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

AIM: 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. METHODS: Adult female Sprague-Dawley rats aged 2 months were randomly assigned to control and exercise groups. Animals in exercise group were kept in cages equipped with free access running wheels to perform habitual exercise for 5 months. Animals in the control group were caged in the absence of running wheels. Animals were sacrificed after the 5-month experimental period. Plantaris muscle tissues were harvested for analysis. RESULTS: We showed that long-term habitual exercise enhanced basal autophagy, but without altering expressions of autophagy proteins in plantaris muscle. Interestingly, sirtuin protein, a possible regulator of autophagy, was upregulated in plantaris muscle. Furthermore, we suspected that different types of muscle fibre adapted to chronic exercise in different ways. Long-term habitual exercise resulted in fibre-type shifting from type IIX to IIA in both gastrocnemius muscle and plantaris muscle. Intriguingly, our analysis demonstrated that LC3-II protein abundance is positively correlated with the proportion of type IIA fibre whereas it was negatively correlated with the proportion of type IIX fibre in plantaris muscle. PGC-1α protein abundance was positively associated with the proportion of type IIA fibre and LC3-II in plantaris muscle. CONCLUSION: These results suggest that basal autophagy is enhanced in plantaris muscle after long-term habitual exercise and associated with fibre-type shifting.

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.

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

AIM: 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. METHODS: Adult C57BL/6 mice were randomly assigned to saline control (CON), doxorubicin (DOX), doxorubicin with treatment of acylated ghrelin (DOX+Acylated Ghrelin) and doxorubicin with treatment of unacylated ghrelin (DOX+Unacylated Ghrelin). Mice in all groups that involved DOX were intraperitoneally injected with 15 mg of doxorubicin per kg body weight, whereas mice in CON group received saline as placebo. Gastrocnemius muscle tissues were harvested after the experimental period for analysis. RESULTS: The elevation of apoptotic DNA fragmentation and number of TUNEL-positive nuclei were accompanied with the upregulation of Bax in muscle after exposure to doxorubicin, but all these changes were neither seen in the muscle treated with acylated ghrelin nor unacylated ghrelin after doxorubicin exposure. Protein abundances of autophagic markers including LC3 II-to-LC3 I ratio, Atg12-5 complex, Atg5 and Beclin-1 were not altered by doxorubicin but were upregulated by the treatment of either acylated or unacyated ghrelin. Histological analysis revealed that the amount of centronucleated myofibres was elevated in doxorubicin-treated muscle while muscle of others groups showed normal histology. CONCLUSIONS: Collectively, our data demonstrated that acylated ghrelin administration suppresses the doxorubicin-induced activation of apoptosis and enhances the cellular signalling of autophagy. The treatment of unacylated ghrelin has similar effects as acylated ghrelin on apoptotic and autophagic signalling, suggesting that the effects of ghrelin are probably mediated through a signalling pathway that is independent of GHSR-1a. These findings were consistent with the hypothesis that acylated ghrelin inhibits doxorubicin-induced upregulation of apoptosis in skeletal muscle while treatment of unacylated ghrelin can achieve similar effects as the treatment of acylated ghrelin. The inhibition of apoptosis and enhancement of autophagy induced by acylated and unacylated ghrelin might exert myoprotective effects on doxorubicin-induced toxicity in skeletal muscle.

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. METHODS: Adult Sprague-Dawley rats were subjected to an experimental model of pressure-induced deep tissue injury. Static pressure of 100 mmHg was applied to an area of 1.5 cm(2) over the mid-tibialis region of right limb of rats for one single session of 6-h compression (1D) or two sessions of 6-h compression over two consecutive days with rats sacrificed one day (2D) or immediately after (2D-IM) the compression. The left uncompressed limb served as the intra-animal control. Muscle tissues underneath compression region were collected for analysis. RESULTS: Our histological analysis indicated that pathohistological characteristics including rounding contour of myofibres and massive nuclei accumulation were apparently demonstrated in muscles of 2D and 2D-IM. In contrast, these pathohistological changes were generally not found in muscle following 1D. Apoptotic DNA fragmentation, terminal dUTP nick-end labelling index and caspase-3 protease activity were significantly elevated in compressed muscles of all groups. Caspase-9 enzymatic activity was found to be significantly increased in compressed muscles of 2D and 2D-IM whereas increase in caspase-8 activity was exclusively found in compressed muscle of 1D. According to our immunoblot analysis, FoxO3 was significantly reduced in compressed muscles of all groups whereas Beclin-1 was decreased only in 2D. LC3-I was significantly reduced in compressed muscles of all groups while LC3-II was decreased in 2D and 1D. No significant differences were found in the protein abundance of Akt and phospho-Akt in muscles among all groups. CONCLUSION: These data demonstrate the opposing responses of apoptosis and autophagy to moderate compression in muscle. Moreover, our findings suggest that cellular changes in apoptosis and autophagy have already taken place in the very early stage in which apparent histopathology has yet to develop in the process of compression-induced muscle pathology.