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. 2023 Dec 13;65:365–379. doi: 10.1016/j.jare.2023.12.012

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

Qingxia Huang a,b, Yao Yao b, Yisa Wang b, Jing Li b, Jinjin Chen b, Mingxia Wu b, Chen Guo b, Jia Lou c, Wenzhi Yang c, Linhua Zhao d, Xiaolin Tong d,⁎, Daqing Zhao b,⁎, Xiangyan Li b,⁎
PMCID: PMC11518965  PMID: 38101749

Graphical abstract

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

Highlights

  • •

    Ginsenoside fraction from Panax ginseng decreased the acetylated protein levels of spliceosome in cardiomyocytes.

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    Ginsenoside Rb2 inhibited SF3A2 acetylation at lysine 10 to promote mitochondrial function against myocardial ischemic/reperfusion injury.

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    Ginsenoside Rb2 promoted Fscn1 alternative splicing to enhance mitochondrial respiration.

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    The decreased acetylation of SF3A2 (K10) by ginsenoside Rb2 was mediated by p300 inhibition.

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.

Introduction

Myocardial ischemia and its secondary ischemia reperfusion (I/R) injury are the principal pathophysiological step of various cardiovascular diseases, which eventually leads to increased death rates and heavy health care costs [1]. The current anti-myocardial ischemic injury strategies are mainly aimed at cardiomyocyte protection by restricting apoptosis, scavenging reactive oxygen species, and anti-inflammation [2]. Increased mitochondrial function helps cardiomyocyte cope with cellular ischemic stresses and subsequently promotes cell survival [3]. With the development of mass spectrometry-based acetylome technologies, recent works have identified the critical role of reversible non-histone acetylation in maintaining mitochondrial function both in the normal and ischemic heart [4]. Intriguingly, there are reports demonstrating that an important part of these non-histone proteins is the splicing factor, which can quickly and reversibly regulate the mitochondria under stress state to normal condition by regulating pre-mRNA alternative splicing (AS) after the acetylation [5]. Previous studies have shown that the AS is dramatically changed at a global scale in both human patients and mouse myocardial ischemic models [6], [7]. In addition, those mitochondrial function-related proteins are encoded by a single gene for producing multiple isoforms through AS [8]. Therefore, the strategies that modulate the acetylation of non-histone proteins, especially splicing factors to promote mitochondrial function, which might be a critical and effective way for the prevention and treatment of myocardial ischemic injury.

Lysine acetylation is one of protein post-translational modifications that mainly are controlled by two classes of enzymes: lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) [9]. More recently, numerous studies have shown that the KATs/KDACs were recruited to several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1), SF3A2 (splicing factor 3a subunit 2), and PHD finger protein 5A to alter the pre-mRNA AS in response to multiple cellular stresses [10], [11], [12]. In the aberrant of KDAC or p300, splicing factors cannot be modified by acetylation, resulting in the inhibition of spliceosome assembly and dysregulated RNA splicing [11], [13]. Thus, targeting KATs/KDACs for regulating the acetylation of splicing factor may provide a new method for treating myocardial ischemic diseases.

The ginsenosides from the herbs of Traditional Chinese Medicine, such as Panax ginseng C. A. Meyer, Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow, or Astragalus membranaceus (Fisch.) Bunge) exerts cardioprotective effects in ischemia injury [14]. Ginsenosides (GS) fraction is a type of steroid saponins derived from Panax ginseng, which is known to effectively mitigates myocardial injury via anti-oxidative, anti-inflammatory and maintaining mitochondrial function properties [15]. More importantly, the latest research suggests that ginsenoside Rd, Rg3, and Rh2 can activate SIRT1/SIRT3 or block CREB-binding protein (CBP)/p300 pathway to regulate the deacetylation of LKB1/Cyclophilin D/FOXO3a, thereby reverse oxidative stress, inflammation and mitochondrial dysfunction [16], [17], [18]. Our previous study also observed that ginsenoside Rc reduces mitochondrial damage and apoptosis through SIRT1-mediated acetylation in the I/R-induced cardiac injury models [19]. These findings definitely suggest that ginsenosides can regulate protein acetylation for cardioprotective function, but its modulated acetylated proteins, possible KATs/KDACs, and which ginsenoside monomer for regulating acetylation modification should be further explored in the in vitro and in vivo experiments.

In this study, we provide the quantitative acetylomics data on the inventory of the acetyl-lysine proteins and acetylated peptides on the myocardial protection effect of GS. We found that the deacetylation of SF3A2, an essential component of spliceosome U2 snRNP at lysine 10 was directly related to the myocardial protective effect of GS in alleviating ischemic injury. Ginsenoside Rb2 was identified as a key ginsenoside monomer for inhibiting the acetylation of SF3A2 (K10) to promote mitochondrial respiration against myocardial ischemic injury in vivo and in vitro. Furthermore, RNA-seq analysis showed that ginsenoside Rb2 upregulated the expression of fascin actin-bundling protein 1 (Fscn1) through AS to promote mitochondrial function for cardioprotective effect. Importantly, we demonstrated for the first time that ginsenoside Rb2 can directly interact with p300 to inhibit its activity and reduce the acetylation of SF3A2 (K10). These findings provide a novel mechanism underlying cardiomyocyte protection of ginsenoside Rb2 by regulating p300-SF3A2 acteylation-Fscn1 signaling, which might be a promising approach for myocardial ischemic prevention and treatment.

