Role of protein kinase C and NF-κB in proteolysis-inducing factor-induced proteasome expression in C2C12 myotubes

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin–proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4alpha-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 muM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 muM). Proteolysis-inducing factor-induced activation of PKCalpha, with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKCalpha (pCO2) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKCalpha (pKS1), which showed no activation of PKCalpha in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-kappaB (NF-kappaB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-kappaBalpha and an increase in nuclear binding of NF-kappaB in pCO2, but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-kappaBalpha, induced by PIF, necessary for the release of NF-kappaB from its inactive cytosolic complex

Transcriptomic and Epigenetic Regulation of Disuse Atrophy and the Return to Activity in Skeletal Muscle

Physical inactivity and disuse are major contributors to age-related muscle loss. Denervation of skeletal muscle has been previously used as a model with which to investigate muscle atrophy following disuse. Although gene regulatory networks that control skeletal muscle atrophy after denervation have been established, the transcriptome in response to the recovery of muscle after disuse and the associated epigenetic mechanisms that may function to modulate gene expression during skeletal muscle atrophy or recovery have yet to be investigated. We report that silencing the tibialis anterior muscle in rats with tetrodotoxin (TTX)—administered to the common peroneal nerve—resulted in reductions in muscle mass of 7, 29, and 51% with corresponding reductions in muscle fiber cross-sectional area of 18, 42, and 69% after 3, 7, and 14 d of TTX, respectively. Of importance, 7 d of recovery, during which rodents resumed habitual physical activity, restored muscle mass from a reduction of 51% after 14 d TTX to a reduction of only 24% compared with sham control. Returning muscle mass to levels observed at 7 d TTX administration (29% reduction). Transcriptome-wide analysis demonstrated that 3714 genes were differentially expressed across all conditions at a significance of P ≤ 0.001 after disuse-induced atrophy. Of interest, after 7 d of recovery, the expression of genes that were most changed during TTX had returned to that of the sham control. The 20 most differentially expressed genes after microarray analysis were identified across all conditions and were cross-referenced with the most frequently occurring differentially expressed genes between conditions. This gene subset included myogenin (MyoG), Hdac4, Ampd3, Trim63 (MuRF1), and acetylcholine receptor subunit α1 (Chrna1). Transcript expression of these genes and Fboxo32 (MAFbx), because of its previously identified role in disuse atrophy together with Trim63 (MuRF1), were confirmed by real-time quantitative RT-PCR, and DNA methylation of their promoter regions was analyzed by PCR and pyrosequencing. MyoG, Trim63 (MuRF1), Fbxo32 (MAFbx), and Chrna1 demonstrated significantly decreased DNA methylation at key time points after disuse-induced atrophy that corresponded with significantly increased gene expression. Of importance, after TTX cessation and 7 d of recovery, there was a marked increase in the DNA methylation profiles of Trim63 (MuRF1) and Chrna1 back to control levels. This also corresponded with the return of gene expression in the recovery group back to baseline expression observed in sham-operated controls. To our knowledge, this is the first study to demonstrate that skeletal muscle atrophy in response to disuse is accompanied by dynamic epigenetic modifications that are associated with alterations in gene expression, and that these epigenetic modifications and gene expression profiles are reversible after skeletal muscle returns to normal activity

Corticosteroid effects on ventilator-induced diaphragm dysfunction in anesthetized rats depend on the dose administered

