Expression of protein phosphatases during postnatal development of rabbit heart.

Abstract

Protein phosphatases play a major role in the regulation of L-type calcium current (I(Ca)) in heart cells. We previously showed developmental differences in the effects of inhibitors of protein phosphatases (PP's) on the modulation of I(Ca), with greater stimulatory effects on I(Ca) observed in newborn than in adult ventricular cells. We hypothesized that this developmental difference might be due to greater expression and levels of PP 1 and PP 2A in newborn than in adult ventricular cells. We thus determined the mRNA expression of alpha and beta subunits of PP 1 and the a subunit of PP 2A in adult and newborn rabbit ventricles and levels of PP 1 and PP 2A in total homogenates, particulate membranes, and in soluble fraction prepared from isolated ventricular myocytes from adult and newborn rabbits. RT-PCR analysis demonstrated the presence of mRNA of these subunits of PP's in both newborn and adult ventricles. Northern blot analysis using 32P labeled cDNA probes specific for PP 1alpha, PP 1beta and PP 2Aalpha showed that the expression of steady state mRNA levels for PP 1alpha, PP 1beta and PP 2Aalpha were much higher in newborn compared to adult rabbit ventricles. mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in rabbit ventricles were measured as controls. GAPDH did not show significant developmental changes while mRNA for SERCA was higher in adult compared to newborns. Western blot analysis showed that PP 1 and PP 2A protein levels were also much higher in newborn compared to adult rabbit ventricular cells. Immunoblot analysis in particulate membranes and soluble fraction showed that PP1 was mainly membrane bound while PP 2 was present only in soluble fraction. These findings suggest that the two major protein phosphatases (PP 1 and PP 2A) in heart are expressed at much higher levels in newborn and decline to lower levels in adult ventricular myocytes. The presence of high levels of PP's and particularly PP 1 in newborn cells may be responsible for the greater dependence of newborn cells on the inhibition of PP as a mechanism of action of beta-agonist isoproterenol on I(Ca).