Sirt1 Knockout Mice Research Articles

Knockout of Sirtuin 3 in endothelial cells impairs endothelial-dependent relaxation and myogenic response in mice.

Sirtuin 3 has been shown to regulate endothelial function and coronary flow reserve in mice. Knockout of SIRT3 reduced endothelial nitric oxide synthase expression in the mouse hearts. In this study, we investigate whether endothelial SIRT3 regulates vascular function and myogenic responses in distal intramural branches of the left anterior descending coronary artery (CA) and middle cerebral artery (MCA) of mice. Both male and female endothelial SIRT3 knockout (SIRT3ECKO) mice and control SIRT3LoxP mice were used and CA and MCA were dissected and mounted in a myograph system. The myogenic response was evaluated by measuring changes in inner diameter in response to 20 mmHg stepwise increases in intraluminal pressure in PSS (active diameter) and Ca2+-free PSS (passive diameter). Acetylcholine (Ach)-induced endothelial-dependent relaxation (EDR) and sodium nitroprusside (SNP)-induced endothelial-independent relaxation (EIR) were examined. Our results showed that the myogenic responses were significantly impaired in both the CA and MCA of SIRT3ECKO mice. Furthermore, female mice had worsened myogenic response in MCA. In CA, EDR was abolished in both male and female SIRT3ECKO mice. Intriguingly, EIR was only reduced in the female mice. In MCA, EDR was reduced in male SIRT3ECKO mice, whereas EIR was decreased in both male and female mice. Female SIRT3ECKO mice had profound dysfunction in CA, whereas male mice exhibited more dysfunction in MCA. These data revealed a sex and organ-specific role of endothelial SIRT3 in vascular function and myogenic responses. Our study suggests that endothelial SIRT3 is necessary for maintaining vascular function and blood flow autoregulation.

Sirtuin 3 reinforces acylcarnitine metabolism and maintains thermogenesis in brown adipose tissue of aging mice.

Acylcarnitine (ACar) is a novel fuel source for activating thermogenesis in brown adipose tissue (BAT). However, whether ACar metabolism underlies BAT thermogenesis decline with aging remain unclear. Here, the L-carnitine-treated young and aging mice were used to investigate the effects of activation of ACar metabolism on BAT thermogenesis during aging. We showed that long term L-carnitine feeding, which results in an elevation in circulating ACar levels, failed to improve cold sensitivity of aging mice, which still displayed impaired thermogenesis and ACar metabolism in interscapular BAT (iBAT). The RNA-sequencing was used to identify the key regulator for the response of aging mice to LCar induced activation of ACar metabolism in BAT, and we identified Sirt3 as a key regulator for the response of aging mice to L-carnitine induced activation of ACar metabolism in iBAT. Then the adipose-specific Sirt3 knockout (Sirt3 AKO) mice were used to investigate the role of Sirt3 in ACar metabolism and thermogenesis of BAT and explore the underlying mechanism, and the results showed that Sirt3 AKO mice displayed defective ACar metabolism and thermogenesis in iBAT. Mechanically, Sirt3 regulated ACar metabolism via HIF1α-PPARα signaling pathway to promote iBAT thermogenesis, and knockdown or inhibition of HIF1α ameliorated impaired ACar metabolism and thermogenesis of iBAT in the absence of Sirt3. Collectively, we propose that Sirt3 regulated ACar metabolism is critical in maintaining thermogenesis in BAT of aging mice, which can promote the development of anti-aging intervention strategy.

Abstract Tu087: Knockout of TIGAR Rescues Cardiac Dysfunction in SIRT3 Knockout Mice

