Dissimilar effects of stereoisomers and racemic hydroxychloroquine on Ca²⁺ oscillations in human induced pluripotent stem cell‐derived cardiomyocytes

Figures

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  FIGURE 1. Schematic representation of chemical structure and the separation of racemic hydroxychloroquine (HCQ) into its optical isomers.

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  FIGURE 2. Chiral high‐performance liquid chromatography (HPLC) separation of free base racemic hydroxychloroquine (HCQ) on the analytical HPLC column (a), serial injections used for fraction collections on semi‐preparative HPLC column (b), and HPLC image of purified free bases of R(−)HCQ and S(+)HCQ after injection of pure optical isomers onto analytical chiral HPLC column (c). Note that the recordings were performed with different amplification of the signal and recording speed.

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  FIGURE 3. Circular dichroism (CD) spectra of R(−)hydroxychloroquine (HCQ) (2nd—peak on high‐performance liquid chromatography (HPLC) separation, orange trace) and S(+)HCQ sulfate (first peak on HPLC separation, blue trace). Data are presented as molecular ellipticity difference (delta epsilon in M−1 cm−1) for 1 mM concentrations of stereoisomers in water versus wavelength (nm) (for details of the method see Okuom et al., 2015). The R(−) enantiomer has a positive cotton effect and the S(+) enantiomer has a negative cotton effect centered between 200 and 290 nm.

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  FIGURE 4. Effect of R(−)hydroxychloroquine (HCQ) on Ca2+ oscillations and Ca2+ wave width. Sample baseline Ca2+ oscillations acquired from a single microplate well in human induced pluripotent stem cell (iPSC)‐derived cardiomyocytes 5 min after the application of control buffer (a), 30 μM R(−)HCQ (b), 100 μM R(−)HCQ (c) and 300 μM R(−)HCQ (d). Note that oscillation frequency decreased, and wave width increased with HCQ concentrations greater than 100 μM.

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  FIGURE 5. Effect of S(+)hydroxychloroquine (HCQ) on Ca2+ oscillations and Ca2+ wave width. Sample baseline Ca2+ oscillations acquired from a single microplate well in human induced pluripotent stem cell (iPSC)‐derived cardiomyocytes 5 min after the application of control buffer (a), 30 μM S(+)HCQ (b), 100 μM S(+)HCQ (c) and 300 μM S(+)HCQ (d). Note that oscillation frequency decreased, and wave width increased with HCQ concentrations greater than 100 μM.

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  FIGURE 6. Effect of (±)hydroxychloroquine (HCQ) racemate on Ca2+ oscillations and Ca2+ wave width. Sample baseline Ca2+ oscillations acquired from a single microplate well in human induced pluripotent stem cell (iPSC)‐derived cardiomyocytes 5 min after the application of control buffer (a), 30 μM (±)HCQ (b), 100 μM (±)HCQ (c) and 300 μM (±)HCQ (d). Note that oscillation frequency decreased, and wave width increased with HCQ concentrations greater than 100 μM.

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  FIGURE 7. Racemate and enantiomeric hydroxychloroquine (HCQ) concentration‐response relationships of human cardiomyocytes 5‐ (a), 15‐ (b), 30‐ (c), and 45 min (d) following HCQ exposure. Each data point represents the (mean ± SD) oscillation frequency (beats/min) from three independent experiments with 6–8 wells per concentration. The smooth curves were obtained by fitting the data points to the Hill equation. The IC50 values are listed in Table 1.

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  FIGURE 8. Racemate and enantiomeric hydroxychloroquine (HCQ) concentration‐response relationships of human cardiomyocyte Ca2+ peak width for R(−)HCQ (a), S(+)HCQ (b), and ±HCQ compounds. The plots in each panel denote the (mean ± SD) Ca2+ peak width 5‐, 15‐, 30‐, and 45 min after HCQ compound application. Each data point was acquired from three independent experiments with 6–8 wells per concentration. The smooth curves were obtained by fitting the data points to the Hill equation. The IC50 values are listed in Table 2.