Assessment of Sarcoplasmic Reticulum Ca2+ depletion during spontaneous Ca2+ waves in isolated permeabilized rabbit ventricular Cardiomyocytes

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MacQuaide, N. and Dempster, J. and Smith, G.L. (2009) Assessment of Sarcoplasmic Reticulum Ca2+ depletion during spontaneous Ca2+ waves in isolated permeabilized rabbit ventricular Cardiomyocytes. Biophysical Journal, 96 (7). pp. 2744-2754. ISSN 0006-3495 (https://doi.org/10.1016/j.bpj.2008.12.3944)

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Abstract

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 μM during a Ca2+ wave. Reduction of RyR2 Ca2+ sensitivity by tetracaine (50 μM) 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 μM) 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 50 μM.

ORCID iDs

MacQuaide, N., Dempster, J. ORCID logoORCID: https://orcid.org/0000-0003-2199-2945 and Smith, G.L.;
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