Materials and methods

Chemicals and reagents

Total ginsenosides (GS, UV purity ≥ 90 %) from dry root of Panax ginseng (5-year-old, Changbai Mountain, Jilin, China) was prepared as previously described [20] and ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg3, Ra2, R1, and F11 (HPLC purity ≥ 98 %) were purchased from Shanghai Standard Biotech. Co., Ltd (Shanghai, China). Representative LC-MS chromatograms of GS and the contents of 12 ginsenoside monomers in GS were shown in Fig. S1 and Table S1–S2. Antibodies against acetylated-lysine (#9814S), Bax (#2772), Cytochrome C (Cyto-C, #11940), Flag (#14793), p300 (#57625), and β-Actin (#3700) were purchased from Cell Signaling Technology (Beverly, MA, USA). SF3A2 (ab77800), Bcl-2 (ab196495) and SIRT7 (ab259968) antibodies were obtained from Abcam (Cambridge, MA, USA). SF3A2 antibody (15596–1-AP) was purchased from Proteintech (Wuhan, China). Trichostatin A (TSA, HY-15144), nicotinamide (NAM, HY-B0150), A-485 (HY-107455), and anacardic acid (HY-N2020) were purchased from MedChemExpress LLC (Shanghai, China).

Animal experiments and ethics statement

Male Sprague Dawley rats (190–210 g) at 8 weeks of age, were obtained from the Animal Core Facility of Changchun Yisi Experimental Animal Technology Co. Ltd (Changchun, China). All rats were randomly assigned to three groups (Sham, I/R, and I/R + Rb2 groups) with 20 animals per group for different experiments. All animal experiments, the protocols of experimental myocardial I/R model and ginsenoside treatment used in this study were reviewed and approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (Changchun, China, approval No. 2021343). All animal studies were performed strictly in accordance with the ARRIVE guidelines 2.0 and China National Institutes of Healthy Guidelines for the Care and Use of Laboratory Animals [21]. The rats were maintained on a 12 h light/dark cycle at 23–25 °C with access to sterile pellet diet and water ad libitum.

The generation of anti-acetylated (Ac)-K10 of SF3A2 antibody

According to the protein sequence and modification type, two antigenic peptides encompassing K10 of SF3A2 and one unmodified control peptide were designed and synthesized. Six specific pathogen-free experimental New Zealand rabbits were immunized by peptides, then the serum was taken for ELISA analysis and Western blot detection to evaluate the specificity. Antibodies were purified by affinity chromatography and evaluated by ELISA, Dot Blot and Western blot methods, and finally one specific antibody was selected as the anti-SF3A2 (K10) antibody in this study.

Lentivirus infection and selection of stable infected cells

SF3A2 wild-type and mutant (K10Q, K10R) plasmids and lentivirus were constructed by Genechem (Shanghai, China). H9c2 cells were seeded in a six-well plate at about 60 % density for 24 h before infection. The 200 µl lentivirus at a dose of 100 MOI (1 × 108 TU/ml) and 50 µl HiTranG A infection enhancement reagents were added to each well to infect H9c2 cells. After 12 h of incubation, the medium was removed, and cells were cultured in DMEM containing 10 % FBS for continue culturing. After 48 h infection, puromycin at 5 µg/ml or 2.5 µg/ml was used to kill non-infected cells and only resistant clones were grown until all cells in the control group died. The stable infected cells were collected and identified by Western blot for subsequent experiments.

Mitochondrial respiration analysis

The mitochondrial respiration was determined by high-resolution respirometer using a Seahorse XFe24 analyzer (Seahorse Bioscience, Billerica, MA, USA) as reported previously [19]. The cardiomyocytes were plated in Seahorse XFe24 cell culture plate before the pretreatment with ginsenosides and/or oxygen glucose deprivation (OGD), OGD/reperfusion (OGD/R) incubation. The baseline oxygen consumption rate (OCR) was recorded, and then continuous injections with pharmacologic inhibitors: oligomycin (1.5 µM, an inhibitor of ATP synthase); carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP, 2 µM, an uncoupling agent); and a mixture of antimycin A (0.5 µM, mitochondrial complex I inhibitor), and rotenone (0.5 µM, mitochondrial complex III inhibitor). Key parameters for mitochondrial respiration, such as basal OCR, maximal respiration capacity (MRC), and spare respiration capacity (SRC) and ATP production were analyzed.

Co-Immunoprecipitation (co-IP) analysis

Proteins from cardiomyocytes were lysed in NP-40 lysis buffer (Beyotime Biotechnology, Shanghai, China) containing a complete protease inhibitor cocktail (Sigma-Aldrich). After cell homogenates incubation with specified antibodies at 4 °C overnight, the antigen–antibody complex was immunoprecipitated with the A/G agarose beads (MedChemExpress LLC) at room temperature for 1 h. The beads were washed with lysis buffer and then eluted with SDS loading buffer by boiling at 100 °C for 5 min. Finally, the interested proteins of the supernatant were analyzed by Western blot analysis.