Introduction: Heart failure (HF) is the leading cause of death in the United States, despite advances in treatment, it maintains the highest disease mortality rate in the country. Previously it has been established that Sirtuin 3 (SIRT3), a mitochondrial NAD+- dependent deacetylase, and the Tp53-induced glycolysis and apoptosis regulator (TIGAR) are associated with the development of HF. SIRT3 plays a key role in the maintenance of cardiac function. Decreases in SIRT3 are associated with human aging and diabetes, whereas knockout results in cardiac dysfunction in mice. We have shown: (1) overexpression of SIRT3 downregulates TIGAR and attenuates diabetic cardiomyopathy; (2) knockout of TIGAR enhances myocardial glycolysis and reduces pathological remodeling while maintaining cardiac function in pressure-overload-induced heart failure. To date, the direct interactions between TIGAR and SIRT3 in the development of HF remain unexplored. Aim: By crossing TIGAR knockout mice (TIGARKO) with SIRT3 knockout (SIRT3KO) mice, this study tested if genetic targeting of TIGAR could rescue cardiac dysfunction in SIRT3KO mice. Methods and Results: Echocardiography was performed on SIRT3KO, TIGARKO, TIGARKO/SIRT3KO, and SIRT3 Control mice. Analysis of cardiac function showed that SIRT3KO mice had significant decreases in coronary flow reserve (CFR), ejection fraction (EF) SIRT3KO (41.52 ± 4.50) vs SIRT3 Control (60.91 ± 4.91, p<0.001) and fractional shortening (FS) SIRT3KO (20.11 ± 2.48) vs SIRT3 Control (32.07 ± 3.37, p<0.001). The diastolic E’/A’ ratio was decreased; IVRT and MPI were significantly increased. Knockout of TIGAR in mice maintained systolic function by increasing EF (62.25 ± 9.84) vs (41.52 ± 4.50, p<0.001), FS (33.33 ± 7.12 vs 20.11 ± 2.48, p<0.05), and CFR, as well as improved diastolic function by decreasing IVRT, MPI, and increasing E’/A’ ratio in SIRT3KO mice. SIRT3KO and TIGARKO mice had a significant increase in weight/tibia length ratio (HW/TL). Interestingly, the knockout of TIGAR significantly reduced LV mass and HW/TL in SIRT3KO mice. Conclusions: The knockout of TIGAR can rescue the HF phenotype of SIRT3KO mice, but the underlying mechanisms require further investigation to develop potential treatments for HF.

Sex-Related Differences in Oxidant and Antioxidant Profiles of Murine Kidney and Brain: A Focus on Sirt3-Mediated Regulation

Background: Sirt3 is a mitochondrial deacetylase with an important role in maintainance of cellular redox and metabolic homeostasis and mitochondrial function. As growing evidence support the existence of sex-specific responses to metabolic and oxidative stress, we aimed to investigate sex- and organ-specific effects of Sirt3 loss. Materials and methods: Expression of Sirt3, PGC-1a, CuZnSOD, MnSOD and Cat proteins in kidneys and brains of Sirt3-wild type (Sirt3 WT) and Sirt3-knockout (Sirt3 KO) mice was assessed by Western blotting. Protein carbonylation and lipid peroxidation levels were measured using ELISA and fluorometric assays, respectively. SOD and Cat activities were determined using standard enzymatic assays. Results: Significant sex- and organ- specific differences in response to Sirt3 loss were detected. Sirt3 knockout affected kidneys more than brain tissue, with females showing lower levels PC and LPO. In kidneys, female KO showed higher MnSOD, but lower CuZnSOD and Cat activity compared to males. In brains, WT females show higher activities of these enzymes than males, suggesting a sex-specific protection mechanism, but female KO brains show a larger decrease in these parameters. Conclusion: Our study provides comprehensive insights into the complex interplay of Sirt3, oxidative stress, and antioxidant defenses in murine kidney and brain. The observed differences between the two organs and the impact of sex highlight the need for studying Sirt3 function in diverse physiological contexts. The tissue-specific responses and sex-related variations underscore the importance of considering these factors in the development of therapeutic strategies targeting mitochondrial function and redox homeostasis.

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

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. 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. 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. 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.

FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

Mitochondrial dysfunction and oxidative stress are important mechanisms for secondary injury after traumatic brain injury (TBI), which result in progressive pathophysiological exacerbation. Although the Fibronectin type III domain-containing 5 (FNDC5) was reported to repress oxidative stress by retaining mitochondrial biogenesis and dynamics, its possible role in the secondary injury after TBI remain obscure. In present study, we observed that the level of serum irisin (the cleavage product of FNDC5) significantly correlated with the neurological outcomes of TBI patients. Knockout of FNDC5 increased the lesion volume and exacerbated apoptosis and neurological deficits after TBI in mice, while FNDC5 overexpression yielded a neuroprotective effect. Moreover, FNDC5 deficiency disrupted mitochondrial dynamics and function. Activation of Sirtuin 3 (SIRT3) alleviated FNDC5 deficiency-induced disruption of mitochondrial dynamics and bioenergetics. In neuron-specific SIRT3 knockout mice, FNDC5 failed to attenuate TBI-induced mitochondrial damage and brain injuries. Mechanically, FNDC5 deficiency led to reduced SIRT3 expression via enhanced ubiquitin degradation of transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), which contributed to the hyperacetylation and inactivation of key regulatory proteins of mitochondrial dynamics and function, including OPA1 and SOD2. Finally, engineered RVG29-conjugated nanoparticles were generated to selectively and efficiently deliver irisin to the brain of mice, which yielded a satisfactory curative effect against TBI. In conclusion, FNDC5/irisin exerts a protective role against acute brain injury by promoting SIRT3-dependent mitochondrial quality control and thus represents a potential target for neuroprotection after TBI.

