Assessment of Sarcoplasmic Reticulum Ca2+ Depletion During Spontaneous Ca2+ Waves in Isolated Permeabilized Rabbit Ventricular Cardiomyocytes
Assessment of Sarcoplasmic Reticulum Ca2+ Depletion During Spontaneous Ca2+ Waves in Isolated Permeabilized Rabbit Ventricular Cardiomyocytes
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DOI：
10.1016/j.bpj.2008.12.3944
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发表时间：
2009-04-08
影响因子：
3.4
通讯作者：
Smith, G. L.
中科院分区：
文献类型：
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作者：
MacQuaide, N.;Dempster, J.;Smith, G. L.
在这项研究中，从肌浆网 (SR) 内部和兔心肌细胞的胞浆中测量了自发 Ca2+ 波引起的 Ca2+ 释放。这些测量利用 Fluo5N-AM 检测完整细胞的内 SR Ca2+，并利用 Fluo5F 检测透化细胞的胞质溶胶。使用激光扫描共聚焦显微镜解决了受限的亚细胞体积问题。将自发 Ca2+ 释放期间的局部 Ca2+ 信号与快速应用咖啡因诱导的局部 Ca2+ 信号进行比较。 Ca2+ 波期间游离细胞质 [Ca2+] 的增加是咖啡因应用期间观察到的 98.1% +/- 0.3%。转换为总 Ca2+ 释放表明，Ca2+ 波中的 Ca2+ 释放与咖啡因应用期间释放的 Ca2+ 没有显着差异 (104% +/- 6%)。相比之下，Ca2+波期间SR Fluo-5N内荧光的最大减少量是应用咖啡因期间观察到的82.5%+/-2.6%。假设最大游离 [Ca2+] 为 1.1 mM，这意味着 SR 内游离 [Ca2+] 变化 96.2% +/- 0.8%，总 Ca2+ 消耗 91.7% +/- 1.6%。这相当于在 Ca2+ 波期间 SR 内最小游离 Ca2+ 为 46 +/- 7 μM。丁卡因 (50 μM) 降低 RyR2 Ca2+ 敏感性会降低自发 Ca2+ 释放频率，同时增加 Ca2+ 波振幅。这并没有显着改变 SR 的总损耗 (94.5% +/- 1.1%)。在这些 Ca2+ 波期间计算出的最小 [Ca2+] (87 +/- 19 μM) 显着高于对照 (p < 0.05)。计算模型在 Ca2+ 波期间结合了这种 Ca2+ 消耗水平，模拟了丁卡因对自发 Ca2+ 释放的瞬时和持续影响。总之，自发 Ca2+ 释放导致 SR 局部 Ca2+ 大量但不完全耗尽。此外，测量表明，当管腔 [Ca2+] 达到大约 50 μM 时，Ca2+ 释放终止。
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 mu M during a Ca2+ wave. Reduction of RyR2 Ca2+ sensitivity by tetracaine (50 mu M) 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 mu M) 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 similar to 50 mu M.