| Izvleček: | We compared commonly used BAPTA-derived chemical Ca<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> transients derived from BAPTA-like chemical Ca<sup>2+</sup> dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.
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