MONOMERIC PHOSPHOLAMBAN OVEREXPRESSION IN TRANSGENIC MOUSE HEARTS

Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR ) Ca2+-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys(41) to Phe(41), was shown to be as effective as pentameric phosph olamban in inhibiting Ca2+ transport in expression systems. To determi ne whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpressio n of the mutant (Cys(41)-->Phe(41)) phospholamban. Quantitative immuno blotting indicated a 2-fold increase in the cardiac phospholamban prot ein levels compared with wild-type controls, with approximate to 50% o f phospholamban migrating as monomers and approximate to 50% as pentam ers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were ana lyzed in parallel with transgenic hearts overexpressing (2-fold) wild- type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca2-uptake assays revealed that the EC50 values for Ca2+ were as follows: 0.32+/-0.01 mu mol/L in hearts overexpressing monomeric phospholamban ; 0.49+/-0.05 mu mol/L in hearts overexpressing wild-type phospholamba n, and 0.26+/-0.01 mu mol/L in wild-type con trol mouse hearts. Analys is of cardiomyocyte mechanics and Ca2+ kinetics indicated that the inh ibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) a s assessed by depression of the following: (1) shortening fraction (25 % mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), ( 3) rates of relengthening (25% mt versus 50% ov), (4) amplitude of the Ca2+ signal (21% mt versus 40% ov), and (5) time for decay of the Ca2 + signal (25% mt versus 106% ov) compared with control (100%) myocytes . The differences in basal cardiac myocyte mechanics and Ca2+ transien ts among the animal groups overexpressing monomeric or wild-type phosp holamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phosph olamban is important for mediating its optimal regulatory effects on m yocardial contractility in vivo.