Cellular thermal shift assay

After the incubation with ginsenoside Rb2 or dimethyl sulfoxide for 24 h, H9c2 cell proteins were extracted and adjusted to the similar concentrations using the BCA kit. The supernatants of protein extraction were divided into ten equal parts and transferred to the PCR tubes. The PCR tubes were heated according to designated temperatures for 5 min in the PCR instrument, then saved at −80 °C. The levels of p300 and SIRT7 were detected and quantified by Western blot analysis, according to a previous report [22].

Surface plasmon resonance analysis

Surface plasmon resonance (SPR) analysis was performed on Reichert 2SPR system (Buffalo, New York, USA). The surface of carboxymethyl dextran P/N sensor chip (Reichert) was activated by 100 μl of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride mixed with 100 μl of N-hydroxysuccinimide. Flow rate was 25 μl/min for 10 min. Recombinant human p300 protein was diluted with sodium acetate (pH 5.5) to 200 μg/ml, and then immobilized on the sensor chip using a standard amine coupling method. After blockage by 1 M ethanol amine for 10 min and solvent correction, gradient concentrations of various ginsenoside mononer or A-485 (0.78 to 100 μM) in the running buffer were tested for the binding to p300. Kinetic parameters were computed with Reichert evaluation software.

Quantification and statistical analysis

All the in vivo and in vitro experimental groups were designed to establish equal size, blinding and randomization. All group sizes represent the numbers of experimental independent values, and these independent values were used to evaluate statistical analyses. Statistical analyses were undertaken for the experiments where each group sizes (n) ≥ 3. Values are expressed as mean ± SD in in vitro study and mean ± SEM in in vivo study. The unpaired, two-tailed Student’s t test was used to calculate the comparison for two groups, and one-way ANOVA followed by a Tukey’s post hoc test was used for the comparisons among multiple groups. The post hoc tests were conducted only if the F in ANOVA achieved the necessary statistical significance level and there was no significant variance inhomogeneity. The analyses were performed by GraphPad Prism 8.0 (Boston, MA, USA) and P < 0.05 was considered statistically significant.

Data availability

The proteome and acetylome data, and their corresponding raw data, have been all publicly deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org; accession code: PXD043035). The transcriptome data are available at the Gene Expression Omnibus (CEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi; accession code: GSE236471).

Results

The decrease of SF3A2 acetylation might be a potential target of GS against myocardial ischemia and/or reperfusion injury

To further determined the protective effect of GS on cardiomyocyte damage after hypoxia and subsequent reperfusion injury, we investigated the effect of GS on cell viability, apoptosis, ATP content, and oxygen consumption rate in cardiomyocytes pretreated with GS at 2.5, 5, and 10 µg/ml for 48 h and then incubated in hypoxia, OGD or OGD/R for mimicking myocardial ischemia and/or reperfusion injury. In agreement with previous studies [23], we found that GS pretreatment significantly abolished OGD or OGD/R-induced decreased cell growth, apoptosis, and mitochondrial membrane potential (MMP) deficiency in H9c2 cells (Fig. S2A–D). Moreover, OGD or OGD/R-induced decrease of ATP content was obviously ameliorated by the GS pretreatment (Fig. S2E). Additionally, the OCR assay showed that the pretreatment with GS prior to OGD or OGD/R incubation significantly elevated the values of key parameters of mitochondrial respiration, such as basal OCR, MRC, and SRC in primary cardiomyocytes (Fig. S2F–G). These results confirmed the protective effect of GS against OGD or OGD/R-mediated cardiomyocyte injury via the promotion of mitochondrial respiration.

Based on the potential role of protein acetylation in myocardial injury [24], we next employed 4D label-free quantitative proteomic and acetylomic analysis to gain the inventory of lysine-acetylated peptides and proteins for explaining the protective effect of GS against OGD or OGD/R injury (Fig. 1A). In total, 9,755 high-confidence acetylation modification sites in 3,051 proteins (location probability > 0.75) were identified from H9c2 cells treated with GS under different conditions of normal, OGD, or OGD/R induction (Fig. S3A). These acetylation sites showed significant different occupancy between the six groups by PCA analysis (Fig. S3A). The quantitative results of differentially acetylated proteins and modification sites from different compared groups were provided in Table S3 (P < 0.05, fold change > 1.5). Volcano plots showed the global distribution of acetylated proteins in the Ctrl + GS vs Ctrl, OGD + GS vs OGD and OGD/R + GS vs OGD/R comparisons (Fig. S3B–D). The spliceosome signaling was significantly enriched by KEGG analysis in each of comparisons (Fig. S3E–G). Of note, the spliceosome signaling was the top enriched pathways in the deacetylation proteins (Fig. S4A). Many splicing factors interacted with U1, U2, U4/U6, and U5 snRNAs were enriched and found to be involved in these spliceosomes signaling, including U2B, SF3A, SF3B, U2AF, PUF60, PRP43, Prp31, Prp8BP, and others (Fig. S4B). Notably, the total of 57 acetylated proteins were enriched by Venn diagram for these three comparisons (Fig. 1B). KEGG analysis and protein interaction network analysis suggested the 57 acetylated proteins were also highly enriched in spliceosome pathways (Fig. 1C–D). Furthermore, a hierarchical clustering heat-map further depicted these significantly altered proteins in detail, the acetylation levels and acetylated sites of these enriched proteins (Fig. 1E and Table S4-S6). Among them, SF3A2, a component of U2 snRNPs, was the most significant in the downregulated acetylation level in the three comparisons (Ctrl + GS vs Ctrl, OGD + GS vs OGD, and OGD/R + GS vs OGD/R) (Fig. 1E and Table S4-S6). We further confirmed the acetylation of SF3A2 and the co-localization of SF3A2 and acetylated-lysine (Ac-K) in the nucleus by co-immunoprecipitation and immunofluorescence staining. As expected, immunoprecipitation assay showed that GS pretreatment efficiently mitigated the direct binding of SF3A2 with Ac-K (Fig. 1F). Consistently, the evidence from laser scanning confocal microscopy showed that the co-localization of SF3A2 with Ac-Ly was impeded after the pretreatment with GS under different conditions (Fig. 1G and Fig. S5A–B). Our data indicated the decrease in the acetylation of SF3A2 might be a potential target of GS in alleviating myocardial injury after ischemia.

