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A119. Cardioprotective effects of exercise training on the altered SR Ca2+-uptake function in ovariectomized rats
910
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
slope of Gd-DTPA was significantly greater in infarct region than in normal regions, suggesting that less contrast reagent diffused out of the capillaries in infarct region during its first pass. This may be an indication of decreased capillary permeability. It was also found that T1 relaxation times measured during 30-minute contrast perfusion were significantly shorter in infarct than in normal myocardium. This suggests that distribution volume for the contrast reagent was still significantly greater in chronic infarct region than in normal region. Our results demonstrate that both T*- and Tweighted imaging techniques can be used to delineate chronic myocardial infarction. doi:10.1016/j.yjmcc.2006.03.411
A132. The role of chronic exogenous testosterone in myocardial function following acute ischemia-reperfusion Meijing Wang, Paul Crisostomo, Christine Herring, Daniel R. Meldrum. Indiana Univ. School of Medicine, Indianapolis, IN 46202 Objective: Gender differences exist in the myocardial response to acute ischemia-reperfusion (I/R) injury. These differences may be attributable to the effects of the sex hormones estrogen and testosterone. However, little information exists regarding the effect of testosterone on myocardial injury. Based on the negative effect of chronic endogenous testosterone from our previous study, we hypothesized that chronic exogenous testosterone may exert deleterious effects on myocardial function following I/R. Methods: Langendorff perfused rat hearts were subjected to 25 min ischemia and 40 min reperfusion (I/R) and left ventricular developed pressure (LVDP) was recorded. Groups (N = 4 –8/group): castrated males, ovariectomized (OVX) females, senescent females, and all them with chronic 5alphadihydrotestosterone (DHT) replacement therapy. Data were analyzed with ANOVA, P < 0.05 = statistically significant. Results: Chronic 5alpha-dihydrotestosterone replacement therapy did not improve or worsen recovery of LVDP after 40 min reperfusion in castrated males (65.1 T 8.13% vs. 66.3 T 4.54%), OVX females (64.5 T 10.6 vs. 50.2 T 5.97) and senescent females (42.1 T 0.04% vs. 41 T 0.05%). However, it is interesting that DHT treatment resulted in significantly (P < 0.05) increased LVDP after only 10 min reperfusion in castrated males, OVX females and senescent females compared to their untreated counterparts (54.8 T 11.9% vs. 32.9 T 5.75%; 66.7 T 11.5% vs. 43.1 T 8.15%, 53.4 T 10.1% vs. 32.9 T 5.75%, respectively). Conclusion: Chronic exogenous testosterone did not exert deleterious or beneficial effects on myocardial function following 25 min ischemia and 40 min reperfusion. However, chronic DHT treatment may have some protective effect on myocardium against 25 min ischemia. doi:10.1016/j.yjmcc.2006.03.412
A120. Activation of the calcineurin-NFAT pathway in desminopathy mouse hearts: The role of proteasomal malfunction Mingxin Tang a, Katherine Horak a, Hanqiao Zheng a, Jeffery Molkentin b, Xuejun Wang. University of South Dakota. * University of Cincinnati Desminopathy is a myopathy caused by mutations in the desmin gene. It is featured by the presence of abnormal desmin aggregates in myocytes. Mouse models of cardiac desminopathy develop cardiac hypertrophy and dysfunction, recapitulating the human disease. Although desminopathy does not appear to be very common desminopathy mice serve an important model for studying the pathogenesis of aberrant protein aggregation which has been frequently observed in congestive heart failure resulting from common forms of heart disease. We recently discovered that the proteolytic function of the ubiquitin-proteasome system (UPS) in cardiomyocytes is severely impaired by abnormal protein aggregation. The degradation of calcineurin depends upon the UPS. To elucidate the pathogenic role of UPS malfunction, the present study tests the hypothesis that UPS malfunction causes cardiac remodeling and failure by activating or potentiating calcineurin-NFATsignaling. We have found that calcineurin protein levels were significantly increased in desminopathy mouse hearts. To test whether the increased calcineurin actually leads to NFATmediated transcription activation, we cross-bred the desminopathy mice with NFAT reporter mice and we found that NFAT transactivation was significantly increased in desminopathy mouse hearts. To demonstrate that UPS malfunction is sufficient to activate the calcineurin-NFAT pathway, we treated the NFAT reporter mice systemically with a specific proteasome inhibitor (MG-262). This systemic proteasome inhibition significantly increased cardiac NFAT transactivation in the NFAT reporter mice. These new findings suggest that the calcineurin-NFAT pathway is activated in the heart of desminopathy mice and proteasomal malfunction may be responsible. Additional experiments using cultured cardiomyocytes are ongoing to test the hypothesis further. doi:10.1016/j.yjmcc.2006.03.413
