Changes in αB-crystallin,tubulin, and MHC isoforms by hindlimb unloading show different expression patterns in various hindlimb muscles

[Purpose]

αB-crystallin is a small heat shock protein that acts as a molecular chaperone under various stress conditions. Microtubules, which consist of tubulin, are related to maintain the intracellular organelles and cellular morphology. These two proteins have been shown to be related to the properties of different types of myofibers based on their contractile properties. The response of these proteins during muscular atrophy, which induces a myofibril component change, is not clearly understood.

[Methods]

We performed 15 days of hindlimb unloading on rats to investigate the transitions of these proteins by analyzing their absolute quantities. Protein contents were analyzed in the soleus, plantaris, and gastrocnemius muscles of the unloading and control groups (N = 6).

[Results]

All three muscles were significantly atrophied by hindlimb unloading (P < 0.01): soleus (47.5%), plantaris (16.3%), and gastrocnemius (21.3%) compared to each control group. αB-crystallin was significantly reduced in all three examined unloaded hindlimb muscles compared to controls (P < 0.01) during the transition of the myosin heavy chain to fast twitch muscles. α-Tubulin responded only in the unloaded soleus muscle. Muscle atrophy induced the reduction of αB-crystallin and α-tubulin expressions in plantar flexor muscles with a shift to the fast muscle fiber compared to the control.

[Conclusion]

The novel finding of this study is that both proteins, αB-crystallin and α-tubulin, were downregulated in slow muscles (P < 0.01); However, α-tubulin was not significantly reduced compared to the control in fast muscles (P < 0.01).     

Role of mitochondria in apoptosis induced by the 2‐5A system and mechanisms involved

The 2‐5A system (2-5OAS/RNaseL) is composed of the 2′,5′oligoadenylate synthetase 1 (2-5OAS1) and 2-5A-dependent RNase (RNaseL), enzymes that play a key role in antiviral defence mechanisms. Activation of the 2-5A system by double stranded RNA (dsRNA) induces degradation of ribosomal RNAs and apoptosis in mammalian cells. To obtain further information into the molecular mechanisms by which RNaseL induces apoptosis, we expressed human RNaseL and 2-5OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis. In this expression virus-cell system the activation of RNaseL, as index of rRNA degradation, is an upstream event of apoptosis induction. RNaseL induces apoptosis in a caspase-dependent manner (caspases 8, 9 and 2). At the beginning of apoptosis RNaseL and 2-5OAS are localized in the mitochondria and cytosol fractions, while at the onset of apoptosis both enzymes are largely in mitochondria. The 2-5A system induces the release of Cytochrome c from mitochondria to cytosol in a caspase dependent manner. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (ΔΦm), as well as the generation of reactive oxygen species (ROS). Moreover, the activation of RNaseL induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of ΔΦm induced by the activation of RNaseL. These findings provide new insights into the molecular mechanisms of apoptosis induction by the 2-5A system, demonstrating the importance of mitochondria in 2-5OAS/RNaseL-induced apoptosis.     

PGC-1α and FOXO1 mRNA levels and fiber characteristics of the soleus and plantaris muscles in rats after hindlimb unloading

Fifteen-week-old rats were subjected to unloading induced by hindlimb suspension for 3 weeks. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and forkhead box-containing protein O1 (FOXO1) mRNA levels and fiber profiles of the soleus and plantaris muscles in rats subjected to unloading (unloaded group) were determined and compared with those of age-matched control rats (control group). The body weight and both the soleus and plantaris muscle weights were lower in the unloaded group than in the control group. The PGC-1α mRNA was downregulated in the soleus, but not in the plantaris muscle of the unloaded group. The FOXO1 mRNA was upregulated in both the soleus and plantaris muscles of the unloaded group. The oxidative enzyme activity was reduced in the soleus, but not in the plantaris muscle of the unloaded group. The percentage of type I fibers was decreased and the percentages of type IIA and IIC fibers were increased in the soleus muscle of the unloaded group, whereas there was no change in fiber type distribution in the plantaris muscle of the unloaded group. Atrophy of all types of fibers was observed in both the soleus and plantaris muscles of the unloaded group. We conclude that decreased oxidative capacity and fiber atrophy in unloaded skeletal muscles are associated with decreased PGC-1α and increased FOXO1 mRNA levels.     

