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Enhancing Autophagy Protects Against Sepsis-Induced Neuromuscular Dysfunction Associated with Qualitative Changes to Acetylcholine Receptors

Chen, Jingyuan; Min, Su; Xie, Fei; Yang, Jun; Wang, Xin

doi: 10.1097/SHK.0000000000001189
Basic Science Aspects
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ABSTRACT Sepsis-induced myopathy is a heavy burden for patients during respiratory failure as well as after discharge, which could be characterized with qualitative changes to nAChR in a rat model of sepsis, regulated by NRG-1. Autophagy is an innate immune defense mechanism against microbial challenges. We found neuromuscular dysfunction in anterior tibial muscle of male Sprague-Dawley rats 24 h after cecal ligation and puncture (CLP). CLP resulted in increased systemic and local inflammation in anterior tibial muscle tissue. The start-up phase of autophagy, as measured by LC3II, was activated immediately after CLP and continued until 24 h; the degradation phase was suppressed until 24 h, after a brief increase at 4 h (revealed by p62). NRG-1 first increased, and then decreased to a level lower than that in the sham group. Meanwhile, expression of γ- and α7- acetylcholine receptors was detected at 8 and 16 h after CLP; levels increased continuously until 24 h. Subsequently, we investigated the significance of autophagy in CLP-induced neuromuscular dysfunction by treatment with rapamycin or 3-methyladenine, which were classical pharmaceuticals for enhancing or suppressing autophagy. Rapamycin activated autophagy, limited the CLP-induced systemic pro-inflammatory response and blood bacterial load without affecting local inflammatory response, upregulated NRG-1, downregulated γ- and α7-acetylcholine receptors, and improved 7-day neuromuscular function and survival rate. In contrast, 3-methyladenine enhanced local inflammatory response, suppressed autophagy, worsened 7-day neuromuscular function. We conclude that impaired autophagy may contribute to sepsis-induced neuromuscular dysfunction in young male rats. Enhancing autophagy with rapamycin alleviated qualitative changes to acetylcholine receptors without triggering local anti-inflammatory response and improved anterior tibial muscle function in septic early phase (24 h) as well as in septic chronic phase (7d). Enhancing autophagy soon after sepsis is a potential strategy for treatment of sepsis-induced myopathy.

Department of Anesthesiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

Address reprint requests to Prof. Su Min, MD, Department of Anesthesiology, First Affiliated Hospital of Chongqing Medical University, Friendship Road 1#, Yuan Jia Gang, Chongqing 400016, China. E-mail: ms89011068@163.com

Received 28 February, 2018

Revised 14 March, 2018

Accepted 18 May, 2018

J-YC contributed to the design and conduction of this study, data analysis, and writing of this manuscript. SM approved the final manuscript. FX and JY participated in the establishment of the animal model and recording of neuromuscular function. XW recorded the results of molecular biology and analyzed the data. All authors read and approved the final manuscript.

This work was supported by the National Natural Science Foundation of China (No. 81471282), the Foundation of Postgraduate Innovation Project of Chongqing province, China (CYB17102), the Municipal Medical Key Discipline of Chongqing (YuWeiKeJiao No. 2007-2), and the Chongqing education commission (KJ1702027).

The authors report no conflicts of interest.

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INTRODUCTION

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (1), which may progress to septic shock and multiple organ failure and is a leading cause of death in hospitals (2). Global incidence has been estimated at 31.5 million, with 5.3 million deaths annually (3), and hospital mortality due to sepsis was 35.3%, which was significantly higher than general hospital mortality (4). Most importantly, sepsis survivors suffer from impaired health-related quality of life in physical and mental domains after discharge, and nearly half of them cannot return to work in the first year, mainly due to neuromuscular dysfunction after a stay in the intensive care unit (5, 6).

The key mechanism of sepsis-induced neuromuscular dysfunction remains unclear. Previously, we found sepsis-induced myopathy was characterized with qualitative changes to nAChRs in a rat model of sepsis (7). These changes included abnormal emergence of fetal γ-nicotinic acetylcholine receptors (γ-nAChR) and neuronal α7-nAChR (7, 8), which were characterized with low single channel electric conductivity, resulting in poor skeletal muscle excitability. In the early fetal stage, before innervation, γ- and α7-nAChR are scattered throughout the muscle membrane (9, 10). These two isoforms are subsequently downregulated. Denervation and some other pathological states (e.g., burn, immobilization, aging) lead to re-expression of γ- and α7-nAChR (7, 11–13).

