Berberine ameliorates chronic intermittent hypoxia‐induced cardiac remodelling by preserving mitochondrial function, role of SIRT6 signalling

Abstract Chronic intermittent hypoxia (CIH) is associated with an increased risk of cardiovascular diseases. Previously, we have shown that berberine (BBR) is a potential cardioprotective agent. However, its effect and mechanism on CIH‐induced cardiomyopathy remain uncovered. This study was designed to determine the effects of BBR against CIH‐induced cardiac damage and to explore the molecular mechanisms. Mice were exposed to 5 weeks of CIH with or without the treatment of BBR and adeno‐associated virus 9 (AAV9) carrying SIRT6 or SIRT6‐specific short hairpin RNA. The effect of BBR was evaluated by echocardiography, histological analysis and western blot analysis. CIH caused the inactivation of myocardial SIRT6 and AMPK‐FOXO3a signalling. BBR dose‐dependently ameliorated cardiac injury in CIH‐induced mice, as evidenced by increased cardiac function and decreased fibrosis. Notably, SIRT6 overexpression mimicked these beneficial effects, whereas infection with recombinant AAV9 carrying SIRT6‐specific short hairpin RNA abrogated them. Mechanistically, BBR reduced oxidative stress damage and preserved mitochondrial function via activating SIRT6‐AMPK‐FOXO3a signalling, enhancing mitochondrial biogenesis as well as PINK1‐Parkin‐mediated mitophagy. Taken together, these data demonstrate that SIRT6 activation protects against the pathogenesis of CIH‐induced cardiac dysfunction. BBR attenuates CIH‐induced myocardial injury by improving mitochondrial biogenesis and PINK1‐Parkin‐dependent mitophagy via the SIRT6‐AMPK‐FOXO3a signalling pathway.


| INTRODUC TI ON
As a common disorder in the general population, obstructive sleep apnoea (OSA) is characterized by the collapse of the upper airway and chronic intermittent hypoxia (CIH) during sleep. 1 OSAassociated CIH is recognized as an independent risk factor for cardiovascular diseases including atherosclerosis, hypertension, coronary heart disease, myocardial infarction and arrhythmias. 2,3evious studies have revealed that CIH increases the risk of cardiovascular disease owing to the cardiac structural remodelling and cardiac dysfunction by triggering oxidative stress and myocardial fibrosis. 4Nevertheless, the current understanding of the molecular mechanism of CIH-induced cardiac dysfunction remains limited.
The sirtuin (SIRT) family (SIRT1-SIRT7) is a group of conserved nicotinamide adenine dinucleotide (NAD + )-dependent class III histone deacetylases.It has been shown that sirtuin family members are widely distributed in the nucleus, cytoplasm and mitochondria, and play vital roles in maintaining intracellular homeostasis. 5[8] SIRT6, a member of the sirtuin family, is a nuclear protein primarily responsible for gene expression control and DNA repair.In the last decade, SIRT6 has been recognized to play multiple functions in stress resistance, apoptosis, aging, senescence, and inflammation. 9[12] However, the relationship between CIH-induced cardiomyopathy and SIRT6 remains uncovered.
Berberine (BBR) is a major form of isoquinoline alkaloid (Figure 2A) isolated from medicinal plants such as Hydrastis canadensis and Rhizoma coptidis.Due to its powerful antimicrobial, antioxidant and anti-inflammatory activities, numerous studies have reported the pharmaceutical value of BBR in digestive system, immune system, nervous system and critically, cardiovascular diseases. 13,146][17] In particular, studies have shown that SIRT1 and SIRT3 play a critical role in the cardioprotective effects of BBR. 18,19However, the protective effect of BBR on CIH-induced cardiac remodelling and myocardial oxidative stress remain unknown.In addition, it is not yet clear the potential role of SIRT6 in these effects.
Therefore, this study was designed to investigate the role of SIRT6 during the progression of CIH-induced cardiac damage.In addition, we evaluated whether BBR conferred a protective effect against CIH-induced cardiac remodelling and oxidative stress.Next, we explored the molecular mechanisms with a focus on SIRT6adenosine 5′-monophosphate-activated protein kinase (AMPK)forkhead box O3alpha (FOXO3a) signalling and mitochondrial quality control processes.

| Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis
For functional enrichment analysis, an expression profiling dataset of the CIH and control groups (GSE138008) was screened from the Gene Expression Omnibus (GEO) database (https:// www.ncbi.nlm.nih.gov/ geo/ ). 20The series matrix files of GSE138008 were downloaded and processed with the 'limma' R packages by employing R platform (version 4.2.1).Differentially expressed genes (DEGs) were identified using the 'limma' package (absolute Log 2 FC value greater than 0.263, a p-value <0.05). 21We performed GO and KEGG pathway enrichment analyses for these DEGs using the Metascape bioinformatics resources (http:// metas cape.org, version 3.5.20230501).2A, purity ≥95%, B139120, Aladdin, Shanghai, China) was dissolved in the water and administered orally to the mice.The dose of BBR was chosen based on the preliminary experiment in our group and previous publications by our team and others. 17,19,22Hypoxic chambers (ProOx-100; TOW-INT TECH, Shanghai, China) were used to establish the CIH mouse models.The O 2 concentration in these chambers was decreased from 21 to 5-8% by injection of N 2 and then maintained for 15 s, followed by a rapid increase in O 2 concentration to 21% for 60 s.The cycle was repeated for 12 h daily for 5 weeks.Mice in the control group were exposed to normoxic air at all times. 23,24All the animals survived throughout the experimental period.

| Adeno-associated virus infection and experimental design
The recombinant adeno-associated virus 9 (AAV9) carrying a cardiac troponin T (c-TNT) promoter that expressed mouse SIRT6 or SIRT6-

group (CIH-SIRT6
).All the animals survived throughout the experimental period.The virus titre of the AAV9 virus suspension was >10 12 vg/mL, and mice were injected with 100 μL of the solution via the tail vein as described previously. 25,26

| Evaluation of body index and hemodynamic parameter
The mice and their heart mass were weighted on an electronic scale at the end of the experimental period.Hemodynamic parameters and resting heart rate (HR) of each group were measured using a small animal blood pressure meter (BP-2010A; Softron Beijing Biotechnology Co., Ltd, Beijing, China).The blood pressure meter utilized a pressure recording sensor and a tail cuff for the noninvasive evaluation of systolic and diastolic blood pressure in mice as described previously. 27

| Echocardiography
Cardiac function was assessed using a high-resolution doppler ultrasound imaging system for small animals (D700; Vinno Technology Co., Ltd, Suzhou, China) as described previously. 28Blind assessment was ensured during the measurements.Ventricular structure was recorded in the parasternal long-axis view.Heart rate, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and left ventricular end-diastolic posterior wall thickness (LVPWd) were calculated from the recorded M-mode images using a pre-set computer program.

| HE and Masson's trichrome staining
For histological analysis, the mouse hearts were harvested and briefly perfused with 10% potassium chloride. 29Then, the samples were fixed in 4% paraformaldehyde and embedded in paraffin.Paraffin-embedded samples were cut into 5 μm sections for HE and Masson's trichrome staining.H&E staining (KGA224; KeyGEN Biotech, Jiangsu, China) was used to observe histomorphological changes.Masson's trichrome staining (abs9348; Absin, Shanghai, China) was used to assess cardiac fibrosis.Image J software (Image Solutions, Torrance, CA, USA) was used to quantify the data.

