Hydrogen Sulfide Attenuates Angiotensin II-Induced Cardiac Fibroblast Proliferation and Transverse Aortic Constriction-Induced Myocardial Fibrosis through Oxidative Stress Inhibition via Sirtuin 3.

Sirtuin 3 (SIRT3) is critical in mitochondrial function and oxidative stress. Our present study investigates whether hydrogen sulfide (H2S) attenuated myocardial fibrosis and explores the possible role of SIRT3 on the protective effects. Neonatal rat cardiac fibroblasts were pretreated with NaHS followed by angiotensin II (Ang II) stimulation. SIRT3 was knocked down with siRNA technology. SIRT3 promoter activity and expression, as well as mitochondrial function, were measured. Male wild-type (WT) and SIRT3 knockout (KO) mice were intraperitoneally injected with NaHS followed by transverse aortic constriction (TAC). Myocardium sections were stained with Sirius red. Hydroxyproline content, collagen I and collagen III, α-smooth muscle actin (α-SMA), and dynamin-related protein 1 (DRP1) expression were measured both in vitro and in vivo. We found that NaHS enhanced SIRT3 promoter activity and increased SIRT3 mRNA expression. NaHS inhibited cell proliferation and hydroxyproline secretion, decreased collagen I, collagen III, α-SMA, and DRP1 expression, alleviated oxidative stress, and improved mitochondrial respiration function and membrane potential in Ang II-stimulated cardiac fibroblasts, which were unavailable after SIRT3 was silenced. In vivo, NaHS reduced hydroxyproline content, ameliorated perivascular and interstitial collagen deposition, and inhibited collagen I, collagen III, and DRP1 expression in the myocardium of WT mice but not SIRT3 KO mice with TAC. Altogether, NaHS attenuated myocardial fibrosis through oxidative stress inhibition via a SIRT3-dependent manner.

Alterations in mitochondrial dynamics with age-related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility.

Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) protects cardiac function against ischemia/reperfusion (I/R) injury. Mitochondria are critical in response to myocardial I/R injury as disturbance of mitochondrial dynamics contributes to cardiac dysfunction. It is hypothesized that SIRT1 and SIRT3 are critical components to maintaining mitochondria homeostasis especially mitochondrial dynamics to exert cardioprotective actions under I/R stress. The results demonstrated that deficiency of SIRT1 and SIRT3 in aged (24-26 months) mice hearts led to the exacerbated cardiac dysfunction in terms of cardiac systolic dysfunction, cardiomyocytes contractile defection, and abnormal cardiomyocyte calcium flux during I/R stress. Moreover, the deletion of SIRT1 or SIRT3 in young (4-6 months) mice hearts impair cardiomyocyte contractility and shows aging-like cardiac dysfunction upon I/R stress, indicating the crucial role of SIRT1 and SIRT3 in protecting myocardial contractility from I/R injury. The biochemical and seahorse analysis showed that the deficiency of SIRT1/SIRT3 leads to the inactivation of AMPK and alterations in mitochondrial oxidative phosphorylation (OXPHOS) that causes impaired mitochondrial respiration in response to I/R stress. Furthermore, the remodeling of the mitochondria network goes together with hypoxic stress, and mitochondria undergo the processes of fusion with the increasing elongated branches during hypoxia. The transmission electron microscope data showed that cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress. Thus, the age-related deficiency of SIRT1/SIRT3 in the heart affects mitochondrial dynamics and respiration function that resulting in the impaired contractile function of cardiomyocytes in response to I/R.

Protective role of sirtuin3 against oxidative stress and NLRP3 inflammasome in cholesterol accumulation and foam cell formation of macrophages with ox-LDL-stimulation.

