Rosuvastatin exerts cardioprotective effect in lipopolysaccharide-mediated injury of cardiomyocytes in an MG53-dependent manner

Figures

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  Fig. 1. Construction of in vitro myocarditis model of LPS-treated H9C2 cells. H9C2 cells were treated with different concentrations of LPS for 24 h. A Cell viability was analyzed using the MTT assay and was calculated as a percentage, compared with the corresponding blank control. Data are represented as mean ± standard deviation (SD) of six biological replicates. B Mitochondrial membrane potential was determined using JC-1 assay. Representative images from three independent experiments. C Quantification of the mitochondrial membrane potential level indicated using mean fluorescence intensity (MFI). D Cell apoptosis was analyzed through Annexin-V/PI staining via FACS. Representative images from three independent experiments. E Quantification of apoptosis. F Quantitation of the mRNA expression of cytokines, namely IL-1β, IL-6, TNF-α, and MCP-1, as estimated using RT-qPCR. Data are represented as mean ± SD of three technical replicates. G Quantitation of level of cytokines, namely IL-1β, IL-6, TNF-α, and MCP-1, as determined using ELISA. Data are represented as mean ± SD of three biological replicates. Group differences were analyzed using one-way ANOVA with the Tukey’s post-hoc test,…

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  Fig. 2. Effect of LPS on cell viability in H9C2 cells with or without RS. A Cell viability was analyzed and calculated as percentage. H9C2 cells were treated with 10 μg/ml LPS and different concentrations of RS for 24 h. Data are represented as mean ± SD of six biological replicates. RS: Rosuvastatin. B Mitochondrial membrane potential was determined using JC-1 assay. Representative images from three independent experiments. C Quantitation of mitochondrial membrane potential level indicated using MFI. D Cell apoptosis was analyzed using Annexin-V/PI staining via FACS. Representative images from three independent experiments. E Quantitation of apoptotic cells. F Quantitation of mRNA expression of cytokines, namely IL-1β, IL-6, TNF-a, and MCP-1, as estimated by RT-qPCR. Each experiment was performed in triple. G Quantitation of the level of cytokines, namely IL-1β, IL-6, TNF-a, and MCP-1, as determined using ELISA Data are represented as mean ± SD of three biological replicates. H9C2 cells were treated with 10 μg/mL LPS and 1.0 μM RS for 24 h in (B–G). Group differences were analyzed using one-way ANOVA with the Tukey’s post-hoc test. ns, not significant; **P < 0.01; ***P < 0.005; ****P <…

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  Fig. 3. Effect of RS on MG53 expression under conditions of LPS treatment. A RS increased MG53 mRNA level in a concentration-dependent manner. RT-qPCR was performed when H9C2 cells were treated with different concentrations of RS for 24 h. Data are represented as mean ± SD of three technical replicates. B MG53 protein levels were detected using western blotting after treatment with RS for 24 h. Representative image of three independent experiments. C Quantification of (B). D The knockdown efficiencies of siRNAs were determined using RT-qPCR. Data are represented as mean ± SD of three technical replicates. (E) MG53 mRNA expression as analyzed in the following five groups: 1) Control; 2) Treatment with 10 μg/mL LPS for 24 h; 3) Combined treatment with 10 μg/mL LPS and 1.0 μM RS for 24 h; 4) Combined treatment for 24 h after a 12-h transfection with siRNA negative control; and 5) Combined treatment for 24 h after a 12-h transfection with MG53 siRNA. Data are presented as mean ± SD of three technical replicates. F MG53 protein expression as analyzed using western blotting. Representative image of three independent experiments. G Quantification of MG53 protein. Group differences were analyzed…

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  Fig. 4. MG53 deficiency eliminates the protective effect of Rosuvastatin on H9C2 cells. A Cell viability was measured by an MTT assay and was calculated as percentage compared with the corresponding group. Data are represented as mean ± SD of six biological replicates. B Mitochondrial membrane potential was analyzed using JC-1 assay. Representative images from three independent experiments. C Quantitation of the mitochondrial membrane potential level indicated using MFI. D Cell apoptosis was analyzed using Annexin-V/PI. Representative images from three independent experiments. E Quantitation of apoptotic cells. Group differences were analyzed using one-way ANOVA with the Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

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  Fig. 5. The Rosuvastatin-induced decrease in cytokine level was abolished by MG53 knockdown. A Quantitation of the mRNA expression of cytokines, namely IL-1β, IL-6, TNF-a, and MCP-1, as analyzed using RT-qPCR. Data are represented as mean ± SD of three technical replicates. B Quantitation of cytokine levels using ELISA. Data are represented as mean ± SD of three biological replicates. Group differences were analyzed using one-way ANOVA with the Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

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  Fig. 6. Effect of RS on signaling pathways under conditions of LPS treatment. A, C, E Protein expression as analyzed using western blotting. Representative image of three independent experiments. B, D, F Quantitation of proteins. Group differences were analyzed using one-way ANOVA with the Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001