Fig. 1.

Fig. 1

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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.

GS treatment significantly reduced the acetylated level of SF3A2 at lysine 10 acetylation of is attenuated under GS administration

Next, we sought to determine the acetylation sites of SF3A2. Amino acid sequence analysis of protein modification revealed that lysine (K) was most intent to be acetylated when it was preceded by glycine (G) and followed by threonine (T) under GS pretreatment in normal, OGD or OGD/R incubation. (Fig. 2A and Fig. S6A). Conservation analysis of SF3A2 indicated that K10 (GKT) is a highly conserved site from Colobus angolensis palliatus to Homo sapiens (Fig. 2B). In addition, the LC-MS/MS analysis of SF3A2 protein confirmed that the lysine at the +10 position (K10) was the major acetylation site of SF3A2 (Fig. 2C and Fig. S6B–C). To further ascertain the role of GS on the inhibition of SF3A2 acetylation, we generated specific SF3A2 (K10) antibody (Fig. S7 and Table S7) and performed dot blotting and immunoblotting assays to ensure the valence and specificity of this antibody for recognizing the K10 acetylation of SF3A2 (Ab5 was selected, Fig. S8A–B). As expected, the immunoblotting with SF3A2 (K10) antibody revealed that GS pretreatment mitigated K10 acetylation of SF3A2 in H9c2 cells and primary cardiomyocytes, compared to control group (Fig. 2D–E). We also examined the ability of GS to deacetylate SF3A2 (K10) in the OGD or OGD/R incubation and found that the elevated level of acetylation at K10 after ischemic injury was significantly inhibited upon GS pretreatment in both cell models of primary cardiomyocytes and H9c2 cells (Fig. 2F and Fig. S8C–D). Meanwhile, we mutated the lysine at the position of K10 to glutamine (K10Q) and arginine (K10R) in H9c2 cells, which mimicked the hyperacetylation and deacetylated state of protein. As shown in Fig. 2G, both K10R and K10Q mutants resulted in lower acetylation levels compared to Flag-tagged wild-type SF3A2 (WT), which indicated the mutant H9c2 cells can be used to explore the role of GS on the K10 acetylation of SF3A2. Moreover, the effect of GS pretreatment prior to OGD/R incubation on circumventing the expression of SF3A2 (K10) was also confirmed in H9c2 cells with stably transfected WT, K10Q and K10R mutants of SF3A2 vectors (Fig. 2H). These data demonstrated that lysine at the + 10 position is the major site of deacetylation modification of SF3A2 on GS pretreatment.

Fig. 2.

Fig. 2

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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.

The main active ginsenosides in GS were screened by SF3A2 (K10) deacetylation and mitochondrial function phenotypes

To further gain the insight into the role of the main active ginsenosides in GS on SF3A2 acetylation, we performed a series of experiments to investigate the effects of different ginsenosides on the binding of SF3A2 with Ac-K, SF3A2 (K10) level, and mitochondrial function, according to the contents of different ginsenosides in GS (Table S2). The co-IP results and co-localization analysis both revealed that ginsenoside monomers Rb1, Rc or Rb2 at individual concentrations of GS decreased the binding of SF3A2 with Ac-K in normal, OGD or OGD/R incubation (Fig. 3A–B and Fig. S9A–B). For SF3A2 (K10) expression, we found that the upregulation of SF3A2 (K10) level induced by OGD/R incubation was obviously reduced by ginsenosides Rb1 or Rb2 (Fig. 3C). Additionally, the Rb1-, Rc-, or Rb2-treated H9c2 cells has higher basal OCR and ATP production OCR than the control (Fig. 3D and Fig. S10A). Compared with the control, the capacity of maximal respiration was increased by ginsenoside Rc or Rb2 pretreatment (Fig. 3E). Especially, ginsenoside Rb2 obviously increased mitochondrial reserve capacity of H9c2 cells (Fig. S10B). In the H9c2 cell models with OGD condition, ginsenoside Rc or Rb2 pretreatment significantly augmented the degrees of basal OCR, MRC and SRC in cardiomyocytes (Fig. 3F and Fig. S10C). For OGD/R condition, we found that ginsenoside Rb2 pretreatment effectively incremented the basal OCR, MRC, and ATP production OCR in the OGD/R-induced H9c2 cell injury (Fig. 3G and Fig. S10D). These results indicated that the mitochondrial oxidative phosphorylation and its impairment induced by OGD or OGD/R were significantly improved by ginsenoside Rb2. Importantly, ginsenoside Rb2 had a similar role in decreasing OGD/R-induced apoptosis in H9c2 cells as that of GS pretreatment (Fig. S11A–B). Furthermore, the JC-1 assays showed that the potential of mitochondrial membrane was recovered by ginsenoside monomers Rb1, Rc or Rb2, which was similar as that of GS in reducing OGD/R-induced mPTP opening (Fig. S11C–D). Taken together, these data demonstrated the ginsenoside Rb2 was one of active ginsenosides in GS, can obviously reduce SF3A2 (K10) acetylation and improve mitochondrial respiration to protect cardiomyocytes from the injuries induced by OGD or OGD/R.