| Immunohistochemistry
Myocardial SIRT6 levels were determined by immunohistochemical staining.Cardiac paraffin-embedded tissues were sectioned at 5 μm thickness.Sections were subjected to heat-mediated antigen retrieval, peroxidase blocking with 3% hydrogen peroxide and non-specific binding site blocking.All slides were incubated with anti-SIRT6 antibody (1:200 dilution; #12486, Cell Signalling Technology, MA, USA) at 4°C overnight.The sections were then treated with horseradish peroxidaseconjugated antibodies and detected with 3,3′-diaminobenzidine (DAB, KeyGEN BioTECH, Jiangsu, China).5 random fields were captured by using a stereomicroscope (#UB2031; UOP Technology, Chongqing, China).SIRT6 levels were analysed and calculated using Image J software (Image Solutions, Torrance, CA, USA).

| Transmission electron microscopy
The procedures were performed as previously described. 28Briefly, ventricular samples were initially fixed with 2.5% glutaraldehyde and then post-fixed in 1% osmium tetroxide.The cardiac ultrastructure was captured and analysed in a blinded manner with a transmission electron microscope (OLYMPUE, Tokyo, Japan).

| Immunofluorescence
Cross-sectional area of the cardiomyocytes was determined by analysing immunofluorescence images of wheat germ agglutinin Invitrogen, UK).The nuclei were stained with DAPI solution (Beyotime Biotechnology, Shanghai, China).Fluorescence images were captured in 5 random fields using a Nikon C2 Plus confocal microscope (Nikon, Tokyo, Japan).All data were analysed and calculated using Image J software.

| Assessment of glutathione/glutathione disulphide (GSH/GSSG) ratio
To assess cellular redox metabolism, the GSH/GSSG ratio was determined using commercially available kits (S0053; Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions.

| Western blot
The total protein content of ventricular samples was extracted using a lysis buffer containing protease inhibitor cocktail. 30The samples were lysed on ice for 30 min and centrifuged at 12,000 rpm for 30 min.Then, the protein concentration was determined by BCA method (P0012; Beyotime Biotechnology, China).Sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel was used to isolate the protein, which was then transferred to a polyvinylidene fluoride (PVDF, 0.45 μm) membrane (IPVH00010; Millipore Corporation, Billerica, MA, USA).The membranes were blocked with 5% skim milk for 1.5 h and incubated overnight at 4°C using the primary antibodies, includ-

| Data analysis and statistics
All data are presented as mean ± standard deviation (SD).Differences between the groups were compared using the Student's t-test (for two groups) or one-way analysis of variance (ANOVA) followed by the Turkey's comparison test was used to assess the differences among multiple groups.The statistical analyses were performed with the Prism software (GraphPad Prism 8.0; GraphPad Software Inc., CA, USA).The statistical significance was defined as p < 0.05.

| Mitochondrial dysfunction and impaired SIRT6 signalling may play critical roles in the pathogenesis of CIH-induced cardiac injury
The data set of GSE138008 is an atrial mRNA profile of C57BL/6J mice, which includes 3 samples of CIH model and 3 samples from the control group (CON).Mice were housed in customized cages to deliver CIH, during which hypoxic events occurred at a rate of one event per 180 s.Mice in the control group were exposed to normoxic air at all times. 20To investigate the mechanisms of CIHinduced cardiac injury, a total of 18,456 genes were investigated in the bioinformatics analysis of this dataset and 1967 DEGs were identified for GO and KEGG analysis.The top 114 genes, including 30 up-regulated DEGs and 84 down-regulated DEGs were ranked by Log 2 (FC) (Figure 1A).An integrated bioinformatics platform (www.bioin forma tics.com.cn) was employed to visualize the GO and KEGG enrichment.We found that the DEGs were mainly enriched in NADH dehydrogenase complex, inner mitochondrial membrane protein complex and sarcoplasmic reticulum (cellular component, CC).Further analysis showed that membrane repolarization during cardiac muscle cell action potential, protein lipidation involved in autophagosome assembly, mitochondrial respiratory chain complex I assembly, cardiac muscle contraction (biological process, BP) and voltage-gated potassium channel activity involved in ventricular cardiac muscle cell action potential repolarization and oxidoreduction-driven active transmembrane transporter activity (molecular function, MF) were significantly enriched (Figure 1C).KEGG analysis showed significant enrichment of the oxidative phosphorylation and cardiac muscle contraction (organismal systems) (Figure 1B).These results suggested that mitochondrial dysfunction-induced oxidation-reduction imbalance might be a vital contributor to CIH-induced cardiac injury.
Additionally, heatmap of genes involved in the sirtuin family were plotted with TBtools software (version 1.129) (Figure 1D).Although the gene expression differences among all members of the sirtuin family were not significant, we found a marked decrease in myocardial SIRT6 expression in CIH group (Figure 1E,F).Compared to the control group, the levels of AMPK phosphorylation and FOXO3a were also decreased in CIH group (Figure 1E,F), indicating that reduced SIRT6-AMPK-FOXO3a signalling might play a role in CIHassociated cardiac injury.

