Our previous studies, and those of others, provide considerable evidence that oxidant stress (eg, oxyradical production) contributes to the disorders of cardiac rhythm and contractile function which characterize ischemia and reperfusion. The work funded by our previous 3 year NIH grant has led us to question the widely held view that such effects are mediated via membrane lipid peroxidation. Instead, we propose that it is oxidant stress-induced changes in membrane-bound ion translocating proteins that are primarily responsible for the ionic disturbances which lead to myocardial dysfunction. Oxidant stress-induced changes in the redox state of free (glutathione) and protein-bound thiols, which regulate the activity of certain sarcolemmal ion translocators, can lead to the rapid, specific and reversible stimulation or inhibition of the Na/K pump, Na/Ca exchange, the transient outward current and the ATP- sensitive K channel. To test this hypothesis, and identify the mechanism(s) by which oxidant stress-induced changes in redox state contribute to early contractile failure, reperfusion arrhythmias and post-ischemic contractile dysfunction (stunning) we propose to cross- species study (rat, guinea pig and rabbit) employing whole hearts in vivo, isolated blood and crystalloid perfused hearts, isolated myocytes and single channels. We will pursue three Specific Aims:
Specific Aim 1 : To use whole hearts, in vivo and in vitro, to define the extent and time-course of ischemia- and reperfusion-induced changes in the redox state of free and protein-bound thiols and related these changes to the profiles of contractile and electrical injury. In these studies we will also assess the specificity and reversibility of such changes by measuring the extent to which they can be prevented or reversed by anti- oxidants or chemical reductants.
Specific Aim 2 : To use this information in whole hearts to permit us to titrate doses of exogenously generated oxidant stress or other interventions which modify intracellular redox status so that, in subsequent mechanistic studies, we can realistically mimic the degree of stress which arises during ischemia and reperfusion.
Specific Aim 3 : To use single cell and channel recordings in mechanistic studies to define the effect of oxidant stress (applied intracellularly or extracellularly) on four sarcolemmal ion translocators (the Na/K pump, Na/Ca exchange, the ATP-sensitive K channel and the channel responsible for the transient outward current) which we believe to vulnerable to redox modification. We will determine whether they are stimulated or inhibited and the extend to which this, in turn, perturbs intracellular ion activity. Finally, we will assess the specificity and reversibility of oxidant stress-induced changes in the activity of ion translocators by measuring the extent to which they can be reversed by anti-oxidants or chemical reductants - ie: we will assess the potential of anti-oxidant therapy directed at membrane protein activity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037278-05
Application #
2218407
Study Section
Cardiovascular and Renal Study Section (CVB)
Project Start
1987-08-01
Project End
1996-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
St. Thomas Hospital (London, Uk)
Department
Type
DUNS #
City
London
State
Country
United Kingdom
Zip Code
Galinanes, M; Hearse, D J; Shattock, M J (1996) The role of the rate of vascular collapse in ischemia-induced acute contractile failure and decreased diastolic stiffness. J Mol Cell Cardiol 28:519-29
Haddock, P S; Woodward, B; Hearse, D J (1995) Cardiac Na+/K+ ATPase activity and its relation to myocardial glutathione status: studies in the rat. J Mol Cell Cardiol 27:1185-94
Haddock, P S; Shattock, M J; Hearse, D J (1995) Modulation of cardiac Na(+)-K+ pump current: role of protein and nonprotein sulfhydryl redox status. Am J Physiol 269:H297-307
Blasig, I E; Shuter, S; Garlick, P et al. (1994) Relative time-profiles for free radical trapping, coronary flow, enzyme leakage, arrhythmias, and function during myocardial reperfusion. Free Radic Biol Med 16:35-41
Coetzee, W A; Ichikawa, H; Hearse, D J (1994) Oxidant stress inhibits Na-Ca-exchange current in cardiac myocytes: mediation by sulfhydryl groups? Am J Physiol 266:H909-19
Shattock, M J; Matsuura, H (1993) Measurement of Na(+)-K+ pump current in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Inhibition of the pump by oxidant stress. Circ Res 72:91-101
Hearse, D J (1991) Stunning: a radical re-view. Cardiovasc Drugs Ther 5:853-76
Matsuura, H; Shattock, M J (1991) Effects of oxidant stress on steady-state background currents in isolated ventricular myocytes. Am J Physiol 261:H1358-65
Matsuura, H; Shattock, M J (1991) Membrane potential fluctuations and transient inward currents induced by reactive oxygen intermediates in isolated rabbit ventricular cells. Circ Res 68:319-29
Hearse, D J (1991) Reperfusion-induced injury: a possible role for oxidant stress and its manipulation. Cardiovasc Drugs Ther 5 Suppl 2:225-35

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