Haemin attenuates intermittent hypoxia‐induced cardiac injury via inhibiting mitochondrial fission

Obstructive sleep apnoea (OSA) characterized by intermittent hypoxia (IH) is closely associated with cardiovascular diseases. IH confers cardiac injury via accelerating cardiomyocyte apoptosis, whereas the underlying mechanism has remained largely enigmatic. This study aimed to explore the potential mechanisms involved in the IH‐induced cardiac damage performed with the IH‐exposed cell and animal models and to investigate the protective effects of haemin, a potent haeme oxygenase‐1 (HO‐1) activator, on the cardiac injury induced by IH. Neonatal rat cardiomyocyte (NRC) was treated with or without haemin before IH exposure. Eighteen male Sprague‐Dawley (SD) rats were randomized into three groups: control group, IH group (PBS, ip) and IH + haemin group (haemin, 4 mg/kg, ip). The cardiac function was determined by echocardiography. Mitochondrial fission was evaluated by Mitotracker staining. The mitochondrial dynamics‐related proteins (mitochondrial fusion protein, Mfn2; mitochondrial fission protein, Drp1) were determined by Western blot. The apoptosis of cardiomyocytes and heart sections was examined by TUNEL. IH regulated mitochondrial dynamics‐related proteins (decreased Mfn2 and increased Drp1 expressions, respectively), thereby leading to mitochondrial fragmentation and cell apoptosis in cardiomyocytes in vitro and in vivo, while haemin‐induced HO‐1 up‐regulation attenuated IH‐induced mitochondrial fragmentation and cell apoptosis. Moreover, IH resulted in left ventricular hypertrophy and impaired contractile function in vivo, while haemin ameliorated IH‐induced cardiac dysfunction. This study demonstrates that pharmacological activation of HO‐1 pathway protects against IH‐induced cardiac dysfunction and myocardial fibrosis through the inhibition of mitochondrial fission and cell apoptosis.     

Protection of PMS777, a New AChE Inhibitor with PAF Antagonism, Against Amyloid-β-Induced Neuronal Apoptosis and Neuroinflammation

Amyloid-β (Aβ) plays a central role in the neuroinflammation and cholinergic neuronal apoptosis in Alzheimer’s disease, and thus has been considered as a main determinant of this disease. In the previous study, we reported that PMS777, a novel bis-interacting ligand for acetylcholinesterase (AChE) inhibition and platelet-activating factor (PAF) receptor antagonism, could significantly attenuate PAF-induced neurotoxicity. Continuing our efforts, we further investigated the protective effect of PMS777 on Aβ-induced neuronal apoptosis in vitro and neuroinflammation in vivo. PMS777 (1–100 μM) was found to inhibit Aβ-induced human neuroblastoma SH-SY5Y cell apoptosis in a concentration-dependent manner. Concurrently, PMS777 increased ratio of bcl-2 to bax mRNA, and inhibited both mRNA expression and activity of caspase-3 in SH-SY5Y cells after the exposure with Aβ. In vivo experimental study demonstrated that PMS777 could attenuate Aβ-induced microglial and astrocytic activation in the rat hippocampus after systemic administration. These results suggest that PMS777 potently protects against Aβ-induced neuronal apoptosis and neuroinflammation, and warrants further investigations in connection with its potential value in the treatment of Alzheimer’s disease. The authors Juan Li and Jinjia Hu contributed equally to this article.     

DLP1‐dependent mitochondrial fragmentation and redistribution mediate prion‐associated mitochondrial dysfunction and neuronal death

