title: Measurement of subcellular Ca2+ redistribution in cardiac muscle in situ: time resolved rapid freezing and electron probe microanalysis.
abstract: To directly assess the physiological roles of sarcoplasmic reticulum (SR) and mitochondria (MT), we have utilized energy dispersive electron probe microanalysis (EPMA) on ultrathin freeze-dried cryosections from isolated papillary muscles, rapidly frozen at precise time points of the contractile cycle. Using this approach, we can detect redistribution of subcellular Ca2+ during the cardiac contractile cycle. Changes in Ca2+ of less than 1.0 mmol/kg dry wt can be detected. By determining the variability of the Ca2+ measurements in preliminary experiments, we have also demonstrated that it is possible to optimize experimental design, i.e., to predict the number of animals per treatment group and the number of X-ray spectra per animal that are required in order to detect a specified Ca2+ difference. Quantitative EPMA of rapidly frozen contracting papillary muscle has also allowed us to correlate the Ca2+ content of SR and MT with the contractile state of the muscle. Our results show a decrease of 40% in the amount of Ca2+ stored in the junctional SR during a cardiac muscle twitch, thus providing direct evidence for a role of the SR as a primary site of Ca2+ release. In addition, we have demonstrated dissociation between MT Ca2+ uptake and activation of regulatory enzymes, such as pyruvate dehydrogenase, indicating that MT Ca2+ uptake is not required for activation of MT metabolism.
authors.affiliation: Department of Molecular Cardiology, Cleveland Clinic Foundation, OH 44195, USA.
authors.forename: M
authors.initials: M
authors.lastname: Bond
authors.forename: M D
authors.initials: MD
authors.lastname: Schluchter
authors.forename: E
authors.initials: E
authors.lastname: Keller
authors.forename: C S
authors.initials: CS
authors.lastname: Moravec
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D006224:Cricetinae; D004577:Electron Probe Microanalysis / Q000379:methods; D005612:Freeze Drying; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000648:ultrastructure; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010210:Papillary Muscles / Q000378:metabolism* / Q000648:ultrastructure; D012519:Sarcoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D012680:Sensitivity and Specificity
chemical_list: D002118:Calcium