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published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Pharmacology / Doctoral / Doctor of Philosophy

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ABSTRACT To understand the mechanisms underlying cardiac dysfunction during ischemia reperfusion (I/R) injury, we tested the hypothesis that oxidative stress and defects in endothelium play a critical role in the activation of calpain and matrix metalloproteinases (MMP), inhibition of sarcolemmal (SL) Na+-K+-ATPase, and induction of cardiac dysfunction during I/R injury. It was observed that I/R induced depression in cardiac function and SL Na+-K+-ATPase activity was greater in hearts perfused at constant flow than in hearts perfused at constant pressure. Such a difference was associated with increased calpain activity as well as decreased endothelial nitric oxide synthase protein content and in nitric oxide production. The depression in Na+-K+-ATPase activity and decrease in protein content of Na+-K+-ATPase isoforms in I/R hearts were associated with an increase in calpain activity and translocation of calpain isoforms (I and II) from the cytosol to SL membrane as well as changes in the distribution of calpastatin. I/R induced alterations were Ca2+-dependent and were prevented by treatment with calpain inhibitors, MDL28170 and Leupeptin (Leu). These results suggest that depressions in cardiac function and SL Na+-K+-ATPase activity in the I/R hearts may be due to endothelial dysfunction as well as changes in the activity and translocation of calpain. In another set of experiments, we examined the role of oxidative stress in activation of calpain during I/R and its association with changes in the activity of MMP. Our results show depression of cardiac function and Na+-K+-ATPase activity in I/R hearts were associated with increased calpain and MMP activities. These alterations due to I/R were attenuated by ischemic preconditioning as well as treatment with antioxidant, N-acetylcysteine (NAC) and mercaptopropionylglycine (MPG). Treatment of I/R hearts with MMP inhibitor doxycycline (Dox) improved I/R-induced changes in cardiac function and Na+-K+-ATPase activity without affecting the calpain activation while treatment with calpain inhibitors, Leu and MDL 28170, reduced the MMP activity significantly in addition to attenuating the I/R-induced depression in Na+-K+-ATPase activity. These results suggests that alterations in Na+-K+-ATPase activity in I/R hearts are associated with oxidative stress and intracellular Ca2+ overload induced activation of calpain and MMP.

ABSTRACT To understand the mechanisms underlying cardiac dysfunction during ischemia reperfusion (I/R) injury, we tested the hypothesis that oxidative stress and defects in endothelium play a critical role in the activation of calpain and matrix metalloproteinases (MMP), inhibition of sarcolemmal (SL) Na+-K+-ATPase, and induction of cardiac dysfunction during I/R injury. It was observed that I/R induced depression in cardiac function and SL Na+-K+-ATPase activity was greater in hearts perfused at constant flow than in hearts perfused at constant pressure. Such a difference was associated with increased calpain activity as well as decreased endothelial nitric oxide synthase protein content and in nitric oxide production. The depression in Na+-K+-ATPase activity and decrease in protein content of Na+-K+-ATPase isoforms in I/R hearts were associated with an increase in calpain activity and translocation of calpain isoforms (I and II) from the cytosol to SL membrane as well as changes in the distribution of calpastatin. I/R induced alterations were Ca2+-dependent and were prevented by treatment with calpain inhibitors, MDL28170 and Leupeptin (Leu). These results suggest that depressions in cardiac function and SL Na+-K+-ATPase activity in the I/R hearts may be due to endothelial dysfunction as well as changes in the activity and translocation of calpain. In another set of experiments, we examined the role of oxidative stress in activation of calpain during I/R and its association with changes in the activity of MMP. Our results show depression of cardiac function and Na+-K+-ATPase activity in I/R hearts were associated with increased calpain and MMP activities. These alterations due to I/R were attenuated by ischemic preconditioning as well as treatment with antioxidant, N-acetylcysteine (NAC) and mercaptopropionylglycine (MPG). Treatment of I/R hearts with MMP inhibitor doxycycline (Dox) improved I/R-induced changes in cardiac function and Na+-K+-ATPase activity without affecting the calpain activation while treatment with calpain inhibitors, Leu and MDL 28170, reduced the MMP activity significantly in addition to attenuating the I/R-induced depression in Na+-K+-ATPase activity. These results suggests that alterations in Na+-K+-ATPase activity in I/R hearts are associated with oxidative stress and intracellular Ca2+ overload induced activation of calpain and MMP.

A decrease in the apparent diffusion coefficient (ADC) of water is becoming an important tool for the detection of acute and chronic brain disorders, yet it is not known whether changes in myocardial ADCs hold similar potential. Consequently, this work determined whether the ADCs of water or intracellular metabolites could be used to show ischaemia or cell swelling in the isolated rat heart. A modified STEAM pulse sequence was designed to measure the ADCs of the ¹H-NMR detectable metabolites, taurine and creatine, with 4 min time resolution in a 3 mm myocardial slice. Experiments included the measurement of: a) metabolite diffusion coefficients and diffusion tensors in solutions, and ADCs and diffusion tensors in the isolated, KCl-arrested rat heart; b) taurine and creatine content during 32 min total, global ischaemia in the isolated rat heart; c) metabolite and water ADCs before, during and after ischaemia; and d) changes in average cardiomyocyte diameter during perfusion and ischaemia using the taurine ADC measurements. At a diffusion time of 50 ms, the myocardial ADCs were 1.06 x 10^-3 mm²/s for water, 0.29 x 10^-3 mm²/s for taurine and 0.26 x 10"3 mm 2 /s for creatine. Neither taurine nor creatine was lost from the heart during ischaemia, making either suitable for ischaemic diffusion measurements. Contrary to changes in the brain, myocardial water and taurine ADCs remained constant during ischaemia; however, the total creatine ADC increased by 35% which was shown to result from hydrolysis of PCr to creatine. Using the taurine ADC measurements at diffusion times between 50 ms and 1510 ms, the average myocyte diameter was calculated to be 40 μm during perfusion and 27 μm by the end of ischaemia. The decrease in myocyte diameter indicates that the buffer perfused heart is highly oedematous. This is the first time that: 1) metabolite ADCs have been measured in isolated heart, and 2) NMR spectroscopy has been used to determine the myocyte diameter. Thus ADC changes may not have potential for detecting ischaemia in the heart, although the measurement of myocyte diameter using taurine ADCs could indicate myocardial oedema.

Thesis (M.S.)--University of Hawaii at Manoa, 2007. / Includes bibliographical references (leaves 13-17). / vii, 17 leaves, bound ill. 29 cm