S-nitrosylation of paired-related homeobox 1 promotes cardiac remodeling following myocardial infarction

Figures

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  Fig. 1. Myocardial infarction (MI) induces cardiac remodeling through S-nitrosylation in mice. The experimental protocol was illustrated in Online Fig. 1A. (A-C) The heart functions were determined at the 28th postoperative before sacrificed using ultrasound in A. LVEF in B and LVFS in C were calculated. (D and E) Hearts were harvested from mice to perform HE staining and Masson staining in D. Collagen content was calculated in E. (F and G) Protein S-nitrosylation was measured heart tissues using the biotin-switch method in F and quantification in G. (H) The productions of NO and ROS were determined in hearts. N = 10 per group. A one-way ANOVA followed by Tukey's HSD test was used to determine P value between two groups in B, C, E, and G. An unpaired Student's t-test was used to determine P value in H.

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  Fig. 2. Nitric oxide (NO) S-nitrosylates human Prrx1 protein at C207 and activate Prrx1 transcriptional activity. (A) Recombinant human Prrx1 protein was incubated with four NO donors (1 mM) for 2 h in reaction buffers. Reaction products were subjected to determine Prrx1 S-nitrosylation. *P < 0.05 vs. Vehicle. (B) Illustration of human Prrx1 C207 S-nitrosylation identified by mass spectrometry. (C) Purified human Prrx1 (WT, C207R) proteins from E.coli. were incubated with sodium nitroprusside (SNP) for 2 h in reaction buffers. Reaction products were subjected to determine Prrx1 S-nitrosylation. (D and E) Cardiac fibroblasts were transfected with plasmids of Prrx1 (WT, C207R) for 48 h and then incubated with SNP (1 mM) for 2 h. Total cell lysates were subjected to detect Prrx1 S-nitrosylation in D and transcriptional activity using luciferase reporter gene in E. N = 5 per group. A one-way ANOVA followed by Dunnett's test was used to determine P value in A. A one-way ANOVA followed by Tukey's HSD test was used to determine P value between two groups in C, D and E.

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  Fig. 3. Oxygen-glucose deprivation (OGD) induces Prrx1 S-nitrosylation to promote fibroblast-to-myofibroblast differentiation in cardiac fibroblasts. Cardiac fibroblasts were transfected with plasmids of Prrx1 (WT, C207R) for 48 h followed by OGD or glucose deprivation (GD) for 24 h. (A) Cell proliferation was measured using CCK8. (B and C) Total cell lysates were subjected to detect Prrx1 S-nitrosylation in B and transcriptional activity using luciferase reporter gene in C. (D and E) IFC analysis of α-SMA in D and quantification in E were performed. (F–I) Gene expressions of α-SMA in F, Wnt5a in G, Col I in H, and Col III in I were determined using quantitative PCR. N = 5 per group. A one-way ANOVA followed by Tukey's HSD test was used to determine P value between two groups.

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  Fig. 4. Transforming growth factor beta (TGF-β) promotes fibroblast-to-myofibroblast differentiation of cardiac fibroblasts through Prrx1 S-nitrosylation. Cardiac fibroblasts were transfected with plasmids of Prrx1 (WT, C207R) for 48 h and then incubated with TGF-β (10 ng/ml) for 24 h. (A and B) Total cell lysates were subjected to detect Prrx1 S-nitrosylation in A and transcriptional activity using luciferase reporter gene in B. (C) Cell proliferation was measured using CCK8 in C. (D and E) IFC analysis of α-SMA in D and quantification in E were performed. (F–I) Gene expressions of α-SMA in F, Wnt5a in G, Col I in H, and Col III in I were determined using quantitative PCR. N = 5 per group. A one-way ANOVA followed by Tukey's HSD test was used to determine P value between two groups.

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  Fig. 5. TGF-β-induced Prrx1-mediated fibroblast-to-myofibroblast differentiation is Wnt5a dependent in cardiac fibroblasts. (A-C) Cardiac fibroblasts were infected with adenovirus expressing Prrx1-WT plus negative control (NC) or Wnt5a shRNA for 48 h followed by TGF-β (10 ng/ml) treatment for 24 h. Cell proliferation was measured using CCK8 in A. IFC analysis of α-SMA in B and quantification in C were performed. (D-F) Cardiac fibroblasts were infected with adenovirus expressing Prrx1-MT (C207R) plus vector or Wnt5a cDNA for 48 h followed by TGF-β treatment for 24 h. Cell proliferation was measured using CCK8 in D. IFC analysis of α-SMA in E and quantification in F were performed. N = 5 per group. A one-way ANOVA followed by Tukey's HSD test was used to determine P value between two groups.

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  Fig. 6. Enforced expression of Prrx1-C209R improves cardiac remodeling in mice after myocardial infarction (MI). The experimental protocol was illustrated in Online Fig. 6A. (A-D) At the 28th postoperative day, hearts were harvested from mice to perform HE staining and Masson staining. Heart functions were measured before sacrificed using ultrasound. Representative images were shown in A. Collagen intensity in B, LVFS in C, and LVEF in D were calculated. (E) Serum NO concentrations. (F–I) Cardiac fibroblasts isolated from mice were subjected to determine Prrx1 S-nitrosylation using the biotin-switch method in F, cell proliferation using CCK8 in G, α-SMA mRNA in H, and Wnt5a mRNA in I. N = 10 per group. A one-way ANOVA followed by Tukey's HSD test was used to determine P value between two groups.

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  Fig. 7. Fibroblast-specific Prrx1 gene knockout prevents cardiac remodeling in mice following myocardial infarction (MI). The experimental protocol was illustrated in Online Fig. 8A. (A-D) At the 28th postoperative day, heart functions were measured before sacrificed using ultrasound. Hearts were harvested from mice to perform HE staining and Masson staining. Representative images were shown in A. LVEF in B, LVFS in C, collagen content in D were calculated. (E-H) Cardiac fibroblasts isolated from mice were subjected to gene expressions of α-SMA in E, Wnt5a in F, Col I in G, and Col III in H using quantitative PCR. N = 10 per group. An unpaired Student's t-test was used to determine P value.

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  Fig. 8. Increased Prrx1 S-nitrosylation in patients with post myocardial infarction (MI). The demographic data of human subjects were presented in Online Table 1. (A-D) Heart functions were measured using ultrasound in A. LVEF in B and LVFS in C were calculated. Serum level of N-terminal propeptide procollagen type III (PIIINP) was measured using ELISA in D. (E-H) Leucocytes in peripheral blood isolated from patients. Total cell lysates were subjected to determine the levels of Prrx1 S-nitrosylation using the biotin-switch method, α-SMA and Wnt5a proteins using Western blot in E. Quantifications of Prrx1 S-nitrosylation in F, Wnt5a in G, and α-SMA in H were calculated. N = 15 per group. *P < 0.05 vs. Non-MI. An unpaired Student's t-test was used to determine P value.