Sirtuin3 (SIRT3) is involved in reactive oxygen species (ROS), cell metabolism, apoptosis and inflammation. However, the exact role of SIRT3 in macrophages during pathophysiological process of atherosclerosis remains unclear. The present study was to investigate the possible effects and mechanisms of SIRT3 on lipid uptake and foam cells transforming in oxidized low-density lipoprotein (ox-LDL)-stimulated macrophages. Compared with wild-type (WT) mice, SIRT3 deficiency further increased foam cell formation and cellular cholesterol accumulation, exacerbated oxidative stress, impaired mitochondrial permeability potential, decreased optic atrophy 1 (OPA1) but enhanced dynamin-related protein 1 (DRP1) expression, and promoted NLR family pyrin domain-containing protein 3 (NLRP3) activation in ox-LDL-stimulated macrophages from SIRT3 knockout (KO) mice. Dihydromyricetin (DMY), a potential compound to enhance SIRT3 expression, significantly inhibited cellular cholesterol accumulation, suppressed foam cell formation, improved mitochondrial function, attenuated oxidative stress, and alleviated NLRP3 activation in ox-LDL-stimulated macrophages. Moreover, above protective effects of DMY was unavailable in macrophages from SIRT3 KO mice. Collectively, the study demonstrated the protective role of SIRT3 against oxidative stress and NLRP3 inflammasome in cholesterol accumulation and foam cell formation of macrophages with ox-LDL-stimulation, which is beneficial to provide novel strategy for atherosclerosis prevention and treatment.

Hypertrophic Preconditioning Attenuates Myocardial Ischaemia-Reperfusion Injury by Modulating SIRT3-SOD2-mROS-Dependent Autophagy.

BACKGROUND: Ischaemic preconditioning elicited by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischaemic insult. Here, we test the hypothesis that short-term non-ischaemic stimulation of hypertrophy renders the heart resistant to subsequent ischaemic injury. METHODS AND RESULTS: Transient transverse aortic constriction (TAC) was performed for 3 days in mice and then withdrawn for 4 days by aortic debanding, followed by subsequent exposure to myocardial ischaemia-reperfusion (I/R) injury. Following I/R injury, myocardial infarct size and apoptosis were significantly decreased, and cardiac dysfunction was markedly improved in the TAC preconditioning group compared with the control group. Mechanistically, TAC preconditioning markedly suppressed I/R-induced autophagy and preserved autophagic flux by deacetylating SOD2 via a SIRT3-dependent mechanism. Moreover, treatment with an adenovirus encoding SIRT3 partially mimicked the effects of hypertrophic preconditioning, whereas genetic ablation of SIRT3 in mice blocked the cardioprotective effects of hypertrophic preconditioning. Furthermore, in vivo lentiviral-mediated knockdown of Beclin 1 in the myocardium ameliorated the I/R-induced impairment of autophagic flux and was associated with a reduction in cell death, whereas treatment with a lentivirus encoding Beclin 1 abolished the cardioprotective effect of TAC preconditioning. CONCLUSIONS: The present study identifies TAC preconditioning as a novel strategy for induction of an endogenous self-defensive and cardioprotective mechanism against cardiac injury. Specifically, TAC preconditioning reduced myocardial autophagic cell death in a SIRT3/SOD2 pathway-dependent manner.

Chronic High Fat Diet Intake Impairs Hepatic Metabolic Parameters in Ovariectomized Sirt3 KO Mice.

High fat diet (HFD) is an important factor in the development of metabolic diseases, with liver as metabolic center being highly exposed to its influence. However, the effect of HFD-induced metabolic stress with respect to ovary hormone depletion and sirtuin 3 (Sirt3) is not clear. Here we investigated the effect of Sirt3 in liver of ovariectomized and sham female mice upon 10 weeks of feeding with standard-fat diet (SFD) or HFD. Liver was examined by Folch, gas chromatography and lipid hydroperoxide analysis, histology and oil red staining, RT-PCR, Western blot, antioxidative enzyme and oxygen consumption analyses. In SFD-fed WT mice, ovariectomy increased Sirt3 and fatty acids synthesis, maintained mitochondrial function, and decreased levels of lipid hydroperoxides. Combination of ovariectomy and Sirt3 depletion reduced pparα, Scd-1 ratio, MUFA proportions, CII-driven respiration, and increased lipid damage. HFD compromised CII-driven respiration and activated peroxisomal ROS scavenging enzyme catalase in sham mice, whereas in combination with ovariectomy and Sirt3 depletion, increased body weight gain, expression of NAFLD- and oxidative stress-inducing genes, and impaired response of antioxidative system. Overall, this study provides evidence that protection against harmful effects of HFD in female mice is attributed to the combined effect of female sex hormones and Sirt3, thus contributing to preclinical research on possible sex-related therapeutic agents for metabolic syndrome and associated diseases.

