The specific goal of this project is to understand the role of cGMP in the regulation of cardiac L-type calcium current (Ica,pA/pF), particularly in newborn (NB) rabbit heart. Various studies on the modulation of Ica by cGMP in different species shows inconsistency and the role of cGMP remains unclear and controversial. In heart cells, knowledge about specific isoforms of cGMP-dependent Protein Kinase (PKG) and its substrates is very limited. Recently, Dr. Kumar showed that, in NB heart cells, basal Ica was significantly increased by increased levels of cGMP and inhibited by lowered cGMP levels (produced by guanylyl cyclase inhibitors Methylene blue or LY-83583). Effects of Methylene blue were blocked by 8BrcGMP. Basal Ica was not affected by these agents in adult (AD) heart cells. cAMP dependent protein kinase (PKA) inhibitor blocked the stimulatory effect of cAMP but not of 8CPT-cGMP on Ica in NB heart cells. This fundamental difference between NB and AD heart cells, led Dr. Kumar to examine regulation of Ica by cGMP in NB heart cells. He will specifically test the hypotheses that cGMP is an important modulator of Ica in NB cells. Physiological relevance of cGMP stimulation of Ica will be assessed by increasing or lowering cGMP levels. Interactions of cGMP and cAMP in the regulation of Ica will be examined in the presence of modulators specific for kinases, phosphodiesterases and phosphatases. He will test the hypothesis that the roles of cGMP and cAMP in the regulation of Ica in NB cells are not antagonistic. He will investigate the differences in the expression and levels of PKG in AD and NB cells and identify substrates phosphorylated by PKA and PKG. Dr. Kumar will test different hypotheses using multiple experimental approaches: 1) recording whole cell Ica and changing the internal solution by perfusible pipette in isolated NB rabbit ventricular cells, 2) recording single channel activity and its modulation by various interventions, 3) measuring PKG activity and its modulation, subcellular distribution, specific mRNA and isoform types of PKG in NB using different molecular biology techniques, e.g. Western blotting, Northern blotting and molecular cloning, and 4) phosphorylation assays to specifically identify the physiological substrates of PKG and PKA. Understanding these developmental differences in the regulation of Ica by cGMP dependent mechanisms may provide insights into the mechanisms involved in ion channel regulation and may contribute to a better understanding of therapeutic approaches for cardiac dysfunction in infants.
