Sirtuin Control of Mitochondrial Dysfunction, Oxidative Stress, and Inflammation in Chagas Disease Models

Trypanosoma cruzi is a digenetic parasite that requires triatomines and mammalian host to complete its life cycle. T. cruzi replication in mammalian host induces immune-mediated cytotoxic proinflammatory reactions and cellular injuries, which are the common source of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during the acute parasitemic phase. Mitochondrial dysfunction of electron transport chain has been proposed as a major source of superoxide release in the chronic phase of infection, which renders myocardium exposed to sustained oxidative stress and contributes to Chagas disease pathology. Sirtuin 1 (SIRT1) is a class III histone deacetylase that acts as a sensor of redox changes and shapes the mitochondrial metabolism and inflammatory response in the host. In this review, we discuss the molecular mechanisms by which SIRT1 can potentially improve mitochondrial function and control oxidative and inflammatory stress in Chagas disease

Defects of mtDNA replication impaired mitochondrial biogenesis during Trypanosoma cruzi infection in human cardiomyocytes and Chagasic patients: The role of Nrf1/2 and antioxidant response

Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)-regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi-infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)-regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi-infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.Fil: Wan, Xianxiu. University of Texas Medical Branch; Estados UnidosFil: Gupta, Shivali. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Davidson, Mercy M.. Columbia University; Estados UnidosFil: Dousset, Pierre. Hospital San Bernardo; ArgentinaFil: Amoroso, Alejandro. Hospital San Bernardo; ArgentinaFil: Garg, Nisha Jain. University of Texas Medical Branch; Estados Unido

Chemotherapeutic Efficacy of Phosphodiesterase Inhibitors in Chagasic Cardiomyopathy

Molecular mechanisms of Trypanosoma cruzi (Tc)-induced Chagasic cardiomyopathy (CCM) are not well understood. The NO-cGMP-PKG1α pathway maintains cardiac homeostasis and inotropy and may be disturbed due to phosphodiesterase (PDE5)-mediated cGMP catabolism in CCM. To test this, C57BL/6 mice were infected with T. cruzi, and after the control of acute parasitemia (∼45 days post-infection), given sildenafil (SIL) (1 mg/kg) treatment for 3 weeks that ended long before the chronic disease phase (∼150 days post-infection). The PDE5 was increased and cGMP/PKG activity was decreased in chagasic myocardium. Transthoracic echocardiography revealed left ventricular (LV) systolic function, that is, stroke volume, cardiac output, and ejection fraction, was significantly decreased in chagasic mice. SIL treatment resulted in normal levels of PDE5 and cGMP/PKG activity and preserved the LV function. The cardioprotective effects of SIL were provided through inhibition of cardiac collagenosis and chronic inflammation that otherwise were pronounced in CCM. Further, SIL treatment restored the mitochondrial DNA–encoded gene expression, complex I–dependent (but not complex II–dependent) ADP-coupled respiration, and oxidant/antioxidant balance in chagasic myocardium. In vitro studies in cardiomyocytes verified that SIL conserved the redox metabolic state and cellular health via maintaining the antioxidant status that otherwise was compromised in response to T. cruzi infection. We conclude that SIL therapy was useful in controlling the LV dysfunction and chronic pathology in CCM

METTL14 enhances the m6A modification level of lncRNA MSTRG.292666.16 to promote the progression of non-small cell lung cancer

Abstract Background m6A modification has close connection with the occurrence, development, and prognosis of tumors. This study aimed to explore the roles of m6A modification and its related mechanisms in non-small cell lung cancer (NSCLC). Methods NSCLC tissues and their corresponding para-cancerous tissues were collected to determine the m6A levels of total RNA/lncRNAs and the expression of m6A modification-related genes/lncRNAs. Then, A549 cells were transfected with si-METTL14 or oe-METTL14, and the cell transfection efficiency was assessed. Subsequently, the viability, apoptosis, cell colony formation, migration and invasion of the different cells were determined. Finally, the nude mouse tumorigenicity experiments were performed to observe the effects of METTL14 in vivo. Results Compared to the para-NSCLC tissues, the m6A level and METTL14 expression were both significantly increased in the NSCLC tissues (P < 0.05). Based on the expression of METTL14 in the different cell lines, A549 cells were chosen for further experiments. Then, the A549 cells with METTL14 knockdown and overexpression were successfully established, as well as it was found that METTL14 knockdown could inhibit the viability, colony formation, migration, and invasion of A549 cells, while facilitate their apoptosis. In vivo experiments also showed that METTL14 knockdown could inhibit tumor formation and growth. Additionally, the m6A level of MSTRG.292666.16 was higher in the NSCLC tissues; and after METTL14 knockdown, the expression and m6A level of MSTRG.292666.16 were both significantly reduced in A549 cells, and vice versa. Conclusion METTL14 may promote the progression of NSCLC through up-regulating MSTRG.292666.16 and enhance its m6A modification level

