iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

Effects of electroacupuncture at "Jiaji" (EX-B 2) combined with neurodynamic mobilization on gastrocnemius muscle atrophy and expression of NF-κB and MuRF1 in rabbits after sciatic nerve injury / 中国针灸

OBJECTIVES@#To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) combined with neurodynamic mobilization (NM) on the cross-sectional area of the gastrocnemius muscle fibers after sciatic nerve injury in rabbits, and the expression of nuclear factor κB (NF-κB) and muscle-specific ring-finger protein 1 (MuRF1).@*METHODS@#A total of 180 common-grade New Zealand rabbits (half male and half female) were randomly divided into five groups, i.e. a normal control group, a model control group, a NM group, an EA group and a combined intervention group, 36 rabbits in each group. Except in the normal control group, clipping method was used to prepare the model of sciatic nerve injury in the rest groups. On the 3rd day of successful modeling, NM was delivered in the NM group. In the EA group, EA was exerted at bilateral "Jiaji" (EX-B 2) of L4 to L6, stimulated with disperse-dense wave and the frequency of 2 Hz/100 Hz. In the combined intervention group, after EA delivered at bilateral "Jiaji" (EX-B 2) of L4 to L6 , NM was operated. The intervention in each group was delivered once daily, for 6 days a week, and lasted 1, 2 or 4 weeks according to the collection time of sample tissue. After 1, 2 and 4 weeks of intervention, in each group, the toe tension reflex score and the modified Tarlov test score were observed; the morphology of the gastrocnemius muscle was observed by HE staining and the cross-sectional area of muscular fiber was measured; using Western blot method, the expression of NF-κB and MuRF1 of the gastrocnemius muscle was detected.@*RESULTS@#After 1, 2 and 4 weeks of intervention, the toe tension reflex scores and the modified Tarlov scores in the model control group were lower than those of the normal control group (P<0.05), and these two scores in the NM group, the EA group and the combined intervention group were all higher than those of the model control group (P<0.05); the scores in the combined intervention group were higher than those in the EA group and the NM group (P<0.05). The gastrocnemius fibers were well arranged and the myocyte morphology was normal in the normal control group. In the model control group, the gastrocnemius fibers were disarranged, the myocytes were irregular in morphology and the inflammatory cells were infiltrated in the local. In the NM group, the EA group and the combined intervention group, the muscle fibers were regularly arranged when compared with the model control group. After 1, 2 and 4 weeks of intervention, the cross-sectional areas of the gastrocnemius muscle fibers in the model control group were smaller than those of the normal control group (P<0.05). The cross-sectional areas in the NM group, the EA group and the combined intervention group were larger than those of the model control group (P<0.05), and the cross-sectional areas in the combined intervention group were larger than those in the NM group and the EA group (P<0.05). After intervention for 1, 2 and 4 weeks, the protein expressions of NF-κB and MuRF1 in the gastrocnemius muscle were higher in the model control group in comparison of those in the normal control group (P<0.05). In the NM group, the EA group and the combined intervention group, the expressions of NF-κB after intervention for 1, 2 and 4 weeks and the expressions of MuRF1 after 2 and 4 weeks of intervention were lower when compared with those in the model control group (P<0.05). In the combined intervention group, the protein expressions of NF-κB after intervention for 1, 2 and 4 weeks and the expressions of MuRF1 after 2 and 4 weeks of intervention were decreased when compared with those in the NM group and the EA group (P<0.05).@*CONCLUSIONS@#Electroacupuncture at "Jiaji" (EX-B 2) combined with NM may increase the muscle strength and sciatic function and alleviate gastrocnemius muscle atrophy in the rabbits with sciatic nerve injury. The underlying mechanism is related to the inhibition of NF-κB and MuRF1 expression.

