HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury.

RATIONALE: Recent evidence indicates that histone deacetylase enzymes (HDACs) contribute to ischemia reperfusion (I/R) injury, and pan-HDAC inhibitors have been shown to be cardioprotective when administered either before an ischemic insult or during reperfusion. We have shown previously that selective inhibition of class I HDACs provides superior cardioprotection when compared to pan-HDAC inhibition in a pretreatment model, but selective class I HDAC inhibition has not been tested during reperfusion, and specific targets of class I HDACs in I/R injury have not been identified. OBJECTIVE: We hypothesized that selective inhibition of class I HDACs with the drug MS-275 (entinostat) during reperfusion would improve recovery from I/R injury in the first hour of reperfusion. METHODS AND RESULTS: Hearts from male Sprague-Dawley rats were subjected to ex vivo I/R injury±MS-275 class I HDAC inhibition during reperfusion alone. MS-275 significantly attenuated I/R injury, as indicated by improved LV function and tissue viability at the end of reperfusion. Unexpectedly, we observed that HDAC1 is present in the mitochondria of cardiac myocytes, but not fibroblasts or endothelial cells. We then designed mitochondria-restricted and mitochondria-excluded HDAC inhibitors, and tested both in our ex vivo I/R model. The selective inhibition of mitochondrial HDAC1 attenuated I/R injury to the same extent as MS-275, whereas the mitochondrial-excluded inhibitor did not. Further assays demonstrated that these effects are attributable to a decrease in SDHA activity and subsequent metabolic ROS production in reperfusion. CONCLUSIONS: We demonstrate for the first time that HDAC1 is present within the mitochondria of cardiac myocytes, and mitochondrial HDAC1 contributes significantly to I/R injury within the first hour of reperfusion.

GSNO promotes functional recovery in experimental TBI by stabilizing HIF-1α.

Traumatic brain injury (TBI) causes sustained disability due to compromised neurorepair mechanisms. Crucial to neurorepair and functional recovery following both TBI and stroke is hypoxia-inducible factor-1 alpha (HIF-1α). Based on reports that HIF-1α could be stabilized via S-nitrosylation, we tested the hypothesis that the S-nitrosylating agent S-nitrosoglutathione (GSNO) would stabilize HIF-1α, thereby stimulating neurorepair mechanisms and aiding in functional recovery. TBI was induced by controlled cortical impact (CCI) in adult rats. GSNO (0.05mg/kg) was administered at two hours after CCI. The treatment was repeated daily until the 14th day after CCI. Functional recovery was assessed by motor and cognitive functions, and the recovery was compared with the expression of HIF-1α. The mechanisms of GSNO-mediated S-nitrosylation of HIF-1α were determined using brain endothelial cells. While non-treated TBI animals showed sustained neurobehavioral deficits, GSNO treatment of TBI improved neurobehavioral functions. GSNO also increased the expression of HIF-1α and VEGF. The beneficial effects of GSNO on neurobehavioral functions in TBI animals were blocked by treatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME). The stimulatory effect of GSNO on VEGF was reversed not only by 2-ME but also by the denitrosylating agent dithiothreitol, confirming our hypothesis that GSNO's benefits are mediated by the stabilization of HIF-1α via S-nitrosylation. GSNO's S-nitrosylation of HIF-1α was further confirmed using a biotin switch assay in endothelial cells. The data provide evidence that GSNO treatment of TBI aids functional recovery through stabilizing HIF-1α via S-nitrosylation. GSNO is a natural component of the human brain/body, and its exogenous administration has not shown adverse effects in humans. Therefore, the translational potential of GSNO therapy in TBI is high.

Functional Characterization of IPSC-Derived Brain Cells as a Model for X-Linked Adrenoleukodystrophy.

X-ALD is an inherited neurodegenerative disorder where mutations in the ABCD1 gene result in clinically diverse phenotypes: the fatal disorder of cerebral childhood ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). The various models used to study the pathobiology of X-ALD disease lack the appropriate presentation for different phenotypes of cALD vs AMN. This study demonstrates that induced pluripotent stem cells (IPSC) derived brain cells astrocytes (Ast), neurons and oligodendrocytes (OLs) express morphological and functional activities of the respective brain cell types. The excessive accumulation of saturated VLCFA, a "hallmark" of X-ALD, was observed in both AMN OLs and cALD OLs with higher levels observed in cALD OLs than AMN OLs. The levels of ELOVL1 (ELOVL Fatty Acid Elongase 1) mRNA parallel the VLCFA load in AMN and cALD OLs. Furthermore, cALD Ast expressed higher levels of proinflammatory cytokines than AMN Ast and control Ast with or without stimulation with lipopolysaccharide. These results document that IPSC-derived Ast and OLs from cALD and AMN fibroblasts mimic the respective biochemical disease phenotypes and thus provide an ideal platform to investigate the mechanism of VLCFA load in cALD OLs and VLCFA-induced inflammatory disease mechanisms of cALD Ast and thus for testing of new therapeutics for AMN and cALD disease of X-ALD.

