Resveratrol‐enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol‐induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol‐mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long‐term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol‐induced down‐regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H₂O₂ increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1‐dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.     

Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.     

Role of SIRT1 in Streptococcus pneumoniae-induced human β-defensin-2 and interleukin-8 expression in A549 cell

Streptococcus pneumoniae is an important pathogen of pneumonia in human. Human alveolar epithelium acts as an effective barrier and is an active participant in host defense against invasion of bacterial by production of various mediators. Sirtuin 1 (SIRT1), the prototypic class III histone deacetylase, is involved in the molecular control of lifespans and immune responses. This study aimed at examining the role of SIRT1 in mediating S. pneumoniae-induced human β-defensin-2 (hBD2) and interleukin-8(IL-8) expression in the alveolar epithelial cell line A549 and the underlying mechanisms involved. A549 cells were infected with S. pneumoniae for indicated times. Exposure of A549 cells to S. pneumoniae increased the expressions of SIRT1 protein, hBD2 and IL-8 mRNA, and protein. The SIRT1 activator resveratrol enhanced S. pneumoniae-induced gene expression of hBD2 but decreased IL-8 mRNA levels. Blockade of SIRT1 activity by the SIRT1 inhibitors nicotinamide reduced S. pneumoniae-induced hBD2 mRNA expression but increased its stimulatory effects on IL-8 mRNA. S. pneumoniae-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SIRT1 expression was attenuated by selective inhibitors of ERK and p38 MAPK. The hBD2 mRNA production was decreased by pretreatment with p38 MAPK inhibitor but not with ERK inhibitor, whereas the IL-8 mRNA expression was controlled by phosphorylation of ERK. These results suggest that SIRT1 mediates the induction of hBD2 and IL-8 gene expression levels in A549 cell by S. pneumoniae. SIRT1 may play a key role in host immune and defense response in A549.     

Resveratrol Upregulates Cardiac SDF-1 in Mice with Acute Myocardial Infarction through the Deacetylation of Cardiac p53

Aims

We previously demonstrated that resveratrol (RSV) administration causes cardiac stromal cell-derived factor (SDF)-1 upregulation and can enhance the mobilization of stem cells in mice with acute myocardial infarction (AMI). However, the upstream signal transduction involved in SDF-1 regulation in the setting of AMI and RSV administration remains unclear. Because RSV is a sirtuin 1 (SIRT1) activator and SIRT proteins act as deacetylases, we investigated the role of SIRT1 in SDF-1 upregulation and its subsequent effects.

Methods and Results

In vitro experiments with H9C2 cardiomyocytes under hypoxia and serum-deprivation conditions showed that p53 acted upstream of SDF-1. RSV could not regulate SDF-1 effectively after SIRT1 silencing, indicating that it is dependent on SIRT1. Subsequently, male C57BL/6 mice were divided into four groups: 1) sham, 2) MI, 3) MI+RSV, and 4) MI+RSV plus nicotinamide, an inhibitor of the deacetylase activity of SIRT (MI+RSV+NAM). Compared with the sham mice, AMI caused a slight increase in the cardiac p53 level and resulted in significant SIRT1 downregulation and p53 acetylation or activation. Compared with the MI mice, MI+RSV administration improved the cardiac SDF-1 level and reversed the reduction of SIRT1 and the activation of p53. Furthermore, we observed less cardiac dysfunction in MI+RSV mice and determined that NAM abolished the effects of RSV.

Conclusions

RSV enhances cardiac SDF-1 excretion after AMI partially through a SIRT1 normalization/p53 inactivation pathway.     

Tropisetron restores normal expression of BAD,SIRT1, SIRT3, and SIRT7 in the rat pressure overload-induced cardiac hypertrophy model

Tropisetron exerts a protective effect against cardiac complications, particularly cardiac hypertrophy. Oxidative stress and apoptosis are the main contributors to the pathogenesis of cardiac hypertrophy. Sirtuins, a family of histone deacetylases, are connected to cellular oxidative stress signaling and antioxidant defense. Sirtuins are also linked to apoptosis which is an important mechanism in the progression of cardiac hypertrophy to heart failure. Literature also suggests that tropisetron impedes apoptosis, partly mediated through an antioxidant mechanism. Therefore, we examined if tropisetron fights cardiac hypertrophy by adjusting sirtuin family proteins (Sirts) and components of mitochondrial death pathway, Bcl-associated X (BAX), Bcl-2-associated death promoter (BAD). Male Sprague–Dawley rats got divided into four groups, including control (Ctl), tropisetron (Trop), cardiac hypertrophy (Hyp), and hypertrophic rats under tropisetron treatment (Hyp + Trop). Pathological cardiac hypertrophy was induced by surgical abdominal aortic constriction (AAC). The increased expression of brain natriuretic peptide (BNP) in the Hyp group confirms the cardiac hypertrophy establishment. The mRNA levels of SIRT1, SIRT3, SIRT7, and BAD also upregulated in the hypertrophic group (p < 0.001). Postoperational administration of tropisetron for 3 weeks lowered the increased expression of BNP (p < 0.05) and BAD (p < 0.001), though the reduction of BAX expression was statistically insignificant (p > 0.05). Tropisetron treatment also restored the normal level of SIRT1/3/7 genes expression in the Hyp + Trop group (p < 0.05). Present findings suggest that tropisetron can suppress cardiomyocyte hypertrophy progression to heart failure by counteracting BNP, SIRT1, SIRT3, Sirt7, and BAD overexpression-mediated apoptosis in a rat model of cardiac hypertrophy.     

