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YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway
doi: 10.2478/fzm-2024-0006
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Abstract:
Background Myocardial infarction (MI) is associated with higher morbidity and mortality in the world, especially in cold weather. YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis. But YBX1, as an individual RNA-binding protein, regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored. Methods In vivo, the mouse MI model was induced by ligating the left anterior descending coronary artery (LAD), and randomly divided into sham operation group, MI group, MI+ YBX1 knockdown/overexpression group and MI+ negative control (NC) group. The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining. In vitro, mitochondrial-dependent apoptosis was investigated by using CCK8, TUNEL staining, reactive oxygen species (ROS) staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes (NMCMs). Results YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition. Compared to mice induced by MI, YBX1 overexpression mediated by adeno-associated virus serotype 9 (AAV9) vector reduced the infarcted size and improved cardiac function. Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction. In hypoxic cardiomyocytes, YBX1 overexpression decreased lactic dehydrogenase (LDH) release, increased cell viability, and inhibited apoptosis by affecting the expression of apoptosis related proteins, while knockdown of endogenous YBX1 by siRNA had the opposite effect. Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS. In hypoxic NMCMs, YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase (PI3K)/AKT, and the antiapoptosis effect of YBX1 was eliminated t by LY294002, PI3K/AKT inhibitor. Conclusion YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway. It is anticipated that YBX1 may serve as a novel therapeutic target for MI. -
Key words:
- YBX1 /
- PI3K/AKT /
- apoptosis /
- mitochondrial function /
- myocardial infarction
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Figure 1. YBX1 is downregulated in ischemic hearts and hypoxic cardiomyocytes. (A) Volcano plot of differentially expressed mRNAs from the GEO database (
http://www.ncbi.nlm.nih.gov/geo ), showing markedly downregulated YBX1 expression in MI mice compared to the Sham group. (B) The hierarchical clustering heat map. (C, D) YBX1 levels determined by RT-PCR and Western blot. *P < 0.05, **P < 0.01, N = 3-5 duplicates/group. (E, F) Downregulation of mRNA and protein levels of YBX1 in cultured neonatal mouse cardiomyocytes (NMCMs) after hypoxia treatment for 12 h relative to cells cultured under normoxic conditions. *P < 0.05, **P < 0.01, N = 3-5 duplicates/group. (G) Immunofluorescence displaying the changes of YBX1 expression and subcellular distribution (YBX1 in green, nucleus in blue). Scale bars: 10 µm. *P < 0.05, N = 5 duplicates/group. The data are presented as mean ± SEM.
Figure 2. Overexpression of YBX1 improves cardiac function in myocardial infarction (MI) mice. YBX1 was overexpressed by an AAV9 vector engineered to contain the full-length YBX1 gene in MI mice. (A) Experimental procedure. (B) Echocardiographic analysis of left ventricular dimensions and cardiac function in mice. (C) EF: ejection fraction (D) FS: fractional shortening of left ventricular diameter. (E) LVIDs: systolic left ventricular internal diameters. (F) LVIDd: diastolic left ventricular internal diameters. *P < 0.05, N = 5. (H) TTC staining. **P < 0.01, N = 5 duplicates/group. (H, I) Representative bands and statistical data on Bax and Bcl-2 expression, as determined by Western blot analysis. *P < 0.05, **P < 0.01, N = 3 duplicates/group.
Figure 3. Knockdown YBX1 aggravates cardiac dysfunction in myocardial infarction (MI) mice. YBX1 was knocked down via an AAV9 vector carrying a YBX1-specific shRNA in mice. (A) Experimental procedure. (B) Echocardiographic analysis of left ventricular dimensions and cardiac function in mice. (C) EF: ejection fraction (D) FS: fractional shortening of left ventricular diameter. (F) LVIDs: systolic left ventricular internal diameters. (F) LVIDd: diastolic left ventricular internal diameters. *P < 0.05, N = 5. (G) Representative images of TTC staining. **P < 0.01, N = 5 duplicates/group. (H, I) Representative bands and statistical data on Bax and Bcl-2 expression, as determined by Western blot analysis. *P < 0.05, N = 3 duplicates/group.
