Small heat shock proteins in surgically-induced myocardial stunning

手术引起的心肌顿抑中的小热休克蛋白

• 批准号: 8134837
• 负责人: Richard T Clements
• 金额: $ 24.9万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8134837
• 关键词: Acidosis; Applications Grants; Award; Binding; Binding Proteins; Biological Preservation; Blood Circulation; Cardiac; Cardiac Myocytes; Cardiac Surgery procedures; Cardiopulmonary Bypass; Cardiovascular system; Catheters; Cells; Cellular biology; Clinical Research; Collaborations; Complement; Crystallins; Data; Detection; Disease; Doctor of Philosophy; Drug Delivery Systems; Electrophoresis; Endothelial Cells; Ensure; Feeling; Fellowship; Funding; Future; Gene Delivery; Genetic; Goals; Grant; Heart; Heart Arrest; Heat Shock Protein 27; Heat shock proteins; Hypoxia; In Vitro; Individual; Induced Heart Arrest; Injury; Intervention; Investigation; Ischemia; Israel; Laboratories; Literature; Location; MAP Kinase Gene; MAPK14 gene; Manuscripts; Mediating; Medical center; Mentors; Metabolic; Modeling; Molecular; Muscle Cells; Myocardial; Myocardial Stunning; Myocardium; Operative Surgical Procedures; Oxidants; Pathway interactions; Patients; Pattern; Permeability; Phase; Phosphorylation; Phosphorylation Inhibition; Phosphotransferases; Physiology; Play; Preparation; Progress Reports; Prophylactic treatment; Protein Binding; Protein Kinase C Alpha; Proteins; Proteomics; Publications; Publishing; Regulation; Reperfusion Therapy; Research; Role; Science; Signal Transduction; Signal Transduction Pathway; Solutions; Staining method; Stains; Techniques; Testing; Therapeutic Intervention; Time; TimeLine; Tissues; Training; Treatment Protocols; Update; Work; base; design; experience; gene therapy; genetic manipulation; heart cell; in vivo Model; inhibitor/antagonist; insight; liquid chromatography mass spectrometry; medical schools; mutant; natural hypothermia; novel; overexpression; research study; response; success; two-dimensional

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 IlI - 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.

答：个人陈述 拟议研究的目的是研究心肌惊人的新型机制 与心脏手术有关。拟议的工作专门针对小热冲击的作用 在介导心肌惊人方面的蛋白质。我从部门获得了博士学位 2005年，纽约州奥尔巴尼医学院的心血管科学。特别是，我的研究涉及 研究参与内皮细胞收缩和随后增加的信号传递途径 渗透性。这项工作的大多数涉及当前的体外和分子细胞中的高级技术 生物学研究细胞收缩的机制，该机制直接适用于当前研究 心肌收缩的改变。 贝丝以色列女执事医学中心和哈佛医学院的弗兰克·塞克（Frank Selike）博士实验室 扩大了我的体内模型，高级生理技术和更多应用的培训 翻译/临床研究。我的奖学金的早期工作是热的心肌反应 休克蛋白27（HSP27）和AB-晶状体蛋白（CRYAB）接受了心脏外科手术（CP）和 心肺旁路（CPB）（请参阅下面的参考文献2）。这些研究为电流奠定了基础工作 提案调查了总体假设，即非磷酸化HSP27和Cryab的预截图 水平将减少或阻止CP引起的心肌收缩性缺陷。同样在我的团契期间，我获得了 在必要的技术方面的专业知识，以充分完成拟议的研究，包括建模和 量化孤立心脏和细胞中CP诱导的损伤（请参阅参考文献1-附录）。其他建立 与修订的赠款提案中提出的目标相关的专业知识包括必要的技术 执行先进的蛋白质组学技术来确定SHSP结合伙伴。我已经建立了合作 并需要执行二维电泳，差异蛋白检测和LC/MS的技术 分析。这些调查导致发表在流通中的手稿（以下参考文献3）和 目前正在准备的另一个。将来的调查将使用类似的技术来评估缺血 手术期间心脏地章停滞后HSP27和Cryab结合伴侣的特定改变。在 此外，我的博士后奖学金和当前K99/R00赠款的修订阶段非常高 生产性并记录了我的研究努力成功记录。总的来说，我有必要的 专业知识和培训以及有记录的生产研究水平，以确保完成拟议的 目标。 更新的研究计划 提交的K99/R00赠款的原始提案“小热冲击蛋白 手术引起的心肌令人惊叹，”是三个目的： 目的I-确定CP/CPB诱导的HSP27和Cryab的激酶信号传导机制 分离的肌细胞中的磷酸化。 AIM II-确定磷酸突变的SHSP的过表达是否会减少心脏双毛诱导的 收缩在孤立的心肌细胞中定义。 AIM ILI-通过药物确定是否制备非磷酸化SHSP的制备 抑制磷酸化或基于导管的基因递送的磷酸化SHSP的基因递送将 保护心脏免受CP诱导的心肌收缩的定义。 在AIM I和III中，这笔赠款方面取得了很大进展（请 请参阅附件的进度报告，附录）。赠款的工作重点是测试特定 Phannokic抑制剂（即P38-MAPK和PKCDELTA抑制剂）对心脏功能和 在继续评估角色之前 赠款中提出的其他激酶（即PKC Alpha和Erk）。这代表了一点 更改原始赠款中提出的目标时间表，因为将评估每个抑制剂 依次在孤立的细胞和心脏中转到随后的电势SHSP 磷酸化途径。我们的感觉是，这将有助于发布结果 专注于单个途径。利用药物抑制剂的实验将是 在接下来的12个月中进行并完成。使用不可磷酸的遗传操作 HSP27和Alphab-晶状体的磷酸化构建（AIM II和III） 原始提案）将在接下来的一年中启动，并完成 随后的几年资金。作为原始目标的大部分，大部分 AIM III已经完成，我将赠款的最初三个目标重组为 遵循特定目标。 AIM I-确定CP/CPB诱导的HSP27和 哭泣的心脏和肌细胞中的磷酸化。 AIM II»是否通过磷酸化或基于导管的基因递送的磷酸化SHSPSSSSSSP？S的药物点火来确定非磷酸化的SHSP s的保存，将会 保护孤立的心肌细胞和心脏免受CP诱导的心肌收缩的定义。 这两个目标以及参与的更新研究策略涵盖了所有 原始提案中提出的目标。新研究计划省略了那些实验 已经完成（有关完成的研究的描述，请参阅 参加进展报告。） 已经开发了一个新的目标，将在独立阶段完成 项目将增加相当大的新颖性和机械见解来完成上述 目标。简而言之，在原始建议中进行了检查心脏分子指标的实验 收缩（研究心脏收缩的SHSP调节的潜在机制） 组之间没有显着差异，而存在非常明显的正则 对心脏后停滞后心脏功能的影响。因此，第三个目标提议 确定SHSP蛋白结合伴侣中的心脏质心/缺血特异性变化。我已经 有记录的经验和合作者成功完成这些 调查（请参阅初步数据，以及Clements等。2008年。） 在修订的研究策略的第三个目标中划定了实验。简而言之，HSP27 在心脏地杆菌逮捕之前和之后，Alphab-Crystallin将被免疫沉淀 没有已知可以调节心脏功能的干预措施。以下，免疫沉淀将是 进行二维电泳和质量分析以确定差异。