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

The controversial links among calorie restriction, SIRT1, and resveratrol

It has been widely known that slow metabolism induced by calorie restriction (CR) can extend the life span of model organisms though the underlying mechanism remains poorly understood. Accumulated evidence suggests that SIRT1 may be actively involved in CR-induced signaling pathways. As a putative activator of SIRT1, resveratrol, known for the French paradox, can partially mimic the physiological effects of CR. While the deacetylase activity of SIRT1 is important for the beneficial effects of resveratrol, resveratrol-induced SIRT1 activation has recently been challenged by the observations that resveratrol could not induce SIRT1-mediated deacetylation of native substrates in vitro. To resolve the discrepancy of resveratrol-induced activation of SIRT1 deacetylase activity between the in vitro and in vivo assays, a model of indirect SIRT1 activation by resveratrol is proposed. In this review, we will discuss the emerging roles of SIRT1 and resveratrol in CR and focus on debate over the links between SIRT1 and resveratrol.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

Reduction in mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised compared with sedentary mdx mice

In our recent study was shown a significant recovery of damaged skeletal muscle of mice with X-linked muscular dystrophy (mdx) following low-intensity endurance exercise, probably by reducing the degeneration of dystrophic muscle. Consequently, in the present work, we aimed to identify proteins involved in the observed reduction in degenerating fibres. To this end, we used proteomic analysis to evaluate changes in the protein profile of quadriceps dystrophic muscles of exercised compared with sedentary mdx mice. Four protein spots were found to be significantly changed and were identified as three isoforms of carbonic anhydrase 3 (CA3) and superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were significantly up-regulated in quadriceps of sedentary mdx mice and were completely restored to wild–type (WT) mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Protein levels of SODC were down-regulated in quadriceps of sedentary mdx mice and were significantly restored to WT mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Western blot data were in agreement with those obtained using proteomic analysis and revealed the presence of one more CA3 isoform that was significantly changed. Based on data found in the present study, it seems that low-intensity endurance exercise may in part contribute to reduce cell degeneration process in mdx muscles, by counteracting oxidative stress.     

ICS II protects against cardiac hypertrophy by regulating metabolic remodelling,not by inhibiting autophagy

Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization. Therefore, the regulation of ketone body uptake and metabolism may have beneficial effects on heart injuries that induce cardiac remodelling. In this study, we investigated whether icariside II (ICS II) protects against cardiac hypertrophy in mice and cardiomyocytes. To create cardiac hypertrophy animal and cell models, mice were subjected to transverse aortic constriction (TAC), and embryonic rat cardiomyocytes (H9C2) were stimulated with angiotensin II, a neurohumoral stressor. Both the in vivo and in vitro results suggest that ICS II treatment ameliorated pressure overload–induced cardiac hypertrophy and preserved heart function. In addition, apoptosis and oxidative stress were reduced in the presence of ICS II. Moreover, ICS II inhibited excess autophagy in TAC-induced hearts and angiotensin II–stimulated cardiomyocytes. Mechanistically, we found that ICS II administration regulated SIRT3 expression in cardiac remodelling. SIRT3 activation increased ketone body transportation and utilization. Collectively, our data show that ICS II attenuated cardiac hypertrophy by modulating ketone body and fatty acid metabolism, and that this was likely due to the activation of the SIRT3-AMPK pathway. ICS II treatment may provide a new therapeutic strategy for improving myocardial metabolism in cardiac hypertrophy and heart failure.     

Glucocorticoid-treated mice are an inappropriate positive control for long-term preclinical studies in the mdx mouse

Background

Dmd^mdx (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.

Methods and Findings

We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.

Conclusion

Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials.     

miR-145-5p targets paxillin to attenuate angiotensin II-induced pathological cardiac hypertrophy via downregulation of Rac 1, pJNK,p-c-Jun,NFATc3, ANP and by Sirt-1 upregulation

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3′UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3′UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy

     

Resveratrol reduces vascular cell senescence through attenuation of oxidative stress by SIRT1/NADPH oxidase-dependent mechanisms

ObjectiveSenescence of vascular cells contributes to the development of cardiovascular diseases and the overall aging. This study was undertaken to investigate the effects of resveratrol (Res) on amelioration of vascular cell aging and the role of SIRT1/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathway.Methods and ResultsAdult male Wistar rats were treated with a high-fat/sucrose diet (HFS) in the presence or absence of Res for 3 months. HFS and in vitro treatment with high glucose increased the senescence cells and reactive oxygen species production in rat aorta and cultured bovine aortic endothelial cells (BAECs), respectively, which was attenuated by Res treatment. Res protected against HFS- or high-glucose-induced increase in NADPH oxidase p47phox expression and decrease in SIRT1 level. Apocynin, a NADPH oxidase inhibitor, down-regulated p47phox protein expression, but had no influence on SIRT1 protein; sirtinol, a SIRT1 inhibitor, aggravated the decrease in SIRT1 protein level and the increase in p47phox protein expression induced by high glucose.ConclusionOur studies suggested that Res was able to reverse the senescence process in aorta induced by HFS in rats or induced by the exposure to high glucose in cultured BAECs. The underlying mechanism is at least SIRT1/NADPH oxidase pathway dependent.     

ACE2 Is Augmented in Dystrophic Skeletal Muscle and Plays a Role in Decreasing Associated Fibrosis

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.