2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis

Sirtuin 3 (SIRT3) is well known as a conserved nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase located in the mitochondria that may regulate oxidative stress, catabolism and ATP production. Accumulating evidence has recently revealed that SIRT3 plays its critical roles in cardiac fibrosis, myocardial fibrosis and even heart failure (HF), through its deacetylation modifications. Accordingly, discovery of SIRT3 activators and elucidating their underlying mechanisms of HF should be urgently needed. Herein, we identified a new small-molecule activator of SIRT3 (named 2-APQC) by the structure-based drug designing strategy. 2-APQC was shown to alleviate isoproterenol (ISO)-induced cardiac hypertrophy and myocardial fibrosis in vitro and in vivo rat models. Importantly, in SIRT3 knockout mice, 2-APQC could not relieve HF, suggesting that 2-APQC is dependent on SIRT3 for its protective role. Mechanically, 2-APQC was found to inhibit the mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K), c-jun N-terminal kinase (JNK) and transforming growth factor-β (TGF-β)/ small mother against decapentaplegic 3 (Smad3) pathways to improve ISO-induced cardiac hypertrophy and myocardial fibrosis. Based upon RNA-seq analyses, we demonstrated that SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) axis was closely assoiated with HF. By activating PYCR1, 2-APQC was shown to enhance mitochondrial proline metabolism, inhibited reactive oxygen species (ROS)-p38 mitogen activated protein kinase (p38MAPK) pathway and thereby protecting against ISO-induced mitochondrialoxidative damage. Moreover, activation of SIRT3 by 2-APQC could facilitate AMP-activated protein kinase (AMPK)-Parkin axis to inhibit ISO-induced necrosis. Together, our results demonstrate that 2-APQC is a targeted SIRT3 activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis, which may provide a new clue on exploiting a promising drug candidate for the future HF therapeutics.

Fine particulate matter (PM2.5) induces testosterone disruption by triggering ferroptosis through SIRT1/HIF-1α signaling pathway in male mice

Fine particulate matter (PM2.5), a significant component of air pollution particulate matter, is inevitable and closely associated with increasing male reproductive disorder. However, the testicular targets of PM2.5 and its toxicity related molecular mechanisms are still not fully understood. In this study, the conditional knockout (cKO) mice and primary Leydig cells were used to explore the testicular targets of PM2.5 and the related underlying mechanisms. First, apparent the structure impairment of seminiferous tubules, Leydig cells vacuolization, decline of serum testosterone and sperm quality reduction were found in male wild-type (WT) and Sirt1 knockout mice after exposure to PM2.5. Enrichment analyses revealed that differentially expressed genes (DEGs) were enriched in steroid hormone biosynthesis, ferroptosis, and HIF-1 signaling pathway in the mice testes after exposure to PM2.5, which were subsequently verified by the molecular biological analyses. Notably, similar enrichment analyses results were also observed in primary Leydig cells after treatment with PM2.5. In addition, Knockdown of Sirt1 significantly increased PM2.5-induced expression and activation of HIF-1α, which was in parallel to the changes of cellular iron levels, oxidative stress indicators and the ferroptosis markers. In conclusion, this highlights that PM2.5 triggers ferroptosis via SIRT1/HIF-1α signaling pathway to inhibit testosterone synthesis in males. These findings provide a novel research support for the study that PM2.5 causes male reproductive injury.