A119. Cardioprotective effects of exercise training on the altered SR CA2+-uptake function in ovariectomized rats Jitanan Laosiripisan, Jonggonnee Wattanapermpool. Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Hormone replacement therapy in postmenopausal women has been shown to induce many unfavorable effects including the cardiovascular defects which then urge the search of alternatives for prevention and therapy. The present study tested the cardio-protective effects of exercise training on changes in the SR Ca2+-uptake activity in 10-wk ovariectomized (OVX) rats. A nine-wk running program of moderate intensity on a motor-driven treadmill was introduced to the exercise groups one week after surgery. The SR Ca2+-uptake and the SR Ca2+ATPase (SERCA) activities were analysed using left ventricular
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
homogenates and left ventricular SR vesicles, respectively. Significant reductions in maximum SR Ca2+-uptake and maximum SERCA activities with increases in the Ca2+ sensitivity demonstrated in OVX hearts were completely abolished by exercise training. Immunoblot analyses of SERCA and its regulatory protein, phospholamban (PLB), further demonstrated a downregulation of SERCA in OVX hearts, in which could be prevented by exercise training. While there was no change in the expression of PLB, a significant increase in the monomeric PLB, an supra-inhibitory form, was demonstrated in OVX hearts in which exercise training could prevent the change. Quantification of the phosphorylated PLB either at Ser16 or Thr17 site using immunoblot revealed a significant reduction in the phospho-Thr17 PLB in OVX hearts. As expected exercise training could completely prevent the change. These results clearly indicate the cardioprotective effects of exercise training on the altered SR Ca2+-uptake activity in OVX hearts via restorations of both the amount and the activity of SERCA. doi:10.1016/j.yjmcc.2006.03.414
A48. Lysosomotropic and antioxidant mechanisms of protection by beta blockers William B. Weglicki, Jay H. Kramer, Andrei M. Komarov, Joanna J. Chmielinska, I. Tong Mak. Div. Expl. Med., Depts. Med., and Biochem. and Mol. Biol., George Washington Univ., Washington, DC Previous studies have reported greater antioxidant activity of lipophilic vs hydrophilic beta receptor blockers, and attributed this, in part, to biophysical membrane stabilization. Recent data indicate that lipophilic intercalation of propranolol into cellular endosomal/lysosomal compartments may contribute significantly to an important indirect antioxidant property of this agent involving lysosomal iron stabilization. Using cultured endothelial cells, the ability of propranolol to concentrate into acidic vesicles resulted in a more alkaline lysosomal pH due to the effect of the amino side chain of the drug. When these cells were exposed to exogenous reactive oxygen species (ROS), significant preservation against loss of glutathione and cellular viability was observed. Using both in vitro endothelial cell and macrophage models, we found significant inhibition by propranolol (compared to atenolol) of iron dextran uptake and release that correlated with protection against exogenous ROS exposure. Since the non-beta blocking enantiomer, D-propranolol, and D, L-propranolol had equal efficacies, the protective effect must be independent of beta receptor blockade. When isolated, perfused iron-overloaded rat hearts were exposed to ischemia / reperfusion conditions, acute D-propranolol pretreatment significantly prevented lipid peroxidation, tissue iron and lysosomal N-acetyl-beta-glucosaminidase (NAGA) release, and improved recovery of cardiac output. Collectively, these findings support a non-beta blocking, lysosomotropic mechanism of cardioprotection by propranolol, which involves myocardial iron stabilization in the presence of oxidative stress. We submit that clinically-used lipophilic beta blockers having
911
such indirect antioxidant properties, may prove to be substantially protective against cardiovascular disorders in which acute and chronic iron-mediated oxidative stress is part of the pathobiology. Supported in part by NIH grants HL066226 and HL065178. doi:10.1016/j.yjmcc.2006.03.425
