Salbutamol attenuates arrhythmogenic effect of aminophylline in a hPSC-derived cardiac model

Figures

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  Fig. 1. Effect of salbutamol on contraction properties of hPSC-CMs. Scatter plots with indicated means and standard deviation (SD) of the contraction force and beat rate relative responses normalized to the respective baseline control measurement in the case of salbutamol and to mean values of the control measurement in the case of control (n = 11 for controls, n = 15, 15, 23, and 10 for 10 nM, 100 nM, 1 µM, and 10 µM salbutamol, respectively). At least four biological repetitions were used in each column. (A) The contraction force was significantly increased after administration of salbutamol at the concentration of 100 nM (Kruskal-Wallis test with Dunn’s multiple comparisons test, P < 0.05). (B) The beat rate wasn’t significantly elevated after administration of salbutamol (P > 0.05). (C, D) Linear regression of beat rate relative responses and measured concentrations coupled with residual plot. Results show a statistically significant non-zero slope (Wald test, P = 0.0305), proving a positive relationship between concentration and relative response.

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  Fig. 2. Beat rate variability analysis of hPSC-CMs with salbutamol. (A) Scatters plots with indicated means and SD of the standard deviation of successive differences (SDSD) of EBs measured in salbutamol and control (n = 8, 9, 9, and 6 for 10 nM, 100 nM, 1 µM, and 10 µM salbutamol, respectively, and n = 14, 11, 11, and 5 for the respective controls). SDSD with 10 nM and 100 nM salbutamol were significantly lower compared to controls (Brown-Forsythe and Welch ANOVA test with Holm-Sidak multiple comparisons test, control vs. 10 nM salbutamol, P = 0.0115, control vs. 100 nM salbutamol, P = 0.0139). (B) Poincaré plots with SDSD intervals.

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  Fig. 3. Synergic effect of salbutamol and aminophylline on contractile properties of hPSC-CMs. *stands for P ≤ 0.05, **P ≤ 0.01, compared to control, # stands for P ≤ 0.05 and #### stands for P ≤ 0.0001. Scatter plots with indicated means and standard deviation (SD) of the beat rate (A) and contraction force (B) relative responses normalized to the respective baseline control measurement in the case of salbutamol and aminophylline and to mean values of the control measurement in the case of control (n = 17 for controls, n = 13, 13, 11, 10, 10, 15, 15, 23, and 15 for treatments, P values in Table S1 in Supplementary data, Brown-Forsythe and Welch ANOVA test was used for contraction force results, ordinary one-way ANOVA for the beat rate results and Kruskal-Wallis test was applied to compare contraction force and beat rate results of mixes with individual treatment). The legend in A applies to the whole graph. The results of the mixes consist of two separate experiments, same control dataset used based on time of measurement (the first two controls are the same as the third and fourth, relating to 20 min and 40 min of treatment). The results of separate aminophylline administration…

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  Fig. 4. Beat rate variability analysis with combined salbutamol and aminophylline treatments. Scatter plots indicate means and SDs of SDSDs measured with mixed treatments and control (n = 10 for controls, n = 10, 11, 10, 8, 8, 24, 6, 6, and 9 for treatments, p values in Table S1 in Supplementary data, Ordinary one way ANOVA test was used). (A) SDSD results of EBs with indicated concentrations of salbutamol and aminophylline supplemented with separate measurements of both treatments for comparison. The results of the mixes consist of two separate experiments, so the same control dataset was used based on time of measurement (the first two controls are the same as the third and fourth, relating to 20 min and 40 min of treatment). The results of separate aminophylline administration originate from our previous paper6. (B) Poincaré plots with SDSD intervals.

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  Fig. 5. Contractile properties and beat rate variability analysis of hPSC-CMs with salbutamol, aminophylline, and selective β2-AR blocker butoxamine. Scatter plots indicate means and standard deviations (SD) of the beat rate and contraction force relative responses normalized to the respective baseline control measurement in the case of salbutamol and mixed treatments and to mean values of the control measurement. Scatter plots with means and SDs of SDSDs measured with mixed treatments and control means (n and P values) are presented in Table S1 in Supplementary data, ordinary one-way ANOVA test was used. (A, B) The contraction force and beat rate of EBs treated with the mixes of salbutamol, aminophylline, and butoxamine are indicated at the bottom. (C) Analysis of short-term variability SDSD. Control groups were always paired with treatment groups based on the time they were given, so they were repeated from previous figures. (D) Poincaré plots with SDSD. If butoxamine was added to the mix, contractile properties were not significantly different from the effect of aminophylline alone. The anti-arrhythmic potential of salbutamol completely diminished when butoxamine was co-administered.

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  Fig. 6. Beat rate variability analysis of hPSC-CMs with salbutamol, aminophylline, and L-NAME, non-selective inhibitor of NOS. Figure shows relative responses to the respective baseline control. Results shows that L-NAME is co-administrated with mix of salbutamol (10 nM) and aminophylline (1 mM), SDSD values are comparable to results with aminophylline (1mM) and significantly increased to salbutamol and aminophylline mix without the inhibitor (Amino 1 mM vs. Mix, p = 0.0001, Mix + L-NAME vs. Mix, p = 0.0065, Kruskal-Wallis with Dunn’s multiple comparisons test, n = 8 for Amino 1 mM and Mix + L-NAME, n = 10 for Mix).