Losartan protects human stem cell-derived cardiomyocytes from angiotensin II-induced alcoholic cardiotoxicity

Figures

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  Fig. 1. Modeling alcoholic cardiomyopathy using hiPSC-CMs. A Representative immunostaining for the expression of MYL2 (green) and cTNT (red) in hiPSC-CMs. Scale bar = 50 μm. B CCK-8 assay showed that the cell viability decreased to 95.3, 98.9, 93.6, 83.1, 68.5, 59.4, 54.8, and 48.3% after treatment with different concentrations of alcohol (i.e., 20, 40, 60, 80, 100, 150, 200, 250, and 300 mM) for 24 h (n = 4). C Representative images showed that hiPSC-CMs shrink and became round after alcohol treatment. Scale bar = 200 μm. D RTCA was used to monitor the changes in cell index after treating hiPSC-CMs with different concentrations of alcohol (i.e., 100, 200, and 300 mM) for 30 h (n = 30). E–G Changes in vibration amplitude and beat frequency over a 30-h period [F(n = 39); G(n = 41)]. Results are presented as means ± standard error of the mean (*P < 0.05, **P < 0.01, ***P < 0.001; two-sided Student’s t-test or one-way analysis of variance).

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  Fig. 2. Alcohol-induced cardiomyocyte damage. A LDH activity assay showed that 100 mM alcohol can induce the upregulation of LDH expression in hiPSC-CMs 2.5 times (n = 7). B HTS analysis showed that the red-green fluorescence ratio has significantly dropped (n = 7). C The representative immunofluorescence image of JC-1 labeling showed that the green fluorescence expression of hiPSC-CMs significantly increased after treatment with 100-mM alcohol for 2 h. Scale bar = 25 μm. D, E Flow cytometry and quantitative analysis showed that 100-mM alcohol increased early apoptosis of hiPSC-CMs (n = 3). Results are presented as means ± standard error of the mean (**P < 0.01; two-sided Student’s t-test or one-way analysis of variance).

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  Fig. 3. Alcohol-induced changes in the global gene expression profiles of cardiomyocytes. A The enriched terms GO (correlated P value of ≤0.05) of 100 mM alcohol treatment compared with vehicle. B The pathway analysis between the control and hiPSC-CMs treated with 100 mM alcohol is listed (P value of ≤0.05). C The heatmap of 39 differentially expressed RNAs between the 100 mM alcohol group and hiPSC-CMs showing four functional clusters. Each row corresponds to a single differentially expressed gene. Colors ranging from blue to red represent the relative expression levels (Log2(fold change)) of RNAs.

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  Fig. 4. Alcohol-induced ROS and AngII/AT1R activation in cardiomyocytes. A–C Representative immunofluorescence images of CM-H2DCFDA (scale bar = 15 μm) and Texas red labeling (scale bar = 25 μm), and quantitative statistics showed that the level of the oxidative stress and mitochondrial oxidative stress significantly increased after hiPSC-CMs were treated with 100 mM alcohol for 2 h (n = 10). D Western blot showed that AT1R protein expression increased significantly after treatment with 100 mM ethanol for 24 h (n = 5). E RT-PCR showed that the expression of AngII and AT1R expression increased significantly after treatment with 100 mM alcohol for 24 h (n = 5). F RT-PCR showed that the expression of AngII and AT1R expression increased significantly after treatment with 100 mM alcohol for 24 h (n = 5). G Western blot showed that AT1R protein expression increased significantly after treatment with 100 mM alcohol for 24 h (n = 5). H Enzyme-linked immunosorbent assay showed that the level of AngII in the plasma and heart of mice increased after alcohol treatment (n = 6). Results are presented as means ± SEM (**P < 0.01; two-sided Student’s t-test or one-way analysis of variance).

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  Fig. 5. Blocking AT1R prevents the electrophysiological abnormalities and cardiac oxidative stress in hiPSC-CMs models of ACM. A Representative immunofluorescence images of JC-1 labeling showed a reduction in green fluorescence intensity after losartan administration compared with that after alcohol treatment (n = 20). Scale bar = 25 μm. B Representative immunofluorescence images of CM-H2DCFDA showed that losartan restored the level of mitochondrial oxidative stress induced by 100-mM alcohol. (n = 10) C Representative immunofluorescence images of Texas red labeling showed that losartan restored the level of the oxidative stress induced by 100-mM alcohol. (n = 10) D Western blotting and quantitative statistics showed that losartan restored the protein expression level of NOX4 induced by 100-mM alcohol (n = 5). E–G RTCA for dynamically monitoring cell index waveform, amplitude and beat frequency showed that losartan restored the electrophysiological disturbance of hiPSC-CMs induced by 100 mM alcohol [F (n = 39); G (n = 41)]. Results are presented as means ± standard error of the mean (**P < 0.01; two-sided Student’s t-test or one-way analysis of variance).

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  Fig. 6. Schematic illustration of alcohol-induced cardiomyocyte damage in hiPSC-CMs. Alcohol can increase the expression of NOX4 by activating the AngII–AT1R signaling pathway, increasing the production of ROS, and causing cardiomyocyte apoptosis.