A30. Identification of KATP channels as mediators of antihypertrophic effects of adenosine receptor activation Y. Xia, S. Javadov, X.T. Gan, M.A. Cook, M. Karmazyn. Dept. of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada Recent evidence suggests that both adenosine receptor (AR) and KATP channel activation exert antihypertrophic effects in cardiac myocytes. We studied the relative contributions of mitochondrial KATP (mitoKATP) and sarcolemmal KATP(sarcKATP) to the antihypertrophic effects of ARs in cultured neonatal rat ventricular myocytes. The A1R agonist CPA, A2aR agonist CGS21680 and the A3R agonist IB-MECA all prevented phenylephrine (PE)-induced hypertrophy. Glibenclamide, a nonselective KATP channel blocker reversed the antihypertrophic effect of all three AR agonists as determined by cell size and, ANP expression and early c-fos upregulation. In contrast, the mitoKATP blocker 5-HD selectively attenuated the effect of CGS and IBMECA whereas HMR 1098, a specific blocker of sarcKATP, only abolished the antihypertrophic effect of CPA. When used at 5% of the original concentrations a greater than additive antihypertrophic effect was observed when either CGS or IB-MECA were studied in combination with the mitoKATP opener (80 T 13% increase for CGS+ diazoxide and 93 T 10% for IB-MECA+diazoxide) whereas a combination of CPA + diazoxide(36 T 10%) failed to produce effects greater than those seen with either drug alone. Moreover, both CGS and IB-MECA but not CPA decreased the mitochondrial membrane potential when PE was present similarly to that seen with diazoxide and both agents inhibited PE-stimulated elevation in mitochondrial Ca2+. Our data strongly suggest that AR-mediated antihypertrophic effects are mediated by distinct KATP channels with sarcKATP mediating the antihypertrophic effects of A1R activation and mitoKATP mediating the antihypertrophic effects of both A2R and A3R agonists. doi:10.1016/j.yjmcc.2006.03.416
A109. Voltage-dependent hysteresis of human pacemaker channels expressed in hek293 cells Yong-Fu Xiao a, Joshua J. Wilhelm a, Erica M. TenBroek a, Paul A. Iaizzo b, Daniel C. Sigg a. a Medtronic Inc., Minneapolis, MN. b University of Minnesota, Minneapolis, MN Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels in the heart modulate cardiac rhythm via the hyperpolarization-activated cation current (If). To date, four mammalian HCN molecular isoforms (HCN1-4) have been
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
slope of Gd-DTPA was significantly greater in infarct region than in normal regions, suggesting that less contrast reagent diffused out of the capillaries in infarct region during its first pass. This may be an indication of decreased capillary permeability. It was also found that T1 relaxation times measured during 30-minute contrast perfusion were significantly shorter in infarct than in normal myocardium. This suggests that distribution volume for the contrast reagent was still significantly greater in chronic infarct region than in normal region. Our results demonstrate that both T*- and Tweighted imaging techniques can be used to delineate chronic myocardial infarction. doi:10.1016/j.yjmcc.2006.03.411
A132. The role of chronic exogenous testosterone in myocardial function following acute ischemia-reperfusion Meijing Wang, Paul Crisostomo, Christine Herring, Daniel R. Meldrum. Indiana Univ. School of Medicine, Indianapolis, IN 46202 Objective: Gender differences exist in the myocardial response to acute ischemia-reperfusion (I/R) injury. These differences may be attributable to the effects of the sex hormones estrogen and testosterone. However, little information exists regarding the effect of testosterone on myocardial injury. Based on the negative effect of chronic endogenous testosterone from our previous study, we hypothesized that chronic exogenous testosterone may exert deleterious effects on myocardial function following I/R. Methods: Langendorff perfused rat hearts were subjected to 25 min ischemia and 40 min reperfusion (I/R) and left ventricular developed pressure (LVDP) was recorded. Groups (N = 4 –8/group): castrated males, ovariectomized (OVX) females, senescent females, and all them with chronic 5alphadihydrotestosterone (DHT) replacement therapy. Data were analyzed with ANOVA, P < 0.05 = statistically significant. Results: Chronic 5alpha-dihydrotestosterone replacement therapy did not improve or worsen recovery of LVDP after 40 min reperfusion in castrated males (65.1 T 8.13% vs. 66.3 T 4.54%), OVX females (64.5 T 10.6 vs. 50.2 T 5.97) and senescent females (42.1 T 0.04% vs. 41 T 0.05%). However, it is interesting that DHT treatment resulted in significantly (P < 0.05) increased LVDP after only 10 min reperfusion in castrated males, OVX females and senescent females compared to their untreated counterparts (54.8 T 11.9% vs. 32.9 T 5.75%; 66.7 T 11.5% vs. 43.1 T 8.15%, 53.4 T 10.1% vs. 32.9 T 5.75%, respectively). Conclusion: Chronic exogenous testosterone did not exert deleterious or beneficial effects on myocardial function following 25 min ischemia and 40 min reperfusion. However, chronic DHT treatment may have some protective effect on myocardium against 25 min ischemia. doi:10.1016/j.yjmcc.2006.03.412
