Assessment of sarcoplasmic reticulum Ca2+ depletion during spontaneous Ca2+ waves in isolated permeabilized rabbit ventricular cardiomyocytes.
In this study, Ca2+ release due to spontaneous Ca2+ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca2+ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca2+ signals during spontaneous Ca2+ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca2+] increase during a Ca2+ wave was 98.1% +/- 0.3% of that observed during caffeine application. Conversion to total Ca2+ release suggested that Ca2+ release from a Ca2+ wave was not significantly different from that released during caffeine application (104% +/- 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca2+ wave was 82.5% +/- 2.6% of that observed during caffeine application. Assuming a maximum free [Ca2+] of 1.1 mM, this translates to a 96.2% +/- 0.8% change in intra-SR free [Ca2+] and a 91.7% +/- 1.6% depletion of the total Ca2+. This equates to a minimum intra-SR free Ca2+ of 46 +/- 7 microM during a Ca2+ wave. Reduction of RyR2 Ca2+ sensitivity by tetracaine (50 microM) reduced the spontaneous Ca2+ release frequency while increasing the Ca2+ wave amplitude. This did not significantly change the total depletion of the SR (94.5% +/- 1.1%). The calculated minimum [Ca2+] during these Ca2+ waves (87 +/- 19 microM) was significantly higher than control (p < 0.05). A computational model incorporating this level of Ca2+ depletion during a Ca2+ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca2+ release. In conclusion, spontaneous Ca2+ release results in substantial but not complete local Ca2+ depletion of the SR. Furthermore, measurements suggest that Ca2+ release terminates when luminal [Ca2+] reaches approximately 50 microM.
Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom. n.macquaide@bio.gla.ac.uk
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MacQuaide
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Dempster
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Smith
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003198:Computer Simulation; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology*; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010539:Permeability; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013748:Tetracaine / Q000494:pharmacology
D013748:Tetracaine; D002110:Caffeine; D002118:Calcium