STIM-Dependent Signaling in Cardiac Pathophysiology

描述(申请人提供)：这笔K99/R00赠款的目的是帮助曼卡雷拉博士过渡到研究型大学的一个稳定的独立研究职位，在那里他可以进行他在心血管领域的研究。唐纳德·吉尔博士和史蒂文·豪泽博士是细胞信号和心血管科学方面的两位著名专家，他们将在这一过渡期间协助曼卡雷拉博士。在博士后培训期间，这位候选人研究了钙离子传感器STIM1的生物物理性质。这些结果表明，STIM形成了专门的蛋白质网络，能够捕获来自近膜室的钙信号，并将其转化为激活基因转录的信号，该信号发生在信号源的远端。候选人的理论是，STIM1和STIM2-是钙浓度微域所必需的，这些微域与NFAT系统通信，以调节心肌的病理性生长。候选人将结合成像分析、离子通道、生物信息学、蛋白质分析和动物模型的数据，建立一个蛋白质网络，描述蛋白质之间的相互关系如何导致心肌肥厚和心力衰竭。在K99阶段，候选人将描述STIM依赖的新生儿心肌细胞的“钙信号”(AIM I)，并分离STIM激活的离子电流。为此，曼卡雷拉博士将结合遗传学、活细胞成像和电生理学等技术，研究STIM1和STIM2在心脏中的功能作用。随后，(目标2)他将鉴定和表征STIM--控制心脏病理性生长的微域“相互作用体”的蛋白质组成。蛋白质组学分析和生物信息学集成将使系统地评估心脏近膜STIM微域的组成和功能。为了实现他的目标，曼卡雷拉博士将在坦普尔大学蛋白质组学中心接受培训。在R00阶段，曼卡雷拉博士将检验STIM 1和STIM2是心脏病理性生长所必需的假设。实现这一目标的实验方法是基于获得心脏特异性STIM1/2可诱导的双KO小鼠模型；这将通过Cre/Lox技术实现。所产生的菌株将允许在时间和空间上受控地消融心脏中的STIM蛋白。该模型将作为体内模型来验证AIM 1和AIM2的发现，并朝着以下方向发展：(A)研究STIM在成人心功能中的作用(B)研究STIM在胸主动脉缩窄(TAC)引起的压力超负荷中的作用，并检查心脏重构和心功能。利用共聚焦显微镜对分离的心肌细胞进行评价，包括局部和整体水平的形态、功能和钙动态变化。建议的多学科方法(生物化学和生理学)将确保候选人的高质量培训。

DESCRIPTION (provided by applicant): The purpose of this K99/R00 grant is to assist Dr. Mancarella in transitioning to a stable independent research position in a research University where he can conduct his research in the cardiovascular field. Drs. Donald Gill and Steven Houser, two renowned experts in cell signaling and cardiovascular science, will assist Dr. Mancarella during this transition. During his postdoctoral training the candidate has investigated the biophysical properties of the Ca2+ sensors STIM1. These results suggest that STIM forms specialized proteins network able to capture Ca2+ signals from the near-membrane compartment and convert it in signals that activate gene transcription which take place distal from the signal source. The candidate theorized that STIM1 and STIM2-are required for calcium-concentration microdomains which communicate with the NFAT system to regulate the pathological growth of the myocardium. The candidate will combine data from imaging analysis, ion channels, bioinformatics, protein analysis and animal models to build a protein network to describe how inter-relations between proteins lead to cardiac hypertrophy and heart failure. During the K99 phase, the candidate will characterize the STIM-dependent "Ca2+ signature" in neonatal cardiomyocytes (Aim I) and isolate the ionic currents activated by STIM. To this end Dr. Mancarella will implement a combination of techniques such as, genetics, live-cell imaging, and electrophysiology to examine the functional role of STIM1 and STIM2 in the heart. Subsequently, (Aim 2) he will identify and characterize the protein compositions of the STIM-microdomains "Interactome" that govern pathological growth of the heart. Proteomic analysis and bioinformatics integration will allow a systematic assessment of the near-membrane STIM microdomains composition and function in the heart. To achieve his Goals Dr. Mancarella will be trained at Temple University Proteomic Center. During the R00 phase, Dr. Mancarella will test the hypothesis that STIM 1 and STIM2 are required for pathological growth of the heart. The experimental approach to this aim is based on obtain a cardiac specific STIM1/2 inducible double KO mouse model; this will be achieved by Cre/Lox technology. The resultant strain will allow a temporally and spatially controlled ablation of the STIM proteins in the heart. This model will serve as an in vivo model to validate the findings in aim1 and aim2 and expand towards: (a) Examine the role of STIM in adult cardiac functions (b) To investigate the role of STIM during pressure overload induced by thoracic aortic constriction (TAC) and examine, cardiac remodeling and cardiac functions. The evaluation will be extended to the isolated myocytes, including appearance, functions and Ca2+ dynamic at local as well as global level with the use of confocal microscopy. The multidisciplinary approach (biochemical and physiological) proposed will ensure a high quality training of the candidate.