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17β-estradiol on isolated canine basilar and internal carotid arteries

Progesterone and 17β-estradiol induce vasorelaxation through non-genomic mechanisms in several isolated blood vessels; however, no study has systematically evaluated the mechanisms involved in the relaxation induced by 17β-estradiol and progesterone in the canine basilar and internal carotid arteries that play a key role in cerebral circulation. Thus, relaxant effects of progesterone and 17β-estradiol on KCl- and/or PGF_2α-pre-contracted arterial rings were investigated in absence or presence of several antagonists/inhibitors/blockers; the effect on the contractile responses to CaCl₂ was also determined. In both arteries progesterone (5.6–180 μM) and 17β-estradiol (1.8–180 μM): (1) produced concentration-dependent relaxations of KCl- or PGF_2α-pre-contracted arterial rings; (2) the relaxations were unaffected by actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM), potassium channel blockers and ICI 182,780 (only for 17β-estradiol). In the basilar artery the vasorelaxation induced by 17β-estradiol was slightly blocked by tetraethylammonium (10 mM) and glibenclamide (K_ATP; 10 μM). In both arteries, progesterone (10–100 μM), 17β-estradiol (3.1–31 μM) and nifedipine (0.01–1 μM) produced a concentration-dependent blockade of the contraction to CaCl₂ (10 μM–10 mM). These results suggest that progesterone and 17β-estradiol produced relaxation in the basilar and internal carotid arteries by blockade of L-type voltage dependent Ca²⁺ channel but not by genomic mechanisms or production of cAMP/cGMP. Potassium channels did not play a role in the relaxation to progesterone in both arteries or in the effect of 17β-estradiol in the internal carotid artery; meanwhile K_ATP channels play a minor role on the effect of 17β-estradiol in the basilar artery.     

Adult and juvenile dermatomyositis: are the distinct clinical features explained by our current understanding of serological subgroups and pathogenic mechanisms?

Adult and juvenile dermatomyositis share the hallmark features of pathognomic skin rash and muscle inflammation, but are heterogeneous disorders with a range of additional disease features and complications. The frequency of important clinical features such as calcinosis, interstitial lung disease and malignancy varies markedly between adult and juvenile disease. These differences may reflect different disease triggers between children and adults, but whilst various viral and other environmental triggers have been implicated, results are so far conflicting. Myositis-specific autoantibodies can be detected in both adults and children with idiopathic inflammatory myopathies. They are associated with specific disease phenotypes and complications, and divide patients into clinically homogenous subgroups. Interestingly, whilst the same autoantibodies are found in both adults and children, the disease features remain different within autoantibody subgroups, particularly with regard to life-threatening disease associations, such as malignancy and rapidly progressive interstitial lung disease. Our understanding of the mechanisms that underlie these differences is limited by a lack of studies directly comparing adults and children. Dermatomyositis is an autoimmune disease, which is believed to develop as a result of an environmental trigger in a genetically predisposed individual. Age-specific host immune responses and muscle physiology may be additional complicating factors that have significant impact on disease presentation. Further study into this area may produce new insights into disease pathogenesis.     

The FcRβ- and γ-ITAMs play crucial but distinct roles in the full activation of mast cells induced by IgEκ and Protein L

Previous studies suggested that Protein L (PpL), the bacterial Ig-binding protein, activates mast cells. PpL presumably performs the activation by interacting with membrane-bound IgEκ, but the underlying mechanisms behind the process remain unclear. In the current study, we found that cell-surface FcεRI expression is a critical factor participant in PpL-mediated full activation of murine mast cells, which includes cytokine production, the degranulation response, and leukotriene C(4) (LTC(4)) release, and that engagement of the FcεRI with IgEκ and PpL is enough to induce tyrosine phosphorylation of ITAM in the FcRβ- and γ-signaling subunits. Introduction of mutations in two canonical tyrosine residues (Y47F/Y58F) of the FcRγ-ITAM completely abolished the above-mentioned mast cell functions, with the exception of LTC(4) release. Importantly, the FcRβ-ITAM acts as a signal transducer that is responsible for LTC(4) release independently of the FcRγ-ITAM. Taken together, our results suggest crucial and distinct functions for the FcRβ- and γ-ITAMs in the FcεRI-dependent full activation of mast cells induced by IgEκ and PpL.     