Mitochondrial malfunction is a universal and critical step in the pathogenesis of many neurodegenerative diseases including prion diseases. Dynamin‐like protein 1 (DLP1) is one of the key regulators of mitochondrial fission. In this study, we investigated the role of DLP1 in mitochondrial fragmentation and dysfunction in neurons using in vitro and in vivo prion disease models. Mitochondria became fragmented and redistributed from axons to soma, correlated with increased mitochondrial DLP1 expression in murine primary neurons (N2a cells) treated with the prion peptide PrP^106–126 in vitro as well as in prion strain‐infected hamster brain in vivo. Suppression of DLP1 expression by DPL1 RNAi inhibited prion‐induced mitochondrial fragmentation and dysfunction (measured by ADP/ATP ratio, mitochondrial membrane potential, and mitochondrial integrity). We also demonstrated that DLP1 RNAi is neuroprotective against prion peptide in N2a cells as shown by improved cell viability and decreased apoptosis markers, caspase 3 induced by PrP^106–126. On the contrary, overexpression of DLP1 exacerbated mitochondrial dysfunction and cell death. Moreover, inhibition of DLP1 expression ameliorated PrP^106–126‐induced neurite loss and synaptic abnormalities (i.e., loss of dendritic spine and PSD‐95, a postsynaptic scaffolding protein as a marker of synaptic plasticity) in primary neurons, suggesting that altered DLP1 expression and mitochondrial fragmentation are upstream events that mediate PrP^106–126‐induced neuron loss and degeneration. Our findings suggest that DLP1‐dependent mitochondrial fragmentation and redistribution plays a pivotal role in PrP^Sc‐associated mitochondria dysfunction and neuron apoptosis. Inhibition of DLP1 may be a novel and effective strategy in the prevention and treatment of prion diseases.     

The role of thioredoxin reductase 1 in melanoma metabolism and metastasis

Although significant progress has been made in targeted and immunologic therapeutics for melanoma, many tumors fail to respond, and most eventually progress when treated with the most efficacious targeted combination therapies thus far identified. Therefore, alternative approaches that exploit distinct melanoma phenotypes are necessary to develop new approaches for therapeutic intervention. Tissue microarrays containing human nevi and melanomas were used to evaluate levels of the antioxidant protein thioredoxin reductase 1 (TR1), which was found to increase as a function of disease progression. Melanoma cell lines revealed metabolic differences that correlated with TR1 levels. We used this new insight to design a model treatment strategy that creates a synthetic lethal interaction wherein targeting TR1 sensitizes melanoma to inhibition of glycolytic metabolism, resulting in a decrease in metastases in vivo. This approach holds the promise of a new clinical therapeutic strategy, distinct from oncoprotein inhibition.     

Loss of strumpellin in the melanocytic lineage impairs the WASH Complex but does not affect coat colour

The five‐subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin.     

In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry

The neuromuscular junction (NMJ), where a motor neuron intercepts and activates a muscle fiber, is a highly versatile and complex subcellular region. Genomic and proteomic approaches using the large (>1 kg) electric organ of Torpedo californica have helped advancing our understanding of this minute (30–50 μm) electric synapse. However, the majority of these studies have focused on mRNA and proteins, therefore neglecting small signaling molecules involved in muscle-nerve ‘dialogue’. We developed a novel technique, mid-infrared laser ablation electrospray ionization (LAESI) mass spectrometry (MS), with the potential of detecting a diversity of small signaling molecules in vitro. LAESI uses the native water in the tissue as the matrix to couple the laser pulse energy into the target for the ablation process and enables its direct analysis essentially without sample preparation. Here, we report the detection of metabolites from the untreated frozen tissue of the Torpedo electric organ with LAESI MS at atmospheric pressure. A total of 24 metabolites were identified by accurate mass measurements, natural isotope patterns, and tandem mass spectrometry. Most of the identified metabolites were related to the cholinergic function of the electric synapse (acetylcholine and choline), fatty acid metabolism and acetyl transfer (carnitine and acetylcarnitine), the mitigation of osmotic stress (betaine and trimethylamine N-oxide), and energy production (creatine and creatinine). The biosynthetic precursors of these metabolites and their expected degradation products were also detected indicating that LAESI MS is well suited for tissue metabolomics with the ultimate goal of imaging and in vivo studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

PET studies of cerebral metabolism in Parkinson Disease

A defect in cerebral energy production due to dysfunction of the mitochondrial electron transport system (ETS) has been postulated to be important in the pathogenesis of Parkinson Disease (PD). However, direct in vivo measurements of cerebral mitochondrial function are scant and inconsistent. We directly investigated cerebral mitochondrial function in vivo with positron emission tomography (PET) in 12 patients with early, never-medicated PD and 12 age-matched normal controls by combined measurements of the cerebral metabolic rate of oxygen (CMRO₂) and the cerebral metabolic rate of glucose (CMRglc). Instead of the decrease in CMRO₂ and CMRO₂/CMRglc molar ratio characteristic of defects in mitochondrial oxidative metabolism, there was a statistically significant 24% general increase in CMRO₂ and no change in CMRO₂/CMRglc. Since PD symptoms were already manifest, reduced oxidative activity of the mitochondrial ETS cannot be a primary mechanism of neuronal death in early PD. This increase in metabolism could reflect the increased energy requirements of an injured brain or an uncoupling of ATP production from oxidation in the terminal stage of oxidative phosphorylation. Which is the case in early PD and whether these metabolic abnormalities are important in the pathogenesis of PD will require further study.     