Mitochondrial SIRT3 as a protective factor against cyclosporine A-induced nephrotoxicity

Sirtuin3 (SIRT3), a mitochondrial deacetylase, has been shown to be involved in various kidney diseases. In this study, we aimed to clarify the role of SIRT3 in cyclosporine-induced nephrotoxicity and the associated mitochondrial dysfunction. Madin-Darby canine kidney (MDCK) cells were transfected with Flag-tagged SIRT3 for SIRT3 overexpression or SIRT3 siRNA for the inhibition of SIRT3. Subsequently, the cells were treated with cyclosporine A (CsA) or vehicle. Wild-type and SIRT3 knockout (KO) mice were randomly assigned to receive cyclosporine A or olive oil. Furthermore, SIRT3 activator, honokiol, was treated alongside CsA to wild type mice. Our results revealed that CsA treatment inhibited mitochondrial SIRT3 expression in MDCK cells. Inhibition of SIRT3 through siRNA transfection exacerbated apoptosis, impaired the expression of the AMP-activated protein kinase-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (AMPK-PGC1α) pathway, and worsened mitochondrial dysfunction induced by CsA treatment. Conversely, overexpression of SIRT3 through Flag-tagged SIRT3 transfection ameliorated apoptosis, increased the expression of mitochondrial superoxide dismutase 2, and restored the mitochondrial regulator pathway, AMPK-PGC1α. In SIRT3 KO mice, CsA treatment led to aggravated kidney dysfunction, increased kidney tubular injury, and accumulation of oxidative end products indicative of oxidative stress injury. Meanwhile, SIRT3 activation in vivo significantly mitigated these adverse effects, improving kidney function, reducing oxidative stress markers, and enhancing mitochondrial health following CsA treatment. Overall, our findings suggest that SIRT3 plays a protective role in alleviating mitochondrial dysfunction caused by CsA through the activation of the AMPK-PGC1α pathway, thereby preventing further kidney injury.

Hepatic Sirt6 activation abrogates acute liver failure.

Acute liver failure (ALF) is a deadly illness due to insufficient detoxification in liver induced by drugs, toxins, and other etiologies, and the effective treatment for ALF is very limited. Among the drug-induced ALF, acetaminophen (APAP) overdose is the most common cause. However, the molecular mechanisms underlying APAP hepatoxicity remain incompletely understood. Sirtuin 6 (Sirt6) is a stress responsive protein deacetylase and plays an important role in regulation of DNA repair, genomic stability, oxidative stress, and inflammation. Here, we report that genetic and pharmacological activation of Sirt6 protects against ALF in mice. We first observed that Sirt6 expression was significantly reduced in the liver tissues of human patients with ALF and mice treated with an overdose of APAP. Then we developed an inducible Sirt6 transgenic mice for Cre-mediated overexpression of the human Sirt6 gene in systemic (Sirt6-Tg) and hepatic-specific (Sirt6-HepTg) manners. Both Sirt6-Tg mice and Sirt6-HepTg mice exhibited the significant protection against APAP hepatoxicity. In contrast, hepatic-specific Sirt6 knockout mice exaggerated APAP-induced liver damages. Mechanistically, Sirt6 attenuated APAP-induced hepatocyte necrosis and apoptosis through downregulation of oxidative stress, inflammation, the stress-activated kinase JNK activation, and apoptotic caspase activation. Moreover, Sirt6 negatively modulated the level and activity of poly (ADP-ribose) polymerase 1 (PARP1) in APAP-treated mouse liver tissues. Importantly, the specific Sirt6 activator MDL-800 exhibited better therapeutic potential for APAP hepatoxicity than the current drug acetylcysteine. Furthermore, in the model of bile duct ligation induced ALF, hepatic Sirt6-KO exacerbated, but Sirt6-HepTg mitigated liver damage. Collectively, our results demonstrate that Sirt6 protects against ALF and suggest that targeting Sirt6 activation could be a new therapeutic strategy to alleviate ALF.

SIRT3 regulates cardiolipin biosynthesis in pressure overload-induced cardiac remodeling by PPARγ-mediated mechanism.

Cardiac remodeling is the primary pathological feature of chronic heart failure (HF). Exploring the characteristics of cardiac remodeling in the very early stages of HF and identifying targets for intervention are essential for discovering novel mechanisms and therapeutic strategies. Silent mating type information regulation 2 homolog 3 (SIRT3), as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolism. However, whether SIRT3 plays a role in cardiac remodeling by regulating the biosynthesis of mitochondrial cardiolipin (CL) is unknown. In this study, we induced pressure overload in wild-type (WT) and SIRT3 knockout (SIRT3-/-) mice via transverse aortic constriction (TAC). Compared with WT mouse hearts, the hearts of SIRT3-/- mice exhibited more-pronounced cardiac remodeling and fibrosis, greater reactive oxygen species (ROS) production, decreased mitochondrial-membrane potential (ΔΨm), and abnormal mitochondrial morphology after TAC. Furthermore, SIRT3 deletion aggravated TAC-induced decrease in total CL content, which might be associated with the downregulation of the CL synthesis related enzymes cardiolipin synthase 1 (CRLS1) and phospholipid-lysophospholipid transacylase (TAFAZZIN). In our in vitro experiments, SIRT3 overexpression prevented angiotensin II (AngII)- induced aberrant mitochondrial function, CL biosynthesis disorder, and peroxisome proliferator-activated receptor gamma (PPARγ) downregulation in cardiomyocytes; meanwhile, SIRT3 knockdown exacerbated these effects. Moreover, the addition of GW9662, a PPARγ antagonist, partially counteracted the beneficial effects of SIRT3 overexpression. In conclusion, SIRT3 regulated PPARγ-mediated CL biosynthesis, maintained the structure and function of mitochondria, and thereby protected the myocardium against cardiac remodeling.

