The studies proposed seek to gain further understanding of fundamental mechanisms involved in the control of cardiac function at the cellular and molecular level, and how cardiac function is modulated by hormones, neurotransmitters, and particularly by the group of drugs known as cardiac glycosides (digitalis). Using spontaneously beating monolayer cultures of cardiac myocytes, we will test the hypothesis that cardiac glycoside binding to and inhibition of NaK-ATPase in the sarcolemmal membrane are steps requisite to the development of a positive inotropic response. Unidirectional and net Na+, K+ and Ca++ fluxes will be measured at intervals during the inotropic response. To distinguish between NaK pump inhibition and other postulated mechanisms of inotropy, we will alter the number of NaK-ATPase sites in cultured heart cells by growth in low [K+]o media and test inotropic and toxic responsiveness to cardiac glycosides. We will exploit our ability to grow cultured heart cells in chemically defined, serum free media, confirming and extending our finding that these cells are fast channel activated and highly responsive to muscarinic agonists. Cells grown in serum-free or conventional media will be compared with respect to Na+, K+ and Ca++ fluxes and contents; NaK-ATPase properties; beta-adrenergic and muscarinic receptor properties; and membrane composition including cholesterol content. Of particular interest will be studies of voltage-dependent fast sodium and slow calcium channels by radioligand binding and patch clamp analyses. Additional studies using cultured heart cells will test the hypotheses that thyroid hormone directly induces an increase in the number of NaK-ATPase sites, and that the phenomenon of resistance to digitalis in the hyperthyroid state is due to enhanced NaK pump capacity. Studies using high-field Fourier transform NMR will extend our previous work on the delineation of Na+i and Na+o using lanthanide chelates that are confined to the extracellular space and act as isotropic hyperfine shift reagents. Using intact perfused hearts, we will determine the magnitude and time course of changes in transsarcolemmal Na+ distribution in response to cardiac glycosides and other interventions using 23Na NMR. Gating of signal acquisition to the cardiac cycle will be used to test the hypothesis that augmentation of the Na+i transient during the action potential underlies or contributes to the positive inotropic effects of digitalis at subtoxic levels. Other NMR experiments will examine K+i and K+o in the heart using lanthanide shift reagents, and 31P-NMR will be used to delineate E1-P and E2-P phosphoenzyme forms of NaK-ATPase and SR Ca-ATPase.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL036141-13
Application #
2642202
Study Section
Special Emphasis Panel (NSS)
Project Start
1985-09-01
Project End
1999-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
13
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Frantz, Stefan; Ducharme, Anique; Sawyer, Douglas et al. (2003) Targeted deletion of caspase-1 reduces early mortality and left ventricular dilatation following myocardial infarction. J Mol Cell Cardiol 35:685-94
Fukazawa, Ryuji; Miller, Thomas A; Kuramochi, Yukio et al. (2003) Neuregulin-1 protects ventricular myocytes from anthracycline-induced apoptosis via erbB4-dependent activation of PI3-kinase/Akt. J Mol Cell Cardiol 35:1473-9
Frantz, S; Kelly, R A; Bourcier, T (2001) Role of TLR-2 in the activation of nuclear factor kappaB by oxidative stress in cardiac myocytes. J Biol Chem 276:5197-203
Fischer, T A; Ludwig, S; Flory, E et al. (2001) Activation of cardiac c-Jun NH(2)-terminal kinases and p38-mitogen-activated protein kinases with abrupt changes in hemodynamic load. Hypertension 37:1222-8
Feron, O; Zhao, Y Y; Kelly, R A (1999) The ins and outs of caveolar signaling. m2 muscarinic cholinergic receptors and eNOS activation versus neuregulin and ErbB4 signaling in cardiac myocytes. Ann N Y Acad Sci 874:11-9
Hauptman, P J; Kelly, R A (1999) Digitalis. Circulation 99:1265-70
Feron, O; Han, X; Kelly, R A (1999) Muscarinic cholinergic signaling in cardiac myocytes: dynamic targeting of M2AChR to sarcolemmal caveolae and eNOS activation. Life Sci 64:471-7
Sawyer, D B; Fukazawa, R; Arstall, M A et al. (1999) Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circ Res 84:257-65
Frantz, S; Kobzik, L; Kim, Y D et al. (1999) Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest 104:271-80
Zhao, Y Y; Feron, O; Dessy, C et al. (1999) Neuregulin signaling in the heart. Dynamic targeting of erbB4 to caveolar microdomains in cardiac myocytes. Circ Res 84:1380-7

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