Abstract: Objective Exploring the effect of PKM2 neddylation modification on myocardial fibrosis in pulmonary hypertension rats. Methods SD rats were randomly divided into 3 groups: control group, model group, and MLN4924 group. Pulmonary arteries were detected by HE staining, and right ventricular fibrosis was detected by Masson staining. Primary cardiac fibroblasts were isolated from rats and the expression of α-Smooth muscle actin (α-SMA) was measured by immunofluorescence assay. The si-NC, si-PKM2, and pcDNA3. 1-PKM2 were transfected into the primary cardiac fibroblasts and the expression levels of PKM2, fibrosis related proteins Collagen Ⅰ , Collagen Ⅲ , MMP2, and MMP9 were detected by Western blotting. Immunoprecipitation assay was used to detect the PKM2 neddylation modification. Real time fluorescence quantitative PCR method was used to detect the mRNA expression level of PKM2 in the rat heart tissues. The degradation of PKM2 protein was detected by Western blotting, and the half-life of PKM2 was determined. Results  The HE staining results showed that the space between pulmonary arterioles (fibrous layer) and intima (inner transport protein) in the model group was significantly widened and the intermediate layer were thickened. Masson staining showed that the collagen deposition was increased and the fibrosis was more severe in the model group when compared with those in the control group. The expression level of PKM2 protein in the model group was higher than that in the control group, while there was no significant difference in the mRNA expression level. PKM2 underwent neddylation modification in the right ventricular tissues of pulmonary hypertension rats. Knocking down Nedd8 in cardiac fibroblasts or inhibiting neddylation modification with MLN4924 could downregulate the expression levels of PKM2 and fibrosis related proteins Collagen Ⅰ , Collagen Ⅲ , MMP2, MMP9, etc. , promotingthe degradation of PKM2 protein [ (3. 03 ± 0. 23) - (11. 97 ± 0. 66) h, t = - 12. 82, P <0. 001] . However, the overexpression of Nedd8 could increase the expression level of PKM2 protein. The degree of right ventricular fibrosis and the expression level of α-SMA protein in theMLN4924 group were lower than those in the model group. Conclusion Neddylation modificationenhances the protein stability of PKM2, thereby promoting the process of right ventricular fibrosis inthe rat model of pulmonary arterial hypertension.

Key words: pulmonary arterial hypertension, PKM2, neddylation, right ventricular fibrosis