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. 2024 Nov:65:365-379.
doi: 10.1016/j.jare.2023.12.012. Epub 2023 Dec 13.

Ginsenoside Rb2 inhibits p300-mediated SF3A2 acetylation at lysine 10 to promote Fscn1 alternative splicing against myocardial ischemic/reperfusion injury

Affiliations

Ginsenoside Rb2 inhibits p300-mediated SF3A2 acetylation at lysine 10 to promote Fscn1 alternative splicing against myocardial ischemic/reperfusion injury

Qingxia Huang et al. J Adv Res. 2024 Nov.

Abstract

Introduction: Ginsenosides (GS) derived from Panax ginseng can regulate protein acetylation to promote mitochondrial function for protecting cardiomyocytes. However, the potential mechanisms of GS for regulating acetylation modification are not yet clear.

Objectives: This study aimed to explore the potential mechanisms of GS in regulating protein acetylation and identify ginsenoside monomer for fighting myocardial ischemia-related diseases.

Methods: The 4D-lable free acetylomic analysis was employed to gain the acetylated proteins regulated by GS pretreatment. The co-immunoprecipitation assay, immunofluorescent staining, and mitochondrial respiration measurement were performed to detect the effect of GS or ginsenoside monomer on acetylated protein level and mitochondrial function. RNA sequencing, site-specific mutation, and shRNA interference were used to explore the downstream targets of acetylation modificationby GS. Cellular thermal shift assay and surface plasmon resonance were used for identifying the binding of ginsenoside with target protein.

Results: In the cardiomyocytes of normal, oxygen glucose deprivation and/or reperfusion conditions, the acetylomic analysis identified that the acetylated levels of spliceosome proteins were inhibited by GS pretreatment and SF3A2 acetylation at lysine 10 (K10) was significantly decreased as a potential target of GS. Ginsenoside Rb2 was identified as one of the active ginsenoside monomers for reducing the acetylation of SF3A2 (K10), which enhanced mitochondrial respiration against myocardial ischemic injury in in vivo and in vitro experiments. RNA-seq analysis showed that ginsenoside Rb2 promoted alternative splicing of mitochondrial function-related genes and the level of fascin actin-bundling protein 1 (Fscn1) was obviously upregulated, which was dependent on SF3A2 acetylation. Critically, thermodynamic, kinetic and enzymatic experiments demonstrated that ginsenoside Rb2 directly interacted with p300 for inhibiting its activity.

Conclusion: These findings provide a novel mechanism underlying cardiomyocyte protection of ginsenoside Rb2 by inhibiting p300-mediated SF3A2 acteylation for promoting Fscn1 expression, which might be a promising approach for the prevention and treatment of myocardial ischemic diseases.

Keywords: Acetylation; Ginsenoside; Mitochondrial function; Myocardial ischemic/reperfusion injury; P300; SF3A2.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

