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.
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