DESCRIPTION (provided by applicant): Although the prognosis of myocardial ischemia has been dramatically improved by the techniques of early reperfusion, the prevention of irreversible ischemic damage remains a critical aspect of the treatment. An appealing novel therapeutic avenue for the prevention of myocardial ischemia relates to the possibility of a pre- emptive conditioning of the heart, in which an activation of survival pathways could be achieved before potentially lethal ischemia occurs. Ischemic preconditioning represents the "gold standard" method of cardioprotection in vivo but it remains difficult to use in the clinical setting. We propose that H11 kinase/Hsp22 (H11K) is both necessary and sufficient to reproduce the pattern of gene expression that characterizes the cardioprotection conferred by the "delayed" or second window of ischemic preconditioning (SWOP). H11K is a chaperone expressed predominantly in the heart, the expression of which increases in various forms of ischemic heart disease, both in animal models and in patients. Increased expression of H11K in a cardiac- specific transgenic (TG) mouse to a level comparable to that found in models of heart disease is sufficient to provide protection against ischemia that is quantitatively similar to ischemic preconditioning. Based on the Preliminary Data, we have three goals. The first goal (Hypothesis 1: Molecular Mechanisms) is to better define the signaling pathway by which H11K promotes cardiac survival. We propose an original pathway in which the stimulation of the bone morphogenetic protein (BMP) receptor by H11K results in the activation of phosphatidylinositol-3 kinase (PI-3K) and the subsequent activation of the transcription factor NF-:B by Akt and the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). The second goal (Hypothesis 2: Physiological Relevance) is to rely on a knockout (KO) mouse model that we generated to prove that H11K is necessary for cardiac cell survival, and especially for the mechanisms of delayed preconditioning. The third goal (Hypothesis 3: Clinical Potential) is to determine whether short-term delivery of H11K in vivo is sufficient to promote cardiac cell survival in a context of lethal ischemia, which would lay the basis for validation of the concept of cardiac pre-emptive conditioning. We will combine an in vitro system (isolated cardiac myocytes and gene knockdown) with mouse models in vivo (TG and KO) and a pre-clinical model (gene delivery in the swine) to address these goals comprehensively.

PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Although the prognosis of myocardial ischemia has been dramatically improved by the techniques of early reperfusion, the prevention of irreversible ischemic damage remains a critical aspect of the treatment. An appealing novel therapeutic avenue for the prevention of myocardial ischemia relates to the possibility of a pre- emptive conditioning of the heart, in which an activation of survival pathways could be achieved in patients with ischemic heart disease who are at risk for a subsequent lethal ischemia. These patients would include those with unstable angina, or with severe and repetitive ischemic episodes, and patients scheduled for surgical revascularization. In these situations, the pre-emptive activation of survival signaling mechanisms would confer a prophylactic cardioprotection during the following ischemic stress. PROJECT NARRATIVE Although the prognosis of myocardial ischemia has been dramatically improved by the techniques of early reperfusion, the prevention of irreversible ischemic damage remains a critical aspect of the treatment. An appealing novel therapeutic avenue for the prevention of myocardial ischemia relates to the possibility of a pre- emptive conditioning of the heart, in which an activation of survival pathways could be achieved in patients with ischemic heart disease who are at risk for a subsequent lethal ischemia. These patients would include those with unstable angina, or with severe and repetitive ischemic episodes, and patients scheduled for surgical revascularization. In these situations, the pre-emptive activation of survival signaling mechanisms would confer a prophylactic cardioprotection during the following ischemic stress. __SpecificAimsTextDelimiter__ SPECIFIC AIMS The prevention of irreversible damage remains a critical aspect of the treatment of ischemic heart disease. A new avenue for the prevention of myocardial