MYZAP promotes angiogenesis by targeting RND1 after myocardial infarction

Figures

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  Figure 1. The expression of MYZAP was decreased in myocardium after MI (A) Flow chart of animal models and related testing. (B) 14 days and 28 days after surgery, cardiac function continued to decline in mice with MI by echocardiography. ∗∗∗p < 0.001,###p < 0.001 vs. Sham. n = 7–8. (C) Angiogenesis marked by IB4 in mice after MI was gradually decreased using immunofluorescence staining. ∗∗∗p < 0.001, ###p < 0.001, vs Sham. n = 9. (D and E) The protein and mRNA levels of MYZAP were decreased 14 days and 28 days after MI. The expression of MYZAP was significantly reduced in the myocardium after MI. ∗p < 0.05, ∗∗∗p < 0.001, ##p < 0.001vs. Sham. n = 4-7. (F) The expression of MYZAP can be seen in both the nucleus and the membrane using immunofluorescence localization staining. Scale bars, 10 μm or 1μm. (G and H) The protein (n = 6) and mRNA (n = 11–12) expression levels of MYZAP in HUVECs and MAECs in the Hypoxia group were significantly reduced compared with the control (Ctl) group. ∗p < 0.05, ∗∗∗p < 0.001, vs. Ctl, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Ctl. n = 6-12. (I and J) The protein of MYZAP was more abundant in isolated cardiomyocytes than in fibroblasts. ∗∗∗p < 0.001, vs. CF. n =…

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  Figure 2. The impact of endothelial-specific overexpression of MYZAP on cardiac function and angiogenesis in mice with MI (A) Experimental process diagram. (B–D) Endothelial-specific overexpression of MYZAP can significantly improve cardiac function in mice after MI. ∗∗∗p < 0.001 vs. Sham, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. +NC. n = 8–12. (E and F) Representative images and statistical results of IB4 and CD31 immunofluorescence staining. ∗∗∗p < 0.001 vs. Sham, #p < 0.05 vs. +NC. n = 9. Scale bars, 20 μm, 10 μm. (G) Representative three-dimensional high-resolution images scanned by Micro-CT and quantitative analysis of Nb of marginal vessels. ∗∗∗p < 0.001 vs. Sham, ###p < 0.001 vs. +NC. n = 7–8. Data are represented as mean ± SEM. For comparisons among multiple groups, one-way ANOVA was used, with Dunnett’s test or Tukey’s test for pairwise comparisons. +MYZAP: MI + MYZAP; +NC: MI + NC.

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  Figure 3. Overexpression of MYZAP promotes expression of vWF and CD31 in vivo and in vitro (A) Immunofluorescent co-localization of the endothelial markers vWF and CD31 revealed that endothelial-specific MYZAP overexpression promotes angiogenesis within the infarct zone of murine hearts following MI. Scale bars, 20 μm. (B) vWF expression was significantly reduced in the MI group relative to the Sham group, whereas it was markedly elevated in the MYZAP group compared to the +NC group. ∗∗∗p < 0.001 vs. Sham, ###p < 0.001 vs. +NC. n = 6. (C) Compared with the Sham group, the MI group showed decreased CD31 expression; conversely, MYZAP overexpression resulted in increased CD31 expression relative to the +NC control. ∗∗∗p < 0.001 vs. Sham, ###p < 0.001 vs. +NC. n = 6. (D) In vitro immunofluorescence analysis revealed that overexpression of MYZAP significantly increased the fluorescence intensities of the endothelial markers vWF and CD31 in HUVECs. Scale bars, 20 μm. (E) Notably, vWF expression decreased in hypoxia-exposed HUVECs versus control cells, yet was substantially augmented upon MYZAP overexpression relative to negative controls. ∗∗∗p < 0.001 vs. Ctl, ###p < 0.001 vs. +NC. n = 8-11. (F)…

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  Figure 4. Overexpression of endothelium-specific MYZAP restores blood flow in mice with left hindlimb ischemia (A) Surgical procedure flowchart. (B) Representative images of hindlimb ischemia. Quantification of blood flow levels in mice after ischemia surgery using MoorFLPIReview revealed that overexpression of endothelium-specific MYZAP significantly restored blood flow levels in the left hindlimb of mice. (C) Overexpression of MYZAP restored blood flow in the left hindlimb of mice at ischemia-3d, 5 days, 7 days, and 14 days. ∗p < 0.05 vs. +NC-3d. ∗∗∗p < 0.001 vs. +NC-5d. ∗∗p < 0.01 vs. +NC-7d. ∗∗∗p < 0.001 vs. +NC-14d. n = 3-4. (D and E) Overexpression of MYZAP restored the area under the curve (AUC) based on the MoorFLPI perfusion data. ∗∗∗p < 0.001 vs. Sham. ###p < 0.001 vs. +NC. n = 3-4. Data are represented as mean ± SEM. For comparisons among multiple groups, one-way ANOVA was used, with Dunnett’s test or Tukey’s test for pairwise comparisons. +MYZAP: Ischemia+MYZAP; +NC: Ischemia+NC.