Fig. 3.

Fig. 3

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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.

Ginsenoside Rb2 promotes SF3A2 deacetylation-mediated mitochondrial function against myocardial ischemia reperfusion injury in rats

The protective effect of ginsenoside Rb2 in a I/R rat model by the reduction of SF3A2 (K10) acetylation was further investigated. After 2-week pretreatment of ginsenoside Rb2 (10 mg/kg daily), 2,3,5-triphenyltetrazolium chloride staining showed that ginsenosides Rb2 effectively reduced the myocardial infarct size induced by the coronary artery ligation for 2 h and loosening for 4 h from 38.58 % to 24.18 % (Fig. 4A–B). H&E staining revealed that the I/R group manifested severe myocardial fiber architecture disruption, myofibrillar contraction bands, destruction of cardiomyocyte membranes, and enlarged interstitial space in the infarct zone, which was significantly repaired by ginsenoside Rb2 pretreatment (Fig. 4C). Consistently, the levels of lactate dehydrogenase and creatine kinase (two key biomarkers of myocardial injury) in serum following I/R injury were significantly alleviated by ginsenoside Rb2 (Fig. 4D). Importantly, the analysis of transmission electron microscopy revealed that I/R -induced the absence and edematous separation of sarcomeres, the vacuolation of mitochondria, the derangement of mitochondrial membrane, and the disruption of mitochondrial cristae in heart tissue were significantly improved by ginsenoside Rb2 pretreatment, compared with these findings of the I/R group (Fig. 4E). Moreover, immunohistochemistry staining and Western blot analysis showed that ginsenoside Rb2 administration mitigated SF3A2 (K10) protein level in the nucleus in heart tissue after I/R induction (Fig. 4F-4G). The protection of ginsenoside Rb2 against cardiac I/R injury was also confirmed by enhancing the ratio of Bcl-2/Bax, and attenuating the expression of Cyto-C (Fig. 4G). Based on these data, ginsenoside Rb2 can reduce SF3A2 (K10) acetylation to alleviate cardiac I/R injury and apoptosis in the rat model.

Fig. 4.

Fig. 4

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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.

The acetylation site of SF3A2 (K10) is critical for the promoting effect of ginsenoside Rb2 on mitochondrial function in the cardiomyocytes with OGD and OGD/R injury

Furthermore, we investigated whether ginsenoside Rb2 promotes mitochondrial function against OGD and OGD/R injury is directly dependent on the deacetylation of SF3A2 at the + 10 position of lysine, K10. In wild-type cells overexpressing Flag-SF3A2, we found that the OGD and OGD/R injury leads to marked binding of SF3A2 with Ac-K in H9c2 and primary cardiomyocytes, which were alleviated by ginsenoside Rb2 pretreatment (Fig. 5A). The exogenous co-immunoprecipitation of SF3A2 with Ac-K showed a significant increase of SF3A2 (K10) acetylation after ginsenoside Rb2 incubation, which was not observed in H9c2 cells overexpressing with SF3A2 mutants (K10Q and K10R) (Fig. 5B). As evidenced by the results of OCR, the increments of mitochondrial respiration parameters (basal OCR, MRC, SRC, and ATP production) by ginsenoside Rb2 in WT H9c2 cells were completely abolished in cells overexpressing SF3A2 K10Q (Fig. 5C–D). Furthermore, the increased ATP levels upon ginsenoside Rb2 pretreatment in H9c2 cells overexpressing SF3A2 were impeded by K10Q and K10R in normal and OGD/R incubation, indicating the effect of ginsenoside Rb2 on ATP production was directly related to SF3A2 (K10) acetylation (Fig. 5E–F). Similarly, the JC-1 fluorescence results revealed that K10Q mutation of SF3A2 abrogated the decreasing effect of ginsenoside Rb2 pretreatment on the potential of mitochondrial membrane under the condition of OGD/R incubation (Fig. 5G). Collectively, these findings suggest that the acetylation of SF3A2 at K10 is critical for the promoting effect of ginsenoside Rb2 on mitochondrial function in the cardiomyocytes with OGD and OGD/R injury.

Fig. 5.

Fig. 5

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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.