| BBR dose-dependently inhibited CIH-induced myocardial structural remodelling and cardiac dysfunction
To examine the effects of BBR (Figure 2A) treatment on CIHinduced cardiac dysfunction and myocardial fibrosis, three gradient concentrations of BBR (50, 100, and 200 mg/kg) groups were established.We found no significant difference in heart rate among all groups (Table 1).Compared with the CON group, blood pressure and heart-to-body mass ratio increased significantly in the CIH group (Table 1 and Figure 2B).BBR treatment ameliorated these alterations in a concentration-dependent manner (Table 1 and Figure 2B).In addition, echocardiographic results showed that BBR significantly attenuated cardiac dys- We found no significant difference in heart rate during echocardiographic measurement among all groups (Figure 2D).Moreover, our results revealed that berberine dose-dependently reduced myocardial fibrosis, which was supported by a decrease in fibrosis area (Figure 2H,I) and reduced expression of fibrosis-related markers (COL1A1, COL3A1, TGFβ and α-SMA , Figure 2L,N-Q).
Additionally, the results of immunohistochemistry and western blot analyses demonstrated the inhibition of SIRT6 expression in the CIH group, while BBR treatment markedly activated SIRT6 signalling in a concentration-dependent manner (Figure 2J-M).

| SIRT6 overexpression attenuated CIH-induced myocardial dysfunction and myocardial structural remodelling
Based on the molecular docking analysis (Figure 3A,B), we found that BBR binds to the active site of SIRT6 by forming a hydrogen bond with an arginine residue (ARG-65), pi-cation with two amino acid residues (PHE-64 and TRP-188) and pi-pi stacking interactions with a nearby amino acid residue (HIS-133).To further investigate the role of SIRT6 in the beneficial effects of BBR, we utilized adenoassociated virus to create cardiac-specific SIRT6-knockdown or overexpressed mice.In view of the data acquired in Figure 2, a concentration of 100 mg/kg BBR was selected in the subsequent experiments.As shown in Table 2, no significant difference in heart rate was found among these groups.Compared with the CON-NC group, blood pressure and heart-to-body mass ratio were comparative in CON-BBR group (Table 2 and Figure 3C).SIRT6 overexpression significantly attenuated CIH-induced elevated blood pressure and increased heart-to-body mass ratio (Table 2 and Figure 3C).In addition, SIRT6 overexpression reduced left ventricular dysfunction (compared with the CIH group, Figure 3D,F-H).Meanwhile, overexpression of SIRT6 reduced myocardial fibrosis as evidenced by a decrease in fibrosis area (Figure 3I,J) as well as fibrosis-related markers (Figure 3L-P).Nevertheless, SIRT6 knockdown inhibited the protective effects of BBR.The cross-sectional area of cardiomyocytes was comparative among all these groups (Figure 3I,K).
No significant changes were found in heart rate during the measurement of cardiac performance among these groups (Figure 3E).
These findings suggested that SIRT6 overexpression ameliorated myocardial dysfunction and structural remodelling triggered by CIH in mice.Additionally, BBR exerted these beneficial effects in a SIRT6-dependent manner.

