RIP1/RIP3/MLKL Mediates Myocardial Function Through Necroptosis in Experimental Autoimmune Myocarditis

Figures

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  Figure 1. Flowchart of rats used in experiments.

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  Figure 2. The level of LVIDd/LVIDs/EF/FS in “early” and “late” stages of myocarditis in control and EAM mice by echocardiography. (A) Control groups (top panel, 3W, I; 8W; II) and EAM mice (bottom panel, 3W,I II; 8W, IV). (B–E) Quantification of LVIDd (B), LVIDs (C), EF (D), and FS (E) in “early” and “late” stages of myocarditis from control groups and EAM mice. n = 4 rat in control group, n = 5 rat per group (B–E). All data are presented as mean ± SEM; a Student's t-test was performed to control group and EAM group. *p < 0.05 vs. control group; **p < 0.01 vs. EAM + DMSO group; Δp > 0.05 vs. EAM group.

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  Figure 3. Protein expression levels of RIP1, RIP3, and MLKL in myocardial tissues of acute and chronic stages detected by Western blot. (A) Western blot analysis of protein expression of RIP1/RIP3/MLKL in rat myocardium during “early” and “late” stages. (B–D) Quantification of RIP1 (B), RIP3 (C), MLKL (D) in (A). (E) Western blot analysis of marker protein expression of autophagosome LC-3 I and LC-3 II in rat myocardium during “early” and “late” stages. (F) Quantification of LC-3 I and LC-3 II in e. All data are presented as mean ± SEM. A Student's t-test was performed. **p < 0.05 means compared with control group (3W); *p < 0.05 means vs. control Group (8 weeks) comparison.

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  Figure 4. The level of LVIDd/LVIDs/EF/FS in “early” stage of myocarditis in EAM mice with inhibitors by echocardiography. (A) Echocardiographic images of control and EAM mice (3W) (I—control, II—EAM, III—EAM + DMSO, IV—EAM + Nec-1, V—EAM + zVAD-fmk, VI—EAM+3Ma). (B–E) Quantification of LVIDd, LVIDs, EF, and FS in the “early” stage of myocarditis from control groups. A horizontal bar is shown to indicate compared groups. All data are presented as mean ± SEM. A Student's t-test was performed for the control group and the EAM group, *p < 0.05 vs. control group **p < 0.01 vs. EAM + DMSO group, Δp > 0.05 vs. EAM group.

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  Figure 5. The relative expression of mRNA in myocardial tissue of each group detected by quantitative real-time PCR. (A–C) Real-time PCR analysis of RIP1, RIP3, and MLKL in each group, and inhibited by necrosis inhibitor Nec-1, and 3-MA but increased by zVAD-fmk. For EAM, 3W rats are used. (D–F) Real-time PCR analysis of Caspase 3, Bax, and Bcl2 in each group. The necrosis inhibitor Nec-1 and 3-MA increase the mRNA expression levels of Caspase 3/Bax mRNA in myocardial tissues. *p < 0.05 vs. control group; **p < 0.01 vs. EAM + DMSO group, Δp > 0.05 vs. EAM group, ΔΔp > 0.05 vs. EAM + DMSO group. A Student's t-test was performed for the control group and the EAM group, one-way analysis of variance (ANOVA) followed by the LSD test; data are presented as mean ± SEM.

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  Figure 6. Nec-I inhibits but 3-MA increases the expression levels of RIP1, RIP3, and MLKL in EAM. (A) Western analysis of protein expression levels of RIP1, RIP3, and MLKL in myocardial tissues from the control group; EAM (3W) group; EAM + DMSO group; and EAM + Nec-I, EAM + zVAD-fmk, and EAM + 3MA drug group. (B–D) Quantification of protein levels of RIP1 in b, RIP3 in c, and MLKL in d. Horizontal lines above bars indicate groups compared. All data are presented as mean ± SEM. A Student's t-test was performed for the control group and EAM group, one-way analysis of variance (ANOVA) followed by the LSD test. *p < 0.05 vs. control group; **p < 0.01 vs. EAM + DMSO group, Δp > 0.05 vs. EAM group. Δp > 0.05 vs. EAM + Nec-1 group, Δp > 0.05 vs. EAM + 3-MA group. (E,F) Quantification of LC-3 I and II. Western analysis of LC-3 I and II expression levels treated by inhibitors of Nec-1 and 3-MA. (D) Quantification of LC-3 I and II of EAM (3W); ΔΔp < 0.05 means compared with the control group (3 weeks); **p > 0.05 means compared with the control group (8 weeks).

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  Figure 7. Immunohistochemical staining of RIP1/RIP3/MLKL and LC3B in rat myocardium. (A) Imunohistochemistry staining images of RIP1/RIP3/MLKL and LC3B. Red arrows point the brown particle deposition. Control: control group; EAM: EAM (3W); IMM + DMSO; EAM + Nec-1: necroptosis inhibitor group; EAM-zVAD-fmk, apoptosis inhibitor group, EAM + 3-MA: autophagy inhibitor group. RIP1, RIP3, MLKL, and LC3B. (B–E) Quantity of brown–red particles' intensity in the cytoplasm of RIP1, RIP3, MLKL, and LC3 I, respectively. The necrosis inhibitor Nec-1 decreased the intensities in RIP1, RIP3, and MLKL, but not in LC3 I and II. However, the autophagy inhibitor 3-MA inhibited the intensity of LC3B. A Student's t-test was performed in each group. *p < 0.05 are obtained for RIP1, when comparing EAM with EAM + Nec-1, EAM + zVAD-fmk, 3-MA; for RIP3, EAM with EAM + Nec-1, EAM + zVAD-fmk, and EAM + 3-MA; for MLKL, EAM with EAM-Nec-1, EAM + zVAD-fmk, and EAM-3-MA; for LC3B, EAM and EAM + Nec-1 and EAM and EAM +3-MA.

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  Figure 8. Pathological evaluations of myocardial tissues of various groups by HE staining. (A) In the heart of control group rat, there was no inflammatory infiltrate. (B,C) HE staining showed dense inflammatory infiltrates in the EAM (B) group (3W) and IMM + DMSO group (C). Treating rat with Nec-1, zVAD-fmk, and 3-MA on day 21 attenuated inflammatory infiltrates. Only little scattered inflammatory infiltrates were detected in the EAM + Nec-1 group (D). (G) Quantification of cellular inflammatory infiltration in (A–F). All data are presented as mean ± SEM. A Student's t-test was performed for the control group and IMM group, one-way analysis of variance (ANOVA) followed by the LSD test. *p < 0.05 vs. control group, **p < 0.01 vs. EAM + DMSO group, p < 0.05 vs. EAM. Δ Compared with EAM group, the EAM_DMSO group has no significance.

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  Figure 9. Hypothetical illustration of cell death in EAM model. In EAM, all three processes of cell death take place, but when necroptosis is blocked by Nec-1, apoptosis increases but there is no effect on autophagy. When autophagy is blocked by 3-MA, both apoptosis and necroptosis are increased. When apoptosis is blocked by zVAD-fmk, it increases both necroptosis and autophagy.