Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by RIPK3 Alleviates Necroptosis in Transverse Arch Constriction-Induced Heart Failure

Figures

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  FIGURE 1. Cardiac dysfunction, increased expressions of ANP and BNP, CaMKIIδ alternative splicing disorder, CaMKII oxidation, and phosphorylation levels are increased in HF mice. Male 8-week-old C57BL/6 mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) Echocardiography. (B) EF and FS. (C) IVS and LVPW. (D) The expression of ANP at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control; (E) The expression of BNP at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. (F) Western blotting analysis was used to detect the expression of OX-CaMKII protein in myocardial tissue, and GAPDH was used as loading control; (G) Western blotting was used to detect the expression of P-CaMKII protein in myocardial tissue, and GAPDH was used as a loading control. (H) Detection of myocardial CaMKIIδ A, CaMKIIδ B, and CaMKIIδ C at the mRNA level by qRT-PCR, and 18S was used as the housekeeping gene. Compared with…

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  FIGURE 2. The expressions of RIPK3 and RIPK1, as well as the levels of MLKL phosphorylation and apoptosis, are increased in HF mice. Male 8-week-old C57BL/6 mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) The expression of RIPK3 at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. (B) The expression of RIPK1 at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. (C) The levels of MLKL and P-MLKL proteins in myocardial tissue were detected by Western blotting analysis, and GAPDH was used as a loading control. (D) The level of apoptosis in myocardial tissue was measured by TUNEL staining, bar = 50 μm; (E) The statistical chart of TUNEL-positive cells. (F) The levels of cleaved caspase 3 and caspase 3 in myocardial tissue were detected by Western blotting analysis, and β-tubulin was used as a loading control. Compared with WT + sham group, *P < 0.05. n = 6.

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  FIGURE 3. Depletion of RIPK3 can alleviate cardiac insufficiency in mice with HF and has no significant improvement in the increased expressions of ANP and BNP. Male 8-week-old WT and RIPK3–/– mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) Echocardiography. (B) EF and FS. (C) IVS and LVPW. (D) The expression of ANP at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control; (E) The expression of BNP at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. Compared with WT + sham group, **P < 0.01; compared with RIPK3–/– + sham group, ##P < 0.01 and #P < 0.05; compared with WT + TAC group, &⁣&P < 0.01 and &P < 0.05. n = 6.

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  FIGURE 4. Depletion of RIPK3 can inhibit the increase of cardiomyocyte cross-sectional area in mice with HF and improve myocardial injury. Male 8-week-old WT and RIPK3–/– mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) The cross-sectional area of mouse cardiomyocytes was observed by WGA staining, bar = 50 μm. (B) Cell area measurements in WGA. (C) Tissue damage area measurements in H&E. (D) H&E staining was used to detect the degree of myocardial injury, bar = 50 μm. Compared with WT + sham group, **P < 0.01; compared with RIPK3–/– + sham group, ##P < 0.01; compared with WT + TAC group, &&P < 0.01. n = 6.

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  FIGURE 5. Depletion of RIPK3 can improve the degree of collagen deposition and myocardial fibrosis in HF mice. Male 8-week-old WT and RIPK3–/– mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) Sirius red staining, bar = 50 μm. (B) Sirius red staining area measurements. (C) Masson staining, bar = 50 μm. (D) Masson staining area measurements. Compared with WT + sham group, **P < 0.01; compared with RIPK3–/– + sham group, ##P < 0.01; compared with WT + TAC group, &&P < 0.01. n = 6.

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  FIGURE 6. Depletion of RIPK3 can alleviate the elevated levels of CaMKII oxidation and phosphorylation, improve the CaMKIIδ alternative splicing disorder in HF mice and the degree of myocardial injury and inflammatory response. Male 8-week-old WT and RIPK3–/– mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) The expression of OX-CaMKII at the protein level in myocardial tissue was detected by Western blotting analysis, and GAPDH was used as a loading control. (B) The expression of P-CaMKII at the protein level in myocardial tissue was detected by Western blotting analysis, and GAPDH was used as a loading control. (C) Detection of myocardial CaMKIIδ A, CaMKIIδ B, and CaMKIIδ C at the mRNA level by qRT-PCR, and 18S was used as the housekeeping gene. (D) The levels of serum LDH, (E) CK, (F) IL-6, and (G) TNF-α. Compared with WT + sham group, **P < 0.01; compared with RIPK3–/– + sham group, ##P < 0.01 and #P < 0.05; compared with WT + TAC group, &⁣&P < 0.01 and &P < 0.05. n = 6.