Neuromodulation protein (neuregulin) mediates neurogenic growth factor signal, which regulates expression, differentiation, and stabilization of postsynaptic acetylcholine receptors (14). Among activity factors that induce nAChRs, neuregulin 1 (NRG-1) plays the primary role. Release of NRG-1 from nerve endings triggers descending pathways, acts on gene promoter sequences, and regulates nAChRs expression (13, 15). In some pathological states (e.g., sepsis, burn, immobilization, aging), down-regulation of NRG-1 leads to up-regulation of γ- and α7-nAChR (7, 10–12).

Autophagy plays an important role in sepsis. In sepsis animal model, enhancing autophagy positively attenuates acute lung and myocardia injuries, improves liver, kidney, and immune functions, and even prolongs survival intervals (16–18). Notably, Eva Masiero et al. (19) blocked autophagy specifically in skeletal muscle in conditional and inducible knockout mice for the critical gene Atg7, and then they observed myopathy. Therefore, there might be some associations between autophagy and sepsis-induced neuromuscular dysfunction, and the potential mechanism remains unknown. In this study, we tested the hypothesis that enhancing autophagy could protect against sepsis-induced neuromuscular dysfunction by reversing qualitative changes in nAChRs, which may provide a new therapeutic strategy for septic myopathy.

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MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (2–3 months old, weight range 200–250 g) were obtained from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). All rats received humane treatment according to the regulations of the Institutional Animal Care and Use Committee of Chongqing Medical University. One week before the experiments, rats were housed in a specific pathogen-free laboratory in an acclimatized room under standard room conditions (25°C ± 2°C, 55% humidity), with a 12-h light–dark cycle. Rats were allowed free access to water and standard chow. All experimental procedures involving animals were approved by the Animal Ethics and Use Committee of Chongqing Medical University.

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Animal models and group assignments

For surgical intervention, all rats were anesthetized with 0.5% sodium pentobarbital (65 mg/kg), administered intraperitoneally. All rats were weighed daily and randomly divided into two groups: a sepsis group, in which cecal ligation and puncture (CLP) was performed; a sham group, in which a sham operation was performed (n = 10). Rats in the sepsis group were assigned to one of the four subgroups: CLP-4 h (n = 10), CLP-8 h (n = 10), CLP-16 h (n = 10), and CLP-24 h (n = 10), based on the time frame of the sepsis model. Animals were sacrificed at 4, 8, 16, or 24 h after surgery. Blood was drawn, and bilateral anterior tibial muscles were removed. Based on the manner of pharmaceutical intervention, rats in the sepsis group were assigned to one of the three subgroups: CLP-DMSO (n = 10), CLP-Rapa (n = 10), and CLP-3-MA (n = 10). In the intervention groups, rats received vehicle (10% dimethyl sulfoxide (DMSO), 4 mL/kg body weight (BW); Sigma, St. Louis, Mo), rapamycin (Rapa 10 mg/kg BW; BioVision, Mountain View, Calif), or 3-methyladenine (3-MA; 15 mg/kg; BioVision, Mountain View, Calif) intraperitoneally at 1 h after CLP. Doses of Rapa and 3-MA were based on previous reports (16, 17). To conduct the survival study, rats in the sepsis group were assigned to one of the four subgroups: CLP (n = 20), CLP-DMSO (n = 20), CLP-Rapa (n = 20), and CLP-3-MA (n = 20). The CLP group was just conducted CLP procedures, while CLP-DMSO, CLP-Rapa, and CLP-3-MA groups were administrated with the respective agents after CLP procedures. The dosage was the same as the above.

Sepsis was induced by CLP, as described previously (20). Midline laparotomy was performed to expose the cecum. The cecum was ligated tightly with a 3-0 silk suture at its base, below the ileocecal valve, and punctured with a 24-G needle, to avoid obstruction of the bowel. A small amount of feces was exteriorized by gentle pressure applied to the ligated cecum. The cecum was then returned to the peritoneal cavity. The abdomen was closed with 3-0 silk. Rats in the sham group received the same anesthesia and surgical manipulation without CLP. Immediately after surgery, each rat received one injection of 0.9% NS (10 mL i.p.) for resuscitation.

