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H2S Preconditioning-Induced PKC Activation Regulates Intracellular Calcium Handling in Rat Cardiomyocytes
S220
Abstracts
ABSTRACTS
BASIC SCIENCE – ELECTROPHYSIOLOGY 499 H2 S Preconditioning-Induced PKC Activation Regulates Intracellular Calcium Handling in Rat Cardiomyocytes Ting Ting Pan ∗ , Jin Song Bian Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117456, Singapore Objective: The present study was aimed to investigate the regulatory effect of Protein Kinase C (PKC) on intracellular Ca2+ handling in hydrogen sulfide (H2 S)-preconditioned cardiomyocytes and its consequent effects upon ischemia challenge. Methods: Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (a H2 S donor, 10−4 M) preconditioning (SP, 30 min). Intracellular Ca2+ was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia/reperfusion insults to indicate the extent of hypercontracture. Results: SP motivated translocation of PKC␣, and ␦ to membrane fraction, but only translocation of PKC and ␦ was abolished by an ATP-sensitive potassium (KATP) channel blocker, glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca2+ ] transients, which were reversed by a selective PKC inhibitor, chelerythrine. These data suggest that SP facilitated Ca2+ removal via both accelerating uptake of Ca2+ into sarcoplasmic reticulum (SR) and enhancing Ca2+ extrusion through Na+ /Ca2+ exchanger (NCX) in a PKC-dependant manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca2+ overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. Conclusion: We demonstrate for the first time that SP activates PKC␣, and ␦ in cardiomyocytes via different signaling mechanism. Such PKC activation, in turn, protects the heart against ischemia/reperfusion insults at least partly by ameliorating intracellular Ca2+ handling. doi:10.1016/j.hlc.2008.05.553 500 Electrophysiological Characterization of the Gregg Effect in the Isolated Perfused Rat Right Ventricle Douglas Kelly ∗ , Lorraine Mackenzie, David Saint University of Adelaide, Adelaide, SA, Australia Background: The Gregg effect (increased myocardial contractility in response to increased coronary pressure) may involve activation of stretch-activated ion channels (SACs). Whilst the inotropic responses are modulated by SAC inhibitors, the electrophysiological changes that may accompany the Gregg effect remain undefined.
Heart, Lung and Circulation 2008;17S:S219–S241
Methods: The right ventricle free wall was dissected from hearts of anaesthetised rats (350–400 g, n = 5) and perfused via the right coronary vessel at various flow rates in random order. Isolated ventricles were paced at 4 Hz with 10 ms, bi-polar pulses whilst contractions were suppressed with 20 mM BDM. Intracellular recordings were made using aluminosilicate glass microelectrodes filled with 3 M KCl having a resistance of >20 M. Results: Perfusion pressures were 21 ± 8 mmHg at 4 ml/min and up to 41 ± 9 mmHg at coronary flows of 16 ml/min. Action potential amplitude (82 ± 3.0 mV) and resting membrane potential (−73 ± 2.5 mV) did not change significantly with alterations in coronary flow (p > 0.05, ANOVA GLM). Action potential durations did not significantly change despite considerable increases in perfusion pressure, remaining at 11 ± 1.6, 25 ± 3.1 and 59 ± 6.3 ms for APD20 , 50 and 80, respectively. Conclusion: In the isolated, perfused rat right ventricle modulation of coronary flow rate (and hence perfusion pressure) was not associated with any electrophysiological changes. This suggests that activation of SACs in cardiac myocytes (at least to the extent which would change action potentials) is not involved in the Gregg effect. doi:10.1016/j.hlc.2008.05.554 CLINICAL CARDIOLOGY – ELECTROPHYSIOLOGY 501 Markers of Endothelial Function and Inflammation in Patients with Atrial Fibrillation: A Comparison Between Peripheral and Intracardiac Blood Samples Ross Roberts-Thomson ∗ , Scott Willoughby, Christopher Wong, Dennis Lau, Lorraine Mackenzie, Anthony Brooks, Glenn Young, Prashanthan Sanders Cardiovascular Research Institute, University of Adelaide, Adelaide, South Australia, Australia Introduction: Increased levels of the endothelial dysfunction/damage marker von Willebrand Factor (vWF) measured in peripheral blood is predictive of stroke in patients with atrial fibrillation (AF). Recently, peripheral levels of soluble CD40L (sCD40L), a platelet activationderived inflammatory marker, has been shown to predict vascular events in AF. Suggesting that interaction between the endothelium and platelets may play a role in the genesis of vascular events in AF. However, thrombus and spontaneous echo contrast per se are more commonly seen in the left atrium (LA). Whether there are site-specific modulations of vWF and sCD40L is not known. Methods: 19 consecutive patients (13 males) with paroxysmal AF undergoing ablation were studied. All patients ceased warfarin 7 days and enoxaparin 12 h prior to the procedure. Anticoagulated blood samples were obtained at the start of the procedure immediately after transseptal puncture from the femoral vein (FV), the left atria (LA) and the right atria (RA), prior to the administration of heparin. Plasma samples were prepared by centrifugation and then
