Inhibition of RNA Helicase Activity Prevents Coxsackievirus B3-Induced Myocarditis in Human iPS Cardiomyocytes

Figures

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  Figure 1. Screening antiviral effect chemical. (A) Anti-enterovirus activity of synthetic chemical was screened by virus replication inhibition assay in expression Renilla-luciferase following Renillar-CVB3 infection. 10 ug/mL, 1 ug/mL, 100 ng/mL, and 10 ng/mL concentration of synthetic chemical treatment reduced Renilla-luciferase expression (Relative luminometer units, RLU) compare to positive agent Pleconaril. SM1 (enterovirus-2C inhibitor (E2CI)) chose best candidate for antiviral chemical. (B) Chemical structure of E2CI.

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  Figure 2. E2CI inhibit CVB3 replication in HeLa cells. (A) E2CI significantly inhibited CVB3 replication. Green fluorescent protein (GFP) was expressed during CVB3 replication with viral protein production. GFP expression was reduced by high dose (10 ng/mL) E2CI treatment (5.6 ± 0.5% vs. 42.3 ± 0.3% GFP positive cells, 10 vs. 0 ng/mL, p < 0.05). (B) CVB3 genome amplification was confirmed in CVB3 infected HeLa cells with E2CI treatment. CVB3 capsid protein VP1 gene positive and negative strand RNA were amplified by reverse transcription PCR. Both strand of VP1 RNA was significantly decreased by E2CI treatment. Data are presented as the mean plus or minus the standard error of the mean from three independent experiments. **, p < 0.01 (Scale bar, 100 μm).

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  Figure 3. Decrease mortality and organ virus titer in murine myocarditis model. (A) In vivo experiment skim in murine viral myocarditis model. Tissue was corrected at Day 3, 7, and 14 p.i. for PFU assay and histological observation. (B) Mice survival was improved by E2CI treatment compare to untreated control group (CVB3 vs. CVB3 + E2CI, 70% vs. 95%, p < 0.01). (C) The live virus titer of the heart and pancreas were measured by PFU assay. E2CI decreased progeny virus production in the heart at day 7 p.i. Data are presented as the mean plus or minus the standard error of the mean from three independent experiments. **, p < 0.01.

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  Figure 4. Histological finding and myocardium damage. (A) CVB3 infection induced heart damage. Inflammation and myocardium damage were observed by H&E and Evans blue stain at 7 days post-infection. Myocardium damage and inflammatory cell infiltration were significantly decreased by E2CI treatment. (B) Heart inflammation was quantified by imageJ software. E2CI treatment decreased inflammation area in the heart compare to untreated control group (CVB3 vs. CVB3 + E2CI, 23.67 ± 1.202% vs. 4.833 ± 1.327%, n = 6). Data are presented as the mean plus or minus the standard error of the mean from three independent experiments. ***, p < 0.001 (scale bar, 100 µm).

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  Figure 5. E2CI prevent LV dysfunction in viral myocarditis model. (A) M-mode echocardiography showed LV chamber dimension. E2CI treatment maintained heart function compare to untreated group. (B) Left Ventricular-systolic dimension (LVIDS, red bar) and diastolic dimension (LVIDD) were maintained in E2CI treated group compare to untreated control group (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVIDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Fractional shortening (FS) was dramatically preserved by E2CI treatment (FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Data are presented from three independent experiments. ***, p < 0.001; **, p < 0.01, *, p < 0.05.

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  Figure 6. E2CI inhibit CVB3 replication in human cardiomyocyte. (A) E2CI significantly inhibited CVB3 replication with viral protein production. GFP expression was reduced by 10 ng/mL E2CI treatment in human iPS cardiomyocyte (1 ± 0.2% vs. 61.3 ± 0.4% GFP positive cells, 10 vs. 0 ng/mL, p < 0.05). (B) CVB3 positive and negative strand RNA were amplified by reverse transcription PCR. Both strand of VP1 capsid protein mRNA was significantly decreased by E2CI treatment. Data are presented as the mean plus or minus the standard error of the mean from three independent experiments. **, p < 0.01 (scale bar, 100 μm).