The SP1/SIRT1/ACLY signaling axis mediates fatty acid oxidation in renal ischemia–reperfusion-induced renal fibrosis

BackgroundRenal ischemia–reperfusion is the primary cause of acute kidney injury (AKI). Clinically, most patients who experience ischemia–reperfusion injury eventually progress gradually to renal fibrosis and chronic kidney disease (CKD). However, the underlying mechanism for AKI to CKD transition remain absent. Our study demonstrated that the downregulation of sirtuin 1 (Sirt1)-mediated fatty acid oxidation (FAO) facilitates IRI-induced renal fibrosis. MethodsThe IRI animal model was established, and ribonucleic acid (RNA) sequencing was used to explore potential differentially expressed genes (DEGs) and pathways. The SIRT1 knockout mice were generated, and a recombinant adeno-associated virus that overexpresses SIRT1 was injected into mice to explore the function of SIRT1 in renal fibrosis induced by renal IRI. In vitro, hypoxia/reoxygenation (H/R) was used to establish the classical model of renal IRI and overexpression or knockdown of SIRT1 to investigate the SIRT1 function through lentiviral plasmids. The underlying molecular mechanism was explored through RNA sequencing, bioinformatics analysis, and chromatin immunoprecipitation assay. ResultsRNA sequencing analysis and western blot demonstrated that the expression of SIRT1 was significantly decreased in IRI mice. Overexpression of SIRT1 improved renal function and reduced lipid deposition and renal fibrosis. On the contrary, knockout of SIRT1 aggravated kidney injury and renal fibrosis. RNA sequencing, bioinformatics analysis, and chromatin immunoprecipitation assay mechanistically revealed that SIRT1 impairs the acetylation of histone H3K27 on the promoter region of ACLY, thereby impeding FAO activity and promoting renal fibrosis. Additionally, SP1 regulated FAO by directly modulating SIRT1 expression. ConclusionOur findings highlight that downregulation of SIRT1-modulated FAO facilitated by the SP1/SIRT1/ACLY axis in the kidney increases IRI, suggesting SIRT1 to be a potential therapeutic target for renal fibrosis induced by renal IRI.

SIRT1 Coordinates Transcriptional Regulation of Neural Activity and Modulates Depression-Like Behaviors in the Nucleus Accumbens

BackgroundMajor depression and anxiety disorders are significant causes of disability and socioeconomic burden. Despite the prevalence and considerable impact of these affective disorders, their pathophysiology remains elusive. Thus, there is an urgent need to develop novel therapeutics for these conditions. We evaluated the role of SIRT1 in regulating dysfunctional processes of reward by using chronic social defeat stress to induce depression- and anxiety-like behaviors. Chronic social defeat stress induces physiological and behavioral changes that recapitulate depression-like symptomatology and alters gene expression programs in the nucleus accumbens, but cell type–specific changes in this critical structure remain largely unknown. MethodsWe examined transcriptional profiles of D1-expressing medium spiny neurons (MSNs) lacking deacetylase activity of SIRT1 by RNA sequencing in a cell type–specific manner using the RiboTag line of mice. We analyzed differentially expressed genes using gene ontology tools including SynGO and EnrichR and further demonstrated functional changes in D1-MSN–specific SIRT1 knockout (KO) mice using electrophysiological and behavioral measurements. ResultsRNA sequencing revealed altered transcriptional profiles of D1-MSNs lacking functional SIRT1 and showed specific changes in synaptic genes including glutamatergic and GABAergic (gamma-aminobutyric acidergic) receptors in D1-MSNs. These molecular changes may be associated with decreased excitatory and increased inhibitory neural activity in Sirt1 KO D1-MSNs, accompanied by morphological changes. Moreover, the D1-MSN–specific Sirt1 KO mice exhibited proresilient changes in anxiety- and depression-like behaviors. ConclusionsSIRT1 coordinates excitatory and inhibitory synaptic genes to regulate the GABAergic output tone of D1-MSNs. These findings reveal a novel signaling pathway that has potential for the development of innovative treatments for affective disorders.

