The cellular imaging core will provide three major services to all four of the projects in this PPG including comprehensive: 1) functional analysis of cardiac cells in wild-type and genetically modified mice 2) ultrastructural analysis of cardiac cells in wild-type and genetically modified mice and 3) evaluation of apoptosis in myocardial 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 system C) Analysis of in vitro cellular calcium (Ca2+) transients in VCs using the fluorescent calcium indicator FURA2-AM. Ultra-structural cellular analysis includes: A) Immunofluorescent confocal microscopy of VCs B) Immunofluorescent and immunohistological microscopic analysis of myocardial tissue C) Epifluorescent analysis of VC morphology Apoptosis evaluation includes: A) Terminal deoxynucleotidyl-transferase mediated dUTP nick end (TUNEL) labeling and related imaging in myocardial sections B) Caspase-3 activity in myocardial extracts C) Hairpin 1 (apoptosis) and Hairpin 2 (necrosis) D) Cardiac Myosin Heavy Chain imaging The laboratories of the Core Co-Directors have considerable experience in a wide-ranging array of methodologies for functional and ultra-structural cellular imaging. In particular, isolation and maintenance of high-quality murine VCs is a crucial prerequisite for subsequent functional and ultra-structural analysis. An enzymatic isolation procedure yielding more than 90% viable VCs has been established by Core Co-Directors and is combined with a low-frequent field-stimulated culture system preventing functional and structural dedifferentiation such as the disintegration of the T-tubular system with subsequent loss of cellular excitability. This innovative approach will enable us to investigate VCs from the same heart in a functional and ultra-structural manner.
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