- Email: [email protected]
Cyclooxygenase-2 does not mediate late preconditioning against infarction induced by adenosine A1 or A3 receptors
MYOCARDIAL COLLAGEN AND FIDRONECTIN LEVELS IN PATIENTS WlTE PULMONARY ATRESIA AND VENTRICULAR SEPTAL DEFECT Lmnart Klompe, Thecdo~s H.F. Peters, Peter de Jong, Act J.J.C. Bogers and Hari S. Sharma.Erasmus University Medical Center, Rotterdam, The Netherlands
pulmonary atresia (PA) with ventricular se@al defect (VSD) and presence of systemic polmonary collateral arteries is an extreme form of teualogy of Fallot with characteristic right ventricular hypertrophy (RVH). To reduce the right ventricular overload sod the cyanotic situation, these children have to undergo corrective surgery to restore pulmonary perfusion by a modified BlalockTaussig shunt followed by the closing of shunt and VSD. It is not yet established whether afler these different interventions, RVH would regressto give rise normal cardiac performance. We studied the degree of fibrosis by analysing the myocarclial expression pattern (at mRNA and protein level) of the extracellular matrix proteins, collagen and fibronectio in biopsies from 14 Patients affected by PAiVSD. Expression analysis by RT-PCR showed significantly higher levels for collagen III @=0.03), whereas collagen Iu Q~0.31) and fibronectin @=0.47) mRNA levels remained unaltered in PNVSD patients as compared to age matched controls. Video image analysis of immooohistochemlcal staining showed unchanged interstitial levels for total collagen @=0.17) as well as for fibronectio @=0.13) in the patients with PANSD. However, peri-vascular staining for collagen @
CYCLOOXYGENASE-2 DOES NOT YEDlATE l&E PRECONDlTlONlNG AGAINST INFARCTION INDUCED BY ADENOSINE AI OR & RECEPTORS E&o Kcdani, Ken Sh!mmura, Yu-Ting Xuan, Hiishi Takano, John A. Auchampaoh, Xian-Liang Tang, Roberto Bolli Division of Cardiology, Univemity of Louisville and Jewish Head and Lung Institute, Louisville, KY
Recentstudies have demonstrated that the adenosine At receptor agonist 2-ohlor&@-oydopentyladenosine (CCPA) and the adenosine k receptor agonist Ns(3-iodobenzyl)adenosine-5’-Kmethyluronamide (IB-MECA) p&~ce a delayed phaseof protection against infarction similar to the late phase of isohemic preccnditioning (PC). However, the mechanism for adenosine AI or A3 receptor-induced late PC remains unknown. The goal of this study was to determine whether the delayed oardionroteothe effects of adenosine AI or A3 receptors are mediated b-y oydwxygenase-2 (COX2) which is an obligatory mediator of ischemic PC. We found that COX-2 protein expression (Western blotting) did not increase 24 h after administration of either CCPA (100 unrkc i.v.) or IB-MECA (306 u&a Lv.) compared with controls. To’ pmba ihe ‘mle of cons&&-CCX-2 expression, oonsdous rabbits were subjeoted to 36-min coronary occlusion followed by 72-h reperfusion. Twenty-four hours prior to the occlusion, the rabbits were pretreated with CCPA (100 pgrkg i.v.) or IB-MECA (300 pgn(g i.v.). Both CCPA and IB-MECA resulted in a marked (-47%) reduction in infarct size vs. controls (36.2*4.0% of the risk region [n=9], 31.2t4.71 [n=9], and 59.5*3.6% [n=9], respectively; P
A60
STRUCTURAL AND FUNCTIONAL PROPERTIES OF NORMAL, FAILING AND FOBTAL HUMAN HEART TROPONIN Adam Knott, Ian Purcell, Steven Marston. Dept of Cardiac Medicine, Imperial College School of Medlclne, National Heart and Lung Institute, London, UK We have directly investigated the StNdIJd and functional properties of trcponin extracted from normal, failing and foetal hearts. Using the in vitro motility assay we found that thin filaments reconstituted with normal heart trcpcnin increased filament velocity by 52k2%, foetal hearts increased thin filament velocity by 194% and failing heart reconstituted thin filaments produced an intermediate increase. Thin filaFenta reconstituted with failing heart trcpcnin ware more Ca sensitive than normal (ApCa50=0.24*0.54 of a pCa unit for the fraction of filaments motile and 0.26kO.15 of a pCa unit for the filament velocity). Thin filamer@ reconstituted with f&cl heart troponin were even more Ca sensitive than normal (ApCaSO= 0.34kO.05 of a pCa unit for the fraction of filaments motile and 0.37kO.16 of a pCa unit for filament velocity). Western blots revealed no difference in troponin T isofon expression in tropcnin from normal and failing hearts, with T3 being the only isoform. PKA phosphorylation of tropcnin I causes a decrease in thin filament calcium sensitivity of filament movement in the in vitro motility assay (ApCa50=0.23 of a pCa unit), but we observed no significant difference in the level of PKA phosphorylation of troponin I from normal and failing hearts: Trcponiri preparatioris contained a variable amount of N-terminally cleaved troponin I, but this product was not incorporated into the thin filament. We conclude that functional differences exist between thin filaments reconstituted with troponin from normal and failing hearts but these differences are not due to troponin T isoform expression differences, decreased PKA phosphorylation of tropcnin I or degradation of troponin I.