Roles of Bak and Sirt3 in Paraquat-Induced Cochlear Hair Cell Damage.

Paraquat, a superoxide generator, can damage the cochlea causing an ototoxic hearing loss. The purpose of the study was to determine if deletion of Bak, a pro-apoptotic gene, would reduce paraquat ototoxicity or if deletion of Sirt3, which delays age-related hearing loss under caloric restriction, would increase paraquat ototoxicity. We tested these two hypotheses by treating postnatal day 3 cochlear cultures from Bak±, Bak-/-, Sirt3±, Sirt3-/-, and WT mice with paraquat and compared the results to a standard rat model of paraquat ototoxicity. Paraquat damaged nerve fibers and dose-dependently destroyed rat outer hair cells (OHCs) and inner hair cells (IHCs). Rat hair cell loss began in the base of the cochlea with a 10 µM dose and as the dose increased from 50 to 500 µM, the hair cell loss increased near the base of the cochlea and spread toward the apex of the cochlea. Rat OHC losses were consistently greater than IHC losses. Unexpectedly, in all mouse genotypes, paraquat-induced hair cell lesions were maximal near the apex of the cochlea and minimal near the base. This unusual damage gradient is opposite to that seen in paraquat-treated rats and in mice and rats treated with other ototoxic drugs. However, paraquat always induced greater OHC loss than IHC loss in all mouse strains. Contrary to our hypothesis, Bak deficient mice were more vulnerable to paraquat ototoxicity than WT mice (Bak-/- > Bak± > WT), suggesting that Bak plays a protective role against hair cell stress. Also, contrary to expectation, Sirt3-deficient mice did not differ significantly from WT mice, possibly due to the fact that Sirt3 was not experimentally upregulated in Sirt3-expressing mice prior to paraquat treatment. Our results show for the first time a gradient of ototoxic damage in mice that is greater in the apex than the base of the cochlea.

SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.

Salmonella typhimurium (S. typhimurium) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1ß. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD+) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation. Methods: We initially tested IL-1ß production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium-induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis. Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo. Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation. Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.

SIRT3 deficiency increases mitochondrial oxidative stress and promotes migration of retinal pigment epithelial cells.

Retinal pigment epithelial cells are closely associated with the pathogenesis of diabetic retinopathy. The mechanism by which diabetes impacts retinal pigment epithelial cell function is of significant interest. Sirtuins are an important class of proteins that primarily possess nicotinamide adenine dinucleotide-dependent deacetylases activity and involved in various cellular physiological and pathological processes. Here, we aimed to examine the role of sirtuins in the induction of diabetes-associated retinal pigment epithelial cell dysfunction. High glucose and platelet-derived growth factor (PDGF) treatment induced epithelial-mesenchymal transition and the migration of retinal pigment epithelial cells, and decreased sirtuin-3 expression. Sirtuin-3 knockdown using siRNA increased epithelial-mesenchymal transition and migration of retinal pigment epithelial cells. In contrast, sirtuin-3 overexpression attenuated the effects caused by high glucose and PDGF on epithelial-mesenchymal transition and migration of retinal pigment epithelial cells, suggesting that sirtuin-3 deficiency contributed to retinal pigment epithelial cell dysfunction induced by high glucose and PDGF. Mechanistically, sirtuin-3 deficiency induced retinal pigment epithelial cell dysfunction by the overproduction of mitochondrial reactive oxygen species. These results suggest that sirtuin-3 deficiency mediates the migration of retinal pigment epithelial cells, at least partially by increasing mitochondrial oxidative stress, and shed light on the importance of sirtuin-3 and mitochondrial reactive oxygen species as potential targets in diabetic retinopathy therapy.

