STIM-Dependent Signaling in Cardiac Pathophysiology

描述（由申请人提供）：此K99/R00补助金的目的是帮助Mancarella博士过渡到研究型大学的稳定独立研究职位，在那里他可以进行心血管领域的研究。唐纳德·吉尔博士和史蒂文·豪泽博士，两位著名的细胞信号和心血管科学专家，将在这一过渡期间协助Mancarella博士。在博士后培训期间，该候选人研究了Ca2+传感器STIM 1的生物物理特性。这些结果表明，STIM形成专门的蛋白质网络，能够从近膜区室捕获Ca2+信号，并将其转化为激活发生在信号源远端的基因转录的信号。该候选人的理论是，STIM1和STIM2-是钙浓度微区所必需的，钙浓度微区与NFAT系统通信以调节心肌的病理生长。候选人将结合来自成像分析、离子通道、生物信息学、蛋白质分析和动物模型的联合收割机数据，构建蛋白质网络，描述蛋白质之间的相互关系如何导致心脏肥大和心力衰竭。在K99阶段，候选人将表征新生儿心肌细胞（Aim I）中的STIM依赖性"Ca2+特征"，并分离由STIM激活的离子电流。为此，Mancarella博士将实施遗传学，活细胞成像和电生理学等技术的组合，以检查STIM1和STIM2在心脏中的功能作用。随后，（目标2）他将鉴定和表征控制心脏病理性生长的STIM微结构域"相互作用组"的蛋白质组成。蛋白质组学分析和生物信息学整合将允许系统评估心脏中的近膜STIM微结构域的组成和功能。为了实现他的目标，Mancarella博士将在坦普尔大学蛋白质组学中心接受培训。在R00阶段，Mancarella博士将检验STIM 1和STIM 2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STIM 1/2诱导型双KO小鼠模型;这将通过Cre/Lox技术实现。所得菌株将允许心脏中STIM蛋白质的时间和空间受控消融。该模型将作为体内模型，以验证aim 1和aim 2中的发现，并扩展到：（a）检查STIM在成人心脏功能中的作用（B）研究STIM在胸主动脉缩窄（TAC）诱导的压力超负荷期间的作用，并检查心脏重塑和心脏功能。评价将扩展到分离的心肌细胞，包括外观，功能和Ca2+动态在局部以及全球水平与共聚焦显微镜的使用。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.