ABCD1 deletion-induced mitochondrial dysfunction is corrected by SAHA: implication for adrenoleukodystrophy.

X-linked Adrenoleukodystrophy (X-ALD), an inherited peroxisomal metabolic neurodegenerative disorder, is caused by mutations/deletions in the ATP-binding cassette transporter (ABCD1) gene encoding peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). Metabolic dysfunction in X-ALD is characterized by the accumulation of very long chain fatty acids ≥ C22:0) in the tissues and plasma of patients. Here, we investigated the mitochondrial status following deletion of ABCD1 in B12 oligodendrocytes and U87 astrocytes. This study provides evidence that silencing of peroxisomal protein ABCD1 produces structural and functional perturbations in mitochondria. Activities of electron transport chain-related enzymes and of citric acid cycle (TCA cycle) were reduced; mitochondrial redox status was dysregulated and the mitochondrial membrane potential was disrupted following ABCD1 silencing. A greater reduction in ATP levels and citrate synthase activities was observed in oligodendrocytes as compared to astrocytes. Furthermore, most of the mitochondrial perturbations induced by ABCD1 silencing were corrected by treating cells with suberoylanilide hydroxamic acid, an Histone deacetylase inhibitor. These observations indicate a novel relationship between peroxisomes and mitochondria in cellular homeostasis and the importance of intact peroxisomes in relation to mitochondrial integrity and function in the cell types that participate in the pathobiology of X-ALD. These observations suggest suberoylanilide hydroxamic acid as a potential therapy for X-ALD. Schematic description of the effects of loss of peroxisomal ATP-binding cassette transporter D1 (ABCD1) gene on cellular Redox and mitochondrial activities and their correction by suberoylanilide hydroxamic acid (SAHA) treatment. Pathogenomic accumulation of very long chain fatty acids (VLCFA) as a result of loss of ABCD1 leads to dysfunctions of mitochondrial biogenesis and its activities. Treatment with SAHA corrects mitochondrial dysfunctions. These studies describe unique cooperation between mitochondria and peroxisome for cellular activities.

Histone deacetylase inhibitor upregulates peroxisomal fatty acid oxidation and inhibits apoptotic cell death in abcd1-deficient glial cells.

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal ß-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

Incidence of Abcd1 level on the induction of cell death and organelle dysfunctions triggered by very long chain fatty acids and TNF-α on oligodendrocytes and astrocytes.

X-linked adrenoleukodystrophy (X-ALD) is characterized by ABCD1 deficiency. This disease is associated with elevated concentrations of very long chain fatty acids (C24:0 and C26:0) in the plasma and tissues of patients. Under its severe form, brain demyelination and inflammation are observed. Therefore, we determined the effects of C24:0 and C26:0 on glial cells:oligodendrocytes, which synthesize myelin, and astrocytes, which participate in immune response. So, 158N murine oligodendrocytes, rat C6 glioma cells, rat primary cultures of neuronal-glial cells, and of oligodendrocytes were treated for various periods of time in the absence or presence of C24:0 and C26:0 used at plasmatic concentrations found in X-ALD patients (1-5 µM) and higher (10, 20, 40 µM). To evaluate the importance of extrinsic and intrinsic factors, the part taken by TNF-α and reduced Abcd1 level was studied. Whatever the cells considered, no effects on cell growth and/or viability were detected at 1-5 µM, more or less pronounced effects were identified at 10 µM, and an induction of cell death with increased permeability to propidium iodide and loss of transmembrane mitochondrial potential was observed at 20-40 µM. On 158N, cell death was characterized by (i) an increased superoxide anion production at the mitochondrial level; (ii) the presence of vacuoles of different sizes and shapes; a destabilization of lysosomal membrane and a cytoplasmic redistribution of lysosomes; (iii) a modulation of Abcd3/PMP70 and Acox-1 protein expression, and a decrease in catalase activity at the peroxisomal level. When TNF-α was combined with C24:0 or C26:0 and used on 158N cells, C6 cells, and on 158N cells after siRNA mediated knockdown of Abcd1, no or slight potentiation was revealed. Thus, on the different cell models used, an induction of cell death with marked cellular dysfunctions at the mitochondrial, lysosomal, and peroxisomal levels were found with C24:0 and C26:0 at 20 µM and higher. However, in our experimental conditions, plasmatic concentrations of these fatty acids were unable to induce cell death, and organelle dysfunctions on oligodendrocytes and astrocytes, and additional intrinsic and environmental factors, such as reduced Abcd1 level and/or TNF-α, were ineffective to potentiate their side effects.

