Involvement of free radicals in the pathophysiology of ischemic heart disease
Authors: Hess Michael L. , Manson Nancy H. , Okabe Eiichiro 
Published: April 2011

Acute myocardial ischemia results in a decrease in developed tension and an increase in resting tension. A breakdown of the excitation–contraction coupling system can explain the behavior of ischemic muscle at a subcellular level. We have identified a specific defect in the sarcoplasmic reticulum (SR) from the ischemic myocardium; i.e., the uncoupling of calcium transport from ATP hydrolysis. The mediators of this excitation–contraction uncoupling process have not been identified. It is now established that the intracellular pH of the ischemic myocardium is in the range of 6.4 but the role of protons and potential role of free radicals have not been identified. We have hypothesized that protons and free radicals may interact to produce the excitation–contraction uncoupling of the ischemic myocardium. Cardiac SR was isolated from the wall of canine left ventricle and calcium uptake velocity and Ca2+-stimulated Mg2+-dependent ATPase activity determined. Increasing proton concentration between pH 7.0 and 6.4 significantly reduced calcium uptake rates (pH 7.0 = 0.95 ± 0.02; 6.4 = 0.50 ± 0.02 μmol Ca2+∙mg protein−1∙min−1; p < 0.01) with no effect on ATPase activity. Calculated coupling ratios (micromoles Ca2+/micromoles Pi) decreased from 0.87 ± 0.06 at pH 7.0 to 0.51 ± 0.05 at pH 6.4. At pH 7.0, the generation of exogenous free radicals from the xanthine (X) – xanthine oxidase (XO) system significantly depressed both calcium uptake rates (control = 0.95 ± 0.02; X + XO = 0.15 ± 0.02) and ATPase activity (control = 1.05 ± 0.02; X + XO = 0.30 ± 0.01 μmol Pi∙mg protein−1∙min−1; p < 0.01). The decreases in calcium uptake and in ATPase activity were completely reversible with superoxide dismutase (SOD). At pH 6.4 in the presence of xanthine and xanthine oxidase, there is a further depression of calcium uptake rates (control = 0.50 ± 0.02; X + XO = 0.11 ± 0.01; p < 0.05) but there is no SOD-reversible component. The addition of SOD + 20 mM mannitol normalized calcium transport at pH 6.4. The calculated coupling ratio at pH 6.4 in the presence of free radicals was 0.13. This value is identical with that reported for the ischemic myocardium. In the absence of an exogenous free-radical generating system, preincubation of whole heart homogenate at pH 6.4 resulted in a significant depression of calcium transport. At 1 min preincubation at pH 6.4, the return of the pH to 7.0 reversed the inhibiting effects of pH 6.4. At 5 min preincubation at pH 6.4, the system required both normalization to pH 7.0 and SOD to reverse the inhibiting effects of acidosis. At 15-min preincubation, the system required both SOD and 20 mM mannitol to reverse the depressant effects of acidosis. It is concluded that at pH 7.0, the xanthine – xanthine oxidase system generates superoxide anion [Formula: see text] which depresses both calcium uptake and ATPase activity. At pH 6.4, this system generates the more lethal hydroxylradical (∙OH) which uncouples calcium transport from ATP hydrolysis in a manner analogous to that observed in the ischemic myocardium. In the whole heart homogenate, acidosis results in a three phase reaction; an initial reversible effect of protons, followed by the production of [Formula: see text] and finally with continued acidosis, the generation of the ∙OH. Combining our observations with the mechanical function of the ischemic myocardium, it is hypothesized that free-radical generation, most probably the ∙OH, may form the link between the reversible and irreversible phase of myocardial ischemia.