Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK; BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK; BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
J Mol Cell Cardiol. 2023 Jul;180:44-57. doi: 10.1016/j.yjmcc.2023.04.008. Epub 2023 Apr 29.
We compared commonly used BAPTA-derived chemical Ca dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca transients derived from BAPTA-like chemical Ca dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.
我们在心脏的单细胞模型中比较了常用的 BAPTA 衍生的化学 Ca 染料（fura2、Fluo-4 和 Rhod-2）与一种新型的基因编码指示剂（R-GECO）。我们评估了它们的性能及其对心肌细胞收缩性的影响，确定了来自成年小鼠和豚鼠的心室原代心肌细胞以及人 iPSC 衍生的心肌细胞中的荧光信号与噪声比和肌节缩短。化学 Ca 染料在所有细胞类型中均表现出剂量依赖性的收缩损伤，并且我们观察到收缩与荧光信号与噪声比之间呈负相关，尤其是对于 fura2 和 Fluo-4。R-GECO 对肌节缩短没有影响。BAPTA 基染料，但不是 R-GECO，抑制了体外肌球蛋白 ATP 酶活性。fura2 的存在突出或减弱了小分子收缩性调节剂和细胞内离子动态平衡调节剂（mavacamten、左西孟旦和氟卡尼）引起的收缩性和 Ca 处理变化，但在成年豚鼠左心室心肌细胞中使用 R-GECO 时并未观察到这种情况。Ca 处理研究对于旨在临床使用的小分子的心脏毒性评估是必要的。在细胞测定中，当解释源自 BAPTA 样化学 Ca 染料的小分子研究评估收缩性效应和 Ca 瞬变时，应谨慎行事，这是心脏毒性测试和心脏疾病生理学和治疗的机制研究的常见平台。