5-Heptadecylresorcinol Improves Aging-Associated Hepatic Fatty Acid Oxidation Dysfunction via Regulating Adipose Sirtuin 3.

Aging-associated hepatic fatty acid (FA) oxidation dysfunction contributes to impaired adaptive thermogenesis. 5-Heptadecylresorcinol (AR-C17) is a prominent functional component of whole wheat and rye, and has been demonstrated to improve the thermogenic capacity of aged mice via the regulation of Sirt3. However, the effect of AR-C17 on aging-associated hepatic FA oxidation dysfunction remains unclear. Here, 18-month-old C57BL/6J mice were orally administered with AR-C17 at a dose of 150 mg/kg/day for 8 weeks. Systemic glucose and lipid metabolism, hepatic FA oxidation, and the lipolysis of white adipose tissues (WAT) were measured. The results showed that AR-C17 improved the hepatic FA oxidation, and especially acylcarnitine metabolism, of aged mice during cold stimulation, with the enhancement of systemic glucose and lipid metabolism. Meanwhile, AR-C17 improved the WAT lipolysis of aged mice, promoting hepatic acylcarnitine production. Furthermore, the adipose-specific Sirt3 knockout mice were used to investigate and verify the regulation mechanism of AR-C17 on aging-associated hepatic FA oxidation dysfunction. The results showed that AR-C17 failed to improve the WAT lipolysis and hepatic FA oxidation of aged mice in the absence of adipose Sirt3, indicating that AR-C17 might indirectly influence hepatic FA oxidation via regulating WAT Sirt3. Our findings suggest that AR-C17 might improve aging-associated hepatic FA oxidation dysfunction via regulating adipose Sirt3.

P53 Acetylation Exerts Critical Roles in Pressure Overload-Induced Coronary Microvascular Dysfunction and Heart Failure in Mice.

Coronary microvascular dysfunction (CMD) has been shown to contribute to cardiac hypertrophy and heart failure (HF) with preserved ejection fraction. At this point, there are no proven treatments for CMD. We have shown that histone acetylation may play a critical role in the regulation of CMD. By using a mouse model that replaces lysine with arginine at residues K98, K117, K161, and K162R of p53 (p534KR), preventing acetylation at these sites, we test the hypothesis that acetylation-deficient p534KR could improve CMD and prevent the progression of hypertensive cardiac hypertrophy and HF. Wild-type and p534KR mice were subjected to pressure overload by transverse aortic constriction to induce cardiac hypertrophy and HF. Echocardiography measurements revealed improved cardiac function together with a reduction of apoptosis and fibrosis in p534KR mice. Importantly, myocardial capillary density and coronary flow reserve were significantly improved in p534KR mice. Moreover, p534KR upregulated the expression of cardiac glycolytic enzymes and Gluts (glucose transporters), as well as the level of fructose-2,6-biphosphate; increased PFK-1 (phosphofructokinase 1) activity; and attenuated cardiac hypertrophy. These changes were accompanied by increased expression of HIF-1α (hypoxia-inducible factor-1α) and proangiogenic growth factors. Additionally, the levels of SERCA-2 were significantly upregulated in sham p534KR mice, as well as in p534KR mice after transverse aortic constriction. In vitro, p534KR significantly improved endothelial cell glycolytic function and mitochondrial respiration and enhanced endothelial cell proliferation and angiogenesis. Similarly, acetylation-deficient p534KR significantly improved coronary flow reserve and rescued cardiac dysfunction in SIRT3 (sirtuin 3) knockout mice. Our data reveal the importance of p53 acetylation in coronary microvascular function, cardiac function, and remodeling and may provide a promising approach to improve hypertension-induced CMD and to prevent the transition of cardiac hypertrophy to HF.

The SIRT3-ATAD3A axis regulates MAM dynamics and mitochondrial calcium homeostasis in cardiac hypertrophy.

