Mouse phospholamban gene expression during development in vivo and in vitro

To establish a murine model that may allow for definition of the precise role of phospholamban in myocardial contractility through selective perturbations in the phospholamban gene, we initiated studies on the role of phospholamban in the murine heart. Intact beating hearts were perfused in the absence or presence of isoproterenol, and quantitative measurements of cardiac performance were obtained. Isoproterenol stimulation was associated with increases in the affinity of the sarcoplasmic reticulum Ca2+ pump for Ca2+ that were due to phospholamban phosphorylation. To assess the regulation of phospholamban gene expression during murine development, Northern blot and polymerase chain reaction analyses were used. Phospholamban mRNA was first detected in murine embryos on the ninth day of development (the time when the cardiac tube begins to contract). In murine embryoid bodies, which have been shown to recapitulate several aspects of cardiogenesis, phospholamban mRNA was detected on the seventh day (the time when spontaneous contractions are first observed). Only those embryoid bodies that exhibited contractions expressed phospholamban transcripts, and these were accompanied by expression of the protein, as revealed by immunofluorescence microscopy. Sequence analysis of the cDNA encoding phospholamban in embryoid bodies indicated complete homology to that in adult hearts. The deduced amino acid sequence of murine phospholamban was identical to rabbit cardiac phospholamban but different from dog cardiac and human cardiac phospholamban by one amino acid. These data suggest that phospholamban, the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, is present very early in murine cardiogenesis in utero and in vitro, and this may constitute an important determinant for proper development of myocardial contractility.