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KATP channel openers preserve mitochondria from reoxygenation injury
SlOOB MODULATES CARDIOVASCULAR CATECHOLAMINE
THE ACUTE RESPONSE STIMULATION.
AND TO
CHRONIC
Christopher B. Overgaard, Hahn Hoe Kim, James Alexander Marks, and Thomas G. Parker. University Toronto, Canada
N. Tsoporis, of Toronto,
The calcium binding protein S 1008, normally expressed in neuronal tissue is induced in the heart and vasculature by catecholamine stimulation. Overexpression of SlOOB in the heart in a transgenic (TG) mouse model inhibits the normal hypertrophic response to catecholamine stimulation. In this study we examined the role of SIOOB in the acute and chronic hemodynamic response to norepinephrine (NE) stimulation. Knockout (KO) mice devoid of the SIOOB gene, TG mice with forced over-expression of SIOOB, and control CD1 mice were injected daily with NE for 4 weeks. Acute and chronic blood pressure and heart rate responses were measured by carotid arterial cannulation and tail cuff recordings, respectively. Left ventricular (LV)/body weight (BW) ratios were measured to assess cardiac hypertrophy. NE significantly increased LV/BW ratios in CD1 (20 %) and KO (25 %) mice. but not in TG mice. Chronic NE treatment r<ed in a sig&ant increase in tail cuff mean arterial pressure in CD1 mice [112+4 (NE) vs. 9055 (vehicle) mmHg, p=O.O08, n=6), but was not increased in KO mice [89+9 (NE) vi. 92+4 (vehicle) mmHg p=O.82, n=6). Furthermore, the acute increase in blood pressure in response to NE was significantly attenuated in KO mice (35+8 % increase, n=6) compared with CDI mice (77+14 % increase, n=8) [p
A88
FLWASlAllN IMPROVES THE THROMBOTlC AND FIBRlNOLYllC BALANCE IN VASCULAR ENDOTHELlAL CELLS Kazuyukl Ozakl, Tarhi Yamamoto’, Takaharu lshlbashi t , Taku Matsubam, Matomo Nlshlo t & YashlWa Aizawa. Fimt Depfartmnt of Internal Mdlclne, Weqartment oi Renal Pathology, Instltuta of Nephrology, Bchool of Medlclne, Nllgata Unlvemlty, Nllgata, t Depaltment of Pharmacology, Kanazawa MedIcal Unhmmlty, Iahlkawa, Japan. Benefiil effect of fluvastatin (FLU), a HMG-CoA reduotase inhibitor, on balance betmeen endotheliil thrombotfo and fibrinolytic properties was investigated using human umbilical vein endothellal cells (HUVECs). HUVECs were incubated with 0.01, 0.1, or 1.0 pm&L of FLU or vehicb for 12 hours. As anti-thrombotii properties, eNOS mRNA expression in HUVECs and nitrite accumulation in culture medium was examined by RNase protediin assay and the HPLC-Griess system, respectively. PAIproduction as a thrombotic measure, and tPA, TM and TFPI production as fibrinofytii measures were determined by ELISA. Expression of eNOS mRNA and nitrite accumulaliin were significantly increased by FLU (276*38% and 165+359/o of the control at 1.0 ptnolh, respectively), while PAI-I production was signifiintly decreased (SOf8.2?~ and 82*&l% of the control at 0.1 and 1.0 pmol/L, respectively). tPA and TM produotiin was significantly increased (115_+10% and 134*2Pb of the control at 1.0 flmolA, respectively), and TFPI production was significantly decreased by FLU in a concentration-dependent manner (74-1 7.5% of the oontrol at 1 .O pmoWL). FLU increased anti-thrombotii substance (NO) and fibrinolytic substances (tPA and TM), while it decreased thrombotic substance (PA&l). These results indicate that FLU improved endotheliil thrombotic and fibrinolytic balance. Decreased TFPI may reflect improvement of endothelial function in the present situation.
