The molecular and cellular imaging and histology core (Core C) will provide three main services to each of the three projects described: 1) isolation and functional analysis of murine adult ventricular cardiomyocytes (VC), 2) microscopy analysis of VC and myocardial tissue sections, and 3) evaluation of cell death in VC and myocardial samples. 1) Enzymatic isolation and electrical-field stimulated culture of VCs will be performed in Core C to provide reliable and reproducibly viable VC for analysis of VC function under conditions specific to each project. Functional analyses include assessment of in vitro contractility and intracellular calcium (Ca2+) transients using a digitalized video-edge detection system and FURA2 detection, respectively. 2) A number of microscopy-based assays will be available to provide detailed immunohistological and fluorescence assessment of VC and myocardial tissue sections specific to each project. These analyses include immunohistological and morphological analyses of VC and myocardial tissue sections, fluorescence analysis of VCs and myocardial tissue sections and fluorescence/forster resonance energy transfer (FRET) analysis of 2nd messenger generation and protein interactions in VCs. 3) To evaluate cellular death in VC and myocardial samples. Core C uses assays to detect both apoptotic and necrotic cell death. Each project will have access to analyses of terminal deoxynucleotidyl-transferase mediated dUTP nick end (TUNEL) labeling, caspase-3 activity and Annexin V staining isolated VC and/or myocardial sections/extracts for evaluation of apoptosis, as well as ethidium homodimer III (necrosis) and Hoechst (living cells) staining of VC and myosin heavy chain staining as an indicator or necrosis in myocardial tissue sections. This core will continue to be utilized by all four projects to provide reproducible functional analyses of isolated VC, as well as high quality microscopy and cell death analyses of VC and myocardial tissue.
Attaining a better understanding of the changes in structure, function and survival of VC and myocardium upon manipulation of the signaling pathways being explored within each project, normally and under different conditions of cardiac injury, is a key goal of all three projects. With the essential equipment and expertise on-hand. Core C is available to each project to perform the functional, microscopy and survival assays required.
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