Pentose Phosphate Shunt Modulates Reactive Oxygen Species and Nitric Oxide Production Controlling Trypanosoma cruzi in Macrophages

Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (Mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical Mφs. How Mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived Mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classical Mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc + IFN-γ stimulated Mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in Mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected Mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by Mφs

SIRT1-PGC1α-NFκB Pathway of Oxidative and Inflammatory Stress during <i>Trypanosoma cruzi</i> Infection: Benefits of SIRT1-Targeted Therapy in Improving Heart Function in Chagas Disease

<div><p>Chronic chagasic cardiomyopathy (CCM) is presented by increased oxidative/inflammatory stress and decreased mitochondrial bioenergetics. SIRT1 senses the redox changes and integrates mitochondrial metabolism and inflammation; and SIRT1 deficiency may be a major determinant in CCM. To test this, C57BL/6 mice were infected with <i>Trypanosoma cruzi</i> (<i>Tc</i>), treated with SIRT1 agonists (resveratrol or SRT1720), and monitored during chronic phase (~150 days post-infection). Resveratrol treatment was partially beneficial in controlling the pathologic processes in Chagas disease. The 3-weeks SRT1720 therapy provided significant benefits in restoring the left ventricular (LV) function (stroke volume, cardiac output, ejection fraction etc.) in chagasic mice, though cardiac hypertrophy presented by increased thickness of the interventricular septum and LV posterior wall, increased LV mass, and disproportionate synthesis of collagens was not controlled. SRT1720 treatment preserved the myocardial SIRT1 activity and PGC1α deacetylation (active-form) that were decreased by 53% and 9-fold respectively, in chagasic mice. Yet, SIRT1/PGC1α-dependent mitochondrial biogenesis (i.e., mitochondrial DNA content, and expression of subunits of the respiratory complexes and mtDNA replication machinery) was not improved in chronically-infected/SRT1720-treated mice. Instead, SRT1720 therapy resulted in 2-10-fold inhibition of <i>Tc</i>-induced oxidative (H₂O₂ and advanced oxidation protein products), nitrosative (inducible nitric oxide synthase, 4-hydroxynonenal, 3-nitrotyrosine), and inflammatory (IFNγ, IL1β, IL6 and TNFα) stress and inflammatory infiltrate in chagasic myocardium. These benefits were delivered through SIRT1-dependent inhibition of NFκB transcriptional activity. We conclude that <i>Tc</i> inhibition of SIRT1/PGC1α activity was not a key mechanism in mitochondrial biogenesis defects during Chagas disease. SRT1720-dependent SIRT1 activation led to suppression of NFκB transcriptional activity, and subsequently, oxidative/nitrosative and inflammatory pathology were subdued, and antioxidant status and LV function were enhanced in chronic chagasic cardiomyopathy.</p></div

Table_2.DOCX

<p>Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (Mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical Mφs. How Mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived Mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classical Mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc + IFN-γ stimulated Mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in Mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected Mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by Mφs.</p

Image_2.TIF

<p>Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (Mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical Mφs. How Mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived Mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classical Mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc + IFN-γ stimulated Mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in Mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected Mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by Mφs.</p

Effects of SRT1720 on cardiac remodeling in chagasic disease.

<p>Mice were infected with <i>T</i>. <i>cruzi</i>, treated with SRT1720, and examined at 150 days pi. <b>(A)</b> Cardiac structural changes were analyzed by echocardiography using a Vevo 2100 System. Shown are systolic (-s) and diastolic (-d) values for inter-ventricular septum (IVS) thickness (a&b), LV posterior wall (LVPW) thickness (c), LV mass (d), and LV area (e) in chronically infected mice. <b>(B)</b> Representative images show Masson's Trichrome staining of the heart tissue sections from (a) normal control, (b) <i>Tc</i>-infected, and (c) infected/ SRT1720-treated mice. <b>(C)</b> Quantitative real-time RT-PCR for collagen isoforms (COLI and COLIII, panel a&b) and alpha smooth muscle actin (αSMA, panel c). Data were normalized to GAPDH mRNA. Significance was calculated and presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005954#ppat.1005954.g001" target="_blank">Fig 1</a>.</p