| BBR treatment enhanced the myocardial antioxidant capacity in CIH-induced mice via SIRT6 signalling
In comparison to the control group, CIH notably increased myocardial oxidative stress levels, as evidenced by increased fluorescence intensity of DHE and MitoSOX (Figure 4A,B,D,E), as well as decreased GSH/GSSG ratio (Figure 4C).BBR and SIRT6 overexpression obviously reversed these adverse effects of CIH, and these beneficial effects of BBR were abolished by SIRT6 knockdown (Figure 4A-E).Next, we detected the changes in the protein levels of antioxidant enzymes.Western blot analysis showed that the expression levels of SOD1, SOD2 and Catalase were significantly down-regulated in CIH group (Figure 4F-H), which were reversed by BBR treatment or SIRT6 overexpression.In contrast, the beneficial effects of BBR were inhibited by SIRT6 knockdown (Figure 4F-H).These data suggested that BBR protected against oxidative stress in a SIRT6dependent manner.(E-G) Left ventricular enddiastolic posterior wall thickness (LVPWd), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (n = 5/ group).(H, I) Representative Masson's trichrome staining images (scale bar = 50 μm) and quantitative analysis of interstitial fibrosis (n = 5/ group).(J, K) Representative immunohistochemical staining images of SIRT6 (scale bar = 50 μm) and quantitative analysis of SIRT6 (n = 5/ group).(L-Q) Representative western blot image and relative expression of myocardial SIRT6, COL1A1, COL3A1, α-SMA and TGFβ (n = 4/ group).Data were presented as Mean ± SD. **p < 0.01 and ***p < 0.001 versus CON; # p < 0.05, ## p < 0.01 and ### p < 0.001 versus CIH.BBR L, BBR M and BBR H indicated that the mice were treated with BBR at the doses of 50, 100 and 200 mg/kg, respectively.ns, not significant.

| BBR treatment activated myocardial AMPK-FOXO3a pathway via SIRT6 signalling
To investigate the underlying mechanisms, we determined the potential downstream targets of SIRT6 by employing AAV9 vector carrying SIRT6 or SIRT6 shRNA (directed by the cardiac-specific c-TNT promoter) as described in our previous study. 12,25,26As

| BBR promoted myocardial microvascular formation via SIRT6 signalling
Previous study has indicated that hypoxia promotes the formation of small arteries and capillaries.Here, we evaluated the arterio-genesis

| BBR improved mitochondrial autophagy and biogenesis via SIRT6 signalling
Considering the critical role of mitochondria in sustaining the energy requirements of cardiomyocytes, 31 we further evaluated myocardial mitochondrial function with a focus on mitochondrial morphology, mitophagy and mitochondrial biogenesis.Electron microscopic examination showed that the control group displayed well-developed mitochondria with integral membrane and cristae (Figure 5J).However, the CIH group displayed obvious degenerative changes in mitochondrial morphology, including abnormal shape, swelling and disorganized cristae (Figure 5I).As shown in   These results indicated that BBR effectively improved myocardial mitophagy and mitochondrial biogenesis through SIRT6 signalling.

