Cytoprotective regulation of intracellular calcium in myocardium by particulate and soluble guanylate cyclases
Introduction
Background:
cGMP is an intracellular second messenger derived from GTP through the action of particulate and soluble guanylyl cyclases (pGCs and sGC). pGCs are membrane-associated receptors for a group of autocrine/paracrine and endocrine mediators called natriuretic peptides [1] whereas sGC is activated physiologically by nitric oxide (NO). Our previous work has identified that elevation of intracellular cGMP via activation of pGC by B-type natriuretic peptide (BNP) confers a marked cytoprotective effect on myocardium under the cytotoxic conditions of ischaemia-reperfusion [2, 3]. Preliminary evidence suggests that this protective effect of pGC is at least partially mediated through mechanisms related to sarcoplasmic reticulum ATPase activation which would promote sarcoplasmic reticulum Ca2+ uptake and decrease cytoplasmic Ca2+ overload, likely through activation of protein kinase G [4]. Whether activation of sGC by NO exerts similar effects on Ca2+ handling is unclear. There is increasing evidence that cGMP from pGC and sGC may be differently compartmentalised within cells and exert different physiological actions.
Aim and hypothesis:
The aim of this project is to characterise the effects of pGC and sGC activation on sarcoplasmic reticulum Ca2+ handling in cardiac myocytes. The primary hypothesis is that activation of pGC and sGC result in the generation of differently compartmentalised cGMP pools, resulting in different patterns of intracellular Ca2+ regulation.
Methodological Approach:
(1) The student will initially undertake studies to characterise the expression of pGC and sGC in myocardium. Using PCR, Western blotting and immunohistochemistry with cell-specific markers, the expression and localisation of sGC and pGC will be undertaken. It is expected that both sGC and pGC will be confirmed in cardiac myocytes and coronary vascular cells but expression patterns in other cells such as cardiac fibroblasts can not be predicted at present. (2) A direct comparison will be made of the effects of pGC and sGC stimulation in an experimental model of irreversible tissue injury during myocardial ischaemia-reperfusion. For these studies the effects of natriuretic peptides (BNP and CNP) which stimulate two different pGC isoforms will be compared with the effects of sGC activators (the NO donor DETA-NO, and the pharmacological sGC activator BayK12576) in an experimental model system of ischaemia-reperfusion injury. In each case, if a protective effect against injury is observed, the involvement of cGMP elevation will be confirmed by ELISA, the contribution of protein kinase G activation will be confirmed by using the selective pharmacological inhibitor KT5827, and the contribution of sarcoplasmic reticulum ATPase activation will be examined with the inhibitor cyclopiazonic acid. (3) To confirm direct effects of cGMP on sarcoplasmic reticulum ATPase activation studies will be undertaken in cultured cardiac myocytes in collaboration with Dr Chris George (Wales Heart Research Institute). By applying analysis of intracellular Ca2+ release and uptake with confocal imaging the student will be able to compare the effects of sGC and pGC activation on intracellular Ca2+ handling. These studies in isolated cells will permit the corroboration of pharmacological findings by complementary molecular techniques including gene transfection and gene silencing by siRNA.
References:
[1] D'Souza SP et al., Pharmacol Ther 2004,101,113-29;;
[2] D'Souza et al., Am J Physiol 2003, 284, H1592-600;
[3] Burley DS & Baxter GF, Basic Res Cardiol 2007, 102, 529-41;
[4] Burley DS, PhD thesis, University of London 2008