Inflammatory cytokines IL-1β and TNF-α regulate p75NTR expression in CNS neurons and astrocytes by distinct cell-type-specific signalling mechanisms

The p75^NTR (where NTR is neurotrophin receptor) can mediate many distinct cellular functions, including cell survival and apoptosis, axonal growth and cell proliferation, depending on the cellular context. This multifunctional receptor is widely expressed in the CNS (central nervous system) during development, but its expression is restricted in the adult brain. However, p75^NTR is induced by a variety of pathophysiological insults, including seizures, lesions and degenerative disease. We have demonstrated previously that p75^NTR is induced by seizures in neurons, where it induces apoptosis, and in astrocytes, where it may regulate proliferation. In the present study, we have investigated whether the inflammatory cytokines IL (interleukin)-1β and TNF-α (tumour necrosis factor-α), that are commonly elevated in these pathological conditions, mediate the regulation of p75^NTR in neurons and astrocytes. We have further analysed the signal transduction pathways by which these cytokines induce p75^NTR expression in the different cell types, specifically investigating the roles of the NF-κB (nuclear factor κB) and p38 MAPK (mitogen-activated protein kinase) pathways. We have demonstrated that both cytokines regulate p75^NTR expression; however, the mechanisms governing this regulation are cytokine- and cell-type specific. The distinct mechanisms of cytokine-mediated p75^NTR regulation that we demonstrate in the present study may facilitate therapeutic intervention in regulation of this receptor in a cell-selective manner.     

Can prenatal exposure to a 900 MHz electromagnetic field affect the morphology of the spleen and thymus,and alter biomarkers of oxidative damage in 21-day-old male rats?

We investigated the effects of a 900 Megahertz (MHz) electromagnetic field (EMF), applied during the prenatal period, on the spleen and thymus of 21-day-old male rat pups. Pregnant Sprague-Dawley rats were divided into control and EMF groups. We applied 900 MHz EMF for 1 h/day to the EMF group of pregnant rats. Newborn male rat pups were removed from their mothers and sacrificed on postnatal day 21. Spleen and thymus tissues were excised and examined. Compared to the control group, thymus tissue malondialdehyde levels were significantly higher in the group exposed to EMF, while glutathione levels were significantly decreased. Increased malondialdehyde and glutathione levels were observed in splenic tissue of rats exposed to EMF, while a significant decrease occurred in superoxide dismutase values compared to controls. Transmission electron microscopy showed pathological changes in cell morphology in the thymic and splenic tissues of newborn rats exposed to EMF. Exposure to 900 MHz EMF during the prenatal period can cause pathological and biochemical changes that may compromise the development of the male rat thymus and spleen.     

Rat hindlimb unloading: Soleus and Extensor Digitorum Longus histochemistry,mitochondrial DNA content and mitochondrial DNA deletions

Mitochondrial phenotypic alterations, mitochondrial DNA content and mitochondrial DNA deletions in a slow, Soleus, and a fast, Extensor Digitorum Longus, skeletal muscle of 3- and 15-month-old hindlimb suspended rats have been studied. Cytochrome c oxidase-negative fibers appeared after unloading in all examined animals and their percentage increased with increasing unloading time. After 14 days of suspension the mitochondrial DNA content did not change in 3-month-old but decreased significantly in 15-month-old rats. Soleus was much more affected by unloading than Extensor Digitorum Longus. The mitochondrial DNA deletion of 4834 bp as well as other mtDNA deletions, researched with Long Distance-PCR, were absent in both studied muscles before and after unloading.     

Influence of hindlimb unloading on the modulation of neurotransmitter secretion through the autoreceptor system

In experiments on neuromuscular junctions of fast (m. extensor digitorum longus, EDL) and slow (m. soleus) muscles of rats under hindlimb unloading of varied duration, we compared the intensity of spontaneous quantal secretion of neurotransmitter in response to potassium depolarization and activation of presynaptic cholinoreceptors with a nonhydrolyzable acetylcholine analog. Secretion was assessed by the mean frequency of miniature endplate potentials. In the controls, carbachol raised this index by 363% in EDL and by 62% in soleus. Secretion in the fast muscle was also more sensitive to [K⁺]. Hindlimb unloading abolished the sensitivity to carbachol in EDL while in soleus it did not change. Preservation of the sensitivity of the fast muscle to potassium depolarization suggested that unloading reduced the number of functional presynaptic receptors.     

Cardiomyocyte death induced by ischaemic/hypoxic stress is differentially affected by distinct purinergic P2 receptors

Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown. In this study, we aimed at investigating the role of ATP and specific P2 receptors in the appearance of cell injury in a cardiac model of ischaemic/hypoxic stress. Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N₂ for 16 hrs in a glucose‐free medium. In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone‐associated‐DNA‐fragments, a marker of apoptosis. Addition of either apyrase, which degrades extracellular ATP, or various inhibitors of ATP release via connexin hemichannels fully abolished ischaemic/hypoxic stress‐associated apoptosis. To dissect the role of specific P2 receptor subtypes, we used a combined approach: (i) non‐selective and, when available, subtype‐selective P2 antagonists, were added to cardiomyocytes before ischaemic/hypoxic stress; (ii) selected P2 receptors genes were silenced via specific small interfering RNAs. Both approaches indicated that the P2Y₂ and P2χ₇ receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y₄ receptor has a protective effect. Overall, these findings indicate a role for ATP and its receptors in modulating cardiomyocyte damage during ischaemic/hypoxic stress.     

