced by Ad.PL showed a 2.8-fold increase in PL compared with hearts transduced by Ad.␤gal. Two days after infection, rat hearts transduced with Ad.PL had lower peak left ventricular pressure (58.3 ؎ 12.9 mmHg, n ‫؍‬ 8) compared with uninfected hearts (92.5 ؎ 3.5 mmHg, n ‫؍‬ 6) or hearts infected with Ad.␤gal (92.6 ؎ 5.9 mmHg, n ‫؍‬ 6). Both peak rate of pressure rise and pressure fall (؉3, 210 ؎ 298 mmHg͞s, ؊2, 117 ؎ 178 mmHg͞s, n ‫؍‬ 8) were decreased in hearts overexpressing PL compared with uninfected hearts (؉5, 225 ؎ 136 mmHg͞s, ؊3, 805 ؎ 97 mmHg͞s, n ‫؍‬ 6) or hearts infected with Ad.␤gal (؉5, 108 ؎ 167 mmHg͞s, ؊3, 765 ؎ 121 mmHg͞s, n ‫؍‬ 6). The time constant of left ventricular relaxation increased significantly in hearts overexpressing PL (33.4 ؎ 3.2 ms, n ‫؍‬ 8) compared with uninfected hearts (18.5 ؎ 1.0 ms, n ‫؍‬ 6) or hearts infected with Ad.␤gal (20.8 ؎ 2.1 ms, n ‫؍‬ 6). These differences in ventricular function were maintained 7 days after infection. These studies open the prospect of using somatic gene transfer to modulate overall cardiac function in vivo for either experimental or therapeutic applications.The regulation of intracellular calcium is intimately related to the systolic and diastolic function of cardiac cells (1, 2). The sarcoplasmic reticulum (SR), which releases calcium during systole and takes it up during diastole, plays an integral part in controlling the synchronized movement of calcium in myocardial cells. The SR Ca 2ϩ ATPase (SERCA2a) pump regulates the uptake of Ca 2ϩ into the SR during diastole. The function of the SERCA2a pump is regulated in turn by phospholamban (PL) (3). In its unphosphorylated form, PL inhibits the SERCA2a pump whereas in its phosphorylated form, this inhibition is relieved. A decrease in SERCA2a activity has been identified in a number of animal models of heart failure and in human heart failure and an increase in the relative ratio of PL to SERCA2a appears to be an important characteristic of both experimental and human heart failure (3, 4). We have previously modeled such alteration in the PL͞SERCA2a ratio by using adenoviral gene transfer to cardiocytes in vitro. Adenoviral overexpression of PL in vitro recapitulates many of the physiological abnormalities seen in heart failure, including prolonged relaxation and decreased contractile function. In contrast, overexpression of SERCA2a enhances relaxation and contractility of normal cardiomyocytes and rescues myocytes overexpressing PL from their abnormal phenotype (5, 6). Cardiac gene transf...

Background-Myocardial cells from failing human hearts are characterized by abnormal calcium handling, a negative force-frequency relationship, and decreased sarcoplasmic reticulum Ca 2ϩ ATPase (SERCA2a) activity. In this study, we tested whether contractile function can be improved by decreasing the inhibitory effects of phospholamban on SERCA2a with adenoviral gene transfer of antisense phospholamban (asPL). Methods and Results-Myocardial cells isolated from 9 patients with end-stage heart failure and 18 donor nonfailing hearts were infected with adenoviruses encoding for either the antisense of phospholamban (Ad.asPL), the SERCA2a gene (Ad.SERCA2a), or the reporter genes ␤-galactosidase and green fluorescent protein (Ad.␤gal-GFP ATPase pump (SERCA2a), the activity of which is reduced in heart failure. [3][4][5] SERCA2a is regulated by phospholamban, the phosphorylation of which relieves the inhibition of SERCA2a. The phospholamban/SERCA2a interaction controls the calcium content of the SR and ultimately controls cardiac contractility. 6,7 A decrease in the phosphorylation of phospholamban, along with an increase in the phoshpholamban/ SERCA2a ratio, contributes to contractile dysfunction in heart failure. 3,5 Contractility, calcium handling, and the frequency response were restored in isolated failing human cardiomyocytes by restoring this ratio by means of a gene transfer that caused the overexpression of SERCA2a. 8 Another approach to restoring the phospholamban/SERCA2a ratio in failing hearts to normal would be to decrease levels of phospholamban through the use of antisense strategies.In the present study, we have used such a strategy by generating adenoviral vectors expressing antisense of phospholamban and examining their effects on phospholamban expression, SERCA2a activity, and myocyte function in failing human cardiomyocytes. MethodsFailing human ventricular myocardial tissue was obtained from 9 explanted hearts (5 ischemic and 4 with dilated cardiomyopathy), and nonfailing tissue was obtained from 18 donor hearts. Myocytes were isolated from 1 g of myocardial tissue removed from the free wall of the left ventricle by enzymatic digestion, as described previously. 8 The proportion of rod-shaped viable cells at the time of isolation was 30% to 50% (nϭ9) for failing and 40% to 60% (nϭ18) for nonfailing cardiomyocytes. After isolation, the cells were resuspended in M199, 50 U/mL penicillin, and 50 U/mL streptomycin; equilibrated to pH 7.4; and infected with the adenoviruses at a multiplicity of infection of 100. The antisense cDNA of phospholamban was first cloned into a shuttle vector, pAdTrack-CMV. 9 The resultant plasmid was linearized with restriction endonuclease PmeI and subsequently was cotransformed into E coli BJ5183 cells with an adenoviral backbone plasmid, pAdEasy-1. Recombinants were selected for kanamycin resistance, and recombination was confirmed by multiple-restriction endonuclease analyses. The linearized recombinant plasmid was transfected into adenovirus-packaging cell lines (293 cells...