Ginsenoside Rb2 augmented SF3A2 (K10) acetylation-mediated Fscn1 alternative splicing to promote mitochondria function

Given that SF3A2 regulates alternative splicing, we collected H9c2 cells untreated and treated with ginsenoside Rb2 for total RNA extraction and RNA sequencing to identify the changes of alternative splicing events in both cell groups. We found that a total of 1,772 differential genes were identified between control and ginsenoside Rb2, of which 1,106 genes was up-regulated and 666 genes were down-regulated (Fig. S12A). As expected, the gene set enrichment analysis demonstrated that multiple metabolic pathways were significantly affected by ginsenoside Rb2 pretreatment, including central carbon metabolism, amino acids metabolism, glucose metabolism, and purine metabolism (Fig. S12B). The rMATS software was used to detect differential splicing events with the default parameters and it detected 318 differential splicing events in 166 genes (cut-off set at 1.5-fold) after ginsenoside Rb2 incubation (Fig. 6A). Of all the differentially spliced genes, we observed the majority of ginsenoside Rb2-regulated differential AS events correspond to intron retention (IR, 203 events), followed by intron retention of overlapping region (98 events) and alternative 3′ splice site (5 events, A3SS) (Fig. 6A). Venn diagram analysis showed that 19 genes had both differentially AS and expressed genes (Fig. S12C). Furthermore, a heat-map depicted the expressions of 19 genes in the both control and Rb2-treated H9c2 cells (Fig. 6B). Based on these findings, we focused and validated mitochondrial function-related genes with AS events and differentially expressed genes, including C-terminal binding protein-1 (Ctbp1), solute carrier family 7 member 1 (Slc7a1), pyruvate kinase muscle (PKM), fragile X-related protein-1 (Fxr1), and fascin actin-bundling protein-1 (Fscn1) by qPCR analysis (Table S8). As shown in Fig. 6C, ginsenoside Rb2 augmented the mRNA expression levels of Fxr1 and Fscn1 in cardiomyocytes, compared to control groups. Meanwhile, the qPCR assay showed that ginsenoside Rb2 treatment elevated the OGD/R-induced mRNA level reduction of Fscn1 gene in cardiomyocytes and had no obvious effect on the OGD/R-induced Fxr1 mRNA level (Fig. 6D). As shown in rMATS software analysis, the differential expression of Fscn1 may be due to AS mediated by intron retention (Fig. 6E). Next, we checked whether SF3A2 (K10) is necessary for ginsenoside Rb2 to regulate Fscn1 alternative splicing in OGD/R cardiomyocyte injury, and found that ginsenoside Rb2 ameliorated the mRNA expression of Fscn1 with dependence only on SF3A2 overexpression, but not the SF3A2 K10Q mutation (Fig. 6F). Importantly, we used Fscn1 shRNA interference to validate whether Fscn1 expression is critical for ginsenoside Rb2 treatment on improved mitochondrial respiration in cardiomyocytes. The results from Seahorse analyzer showed that those key parameters of mitochondrial respiration caused by OGD/R was obviously ameliorated in the ginsenoside Rb2 group, and this improvement was blocked by Fscn1 knockdown (Fig. 6G–H and Fig. S12D). These data collectively supported that the AS of Fscn1 is a key link between SF3A2 acetylation and mitochondrial function for the protection of ginsenoside Rb2 from cardiomyocyte I/R injury.

Fig. 6.

Fig. 6

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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.)

p300 is the key regulator of ginsenoside Rb2 on SF3A2 (K10) acetylation

To further confirm the effect of ginsenoside Rb2 on SF3A2 (K10) acetylation is mediated by KATs or KDACs, we first combined ginsenoside Rb2 with two classic inhibitors, trichostatin A (TSA, a class I/II histone deacetylase inhibitor), and nicotinamide (NAM, an inhibitor of sirtuins) to investigate the change of lysine acetylation in Flag-SF3A2 overexpressing cells. The co-IP analysis demonstrated that the binding of SF3A2 with Ac-K was not inhibited by the combination of ginsenoside Rb2 with TSA or NAM, indicating that the effect of Rb2 on acetylated SF3A2 level might be medicated by deacetylase (Fig. 7A). Furthermore, the reduction in the binding of SF3A2 with lysine acetylation in ginsenoside Rb2-treated group was not detected in the combined groups, Rb2 + A-485, a selective p300/CREB-binding protein (CBP) histone acetyltransferase inhibitor and Rb2 + anacardic acid, a p300/CBP-associated factor (PACF) inhibitor in the cardiomyocyte model with OGD/R injury, which indicated that ginsenoside Rb2 also could regulate acetyltransferase to influence the acetylation level (Fig. 7B). Continuely, different shRNAs targeting KATs including p300, CBP, p300/CBP-associated factor (PCAF), α-Tubulin acetyltransferase 1 (Atat1), and Tat interactive protein 60 kDa (TIP60) or KDACs including histone deacetylase 5 (HDAC5), histone deacetylase 9 (HDAC9), sirtuin 1 (SIRT1), and sirtuin 7 (SIRT7) were used to establish the H9c2 cell models with an effective key gene knockdown (Fig. S13), in which were further investigated to identify lysine acetylation/deacetylation enzyme for ginsenoside Rb2-mediated reduction of SF3A2 acetylation for reducing OGD/R injury. The co-IP analysis showed that the knockdown of p300 or SIRT7 abrogated the effect of ginsenoside Rb2 on the decrease of SF3A2 acetylation in OGD/R-stressed cells and other gene knockdown had no effect on the decreased SF3A2 acetylation by ginsenoside Rb2 (Fig. 7C–D). Importantly, the decreased expression of SF3A2 (K10) acetylation by ginsenoside Rb2 was abolished by p300 or SIRT7 knockdown in OGD/R-induced cell injury (Fig. 7E–F). Additionally, we found that the inhibitory effect of ginsenoside Rb2 on the increment of MMP by OGD/R was eliminated by p300 or SIRT7 knockdown in H9c2 cells (Fig. 7G). Taken together, these results demonstrated that ginsenoside Rb2 might cooperatively regulate p300 and SIRT7 to suppress SF3A2 acetylation at K10 in response to OGD/R stress.