Effect of AMPK/PGC1 alpha on improving skeletal muscle atrophy in type 2 diabetic rats by aerobic exercise / 中国组织工程研究

BACKGROUND: The regulation of mitochondrial energy metabolism by adenylate activated protein kinase (AMPK) is an important cause of fat accumulation in obese and type 2 diabetic patients. Chronic inflammation will further induce skeletal muscle atrophy. Aerobic exercise can increase the activity of AMPK and regulate energy metabolism, but the mechanism of aerobic exercise in improving skeletal muscle atrophy in type 2 diabetes by increasing AMPK is unclear. OBJECTIVE: To explore the effect of aerobic exercise on skeletal muscle atrophy in type 2 diabetic rats and the role of AMPK. METHODS: The model of type 2 diabetic rats was established by high fat feeding and streptozotocin injection, and the rats were divided into four groups: control group (n=6), exercise group (n=9), diabetic control group (n=8) and diabetic exercise group (n=12). The control group and the diabetic control group were kept for 4 weeks, and the exercise group and the diabetic exercise group were given aerobic exercise intervention for 4 weeks. After 4 weeks of aerobic exercise (running speed 16 m/min, 60 min/d, 5 days/week), the muscle atrophy of soleus was observed by immunohistochemical staining. The expression levels of AMPK, PGC-1 α, MAFbx and MuRF1 were detected by western blot assay. The study protocol was approved by the Ethical Committee of School of Sport Science, Beijing Sport University in China on June 25, 2016, with approval No. 2016014. RESULTS AND CONCLUSION: Blood glucose of type 2 diabetes rats was significantly increased, and body weight and insulin levels of type 2 diabetes rats were significantly decreased (P < 0.01). The mean cross sectional area of soleus fiber in the diabetic group was significantly lower than that in the control group (P < 0.01), and the cross sectional area of soleus muscle fiber in the diabetic exercise group was significantly higher than that in the diabetic group (P < 0.01). The expression levels of AMPK and PGC-1 α in the soleus muscle of diabetic rats were significantly lower than those in the control group, and the expression levels of MAFbx and MuRF1 were significantly higher than those in the control group (P < 0.01). The expression levels of AMPK, MAFbx and MuRF1 in the diabetic exercise group were significantly higher than those in the diabetic group (P < 0.01). These results suggest that aerobic exercise can improve mitochondrial function, inhibit the expression of MAFbx and MuRF1, improve skeletal muscle atrophy and restore the metabolic balance of type 2 diabetes mellitus to some extent by activating AMPK/PGC-1α signaling pathway.

Expression of MuRF1 and MAFbx in donor and recipient muscles after musculocutaneous nerve transection and partial pectoralis major muscle transfer for reconstruction of elbow flexion in rats / Expresión de MuRF1 y MAFbx en músculos donantes y receptores después de transección del nervio musculocutáneo y transferencia parcial del músculo pectoral mayor para la reconstrucción de flexión de codo en ratas

The expression of MuRF1 and MAFbx in a denervated muscle has previously been studied. However, the expression of MuRF1 and MAFbx in the recipient and donor muscles after muscle transfer for reconstruction of joint function has not been sufficiently investigated. Forty-two adult Sprague-Dawley rats were divided into 7 groups: normal, 1 w post-, 2 w post-, and 4 w post-musculocutaneous nerve transection; and 1 w post-, 2 w post-, and 4 w post-reconstruction of elbow flexion. Muscle wet weights were assessed, and MuRF1 and MAFbx mRNA expressions were detected by polymerase chain reaction. The length of the oblique part of the pectoralis major of an SD rat is sufficient for suture to the insertion of the biceps brachii tendon. The muscle wet weight and the wet weight retention rate of the biceps brachii continued to decline after musculocutaneous nerve transection and a gradual increase was noted after the oblique part of the pectoralis major was transferred for reconstruction of elbow flexion. The oblique part of the pectoralis major showed a decrease of only 2­6%. The upregulated expression of MuRF1 and MAFbx in the biceps brachii reached a peak 2 w after denervation and 1 w after elbow flexion reconstruction, with an increase of 15% and 4%, respectively. This was followed by downregulation; however, the expression had not normalized at postoperative 4 w. The increased expression of MuRF1 (17%) and MAFbx (1%) in the oblique part of the pectoralis major at postoperative 1 w had decreased to below normal levels at postoperative 4 w. The transfer of the oblique part of the pectoralis major for elbow flexion reconstruction after musculocutaneous nerve transection can downregulate the expression of MuRF1 and MAFbx in the recipient muscle and causes only transient damage to the donor muscle in rats.