ischemia relates to the pre-emptive conditioning of the heart, in which an activation of survival pathways could be achieved in patients who are at risk for a subsequent lethal ischemia. Ischemic preconditioning represents the "gold standard" method of cardioprotection in vivo but it remains difficult to use in the clinical setting. We propose that H11 kinase/Hsp22 (H11K) is both necessary and sufficient to reproduce the pattern of gene expression that characterizes the cardioprotection conferred by the "delayed" or second window of ischemic preconditioning (SWOP). H11K is a chaperone expressed predominantly in the heart, the expression of which increases in various forms of ischemic heart disease, both in animal models and in patients. Increased expression of H11K in a cardiac-specific transgenic (TG) mouse to a level comparable to that found in models of heart disease is sufficient to provide protection against ischemia that is quantitatively similar to ischemic preconditioning. Based on the Preliminary Data, we have three goals. The first goal (Hypothesis 1: Molecular Mechanisms) is to better define the signaling pathway by which H11K promotes cardiac survival. We propose an original pathway (Figure 1) in which the stimulation of the bone morphogenetic protein (BMP) receptor by H11K results in the activation of phosphatidylinositol-3 kinase (PI-3K) and the subsequent activation of the transcription factor NF-¿B by Akt and the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). The second goal (Hypothesis 2: Physiological Relevance) is to rely on a knockout (KO) mouse model that we generated to prove that H11K is necessary for cardiac cell survival. The third goal (Hypothesis 3: Clinical Potential) is to determine whether inducible, short-term delivery of H11K in vivo is sufficient to promote cardiac cell survival in a context of lethal ischemia. " Hypothesis 1: Molecular Mechanisms. H11K activates a survival signaling pathway triggered by the BMP receptor that includes PI-3K, Akt, mTORC2 and NF-¿B, and which reproduces the second window of preconditioning (SWOP). H11K activates PI-3K, translocates Akt to the nuclear membrane where it is phosphorylated by the mTOR/rictor complex (mTORC2), thereby promoting the transcription of NF- ¿B-responsive genes, including the inducible isoform of nitric oxide synthase (iNOS), mediating the SWOP. Specific Aim 1.A.To determine whether the activation of PI-3K by H11K is mediated by the BMP receptor Specific Aim 1.B.To determine whether the mTORC2/Akt complex promotes cardiac cell survival mediated by H11K Specific Aim 1.C.To determine whether the mTORC2/Akt complex activates NF-¿B Specific Aim 1.D.To determine whether survival by H11K requires an activation of the valosin-containing protein (VCP) by mTORC2/Akt Specific Aim 1.E.To determine whether H11K activates an NF-¿B- mediated gene response that corresponds to the SWOP " Hypothesis 2: Physiological Relevance. Deletion of H11K impairs cardiac cell survival. Loss of H11K in cardiomyocytes and in a KO model that we generated impairs survival during ischemia. Specific Aim 2.A.To determine whether siRNA knockdown of H11K impairs cardioprotection in vitro Specific Aim 2.B.To determine in a KO mouse whether deletion of H11K increases irreversible damage during ischemia/reperfusion and impairs the mechanisms of cardioprotection conferred by preconditioning Specific Aim 2.C.To determine in a KO mouse whether genetic disruption of H11K impairs the genomic response to the SWOP " Hypothesis 3: Clinical Potential. Short-term delivery of H11K confers pre-emptive conditioning of the ischemic heart. Short-term induction of H11K to a level comparable to that found in ischemic heart disease reduces infarct size during lethal ischemia. Specific Aim 3.A.To determine whether short-term over-expression of H11K by gene delivery in the swine heart recapitulates the activation of pro-survival signaling pathways Specific Aim 3.B.To determine whether gene delivery of H11K in the swine heart decreases irreversible ischemic damage during lethal ischemia in a nitric oxide-dependent pathway a. Previous observations in the TG mouse H11K PI-3K Akt Akt/mTORC2 iNOS HIF-1¿ VEGF glucose Survival b. Integrated Hypothesis BMPr PI-3K Akt mTORC2 H11K Akt/mTORC2 NF¿B/I¿B¿ P-I¿B¿-VCP NF¿B proteasome COX2 iNOS HIF-1¿ SWOP VEGF glucose Figure 1. Overview of the hypothesis. Circled molecules are those not studied before in cardiac survival mechanisms.