Fig. 7.

Fig. 7

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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.

Ginsenoside Rb2 strongly binds to p300 to inhibit its activity for reducing SF3A2 acetylation

Biophysical analysis of the interaction between p300/SIRT7 and ginsenoside Rb2 can provide more insights on the potential targets of ginsenoside Rb2 for the protection of cardiomyocyte I/R injury. The temperature cellular thermal shift assays demonstrated that ginsenoside Rb2 augmented the thermal stability of p300, but not that of SIRT7 (Fig. 8A), supporting that there may be a direct interaction between ginsenoside Rb2 and p300. To further verify that there is a binding between ginsenoside Rb2 and p300, we purified p300 protein for detecting its affinity and enzymatic activity. As shown in Fig. S14A, we analyzed the binding affinities of 19 ginsenosides from Panax ginseng roots containingRg3, Rb3, F5, Rg1, PPT, Rh1, Rb2, F1, Rc, Rg2, Rh2, compound K, (20S)-PPT, Re, PPD, F2, Rb1, oleanolic acid, and Ro with p300 and found that ginsenosides Rb2, Ro, (20S)-PPT, Rg3, F2 and A-485 as a positive control had much higher binding signal with p300 protein with the smaller KD (equilibrium dissociation constant) values (Fig. 8B and Fig. S14B). Among them, ginsenoside Rb2 dose-dependently bound to p300 with a KD value of 24.2 μM (Fig. 8B). Critically, p300 activity was measured to further confirm the direct binding of these ginsenosides with smaller KD values to p300 protein by quantitatively detecting the production of coenzyme A from acetyl-CoA substrates. In this assay, ginsenoside Rb2 incubation showed a dose-dependent decrease in p300 activity, which was better than other ginsenosides (Fig. 8C). These results indicated that ginsenoside Rb2 could strongly bind to p300 for inhibiting its activity to reduce the acetylation of downstream target, SF3A2.

Fig. 8.

Fig. 8

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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.

Discussion

The non-histone protein acetylation is considered an important sensor and regulator of ischemic injury [25]. Persistent hyper-acetylation in the heart resulted in increased sensitivity to the stress, and the inhibition of hyper-acetylation in hearts significantly reduced infarct size and I/R-induced cardiac dysfunction [26]. Inspired by the remarkable role of GS in cardiac protection and acetylation modification [14], [17], [18], [19], [27], we for the first time performed a quantitative acetylome profiling study to identify the changed acetylated proteins as the potential targets of GS in the cardiomyocytes of normal, OGD or OGD/R incubation. A series of acetylated proteins for spliceosome was enriched, which contained different subunits of small nuclear ribonucleoprotein U1, U2, U4/U6, and U5 (SNRPB2, SNRNP70), RNA binding motif proteins (RBM22), and splicing factors (SF3A2, U2AF2, SRSF5). These acetylated spliceosome proteins were upregulated by OGD or OGD/R incubation in cardiomyocytes, which were significantly inhibited by GS pretreatment. In this study, we mainly investigate the role of GS or ginsenoside monomer on one of the decreased acetylated proteins, SF3A2, participates in spliceosome assembly within the mature U2 small nuclear ribonucleoprotein particle for displacing from the spliceosome to initiate the first step of the AS reaction [28]. Our data confirmed the inhibitory effects of GS on I/R-induced K10 acetylation of SF3A2 by specific antibody recognition and lysine mutation methods. After screening by measuring acetylated SF3A2 level at K10 and mitochondrial respiration, three ginsenoside monomers including ginsenoside Rb2, Rc, and Rb1, can significantly reduce the acetylated level of SF3A2 (K10) to promote mitochondrial function for the protection of myocardial I/R damage. Importantly, ginsenoside Rb2 had a similar role on SF3A2 (K10) acetylation as that of GS, which might be an important component of ginsenoside fraction from Panax ginseng root for treating cardiac-related diseases. However, other acetylated proteins regulated by GS pretreatment should be further investigated and validated in future. Meanwhile, the downstream targets of other ginsenosides, Rc and Rb1-meidated SF3A2 acetylation modification also need to be explored.

Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control [29], [30]. Mitochondrial dysfunction is involved in distinct and diverse mechanisms in the pathology and etiology of ischemic injury, such as the mitochondrial energetics, mitophagy-mediated quality control, morphology, mtDNA, mitochondrial ROS, AS regulation, and lysine acetylation [15], [25]. Specifically, we found that SF3A2-mediated AS of mitochondrial function-related genes may be one of the complementary mechanisms of mitochondrial adaptation in ischemic injury. For example, Fscn1, a highly-conserved actin-bundling protein, is responsible for promoting metabolic stress resistance by remodeling mitochondrial actin filaments and regulating the homeostasis of mitochondrial DNA [31]. Here, we found an AS form of Fscn1 was changed after ginsenoside Rb2 pretreatment in cardiomyocytes, suggesting a close relationship between the Fscn1 alternative splicing and ginsenoside Rb2-mediated myocardial protection. Additionally, our results support that Fscn1 knockdown blocked the effect of ginsenoside Rb2 for enhancing mitochondrial respiration against OGD/R injury. Furthermore, in the mutant K10Q SF3A2 cells, the pretreatment with ginsenoside Rb2 had no effect on the mRNA expression of Fscn1, which suggest that this role of ginsenoside Rb2 for increased Fscn1 expression was dependent on SF3A2 acetylation. These findings support the conclusion that ginsenoside Rb2 can inhibit SF3A2 (K10) acetylation to mediate Fscn1 alternative splicing for the promotion of mitochondrial function. Further investigation is warranted to determine how Fscn1 plays different roles in mitochondrial function with different AS forms, and to elucidate their relevance with pathological procedures of cardiac ischemic and I/R injury.