Sirt3 Protects Against Ischemic Stroke Injury by Regulating HIF-1α/VEGF Signaling and Blood-Brain Barrier Integrity.

Sirtuin 3 (Sirt3) is a member of the Sirtuin family proteins and known to regulate multiple physiological processes such as metabolism and aging. As stroke is an aging-related disease, in this work, we attempt to examine the role and potential mechanism of Sirt3 in regulating ischemic stroke by using a permanent middle cerebral artery occlusion (pMCAO) model in wild type (WT) and Sirt3 knockout (KO) mice, coupled with oxygen glucose deprivation (OGD) experiments in cultured primary astrocytes. Sirt3 deficiency aggravated neuronal cell apoptosis and neurological deficits after brain ischemia. In addition, Sirt3 KO mice showed more severe blood-brain barrier (BBB) disruption and inflammatory responses compared with WT group in the acute phase. Furthermore, specific overexpression of Sirt3 in astrocytes by injecting glial fibrillary acidic protein (GFAP)::Sirt3 virus in ischemic region showed protective effect against stroke-induced damage. Mechanistically, Sirt3 could regulate vascular endothelial growth factor (VEGF) expression by inhibiting hypoxia inducible factor-1α (HIF-1α) signaling after ischemia (OGD). Our results have shown that Sirt3 plays a protective role in ischemic stroke via regulating HIF-1α/VEGF signaling in astrocytes, and reversal of the Sirt3 expression at the acute phase could be a worthy direction for stroke therapy.

Glutaredoxin-2 and Sirtuin-3 deficiencies impair cardiac mitochondrial energetics but their effects are not additive.

Altered redox biology and oxidative stress have been implicated in the progression of heart failure. Glutaredoxin-2 (GRX2) is a glutathione-dependent oxidoreductase and catalyzes the reversible deglutathionylation of mitochondrial proteins. Sirtuin-3 (SIRT3) is a class III histone deacetylase and regulates lysine acetylation in mitochondria. Both GRX2 and SIRT3 are considered as key in the protection against oxidative damage in the myocardium. Knockout of either contributes to adverse heart pathologies including hypertrophy, hypertension, and cardiac dysfunction. Here, we created and characterized a GRX2 and SIRT3 double-knockout mouse model, hypothesizing that their deletions would have an additive effect on oxidative stress, and exacerbate mitochondrial function and myocardial structural remodeling. Wildtype, single-gene knockout (Sirt3-/-, Grx2-/-), and double-knockout mice (Grx2-/-/Sirt3-/-) were compared in heart weight, histology, mitochondrial respiration and H2O2 production. Overall, the hearts from Grx2-/-/Sirt3-/- mice displayed increased fibrosis and hypertrophy versus wildtype. In the Grx2-/- and the Sirt3-/- we observed changes in mitochondrial oxidative capacity, however this was associated with elevated H2O2 emission only in the Sirt3-/-. Similar changes were observed but not worsened in hearts from Grx2-/-/Sirt3-/- mice, suggesting that these changes were not additive. In human myocardium, using genetic and histopathological data from the human Genotype-Tissue Expression consortium, we confirmed that SIRT3 expression correlates inversely with heart pathology. Altogether, GRX2 and SIRT3 are important in the control of cardiac mitochondrial redox and oxidative processes, but their combined absence does not exacerbate effects, consistent with the overall conclusion that they function together in the complex redox and antioxidant systems in the heart.

SIRT3 Deficiency Sensitizes Angiotensin-II-Induced Renal Fibrosis.