Abstracts
ABSTRACTS
BASIC SCIENCE – ELECTROPHYSIOLOGY 499 H2 S Preconditioning-Induced PKC Activation Regulates Intracellular Calcium Handling in Rat Cardiomyocytes Ting Ting Pan ∗ , Jin Song Bian Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117456, Singapore Objective: The present study was aimed to investigate the regulatory effect of Protein Kinase C (PKC) on intracellular Ca2+ handling in hydrogen sulfide (H2 S)-preconditioned cardiomyocytes and its consequent effects upon ischemia challenge. Methods: Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (a H2 S donor, 10−4 M) preconditioning (SP, 30 min). Intracellular Ca2+ was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia/reperfusion insults to indicate the extent of hypercontracture. Results: SP motivated translocation of PKC␣, and ␦ to membrane fraction, but only translocation of PKC and ␦ was abolished by an ATP-sensitive potassium (KATP) channel blocker, glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca2+ ] transients, which were reversed by a selective PKC inhibitor, chelerythrine. These data suggest that SP facilitated Ca2+ removal via both accelerating uptake of Ca2+ into sarcoplasmic reticulum (SR) and enhancing Ca2+ extrusion through Na+ /Ca2+ exchanger (NCX) in a PKC-dependant manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca2+ overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. Conclusion: We demonstrate for the first time that SP activates PKC␣, and ␦ in cardiomyocytes via different signaling mechanism. Such PKC activation, in turn, protects the heart against ischemia/reperfusion insults at least partly by ameliorating intracellular Ca2+ handling. doi:10.1016/j.hlc.2008.05.553 500 Electrophysiological Characterization of the Gregg Effect in the Isolated Perfused Rat Right Ventricle Douglas Kelly ∗ , Lorraine Mackenzie, David Saint University of Adelaide, Adelaide, SA, Australia Background: The Gregg effect (increased myocardial contractility in response to increased coronary pressure) may involve activation of stretch-activated ion channels (SACs). Whilst the inotropic responses are modulated by SAC inhibitors, the electrophysiological changes that may accompany the Gregg effect remain undefined.
Heart, Lung and Circulation 2008;17S:S219–S241
Methods: The right ventricle free wall was dissected from hearts of anaesthetised rats (350–400 g, n = 5) and perfused via the right coronary vessel at various flow rates in random order. Isolated ventricles were paced at 4 Hz with 10 ms, bi-polar pulses whilst contractions were suppressed with 20 mM BDM. Intracellular recordings were made using aluminosilicate glass microelectrodes filled with 3 M KCl having a resistance of >20 M. Results: Perfusion pressures were 21 ± 8 mmHg at 4 ml/min and up to 41 ± 9 mmHg at coronary flows of 16 ml/min. Action potential amplitude (82 ± 3.0 mV) and resting membrane potential (−73 ± 2.5 mV) did not change significantly with alterations in coronary flow (p > 0.05, ANOVA GLM). Action potential durations did not significantly change despite considerable increases in perfusion pressure, remaining at 11 ± 1.6, 25 ± 3.1 and 59 ± 6.3 ms for APD20 , 50 and 80, respectively. Conclusion: In the isolated, perfused rat right ventricle modulation of coronary flow rate (and hence perfusion pressure) was not associated with any electrophysiological changes. This suggests that activation of SACs in cardiac myocytes (at least to the extent which would change action potentials) is not involved in the Gregg effect. doi:10.1016/j.hlc.2008.05.554 CLINICAL CARDIOLOGY – ELECTROPHYSIOLOGY 501 Markers of Endothelial Function and Inflammation in Patients with Atrial Fibrillation: A Comparison Between Peripheral and Intracardiac Blood Samples Ross Roberts-Thomson ∗ , Scott Willoughby, Christopher Wong, Dennis Lau, Lorraine Mackenzie, Anthony Brooks, Glenn Young, Prashanthan Sanders Cardiovascular Research Institute, University of Adelaide, Adelaide, South Australia, Australia Introduction: Increased levels of the endothelial dysfunction/damage marker von Willebrand Factor (vWF) measured in peripheral blood is predictive of stroke in patients with atrial fibrillation (AF). Recently, peripheral levels of soluble CD40L (sCD40L), a platelet activationderived inflammatory marker, has been shown to predict vascular events in AF. Suggesting that interaction between the endothelium and platelets may play a role in the genesis of vascular events in AF. However, thrombus and spontaneous echo contrast per se are more commonly seen in the left atrium (LA). Whether there are site-specific modulations of vWF and sCD40L is not known. Methods: 19 consecutive patients (13 males) with paroxysmal AF undergoing ablation were studied. All patients ceased warfarin 7 days and enoxaparin 12 h prior to the procedure. Anticoagulated blood samples were obtained at the start of the procedure immediately after transseptal puncture from the femoral vein (FV), the left atria (LA) and the right atria (RA), prior to the administration of heparin. Plasma samples were prepared by centrifugation and then