A120. Activation of the calcineurin-NFAT pathway in desminopathy mouse hearts: The role of proteasomal malfunction Mingxin Tang a, Katherine Horak a, Hanqiao Zheng a, Jeffery Molkentin b, Xuejun Wang. University of South Dakota. * University of Cincinnati Desminopathy is a myopathy caused by mutations in the desmin gene. It is featured by the presence of abnormal desmin aggregates in myocytes. Mouse models of cardiac desminopathy develop cardiac hypertrophy and dysfunction, recapitulating the human disease. Although desminopathy does not appear to be very common desminopathy mice serve an important model for studying the pathogenesis of aberrant protein aggregation which has been frequently observed in congestive heart failure resulting from common forms of heart disease. We recently discovered that the proteolytic function of the ubiquitin-proteasome system (UPS) in cardiomyocytes is severely impaired by abnormal protein aggregation. The degradation of calcineurin depends upon the UPS. To elucidate the pathogenic role of UPS malfunction, the present study tests the hypothesis that UPS malfunction causes cardiac remodeling and failure by activating or potentiating calcineurin-NFATsignaling. We have found that calcineurin protein levels were significantly increased in desminopathy mouse hearts. To test whether the increased calcineurin actually leads to NFATmediated transcription activation, we cross-bred the desminopathy mice with NFAT reporter mice and we found that NFAT transactivation was significantly increased in desminopathy mouse hearts. To demonstrate that UPS malfunction is sufficient to activate the calcineurin-NFAT pathway, we treated the NFAT reporter mice systemically with a specific proteasome inhibitor (MG-262). This systemic proteasome inhibition significantly increased cardiac NFAT transactivation in the NFAT reporter mice. These new findings suggest that the calcineurin-NFAT pathway is activated in the heart of desminopathy mice and proteasomal malfunction may be responsible. Additional experiments using cultured cardiomyocytes are ongoing to test the hypothesis further. doi:10.1016/j.yjmcc.2006.03.413
A119. Cardioprotective effects of exercise training on the altered SR CA2+-uptake function in ovariectomized rats Jitanan Laosiripisan, Jonggonnee Wattanapermpool. Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Hormone replacement therapy in postmenopausal women has been shown to induce many unfavorable effects including the cardiovascular defects which then urge the search of alternatives for prevention and therapy. The present study tested the cardio-protective effects of exercise training on changes in the SR Ca2+-uptake activity in 10-wk ovariectomized (OVX) rats. A nine-wk running program of moderate intensity on a motor-driven treadmill was introduced to the exercise groups one week after surgery. The SR Ca2+-uptake and the SR Ca2+ATPase (SERCA) activities were analysed using left ventricular
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
homogenates and left ventricular SR vesicles, respectively. Significant reductions in maximum SR Ca2+-uptake and maximum SERCA activities with increases in the Ca2+ sensitivity demonstrated in OVX hearts were completely abolished by exercise training. Immunoblot analyses of SERCA and its regulatory protein, phospholamban (PLB), further demonstrated a downregulation of SERCA in OVX hearts, in which could be prevented by exercise training. While there was no change in the expression of PLB, a significant increase in the monomeric PLB, an supra-inhibitory form, was demonstrated in OVX hearts in which exercise training could prevent the change. Quantification of the phosphorylated PLB either at Ser16 or Thr17 site using immunoblot revealed a significant reduction in the phospho-Thr17 PLB in OVX hearts. As expected exercise training could completely prevent the change. These results clearly indicate the cardioprotective effects of exercise training on the altered SR Ca2+-uptake activity in OVX hearts via restorations of both the amount and the activity of SERCA. doi:10.1016/j.yjmcc.2006.03.414
