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.
Intracellular calcium regulates a variety of cell functions during cardiac hypertrophy and heart failure. We will use genetic mouse models to study how calcium sensors STIM proteins, regulate the activity of calcium ion channels and intracellular calcium levels in healthy and hypertrophic myocardium. These studies may facilitate the development of new pharmacological approaches for the treatment of cardiac hypertrophy and heart failure, the leading cause of death in the United States.