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Western blot analysis

Tissues were homogenized using lysis buffer (Beyotime, China), and supernatants were collected after centrifugation at 12,000 × g for 15 min at 4°C. After quantitative analysis of protein concentration, total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, Mass), blocked with 5% non-fat milk in Tris-buffered saline for 1 h at 37°C, and then incubated overnight at 4°C with primary antibodies. After incubation for 1 h at 37°C with secondary antibody (1:2,000; Beyotime), bands were seen using the enhanced chemilumine-scence kit (Beyotime) according to the manufacturer's protocol. The primary antibodies used were as follows: anti-neuregulin-1 (1:500, sc-28916, Santa Cruz Biotechnology Inc, Dallas, Tex), anti-γ-nAChR (1:500, sc-13998, Santa Cruz Biotechnology Inc, Dallas, Tex), anti-α7-nAChR (1:1,000, ab10096, Abcam, Ltd, Cambridge, Mass), anti-LC3II (1:500, 4108, Cell Signaling Technology, Danvers, Mass), anti-p62 (1:500, 5114, Cell Signaling Technology, Danvers, Mass), anti-GAPDH (1:1,000, AF1186, Beyotime Institute of Biotechnology, Jiangsu Province, China). All results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.

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Immunofluorescence

Four-micrometer-thick transverse and longitudinal cryosections of the anterior tibial muscle were incubated in a blocking solution (10% normal goat serum in phosphate-buffered saline, pH 7.4) for 10 min at room temperature. The sections were incubated with a rabbit anti-a7-nAChR polyclonal antibody (1:500, ab10096, Abcam Ltd, Cambridge, Mass) or a rabbit antiα7-nAChR (1:200, sc-13998, Santa Cruz Biotechnology Inc, Dallas, Tex) overnight at 4°C. After washing in phosphate-buffered saline, we incubated the sections with a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (1:100, sc-2012, Santa Cruz Inc, Santa Cruz, Calif) to stain for α7-nAChR or with a Cy3-conjugated goat anti-rabbit IgG antibody (1:1,000, ab6939, Abcam Ltd, Cambridge, Mass) to stain for α7- nAChR at room temperature for 1 h. The sections were mounted and examined under an Olympus IX70 inverted microscope with a Xuorescent attachment (Olympus, Tokyo, Japan) and a Photonic Science CCD camera (Olympus DP50, Tokyo, Japan), controlled by the VueMinder Lite (version 1.0) software (Pixera Corporation, Kawasaki, Japan).

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Evaluation of neuromuscular function

Electromyography (EMG) recordings were measured at each group. The data were obtained from the right sciatic nerve that was stimulated supramaximally (intensity, 2 V; duration, 0.2 ms; and frequency, 1 Hz) by a direct stimulation electrode (RM6240 Systems Inc, Cheng Du, China) from the sciatic nerve. Compound muscle action potential (CMAP) was recorded using a superficial disc electrode located on the tibialis anterior muscle before and at different times after surgery, as described previously. Electromyographical analysis used the RM6240USB2.0S (I) version 1.0.2 software (RM6240 Systems, Chengdu Instrument Company, Chengdu, China), with latency, amplitude, and duration of CMAP as the parameters. The nerve conduction velocity (NCV) was calculated as the distance of conduction/latency time. The temperature of each rat was kept at 36–37°C using a heating light. Neuromuscular dysfunction was defined as a decrease of ≥ 20% of the lower limit of the normal CMAP amplitude (7).

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Biochemical and cytokine analysis

C-reactive protein in serum was measured using commercially available Enzyme-linked immunosorbent assay (ELISA) kits (C-reactive protein; BioVendor Laboratory Medicine, Czech Republic). The concentrations of interleukin 6 (IL-6) in the serum and muscle tissue homogenates were measured using commercially available cytometric bead array inflammation kits (BD Biosciences, Franklin Lakes, NJ). Data analysis was carried out using CellQuest Pro and FCAP Array software (BD Biosciences, Franklin Lakes, NJ).

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Bacterial load

Bacterial count was assessed as previously described (21). Twenty-four hours after sham or CLP procedures, rats were anesthetized, and blood and muscle were collected. The muscle of sham and CLP rats were weighed and homogenized in 1 mL of 0.9% saline using a tissue homogenizer. The solid tissue was allowed to sediment for 10 min at room temperature, and the supernatants were serially diluted. Aliquots (20 mL) of each sample were plated on tryptic soy agar plates (Sigma-Aldrich, St. Louis, Mo). The number of colonies was enumerated after incubation at 37 °C overnight.

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Survival studies

To determine the role of autophagic agents on mortality from CLP-induced sepsis, survival studies were performed. Following sham or CLP procedures, rats were observed every 24 h for up to 7 days. All rats had free access to water and food. Rats mortality was monitored by operators blinded to strain information.

