Skeletal Muscle SIRT3 Deficiency Contributes to Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) is a major complication linked to adverse outcomes in heart failure with preserved ejection fraction (HFpEF), yet no specific therapies exist for PH associated with HFpEF (PH-HFpEF). We have recently reported on the role of skeletal muscle SIRT3 (sirtuin-3) in modulation of PH-HFpEF, suggesting a novel endocrine signaling pathway for skeletal muscle modulation of pulmonary vascular remodeling. METHODS: Using skeletal muscle-specific Sirt3 knockout mice (Sirt3skm-/-) and mass spectrometry-based comparative secretome analysis, we attempted to define the processes by which skeletal muscle SIRT3 defects affect pulmonary vascular health in PH-HFpEF. RESULTS: Sirt3skm-/- mice exhibited reduced pulmonary vascular density accompanied by pulmonary vascular proliferative remodeling and elevated pulmonary pressures. Comparative analysis of secretome by mass spectrometry revealed elevated secretion levels of LOXL2 (lysyl oxidase homolog 2) in SIRT3-deficient skeletal muscle cells. Elevated circulation and protein expression levels of LOXL2 were also observed in plasma and skeletal muscle of Sirt3skm-/- mice, a rat model of PH-HFpEF, and humans with PH-HFpEF. In addition, expression levels of CNPY2 (canopy fibroblast growth factor signaling regulator 2), a known proliferative and angiogenic factor, were increased in pulmonary artery endothelial cells and pulmonary artery smooth muscle cells of Sirt3skm-/- mice and animal models of PH-HFpEF. CNPY2 levels were also higher in pulmonary artery smooth muscle cells of subjects with obesity compared with nonobese subjects. Moreover, treatment with recombinant LOXL2 protein promoted pulmonary artery endothelial cell migration/proliferation and pulmonary artery smooth muscle cell proliferation through regulation of CNPY2-p53 signaling. Last, skeletal muscle-specific Loxl2 deletion decreased pulmonary artery endothelial cell and pulmonary artery smooth muscle cell expression of CNPY2 and improved pulmonary pressures in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: This study demonstrates a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH-HFpEF. This study suggests a new endocrine signaling axis that links skeletal muscle health and SIRT3 deficiency to remote CNPY2 regulation in the pulmonary vasculature through myokine LOXL2. Our data also identify skeletal muscle SIRT3, myokine LOXL2, and CNPY2 as potential targets for the treatment of PH-HFpEF.

NAD deficiency contributes to progressive kidney disease in HIV-nephropathy mice.

HIV disease remains prevalent in the United States and is particularly prevalent in sub-Saharan Africa. Recent investigations revealed that mitochondrial dysfunction in kidney contributes to HIV-associated nephropathy (HIVAN) in Tg26 transgenic mice. We hypothesized that nicotinamide adenine dinucleotide (NAD) deficiency contributes to energetic dysfunction and progressive tubular injury. We investigated metabolomic mechanisms of HIVAN tubulopathy. Tg26 and wild-type (WT) mice were treated with the farnesoid X receptor (FXR) agonist INT-747 or nicotinamide riboside (NR) from 6 to 12 wk of age. Multiomic approaches were used to characterize kidney tissue transcriptomes and metabolomes. Treatment with INT-747 or NR ameliorated kidney tubular injury, as shown by serum creatinine, the tubular injury marker urinary neutrophil-associated lipocalin, and tubular morphometry. Integrated analysis of metabolomic and transcriptomic measurements showed that NAD levels and production were globally downregulated in Tg26 mouse kidneys, especially nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway. Furthermore, NAD-dependent deacetylase sirtuin3 activity and mitochondrial oxidative phosphorylation activity were lower in ex vivo proximal tubules from Tg26 mouse kidneys compared with those of WT mice. Restoration of NAD levels in the kidney improved these abnormalities. These data suggest that NAD deficiency might be a treatable target for HIVAN.NEW & NOTEWORTHY The study describes a novel investigation that identified nicotinamide adenine dinucleotide (NAD) deficiency in a widely used HIV-associated nephropathy (HIVAN) transgenic mouse model. We show that INT-747, a farnesoid X receptor agonist, and nicotinamide riboside (NR), a precursor of nicotinamide, each ameliorated HIVAN tubulopathy. Multiomic analysis of mouse kidneys revealed that NAD deficiency was an upstream metabolomic mechanism contributing to HIVAN tubulopathy.

