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 Cys⁴¹ to Phe⁴¹, was shown to be as effective as pentameric phospholamban in inhibiting Ca²⁺ transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys⁴¹→Phe⁴¹) phospholamban. Quantitative immunoblotting indicated a 2- fold increase in the cardiac phospholamban protein levels compared with wild- type controls, with ≃50% of phospholamban migrating as monomers and ≃50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed 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 μmol/L in hearts overexpressing monomeric phospholamban, 0.49±0.05 μmol/L in hearts overexpressing wild type phospholamban, and 0.26±0.01 μmol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca²⁺ kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as 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⁺ transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.