Pro-apoptotic versus anti-apoptotic properties of dietary resveratrol on tumoral and normal cardiac cells.

Resveratrol is a natural dietary polyphenol found in grape skin, red wine, and various other food products. Resveratrol has proved to be an effective chemopreventive agent for different malignant tumors. It has also been shown to prevent vascular alterations such as atherosclerosis and inflammatory-associated events. In view of these observations, we investigated the anti-proliferative and pro-apoptotic activities of resveratrol on a tumoral cardiac cell line (HL-1 NB) derived from mouse tumoral atrial cardiac myocytes. These effects were compared with those found on normal neonatal mouse cardiomyocytes. HL-1 NB cells and neonatal cardiomyocytes were treated with resveratrol (5, 30, and/or 100 µM) for different times of culture (24, 48, and/or 72 h). Resveratrol effects were determined by various microscopical and flow cytometric methods. After resveratrol treatment, a strong inhibition of tumoral cardiac HL1-NB cell growth associated with a loss of cell adhesion was observed. This cell proliferation arrest was associated with an apoptotic process revealed by an increased percentage of cells with fragmented and/or condensed nuclei (characteristic of apoptotic cells) identified after staining with Hoechst 33342 and by the presence of cells in subG1. At the opposite, on normal cardiomyocytes, no cytotoxic effects of resveratrol were observed, and a protective effect of resveratrol against norepinephrine-induced apoptosis was found on normal cardiomyocytes. Altogether, the present data demonstrate that resveratrol (1) induces apoptosis of tumoral cardiac HL1-NB cells, (2) does not induce cell death on normal cardiomyocytes, and (3) prevents norepinephrine-induced apoptosis on normal cardiomyocytes.

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes.

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 µg/mL corresponding to 50 µM, 24-48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3ß), and with degradation of Mcl-1. With α-tocopherol (400 µM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3ß dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3ß/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.

Impact of 7-ketocholesterol and very long chain fatty acids on oligodendrocyte lipid membrane organization: evaluation via LAURDAN and FAMIS spectral image analysis.

In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 µM), C24:0 and C26:0 (10, 20 µM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 µM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 µM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 µM), C24:0 and C26:0 (20 µM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono- and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 µM) is more efficient than C24:0 and C26:0 (20 µM), to trigger lateral lipid membrane disorganization.

Iron nanoparticles increase 7-ketocholesterol-induced cell death, inflammation, and oxidation on murine cardiac HL1-NB cells.

OBJECTIVE: To evaluate the cytotoxicity of iron nanoparticles on cardiac cells and to determine whether they can modulate the biological activity of 7-ketocholesterol (7KC) involved in the development of cardiovascular diseases. Nanoparticles of iron labeled with Texas Red are introduced in cultures of nonbeating mouse cardiac cells (HL1-NB) with or without 7-ketocholesterol 7KC, and their ability to induce cell death, pro-inflammatory and oxidative effects are analyzed simultaneously. STUDY DESIGN: Flow cytometry (FCM), confocal laser scanning microscopy (CLSM), and subsequent factor analysis image processing (FAMIS) are used to characterize the action of iron nanoparticles and to define their cytotoxicity which is evaluated by enhanced permeability to SYTOX Green, and release of lactate deshydrogenase (LDH). Pro-inflammatory effects are estimated by ELISA in order to quantify IL-8 and MCP-1 secretions. Pro-oxidative effects are measured with hydroethydine (HE). RESULTS: Iron Texas Red nanoparticles accumulate at the cytoplasmic membrane level. They induce a slight LDH release, and have no inflammatory or oxidative effects. However, they enhance the cytotoxic, pro-inflammatory and oxidative effects of 7KC. The accumulation dynamics of SYTOX Green in cells is measured by CLSM to characterize the toxicity of nanoparticles. The emission spectra of SYTOX Green and nanoparticles are differentiated, and corresponding factor images specify the possible capture and cellular localization of nanoparticles in cells. CONCLUSION: The designed protocol makes it possible to show how Iron Texas Red nanoparticles are captured by cardiomyocytes. Interestingly, whereas these fluorescent iron nanoparticles have no cytotoxic, pro-inflammatory or oxidative activities, they enhance the side effects of 7KC.

Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes.

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l-peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, Abcd1, Abcd2, Abcd3, and Acox1) involved in peroxisomal transport or beta-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l-peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.

Analysis of the capture and influence of nanoparticles on the cytotoxic effects of 7-ketocholesterol on cardiac cells: investigation by flow cytometry and by dynamic and spectral imaging microscopy associated with factor analysis of medical image sequences.

OBJECTIVE: To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties. STUDY DESIGN: Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine. RESULTS: Whereas the nanoparticles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nanoparticles were visualized to differentiate the emission spectra of SYTOX Green and nanoparticles and to precisely determine the cellular localization of nanoparticles. CONCLUSION: The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC.