Xiao M. Pei

Muscle apoptosis is induced in pressure-induced deep tissue injury.

Pressure ulcer is a complex and significant health problem. Although the factors including pressure, shear, and ischemia have been identified in the etiology of pressure ulcer, the cellular and molecular mechanisms that contribute to the development of pressure ulcer are unclear. This study tested the hypothesis that the early-onset molecular regulation of pressure ulcer involves apoptosis in muscle tissue. Adult Sprague-Dawley rats were subjected to an in vivo protocol to mimic pressure-induced deep tissue injury. Static pressure was applied to the tibialis region of the right limb of the rats for 6 h each day on two consecutive days. The compression force was continuously monitored by a three-axial force transducer equipped in the compression indentor. The contralateral uncompressed limb served as intra-animal control. Tissues underneath the compressed region were collected for histological analysis, terminal dUTP nick-end labeling (TUNEL), cell death ELISA, immunocytochemical staining, and real-time RT-PCR gene expression analysis. The compressed muscle tissue generally demonstrated degenerative characteristics. TUNEL/dystrophin labeling showed a significant increase in the apoptotic muscle-related nuclei, and cell death ELISA demonstrated a threefold elevation of apoptotic DNA fragmentation in the compressed muscle tissue relative to control. Positive immunoreactivities of cleaved caspase-3, Bax, and Bcl-2 were evident in compressed muscle. The mRNA contents of Bax, caspase-3, caspase-8, and caspase-9 were found to be higher in the compressed muscle tissue than control. These results demonstrated that apoptosis is activated in muscle tissue following prolonged moderate compression. The data are consistent with the hypothesis that muscle apoptosis is involved in the underlying mechanism of pressure-induced deep tissue injury.

Acylated and unacylated ghrelin inhibit doxorubicin-induced apoptosis in skeletal muscle.

Doxorubicin, a potent chemotherapeutic drug, has been demonstrated previously as an inducer of apoptosis in muscle cells. Extensive induction of apoptosis may cause excessive loss of muscle cells and subsequent functional decline in skeletal muscle. This study examined the effects of acylated ghrelin, a potential agent for treating cancer cachexia, on inhibiting apoptotic signalling in doxorubicin‐treated skeletal muscle. Unacylated ghrelin, a form of ghrelin that does not bind to GHSR‐1a, is also employed in this study to examine the GHSR‐1a signalling dependency of the effects of ghrelin.

Desacyl ghrelin prevents doxorubicin-induced myocardial fibrosis and apoptosis via the GHSR-independent pathway

Doxorubicin is an effective chemotherapeutic agent used to treat malignancies, but it causes cardiomyopathy. Preliminary evidence suggests that desacyl ghrelin might have protective effects on doxorubicin cardiotoxicity. This study examined the cellular effects of desacyl ghrelin on myocardial fibrosis and apoptosis in a doxorubicin cardiomyopathy experimental model. Adult C57BL/6 mice received an intraperitoneal injection of doxorubicin to induce cardiomyopathy, followed by 4-day treatment of saline (control) or desacyl ghrelin with or without [d-Lys3]-GHRP-6 (a growth hormone secretagogue receptor or GHSR1a antagonist). Ventricular structural and functional parameters were evaluated by transthoracic echocardiography. Molecular and cellular measurements were performed in ventricular muscle to examine myocardial fibrosis and apoptosis. Cardiac dysfunction was induced by doxorubicin, as indicated by significant decreases in ventricular fractional shortening and ejection fraction. This doxorubicin-induced cardiac dysfunction was prevented by the treatment of desacyl ghrelin no matter with or without the presence of [d-Lys3]-GHRP-6. Doxorubicin induced fibrosis (accumulated collagen deposition and increased CTGF), activated apoptosis (increased TUNEL index, apoptotic DNA fragmentation, and caspase-3 activity and decreased Bcl-2/Bax ratio), and suppressed phosphorylation status of prosurvival signals (ERK1/2 and Akt) in ventricular muscles. All these molecular and cellular alterations induced by doxorubicin were not found in the animals treated with desacyl ghrelin. Notably, the changes in the major markers of apoptosis, fibrosis, and Akt phosphorylation were found to be similar in the animals following the treatment of desacyl ghrelin with and without GHSR antagonist [d-Lys3]-GHRP-6. These findings demonstrate clearly that desacyl ghrelin protects the cardiomyocytes against the doxorubicin-induced cardiomyopathy by preventing the activation of cardiac fibrosis and apoptosis, and the effects are probably mediated through GHSR-independent mechanism.

