Vu Thi Thu

Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts.

Glutathione peroxidase 1 (GPx1) plays an important role in preventing cardiac dysfunction following ischemia-reperfusion injury. However, its role in protecting cardiac mitochondria against reoxygenation-induced reactive oxygen species (ROS) generation in vivo is unclear. We examined the role of GPx1 in protecting cardiac mitochondria against hypoxia–reoxygenation (HR) damage by testing for alterations in cardiac mitochondrial function. We used a two-dimensional gel electrophoresis proteomics analysis to examine the effects of reoxygenation on cardiac protein in wild-type (GPx1+/+) and GPx1 knockout (GPx1−/−) mouse hearts. We identified 42 protein spots showing differential expression in the two groups. Sixteen of the proteins identified were located in mitochondria and were involved in a number of key metabolic pathways. To verify our proteomics findings functionally, we performed NADH autofluorescence measurements and ATP production assays. The reduced expression of oxidative phosphorylation proteins in GPx1−/− mice following HR treatment resulted in loss of the mitochondrial membrane potential and decreased mitochondrial respiration. Mitochondrial ROS production and oxidative mtDNA damage were increased markedly during reoxygenation in GPx1−/− hearts. We also found morphological abnormalities in cardiac mitochondria and myocytes in HR-treated GPx1−/−. This is the first report of the role of GPx1 in protecting cardiac mitochondria against reoxygenation damage in vivo. These findings will help clarify the mechanisms of HR injury and will aid in the development of antioxidant therapies to prevent cardiac mitochondrial dysfunction associated with reoxygenation.

NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter

AIMS Preservation of mitochondrial function is essential to limit myocardial damage in ischaemic heart disease. We examined the protective effects and mechanism of a new compound, NecroX-5, on rat heart mitochondria in a hypoxia/reoxygenation (HR) model. METHODS AND RESULTS NecroX-5 reduced mitochondrial oxidative stress, prevented the collapse in mitochondrial membrane potential, improved mitochondrial oxygen consumption, and suppressed mitochondrial Ca(2+) overload during reoxygenation in an in vitro rat heart HR model. Furthermore, NecroX-5 reduced the ouabain- or histamine-induced increase in mitochondrial Ca(2+). CONCLUSION These findings suggest that NecroX-5 may act as a mitochondrial Ca(2+) uniporter inhibitor to protect cardiac mitochondria against HR damage.

Effects of the novel angiotensin II receptor type I antagonist, fimasartan on myocardial ischemia/reperfusion injury.

BACKGROUND The aim of this study was to investigate the cardioprotective effect of fimasartan, a newly developed angiotensin II receptor type I blocker (ARB), against myocardial ischemia/reperfusion (I/R) injury and to identify the mechanism by which it reduces mitochondrial damage. METHODS Fimasartan was administered intravenously to Sprague-Dawley rats (3mg/kg), cardiomyocytes (50 μM), and H9c2 cells (50 μM) before ischemia or hypoxia. Myocardial infarction (MI), echocardiograms, DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling, immunoblotting, oxygen consumption, confocal microscopic appearance, and L-type Ca(2+) current (ICa,L) were then assessed. RESULTS Fimasartan pretreatment remarkably reduced the rate of MI and improved cardiac performance well after I/R (n = 9/group). Fimasartan also reduced apoptotic cell death both in vivo and in hypoxia/reoxygenation (H/R)-treated H9c2 cells (n = 5~8/group). H/R-induced mitochondrial O2(-) production and collapse of membrane potential were markedly attenuated in fimasartan-treated cardiomyocytes (n = 4 ~ 6/group). Additionally, mitochondrial Ca(2+) overload during reoxygenation was suppressed by fimasartan (n = 4~6/group), and this was found to be possibly related to the inhibition of ICa,L and mitochondrial Ca(2+) uniporter. Furthermore, fimasartan pretreatment increased phosphorylations of Akt and glycogen synthase kinase-3β (n = 5 ~ 7/group), decreased pro-apoptotic p53 levels, and increased anti-apoptotic Bcl-2 levels (n = 4) during reperfusion. CONCLUSIONS Fimasartan preconditioning has the potential to modulate Bcl-2 and suppress I/R-induced Ca(2+) overload by inhibiting ICa,L and MCU. These beneficial effects could prevent the mitochondrial dysfunction and apoptosis accompanied by I/R.

Cardiac proteomic responses to ischemia-reperfusion injury and ischemic preconditioning.

Cardiac ischemia and ischemia–reperfusion (I/R) injury are major contributors to morbidity and mortality worldwide. Pathological mechanisms of I/R and the physiological mechanisms of ischemic preconditioning (IPC), which is an effective cardiac protective response, have been widely investigated in the last decade to search for means to prevent or treat this disease. Proteomics is a powerful analytical tool that has provided important information to identify target proteins and understand the underlying mechanisms of I/R and IPC. Here, we review the application of proteomics to I/R injury and IPC to discover target proteins. We analyze the functional meaning of the accumulated data on hundreds of proteins using various bioinformatics applications. In addition, we review exercise-induced proteomic alterations in the heart to understand the potential cardioprotective role of exercise against I/R injury. Further developments in the proteomic field that target specialized proteins will yield new insights for optimizing therapeutic targets and developing a wide range of therapeutic agents against ischemic heart disease.