A48. Lysosomotropic and antioxidant mechanisms of protection by beta blockers William B. Weglicki, Jay H. Kramer, Andrei M. Komarov, Joanna J. Chmielinska, I. Tong Mak. Div. Expl. Med., Depts. Med., and Biochem. and Mol. Biol., George Washington Univ., Washington, DC Previous studies have reported greater antioxidant activity of lipophilic vs hydrophilic beta receptor blockers, and attributed this, in part, to biophysical membrane stabilization. Recent data indicate that lipophilic intercalation of propranolol into cellular endosomal/lysosomal compartments may contribute significantly to an important indirect antioxidant property of this agent involving lysosomal iron stabilization. Using cultured endothelial cells, the ability of propranolol to concentrate into acidic vesicles resulted in a more alkaline lysosomal pH due to the effect of the amino side chain of the drug. When these cells were exposed to exogenous reactive oxygen species (ROS), significant preservation against loss of glutathione and cellular viability was observed. Using both in vitro endothelial cell and macrophage models, we found significant inhibition by propranolol (compared to atenolol) of iron dextran uptake and release that correlated with protection against exogenous ROS exposure. Since the non-beta blocking enantiomer, D-propranolol, and D, L-propranolol had equal efficacies, the protective effect must be independent of beta receptor blockade. When isolated, perfused iron-overloaded rat hearts were exposed to ischemia / reperfusion conditions, acute D-propranolol pretreatment significantly prevented lipid peroxidation, tissue iron and lysosomal N-acetyl-beta-glucosaminidase (NAGA) release, and improved recovery of cardiac output. Collectively, these findings support a non-beta blocking, lysosomotropic mechanism of cardioprotection by propranolol, which involves myocardial iron stabilization in the presence of oxidative stress. We submit that clinically-used lipophilic beta blockers having
911
such indirect antioxidant properties, may prove to be substantially protective against cardiovascular disorders in which acute and chronic iron-mediated oxidative stress is part of the pathobiology. Supported in part by NIH grants HL066226 and HL065178. doi:10.1016/j.yjmcc.2006.03.425
A30. Identification of KATP channels as mediators of antihypertrophic effects of adenosine receptor activation Y. Xia, S. Javadov, X.T. Gan, M.A. Cook, M. Karmazyn. Dept. of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada Recent evidence suggests that both adenosine receptor (AR) and KATP channel activation exert antihypertrophic effects in cardiac myocytes. We studied the relative contributions of mitochondrial KATP (mitoKATP) and sarcolemmal KATP(sarcKATP) to the antihypertrophic effects of ARs in cultured neonatal rat ventricular myocytes. The A1R agonist CPA, A2aR agonist CGS21680 and the A3R agonist IB-MECA all prevented phenylephrine (PE)-induced hypertrophy. Glibenclamide, a nonselective KATP channel blocker reversed the antihypertrophic effect of all three AR agonists as determined by cell size and, ANP expression and early c-fos upregulation. In contrast, the mitoKATP blocker 5-HD selectively attenuated the effect of CGS and IBMECA whereas HMR 1098, a specific blocker of sarcKATP, only abolished the antihypertrophic effect of CPA. When used at 5% of the original concentrations a greater than additive antihypertrophic effect was observed when either CGS or IB-MECA were studied in combination with the mitoKATP opener (80 T 13% increase for CGS+ diazoxide and 93 T 10% for IB-MECA+diazoxide) whereas a combination of CPA + diazoxide(36 T 10%) failed to produce effects greater than those seen with either drug alone. Moreover, both CGS and IB-MECA but not CPA decreased the mitochondrial membrane potential when PE was present similarly to that seen with diazoxide and both agents inhibited PE-stimulated elevation in mitochondrial Ca2+. Our data strongly suggest that AR-mediated antihypertrophic effects are mediated by distinct KATP channels with sarcKATP mediating the antihypertrophic effects of A1R activation and mitoKATP mediating the antihypertrophic effects of both A2R and A3R agonists. doi:10.1016/j.yjmcc.2006.03.416
A109. Voltage-dependent hysteresis of human pacemaker channels expressed in hek293 cells Yong-Fu Xiao a, Joshua J. Wilhelm a, Erica M. TenBroek a, Paul A. Iaizzo b, Daniel C. Sigg a. a Medtronic Inc., Minneapolis, MN. b University of Minnesota, Minneapolis, MN Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels in the heart modulate cardiac rhythm via the hyperpolarization-activated cation current (If). To date, four mammalian HCN molecular isoforms (HCN1-4) have been