STRUCTURAL CORRELATE OF ATRIAL FIBRILLATION IN HUMAN HEARTS Sawa Kostin, Gabi Klein, Zoltan Szalay*, Erwin P. Bauer*, Jutta Shaper, Max Planck Institute, *Kerckhoff-Clinic Bad Nauheim, Germany The pathomechanism and structural correlate of atria1 fibrillation are largely unknown. We tested the hypothesis that changes in the gap junction (GJ) proteins, connexin 43 (Cx43) and Cx40, and an increase in fibrosis might be the cause for arrhythmias. In 31 patients (pts) undergoing a MAZE procedure, biopsies were taken intraoperatively from the right atrium or the auricular appendage, pts with sinus rhythm served as control. The ouantitv of Cx43 and Cx40 was measured in the confocal micrbscope as number of positive pixels expressed as % per cell area. The degree of fibrosis was quantified by morphometry using collagen I staining. Cx43 was decreased 25% in MAZE nts as comnared to
control (control 4.44%, MAZE 3:34%). (k-40 was
reduced 10% (5.38% in control, 4.9% in MAZE). The number of lateral Cx43 and Cx40 GJ was increased in
MAZE pts as compared to control. Quantitative differences between atria and auricular tissue were absent in both groups. Collagen I was 52% higher in MAZE pts than in control (4.74% in control, 7.21% in MAZE). Conclusion: The structural correlate of atria1 fibrillation is two-compartimental: 1. Intracellular, characterized by reduction of Cx43 and Cx40, snatial redistribution of GJ. 2. Extracellular. arc
and bv
augmentation of fibrosis. Both ‘alterations contribute to self-perpetuating re-entry pathways and atria1 arrhythmias.
pulmonary atresia (PA) with ventricular se@al defect (VSD) and presence of systemic polmonary collateral arteries is an extreme form of teualogy of Fallot with characteristic right ventricular hypertrophy (RVH). To reduce the right ventricular overload sod the cyanotic situation, these children have to undergo corrective surgery to restore pulmonary perfusion by a modified BlalockTaussig shunt followed by the closing of shunt and VSD. It is not yet established whether afler these different interventions, RVH would regressto give rise normal cardiac performance. We studied the degree of fibrosis by analysing the myocarclial expression pattern (at mRNA and protein level) of the extracellular matrix proteins, collagen and fibronectio in biopsies from 14 Patients affected by PAiVSD. Expression analysis by RT-PCR showed significantly higher levels for collagen III @=0.03), whereas collagen Iu Q~0.31) and fibronectin @=0.47) mRNA levels remained unaltered in PNVSD patients as compared to age matched controls. Video image analysis of immooohistochemlcal staining showed unchanged interstitial levels for total collagen @=0.17) as well as for fibronectio @=0.13) in the patients with PANSD. However, peri-vascular staining for collagen @
CYCLOOXYGENASE-2 DOES NOT YEDlATE l&E PRECONDlTlONlNG AGAINST INFARCTION INDUCED BY ADENOSINE AI OR & RECEPTORS E&o Kcdani, Ken Sh!mmura, Yu-Ting Xuan, Hiishi Takano, John A. Auchampaoh, Xian-Liang Tang, Roberto Bolli Division of Cardiology, Univemity of Louisville and Jewish Head and Lung Institute, Louisville, KY
Recentstudies have demonstrated that the adenosine At receptor agonist 2-ohlor&@-oydopentyladenosine (CCPA) and the adenosine k receptor agonist Ns(3-iodobenzyl)adenosine-5’-Kmethyluronamide (IB-MECA) p&~ce a delayed phaseof protection against infarction similar to the late phase of isohemic preccnditioning (PC). However, the mechanism for adenosine AI or A3 receptor-induced late PC remains unknown. The goal of this study was to determine whether the delayed oardionroteothe effects of adenosine AI or A3 receptors are mediated b-y oydwxygenase-2 (COX2) which is an obligatory mediator of ischemic PC. We found that COX-2 protein expression (Western blotting) did not increase 24 h after administration of either CCPA (100 unrkc i.v.) or IB-MECA (306 u&a Lv.) compared with controls. To’ pmba ihe ‘mle of cons&&-CCX-2 expression, oonsdous rabbits were subjeoted to 36-min coronary occlusion followed by 72-h reperfusion. Twenty-four hours prior to the occlusion, the rabbits were pretreated with CCPA (100 pgrkg i.v.) or IB-MECA (300 pgn(g i.v.). Both CCPA and IB-MECA resulted in a marked (-47%) reduction in infarct size vs. controls (36.2*4.0% of the risk region [n=9], 31.2t4.71 [n=9], and 59.5*3.6% [n=9], respectively; P