| DISCUSS ION
Several major findings have been achieved in this study.First, downregulation of SIRT6 signalling was found in CIH-injured myocardium.
Second, cardiac SIRT6 signalling was shown to play an important role in protecting myocardium against CIH-induced myocardial oxidative stress and fibrosis.Third, BBR attenuated CIH-induced cardiac damage by promoting mitochondrial autophagy and biogenesis via the SIRT6-AMPK-FOXO3a signalling pathway (Figure 6).
3][34] To date, there is still a lack of efficient therapeutic approaches.Thus, it is necessary to further elucidate the molecular mechanism of CIH in inducing myocardial injury and to develop new therapeutic targets.
Members of the VEGF family and their receptors play a crucial role as regulators of angiogenesis and vessel maintenance. 35It has been revealed that CIH up-regulates the expression of VEGF via the activation of the oxygen-dependent transcription factor HIF-1α, which in turn induces vascular smooth muscle cell proliferation and vascular remodelling. 36A prior study by Visniauskas et al. showed that myocardial angiogenesis and capillary density were both decreased from the third to the fifth weeks of CIH exposure, accompanied by delayed increase in both gene and protein expression of VEGF. 37ey further demonstrated that this discrepancy was related to the classic interaction between VEGF and kallikrein-kinin system (KKS) components, and their temporal expressions were not sufficient to stimulate the formation of new blood vessels.This could partially explain our results.In the present study, we showed that CIH obviously decreased the arteriolar and capillary density, while the expression of VEGF was significantly increased in CIH group.Previous study has also showed that VEGF expression was notably increased in hypoxia mice while the numbers of α-SMA+ and CD31+/vWF+ positive cells were significantly decreased, which was consistent with our results. 38The interplay of myocardial arterio-genesis and angiogenesis as well as angiogenic factors during CIH exposure warrants further investigation.
Among the sirtuin family members, SIRT1, SIRT3 and SIRT6 have attracted much attention as the potential cardioprotective modulators against inflammation, vascular remodelling, cardiomyopathy and the development of atherosclerotic plaques. 39Previously, we have reported the critical role of SIRT6-AMPK axis in reducing mitochondrial division, enhancing mitochondrial biogenesis and mitochondrial autophagy in diabetic cardiomyopathy (DCM). 12In addition, Wang et al. found that SIRT6 reduced the level of cellular oxidative stress to protect myocardial infarction in ischemic heart disease by upregulating AMPK-FOXO3a axis, and then activating the expression of downstream antioxidant coding genes. 10Numerous studies have shown that AMPK-FOXO3a signalling is involved in the upregulation of antioxidant enzymes in response to oxidative stress. 40,41Here, we found that SIRT6-AMPK-FOXO3a signalling pathway were downregulated in CIH-induced mouse ventricular tissue (Figure 1), while in the bioinformatics analysis, there were no significant differences in mRNA levels among seven members of the sirtuin family (SIRT1-SIRT7).We speculate that the inconsistency may be due to the regulatory mechanisms regarding the translational or post-translational modifications of these key molecules.Besides, we noticed a prior RNA-seq study by Castro-Grattoni et al. showed that CIH-induced upregulation of stress-response genes were involved in cardioprotection (including SIRT6) in young female mice instead of aged ones. 42It is noted that there are real differences between males and females in the presentation, pathophysiology and comorbidities regarding to obstructive sleep apnoea (OSA).Some published studies suggest a higher risk of cardiovascular comorbidity 43 and type 2 diabetes 44 in males with OSA.Lin et al. revealed that atrial fibrillation burden is a sex-specific risk factor for OSA and is limited to male. 45reover, women with both AF and OSA have a lower AF burden than men, despite being older and having similar OSA severity and body habitus. 45Thus, we think this could also result in the difference of these studies.Besides, the experimental procedures and duration of CIH are also different between our study and the work by Castro-Grattoni. 42Nonetheless, we found that SIRT6 overexpression attenuated CIH-induced myocardial dysfunction, myocardial structural remodelling and enhanced the myocardial antioxidant capacity.These data demonstrated that impaired SIRT6-AMPK signalling contributed greatly to CIH-induced cardiac dysfunction and remodelling.The influence of age and sex on the cardiac response to CIH exposure deserves further investigation.