Mitochondria are energy-producing organelles that are mobile and harbor dynamic network structures. Although mitochondria and endoplasmic reticulum (ER) play distinct cellular roles, they are physically connected to maintain functional homeostasis. Abnormal changes in this interaction have been linked to pathological states, including cardiac hypertrophy. However, the exact regulatory molecules and mechanisms are yet to be elucidated. Here, we report that ATPase family AAA-domain containing protein 3A (ATAD3A) is an essential regulator of ER-mitochondria interplay within the mitochondria-associated membrane (MAM). ATAD3A prevents isoproterenol (ISO)-induced mitochondrial calcium accumulation, improving mitochondrial dysfunction and ER stress, which preserves cardiac function and attenuates cardiac hypertrophy. We also find that ATAD3A is a new substrate of NAD+-dependent deacetylase Sirtuin 3 (SIRT3). Notably, the heart mitochondria of SIRT3 knockout mice exhibited excessive formation of MAMs. Mechanistically, ATAD3A specifically undergoes acetylation, which reduces self-oligomerization and promotes cardiac hypertrophy. ATAD3A oligomerization is disrupted by acetylation at K134 site, and ATAD3A monomer closely interacts with the IP3R1-GRP75-VDAC1 complex, which leads to mitochondrial calcium overload and dysfunction. In summary, ATAD3A localizes to the MAMs, where it protects the homeostasis of ER-mitochondria contacts, quenching mitochondrial calcium overload and keeping mitochondrial bioenergetics unresponsive to ER stress. The SIRT3-ATAD3A axis represents a potential therapeutic target for cardiac hypertrophy.

N-acetylcysteine protects septic acute kidney injury by inhibiting SIRT3-mediated mitochondrial dysfunction and apoptosis.

To investigate the protective effect of N-acetylcysteine (NAC) on septic acute kidney injury (SAKI) via regulating Sirtuin3 (SIRT3)-mediated mitochondrial dysfunction and apoptosis. By constructing SIRT3 knockout mice and culturing kidney tubular epithelial cells (KTECs), we assessed the changes of renal function and detected the protein expression of adenine nucleotide translocator (ANT), cyclophilin (CypD) and voltage-dependent anion channel (VDAC) using western-blotting, and simultaneously detected toll-like receptor 4 (TLR4), inhibitor of kappa B kinase (IKKβ), inhibitor of Kappa Bα (IκBα), and p65 protein expression. We observed mitochondrial damage of KTECs using a transmission electron microscope and assessed apoptosis by TdT-mediated dUTP Nick-End Labeling and flow cytometry. SIRT3 deficiency led to the deterioration of renal function, and caused a significant increase in inducible nitric oxide synthase production, a decrease in mitochondrial volume, up-regulation of TLR4, IκBα, IKKβ, and p65 proteins, and up-regulation of ANT, CypD and VDAC proteins. However, NAC significantly improved renal function and down-regulated the expression of TLR4, IκBα, IKKβ, and p65 proteins. Furthermore, SIRT3 deficiency led to a significant increase in KTEC apoptosis, while NAC up-regulated the expression of SIRT3 and inhibited apoptosis. NAC has a significant protective effect on SAKI by inhibiting SIRT3-mediated mitochondrial dysfunction and apoptosis of KTECs.

Endogenous SIRT6 in platelets negatively regulates platelet activation and thrombosis.

Thromboembolism resulting from platelet dysfunction constitutes a significant contributor to the development of cardiovascular disease. Sirtuin 6 (SIRT6), an essential NAD+-dependent enzyme, has been linked to arterial thrombosis when absent in endothelial cells. In the present study, we have confirmed the presence of SIRT6 protein in anucleated platelets. However, the precise regulatory role of platelet endogenous SIRT6 in platelet activation and thrombotic processes has remained uncertain. Herein, we present compelling evidence demonstrating that platelets isolated from SIRT6-knockout mice (SIRT6-/-) exhibit a notable augmentation in thrombin-induced platelet activation, aggregation, and clot retraction. In contrast, activation of SIRT6 through specific agonist treatment (UBCS039) confers a pronounced protective effect on platelet activation and arterial thrombosis. Moreover, in platelet adoptive transfer experiments between wild-type (WT) and SIRT6-/- mice, the loss of SIRT6 in platelets significantly prolongs the mean thrombus occlusion time in a FeCl3-induced arterial thrombosis mouse model. Mechanistically, we have identified that SIRT6 deficiency in platelets leads to the enhanced expression and release of proprotein convertase subtilisin/kexin type 9 (PCSK9), subsequently activating the platelet activation-associated mitogen-activated protein kinase (MAPK) signaling pathway. These findings collectively unveil a novel protective role of platelet endogenous SIRT6 in platelet activation and thrombosis. This protective effect is, at least in part, attributed to the inhibition of platelet PCSK9 secretion and mitogen-activated protein kinase signaling transduction. Our study provides valuable insights into the intricate interplay between SIRT6 and platelet function, shedding light on potential therapeutic avenues for managing thrombotic disorders.