<p>Abstract</p> <p>Background</p> <p>High dose of corticosteroids has been previously shown to protect against controlled mechanical ventilation (CMV)-induced diaphragmatic dysfunction while inhibiting calpain activation. Because literature suggests that the calpain inhibiting effect of corticosteroid depends on the dose administered, we determined whether lower doses of corticosteroids would also provide protection of the diaphragm during CMV. This may be important for patients undergoing mechanical ventilation and receiving corticosteroids.</p> <p>Methods</p> <p>Rats were assigned to controls or to 24 hours of CMV while being treated at the start of mechanical ventilation with a single intramuscular administration of either saline, or 5 mg/kg (low MP) or 30 mg/kg (high MP) of methylprednisolone.</p> <p>Results</p> <p>Diaphragmatic force was decreased after CMV and this was exacerbated in the low MP group while high MP rescued this diaphragmatic dysfunction. Atrophy was more severe in the low MP group than after CMV while no atrophy was observed in the high MP group. A significant and similar increase in calpain activity was observed in both the low MP and CMV groups whereas the high dose prevented calpain activation. Expression of calpastatin, the endogenous inhibitor of calpain, was decreased in the CMV and low MP groups but its level was preserved to controls in the high MP group. Caspase-3 activity increased in all CMV groups but to a lesser extent in the low and high MP groups. The 20S proteasome activity was increased in CMV only.</p> <p>Conclusions</p> <p>Administration of 30 mg/kg methylprednisolone during CMV protected against CMV-induced diaphragm dysfunction while 5 mg/kg was more deleterious. The protective effect is due mainly to an inhibition of the calpain system through preservation of calpastatin levels and to a lesser extent to a caspase-3 inhibition.</p

Activation of the dopamine 1 and dopamine 5 receptors increase skeletal muscle mass and force production under non-atrophying and atrophying conditions

<p>Abstract</p> <p>Background</p> <p>Control of skeletal muscle mass and force production is a complex physiological process involving numerous regulatory systems. Agents that increase skeletal muscle cAMP levels have been shown to modulate skeletal muscle mass and force production. The dopamine 1 receptor and its closely related homolog, the dopamine 5 receptor, are G-protein coupled receptors that are expressed in skeletal muscle and increase cAMP levels when activated. Thus we hypothesize that activation of the dopamine 1 and/or 5 receptor will increase skeletal muscle cAMP levels thereby modulating skeletal muscle mass and force production.</p> <p>Methods</p> <p>We treated isolated mouse tibialis anterior (TA) and medial gastrocnemius (MG) muscles in tissue bath with the selective dopamine 1 receptor and dopamine 5 receptor agonist SKF 81297 to determine if activation of skeletal muscle dopamine 1 and dopamine 5 receptors will increase cAMP. We dosed wild-type mice, dopamine 1 receptor knockout mice and dopamine 5 receptor knockout mice undergoing casting-induced disuse atrophy with SKF 81297 to determine if activation of the dopamine 1 and dopamine 5 receptors results in hypertrophy of non-atrophying skeletal muscle and preservation of atrophying skeletal muscle mass and force production.</p> <p>Results</p> <p>In tissue bath, isolated mouse TA and MG muscles responded to SKF 81297 treatment with increased cAMP levels. Treating wild-type mice with SKF 81297 reduced casting-induced TA and MG muscle mass loss in addition to increasing the mass of non-atrophying TA and MG muscles. In dopamine 1 receptor knockout mice, extensor digitorum longus (EDL) and soleus muscle mass and force was not preserved during casting with SKF 81297 treatment, in contrast to significant preservation of casted wild-type mouse EDL and soleus mass and EDL force with SKF 81297 treatment. Dosing dopamine 5 receptor knockout mice with SKF 81297 did not significantly preserve EDL and soleus muscle mass and force although wild-type mouse EDL mass and force was significantly preserved SKF 81297 treatment.</p> <p>Conclusions</p> <p>These data demonstrate for the first time that treatment with a dopamine 1/5 receptor agonist results in (1) significant preservation of EDL, TA, MG and soleus muscle mass and EDL muscle force production during periods of atrophy and (2) hypertrophy of TA and MG muscle. These effects appear to be mainly mediated by both the dopamine 1 and dopamine 5 receptors.</p

Physiotherapists' experiences of physiotherapy interventions in scientific physiotherapy publications focusing on interventions for children with cerebral palsy: a qualitative phenomenographic approach

