In myocardium, Na+,K+-ATPase maintains the electrochemical gradient of the cardiac cells, hence, directly and indirectly modulates electrical and contractile activity of the heart. Na+,K+-ATPase consists of an alpha- and a beta-subunit to which multiple isoforms exist, although their physiological function remains unclear. The overall hypothesis to be tested is that during cardiac growth expression of cardiac Na+,K+- ATPase isozyme is differentially regulated, and that individual isozymes serve unique function in regulating intracellular Na+ concentration ([Na]i). Ferret myocardium expresses alpha3 and alpha1 isoforms, in contrast to the commonly studied adult rat heart which expresses predominately alpha1 and alpha2 isoforms. Thus, ferrets provide a useful model to study expression and function of alpha3, which is expressed in human heart along with alpha1 and alpha2. Using ferrets and rats as animal models, the following objectives will be studied: 1) Expression of alpha3 isoform is upregulated during early postnatal development. In neonatal ferret heart, thyroid hormone (TH) treatment preferentially upregulates the alpha3 isoform. The hypothesis will be tested that TH is a physiological regulator in expression of the alpha3 isoform during developmental cardiac growth. Underlying transcriptional and/or post- transcriptional mechanisms will be examined. 2) Myocardium consists of functionally heterogenous myocardial cells. Localized distribution of the isoforms may reveal specialized function of the isozymes. Using in situ hybridization and immunocytochemistry, distribution of the Na+,K+-ATPase isoforms during developmental growth of the myocardium in rats and ferrets will be established. 3) Relative contribution of the pump isoforms to overall pump activity will be determined, under conditions when intracellular Na+ load is altered, by studying Na-pump activity by 86Rb+ uptake, and [Na]i by fluorescence microscopy. Na affinity of the isozymes will be examined directly in isolated myocytes by measuring Na- dependent pump activity using 86Rb+ uptake. Functional consequences in altered isoform expression during development will be examined. A complete understanding in regulation, distribution, and function of the Na+,K+-ATPase isozymes during cardiac growth may prove helpful not only in understanding the physiology and pathophysiology of the myocardium, but also in developing better clinical strategies for use of cardiac glycosides, to which Na+,K+-ATPase is the only known receptor.