B7-H4 downregulation induces mitochondrial dysfunction and enhances doxorubicin sensitivity via the cAMP/CREB/PGC1-α signaling pathway in HeLa cells

B7-H4 is a B7 family coregulatory protein that inhibits T cell-mediated immunity. B7-H4 is overexpressed in various cancers; however, the functional role of B7-H4 in cancer metabolism is poorly understood. Because mitochondria play pivotal roles in development, proliferation, and death of cancer cells, we investigated molecular and functional alterations of mitochondria in B7-H4-depleted HeLa cells. In a human study, overexpression of B7-H4 was confirmed in the cervices of adenocarcinoma patients (n = 3) compared to noncancer patients (n = 3). In the cell line model, B7-H4 depletion was performed by transfection with small interfering RNA (siRNA). B7-H4 depletion suppressed oxygen consumption rate, ATP production, and mitochondrial membrane potential and mass and increased reactive oxygen species production. In particular, electron transport complex III activity was significantly impaired in siB7-H4-treated cells. Coincidently, depletion of B7-H4 suppressed major mitochondrial regulators (peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC1-α] and mitochondrial transcription factor A), a component of oxidative phosphorylation (ubiquinol-cytochrome c reductase core protein 1), and an antiapoptosis protein (Bcl-XL). Mitochondrial dysfunction in siRNA-treated cells significantly augmented oxidative stress, which strongly activated the JNK/P38/caspase axis in the presence of doxorubicin, resulting in increased apoptotic cell death. Investigating the mechanism of B7-H4-mediated mitochondrial modulation, we found that B7-H4 depletion significantly downregulated the cAMP/cAMP response element-binding protein/PGC1-α signaling pathway. Based on these findings, we conclude that B7-H4 has a role in the regulation of mitochondrial function, which is closely related to cancer cell physiology and drug sensitivity.

NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1α expression levels during hypoxia/reoxygenation injury

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 µM) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC1α) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries.

NecroX-5 exerts anti-inflammatory and anti-fibrotic effects via modulation of the TNFα/Dcn/TGFβ1/Smad2 pathway in hypoxia/reoxygenation-treated rat hearts

Inflammatory and fibrotic responses are accelerated during the reperfusion period, and excessive fibrosis and inflammation contribute to cardiac malfunction. NecroX compounds have been shown to protect the liver and heart from ischemia-reperfusion injury. The aim of this study was to further define the role and mechanism of action of NecroX-5 in regulating infl ammation and fi brosis responses in a model of hypoxia/reoxygenation (HR). We utilized HR-treated rat hearts and lipopolysaccharide (LPS)-treated H9C2 culture cells in the presence or absence of NecroX-5 (10 µmol/L) treatment as experimental models. Addition of NecroX-5 signifi cantly increased decorin (Dcn) expression levels in HR-treated hearts. In contrast, expression of transforming growth factor beta 1 (TGFβ1) and Smad2 phosphorylation (pSmad2) was strongly attenuated in NecroX-5-treated hearts. In addition, signifi cantly increased production of tumor necrosis factor alpha (TNFα), TGFβ1, and pSmad2, and markedly decreased Dcn expression levels, were observed in LPS-stimulated H9C2 cells. Interestingly, NecroX-5 supplementation effectively attenuated the increased expression levels of TNFα, TGFβ1, and pSmad2, as well as the decreased expression of Dcn. Thus, our data demonstrate potential antiinflammatory and anti-fibrotic effects of NecroX-5 against cardiac HR injuries via modulation of the TNFα/Dcn/TGFβ1/Smad2 pathway.

Does strong hypertrophic condition induce fast mitochondrial DNA mutation of rabbit heart

Homo- and heteroplasmic mitochondrial DNA (mtDNA) mutations were observed and identified in an isoproterenol-induced rabbit model of cardiac hypertrophy. Genes encoding proteins essential for catalyzing mitochondrial electron transfer and for generating the proton motive force, such as NADH dehydrogenases (ND2, ND3, ND4, and ND6), cytochrome b, and ATPase 8, showed increased susceptibility for mutation. Specifically, five mutations caused amino acid changes and were located in Complex I and Complex V gene clusters. To our knowledge, this is the first demonstration of a relationship between cardiac hypertrophy induced by a strong sympathetic load and rapid mtDNA mutations.

Acute and Chronic Exercise in Animal Models

Numerous animal cardiac exercise models using animal subjects have been established to uncover the cardiovascular physiological mechanism of exercise or to determine the effects of exercise on cardiovascular health and disease. In most cases, animal-based cardiovascular exercise modalities include treadmill running, swimming, and voluntary wheel running with a series of intensities, times, and durations. Those used animals include small rodents (e.g., mice and rats) and large animals (e.g., rabbits, dogs, goats, sheep, pigs, and horses). Depending on the research goal, each experimental protocol should also describe whether its respective exercise treatment can produce the anticipated acute or chronic cardiovascular adaptive response. In this chapter, we will briefly describe the most common kinds of animal models of acute and chronic cardiovascular exercises that are currently being conducted and are likely to be chosen in the near future. Strengths and weakness of animal-based cardiac exercise modalities are also discussed.