Haviland Sarah, Cleemann Lars, Kettlewell Sarah, Smith Godfrey L, Morad Martin
Calcium Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, SC 29425, USA.
Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Cell Calcium. 2014 Sep;56(3):133-46. doi: 10.1016/j.ceca.2014.06.001. Epub 2014 Jun 14.
I(Ca)-gated Ca(2+) release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca(2+), but the extent to which mitochondria contribute to the overall Ca(2+) signaling remains controversial. To examine the possible role of mitochondria in Ca(2+) signaling, we developed a low affinity mitochondrial Ca(2+) probe, mitycam-E31Q (300-500 MOI, 48-72h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca(2+) in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca(2+) while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca(2+)) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na(+) increased global cytosolic and mitochondrial Ca(2+) signals in one population of mitochondria, but unexpectedly decreased it (release of Ca(2+)) in another mitochondrial population. Such mitochondrial Ca(2+) release signals were seen not only during long lasting Na(+) withdrawal, but also when Ca(2+) loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca(2+) transients appear to activate with a delay following the cytosolic rise of Ca(2+) and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca(2+) signaling.
心肌肌浆网中由I(Ca)门控的Ca(2+)释放（CICR）是介导胞质Ca(2+)升高的主要机制，但线粒体对整体Ca(2+)信号传导的贡献程度仍存在争议。为了研究线粒体在Ca(2+)信号传导中的可能作用，我们开发了一种低亲和力的线粒体Ca(2+)探针mitycam-E31Q（300 - 500 MOI，48 - 72小时），并将其与Fura-2AM结合使用，以获取新生大鼠心肌细胞中线粒体和胞质Ca(2+)的同步全内反射荧光（TIRF）图像。成年猫心肌细胞的mitycam-E31Q染色显示出典型的线粒体纵向荧光带，类似于TMRE染色，而新生大鼠心肌细胞则呈现出紊乱的管状或点状外观。咖啡因微喷射使胞质Ca(2+)迅速增加，同时测量的整体mitycam-E31Q信号下降更缓慢（线粒体Ca(2+)增加），然后衰减至基线水平。在自发起搏细胞中观察到类似但振荡的mitycam-E31Q信号。去除Na(+)使一群线粒体中的整体胞质和线粒体Ca(2+)信号增加，但出乎意料的是，在另一群线粒体中却使其降低（Ca(2+)释放）。这种线粒体Ca(2+)释放信号不仅在长时间去除Na(+)期间出现，而且当Ca(2+)负载的细胞暴露于咖啡因微喷射时以及在自发节律性搏动期间也会出现。因此，线粒体Ca(2+)瞬变似乎在胞质Ca(2+)升高后延迟激活，并在线粒体亚群中表现出多样性，这可能有助于线粒体Ca(2+)信号传导的可塑性。