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  Figure 5. Overexpression of MYZAP improves endothelial cell dysfunction (A and B) In vitro scratch assays demonstrated that MYZAP markedly enhanced the migratory capacity of HUVECs and MAECs. ∗∗∗p < 0.001 vs. Ctl. #p < 0.05, ##p < 0.01 vs. +NC. n = 5-6. Scale bars, 100 μm. (C and D) Transwell migration and invasion assays revealed that MYZAP overexpression significantly promoted the migratory and invasive capacities of both HUVECs and MAECs. ∗∗∗p < 0.001 vs. Ctl. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. +NC. n = 6. Scale bars, 100 μm. (E and F) Tube formation analysis of HUVECs demonstrated that MYZAP promoted cellular migration. Scale bars, 200 μm. n = 3.∗∗∗p < 0.001 vs. Ctl. #p < 0.05, ##p < 0.01 vs. +NC. Data are represented as mean ± SEM. For comparisons among multiple groups, one-way ANOVA was used, with Dunnett’s test or Tukey’s test for pairwise comparisons. +MYZAP: Hypoxia+MYZAP; +NC: Hypoxia+NC.

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  Figure 6. Knockdown of MYZAP inhibits endothelial cell function (A and B) MYZAP knockdown significantly suppressed the wound-healing ability of HUVECs and MAECs. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. Ctl, ##p < 0.01, ###p < 0.001 vs. si-NC. n = 6. Scale bars, 100 μm. (C and D) MYZAP silencing resulted in a pronounced inhibition of HUVEC and MAEC migration and invasion and MAECs. ∗∗∗p < 0.001 vs. Ctl, ###p < 0.001 vs. si-NC. n = 5–6. Scale bars, 100 μm. (E and F) The tube formation ability was significantly decreased in HUVECs and MAECs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. Ctl, #p < 0.05 vs. si-NC. n = 3. Scale bars, 200 μm. Data are represented as mean ± SEM. For comparisons among multiple groups, one-way ANOVA was used, with Dunnett’s test or Tukey’s test for pairwise comparisons.

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  Figure 7. Interaction between MYZAP and RND1 (A–C) Western blot results show downregulation of RND1 after overexpression of MYZAP. ∗p < 0.05 vs. Sham, ##p < 0.01, ###p < 0.001 vs. +NC. n = 6. (D)The results of the immunoprecipitation experiment showed an interaction between MYZAP and RND1. (E) Molecular docking results also revealed multiple binding sites between MYZAP and RND1. (F) MYZAP inhibits the stability of RND1 protein by interfering with its degradation process. Statistical results of CETSA: the protein level of RND1 at different temperatures after treatment with MYZAP and NC. n = 3. (G) Protein mass spectrum pulled down by MYZAP antibody showed that TRIM21, a kind of ubiquitin ligase, was the most likely target of MYZAP. Ubiquitination assays revealed that TRIM21 promotes the ubiquitination of RND1, leading to its degradation. Data are represented as mean ± SEM. For comparisons among multiple groups, one-way ANOVA was used, with Dunnett’s test or Tukey’s test for pairwise comparisons. +MYZAP: MI + MYZAP; +NC: MI + NC.

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  Figure 8. Endothelial-specific knockdown of RND1 restores cardiac function post-MI by promoting angiogenesis after MI (A and B) Administration of endothelial-specific AAV9-mTie-sh-RND1 facilitates the recovery of cardiac function in mice following MI. EF and FS values were significantly reduced in the MI group compared to the Sham group. Conversely, in vivo knockdown of endothelial RND1 significantly improved EF and FS compared to the NC group. ∗∗∗p < 0.001 vs. Sham, ###p < 0.001 vs. +sh-NC. n = 8. (C and D) Immunofluorescence staining of MI heart sections reveals a decrease in the expression of angiogenic markers vWF and CD31 within the infarct area. RND1 knockdown significantly upregulated the expression of these markers compared to the NC group. ∗∗∗p < 0.001 vs. Sham, ###p < 0.001 vs. +sh-NC. n = 11–12. Scale bars, 20 μm. (E–H) Silencing of RND1 upregulates the protein and mRNA expression of PI3K and the mRNA levels of VEGF-A in the vascular endothelium, thereby activating angiogenesis-related pathways. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. +sh-NC. n = 5–11. Data are represented as mean ± SEM. Student’s t test was used for comparisons between two groups. For comparisons among multiple…

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  Figure 9. The pro-angiogenic effect and cardiac function of MYZAP overexpression were reversed by RND1 overexpression and a PI3K inhibitor (LY294002), both in vitro and in vivo (A and B) MYZAP overexpression significantly enhanced the wound-healing ability of HUVECs exposed to hypoxic conditions. RND1 overexpression and PI3K inhibitor (LY294002) counteract the effect of MYZAP. ∗∗∗p < 0.001 vs. Ctl; &&&, ααα, ###p < 0.001 vs. +MYZAP. n = 15. Scale bars, 100 μm. (C and D) MYZAP overexpression reduced the inhibition of HUVEC migration exposed to hypoxic conditions. And RND1 overexpression and PI3K inhibitor (LY294002) counteract the effect of MYZAP. ∗∗∗p < 0.001 vs. Ctl; &&&, ααα, ###p < 0.001 vs. +MYZAP. n = 6. Scale bars, 100 μm. (E–G) The tube formation ability was significantly decreased in HUVECs exposed to hypoxic conditions. MYZAP overexpression increased the ability of tube formation. RND1 overexpression and PI3K inhibitor (LY294002) counteract the effect of MYZAP. ∗∗∗p < 0.001 vs. Ctl, &, α, #p < 0.05, ##p < 0.01, &&&, ααα, ###p < 0.001 vs. +MYZAP. n = 6. Scale bars, 200 μm. (H and I) MYZAP overexpression increased the protein level of CD31 in MI mice, and RND1 overexpression and the…