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Graphical abstract
Fig. 1
Fig. 1
The quantitative acetylomic profiling of cardiomyocytes after GS pretreatment. (A) Graphical representation of 4D-label free quantitative proteomic analysis of lysine acetylome for H9c2 cells untreated and treated with GS in triplicate under normal, OGD, or OGD/R conditions. (B) Venn diagrams comparing acetylated proteins enriched in GS versus Ctrl (control), OGD + GS versus OGD, and OGD/R + GS versus OGD/R. (C) KEGG pathways from the 57 acetylated proteins enriched in the three comparisons were analyzed and eight acetylated proteins in spliceosome were obtained. (D) Protein-protein interactions of these acetylated proteins were analyzed by the STRING software. (E) Hierarchically clustered heatmap of differentially acetylated proteins associated with spliceosome pathways. (F) After GS pretreatment for 48 h, SF3A2 and Ac-K antibodies were used to perform co-IP and immunoblot analysis for detecting the acetylated level of SF3A2 in H9c2 cells under the conditions of normal, OGD, or OGD/R. The input represents the 10 % total protein extracts used for immunoblot. (G) Immunofluorescence and co-localization analysis of SF3A2 with acetylated lysine in the nucleus of H9c2 cells. Scale bar: 5 µm. OGD: oxygen glucose deprivation, OGD/R: oxygen glucose deprivation and reperfusion; GS: total ginsenosides; Ac-K: acetylated-lysine.
Fig. 2
Fig. 2
Analysis of the acetylation sites of SF3A2 in the presence of GS pretreatment. (A) The motif analysis predicted acetylated amino acid sites after GS incubation. (B) Conservation analysis of SF3A2 at lysine 10 for different species. (C) The LC-MS/MS analysis of SF3A2 acetylation sites. (D-E) Western blot analysis and the quantification of SF3A2 (K10) acetylation in H9c2 cells and primary cardiomyocytes, after GS pretreatment for 48 h. (F) The level of K10 acetylated SF3A2 in untreated and GS-treated H9c2 cells and primary cardiomyocytes with OGD or OGD/R injury was analyzed by Western blot method. (G) Based on the analysis for SF3A2 (K10) acetylation, the wild-type (WT), K10Q, or K10R mutant H9c2 cells were established by lentivirus infection. (H) In the different conditions of the normal, OGD or OGD/R incubation, the expression of SF3A2 (K10) acetylation was analyzed by Western blot in the WT or mutant cardiomyocytes. *P < 0.05 and ***P < 0.001. OGD: oxygen glucose deprivation, OGD/R: oxygen glucose deprivation and reperfusion; GS: total ginsenosides; Ac-K: acetylated-lysine.
Fig. 3
Fig. 3
Screening of the main active ginsenoside monomer for SF3A2 (K10) acetylation and mitochondrial respiration rate. (A) In different conditions, H9c2 cells were treated with GS or various ginsenoside monomer for 48 h for determining the interaction between SF3A2 and acetylated-lysine by co-IP analysis. (B) Western blot analysis and quantification for the expression of SF3A2 (K10) acetylation in OGD/R-induced H9c2 cells were performed, after GS, Rb1, Rc, or Rb2 pretreatment. (C) After ginsenoside Rb1, Rc or Rb2 pretreatment for 48 h prior to OGD/R incubation, SF3A2 and Ac-K antibodies were used to perform immunofluorescence staining and co-localization analysis by confocal microscopy. Scale bar = 20 μm. DAPI and β-Actin is the specific staining for nuclear counterstain and cytoskeleton structure, respectively. (D) At different time points, electron transfer chain inhibitors were sequentially added to induce mitochondrial pressure, and the effect of different ginsenoside monomer on oxygen consumption rate (OCR) was observed by Seahorse multi-function energy metabolism detector. (F-G) After ginsenoside Rb1, Rc or Rb2 pretreatment for 48 h prior to OGD or OGD/R incubation, mitochondrial respiration of H9c2 cells was measured by Seahorse XFe24 analyzer. MRC: maximal respiration capacity. OGD: oxygen glucose deprivation, OGD/R: oxygen glucose deprivation and reperfusion. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 4
Fig. 4
Ginsenoside Rb2 can reduce rat myocardial injury after reperfusion through the inhibition of SF3A2 (K10) acetylation. (A-B) Representative photographs and quantitative data for heart infarct volume (Rb2 administration for 2 weeks prior to 2 h ischemia followed by 6 h reperfusion). Scale bar: 0.5 cm. (C) H&E staining of heart section after 6 h of reperfusion. Scale bar: 2 mm or 100 µm. (D) The serum LDH and CK of rats in the Sham, I/R or I/R + Rb2 groups. (E) The analysis from Transmission electron microscopy of mitochondrial structure in heart tissue. Scale bar: 2 µm or 500 nm. (F) Representative images of Immunohistochemistry staining for K10 acetylated SF3A2 (left) and the quantification analysis (right) in heart tissues from different groups. Scale bar: 100 µm. (G) The ratio of Bcl-2/Bax and the protein levels of SF3A2 (K10) acetylation and Cyto C were analyzed by Western blot. (n = 5 rat/group). *P < 0.05, **P < 0.01 and ***P < 0.001. LDH: lactate dehydrogenase; CK: creatine kinase; I/R: ischemic reperfusion; Cyto-C: Cytochrome C.
Fig. 5
Fig. 5
The inhibitory effect of ginsenoside Rb2 on SF3A2 (K10) acetylation was confirmed in the K10Q or K10R mutant cells. (A-B) The bindings of SF3A2 with Ac-K in H9c2 and primary cardiomyocytes were analyzed by co-IP assay. (C-D) The cell respiration was analyzed by Seahorse XFe24 in WT and K10Q mutant H9c2 cells after ginsenoside Rb2 incubation. (E-F) The ATP levels were detected and analyzed by luciferase method. (G) The MMP level was detected and analyzed by FCM. Basal: Basal OCR; MRC: maximal respiration capacity; SPC: spare respiratory capacity; ATP-pro: ATP production OCR; Ac-K: acetylated-lysine. **P < 0.01 and ***P < 0.001.
Fig. 6
Fig. 6
Transcriptomic analysis of the difference genes in alternative splicing under ginsenoside Rb2 pretreatment. (A) Pie chart showing the global RNA splicing analysis by rMATS software in Rb2 treated-cardiomyocytes. (B) Heat map showing the higher abundance of genes and alternative splicing (in red) in Rb2-treated sample compared to the control. (C-D) The different mRNA expression of mitochondrial function-related genes in transcriptomics were verified by qPCR analysis in the normal or OGD/R conditions. (E) The diagram of the Fscn1 alternative splicing mediated by ginsenoside Rb2. (F) The Fscn1 mRNA level was analyzed in the wild-type or K10Q mutant cell by qPCR analysis. (G-H) The cell respiration was measured and quantified by Seahorse XFe24 analyzer in Fscn1 knockdown H9c2 cells prior to ginsenoside Rb2 pretreatment and OGD/R incubation. *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7
P300/sirt7 is the key regulator of ginsenoside rb2 on sf3a2 (K10) acetylation. (A-B) The effect of NAM, TSA, A-485 or anacardic acid combined with ginsenoside Rb2 on the binding of SF3A2 with acetylated-lysine was observed by co-IP analysis. (C-D) After the knockdown with RNA interferences targeting different KATs and KDACs, the SF3A2 acetylation was observed in OGD/R-stress H9c2 cells. (E) After Rb2 incubation or p300, SIRT7 shRNA knockdown prior to OGD/R injury, the MMP were analyzed by FCM. (F-G) After shRNA knockdown for p300 or SIRT7, the effect of Rb2 on the level of SF3A2 (K10) acetylation was analyzed and quantified by Western blot in H9c2 cells. *P < 0.05, **P < 0.01 and ***P < 0.001. TSA: trichostatin A, NAM: nicotinamide.
Fig. 8
Fig. 8
The analysis for the binding ability of different ginsenoside monomers on p300 protein. (A) Biophysical analysis of the interaction between p300/SIRT7 and ginsenoside Rb2 using a cellular thermal shift assay. (B) SPR analysis of ginsenoside Rb2 and A-485 binding to p300 protein and KD value was calculated. (C) The inhibitory effect of ginsenosides Rb2, Ro, Rg3, 20S-(PPT), or A-485 on p300 activity were assessed with a commercial kit.

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