A60
STRUCTURAL AND FUNCTIONAL PROPERTIES OF NORMAL, FAILING AND FOBTAL HUMAN HEART TROPONIN Adam Knott, Ian Purcell, Steven Marston. Dept of Cardiac Medicine, Imperial College School of Medlclne, National Heart and Lung Institute, London, UK We have directly investigated the StNdIJd and functional properties of trcponin extracted from normal, failing and foetal hearts. Using the in vitro motility assay we found that thin filaments reconstituted with normal heart trcpcnin increased filament velocity by 52k2%, foetal hearts increased thin filament velocity by 194% and failing heart reconstituted thin filaments produced an intermediate increase. Thin filaFenta reconstituted with failing heart trcpcnin ware more Ca sensitive than normal (ApCa50=0.24*0.54 of a pCa unit for the fraction of filaments motile and 0.26kO.15 of a pCa unit for the filament velocity). Thin filamer@ reconstituted with f&cl heart troponin were even more Ca sensitive than normal (ApCaSO= 0.34kO.05 of a pCa unit for the fraction of filaments motile and 0.37kO.16 of a pCa unit for filament velocity). Western blots revealed no difference in troponin T isofon expression in tropcnin from normal and failing hearts, with T3 being the only isoform. PKA phosphorylation of tropcnin I causes a decrease in thin filament calcium sensitivity of filament movement in the in vitro motility assay (ApCa50=0.23 of a pCa unit), but we observed no significant difference in the level of PKA phosphorylation of troponin I from normal and failing hearts: Trcponiri preparatioris contained a variable amount of N-terminally cleaved troponin I, but this product was not incorporated into the thin filament. We conclude that functional differences exist between thin filaments reconstituted with troponin from normal and failing hearts but these differences are not due to troponin T isoform expression differences, decreased PKA phosphorylation of tropcnin I or degradation of troponin I.
STRUCTURAL CORRELATE OF ATRIAL FIBRILLATION IN HUMAN HEARTS Sawa Kostin, Gabi Klein, Zoltan Szalay*, Erwin P. Bauer*, Jutta Shaper, Max Planck Institute, *Kerckhoff-Clinic Bad Nauheim, Germany The pathomechanism and structural correlate of atria1 fibrillation are largely unknown. We tested the hypothesis that changes in the gap junction (GJ) proteins, connexin 43 (Cx43) and Cx40, and an increase in fibrosis might be the cause for arrhythmias. In 31 patients (pts) undergoing a MAZE procedure, biopsies were taken intraoperatively from the right atrium or the auricular appendage, pts with sinus rhythm served as control. The ouantitv of Cx43 and Cx40 was measured in the confocal micrbscope as number of positive pixels expressed as % per cell area. The degree of fibrosis was quantified by morphometry using collagen I staining. Cx43 was decreased 25% in MAZE nts as comnared to
control (control 4.44%, MAZE 3:34%). (k-40 was
reduced 10% (5.38% in control, 4.9% in MAZE). The number of lateral Cx43 and Cx40 GJ was increased in
MAZE pts as compared to control. Quantitative differences between atria and auricular tissue were absent in both groups. Collagen I was 52% higher in MAZE pts than in control (4.74% in control, 7.21% in MAZE). Conclusion: The structural correlate of atria1 fibrillation is two-compartimental: 1. Intracellular, characterized by reduction of Cx43 and Cx40, snatial redistribution of GJ. 2. Extracellular. arc
and bv
augmentation of fibrosis. Both ‘alterations contribute to self-perpetuating re-entry pathways and atria1 arrhythmias.