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. 2024 Jan 20;27(2):108982.
doi: 10.1016/j.isci.2024.108982. eCollection 2024 Feb 16.

Circ-CIMIRC inhibition alleviates CIH-induced myocardial damage via FbxL4-mediated ubiquitination of PINK1

Runhua Wu  1 , Fengsheng Xu  1 , Jingyi Li  1 , Feng Wang  1 , Naijie Chen  1 , Xiaoting Wang  2 , Qin Chen  2
Affiliations

Circ-CIMIRC inhibition alleviates CIH-induced myocardial damage via FbxL4-mediated ubiquitination of PINK1

Runhua Wu et al. iScience. .

Abstract

Obstructive sleep apnea (OSA) is a common sleep disordered breathing diseases that characterized by chronic intermittent hypoxia (CIH). This work aimed to explore the role of circ-CIMIRC in CIH-induced myocardial injury. CIH aggravated myocardial tissue damage in rats. Circ_CIMIRC overexpression promoted apoptosis and reduced the colocalization of Tom20 and Parkin and mitophagy in CIH-treated H9c2 cells. Additionally, FbxL4 interacted with PINK1, FbxL4 silencing reduced PINK1 ubiquitination in H9c2 cells. Two major ubiquitination sites (K319 and K433) were responsible for ubiquitination of PINK1. Circ_CIMIRC promoted FbxL4-mediated ubiquitination and degradation of PINK1. Furthermore, circ_CIMIRC inhibition alleviated the pathological damage, fibrosis and apoptosis of myocardial tissues, reduced oxidative stress in CIH rats. In conclusion, circ_CIMIRC silencing repressed FbxL4-mediated ubiquitination and degradation of PINK1 and then enhanced PINK1/Parkin-mediated mitophagy, thereby alleviating myocardial damage in CIH rats. Thus, circ_CIMIRC may be a potential strategy to alleviate CIH-induced myocardial damage.

Keywords: Cell biology; Molecular biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
FbxL4 was upregulated, PINK1 and Parkin were downregulated in CIH rats (A) Heart weight/body weight of CIH and normal rats. H&E (B), Masson (C), and TUNEL (D and E) staining assessed the pathological changes, fibrosis and apoptosis of myocardial tissues in CIH and normal rats. (F) Western blotting examined the expression of cleaved caspase-3, p62 and Bcl-2 in myocardial tissues of CIH and normal rats. (G) The expression of FbxL4 in iTRAQ quantification. (H and I) Western blotting detected FbxL4, PINK1, and Parkin expression in myocardial tissues of CIH and normal rats. (J) Western blotting assessed the levels of PINK1 ubiquitination in myocardial tissues of CIH and normal rats. ∗p < 0.05, ∗∗p < 0.01 vs. Normal group.
Figure 2
Figure 2
FbxL4 regulated apoptosis and mitophagy in CIH-treated H9c2 cells H9c2 cells were subjected to CIH treatment, and then transfected with pcDNA-FbxL4, Vector, si-FbxL4 or si-NC. qRT-PCR (A) and western blotting (B) detected the expression of FbxL4 in H9c2 cells. (C) TUNEL staining examined cell apoptosis. (D) IF staining assessed the levels of GFP-LC3 puncta. (E) Western blotting assessed the expression of LC3-I, LC3-II, Mfn2, and Drp1 in H9c2 cells. ∗p < 0.05, ∗∗p < 0.01 vs. Control; #p < 0.05, ##p < 0.01 vs. CIH+Vector; $p < 0.05, $$p < 0.01 vs. CIH+si-NC.
Figure 3
Figure 3
FbxL4 interacted with PINK1, and FbxL4 silencing inhibited the ubiquitination and degradation of PINK1 (A) STRING predicated the interaction between FbxL4 and PINK1. (B) UbiBrowser uncovered the potential ubiquitination sites of PINK1. (C) Co-IP verified the interaction between FbxL4 and PINK1. (D–F) Western blotting examined the levels of PINK1 and PINK1 ubiquitination in H9c2 cells in the presence of si-NC or si-FbxL4. (G and H) Western blotting detected the levels of PINK1 and PINK1 ubiquitination in H9c2 cells in the presence of pcDNA-FbxL4, Vector, and MG132. ∗p < 0.05, ∗∗p < 0.01 vs. si-NC; #p < 0.05, ##p < 0.01 vs. Vector; $p < 0.05 vs. FbxL4.
Figure 4
Figure 4
Circ_CIMIRC repressed PINK1 and Parkin expression, and affected apoptosis and mitophagy of CIH-treated H9c2 cells H9c2 cells were subjected to CIH treatment, and then transfected with pcDNA-circ_CIMIRC, Vector, si-circ_CIMIRC, or si-NC. (A) TUNEL staining examined cell apoptosis. (B–G) Western blotting assessed the expression of FbxL4, PINK1, Parkin, LC3-I, LC3-II, Mfn2, and Drp1 in H9c2 cells. (H) IF staining examined the levels of mitophagy in H9c2 cells. ∗p < 0.05, ∗∗p < 0.01 vs. Vector; #p < 0.05, ##p < 0.01 si-NC.
Figure 5
Figure 5
Circ_CIMIRC interacted with FbxL4 and PINK1, and promoted ubiquitination of PINK1 (A) qRT-PCR detected the expression of circ-CIMIRC in the cytoplasm and nucleus of H9c2 cells. $$p < 0.01 vs. nuclear fraction. (B and C) RIP verified the interaction among circ-CIMIRC, FbxL4 and PINK1. &&p < 0.01 vs. Anti-IgG. (D and E) Western blotting examined the levels of PINK1 and PINK1 ubiquitination in H9c2 cells. ∗p < 0.05 vs. si-NC; %%p < 0.01 vs. Vector; ˆˆp < 0.01 vs. circ-CIMIRC.
Figure 6
Figure 6
Circ_CIMIRC deficiency inhibited apoptosis of CIH-treated H9c2 cells by regulating mitophagy H9c2 cells were subjected to CIH treatment, and then transfected with si-circ_CIMIRC or si-NC or combined with Mdivi-1 treatment. (A) TUNEL staining examined apoptosis of H9c2 cells. (B–E) Western blotting assessed the expression of PINK1, Parkin, LC3-I, and LC3-II in H9c2 cells. (F) IF staining examined the levels of mitophagy in H9c2 cells. ∗∗p < 0.01 vs. si-NC; #p < 0.05 vs. si-circ-CIMIRC.
Figure 7
Figure 7
Circ_CIMIRC inhibition reduced myocardial damage in CIH rats (A) Heart weight/body weight of rats. HE (B and C), Masson (D and E) and TUNEL (F and G) staining assessed the pathological changes, fibrosis and apoptosis of myocardial tissues. (H) Western blotting examined the expression of cleaved caspase-3, p62, and Bcl-2 in myocardial tissues. ∗∗p < 0.01 vs. Normal; #p < 0.05, ##p < 0.01 vs. sh-NC.
Figure 8
Figure 8
Circ_CIMIRC inhibition enhanced the expression of PINK1 and Parkin in CIH rats (A) qRT-PCR detected the expression of circ-CIMIRC in myocardial tissues. (B) Western blotting assessed the expression of PINK1, Parkin, LC3-I, and LC3-II in myocardial tissues. (C) IHC detected the expression of FbxL4 in myocardial tissues. The levels of MDA (D), SOD (E), and ROS (F) in myocardial tissues. ∗p < 0.05, ∗∗p < 0.01 vs. Normal; #p < 0.05, ##p < 0.01 vs. sh-NC.
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