La expresión de MuRF1 y MAFbx en un músculo denervado ha sido estudiada previamente. Sin embargo, la expresión de MuRF1 y MAFbx en los músculos receptores y donantes después de la transferencia del músculo para la reconstrucción de la función articular no se ha investigado lo suficiente. Cuarenta y dos ratas adultas Sprague-Dawley fueron divididas en 7 grupos: normales, 1 semana post-, 2 semanas post- y 4 semanas post-transección del nervio musculocutáneo; y 1 semana post-, 2 semanas post-, y 4 semanas post-reconstrucción de la flexión del codo. Se evaluó el peso de los músculos húmedos, y las expresiones de MuRF1 y MAFbx mRNA fueron detectadas a través de reacción en cadena de la polimerasa. La longitud de la parte oblicua del músculo pectoral mayor de una rata Sprague-Dawley es suficiente para realizar la sutura en la inserción del tendón de músculo bíceps braquial. El peso húmedo del músculo bíceps braquial y su tasa de retención siguieron disminuyendo después de la sección del nervio musculocutáneo y un aumento gradual se observó después de la transferencia de la parte oblicua del músculo pectoral mayor para la reconstrucción de la flexión del codo. La parte oblicua del músculo pectoral mayor mostró una disminución de sólo 2-6%. La expresión regulada por incremento de MuRF1 y MAFbx en el bíceps braquial alcanzó un peak 2 semanas después de la denervación y 1 semana después de la reconstrucción de la flexión del codo, con un incremento del 15% y el 4%, respectivamente. Esto fue seguido por un regulación en baja. Sin embargo, la expresión no se normalizó en el postoperatorio de las 4 semanas. El aumento de la expresión de MuRF1 (17%) y MAFbx (1%) en la parte oblicua del músculo pectoral fue mayor en el postoperatorio de 1 semana, mientras que se encontró por debajo de los niveles normales en el postoperatorio de 4 semanas. La transferencia de la parte oblicua del músculo pectoral mayor para la reconstrucción de la flexión del codo después de la sección del nervio musculocutáneo puede regular a la baja la expresión de MuRF1 y MAFbx en el músculo receptor y provocar solo un daño transitorio en el músculo donado en ratas.

Effect of electro-acupuncture on skeletal muscle atrophy in mice / 全日本鍼灸学会雑誌

[Objective]Skeletal Muscle atrophy is induced in response to unloading by Hindlimb Suspension (HS). Numerous studies have been performed to prevent Skeletal Muscle atrophy. However, the molecular mechanisms underlying Electro-acupuncture (EA) on skeletal muscle have not been identified, and the effect of EA to prevent skeletal muscle atrophy is unknown. Therefore, we aimed to determine the effect of EA and Chishin (where the needles are kept in muscle for thirty minutes) on skeletal muscle atrophy in an animal experiment.<BR>[Methods]Twenty mice (8 week, C57BL6) were randomly grouped into 4 groups;Normal group (N group), Hindlimb Suspension Group (HS group), HS +Chishin group (Chishin group), and HS +EA 1Hz group (1Hz group) (n = 5/group, respectively). Acupuncture interventions were conducted on the gastrocnemius of the Chishin and 1Hz groups. We measured the muscle mass, the cross sectional area, the percentage of Antrogin-1 and MuRF1 (Atrophy related E3ubiquitin ligases) expressions of the soleus 0f each mouse at 2 weeks after acupuncture interventions.<BR>[Result]The weight and Cross sectional area of the soleus of the 1Hz group were significantly greater than the HS group (P<0.01, P<0.01, respectively). On the contrary, the percentage of Atrogin-1 and MuRF1 expressions of the 1Hz group was significantly less than the HS groups (P<0.01, P<0.05, respectively).<BR>[Conclusion]These results suggest that EA and Chishin may influence the activity of Atrogin-1 &MuRF1 expression.

p38 MAPK Participates in Muscle-Specific RING Finger 1-Mediated Atrophy in Cast-Immobilized Rat Gastrocnemius Muscle

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.