Core Functions:The cellular imaging core will provide three major services to all four of the projects in this PPG includingcomprehensive: 1) functional analysis of cardiac cells in wild-type and genetically modified mice 2) ultrastructuralanalysis of cardiac cells in wild-type and genetically modified mice and 3) evaluation of apoptosis inmyocardial tissue.Functional cellular analysis includes:A) Enzymatic isolation and electrical-field stimulated culture of murine adult ventricular cardiomyocytes(VCs)B) Assessment of in vitro contractile function of VCs using a digitalized video-edge detection systemC) Analysis of in vitro cellular calcium (Ca2+) transients in VCs using the fluorescent calcium indicatorFURA2-AM.Ultra-structural cellular analysis includes:A) Immunofluorescent confocal microscopy of VCsB) Immunofluorescent and immunohistological microscopic analysis of myocardial tissueC) Epifluorescent analysis of VC morphologyApoptosis evaluation includes:A) Terminal deoxynucleotidyl-transferase mediated dUTP nick end (TUNEL) labeling and related imaging inmyocardial sectionsB) Caspase-3 activity in myocardial extractsC) Hairpin 1 (apoptosis) and Hairpin 2 (necrosis)D) Cardiac Myosin Heavy Chain imagingThe laboratories of the Core Co-Directors have considerable experience in a wide-ranging array ofmethodologies for functional and ultra-structural cellular imaging. In particular, isolation and maintenance ofhigh-quality murine VCs is a crucial prerequisite for subsequent functional and ultra-structural analysis. Anenzymatic isolation procedure yielding more than 90% viable VCs has been established by Core Co-Directorsand is combined with a low-frequent field-stimulated culture system preventing functional and structuraldedifferentiation such as the disintegration of the T-tubular system with subsequent loss of cellularexcitability. This innovative approach will enable us to investigate VCs from the same heart in a functionaland ultra-structural manner.
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