Habitual exercise increases resistance of lymphocytes to oxidant‐induced DNA damage by upregulating expression of antioxidant and DNA repairing enzymes

The underlying mechanisms of adaptation from staying physically active are not completely revealed. This study examined the effects of 8 and 20 weeks of habitual voluntary exercise on the susceptibility of lymphocytes to oxidant‐induced DNA damage, antioxidant enzyme activities in cardiac and skeletal muscles, and circulatory antioxidant profile. Forty young adult rats were randomly assigned to sedentary control and exercise groups for an experimental period of 8 or 20 weeks. Animals assigned to exercise groups were subjected to 24 h daily free access to an in‐cage running wheel with circumference of 1.19 m. A magnetic digital counter was attached to the running wheel to record daily exercise distance run by the animals. Control rats were housed in cages without a running wheel, located next to the exercised animals. Body weight and food intake were recorded weekly. After the experimental periods of 8 and 20 weeks, blood, left ventricle, soleus and plantaris muscles were collected for analysis. No significant difference was found in plasma total antioxidant capacity between exercised and control animals in the 8 and 20 week groups according to our ferric reducing/antioxidant power (FRAP) analysis. However, modified FRAP for ascorbic acid (FRASC) analysis indicated that plasma ascorbic acid content was significantly increased by 46 and 34% in 8 and 20 week exercise groups, respectively, when compared with the corresponding control groups. Superoxide dismutase (SOD) activity was significantly elevated by 39% in erythrocytes of animals exercised for 8 weeks relative to control animals. In the 20 week exercise group, Glutathione peroxidase (GPx) activity in ventricle and plantaris was significantly upregulated by 477 and 290%, respectively, relative to control values. As demonstrated by comet assay, the oxidant‐induced DNA damage was significantly reduced by 21 and 45% in lymphocytes of animals exercised for 8 and 20 weeks, respectively, when compared with the corresponding control lymphocytes. Our qRT‐PCR analysis showed that the transcript expression of SOD2 was significantly elevated by 939% in lymphocytes of animals exercised for 8 weeks relative to control animals. Increased expressions of SOD2 (by 19%), catalase (25%), APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1; 46%), Protein kinase, DNA‐activated, catalytic polypeptide (Prkdc; 9%) and O‐6‐methylguanine‐DNA methyltransferase (Mgmt; 26%) were found in lymphocytes of animals exercised for 20 weeks relative to control rats. These results demonstrate that habitual exercise confers increased resistance of lymphocytes to oxidant‐induced DNA damage, and this protective effect is possibly attributed to the regular exercise‐induced elevated expression of antioxidant and DNA repairing enzymes.

Autophagic adaptation is associated with exercise‐induced fibre‐type shifting in skeletal muscle

Acute exercise is known to activate autophagy in skeletal muscle. However, little is known about how basal autophagy in skeletal muscle adapts to chronic exercise. In the current study we aim to, firstly, examine whether long‐term habitual exercise alters the basal autophagic signalling in plantaris muscle and, secondly, examine the association between autophagy and fibre‐type shifting.

Opposing responses of apoptosis and autophagy to moderate compression in skeletal muscle

Aim:  The molecular mechanism that contributes to the pathogenesis of deep pressure ulcer remains to be elucidated. This study tested the hypotheses that: (1) apoptosis and autophagy are activated in compression‐induced muscle pathology and (2) apoptotic and autophagic changes precede pathohistological changes in skeletal muscle in response to prolonged moderate compression.

Unacylated ghrelin restores insulin and autophagic signaling in skeletal muscle of diabetic mice

Impairment of insulin signaling in skeletal muscle detrimentally affects insulin-stimulated disposal of glucose. Restoration of insulin signaling in skeletal muscle is important as muscle is one of the major sites for disposal of blood glucose. Recently, unacylated ghrelin (UnAG) has received attention in diabetic research due to its favorable actions on improving glucose tolerance, glycemic control, and insulin sensitivity. The investigation of UnAG has entered phase Ib clinical trial in type 2 diabetes and phase II clinical trial in hyperphagia in Prader-Willi syndrome. Nonetheless, the precise mechanisms responsible for the anti-diabetic actions of UnAG remain incompletely understood. In this study, we examined the effects of UnAG on restoring the impaired insulin signaling in skeletal muscle of db/db diabetic mice. Our results demonstrated that UnAG effectively restored the impaired insulin signaling in diabetic muscle. UnAG decreased insulin receptor substrate (IRS) phosphorylation, increased protein kinase B (Akt) phosphorylation, and, hence, suppressed mTOR signaling. Consequently, UnAG enhanced Glut4 localization and increased PDH activity in the diabetic skeletal muscle. Intriguingly, our data indicated that UnAG normalized the suppressed autophagic signaling in diabetic muscle. In conclusion, our findings illustrated that UnAG restored the impaired insulin and autophagic signaling in skeletal muscle of diabetic mice, which are valuable to understand the underlying mechanisms of the anti-diabetic action of UnAG at peripheral skeletal muscle level.