Mayhem of the multiple mechanisms: modelling neurodegeneration in prion disease

This review examines recent attempts to advance the understanding of the mechanism by which neurones die in prion disease. Prion diseases or transmissible spongiform encephalopathies are characterized by the conversion of a normal glycoprotein, the prion protein, to a protease-resistant form that is suggested to be both the infectious agent and the cause of the rapid neurodegeneration in the disease. Death of the patient results from this widespread neuronal loss. Thus understanding the mechanism by which the abnormal form of the prion protein causes neuronal death might lead to treatments that would prevent the life-threatening nature of these diseases.     

Mechanical stimulation of the plantar foot surface attenuates soleus muscle atrophy induced by hindlimb unloading in rats.

Unloading-induced muscle atrophy occurs in the aging population, bed-ridden patients, and astronauts. This study was designed to determine whether dynamic foot stimulation (DFS) applied to the plantar surface of the rat foot can serve as a countermeasure to soleus muscle atrophy normally observed in hindlimb unloaded (HU) rats. Forty-four mature (6 mo old), male Wistar rats were randomly assigned to ambulatory control, HU alone, HU with active DFS (i.e., plantar contact with active inflation), HU with passive DFS (i.e., plantar contact without active inflation), and HU while wearing a DFS boot with no plantar contact groups. Application of active DFS during HU significantly counteracted the atrophic response by preventing approximately 85% of the reduction in type I myofiber cross-sectional area (CSA) in the soleus while preventing approximately 57% of the reduction in type I myofiber CSA and 43% of the reduction in type IIA myofiber CSA of the medial gastrocnemius muscle. Wearing of a DFS boot without active inflation prevented myofiber atrophy in the soleus of HU animals in a fashion similar to that observed in HU animals that wore an actively inflated DFS boot. However, when a DFS boot without plantar surface contact was worn during HU, no significant protection from HU-induced myofiber atrophy was observed. These results illustrate that the application of mechanical foot stimulation to the plantar surface of the rat foot is an effective countermeasure to muscle atrophy induced by HU.     

Modulation of caspase-3 activity by zinc ions and by the cell redox state

It is known that DNA fragmentation during apoptosis is controlled by a number of factors, a crucial step being the caspase-operated cleavage of ICAD, the DNase inhibitor. We have previously demonstrated that hydrogen peroxide-treated lymphocytes undergo apoptosis without formation of a DNA ladder; however, the use of micromolar amounts of a Zn(2+) chelator allowed DNA cleavage at internucleosomal sites. Such results were extended in the present work, thus allowing their framing into the events related to alterations in the redox state of the cell. Apoptosis in hydrogen peroxide-treated lymphocytes was found to occur with caspase-3 activation, but the enzyme activity was found to be impaired, thus affecting internucleosomal fragmentation as well as nuclear morphology. Caspase-3 activity was found to resume upon mild Zn(2+) chelation. These results provide as well an experimental model from which apoptotic events upstream and downstream of caspase-3 activity can be examined.     

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes

Alterations in intracellular Zn²⁺ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn²⁺ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn²⁺ for Ca²⁺ and Mg²⁺ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential ( m). Therefore, cytoplasmic and mitochondrial events are critical to this process.     

Hypoxia‐induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase‐1

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia‐induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl₂). Both the hypoxic gas mixture (1% O₂) and chemical hypoxia‐induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia‐mediated activation of caspase‐1. Active caspase‐1 induced the classical caspase‐dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase‐3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase‐1/classical caspase‐dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase‐3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.     

Renal cell apoptosis induced by nephrotoxic drugs: cellular and molecular mechanisms and potential approaches to modulation

Apoptosis plays a central role not only in the physiological processes of kidney growth and remodeling, but also in various human renal diseases and drug-induced nephrotoxicity. We present in a synthetic fashion the main molecular and cellular pathways leading to drug-induced apoptosis in kidney and the mechanisms regulating it. We illustrate them using three main nephrotoxic drugs (cisplatin, gentamicin, and cyclosporine A). We discuss the main regulators and effectors that have emerged as key targets for the design of therapeutic strategies. Novel approaches using gene therapy, antisense strategies, recombinant proteins, or compounds obtained from both classical organic and combinatorial chemistry are examined. Finally, key issues that need to be addressed for the success of apoptosis-based therapies are underlined.     

Thrombocytopenia induced by the histone deacetylase inhibitor abexinostat involves p53-dependent and -independent mechanisms

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34⁺ cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.