BBR is recognized for its multiple cardiovascular protective effects and can be used to treat diabetes, hypertension, arrhythmias and other conditions. 14,16An increasing number of studies suggest that BBR possesses profound antioxidative property. 158][49] Previous work by our group has reported that BBR improves myocardial ischemia reperfusion injury by reducing oxidative stress and inflammation response in a SIRT1-dependent manner. 19Other researches have also shown that BBR can increase the protein levels of SIRT1 and SIRT3 to regulate autophagy and mitochondrial biogenesis in cardiomyocytes, which maintains a balance in mitochondrial quality control and reduces DOX-induced cardiotoxicity. 18Nevertheless, the potential cardioprotective effects of BBR on CIH and the role of SIRT6 signalling remain uncovered.Here, we showed that BBR treatment ameliorated myocardial damage and structural remodelling caused by CIH (Figure 2).Furthermore, SIRT6 knockdown restricted the AMPK-FOXO3a signalling activity and the benefits of BBR administration (Figures 3-5).These findings were in line with the previous reports showing the regulatory effect of SIRT6 on AMPK signalling, 12,50 suggesting that AMPK-FOXO3a signalling at least in part, mediated the beneficial effects of BBR-induced SIRT6 activation.
2][53] Our recent study demonstrated that impaired SIRT6-AMPK signalling played a key role in cardiac dysfunction by damaging mitochondrial dynamics and mitophagy. 25Additionally, several studies have highlighted the role of FOXO3a in maintaining mitochondrial homeostasis by regulating PINK-Parkin-mediated mitophagy. 54It is worth noting that CIH-induced myocardial oxidative stress damage is associated with the malfunction of mitophagy and mitochondrial biogenesis.Clinical data indicated that patients with OSA exhibited elevated levels of oxidative stress markers and reduced mitochondrial DNA copy numbers, which correlated with the severity of the condition. 55,56Meanwhile, it has been found that myocardial cells injured by CIH are susceptible to oxidative stress and mitochondrial dysfunction, which results in the accumulation of excessive iron, while activation of mitochondrial autophagy could explicitly lower iron accumulation in the mitochondria, thereby attenuating CIH-induced cardiac dysfunction and structural remodelling. 57On the other hand, enhancing early adaptive changes in mitochondrial biogenesis through activation of the PGC1α/Akt signalling pathway exerts a protective effect against CIH-induced cardiac injury. 58In the current study, we found that the level of AMPK phosphorylation and the expression of FOXO3a, mitophagyrelated molecules (PINK1 and Parkin), as well as mitochondrial biogenesis-related markers (PGC1α, NRF-1 and TFAM) were upregulated by SIRT6 overexpression and BBR treatment (Figure 5).
Conversely, SIRT6 knockdown abrogated the beneficial effects of BBR on mitochondrial function.These data revealed that BBR promoted mitochondrial quality control through a SIRT6-dependent pathway, and AMPK-FOXO3a axis probably participated in this process.Additionally, previous studies showed that BBR promotes the expression of VEGF by inhibiting the macrophage Wnt5a/βcatenin pathway, and thus inducing angiogenesis in the infarcted heart. 59Another recent study suggested that overexpression of SIRT6 down-regulated the acetylation level of H3K9ac, promoted myocardial angiogenesis, ameliorated mitochondrial damage and then improved myocardial remodelling. 60However, it is unclear whether BBR promotes angiogenesis and improves cardiac remodelling in CIH-induced myocardium through SIRT6 signalling.In the present study, we showed that BBR treatment promoted myocardial angiogenesis, while the effects were abolished by SIRT6 knockdown (Figure 5).These results were partially coincided with the previous research showing that the long-term dietary intake of pasta containing 3% barley β-D-glucan (P-BBG) may simultaneously promote the angiogenesis and higher tolerance to oxidative stress due to enhanced endothelial expression of VEGF/MnSOD signalling and Parkin. 61Meanwhile, it has been observed that enhancing the overall cellular antioxidant capacity may also result in the upregulation of VEGF and Parkin within the cardiovascular system, as indicated by the positive regulatory influence of MnSOD on VEGF or Parkin signalling. 61,62The intricate interaction among these molecules deserves further investigation.
We acknowledge that there are several limitations in this study.Despite our results that SIRT6 plays a protective effect in the therapeutic actions of BBR, further research is required to in-

