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Modulation of Cardiac Contraction by Reactive Nitrogen Species

by Kohr, Mark Jeffrey

Abstract (Summary)
Reactive nitrogen species, including peroxynitrite and nitroxyl, have been demonstrated to affect myocardial contractility. Many of these reactive nitrogen species are produced endogenously within cardiac myocytes and serve as critical regulators of myocyte function. Alterations in the production of these reactive species may be responsible for part of the dysfunction that is observed during many pathophysiological conditions of the myocardium. Although studies have partially documented the contractile effects of many different reactive nitrogen species, few have examined the specific mechanism of action. Therefore, in order to address deficiencies in regards to the physiological and pathophysiological regulation of myocardial function by reactive nitrogen species, we set out to characterize the contractile effects and specific mechanism of action for two distinct reactive nitrogen species, peroxynitrite and nitroxyl. We first characterized the effects of both high and low concentrations of peroxynitrite on cardiac myocyte function during three different contractile states (basal, submaximal and maximal beta-adrenergic stimulation). High peroxynitrite produced a negative effect on myocyte function that became more pronounced as the contractile state in the cardiac myocyte was increased. Conversely, low peroxynitrite produced a positive effect on myocyte function that was greatest during basal contraction and diminished as the contractile state in the cardiac myocyte was increased. Interestingly, the effects of both high and low peroxynitrite were completely absent during all contractile states in phospholamban knockout myocytes. These results indicate that high and low peroxynitrite both exert effects on cardiac myocyte contraction by targeting the critical excitation-contraction coupling phosphoprotein, phospholamban. In order to elucidate the specific signaling pathway of high peroxynitrite, we examined phospholamban phosphorylation at the cAMP-dependent kinase site (serine 16) and observed that high peroxynitrite decreased beta-adrenergic-stimulated phospholamban phosphorylation. However, protein phosphatase inhibition (okadaic acid) inhibited the negative effects of high peroxynitrite on myocyte contraction and phospholamban phosphorylation. High peroxynitrite also increased total protein phosphatase activity and promoted the interaction between phospholamban and protein phosphatase 2a. Therefore, high levels of peroxynitrite decrease beta-adrenergic-stimulated myocyte contraction by inducing the dephosphorylation of phospholamban via protein phosphatase 2a activation. We next examined the specific signaling pathway underlying the effects of low peroxynitrite. Since we determined that the effects of high peroxynitrite occurred through a protein phosphatase-dependent mechanism, we repeated our functional measurements in the presence of protein phosphatase inhibition. However, protein phosphatase inhibition did not alter the positive effect of low peroxynitrite on myocyte function. Low peroxynitrite also had no effect on total protein phosphatase activity, indicating that low peroxynitrite exerts effects through a protein phosphatase independent mechanism. Therefore, we examined the cAMP-dependent protein kinase (PKA) as a potential target of low peroxynitrite. Inhibition of PKA (KT5720) completely inhibited the positive effect of low peroxynitrite on myocyte contraction. Low peroxynitrite also significantly increased PKA activity in cardiac homogenates and in purified preparations of PKA. Therefore, low levels of peroxynitrite increase basal and beta-adrenergic-stimulated myocyte contraction through the direct activation of PKA. Finally, we examined the mechanism underlying the effects of nitroxyl on cardiac myocyte function. After confirming the positive effect of nitroxyl on cardiac myocyte function, we examined the effect of nitroxyl on action potential waveform. Resting membrane potential and the action potential duration to 20% repolarization remained unchanged upon treatment with nitroxyl, but the action potential duration to 90% repolarization was slightly prolonged. Therefore, we examined basal and beta-adrenergic-stimulated L-type Ca^2+ current, but the L-type Ca^2+ current was not altered upon exposure to nitroxyl. Next, we further investigated the role of the sarcoplasmic reticulum by examining the effect of nitroxyl on myocyte [Ca^2+]i transients during inhibition of sarcoplasmic reticulum Ca^2+-cycling. The positive effect of nitroxyl on myocyte [Ca^2+]i transients was completely abolished with the inhibition of sarcoplasmic reticulum Ca^2+-cycling. These results indicate that nitroxyl enhances myocyte contraction by exclusively targeting sarcoplasmic reticulum Ca^2+-cycling and does not rely on the recruitment of additional extracellular Ca^2+. The results contained herein provide conclusive evidence as to the regulation of cardiac myocyte function by reactive nitrogen species. Further, the results of these pharmacological studies lend critical insight into the physiological and pathophysiological regulation of myocardial function by reactive nitrogen species, and may have important therapeutic ramifications for heart disease.
Bibliographical Information:

Advisor:

School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:nitric oxide peroxynitrite nitroxyl

ISBN:

Date of Publication:06/26/2009

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