Calpain regulates CVB3 induced viral myocarditis by promoting autophagic flux upon infection

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting.     

Nitric oxide and mitochondrial respiration.

Nitric oxide (NO) and its derivative peroxynitrite (ONOO-) inhibit mitochondrial respiration by distinct mechanisms. Low (nanomolar) concentrations of NO specifically inhibit cytochrome oxidase in competition with oxygen, and this inhibition is fully reversible when NO is removed. Higher concentrations of NO can inhibit the other respiratory chain complexes, probably by nitrosylating or oxidising protein thiols and removing iron from the iron-sulphur centres. Peroxynitrite causes irreversible inhibition of mitochondrial respiration and damage to a variety of mitochondrial components via oxidising reactions. Thus peroxynitrite inhibits or damages mitochondrial complexes I, II, IV and V, aconitase, creatine kinase, the mitochondrial membrane, mitochondrial DNA, superoxide dismutase, and induces mitochondrial swelling, depolarisation, calcium release and permeability transition. The NO inhibition of cytochrome oxidase may be involved in the physiological regulation of respiration rate, as indicated by the finding that isolated cells producing NO can regulate cellular respiration by this means, and the finding that inhibition of NO synthase in vivo causes a stimulation of tissue and whole body oxygen consumption. The recent finding that mitochondria may contain a NO synthase and can produce significant amounts of NO to regulate their own respiration also suggests this regulation may be important for physiological regulation of energy metabolism. However, definitive evidence that NO regulation of mitochondrial respiration occurs in vivo is still missing, and interpretation is complicated by the fact that NO appears to affect tissue respiration by cGMP-dependent mechanisms. The NO inhibition of cytochrome oxidase may also be involved in the cytotoxicity of NO, and may cause increased oxygen radical production by mitochondria, which may in turn lead to the generation of peroxynitrite. Mitochondrial damage by peroxynitrite may mediate the cytotoxicity of NO, and may be involved in a variety of pathologies.     

Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by β-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGL^AdpKO). Here, we find that upon activation of β-adrenergic receptors in vivo, ATGL^AdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGL^fl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4^AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGL^AdpKO mice was indeed from the adipocytes. ATGL^AdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGL^AdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.     

Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.     

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

Exosomes from mesenchymal stem cells expressing miR‐125b inhibit neointimal hyperplasia via myosin IE

Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome‐mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC‐derived exosomes (MSC‐Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon‐induced vascular injury. Our results showed that MSC‐Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR‐125b was enriched in MSC‐Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3? untranslated region. Additionally, MSC‐Exo and exosomally transferred miR‐125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC‐Exo can transfer miR‐125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR‐125b may be a therapeutic target in the treatment of vascular diseases.     

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome

So‐called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in‐depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.     

Identification of c-type cytochromes involved in anaerobic, bacterial U(IV) oxidation

Anaerobic, bacterial reduction of water-soluble U(VI) complexes to the poorly soluble U(IV) mineral uraninite has been intensively studied as a strategy for in situ remediation of uranium-contaminated groundwater. A novel and potentially counteracting metabolic process, anaerobic, nitrate-dependent U(IV) oxidation, has recently been described in two bacterial species (Geobacter metallireducens and Thiobacillus denitrificans), but the underlying biochemistry and genetics are completely unknown. We report here that two diheme, c-type cytochromes (putatively c ₄ and c ₅ cytochromes) play a major role in nitrate-dependent U(IV) oxidation by T. denitrificans. Insertion mutations in each of the two genes encoding these cytochromes resulted in a greater than 50% decrease in U(IV) oxidation activity, and complementation in trans restored activity to wild-type levels. Sucrose-density-gradient ultracentrifugation confirmed that both cytochromes are membrane-associated. Insertion mutations in genes encoding other membrane-associated, c-type cytochromes did not diminish U(IV) oxidation. This is the first report of proteins involved in anaerobic U(IV) oxidation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration

Non‐alcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen‐activated kinases (p38γ and p38δ) have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of obese patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to diet‐induced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ‐deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild‐type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential target for NAFLD therapy.