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Statistical analysis

Statistical analyses were performed using 1-way analysis of variance (ANOVA) and Tukey post-hoc test or the Student t test. The data are represented as mean values ± standard error of the mean. Survival analysis was performed using the Kaplan–Meier method and the log-rank test. SPSS software (version 17.0, SPSS, RRID: SCR_002865) was used for all analyses. All calculations were 2-tailed.

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RESULTS

Sepsis model

In survival study, all sham-operated rats survived through the end of the study (100%), and 7-day survival rate arrived 40% (P = 0.017; Fig. 1A). Death occurred in the first 3 days, mostly the first day. CRP, serum IL-6, and muscle tissue IL-6 were significantly increased to a high level in CLP-16 h and CLP-24 h groups, as compared with that in the sham group (P < 0.05; Fig. 1, B–D). After laparotomy, most CLP animals showed crouching, piloerection, decreased spontaneous locomotor activity, and exudation in the peri-orbital area. Necropsy of dead animals showed that the peritoneal cavity contained abundant bloody and malodorous fluid, and the cecum was distended and gangrenous. Surviving CLP rats developed diarrhea.

Fig. 1

Fig. 1

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Neuromuscular dysfunction

Compound muscle action potentials (CMAP) recorded for sham and CLP groups were shown in Figure 2. As compared with sham group, latency period in CLP-24 h group was significantly prolonged (P < 0.05; Fig. 2B), while other groups did not reach significance (P > 0.05; Fig. 2B). The duration of CMAP gradually increased from 4.24 ± 0.12 ms in the sham group to 4.31 ± 0.12 ms at 4 h (P > 0.05), 4.63 ± 0.14 ms at 8 h (P < 0.05), 5.04 ± 0.15 ms at 16 h (P < 0.05), and 6.12 ± 0.11 ms at 24 h (P < 0.05) in the CLP groups (Fig. 2C). The opposite tendency was observed in the amplitude of CMAP. The CMAP amplitude dropped from 17.61 ± 0.65 mV in the sham group to 15.97 ± 1.37 mV at 4 h (P > 0.05), 14.13 ± 1.11 mV at 8 h (P < 0.05), 8.66 ± 1.08 mV at 16 h (P < 0.05), and 7.01 ± 0.58 mV at 24 h (P < 0.05) in the CLP groups (Fig. 2D). Notably, CMAP amplitude decreased significantly in the CLP-16 h and CLP-24 h groups (>20%, P < 0.05; Fig. 2D), compared with the sham group, which indicated neuromuscular dysfunction. Similar results were observed in the nerve conduction velocity (Fig. 2E).

Fig. 2

Fig. 2

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Altered nAChRs activity in anterior tibial muscle

Expression of NRG-1, γ- and α7- nAChR was detected by western blot (Fig. 3A). Compared with the sham group, NRG-1 decreased significantly at 24 h (P < 0.05) after an initial increase and peaked at 8 h (P < 0.05; Fig. 3, A and B). γ- and α7- nAChR levels increased significantly at CLP 8 h and 16 h, respectively, then increased continuously until 24 h (P < 0.05; Fig. 3, A, C, and D).

Fig. 3

Fig. 3

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Impaired autophagy in anterior tibial muscle

Protein expression levels of microtubule-associated protein light chain 3-II (LC3-II) and sequestosome1 (p62) were quantified by western blot analysis to evaluate regulation of autophagy. The results showed that expression of LC3-II increased significantly at 4 and 8 h, then gradually decreased until 24 h after CLP; however, levels consistently remained higher than observed in the sham group (P < 0.05; Fig. 4, A and B). After a brief reduction at 4 h, levels of p62 dramatically increased until 24 h (P < 0.05; Fig. 4, A and C). Thus, impaired autophagy was accompanied by the emergence of γ- and α7- nAChR. We speculated that impaired autophagy may play a role in CLP-induced qualitative changes to nAChRs.

Fig. 4

Fig. 4

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Effects of regulating autophagy on 7-day survival rates, and 24-h inflammation and bacterial load

In survival study, 7-day survival rate was 100% in the sham group and 40% in the DMSA group, and administration of Rapa after CLP significantly improved 7-day survival rate to 65% while treatment with 3-MA decreased 7-day survival rate to 35% (P = 0.02; Fig. 5A). To investigate the systemic inflammatory response and systemic bacterial load, blood samples were collected at 24 h after CLP and analyzed for CRP, IL-6, and bacterial count. Administration of Rapa after CLP significantly decreased blood levels of CRP, IL-6, and bacterial count, indicating that Rapa may alleviate systemic inflammation and bacteremia in sepsis. In contrast, treatment with 3-MA showed no such salutary effect (P < 0.05; Fig. 5, B, C, and E). Anterior tibial muscle was collected at 24 h after CLP and analyzed for IL-6, a marker of local inflammation; and bacterial count, reflecting local bacterial load. Administration of 3-MA after CLP significantly increased muscle tissue levels of IL-6 (P < 0.05; Fig. 5D). However, no difference between the DMSO and Rapa groups was identified (P > 0.05; Fig. 5D). Muscle bacterial count showed no statistical significance among the four groups (P > 0.05; Fig. 5F).

