Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice

Phospholamban is the regulator of the cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity and an important modulator of basal contractility in the heart. To determine whether all the SR Ca²⁺-ATPase enzymes are subject to regulation by phospholamban in vivo, transgenic mice were generated which overexpressed phospholamban in the heart, driven by the cardiac-specific α- myosin heavy chain promoter. Quantitative immunoblotting revealed a twofold increase in the phospholamban protein levels in transgenic hearts compared to wild type littermate hearts. The transgenic mice showed no phenotypic alterations and no changes in heart/body weight, heart/lung weight, and cardiomyocyte size. Isolated unloaded cardiac myocytes from transgenic mice exhibited diminished shortening fraction (63%) and decreased rates of shortening (64%) and relengthening (55%) compared to wild type (100%) cardiomyocytes. The decreases in contractile parameters of transgenic cardiomyocytes reflected decreases in the amplitude (83%) of the Ca²⁺ signal and prolongation (131%) in the time for decay of the Ca²⁺ signal, which was associated with a decrease in the apparent affinity of the SR Ca²⁺-ATPase for Ca²⁺ (56%), compared to wild type (100%) cardiomyocytes. In vivo analysis of left ventricular systolic function using M mode and pulsed-wave Doppler echocardiography revealed decreases in fractional shortening (79%) and the normalized mean velocity of circumferential shortening (67%) in transgenic mice compared to wild type (100%) mice. The differences in contractile parameters and Ca²⁺ kinetics in transgenic cardiomyocytes and the depressed left ventricular systolic function in transgenic mice were abolished upon isoproterenol stimulation. These findings indicate that a fraction of the Ca²⁺-ATPases in native SR is not under regulation by phospholamban. Expression of additional phospholamban molecules results in: (a) inhibition of SR Ca²⁺ transport; (b) decreases in systolic Ca²⁺ levels and contractile parameters in ventricular myocytes; and (c) depression of basal left ventricular systolic function in vivo.