ACE2 deficiency inhibits thoracic aortic dissection by enhancing SIRT3 mediated inhibition of inflammation and VSCMs phenotypic switch.

BACKGROUND: Thoracic aortic dissection (TAD) is an irreversible cardiovascular disorder with high mortality and morbidity. However, the molecular mechanisms remain elusive. Thus, identifying an effective therapeutic target to prevent TAD is especially critical. The purpose of this study is to elucidate the potential mechanism of inflammation and vascular smooth muscle cell (VSMCs) phenotypic switch in ß-aminopropionitrile fumarate (BAPN)-induced TAD. METHODS: A mouse model of TAD induced by BAPN and IL-1ß -stimulated HVSMCs in vivo and in vitro models, respectively. ACE2 Knockdown mice treated with BAPN or without, and the TAD mouse model was treated with or without AAV-ACE2. Transthoracic ultrasound was conducted for assessment the maximum internal diameter of the thoracic aorta arch. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Western blot were used to detect the expression of MMP2, MMP9, ACE2, SIRT3, OPN, SM22α and other inflammatory markers. The circulating levels of ACE2 was measured by ELISA assay. Histological changes of thoracic aorta tissues were assessed by H&E, EVG and IHC analysis. RESULTS: We found that circulating levels of and the protein levels of ACE2 were increased in the TAD mouse model and in patients with TAD. For further evidence, ACE2 deficiency decelerated the formation of TAD. However, overexpression of ACE2 aggravated BAPN-induced aortic injury and VSMCs phenotypic switch via lowered SIRT3 expression and elevated inflammatory cytokine expression. CONCLUSION: ACE2 deficiency prevented the development of TAD by inhibiting inflammation and VSMCs phenotypic switch in a SIRT3-dependent manner, suggesting that the ACE2/SIRT3 signaling pathway played a pivotal role in the pathological process of TAD and might be a potential therapeutical target.

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 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.

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.

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.

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.

Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.

Berberine (BBR) shows promising effects in the treatment of nonalcoholic fatty liver disease (NAFLD) by influencing various metabolic aspects. Inhibition of mitochondrial ß-oxidation (ß-OX) participates in the pathogenesis of NAFLD. Silent mating-type information regulation 2 homolog 3 (SIRT3) has been reported to regulate mitochondrial ß-OX by deacetylating its substrate, long-chain acyl-coenzyme A dehydrogenase (LCAD). This study aimed to explore whether BBR can promote mitochondrial ß-OX and the role of SIRT3 as well as the mechanisms underlying the effects of BBR on hepatic lipid metabolism in mice fed a high-fat diet (HFD). BBR can significantly improve systematic and hepatic lipid metabolism in HFD-fed mice. Metabolomics analysis revealed that ß-OX was inhibited in HFD-induced mice, as indicated by the reduced production of short and medium carbon chain acyl-carnitines, the activated form of free fatty acids, via ß-OX, which was reversed by BBR intervention. Exploration of the mechanism found that BBR intervention reversed the down-regulation of SIRT3 and decreased the LCAD hyperacetylation level in HFD-fed mice. SIRT3 knockout (KO) mice were used to identify the role of SIRT3 in the BBR's influence of ß-OX. The beneficial effects of BBR on systemic and hepatic metabolism were profoundly attenuated in KO mice. Moreover, the promotive effect of BBR on ß-OX in HFD-induced mice was partially abolished in KO mice. These results suggested that BBR alleviates HFD-induced inhibition of fatty acid ß-OX partly through SIRT3-mediated LCAD deacetylation, which may provide a novel mechanism and support BBR as a promising therapeutic for NAFLD.-Xu, X., Zhu, X.-P., Bai, J.-Y., Xia, P., Li, Y., Lu, Y., Li, X.-Y., Gao, X. Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.