Background: Physiotherapy research concerning interventions for children with CP is often focused on collecting evidence of the superiority of particular therapeutic methods or treatment modalities. Articulating and documenting the use of theory, instrumentation and research design and the assumptions underlying physiotherapy research interventions are important. Physiotherapy interventions focusing on children with Cerebral Palsy should, according to the literature, be based on a functional and environmental perspective with task-specific functional activity, motor learning processes and Family-Centred Service i.e. to enhance motor ability and improve capacity so that the child can perform the tasks necessary to participate actively in everyday life. Thus, it is important to coordinate the norms and values of the physiotherapist with those of the family and child. The aim of this study was to describe how physiotherapists' experiences physiotherapy interventions for children with CP in scientific physiotherapy publications written by physiotherapists. Methods: A qualitative phenomenographic approach was used. Twenty-one scientific articles, found in PubMed, strategically chosen according to year of publication (2001-2009), modality, journals and country, were investigated. Results: Three qualitatively different descriptive categories were identified: A: Making it possible a functional-based intervention based on the biopsychosocial health paradigm, and the role of the physiotherapist as collaborative, interacting with the child and family in goal setting, intervention planning and evaluation, B: Making it work an impairment-based intervention built on a mixed health paradigm (biomedical and biopsychosocial), and the role of the physiotherapist as a coach, leading the goal setting, intervention planning and evaluation and instructing family members to carry out physiotherapist directed orders, and; C: Making it normal an impairment-based intervention built on a biomedical health paradigm, and the role of the physiotherapist as an authoritative expert who determine goals, intervention planning and evaluation. Conclusions: Different paradigms of health and disability lead to different approaches to physiotherapy which influence the whole intervention process regarding strategies for the assessment and treatment, all of which influence Family-Centred Service and the child's motor learning strategies. The results may deepen physiotherapists' understanding of how different paradigms of health influence the way in which various physiotherapy approaches in research seek to solve the challenge of CP

Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact

Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin–proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions

Variability of the chronic obstructive pulmonary disease key epidemiological data in Europe: systematic review

<p>Abstract</p> <p>Background</p> <p>Chronic obstructive pulmonary disease (COPD) is predicted to become a major cause of death worldwide. Studies on the variability in the estimates of key epidemiological parameters of COPD may contribute to better assessment of the burden of this disease and to helpful guidance for future research and public policies. In the present study, we examined differences in the main epidemiological characteristics of COPD derived from studies across countries of the European Union, focusing on prevalence, severity, frequency of exacerbations and mortality, as well as on differences between the studies' methods.</p> <p>Methods</p> <p>This systematic review was based on a search for the relevant literature in the Science Citation Index database via the Web of Science and on COPD mortality rates issued from national statistics. Analysis was finally based on 65 articles and Eurostat COPD mortality data for 21 European countries.</p> <p>Results</p> <p>Epidemiological characteristics of COPD varied widely from country to country. For example, prevalence estimates ranged between 2.1% and 26.1%, depending on the country, the age group and the methods used. Likewise, COPD mortality rates ranged from 7.2 to 36.1 per 10⁵inhabitants. The methods used to estimate these epidemiological parameters were highly variable in terms of the definition of COPD, severity scales, methods of investigation and target populations. Nevertheless, to a large extent, several recent international guidelines or research initiatives, such as GOLD, BOLD or PLATINO, have boosted a substantial standardization of methodology in data collection and have resulted in the availability of more comparable epidemiological estimates across countries. On the basis of such standardization, severity estimates as well as prevalence estimates present much less variation across countries. The contribution of these recent guidelines and initiatives is outlined, as are the problems remaining in arriving at more accurate COPD epidemiological estimates across European countries.</p> <p>Conclusions</p> <p>The accuracy of COPD epidemiological parameters is important for guiding decision making with regard to preventive measures, interventions and patient management in various health care systems. Therefore, the recent initiatives for standardizing data collection should be enhanced to result in COPD epidemiological estimates of improved quality. Moreover, establishing international guidelines for reporting research on COPD may also constitute a major contribution.</p

The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

BACKGROUND: By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. METHODS: Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. RESULTS: Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. CONCLUSION: We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132