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  FIGURE 7. Depletion of RIPK3 can reduce the expressions of RIPK3 and RIPK1, as well as the MLKL phosphorylation level and cardiomyocyte apoptosis in HF mice and improve antioxidant capacity. Male 8-week-old WT and RIPK3–/– mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) The expression of RIPK3 at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. (B) Western blotting analysis was used to detect the expression of RIPK1 at the protein level in myocardial tissue, and GAPDH was used as a loading control. (C) Western blotting analysis was used to detect the expressions of MLKL and P-MLKL at the protein level in myocardial tissue, and GAPDH was used as a loading control. (D) The expressions of cleaved caspase 3 and caspase 3 at the protein level in myocardial tissue were detected by Western blotting analysis, and β-tubulin was used as a loading control. (E) TUNEL staining was used to determine the level of apoptosis in myocardial tissue, bar = 50 μm. (F) The…

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  FIGURE 8. Depletion of RIPK3 can reduce ROS accumulation in myocardial tissue of HF mice and improve antioxidant capacity and mitochondrial structural abnormalities. Male 8-week-old WT and RIPK3–/– mice were treated with TAC using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) DHE staining, bar = 50 μm. (B) Average fluorescence intensity measurements of DHE. (C) Mitochondrial damage area. (D) The ultrastructure of LV mitochondria was observed by transmission electron microscope, bar = 2 μm. Lower picture bar = 0.5 μm. Compared with WT + sham group, **P < 0.01; compared with RIPK3–/– + sham group, ##P < 0.01; compared with WT + TAC group, &&P < 0.01. n = 6.

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  FIGURE 9. Interference with RIPK3 can alleviate cardiac insufficiency in HF mice and has no significant improvement in the up-regulation of ANP and BNP. Male 8-week-old WT mice were pretreated with AAV-vector and AAV-shRNA through the tail vein, and TAC was performed using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) Echocardiography. (B) EF and FS. (C) IVS and LVPW. (D) The expression of ANP at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. (E) The expression of BNP at the protein level in myocardial tissue was detected by Western blotting analysis, and β-tubulin was used as a loading control. Compared with WT + sham group, **P < 0.01 and *P < 0.05; compared with WT + TAC group, #P < 0.05; compared with WT + AAV-vector group, &P < 0.05. n = 6.

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  FIGURE 10. Interference with RIPK3 can inhibit the increase of cardiomyocyte cross-sectional area in HF mice and alleviate myocardial injury. Male 8-week-old WT mice were pretreated with AAV-vector and AAV-shRNA through the tail vein, and TAC was performed using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) The cross-sectional area of mouse cardiomyocytes was observed by WGA staining, bar = 50 μm. (B) Cell area measurements in WGA staining. (C) Tissue damage area measurements in H&E staining. (D) H&E staining was used to detect the degree of myocardial injury, bar = 50 μm. Compared with WT + sham group, **P < 0.01; compared with WT + TAC group, ##P < 0.01; compared with WT + AAV-vector group, &&P < 0.01. n = 6.

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  FIGURE 11. Interference with RIPK3 can improve the degree of collagen deposition and myocardial fibrosis in HF mice. Male 8-week-old WT mice were pretreated with AAV-vector and AAV-shRNA through the tail vein, and TAC was performed using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) Sirius red staining, bar = 50 μm. (B) Sirius red staining area measurements. (C) Masson staining area measurements. (D) Masson staining, bar = 50 μm. Compared with WT + sham group, **P < 0.01; compared with WT + TAC group, ##P < 0.01; compared with WT + AAV-vector group, &&P < 0.01. n = 6.

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  FIGURE 12. Interference with RIPK3 can alleviate the elevated levels of CaMKII oxidation and phosphorylation, improve CaMKIIδ alternative splicing disorder, the degree of myocardial injury, and inflammatory response in HF mice. Male 8-week-old WT mice were pretreated with AAV-vector and AAV-shRNA through the tail vein, and TAC was performed using a 28-g needle. The control group received sham surgery. All groups of mice were given continuous feeding for 6 weeks. Hearts were then obtained for correlative testing. (A) The expression of OX-CaMKII at the protein level in myocardial tissue was detected by Western blotting analysis, and GAPDH was used as a loading control. (B) The expression of P-CaMKII at the protein level in myocardial tissue was detected by Western blotting analysis, and GAPDH was used as a loading control. (C) Detection of myocardial CaMKIIδ A, CaMKIIδ B, and CaMKIIδ C at the mRNA level by qRT-PCR, and 18S was used as the housekeeping gene. (D) The levels of serum LDH, (E) CK, (F) IL-6, and (G) TNF-α. Compared with WT + sham group, **P < 0.01; compared with WT + TAC group, ##P < 0.01 and #P < 0.05; compared with WT + AAV-vector group, &⁣&P < 0.01 and &P < 0.05. n = 6.