Proteasome inhibition alleviates prolonged moderate compression-induced muscle pathology

BackgroundThe molecular mechanism initiating deep pressure ulcer remains to be elucidated. The present study tested the hypothesis that the ubiquitin proteasome system is involved in the signalling mechanism in pressure-induced deep tissue injury.MethodsAdult Sprague Dawley rats were subjected to an experimental compression model to induce deep tissue injury. The tibialis region of the right hind limb was subjected to 100 mmHg of static pressure for six hours on each of two consecutive days. The compression pressure was continuously monitored by a three-axial force transducer within the compression indentor. The left hind limb served as the intra-animal control. Muscle tissues underneath the compressed region were collected and used for analyses.ResultsOur results demonstrated that the activity of 20S proteasome and the protein abundance of ubiquitin and MAFbx/atrogin-1 were elevated in conjunction with pathohistological changes in the compressed muscle, as compared to control muscle. The administration of the proteasome inhibitor MG132 was found to be effective in ameliorating the development of pathological histology in compressed muscle. Furthermore, 20S proteasome activity and protein content of ubiquitin and MAFbx/atrogin-1 showed no apparent increase in the MG132-treated muscle following compression.ConclusionOur data suggest that the ubiquitin proteasome system may play a role in the pathogenesis of pressure-induced deep tissue injury.

Doxorubicin Induces Inflammatory Modulation and Metabolic Dysregulation in Diabetic Skeletal Muscle.

Anti-cancer agent doxorubicin (DOX) has been demonstrated to worsen insulin signaling, engender muscle atrophy, trigger pro-inflammation, and induce a shift to anaerobic glycolytic metabolism in skeletal muscle. The myotoxicity of DOX in diabetic skeletal muscle remains largely unclear. This study examined the effects of DOX on insulin signaling, muscle atrophy, pro-/anti-inflammatory microenvironment, and glycolysis metabolic regulation in skeletal muscle of db/db diabetic and db/+ non-diabetic mice. Non-diabetic db/+ mice and diabetic db/db mice were randomly assigned to the following groups: db/+CON, db/+DOX, db/dbCON, and db/dbDOX. Mice in db/+DOX and db/dbDOX groups were intraperitoneally injected with DOX at a dose of 15 mg per kg body weight whereas mice in db/+CON and db/dbCON groups were injected with the same volume of saline instead of DOX. Gastrocnemius was immediately harvested, weighed, washed with cold phosphate buffered saline, frozen in liquid nitrogen, and stored at −80°C for later analysis. The effects of DOX on diabetic muscle were neither seen in insulin signaling markers (Glut4, pIRS1Ser636∕639, and pAktSer473) nor muscle atrophy markers (muscle mass, MuRF1 and MAFbx). However, DOX exposure resulted in enhancement of pro-inflammatory favoring microenvironment (as indicated by TNF-α, HIFα and pNFκBp65) accompanied by diminution of anti-inflammatory favoring microenvironment (as indicated by IL15, PGC1α and pAMPKβ1Ser108). Metabolism of diabetic muscle was shifted to anaerobic glycolysis after DOX exposure as demonstrated by our analyses of PDK4, LDH and pACCSer79. Our results demonstrated that there might be a link between inflammatory modulation and the dysregulation of aerobic glycolytic metabolism in DOX-injured diabetic skeletal muscle. These findings help to understand the pathogenesis of DOX-induced myotoxicity in diabetic muscle.

Autophagic adaptations to long-term habitual exercise in cardiac muscle

Autophagy has been shown to be responsive to physical exercise. However, the effects of prolonged habitual exercise on autophagy in cardiac muscle remain unknown. The present study aimed to examine whether long-term habitual exercise alters the basal autophagic signalling in cardiac muscle. Female Sprague-Dawley rats aged 2 months were randomly assigned to control and exercise groups. Animals in exercise group were kept in cages with free access exercise wheels to perform habitual exercise for 5 months. Animals in the control group were placed in cages without exercise wheels. Ventricular muscle tissues were harvested for analysis after 5 months. Phosphorylation statuses of upstream autophagic regulatory proteins and protein expressions of downstream autophagic facts remained unchanged in the cardiac muscle of exercise animals when compared to control animals. Intriguingly, the protein abundance of microtubule-associated protein-1 light chain -3 II (LC3-II), heat shock protein 72 (HSP72) and peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α) were significantly increased in cardiac muscle of exercise rats relative to control rats. 5 months of habitual exercise causes the adaptive increase in LC3-II reserve without altering autophagic flux, which probably contributes to the elevation of cellular autophagic capacity and efficiency of cardiac muscle.