We have recently found that HO-1 serves as an obligatory mediator of three different forms of late preconditioning (PC), suggesting a central cardioprotective role of this protein. Despite this, and despite the fact that HO-1 is one of the most frequently induced proteins known to exist, the regulation of cardiac HO-1 and the mechanism of its cardioprotective actions are unknown. The objective of this proposal is to elucidate the molecular mechanisms underlying the newly discovered cardioprotective role of HO-1 and to use this knowledqe to develop novel therapies. We will test the hypothesis that diverse PC stimuli upregulate HO-1 via eNOS-dependent generation of NO and subsequent activation of a cluster of stress-responsive transcription factors, and that the salubrious effects of HO-1 are due to CO-dependent inhibition of apoptosis and modulation of other co-induced proteins (iNOS, COX-2). We propose that CO, far from being a toxic waste product, plays a fundamental role in protecting the heart against ischemia and apoptosis. Three different forms of late PC elicited by a pathological (ischemia), physiological (exercise), and pharmacological (NO donor) stimulus will be interrogated in an effort to develop a comprehensive pathophysiologic paradigm. A broad multidisciplinary approach will be used that will combine diverse techniques (integrative physiology, protein chemistry, biochemistry, pathology, confocal microscopy, immunohistochemistry, molecular biology, gene targeting, and transgenesis) and will integrate genetic and biochemical information at the molecular level with physiological information at the whole animal level. A total of 16 genetically engineered mouse lines will be studied. The effects of late PC and HO-1 on infarct size, apoptosis, and necrosis will be determined using HO-1-/- and HO-1 transgenic mice in conjunction with state-of-the-art pathology, immunohistochemistry, and confocal microscopy techniques. The effects of CO on myocardial ischemia/reperfusion injury and apoptosis will be determined by using a novel class of compounds (CO-releasing molecules) that have potential therapeutic applications. The role of specific transcription factors (NF-kappaB, HIF-1alpha, HSF1, STAT1, and STAT3) in HO-1 upregulation will be established by the use of a novel transdominant IkappaBalpha mutant mouse with cardiac-specific repression of NF-kappaB activity, knockouts for HIF-1alpha, HSF1, and three specific NF-kappaB subunits (p50, RelB, and c-Rel), and a novel conditional STAT3 knockout mouse. The functional interactions of HO-1 with the other two co-mediators of late PC (iNOS and COX-2) will be deciphered in vivo by cardiac-specific overexpression and targeted disruption of each of these three genes. This project should produce important new insights into the role of apoptosis vis-a-vis necrosis in late PC and the mechanism of HO-1-dependent cytoprotection. If the studies of CO donors confirm our preliminary data, they may pave the way for the development of novel antiischemic and antiapoptotic therapies predicated upon the supplementation of exogenous CO to patients with coronary artery disease.
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