Specific inhibition of mitochondrial oxidative stress suppresses inflammation and improves cardiac function in a rat pneumonia-related sepsis model

Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10(6) colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 μmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H(2)O(2) generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H(2)O(2) levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy (P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.     

Genistein improves neuropathology and corrects behaviour in a mouse model of neurodegenerative metabolic disease

Background

Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein.

Methodology/Principal Findings

We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed.

Conclusions/Significance

Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.     

Alterations in mitochondrial function in a mouse model of hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by varying degrees of ventricular hypertrophy and myofibrillar disarray. Mutations in cardiac contractile proteins cause HCM. However, there is an unexplained wide variability in the clinical phenotype, and it is likely that there are multiple contributing factors. Because mitochondrial dysfunction has been described in heart disease, we tested the hypothesis that mitochondrial dysfunction contributes to the varying HCM phenotypes. Mitochondrial function was assessed in two transgenic models of HCM: mice with a mutant myosin heavy chain gene (MyHC) or with a mutant cardiac troponin T (R92Q) gene. Despite mitochondrial ultrastructural abnormalities in both models, the rate of state 3 respiration was significantly decreased only in the mutant MyHC mice by approximately 23%. Notably, this decrease in state 3 respiration preceded hemodynamic dysfunction. The maximum activity of alpha-ketogutarate dehydrogenase as assayed in isolated disrupted mitochondria was decreased by 28% compared with isolated control mitochondria. In addition, complexes I and IV were decreased in mutant MyHC transgenic mice. Inhibition of beta-adrenergic receptor kinase, which is elevated in mutant MyHC mouse hearts, can prevent mitochondrial respiratory impairment in mutant MyHC mice. Thus our results suggest that mitochondria may contribute to the hemodynamic dysfunction seen in some forms of HCM and offer a plausible mechanism responsible for some of the heterogeneity of the disease phenotypes.     

Metformin improves cardiac function in a nondiabetic rat model of post-MI heart failure

Metformin is the first choice drug for the treatment of patients with diabetes, but its use is debated in patients with advanced cardiorenal disease. Epidemiological data suggest that metformin may reduce cardiac events, in patients both with and without heart failure. Experimental evidence suggests that metformin reduces cardiac ischemia-reperfusion injury. It is unknown whether metformin improves cardiac function (remodeling) in a long-term post-MI remodeling model. We therefore studied male, nondiabetic, Sprague-Dawley rats that were subjected to either myocardial infarction (MI) or sham operation. Animals were randomly allocated to treatment with normal water or metformin-containing water (250 mg·kg(-1)·day(-1)). At baseline, 6 wk, and 12 wk, metabolic parameters were analyzed and oral glucose tolerance tests (OGTT) were performed. Echocardiography and hemodynamic parameters were assessed 12 wk after MI. In the MI model, infarct size was significantly smaller after 12-wk metformin treatment (29.6 ± 3.2 vs. 38.0 ± 2.2%, P < 0.05). Moreover, metformin resulted in less left ventricular dilatation (6.0 ± 0.4 vs. 7.6 ± 0.6 mm, P < 0.05) and preservation of left ventricular ejection fraction (65.8 ± 3.7% vs. 48.6 ± 5.6%, P < 0.05) compared with MI control. The improved cardiac function was associated with decreased atrial natriuretic peptide mRNA levels in the metformin-treated group (50% reduction compared with MI, P < 0.05). Insulin resistance did not occur during cardiac remodeling (as indicated by normal OGTT) and fasting glucose levels and the pattern of the OGTT were not affected by metformin. Molecular analyses suggested that altered AMP kinase phosphorylation status and low insulin levels mediate the salutary effects of metformin. Altogether our results indicate that metformin may have potential to attenuate heart failure development after myocardial infarction, in the absence of diabetes and independent of systemic glucose levels.     

Carbon monoxide improves adaptation of Arabidopsis to iron deficiency

Carbon monoxide (CO) is an endogenous gaseous molecule and regulates a variety of biological processes in animals. However, whether CO regulates nutrient stress responses in plants is largely unknown. In this paper, we described an observation that CO can regulate iron-homeostasis in iron-starved Arabidopsis. Exogenous CO at 50 μ m was able to prevent the iron deficient-induced chlorosis and improve chlorophyll accumulation. Expression of AtIRT1 , AtFRO2 , AtFIT1 and AtFER1 was up-regulated by CO exposure in iron-deficient seedlings. CO-regulated iron homeostasis could also be found in monocot maize and green alga Chlamydomonas reinhardtii . Treatment with external CO increased iron accumulation in iron-deficient Arabidopsis and C. reinhardtii , and restored leaf greening in Maize ys1 and ys3 mutants (defective in Fe uptake). Moreover, endogenous CO level was increased in Arabidopsis under iron-deficiency. Finally, CO exposure induced NO accumulation in root tips. However, such an action could be blocked by NO scavenger cPTIO. These results indicate that CO may play an important role in improving plant adaptation to iron deficiency or cross-talking with NO under the iron deficiency.     

Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse

Cyclooxygenase (COX)-2 is expressed in the heart in animal models of ischemic injury. Recent studies have suggested that COX-2 products are involved in inflammatory cell infiltration and fibroblast proliferation in the heart. Using a mouse model, we questioned whether 1). myocardial infarction (MI) in vivo induces COX-2 expression chronically, and 2). COX-2 inhibition reduces collagen content and improves cardiac function in mice with MI. MI was produced by ligation of the left anterior descending coronary artery in mice. Two days later, mice were treated with 3 mg/kg NS-398, a selective COX-2 inhibitor, or vehicle in drinking water for 2 wk. After the treatment period, mice were subjected to two-dimensional M-mode echocardiography to determine cardiac function. Hearts were then analyzed for determination of infarct size, interstitial collagen content, brain natriuretic peptide (BNP) mRNA, myocyte cross-sectional area, and immunohistochemical staining for transforming growth factor (TGF)-beta and COX-2. COX-2 protein, detected by immunohistochemistry, was increased in MI versus sham hearts. MI resulted in increased left ventricular systolic and diastolic dimension and decreased ejection fraction, fractional shortening, and cardiac output. NS-398 treatment partly reversed these detrimental changes. Myocyte cross-sectional area, a measure of hypertrophy, was decreased by 30% in the NS-398 versus vehicle group, but there was no effect on BNP mRNA. The interstitial collagen fraction increased from 5.4 +/- 0.4% in sham hearts to 10.4 +/- 0.9% in MI hearts and was decreased to 7.9 +/- 0.6% in NS-398-treated hearts. A second COX-2 inhibitor, rofecoxib (MK-0966), also decreased myocyte cross-sectional area and interstitial collagen fraction. TGF-beta, a key regulator of collagen synthesis, was increased in MI hearts. NS-398 treatment reduced TGF-beta immunostaining by 40%. NS-398 treatment had no effect on infarct size. These results suggest that COX-2 products contribute to cardiac remodeling and functional deficits after MI. Thus selected inhibition of COX-2 may be a therapeutic target for reducing myocyte damage after MI.     

Chrysin attenuates interstitial fibrosis and improves cardiac function in a rat model of acute myocardial infarction

Interstitial fibrosis after acute myocardial infarction (MI) leads to cardiac structural remodeling and dysfunction. The peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist chrysin has been shown to protect injured myocardium through suppression of oxidative stress and inflammation. This study was designed to investigate the effect and mechanism of chrysin on myocardial fibrosis. A rat MI model was created by ligating the left coronary artery. The rats with MI were treated with chrysin (40 mg/kg/day) or 0.5% carboxymethylcellulose sodium by intragastric administration for 4 weeks after operation. The effect of chrysin on cardiac fibroblasts (CFs) were also assessed in vitro. Echocardiography showed that cardiac function was significantly improved after chrysin treatment. Chrysin reduced the levels of MDA and SOD and GSH-Px in myocardial tissue. Chrysin attenuated the interstitial and perivascular fibrosis and the expression of collagenlin the peri-infarcted zone and remarkably decreased the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. Chrysin up-regulated PPAR-γ and inhibited the nuclear factor-kappa B (NF-κB) pathway by suppressing inhibitor kappa B kinase β phosphorylation. Immunohistochemistry analysis and PCR detected downregulated expression of AP-1 after chrysin treatment. Chrysin also markedly reduced the increased α-SMA, typeland type III collagen expression of CFs mediated by AngII in vitro. In conclusion, chrysin has an antifibrosis cardioprotective effect on the infarct peripheral zone after MI. The underlined mechanism may be the up-regulation of PPAR-γ and inhibition of the NF-κB and AP1 pathway.     

Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome

Non-alcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome characterized by dislipidemia and insulin resistance. We hypothesized that ezetimibe, an inhibitor of NPC1L1, improves these metabolic disorders in Zucker obese fatty rats (ZOF). Ezetimibe significantly lowered total cholesterol and triglycerides in ZOF with prominent reduction in the remnant lipoprotein fraction and small dense low density lipoprotein fraction. Moreover, lipid deposition and fibrosis of liver were decreased by ezetimibe. Interestingly, ezetimibe improved insulin and plasma glucose response after intraperitoneal glucose injection. Further, ezetimibe enhanced insulin signaling in cultured hepatocytes. Our results indicate the potential of ezetimibe in treating the metabolic syndrome and NAFLD.     

Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest

The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (T_c) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while T_c were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.     

Evaluation of mitochondrial function and metabolic reprogramming during tumor progression in a cell model of skin carcinogenesis

Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression.     

Preconditioning improves postischemic mitochondrial function and diminishes oxidation of mitochondrial proteins

This study examines the hypothesis that ischemic or pharmacologic preconditioning improves postischemic mitochondrial function by attenuating oxidation of mitochondrial proteins. Isolated rat hearts were perfused for 38 min preischemia, followed by 25 min global ischemia and then 60 min reperfusion. Hearts were preconditioned by two episodes of 3 min global ischemia, followed by 2 min of reflow (IP), or by perfusion with 50 micromol/l nicorandil (Nic) for 10 min, followed by 10 min washout. IP and Nic significantly (p <.05) improved postischemic function, which was abolished by bracketing the protocols with 200 micromol/l 5-hydroxydecoanate (5HD) or 300 micromol/l alpha-mercaptopropionylglycine (MPG). After isolation of cardiac mitochondria, the respiratory control index (RCI) was calculated from State 3 and State 4 respiration. Both IP and Nic significantly (p <.05) improved postischemic RCI, which was depressed 71% from preischemic values in control hearts. The protective effects of IP and Nic were partially abolished by bracketing with 5HD or MPG. Furthermore, mitochondria from ischemic hearts had significantly (p <.05) less ability to resist swelling on Ca2+ loading, which was improved by both IP and Nic. By use of an immunoblot technique, carbonyl content of multiple bands of mitochondrial proteins was observed to be elevated after 25 min ischemia, and still elevated by the end of 60 min reperfusion. Both IP and Nic attenuated the increased protein oxidation observed at the end of ischemia. The protective effect of IP was almost completely abolished by MPG and partially by 5HD, which also partially abolished the protective effect of Nic. These studies support the conclusion that one mechanism for enhanced postischemic function in the preconditioned heart is improved mitochondrial function as a result of decreased oxidation of mitochondrial proteins.