MITOCHONDRIAL PRODUCTION OF FREE RADICALSBLUNTEDBY K~rp CHANNELOPENERS Cevher Ozcan, Petras P. Dzeja, Martin Blenengraeber, Andre Terzic.Mayo Clinic, Rochester,MN, USA
k,TPCHANNELOPENERSPRESERVE YITOCHONDRIA FROMREOXYGENATION INJURY CevherOzcan,Martin Bienengraeber,Andre Tenic. Mayo Clinic, Rochester,MN, USA
Excessiveproductionof reactiveoxygenspecies(ROS)by the mitochondrialrespiratorychain precipitatescell injury. KATPchannel openers which target mitochondriapossess cardioprotectiveproperties,but it is controversialwhether they regulate ROS production. Here, mitochondriawere isolatedfrom rat hearts, and subjectedto 20-min anoxia followedby 20-min reoxygenation.Generationof ROS was demonstrated by DCFH fluorescence, and release of cytochrome c, a marker of mitochondrial injury, by immunoblotting. Anoxialreoxygenation suppressed mitochondrial respiration and ATP production, while increasinggenerationof ROS and releaseof cytochromec. %TP channel openers, diazoxideor nicorandil,prevented ROS production, reduced cytochrome c release, and presentedrespirationand ATP.The KNPchannelblocker,5hydroxydecanoicacid, abolished the effects of openers. Conversely, the protective effects of openers were maintained in K+ free medium and in the presence or absenceof the K+ ionophore,valinomycin.Opener effects were mimickedby ROS scavengers,superoxidedismutase and catalase, as well as by malonate, an inhibitor of succinate dehydrogenase. Thus, modulation of ROS production by bTP channel openers may contribute to mitochondrialprotection.
This studyevaluatesthe protectiveefficacyof bTP channel openers on mitochondrialoxidative phosphorylationand structural integrity following anoxialreoxygenation, Mitochondriawere isolatedfrom rat hearts,and subjectedto anoxia followed by reoxygenation (up to 20 min). Anoxialreoxygenation decreasedthe rate of ADP-stimulated oxygen consumption and inhibited ATP production with aggravationof energeticinjuryproportionalto the durationof reoxygenation. k&P channel openers, diazoxide and nicorandil,present throughout anoxialreoxygenation, or at the onset of reoxygenation, preserved ADP-stimulated respirationand ATP production from oxidative damage. Following anoxialreoxygenation,the majority (91%) of mitochondriawere damaged or swollen, with openers protectingthe integrityof up to 65% of the mitochondrial population.5Hydroxydecanoicacidabolishedthe protective effect of diazoxideand nicorandil.The presentstudy, thus, demonstrates that KATP channel openers prevent reoxygenation-induced functionaland structuraldeterioration of cardiac mitochondria. By increasing mitochondrial resistance, KNP channel openers may provide cardioprotectionunderoxidativestress.
THE ACUTE RESPONSE STIMULATION.
AND TO
CHRONIC
Christopher B. Overgaard, Hahn Hoe Kim, James Alexander Marks, and Thomas G. Parker. University Toronto, Canada
N. Tsoporis, of Toronto,
The calcium binding protein S 1008, normally expressed in neuronal tissue is induced in the heart and vasculature by catecholamine stimulation. Overexpression of SlOOB in the heart in a transgenic (TG) mouse model inhibits the normal hypertrophic response to catecholamine stimulation. In this study we examined the role of SIOOB in the acute and chronic hemodynamic response to norepinephrine (NE) stimulation. Knockout (KO) mice devoid of the SIOOB gene, TG mice with forced over-expression of SIOOB, and control CD1 mice were injected daily with NE for 4 weeks. Acute and chronic blood pressure and heart rate responses were measured by carotid arterial cannulation and tail cuff recordings, respectively. Left ventricular (LV)/body weight (BW) ratios were measured to assess cardiac hypertrophy. NE significantly increased LV/BW ratios in CD1 (20 %) and KO (25 %) mice. but not in TG mice. Chronic NE treatment r<ed in a sig&ant increase in tail cuff mean arterial pressure in CD1 mice [112+4 (NE) vs. 9055 (vehicle) mmHg, p=O.O08, n=6), but was not increased in KO mice [89+9 (NE) vi. 92+4 (vehicle) mmHg p=O.82, n=6). Furthermore, the acute increase in blood pressure in response to NE was significantly attenuated in KO mice (35+8 % increase, n=6) compared with CDI mice (77+14 % increase, n=8) [p
A88
