The long term objective of this application is to define the molecular mechanisms of the physiologic roles of the different sodium, potassium- adenosine triphosphatase (Na, K-ATPase) isoforms in the cardiovascular system. This is mandated by the intrinsic importance of the Na, K-ATPase in the maintenance of the Na+/K+ gradient and membrane potential, and by the complexity of this enzyme system being revealed by ongoing molecular analyses by us and different research groups. The overall hypothesis to be tested is that Na, K-ATPase alpha subunit isoforms have specific functional characteristics that contribute to cell-specific functions as selected through evolution. As corollary, the dissection of isoform- specific functions is critical in the understanding of physiologic mechanisms in complex organ systems.
The specific aims prioritized for the ensuing 5-year period of study: 1) to analyze cell-specific expression in cardiac development and pathology by isoform-specific in situ hybridization defining the pattern of alpha 1,2,3/beta 1,2 subunit isoform pairing, cardiac regional and vessel-type expression, and patterns of modulation to various pathophysiologic stimuli; 2) to analyze functional characteristics and structure-function relationships of Na, K-ATPase alpha subunit isoforms (alpha 1, alpha 2, and alpha 3) by analysis of kinetics of ion transport and ATPase activity in tissue culture cells with transfected expressed exogenous rat Na, K-ATPase isoforms and strategic site-directed mutants of said rat alpha isoforms; and 3) to investigate isoform-specific integrated biologic functions in transgenic rats with decreased alpha2 or alpha3 Na,K-ATPases in the heart and aorta induced by targeted isoform-specific anti-sense transgene or knockout genes (the latter depending of developing technology), and assessing resultant perturbations of and effects on development, cell-specific gene expression of the other two non-affected isoforms, and effects on the membrane potential as deduced from measured changes in cardiac contractility and diastolic relaxation, rhythmogenicity, vascular endothelial dependent and independent relaxation and agonist-induced contractility. Dissection of the isoform-specific roles at a molecular level elucidating a) isoform-specific ion transport characteristics, b) ATP affinity and ATPase activity, c) cell-specific expression and modulation in development and pathology, and d) deduced integrated biologic roles as proposed in this application, would contribute incisively to the understanding of mechanisms mediated by Na, K-ATPase that modulate myocardial contractility and rhythmogenicity, vascular resistance, and that underlie cardiovascular responses in pathophysiologic situations such as hypertension, and cardiac hypertrophy and ischemia. Furthermore, the molecular analysis of Na, K-ATPase roles in the cardiovascular system will also give critical insight into the physiologic roles of this important enzyme in other organ systems.
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