BACKGROUND: Sirtuin 3 (SIRT3) has a crucial role in the cardiovascular diseases. Our previous study revealed that SIRT3 knockout (SIRT3KO) promoted cardiac pericyte-fibroblast transition. In this study, we investigated the involvement of pericyte and iron in angiotensin II (Ang-II)-mediated renal fibrosis in the SIRT3KO mice. METHODS AND RESULTS: NG2-DsRed mice and NG2-DsRed-SIRT3 knockout (SIRT3KO) mice were infused with saline or Ang-II (1000 ng/kg/min) for 4 weeks. Renal fibrosis, iron content and reactive oxygen species (ROS) were measured. Masson's trichrome staining showed that SIRT3KO enhanced Ang-II-induced renal fibrosis. Immunostaining showed that Ang-II treatment increased the number of NG2-DsRed+ cells in the kidney, and SIRT3KO further enhanced NG2-DsRed+ cells. Moreover, SIRT3KO promoted pericyte differentiation into fibroblasts as evidenced by co-staining NG2-DsRed/FSP-1. Furthermore, DsRed/FSP-1+ and DsRed/transforming growth factor-ß1 (TGF-ß1)+ fibroblasts were elevated by SIRT3KO after Ang-II infusion. Ang-II-induced collagen I and TGF-ß1 expression was also enhanced in the SIRT3KO mice. SIRT3KO significantly exacerbated Ang-II-induced iron accumulation. This was accompanied by an increase in acetyl-p53, HO-1 and FPN expression. Further, SIRT3KO sensitized Ang-II-induced upregulation of p47phox and gp91phox together with increased ROS formation in the kidney. CONCLUSION: Our study suggests that SIRT3 deficiency sensitized Ang-II-induced renal fibrosis by the mechanisms involved in promoting differentiation of pericytes into fibroblasts, exacerbating iron overload and accelerating NADPH oxidase-derived ROS formation.

SIRT3 Ablation Deteriorates Obesity-Related Cardiac Remodeling by Modulating ROS-NF-κB-MCP-1 Signaling Pathway.

Obesity and the associated complications are a major public health issue as obesity incidence increases yearly, worldwide. Effects of obesity on heart failure have been reported previously. Obesity-related cardiac remodeling includes structural and functional dysfunctions, in which cardiac inflammation and fibrosis play a key role. The main mitochondrial deacetylase, SIRT3 participates in numerous cellular processes; however, its role in obesity-related cardiac remodeling remains unclear. In our study, high-fat diet (HFD) feeding induced downregulation of SIRT3 protein level in mice. SIRT3-KO mice fed on HFD exhibited higher cardiac dysfunction and cardiac remodeling compared with the wild-type controls. Further study revealed increases in collagen accumulation and inflammatory cytokine expression including MCP-1, IL-6, TGF-ß, TNF-α in mice fed on HFD compared with chow diet, with higher levels observed in SIRT3-KO mice. Furthermore, significantly high levels of cardiac MCP-1 expression and macrophage infiltration, and ROS generation and activated NF-κB were observed in HFD-fed SIRT3-KO mice. We presumed that SIRT3 ablation-mediated MCP-1 upregulation is attributed to ROS-NF-κB activation. Thus, we concluded that SIRT3 prevents obesity-related cardiac remodeling by attenuating cardiac inflammation and fibrosis, through modulation of ROS-NF-κB-MCP-1 pathway.

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure.

RATIONALE: Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. OBJECTIVE: This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. METHODS AND RESULTS: A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)-enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively-was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction-induced pressure overload. CONCLUSIONS: The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.

SIRT3 promotes auditory function in young adult FVB/nJ mice but is dispensable for hearing recovery after noise exposure.

