The aim of this project is to continue mechanistic investigation of a canine model of acute myocardial ischemia. This is a remarkably stable model of inducible ventricular tachycardia (VT) and ventricular fibrillation (VF);focal endocardial or Purkinje VT results in half of the experiments and epicardial reentry in the other. Tissue excised from the endocardial focus is studied in vitro with pacing protocols generating cellular calcium based oscillations termed delayed afterdepolarization (DADs) giving rise to an arrhythmia called triggered activity (TA). Clinical laboratories have confirmed focal VT by recording endocardial signals at the origin of VT and VF in patients with acute myocardial ischemia. Moreover, prevention of focal VT/VF with point source endocardial ablation has been proven. Thus this model is a relevant model for human ischemic VT, which may be either focal or reentry. Experiments from the prior funding period have shown that focal endocardial VT is prevented with agents which also prevent TA, including HMG co-A reductase inhibitors (statins) and PD123319 (angiotensin AT-2 receptor antagonist.) Furthermore the effect of statins may result from inhibition of reactive oxygen species (ROS) since a ROS scavenger called TEMPO blocks both VT and TA in a similar manner while decreasing tissue measurements of ROS. ROS may also result in oxidized calcium calmodulin kinase II (CaMKII), a fundamental contributor to VT/VF including reentry in small and large animal models. The overall aim is to test the hypothesis that ROS linked to oxidized CaMKII are basic mechanisms resulting in focal and reentrant VT/VF in a clinically relevant canine model of VT/VF. Investigation centers on two specific aims. Aim 1: Determine whether the highest levels of the ischemic tissue ROS occur at foci of VT/VF and whether decreasing ROS by the scavenger TEMPOL or by inhibition of various enzyme systems that promote ROS including NADH oxidase, mitochondrial or xanthine oxidase more effectively prevents VT/VF. Aim 2: Determine whether the highest levels of oxidized CaMKII activity occur at the foci of VT/VF and if CaMKII inhibition by KN-93 or KN-92 can prevent ischemic focal VT/VF. In vivo computerized activation mapping of multiple transmural bipolar electrograms incorporating Purkinje signals taken from 16 pole needles placed in the risk zone of the anterior descending coronary artery allow 3-D maps of VT/VF produced by extra-stimuli after coronary occlusion. Drugs to be infused to block VT/VF include TEMPO (30 mg/kg,) apocynin (4 mg/kg,) diphenyleneiodine (7.6 mg/kg,) rotenone (9.5 mg/kg,) oxipurinol (3.7 mg/kg,) KN-93 (0.2 <g/kg,) KN-92 (0.2 <g/kg) administered randomly. After activation mapping, tissue at the mechanistic origin of VTVF will be excised for measurements of ROS, CaMKII, RyR2, phospholamban, SERCA2a, and NCX and in vitro microelectrode testing. All the techniques required for these studies are in hand in the laboratories where this preliminary work was done. These studies will utilize Fishers exact test and analysis of variance for quantitative data. Standard microelectrode technique and tissue assays will fulfill the aims. The ultimate goal will be to develop new, effective therapies to prevent ischemic VT/VF in patients with coronary disease, particularly in Veterans. PUBLIC HEALTH RELEVANCE: Veterans have a high frequency of severe coronary heart disease, making dangerous heart rhythms likely. Anti-arrhythmic drugs do not prevent these rhythms and are harmful. Drugs that inhibit mechanistic pathways leading to elevated intracellular calcium are attractive if they don't interfere with fundamental cell function. Therapies acting on such pathways used together may prevent sudden death. Mechanisms studied herein could be pursued immediately in clinical trials since readily available drugs, statins or angiotensin converting enzyme inhibitors, may be combined for their oxidant and calcium/calmodulin kinase blocking effects to prevent sudden death in patients. Similarly such drugs may be combined with other antioxidants to prevent dangerous heart rhythms.
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NIH ExPorter