Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime

Figures

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  Figure 1. Two µmol/L ouabain and 10 µmol/L istaroxime are equi‐inotropic but show a differential lusitropic effect. A, Representative traces of 1 Hz paced ventricular myocytes before and after 10 minutes of treatment with ouabain or istaroxime. B, Average results of cell shortening after 10 minutes of treatment expressed as percentage of pre‐treatment value. C, Average results of relaxation TAU after 10 minutes of treatment expressed as percentage of pre‐treatment value. Data presented as mean±SEM, individual points represent the data from 1 myocyte (8–10 different hearts). Unpaired Student t test was used to determine statistical significance; *P<0.05.

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  Figure 2. Differential impact of ouabain and istaroxime on cardiomyocyte viability and activation of cardiomyocyte apoptosis. A, Primary cardiomyocytes culture was maintained for 24 hours and morphometric analysis used as an index of cardiomyocytes viability. B, Immunoblotting was performed to study the ratio between proapoptotic protein Bax and antiapoptotic protein Bcl‐2. Data presented as mean±SEM; individual points represent the data from 1 heart. One‐way ANOVA with post hoc Tukey's test was used to determine statistical significance; *P<0.05, **P<0.01, and ***P<0.001.

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  Figure 3. Ouabain activates CaMKII but istaroxime does not. A, Representative western blot and averaged data for phospho‐Thr287 CaMKII (p‐CaMKII), B, oxidation of CaMKII (ox‐CaMKII) at M281/282 and C, phosphorylation of phospholamban at Thr17. Data presented as mean±SEM, individual points represent the data from 1 heart. One‐way ANOVA with post hoc Tukey's test was used to determine statistical significance; *P<0.05, **P<0.01.

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  Figure 4. Istaroxime does not significantly increase Ca2+ spark and wave frequency. Representative 2‐dimensional Fluo‐4 plots showing cytosolic Ca2+ signal in control and ouabain‐ and istaroxime‐treated ventricular myocytes. Data presented as mean±SEM; individual points represent the data from 1 myocyte (from 6 hearts). One‐way ANOVA with post hoc Tukey's test was used to determine statistical significance; *P<0.05.

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  Figure 5. Propagation of Ca2+ waves. A, Representative 2‐dimensional (space‐time) and 1‐dimensional whole cell temporal profile of Fluo‐4 emitted signal. B, Ca2+ waves propagation velocity; and C, percentage of aborted Ca2+ waves (mini‐waves) observed in absence or presence of ouabain and istaroxime is shown. Data presented as mean±SEM, individual points represent the data from 1 myocyte (from 6 hearts). ANOVA with post hoc Tukey's test was performed to compare propagation velocity and Fisher's exact test was used to determine the statistical significance between groups when comparing incidence of mini‐waves. ****P <0.0001.

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  Figure 6. The differential arrhythmogenicity between ouabain and istaroxime remains present in phospholamban‐knockout (PLN‐KO) cardiomyocytes. A, Representative images and B, average results showing Ca2+ wave (whole cell or mini‐wave) incidence in control and ouabain‐ or istaroxime‐treated cells. C, Aborted Ca2+ waves as percentage of total waves incidence. Data presented as mean±SEM, individual points represent the data from 1 myocyte (from 5 hearts). In B, ANOVA with Tukey's post hoc test was used; in C, indicates significance vs control calculated with Fisher's exact test using absolute values. *P<0.05.