Noise-induced hearing loss (NIHL) affects millions of people worldwide and presents a large social and personal burden. Pharmacological activation of SIRT3, a regulator of the mitochondrial oxidative stress response, has a protective effect on hearing thresholds after traumatic noise damage in mice. In contrast, the role of endogenously activated SIRT3 in hearing recovery has not been established. Here we tested the hypothesis that SIRT3 is required in mice for recovery of auditory thresholds after a noise exposure that confers a temporary threshold shift (TTS). SIRT3-specific immunoreactivity is present in outer hair cells, around the post-synaptic regions of inner hair cells, and faintly within inner hair cells. Prior to noise exposure, homozygous Sirt3-KO mice have slightly but significantly higher thresholds than their wild-type littermates measured by the auditory brainstem response (ABR), but not by distortion product otoacoustic emissions (DPOAE). Moreover, homozygous Sirt3-KO mice display a significant reduction in the progression of their peak 1 amplitude at higher frequencies prior to noise exposure. After exposure to a single sub-traumatic noise dose that does not permanently reduce cochlear function, compromise cell survival, or damage synaptic structures in wild-type mice, there was no difference in hearing function between the two genotypes, measured by ABR and DPOAE. The numbers of hair cells and auditory synapses were similar in both genotypes before and after noise exposure. These loss-of-function studies complement previously published gain-of-function studies and help refine our understanding of SIRT3's role in cochlear homeostasis under different damage paradigms. They suggest that SIRT3 may promote spiral ganglion neuron function. They imply that cellular mechanisms of homeostasis, in addition to the mitochondrial oxidative stress response, act to restore hearing after TTS. Finally, we present a novel application of a biomedical statistical analysis for identifying changes between peak 1 amplitude progressions in ABR waveforms after damage.

Sirt3 is a novel target to treat sepsis induced myocardial dysfunction by acetylated modulation of critical enzymes within cardiac tricarboxylic acid cycle.

Sepsis induced myocardial dysfunction (SIMD) results in high morbidity and mortality. However, the effective therapeutic strategies for SIMD treatment remain limited. Sirt3 is the main mitochondrial Sirtuin member and is a key modulator of mitochondrial metabolism and function. In this study, we aimed to investigate the effect and mechanism of Sirt3 on SIMD. SIMD was induced by 20 mg/kg Lipopolysaccharides (LPS) injection for 6 h in mice. Sepsis could induce the reduction of cardiac Sirt3 expression and global deficiency of Sirt3 exacerbated cardiac function. Quantitative acetyl-proteomics and cardiac metabolomics analysis revealed that loss of Sirt3 led to hyper-acetylation of critical enzymes within cardiac tricarboxylic acid (TCA) cycle and generation of lactate and NADH, subsequently promotion of cardiac dysfunction after sepsis. Additionally, to evaluate whether Emodin could be utilized as a potential Sirt3 modulator to treat SIMD, male wild type mice (WT mice) or global Sirt3 deficient mice (Sirt3-/- mice) were intraperitoneally injected with 40 mg/kg Emodin for 5 days followed by 20 mg/kg LPS administration for another 6 h and observed that exogenous administration of Emodin could attenuate myocardial dysfunction in septic WT mice. However, septic Sirt3-/- mice can not gain benefit on cardiac performance from Emodin infusion. In conclusion, this study presented the protective role of Sirt3 targeting SIMD, which may provide a potential novel approach to maintain normal cardiac performance after sepsis.

Endothelial Sirtuin 3 Dictates Glucose Transport to Cardiomyocyte and Sensitizes Pressure Overload-Induced Heart Failure.