Given the critical role of lysine acetylation in the development of ischemic injury in eukaryotic cells, the development of small-molecule inhibitors targeting the KATs/KDACs-mediated reversible lysine acetylation have been highly pursued by academia in recent years [32]. However, different KATs/KDACs seem to have different regulatory mechanisms. Class I KDACs were regarded as be deleterious to cardiac function, while class II and III KDACs were protective against cardiac ischemic injury [26], [32]. Previous studies indicated that the increment of acetylation level offer cardioprotection against I/R injury in vivo and in vitro [33]. On the other side, the enhanced protein acetylation increased infarct size, impeded cardiac function, and accelerated heart failure [25], [34]. Similarly, the deacetylation enhanced mitochondrial bioenergetics and promoted cell survival in myocardial hypoxia and OGD/R cell injury [35]. In this study, we found that the effects of ginsenoside Rb2 on deacetylation was dependent on p300 in OGD/R-stress cells. The p300, a GCN5-related N-terminal acetyltransferase that localizes to the nucleus, has emerged as a potential therapeutic target for myocardial I/R injury, cardiac hypertrophy, heart failure, and cardiac fibrosis [36], [37]. In particular, our thermodynamic and kinetic studies demonstrated that there may be a direct interaction between ginsenoside Rb2 and p300, but not with the SIRT7. Concomitantly, our SPR and enzyme activity results indicated that ginsenoside Rb2 dose-dependently bound to p300 to inhibit its activity. This finding indicated that ginsenoside Rb2 directly interacted with p300 to block SF3A2 (K10) acetylation for mitigating myocardial ischemia cell injury. Although we have speculated the interaction of ginsenoside Rb2 with p300 by thermal shift and SPR analysis, co-crystal structure analysis needs to be further investigated for exploring the direct binding site of ginsenoside Rb2 on p300. Additionally, ginsenoside Rb2 might regulate SIRT7 expression at the mRNA and protein level to influence its activity, not mediated by direct interaction, which should be further determined by future experiments.

Conclusions

Here, we have established a novel mechanism in the cardiomyocyte protection of ginsenosides against ischemic injury whereby ginsenoside Rb2 inhibits p300-mediated SF3A2 acetylation at lysine 10 to promote Fscn1 alternative splicing against myocardial ischemic/reperfusion injury in in vitro studies. The in vivo results also confirmed that ginsenoside Rb2 promotes SF3A2 (K10) deacetylation-mediated mitochondrial function against myocardial ischemic injury. These findings might provide a new insight on the protective mechanism of ginsenoside Rb2 and a potential therapeutic approach against myocardial ischemic injury-related diseases. Further preclinical and clinical investigations are required to thoroughly elucidate the clinical applications of ginsenosides in myocardial ischemic disease.

CRediT authorship contribution statement

Qing-Xia Huang: Conceptualization, Investigation, Project administration, Writing-original draft, Funding acquisition. Yao Yao: Methodology, Project administration, Data curation, Writing-original draft. Yi-Sa Wang: Project administration, Formal analysis, Validation. Jing Li: Formal analysis, Data curation, Validation. Jin-Jin Chen: Software, Data curation. Ming-Xia Wu: Visualization, Software. Chen Guo: Visualization, Validation. Jia Lou: Validation, Software. Wen-Zhi Yang: Investigation. Lin-Hua Zhao: Formal analysis. Xiao-Lin Tong: Conceptualization, Project administration, Writing-review & editing. Da-Qing Zhao: Conceptualization, Supervision, Funding acquisition, Resources. Xiang-Yan Li: Conceptualization, Resources, Funding acquisition, Supervision, Writing-review & editing.

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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China, China (Grant No. 82374078 and U19A2013); Science and Technology Development Plan Project of Jilin Province, China (Grant No. YDZJ202201ZYTS270), Youth Excellent Discipline Talent Training Project, China (Grant No. 202331), and Innovation and Entrepreneurship Talent Funding Project of Jilin Province, China (2022ZY10).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2023.12.012.

Contributor Information

Xiaolin Tong, Email: tongxiaolin@vip.163.com.

Daqing Zhao, Email: zhaodaqing1963@163.com.

Xiangyan Li, Email: xiangyan_li1981@163.com.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (16.7MB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.docx (16.7MB, docx)

Data Availability Statement

The proteome and acetylome data, and their corresponding raw data, have been all publicly deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org; accession code: PXD043035). The transcriptome data are available at the Gene Expression Omnibus (CEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi; accession code: GSE236471).

Articles from Journal of Advanced Research are provided here courtesy of Elsevier

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