Reduced oxygen intermediates have been implicated in the initiation and progression of various disease states such as ischemia/reperfusion injury, the inflammatory response of myocardium and the toxic action of drugs (e.g., adriamycin). We have hypothesized that the major target organelles injured by free radical attack are the sarcolemma and sarcoplasmic reticulum which are the key regulators of myocardial contractility. The purpose of this project is therefore to define the underlying biochemical, physiological and morphological changes that occur in these organelles as a result of attack by oxygen free radicals and neutrophil derived oxidants.
The specific aim of this project is to answer the following three hypotheses. A combination of in vitro and in vivo models will be used to evaluate these hypotheses. The primary hypothesis is if hydrogen peroxide, hydroxyl radicals and neutrophil derived oxidants such as hypochlorous acid, monochloramine and taurine monochloramine cause dysfunctions of isolated sarcolemma and sarcoplasmic reticulum. We will characterize the important biochemical parameters of sarcolemma such as Na+-Ca2+ exchange, Na+, K+-ATPase, muscarinic and adrenergic receptors and (Ca2+-Mg2+)-ATPase, calcium transport of sarcoplasmic reticulum, and morphological changes in these isolated organelles following in vitro attack by oxidizing species. The second hypothesis is that oxygen free radicals and oxidants inactivate proteins by modification of amino acid residues or initiation of oxidation of sulfhydryl groups which would result in alteration of conformation or cleavage of molecules. We will test this hypothesis on purified (Ca2+-Mg2+)- ATPase of sarcoplasmic reticulum and determine amino acid residues by Pico-tag HPLC in control and oxidants treated enzyme. The third hypothesis is if oxygen radicals bring about biochemical dysfunctions in isolated rat heart. We will perfuse the isolated working heart with free radicals and oxidants and study the myocardial contractility, Na+-Ca2+ exchange, Na+, K+- ATPase and receptor functions in isolated sarcolemma and (Ca2+- Mg2+)-ATPase and calcium uptakes in homogenates. We will also study the in vivo lipid peroxidation following these interventions by HPLC detection of diene-conjugates either in perfusate or muscle extract. This integrated approach should result in more meaningful understanding of our knowledge of free radicals and neutrophil derived oxidants attack on cardiac muscle and function. A better understanding of the mechanism of damage to these membranes is important because it may suggest rational therapies to improve contractility following reperfusion.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL040111-02
Application #
3471887
Study Section
Cardiovascular and Pulmonary Research B Study Section (CVB)
Project Start
1988-07-01
Project End
1991-06-03
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Kinnula, Vuokko L; Crapo, James D (2003) Superoxide dismutases in the lung and human lung diseases. Am J Respir Crit Care Med 167:1600-19
Hess, M L; Kukreja, R C (1994) Myocardial stunning. J Card Surg 9:382-6
Kukreja, R C; Loesser, K E; Kearns, A A et al. (1993) Protective effects of histidine during ischemia-reperfusion in isolated perfused rat hearts. Am J Physiol 264:H1370-81
Okabe, E; Takahashi, S; Norisue, M et al. (1993) The effect of hypochlorous acid and hydrogen peroxide on coronary flow and arrhythmogenesis in myocardial ischemia and reperfusion. Eur J Pharmacol 248:33-9
Vinnikova, A K; Kukreja, R C; Hess, M L (1992) Singlet oxygen-induced inhibition of cardiac sarcolemmal Na+K(+)-ATPase. J Mol Cell Cardiol 24:465-70
Kukreja, R C; Kearns, A A; Zweier, J L et al. (1991) Singlet oxygen interaction with Ca(2+)-ATPase of cardiac sarcoplasmic reticulum. Circ Res 69:1003-14
Loesser, K E; Kukreja, R C; Kazziha, S Y et al. (1991) Oxidative damage to the myocardium: a fundamental mechanism of myocardial injury. Cardioscience 2:199-216
Kukreja, R C; Kontos, H A; Hess, M L (1990) Captopril and enalaprilat do not scavenge the superoxide anion. Am J Cardiol 65:24I-27I
Scott, R B; Wood, H E; Matin, S et al. (1990) Superoxide radical production after phorbol ester stimulation in neutrophils of aged donors. Exp Gerontol 25:523-32
Kontos, H A; Wei, E P; Kukreja, R C et al. (1990) Differences in endothelium-dependent cerebral dilation by bradykinin and acetylcholine. Am J Physiol 258:H1261-6

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