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(Circulation Research. 1997;81:485-492.)
© 1997 American Heart Association, Inc.

Articles

Monomeric Phospholamban Overexpression in Transgenic Mouse Hearts

Guoxiang Chu, Gerald W. Dorn, II, Wusheng Luo, Judy M. Harrer, Vivek J. Kadambi, Richard A. Walsh, , Evangelia G. Kranias

From the Department of Pharmacology & Cell Biophysics (G.C., W.L., J.M.H., V.J.K., E.G.K.) and the Division of Cardiology of the Department of Internal Medicine (G.W.D., R.A.W.), University of Cincinnati (Ohio), College of Medicine.

Correspondence to Evangelia G. Kranias, PhD, Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave, PO Box 670575, Cincinnati, OH 45267-0575.

Abstract 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 (Cys⁴¹Phe⁴¹) 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.

Key Words: phospholamban • transgenic mouse • Ca²⁺-ATPase • cardiomyocyte • Ca²⁺ transient

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