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.

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.

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.

Sirtuin 3 silencing impairs mitochondrial biogenesis and metabolism in colon cancer cells.

Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase and targets several crucial enzymes against oxidative stress. Recent reports suggest that SIRT3 could also participate in the quality and quantity control of mitochondria. The aim of this study was to analyze whether SIRT3 silencing in colon cancer cells could affect mitochondrial biogenesis and impair mitochondrial function. For this purpose, metastatic colon cancer cell line SW620 was transfected with a specific shRNA against SIRT3 to obtain a stable knockdown. Gene expression and protein levels of several proteins related to mitochondrial biogenesis and function were determined by RT-qPCR and Western blotting. Mitochondrial function was studied by analyzing COX, ATPase, and LDH enzymatic activities, oxygen consumption, superoxide levels, and mitochondrial membrane potential. Confocal images were also taken to study mitochondrial morphology, and cell motility and clonogenicity were also studied. SIRT3 silencing resulted in a reduced mitochondrial biogenesis and function, as evidenced by the decrease in proteins such as PGC-1α and mitochondrial transcription factor A and lower levels of OXPHOS complexes. Furthermore, COX activity and oxygen consumption were also diminished after SIRT3 knockdown. Finally, SIRT3-silenced cells showed mitochondrial aggregation compared with control cells as well as reduced motility and colony formation ability. In conclusion, SIRT3 silencing in SW620 cancer cells leads to decreased mitochondrial biogenesis and mitochondrial dysfunction, ultimately affecting cell viability and could be a therapeutic strategy to render cells more sensitive to treatment.

MicroRNA-195 Regulates Metabolism in Failing Myocardium Via Alterations in Sirtuin 3 Expression and Mitochondrial Protein Acetylation.

BACKGROUND: Heart failure leads to mitochondrial dysfunction and metabolic abnormalities of the failing myocardium coupled with an energy-depleted state and cardiac remodeling. The mitochondrial deacetylase sirtuin 3 (SIRT3) plays a pivotal role in the maintenance of mitochondrial function through regulating the mitochondrial acetylome. It is interesting to note that unique cardiac and systemic microRNAs have been shown to play an important role in cardiac remodeling by modulating key signaling elements in the myocardium. METHODS: Cellular signaling was analyzed in human cardiomyocyte-like AC16 cells, and acetylation levels in rodent models of SIRT3-/-and transgenic microRNA-195 (miR-195) overexpression were compared with wild type. Luciferase assays, Western blotting, immunoprecipitation assays, and echocardiographic analysis were performed. Enzymatic activities of pyruvate dehydrogenase (PDH) and ATP synthase were measured. RESULTS: In failing human myocardium, we observed induction of miR-195 along with decreased expression of the mitochondrial deacetylase SIRT3 that was associated with increased global protein acetylation. We further investigated the role of miR-195 in SIRT3-mediated metabolic processes and its impact on regulating enzymes involved in deacetylation. Proteomic analysis of the total acetylome showed increased overall acetylation, and specific lysine acetylation of 2 central mitochondrial metabolic enzymes, PDH and ATP synthase, as well. miR-195 downregulates SIRT3 expression through direct 3'-untranslated region targeting. Treatments with either sirtuin inhibitor nicotinamide, small interfering RNA-mediated SIRT3 knockdown or miR-195 overexpression enhanced acetylation of PDH complex and ATP synthase. This effect diminished PDH and ATP synthase activity and impaired mitochondrial respiration.SIRT3-/- and miR-195 transgenic mice consistently showed enhanced global protein acetylation, including PDH complex and ATP synthase, associated with decreased enzymatic activity. CONCLUSIONS: Altogether, these data suggest that increased levels of miR-195 in failing myocardium regulate a novel pathway that involves direct SIRT3 suppression and enzymatic inhibition via increased acetylation of PDH and ATP synthase that are essential for cardiac energy metabolism.

