Joel P. French

Exercise training provides cardioprotection against ischemia-reperfusion induced apoptosis in young and old animals.

Endurance exercise provides cardioprotection against ischemia-reperfusion (IR)-induced necrotic cell death in young animals. However, whether exercise-induced cardioprotection prevents IR-induced apoptosis in young and old animals is unknown. We tested the hypothesis that endurance exercise training will attenuate IR-induced myocardial apoptosis in young (4 months) and old (24 months) male F344 rats. Young and old rats remained sedentary or performed multiple bouts of moderate intensity running exercise. To induce apoptosis, isolated working hearts were exposed to 45 min of ischemia followed by 90 min of reperfusion. Assessment of myocardial levels of caspase-3 cleaved alpha-spectrin and TUNEL labeled nuclei revealed that IR resulted in apoptosis in hearts from both young and old animals. Importantly, independent of age, exercise attenuated the IR-induced apoptosis of cardiac myocytes. Moreover, exercise attenuated IR-induced calpain activation in the hearts of both young and old animals. These experiments for the first time demonstrate that exercise attenuates IR-induced myocardial apoptosis in both young and old animals. Potential mechanisms for this exercise-induced cardioprotection against IR-induced apoptosis include improved myocardial antioxidant capacity and prevention of calpain and caspase-3 activation.

Exercise-induced protection against myocardial apoptosis and necrosis: MnSOD, calcium-handling proteins, and calpain

Exercise provides protection against myocardial ischemia‐reperfusion (IR) injury. Understanding the mechanisms of this protection may lead to new interventions for the prevention and/or treatment of heart disease. Although presently these mechanisms are not well understood, reports suggest that manganese superoxide dismutase (MnSOD) and calpain may be critical mediators of this protection. We hypothesized that an exercise‐induced increase in MnSOD would provide cardioprotection by attenuating IR‐induced oxidative modification to critical Ca2+‐handling proteins, thereby decreasing calpain‐mediated cleavage of these and other proteins attenuating cardiomyocyte death. After IR, myocardial apoptosis and infarct size were significantly reduced in hearts of exercised animals compared with sedentary controls. In addition, exercise prevented IR‐induced calpain activation as well as the oxidative modification and calpain‐mediated degradation of myocardial Ca2+‐handling proteins (L‐type Ca2+ channels, phospholamban, and sarcoplasmic/endoplasmic reticulum calcium ATPase). Further, IR‐induced activation of proapoptotic proteins was attenuated in exercised animals. Importantly, prevention of the exercise‐induced increase in MnSOD activity via antisense oligonucleotides greatly attenuated the cardioprotection conferred by exercise. These results suggest that MnSOD provides cardioprotection by attenuating IR‐induced oxidation and calpain‐mediated degradation of myocardial Ca2+‐handling proteins, thereby preventing myocardial apoptosis and necrosis.—French, J. P., Hamilton, K L., Quindry, J. C., Lee, Y., Upchurch, P. A., Powers, S. K. Exercise‐induced protection against myocardial apoptosis and necrosis: MnSOD, calcium‐handling proteins, and calpain. FASEB J. 22, 2862–2871 (2008)

Extracellular Superoxide Dismutase Deficiency Exacerbates Pressure Overload–Induced Left Ventricular Hypertrophy and Dysfunction

Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)–induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload.

Adenosine A3 receptor deficiency exerts unanticipated protective effects on the pressure overloaded left ventricle

Background— Endogenous adenosine can protect the overloaded heart against the development of hypertrophy and heart failure, but the contribution of A1 receptors (A1R) and A3 receptors (A3R) is not known. Methods and Results— To test the hypothesis that A1R and A3R can protect the heart against systolic overload, we exposed A3R gene–deficient (A3R knockout [KO]) mice and A1R KO mice to transverse aortic constriction (TAC). Contrary to our hypothesis, A3R KO attenuated 5-week TAC-induced left ventricular hypertrophy (ratio of ventricular mass/body weight increased to 7.6±0.3 mg/g in wild-type mice compared with 6.3±0.4 mg/g in KO mice), fibrosis, and dysfunction (left ventricular ejection fraction decreased to 43±2.5% and 55±4.2% in wild-type and KO mice, respectively). A3R KO also attenuated the TAC-induced increases of myocardial atrial natriuretic peptide and the oxidative stress markers 3′-nitrotyrosine and 4-hydroxynonenal. In contrast, A1R KO increased TAC-induced mortality but did not alter ventricular hypertrophy or dysfunction compared with wild-type mice. In mice in which extracellular adenosine production was impaired by CD73 KO, TAC caused greater hypertrophy and dysfunction and increased myocardial 3′-nitrotyrosine. In neonatal rat cardiomyocytes induced to hypertrophy with phenylephrine, the adenosine analogue 2-chloroadenosine reduced cell area, protein synthesis, atrial natriuretic peptide, and 3′-nitrotyrosine. Antagonism of A3R significantly potentiated the antihypertrophic effects of 2-chloroadenosine. Conclusions— Adenosine exerts protective effects on the overloaded heart, but the A3R acts counter to the protective effect of adenosine. The data suggest that selective attenuation of A3R activity might be a novel approach to treat pressure overload–induced left ventricular hypertrophy and dysfunction.

Adenosine regulation of microtubule dynamics in cardiac hypertrophy

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.