| 7 of 17 ZHOU
function, as evidenced by the concentration-dependent improvement in left ventricular ejection fraction (LVEF), left ventricular F I G U R E 1 Mitochondrial dysfunction and impaired SIRT6 signalling might play critical roles in the pathogenesis of CIH-induced cardiac injury.(A) Heatmap representing the selected DEGs (CIH vs. CON).A total of 114 top genes were ranked by Log 2 (FC).(B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs.(C) Dot-plot representing the Gene Ontology (GO) analysis of DEGs.The longitudinal axis represents enriched GO function classifications, which were divided into three major categories: biological process (BP), molecular function (MF) and cellular component (CC).(D) Heatmap representing the gene of sirtuin (SIRT) family members (CIH vs. CON).(E, F) Western blotting and quantitative analysis of myocardial SIRT6, p-AMPK/AMPK and FOXO3a in mice (n = 5/group).Data were presented as Mean ± SD. **p < 0.01 and ***p < 0.001 versus Normoxia.et al. fractional shortening (LVFS) and left ventricular diastolic posterior wall thickness (LVPWd) in BBR-treated group (Figure 2C-G).
and angiogenesis by analysing smooth muscle cell marker α-SMA and endothelial cell marker CD31 immunofluorescence.The level of vascular endothelial growth factor (VEGF) was also quantified.Compared with the CON-NC group, CIH increased the protein expression of VEGF (Figure5E,F).Interestingly, BBR or SIRT6 overexpression both significantly increased the expression of VEGF compared with the CIH-NC group, while this effect was abolished by SIRT6 knockdown (Figure5E,F).In comparison to the CON-NC group, the numbers of both α-SMA-and CD31-positive vessels were decreased in the CIH group (Figure5G-I).BBR and SIRT6 overexpression notably reversed the adverse effect of CIH, and the ameliorative effect of BBR was abolished by SIRT6 knockdown (Figure5G-I).These data indicated that BBR improved microvascular formation possibly though SIRT6 signalling.
vestigate whether the effects of BBR in CIH-induced cardiac injury are related to other members of the sirtuin family (especially SIRT1 and SIRT3).On the other hand, while the AAV9 virus effectively regulated cardiac SIRT6 expressions, we did not assess the transduction efficiency of the virus.Meanwhile, the potential for non-specific effects of the virus on different cardiac cell types or even other organs cannot be discounted.Animal models featuring cardiac-specific overexpression or knockout of SIRT6 are unquestionably necessary to substantiate the conclusion.Additionally, although the present study provides in vivo evidence that BBR F I G U R E 5 BBR treatment promoted microvascular formation, improved mitochondrial autophagy and biogenesis via SIRT6 signalling.(A-D) Representative western blot image and the associated quantitative analysis of myocardial SIRT6 expression, the AMPK phosphorylation level and FOXO3a expression (n = 4/group).(E, F) Representative western blot image and relative expression of myocardial VEGF (n = 4/group).(G-I) Representative fluorescence images of α-SMA (green)-and CD31 (red)-positive vessels in myocardial tissue (upper bar = 100 μm, lower bar = 50 μm) and the quantitative analysis (n = 5/group).(J) TEM images of myocardial tissue.Green arrowheads: damaged mitochondria.Red arrowheads: autophagosomes carrying mitochondria (upper scale = 2 μm, lower scale = 1 μm, n = 5/group).(K-M) Representative western blot image and relative expression of myocardial PINK1 and Parkin (n = 4/group).(N-Q) Representative western blot image and relative myocardial protein expressions of PGC1α, NRF-1 and TFAM (n = 4/group).Data were presented as Mean ± SD. **p <0.01 and ***p < 0.001 versus CON + CN; # p < 0.05, ## p < 0.01 and ### p < 0.001 versus CIH + NC; & p < 0.05 and &&& p < 0.001 versus CIH + BBR.
Male C57/BL6 mice (8 weeks of age) were obtained from Beijing HUAFUKANG Bioscience Co., LTD (Beijing, China).In the present study, the experiment was conducted in strict compliance with Institutional Animal Care and Use Protocols and approved by the Animal Care Committee of the General Hospital of Northern Theatre Command (No. 2021-14, 17 December 2021).Mice were rand- ). Physiological characteristics.