FLWASlAllN IMPROVES THE THROMBOTlC AND FIBRlNOLYllC BALANCE IN VASCULAR ENDOTHELlAL CELLS Kazuyukl Ozakl, Tarhi Yamamoto’, Takaharu lshlbashi t , Taku Matsubam, Matomo Nlshlo t & YashlWa Aizawa. Fimt Depfartmnt of Internal Mdlclne, Weqartment oi Renal Pathology, Instltuta of Nephrology, Bchool of Medlclne, Nllgata Unlvemlty, Nllgata, t Depaltment of Pharmacology, Kanazawa MedIcal Unhmmlty, Iahlkawa, Japan. Benefiil effect of fluvastatin (FLU), a HMG-CoA reduotase inhibitor, on balance betmeen endotheliil thrombotfo and fibrinolytic properties was investigated using human umbilical vein endothellal cells (HUVECs). HUVECs were incubated with 0.01, 0.1, or 1.0 pm&L of FLU or vehicb for 12 hours. As anti-thrombotii properties, eNOS mRNA expression in HUVECs and nitrite accumulation in culture medium was examined by RNase protediin assay and the HPLC-Griess system, respectively. PAIproduction as a thrombotic measure, and tPA, TM and TFPI production as fibrinofytii measures were determined by ELISA. Expression of eNOS mRNA and nitrite accumulaliin were significantly increased by FLU (276*38% and 165+359/o of the control at 1.0 ptnolh, respectively), while PAI-I production was signifiintly decreased (SOf8.2?~ and 82*&l% of the control at 0.1 and 1.0 pmol/L, respectively). tPA and TM produotiin was significantly increased (115_+10% and 134*2Pb of the control at 1.0 flmolA, respectively), and TFPI production was significantly decreased by FLU in a concentration-dependent manner (74-1 7.5% of the oontrol at 1 .O pmoWL). FLU increased anti-thrombotii substance (NO) and fibrinolytic substances (tPA and TM), while it decreased thrombotic substance (PA&l). These results indicate that FLU improved endotheliil thrombotic and fibrinolytic balance. Decreased TFPI may reflect improvement of endothelial function in the present situation.
MITOCHONDRIAL PRODUCTION OF FREE RADICALSBLUNTEDBY K~rp CHANNELOPENERS Cevher Ozcan, Petras P. Dzeja, Martin Blenengraeber, Andre Terzic.Mayo Clinic, Rochester,MN, USA
k,TPCHANNELOPENERSPRESERVE YITOCHONDRIA FROMREOXYGENATION INJURY CevherOzcan,Martin Bienengraeber,Andre Tenic. Mayo Clinic, Rochester,MN, USA
Excessiveproductionof reactiveoxygenspecies(ROS)by the mitochondrialrespiratorychain precipitatescell injury. KATPchannel openers which target mitochondriapossess cardioprotectiveproperties,but it is controversialwhether they regulate ROS production. Here, mitochondriawere isolatedfrom rat hearts, and subjectedto 20-min anoxia followedby 20-min reoxygenation.Generationof ROS was demonstrated by DCFH fluorescence, and release of cytochrome c, a marker of mitochondrial injury, by immunoblotting. Anoxialreoxygenation suppressed mitochondrial respiration and ATP production, while increasinggenerationof ROS and releaseof cytochromec. %TP channel openers, diazoxideor nicorandil,prevented ROS production, reduced cytochrome c release, and presentedrespirationand ATP.The KNPchannelblocker,5hydroxydecanoicacid, abolished the effects of openers. Conversely, the protective effects of openers were maintained in K+ free medium and in the presence or absenceof the K+ ionophore,valinomycin.Opener effects were mimickedby ROS scavengers,superoxidedismutase and catalase, as well as by malonate, an inhibitor of succinate dehydrogenase. Thus, modulation of ROS production by bTP channel openers may contribute to mitochondrialprotection.
This studyevaluatesthe protectiveefficacyof bTP channel openers on mitochondrialoxidative phosphorylationand structural integrity following anoxialreoxygenation, Mitochondriawere isolatedfrom rat hearts,and subjectedto anoxia followed by reoxygenation (up to 20 min). Anoxialreoxygenation decreasedthe rate of ADP-stimulated oxygen consumption and inhibited ATP production with aggravationof energeticinjuryproportionalto the durationof reoxygenation. k&P channel openers, diazoxide and nicorandil,present throughout anoxialreoxygenation, or at the onset of reoxygenation, preserved ADP-stimulated respirationand ATP production from oxidative damage. Following anoxialreoxygenation,the majority (91%) of mitochondriawere damaged or swollen, with openers protectingthe integrityof up to 65% of the mitochondrial population.5Hydroxydecanoicacidabolishedthe protective effect of diazoxideand nicorandil.The presentstudy, thus, demonstrates that KATP channel openers prevent reoxygenation-induced functionaland structuraldeterioration of cardiac mitochondria. By increasing mitochondrial resistance, KNP channel openers may provide cardioprotectionunderoxidativestress.