Background Alterations of energetic metabolism are suggested to be an important contributor to pressure overload (PO)-induced heart failure. Our previous study reveals that knockout of endothelial Sirtuin 3 (SIRT3) alters glycolysis and impairs diastolic function. We hypothesize that endothelial SIRT3 regulates glucose utilization of cardiomyocytes and sensitizes PO-induced heart failure. Methods and Results SIRT3 endothelial cell knockout mice and their control SIRT3 LoxP mice were subjected to PO by transverse aortic constriction for 7 weeks. The ratio of heart weight to tibia length was increased in both strains of mice, in which SIRT3 endothelial cell knockout mice+transverse aortic constriction exhibited more severe cardiac hypertrophy. Coronary blood flow and systolic function were significantly decreased in SIRT3 endothelial cell knockout mice+transverse aortic constriction compared with SIRT3 LoxP mice+transverse aortic constriction, as evidenced by lower systolic/diastolic ratio, ejection fraction, and fractional shortening. PO-induced upregulation of apelin and glucose transporter 4 were significantly reduced in the hearts of SIRT3 endothelial cell knockout mice. In vitro, levels of hypoxia-inducible factor-1α and glucose transporter 1 and glucose uptake were significantly reduced in SIRT3 knockout endothelial cells. Furthermore, hypoxia-induced apelin expression was abolished together with reduced apelin-mediated glucose uptake in SIRT3 knockout endothelial cells. Exposure of cardiomyocyte with apelin increased expression of glucose transporter 1 and glucose transporter 4. This was accompanied by a significant increase in glycolysis. Supplement of apelin in SIRT3 knockout hypoxic endothelial cell media increased glycolysis in the cardiomyocytes. Conclusions Knockout of SIRT3 disrupts glucose transport from endothelial cells to cardiomyocytes, reduces cardiomyocyte glucose utilization via apelin in a paracrine manner, and sensitizes PO-induced heart failure. Endothelial SIRT3 may regulate cardiomyocyte glucose availability and govern the function of the heart.

SIRT3 deficiency is resistant to autophagy-dependent ferroptosis by inhibiting the AMPK/mTOR pathway and promoting GPX4 levels.

Ferroptosis, an autophagy-dependent cell death, is characterized by lipid peroxidation and iron accumulation, closely associated with pathogenesis of gestational diabetes mellitus (GDM). Sirtuin 3 (SIRT3) has positive regulation on phosphorylation of activated protein kinase (AMPK), related to maintenance of cellular redox homeostasis. However, whether SIRT3 can confer autophagy by activating the AMPK-mTOR pathway and consequently promote induction of ferroptosis is unknown. We used human trophoblastic cell line HTR8/SVneo and porcine trophoblastic cell line pTr2 to deterimine the mechanism of SIRT3 on autophagy and ferroptosis. The expression of SIRT3 protein was significantly elevated in trophoblastic cells exposed to high concentrations of glucose and ferroptosis-inducing compounds. Increased SIRT3 expression contributed to classical ferroptotic events and autophagy activation, whereas SIRT3 silencing led to resistance against both ferroptosis and autophagy. In addition, autophagy inhibition impaired SIRT3-enhanced ferroptosis. On the contrary, autophagy induction had a synergistic effect with SIRT3. Based on mechanistic investigations, SIRT3 depletion inhibited activation of the AMPK-mTOR pathway and enhanced glutathione peroxidase 4 (GPX4) level, thereby suppressing autophagy and ferroptosis. Furthermore, depletion of AMPK blocked induction of ferroptosis in trophoblasts. We concluded that upregulated SIRT3-enhanced autophagy activation by promoting AMPK-mTOR pathway and decreasing GPX4 level to induce ferroptosis in trophoblastic cells. SIRT3 deficiency was resistant to high glucose- and erastin-induced autophagy-dependent ferroptosis and is, therefore, a potential therapeutic approach for treating GDM.

Dietary schizophyllan reduces mitochondrial damage by activating SIRT3 in mice.

