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Cellular pathways of angiotensin II signalling in the hypertrophic myocardium Sunga, Paul S.


The general subject of these studies is the cellular action of angiotensin II (ATII) on the hypertrophic myocardium. The significance of this work is both biological and clinical: biological, because ATII has direct effects on the normal heart muscle and the intracellular signalling pathways activated by ATII are not fully understood. Clinical, because ventricular hypertrophy is a strong predictor of mortality in humans and ATII has been implicated in the process of hypertrophic development. The present work was based on the hypothesis that distinct signal transduction pathways are activated by ATII in the hypertrophic heart, as compared to the normal heart, and that these are identifiable in the heart of the Dahl salt-sensitive rat. The aim of the present study was to determine ATII intra-cellular signalling of the hypertrophic cardiac myocyte. Ventricular hypertrophy in the Dahl SS/Jr (Dahl S) rat was dependent upon the time on 6% NaCl diet. This was confirmed to be partly based on differences in viable myocyte size as determined by Fluorescence Activated Cell Sorting analysis. Dahl SR/Jr (Dahl R) rats showed no cardiac hypertrophy after identical treatment. Isolated ventricular myocytes from normal Dahl R rats and hypertrophic Dahl S rats were probed along the adenylyl cyclase-cAMP axis for effects induced by ATII. ATII induced a concentration and time - dependent inhibition of beta receptor stimulated increase in cAMP concentration in Dahl R ventricular myocytes. However, in hypertrophic Dahl S ventricular myocytes ATII induced a significant enhancement of beta receptor stimulated increase in cAMP concentration. This reversal was evident by stimulating adenylyl cyclase directly with forskolin, and was characterized as being dependent upon cAMP generation, not degradation, as well as displaying pertussis toxin sensitivity. The enhancement of stimulation of cAMP was linked to the degree of ventricular hypertrophy. GTP binding protein Gs alpha and Gi alpha subunits were assessed by ADP ribosylation, and immunodetection of Gs alpha. A diminution in the concentrations of these proteins in the hypertrophic Dahl S ventricular myocytes in comparison to Dahl R ventricular myocytes, was observed. Furthermore, the reversal of cAMP inhibition by ATII was mimicked by carbachol, a muscarinic receptor agonist, indicating that the reversal is likely independent of ATII receptor differences between hypertrophic and normal myocytes. This reversal was blocked by the down regulation of protein kinase C. The basis of these effects was further probed by comparing phosphorylation of hypertrophic Dahl S with Dahl R myocyte in response to ATII.The phosphorylation of a 42 kDa protein was induced by ATII, as well as the phorbol ester, phorbol 12-myristyl 13- acetate (PMA), in the Dahl R and Dahl S cardiac myocyte. Hypertrophic Dahl S myocytes displayed greater phophorylation of pp42 induced by ATII than Dahl R myocytes, although the converse was observed with PMA stimulation, namely greater PMA sensitivity in Dahl R. Examination of mitogen activated protein kinase (MAP) activation by ATII revealed that ATII regulates activity of p44erki and p42"Pk in ventricle. In Dahl R ATII stimulates these MAP kinases, however, in hypertrophic Dahl S ATII inhibits their activity. Both of these effects were mimicked by PMA, and the effects blocked by the protein kinase inhibitor Compound3. Protein kinase C activity assays verified that ATII signalling follows this route in the ventricle, and indicates possible divergences in this component of the pathway as well. In conclusion, firstly, the Dahl rat model of cardiac hypertrophy provided novel information about signal transduction differences between hypertrophic and normal myocardiocytes. Secondly, potentially important differences in ATII signal transduction have been characterized. These were evident in the adenylyl cyclase activation system, phosphorylation of cellular proteins, and the activations of MAP kinase and protein kinase C.

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