FGF21 alleviates endothelial mitochondrial damage and prevents BBB from disruption after intracranial hemorrhage through a mechanism involving SIRT6

BackgroundDisruption of the BBB is a harmful event after intracranial hemorrhage (ICH), and this disruption contributes to a series of secondary injuries. We hypothesized that FGF21 may have protective effects after intracranial hemorrhage (ICH) and investigated possible underlying molecular mechanisms.MethodsBlood samples of ICH patients were collected to determine the relationship between the serum level of FGF21 and the Δ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta$$\\end{document}GCS%. Wild-type mice, SIRT6flox/flox mice, endothelial-specific SIRT6-homozygous-knockout mice (eSIRT6−/− mice) and cultured human brain microvascular endothelial cells (HCMECs) were used to determine the protective effects of FGF21 on the BBB.ResultsWe obtained original clinical evidence from patient data identifying a positive correlation between the serum level of FGF21 and Δ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta$$\\end{document}GCS%. In mice, we found that FGF21 treatment is capable of alleviating BBB damage, mitigating brain edema, reducing lesion volume and improving neurofunction after ICH. In vitro, after oxyhemoglobin injury, we further explored the protective effects of FGF21 on endothelial cells (ECs), which are a significant component of the BBB. Mitochondria play crucial roles during various types of stress reactions. FGF21 significantly improved mitochondrial biology and function in ECs, as evidenced by alleviated mitochondrial morphology damage, reduced ROS accumulation, and restored ATP production. Moreover, we found that the crucial regulatory mitochondrial factor deacylase sirtuin 6 (SIRT6) played an irreplaceable role in the effects of FGF21. Using endothelial-specific SIRT6-knockout mice, we found that SIRT6 deficiency largely diminished these neuroprotective effects of FGF21. Then, we revealed that FGF21 might promote the expression of SIRT6 via the AMPK–Foxo3a pathway.ConclusionsWe provide the first evidence that FGF21 is capable of protecting the BBB after ICH by improving SIRT6-mediated mitochondrial homeostasis.

Exogenous NADPH exerts a positive inotropic effect and enhances energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure

Therapies are urgently required to ameliorate pathological cardiac hypertrophy and enhance cardiac function in heart failure. Our preliminary experiments have demonstrated that exogenous NADPH exhibits a positive inotropic effect on isolated heart. This study aims to investigate the positive inotropic effects of NADPH in pathological cardiac hypertrophy and heart failure, as well as the underlying mechanisms involved. Endogenous plasma NADPH contents were determined in patients with chronic heart failure and control adults. The positive inotropic effects of NADPH were investigated in isolated toad heart or rat heart. The effects of NADPH were investigated in isoproterenol (ISO)-induced cardiac hypertrophy or transverse aortic constriction (TAC)-induced heart failure. The underlying mechanisms of NADPH were studied using SIRT3 knockout mice, echocardiography, Western blotting, transmission electron microscopy, and immunoprecipitation. The endogenous NADPH content in the blood of patients and animals with pathological cardiac hypertrophy or heart failure was significantly reduced compared with age-sex matched control subjects. Exogenous NADPH showed positive inotropic effects on the isolated normal and failing hearts, while antagonism of ATP receptor partially abolished the positive inotropic effect of NADPH. Exogenous NADPH administration significantly reduced heart weight indices, and improved cardiac function in the mice with pathological cardiac hypertrophy or heart failure. NADPH increased SIRT3 expression and activity, deacetylated target proteins, improved mitochondrial function and facilitated ATP production in the hypertrophic myocardium. Importantly, inhibition of SIRT3 abolished the positive inotropic effect of NADPH, and the anti-heart failure effect of NADPH was significantly reduced in the SIRT3 Knockout mice. Exogenous NADPH shows positive inotropic effect and improves energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure. NADPH thus may be one of the potential candidates for the treatment of pathological cardiac hypertrophy or heart failure. This work was supported by grants from the National Natural Science Foundation of China (No. 81973315, 82173811, 81730092), Natural Science Foundation of Jiangsu Higher Education (20KJA310008), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).