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首页> 美国卫生研究院文献>Biophysical Journal >Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores.
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Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores.

机译：在成年兔心肌细胞中的线粒体钙瞬变：钌红的抑制和由Ca（2+）指示荧光体的溶酶体负载引起的伪影。

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摘要

A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca(2+) uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.
机译：使用冷/热加载方案将成年兔心肌细胞的Rhod 2酯加载到线粒体和其他细胞器中，将Fluo 3酯加载到成年兔心肌细胞的细胞溶质中进行共聚焦荧光成像。电刺激后，细胞质Fluo 3和线粒体Rhod 2荧光均发生瞬时增加。钌红，线粒体Ca（2+）单向阻滞剂，抑制线粒体Rhod 2荧光瞬变，但不抑制胞质Fluo 3瞬变。因此，钌红敏感的线粒体Ca（2+）单向转运蛋白在线粒体游离Ca（2+）的逐跳瞬变过程中催化Ca（2+）的摄取，这反过来可能有助于使线粒体ATP的产生与心肌ATP的需求相匹配。酯加载后，大量的Ca（2+）指示荧光团定位到酸性溶酶体/内体隔室。该溶酶体荧光不响应电刺激。由于冷加载/温育过程后荧光主要来自溶酶体，因此总细胞荧光未能追踪线粒体荧光的逐节拍变化。只有三维分辨共聚焦成像才能将较弱的线粒体信号与明亮的溶酶体荧光区分开。

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