Cellular and Molecular Mechanisms of Oxidative Stress and Anti-Oxidant Agents on the Electrophysiology and Calcium Regulation in Atrium and Pulmonary Vein Cardiomyocytes

Project: A - Government Institutionb - Ministry of Science and Technology

Project Details

Description

Atrial fibrillation (AF) was the most important clinical arrhythmias and would increase mortality and morbidity. But, the mechanisms underlying the arrhythmogenesis of AF are still not completely understood. Oxidative stress was suggested to play an important role in the pathophysiology of AF and has direct effects on ionic channels or calcium regulation. However, the cellular and molecular mechanism of oxidative stress and antioxidant agents in the pathophysiology of AF was not clear. Pulmonary veins (PVs) play an important pathological role in the genesis of AF with a high arrhythmogenic activity. Left atrium was demonstrated to have different electrophysiological characteristics between anterior and posterior wall and play a critical role in the maintenance of AF. Abnormal calcium regulation may cause the high arrhythmogenic activity of PVs and left atrium regional wall differences. However, it is not clear about the effects of oxidative stress on PV and left atrium regional differences. Oxidative stress has been suggested to induce inflammation. Nevertheless, it is also not elucidated whether such effects may arise from the NFκB pathway. The purposes of this study were to investigate the effects and molecular mechanisms of oxidative stress, antioxidant agents and NF-κB inhibitor on the PV and left atrium electrophysiological characteristics and calcium regulation. We will evaluate the effects of oxidative stress on the PV arrhythmogenesis and regional differences of left atrium (in the first year); will investigate the direct effects of oxidative stress (H2O2) with and without anti-oxidant agents on the electrophysiology (action potential, ionic currents) in atrial and PV cardiomyocytes (in the second year), and use the confocal microscopy to study the calcium regulation (calcium wave and calcium spark) through immunofluroence and use the RT-PCR and immunoblotting to investigate the effects of oxidative stress (H2O2) and anti-oxidant agents on the expression of calcium regulation proteins in PV and atrial cardiomyocytes (in the third year). Methods: First year experiment: Through traditional microelectrodes, we record the electrical activity and contractility in PV and left atrium anterior and posterior wall and right atrium before and after the administration of H2O2 (20 μM, 200 μM, 2 mM) for 30 minute with and without pretreatment with antioxidant (ascorbic acid of 1 mM, N-MPG 10 mM, N-acetylcystein 10 mM), or NFκB inhibitor (SN 50, 10 μM). In order to evaluate the treatment effects, (Ascorbic acid of 1 mM, N-MPG 10 mM, N-acetylcystein 10 mM), or NFκB inhibitor (SN 50, 10 μM) was also administrated in the presence of H2O2 (2 mM). Second year experiment: Single cardiomyocytes are isolated from rabbit PVs and left and right atrium through perfusion of Tyrode solution containing digestive enzymes. Cardiomyocytes with and without pretreatment of antioxidant (ascorbic acid of 1 mM, N-MPG 10 mM, N-acetylcystein 10 mM), or NFκB inhibitor (SN50, 50 μM) will receive H2O2 (20 μM, 200 μM, 2 mM) for 30 minutes. Whole-cell clamp techniques were used to study the APs and electrical activity, L-type calcium current (ICa-L), transient inward currents, NCX currents, transient outward currents (Ito) and delayed (IK) and inward rectified outward potassium (IK1) current between control and experimental PV and right and left atrial cardiomyocytes. Third year experiment: Single cardiomyocytes are isolated from rabbit PVs and atrum through perfusion of Tyrode solution containing digestive enzymes. Cardiomyocytes with and without pretreatment of antioxidant (ascorbic acid of 1 mM, N-MPG 10 mM, N-acetylcystein 10 mM), or NFκB inhibitor (SN50, 50 μM) will receive H2O2 (20 μM, 200 μM, 2 mM) for 30 minutes. Confocal microscopy was used to measure the intracellular calcium ([Ca2+]i) transient, [Ca2+]i store, and Ca2+ sparks with fluorescence between control and experimental PV and right and left atrial cardiomyocytes. Immunolabeling with confocal microscopy, western blot and PCR were used to detect and measure the expression of proteins and RNA in ryanodine, NCX, SERCA2a, and phospholamban, and NFκB between control and experimental PV, left and right atrial cardiomyocytes.
StatusFinished
Effective start/end date8/1/107/31/11

Keywords

  • Atrial fibrillation
  • oxidative stress
  • pulmonary vein
  • atrium