Figure 4. YBX1 suppresses cardiomyocyte apoptosis induced by hypoxia. (A) Viability of cardiomyocytes. *P < 0.05, N = 6 duplicates/group. (B) LDH content. *P < 0.05, N = 7 duplicates/group. (C, D) Representative Western blot bands and statistical data on Bax and Bcl-2 expression in NMCMs. *P < 0.05, N = 3 duplicates/group. (E) TUNEL photomicrographs of cardiomyocytes. Scale bar: 20 µm; **P < 0.01, N = 5 duplicates/group. The data are presented as mean ± SEM.
Figure 5. YBX1 preserves mitochondrial function in neonatal mouse cardiomyocytes (NMCMs) under hypoxic conditions. (A, B) Representative Western blot bands and statistical data on caspase-9 and caspase-3 expression in NMCMs. *P < 0.05, N = 3 duplicates/group. (C)ATP production. *P < 0.05, N = 6 duplicates/group. (D) Effects of YBX1 overexpression on ROS production. **P < 0.01, N = 6 duplicates/group. (E) Representative images of JC-1 staining for changes in mitochondrial membrane potential in NMCMs. *P < 0.05, N = 5 copies/group. The data are expressed as mean ± SEM.
Figure 6. Silencing YBX1 aggravates hypoxia-induced cardiomyocyte apoptosis. (A) Viability of cardiomyocytes. *P < 0.05, N = 6 duplicates/group. (B) LDH release. *P < 0.05, N = 8 duplicates/group. (C, D) Representative Western blot bands and statistical data on Bax and Bcl-2 expression in NMCMs. *P < 0.05, N = 3 duplicates/group. (E) Typical images of TUNEL staining of cardiomyocytes. Scale bar: 20 µm; *P < 0.05, **P < 0.01, N = 5 duplicates/group. The data are presented as mean ± SEM.
Figure 7. Knockdown of YBX1 aggravates mitochondrial dysfunction in hypoxic neonatal mouse cardiomyocytes (NMCMs). (A, B) Representative Western blot bands and statistical data on caspase-9 and caspase-3 expression in NMCMs. *P < 0.05, N = 3 duplicates/group. (C) ATP production. *P < 0.05, N = 6 duplicates/group. (D) Effects of YBX1 overexpression on ROS production. *P < 0.05, **P < 0.01, N = 6 duplicates/group. (E) Representative images of JC-1 staining in NMCMs. *P < 0.05, N = 5 copies/group. The data are expressed as mean ± SEM.
Figure 8. YBX1 inhibits cardiomyocyte apoptosis via the PI3K/AKT pathway. (A, B) KEGG pathway enrichment analyses on downregulated genes in NMCMs transfected with si-YBX1 relative to control cells. (C) Correlation between YBX1 and PI3K/AKT expressions. (D, E) Representative bands and statistical data on PI3K and AKT expression, as determined by Western blot analysis. The data are presented as fold changes normalized to the sham values. *P < 0.05, **P < 0.01, N = 3 duplicates/group. (F) Viability of cardiomyocytes. *P < 0.05, **P < 0.01, N = 8 duplicates/group. (G) LDH release. *P < 0.05, **P < 0.01, N = 8 duplicates/group. (H, I) Representative bands and statistical data on Bax and Bcl-2 expression, as determined by Western blot analysis. The data are presented as fold changes normalized to the sham values. *P < 0.05, N = 3 duplicates/group. (J) TUNEL assay for investigating the effect of YBX1 on apoptosis. Scale bar: 20 µm; *P < 0.05, **P < 0.01, N = 5. The data are expressed as mean ± SEM.
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