A: Personal Statement The goal of the proposed research is to investigate novel mechanisms of myocardial stunning associated with cardiac surgery. The proposed work focuses specifically on the role of small heat shock proteins in mediating aspects of myocardial stunning. I received my PhD from the department of Cardiovascular Science at Albany Medical College, Albany NY, in 2005. Specifically, my research involved Investigation of signal transduction pathways involved in endothelial cell contraction and subsequent increases in permeability. The majority of this work involved current advanced techniques in in vitro and molecular cell biology to investigate mechanisms of cell contraction, which directly applies to the current proposal examining alterations in myocyte contractile deficits.. Following my graduate studies I took a postdoctoral fellowship in the laboratory of Dr. Frank SelIke at Beth Israel Deaconess Medical Center and Harvard Medical School where I expanded my training with in vivo models, advanced physiology techniques, and more applied translational/clinical research. Early work in my fellowship characterized the myocardial response of heat shock protein 27 (HSP27) and aB-crystallin (cryAB) in patients undergoing surgery with cardioplegia (CP) and cardiopulmonary bypass (CPB) (see reference 2 below). These studies laid the ground work for the current proposal which investigates the overall hypothesis that prevervation of non-phosphorylated HSP27 and cryAB levels will reduce or block CP-induced deficits in myocardial contractility. Also during my fellowship I gained expertise in the necessary techniques to adequately complete the proposed studies, including modeling and quantifying CP-induced injury in isolated hearts and cells (See reference 1 - Appendix). Other established expertise relevant to the aims proposed in the revised grant proposal Includes the necessary techniques to perform advanced proteomic techniques to determine sHSP binding partners. I have established collaborations and required techniques to perform two-dimensional electrophoresis, differential protein detection, and LC/MS analysis. These investigations have resulted in a manuscript published in Circulation (reference 3 below) and another currently in preparation. Similar techniques will be used in future Investigations to evaluate Ischemia specific alterations in HSP27 and cryAB binding partners following cardioplegic arrest during surgery. In addition, my postdoctoral fellowship and the mentored phase of the current K99/R00 grant have been highly productive and document a record of success in my research endeavors. Overall, I have the necessary expertise and training and a documented level of productive research to ensure completion of the proposed aims. Updated Research Plan The original proposal of the submitted K99/R00 grant """"""""Small Heat shock proteins in surgically-induced myocardial stunning"""""""", was in three aims:
Aim I - Determine the kinase signaling mechanism of CP/CPB-induced HSP27 and cryAB phosphorylation in isolated myocytes.
Aim II - Determine if overexpression of phospho-mutant sHSP's will reduce cardioplegia-induced contractile deficits in isolated myocytes.
Aim I lI - Determine if preservation of non-phosphorylated sHSP's, via pharmacological inhibition of phosphorylation or catheter based gene delivery of phospho-mutant sHSP's, will protect hearts from CP-induced myocardial contractile deficits. There has been considerable progress on the grant specifically in Aim I and III (please see attached progress report, Appendix). Work on the grant has focused on testing specific phannacological inhibitors (i.e. p38-MAPK and PKCdelta inhibitors) on cardiac function and sHSP phosphorylation in isolated myocytes and hearts before moving on to assessing the role of other kinases proposed in the grant (i.e. PKC alpha and ERK). This represents a slight change to the timeline of aims proposed in the original grant, as each inhibitor will be assessed sequentially in Isolated cells and hearts before moving on to subsequent potential sHSP phosphorylation pathways. It is our feeling that this will facilitate the publication of results focused on individual pathways. The experiments utilizing pharmacological inhibitors will be conducted and completed over the next 12 months. The genetic manipulations utilizing nonphosphorylatable and phospho-mimic constructs of HSP27 and alphaB-crystallin (Aim II and III of the original proposal) will be initiated in the upcoming year and completed over the subsequent years of funding. As the majority of the original Aim I and considerable portions of Aim III have been completed, I have reorganized the original three aims of the grant into the following specific Aims.
Aim I - Determine the kinase signaling mechanism of CP/CPB-induced HSP27 and cryAB phosphorylation in isolated hearts and myocytes.
Aim II ? Determine if preservation of non-phosphorylated sHSP?s via pharmacological ignition of phosphorylation or catheter-based gene delivery of phosphor-mutant sHSP?s, will protect isolated myocytes and hearts from CP-induced myocardial contractile deficits. These two aims and the accompanying updated research strategy encompass all of the aims presented In the original proposal. The new research plan omits those experiments that have already been completed (for a description of the completed research, please see the accompanying progress report.) A new Aim has been developed, to be completed over the independent phase of the project, which will add considerable novelty and mechanistic insight to complement the above aims. In brief, experiments in the original proposal examining molecular indicators of cardiac contraction (to investigate potential mechanisms of sHSP modulation of cardiac contraction) showed no significant differences between groups, whereas there was a very significant positive effect on cardiac function post-cardioplegic arrest. Therefore, the third aim proposes to determine cardioplegia/ischemia specific changes in sHSP protein binding partners. I already have the documented experience and collaborators to successfully complete these investigations (please see preliminary data, and Clements et al. Circulation 2008.) The experiments are delineated in the third aim of the revised Research Strategy. Briefly, HSP27 and alphaB-crystallin will be immunoprecipitated before and after cardioplegic arrest with and without interventions known to modulate cardiac function. Following, immunoprecipitates will be subjected to 2-dimensional electrophoresis and mass spec analysis to determine differential.