Schizophyllan (SPG), produced by Schizophyllum commune, is an exopolysaccharide with multiple academic and commercial uses, including in the food industry and for various medical functions. We previously demonstrated that SPG conjugated with c-Src peptide exerted a significant therapeutic effect on mouse models of the acute inflammatory diseases polymicrobial sepsis and ulcerative colitis. Here we extended these results by investigating whether SPG exerted a protective effect against mitochondrial damage in the liver via sirtuin 3 (SIRT3) induction, focusing on the deacetylation of succinate dehydrogenase A (SDHA) and superoxide dismutase 2 (SOD2). Liver damage models induced by alcohol or conjugated linoleic acid (CLA, which simulates lipodystrophy) in SIRT3-/-, SOD2-/-, and SDHA-/- mice were used. Results showed that dietary supplementation with SPG induced SIRT3 activation; this was involved in mitochondrial metabolic resuscitation that countered the adverse effects of alcoholic liver disease and CLA-induced damage. The mitochondrial SIRT3 mediated the deacetylation and activation of SOD2 in the liver and SDHA in adipose tissues, suggesting that SPG supplementation reduced ethanol-induced liver damage and CLA-induced adverse dietary effects via SIRT3-SOD2 and SIRT3-SDHA signaling, respectively. Together, these results suggest that dietary SPG has a previously unrecognized role in SIRT3-mediated mitochondrial metabolic resuscitation during mitochondria-related diseases.

SIRT3 deficiency delays diabetic skin wound healing via oxidative stress and necroptosis enhancement.

Sirtuin 3 (SIRT3) plays a vital role in several dermatological diseases. However, the role and detailed mechanism of SIRT3 in diabetic wound healing are unknown well yet. To explore possible involvement of SIRT3 and necroptosis in diabetic skin wound healing, SIRT3 knockout (KO) mice and 129S1/SvImJ wild-type (WT) mice were injected with streptozotocin (STZ), and mice skin fibroblasts were exposed to high glucose (HG). It was found that SIRT3 expression decreased in the skin of diabetic patients. SIRT3 deficiency delayed healing rate, reduced blood supply and vascular endothelial growth factor expression, promoted superoxide production, increased malondialdehyde (MDA) levels, decreased total antioxidant capacity (T-AOC), reduced superoxide dismutase (SOD) activity and aggravated ultrastructure disorder in skin wound of diabetic mice. SIRT3 deficiency inhibited mice skin fibroblasts migration with HG stimulation, which was restored by SIRT3 overexpression. SIRT3 deficiency also suppressed α-smooth muscle actin (α-SMA) expression, enhanced superoxide production but decreased mitochondrial membrane potential with HG stimulation after scratch. SIRT3 deficiency further elevated receptor-interacting protein kinase 3 (RIPK3), RIPK1 and caspase 3 expression both in vitro and in vivo. Collectively, SIRT3 deficiency delayed skin wound healing in diabetes, the mechanism might be related to impaired mitochondria function, enhanced oxidative stress and increased necroptosis. This may provide a novel therapeutic target to accelerate diabetic skin wound healing.

Sirtuin 3 deficiency accelerates Angiotensin II-induced skeletal muscle atrophy.

Background: It has been reported that Angiotensin II (Ang II) induced skeletal muscle atrophy. However, the precise mechanisms remain elusive. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, plays a central role in maintaining cellular metabolic homeostasis. This work aims to determine the role of SIRT3-mediated cellular metabolism in skeletal muscle wasting. Methods and Results: Eight-week-old male wild-type (WT) and SIRT3 knockout (SIRT3 KO) mice were infused with Ang II or saline for 4 weeks. Ang II induces skeletal muscle atrophy by inducing expression of the muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 (MuRF1) and atrogin-1, accompanied by a reduction in SIRT3 in skeletal muscle. SIRT3 deficiency accelerated Ang II-induced loss of lean mass and protein hyper-acetylation, while the activities of mitochondrial oxidative enzymes, such as complex I and complex V, were significantly decreased. Furthermore, SIRT3 deficiency accelerated the Ang II-induced shift from slow-twitch towards fast-twitch fibres. Similarly, the three major rate-limiting enzymes in the glycolytic pathway, hexokinase 2 (HK2), phosphofructokinase-1(PFK) and pyruvate kinase (PK), were upregulated in Ang II-treated SIRT3 KO mice. Conclusion: These studies indicate that SIRT3 deficiency augmented Ang II-induced fibre-type shifting and metabolic reprogramming.