Decreased Neuron Number and Synaptic Plasticity in SIRT3-Knockout Mice with Poor Remote Memory.

The sirtuin family of proteins consists of nicotinamide adenine dinucleotide-dependent deacetylases that are involved in the response to calorie restriction and various physiological phenomena, such as aging and cognition. One of these proteins, sirtuin 3 (SIRT3), is localized in the mitochondria and protects the cell against oxidative or metabolic stress. Sirtuin protein deficiencies have been shown to accelerate neurodegeneration in neurotoxic conditions. The mechanisms underlying the involvement of SIRT3 in cognition remain unclear. Interestingly, SIRT1, another member of the sirtuin family, has been reported to modulate synaptic plasticity and memory formation. To learn more about these proteins, we examined the behavior and cognitive functions of Sirt3-knockout mice. The mice exhibited poor remote memory. Consistent with this, long-term potentiation was impaired in the Sirt3-knockout mice, and they exhibited decreased neuronal number in the anterior cingulate cortex, which seemed to contribute to their memory deficiencies.

Sirtuin3 deficiency exacerbates carbon tetrachloride-induced hepatic injury in mice.

Sirtuin3 (SIRT3) plays an important role in maintaining normal mitochondrial function and alleviating oxidative stress. After carbon tetrachloride (CCl4 ) administration, the expression of SIRT3 decreased in the liver of mice, which indicated that the SIRT3 might play a crucial role during chemical-induced acute hepatic injury. To verify the hypothesis, CCl 4 was given to induce acute hepatic injury in SIRT3 knockout (KO) mice and wild-type (WT) mice. CCl 4 -induced liver injury was more severe in SIRT3 KO mice compared with the WT mice. In addition, the oxidative stress induced by CCl 4 was enhanced in the SIRT3 KO mice. Furthermore, the increased expression of dynamin-related protein 1 was also aggravated in SIRT3 KO mice after CCl 4 administration. In conclusion, our study demonstrated that SIRT3 deficiency exacerbated CCl 4 -induced impairment of the liver in mice, and the mechanism might be related to enhanced oxidative stress.

Sirtuin 3 deficiency aggravates contrast-induced acute kidney injury.

BACKGROUND: Sirtuin 3 (Sirt3) is a key regulator of energy metabolism and oxidative stress. To investigate the role of Sirt3 in contrast-induced acute kidney injury (CIAKI), we established the model both in vivo and in vitro to explore the potential mechanisms. METHODS: In vivo, we established CIAKI models in wild-type (WT) and Sirt3-knockout (Sirt3-KO) mice. Blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by enzyme-linked immunosorbent assay, Glomerular Filtration Rate (GFR) and creatinine clearance were also investigated. We detected the production of reactive oxygen species (ROS) via 2'7'-dichlorodihydro-fluorescein diacetate. The expressions of Sirt3, oxidative stress and apoptosis related markers (MnSOD, Catalase, Acetyl-MnSOD K68, Nox4, Bax, Bcl-2 and Caspase3) were measured and analyzed. In addition, we observed the effect of nicotinamide riboside (NR) on CIAKI in WT and Sirt3-KO mice. In vitro, Sirt3 was knocked out by siRNA transfection method in HK-2 cells. Sirt3, ROS, oxidative stress and apoptosis markers in HK-2 cells were also measured. RESULTS: Our data demonstrated that the levels of Scr and BUN in Sirt3-KO mice were increased while the levels of the GFR and creatinine clearance were decreased in CIAKI mice. In Sirt3-KO or siRNA groups, the activities of MnSOD and Catalase were markedly down-regulated. Also, the expression of Caspase3 were markedly increased and the ratio of Bcl-2/Bax was decreased, while the ROS level was increased in Sirt3 deficiency groups. NR ameliorated CIAKI in WT mice but not in Sirt3-KO mice. CONCLUSION: Our results suggest that Sirt3 deficiency aggravates contrast-induced acute kidney injury. Sirt3 is critical in NR-mediated renoprotection in CIAKI.

Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress.

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3⁻/⁻ livers at 3 months of age. Livers of Sirt3⁻/⁻ mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3⁻/⁻ MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD⁻/⁻ MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3⁻/⁻ MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3⁻/⁻ livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.

Potential mechanisms linking SIRT activity and hypoxic 2-hydroxyglutarate generation: no role for direct enzyme (de)acetylation.

2-Hydroxyglutarate (2-HG) is a hypoxic metabolite with potentially important epigenetic signaling roles. The mechanisms underlying 2-HG generation are poorly understood, but evidence suggests a potential regulatory role for the sirtuin family of lysine deacetylases. Thus, we hypothesized that the acetylation status of the major 2-HG-generating enzymes [lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH)] may govern their 2-HG-generating activity. In vitro acetylation of these enzymes, with confirmation by western blotting, mass spectrometry, reversibility by recombinant sirtuins and an assay for global lysine occupancy, yielded no effect on 2-HG-generating activity. In addition, while elevated 2-HG in hypoxia is associated with the activation of lysine deacetylases, we found that mice lacking mitochondrial SIRT3 exhibited hyperacetylation and elevated 2-HG. These data suggest that there is no direct link between enzyme acetylation and 2-HG production. Furthermore, our observed effects of in vitro acetylation on the canonical activities of IDH, MDH and LDH appeared to contrast with previous findings wherein acetyl-mimetic lysine mutations resulted in the inhibition of these enzymes. Overall, these data suggest that a causal relationship should not be assumed between acetylation of metabolic enzymes and their activities, canonical or otherwise.

Endothelial specific SIRT3 deletion impairs glycolysis and angiogenesis and causes diastolic dysfunction.

Endothelial glycolysis plays a critical role in the regulation of angiogenesis. We investigated the role of Sirtuin 3 (SIRT3) on endothelial cell (EC) glycolytic metabolism, angiogenesis, and diastolic function. Our aim was to test the hypothesis that loss of SIRT3 in ECs impairs endothelial glycolytic metabolism and angiogenesis and contributes to myocardial capillary rarefaction and the development of diastolic dysfunction. Using SIRT3 deficient ECs, SIRT3 was found to regulate a metabolic switch between mitochondrial respiration and glycolysis. SIRT3 knockout (KO)-ECs exhibited higher mitochondrial respiration and reactive oxygen species (ROS) formation. SIRT3 knockout (KO)-ECs exhibited a reduction in the expression of glycolytic enzyme, PFKFB3, and a fall in glycolysis and angiogenesis. Blockade of PFKFB3 reduced glycolysis and downregulated expression of VEGF and Angiopoietin-1 (Ang-1) in ECs. Deletion of SIRT3 in ECs also impaired hypoxia-induced expression of HIF-2α, VEGF, and Ang-1, as well as reduced angiogenesis. In vivo, endothelial-specific SIRT3 KO (ECKO) mice exhibited a myocardial capillary rarefaction together with a reduced coronary flow reserve (CFR) and diastolic dysfunction. Histologic study further demonstrated that knockout of SIRT3 in ECs significantly increased perivascular fibrosis in the coronary artery. These results implicate a role of SIRT3 in modulating endothelial function and cardiac function. Ablation of SIRT3 leads to impairment of EC glycolytic metabolism and angiogenic signaling, which may contribute to coronary microvascular rarefaction and diastolic dysfunction in SIRT3 ECKO mice.