SGLT2 Inhibitor Empagliflozin Modulates Ion Channels in Adult Zebrafish Heart

Figures

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  Figure 1. (A) Representative image of an isolated zebrafish ventricular cardiomyocyte used for patch clamp experiments. Magnification: 40×. (B) Representative image of actin filaments (green) staining using phalloidin in zebrafish cardiomyocytes. Nucleus (blue) was visualized with DAPI. Scale bar is 20 μm. (C) Grouped data on MTT test for cardiomyocytes treated with empagliflozin. Data are expressed as % of value obtained for untreated cells.

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  Figure 2. (A) Representative whole–cell current traces of INa in freshly isolated ventricular cardiomyocytes from zebrafish in control (left) and after 2 h incubation with 5 μM empagliflozin (right). (B) The current density–voltage relationship of the INa in control (n = 13) and in empagliflozin–treated (n = 14) cardiomyocytes. (C) The voltage dependence of steady–state activation and inactivation. The solid and dash lines show least–squares fits to the Boltzmann function. Voltage clamp protocols used to determine activation and inactivation characteristics are shown at the bottom.

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  Figure 3. (A) The voltage dependence of the inactivation fast (triangles) and slow (circles) time constants τfast, τslow of INa in control (filled) and after 2 h incubation with 5 μM empagliflozin (open) cardiomyocytes. (B) The voltage dependence of the time to peak of INa. (C) Grouped data on INa late current as a percentage of INa peak current in control and after 2 h incubation with 5 μM empagliflozin.

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  Figure 4. (A) Representative whole–cell current traces of ICa in freshly isolated ventricular cardiomyocytes from zebrafish in control (left) and after 2 h incubation with 5 μM empagliflozin (right). (B) The current density–voltage relationship of the ICa in control (n = 14) and in empagliflozin–treated (n = 14) cardiomyocytes. Voltage clamp protocols used to estimate ICaT and ICaL are shown at the bottom.

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  Figure 5. (A) Representative whole-cell current traces of IKr in freshly isolated ventricular cardiomyocytes from zebrafish in control (left) and after 2 h incubation with 5 μM empagliflozin (right). (B) The current density–voltage relationship of the IKr step (left) and tail (right) current in control (n = 18) and in empagliflozin–treated (n = 19) cardiomyocytes. * p < 0.05 obtained by Student’s t-test. Voltage clamp protocols used to estimate IKr are shown at the bottom. (C) Concentration–response curve for empagliflozin effect on the IKr tail current density at +10 mV. The solid line shows least-squares fit to the Hill function.

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  Figure 6. (A) Representative whole–cell current traces of IKs in freshly isolated ventricular cardiomyocytes from zebrafish in control (left) and after 2 h incubation with 5 μM empagliflozin (right). (B) The current density–voltage relationship of the IKs in control (n = 18) and in empagliflozin–treated (n = 19) cardiomyocytes. * p < 0.05 obtained by Student’s t-test. (C) Concentration–response curve for empagliflozin effect on the IKs current density at +20 mV. The solid line shows least–squares fit to the Hill function.

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  Figure 7. (A) Representative recording of ventricular AP from ex vivo zebrafish heart. Black line shows AP in control and red in 5 μM empagliflozin–treated heart. (B) Bar graphs of mean results for APD20, APD50, and APD90 in control (n = 5) and empagliflozin–treated (n = 5) heart. ** p < 0.01 obtained by paired Student’s t-test.