Fig. 5

Fig. 5

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Effect of regulating autophagy on 24-h neuromuscular function after CLP

Representative CMAP recordings for each group are presented in (Fig. 6A). Latency period increased significantly in the DMSO group, compared with the sham group. Latency period recovered significantly after Rapa administration but worsened after treatment with 3-MA, compared with the DMSO group (P < 0.05; Fig. 6B). Duration of CMAP was significantly prolonged by treatment with DMSO, compared with sham treatment. No significant difference was found when mice were treated with Rapa, compared with DMSO. However, treatment with 3-MA, compared with DMSO, prolonged the duration of CMAP (P < 0.05; Fig. 6C). In the sham group, CMAP amplitude was 17.60 (0.65) mV. In the DMSO group, CMAP amplitude significantly decreased. However, CMAP amplitude was restored when rats were treated with Rapa after CLP; CMAP amplitude worsened after treatment with 3-MA (P < 0.05; Fig. 6D). Nerve conduction velocity significantly decreased in the DMSO group, compared with the sham group. Nerve conduction velocity recovered significantly after Rapa administration but worsened after treatment with 3-MA (P < 0.05; Fig. 6E).

Fig. 6

Fig. 6

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Effect of regulating autophagy on nAChRs activity in anterior tibial muscle

In anterior tibial muscle tissue harvested from rats 24 h after the sham procedure or CLP, expression of NRG-1 increased in the Rapa group but decreased in the 3-MA group, compared to the DMSO group (P < 0.05; Fig. 7, A and B). Expression of γ- and α7-nAChR was significantly increased after CLP, compared with sham treatment. However, expression of γ- and α7-nAChR was suppressed when rats were treated with Rapa after CLP. In contrast, treatment with 3-MA showed no such salutary effect (P > 0.05; Fig. 7, A, C, and D).

Fig. 7

Fig. 7

Immunofluorescent (IF) staining showed γ- and α7-nAChR immunoreactivity in the skeletal muscle membrane in the sham group or CLP groups (Fig. 8A). The results were consistent with those obtained by western blots (Fig. 7). In the CLP groups, the immunoreactivity of γ- and α7-nAChR stains was much higher than that in the sham group. In the Rapa group, the number of positive cells stained with anti-γ- or anti-α7-nAChR in the skeletal muscle decreased, compared with those in DMSO group (P < 0.05; Fig. 8, B and C). In contrast, treatment with 3-MA showed no such salutary effect (P > 0.05; Fig. 8, B and C).

Fig. 8

Fig. 8

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Rapa and 3-MA regulate autophagy in anterior tibial muscle after CLP

Anterior tibial muscle was harvested from rats 24 h after the sham procedure or CLP. Levels of LC3-II were significantly higher in the Rapa group but significantly lower in the 3-MA group, compared with the DMSO group. Levels of p62 were significantly lower in the Rapa group, compared with the DMSO group (P < 0.05; Fig. 9).

Fig. 9

Fig. 9

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Effect of regulating autophagy on 7-day neuromuscular function after CLP

Representative CMAP recordings for each group are presented in Fig. 10A. In the CLP groups, latency period increased continuously till 7th day after CLP, higher than that in the sham group. Among the Day 7 groups, the latency period of Rapa group decreased significantly compared with the DMSO group (P < 0.05; Fig. 10B). Duration of CMAP was significantly prolonged in CLP groups compared with Sham group. Among the Day 7 groups, the duration of CMAP of Rapa group decreased significantly, while the 3-MA group increased significantly, compared with DMSO group (P < 0.05; Fig. 10C). CMAP amplitude decreased significantly in CLP groups compared with Sham group. Among the Day 7 groups, the CMAP amplitude of Rapa group increased significantly, while the 3-MA group decreased significantly, compared with DMSO group (P < 0.05; Fig. 10D). Nerve conduction velocity decreased significantly in CLP groups compared with Sham group. Among the Day 7 groups, the nerve conduction velocity of Rapa group increased significantly compared to DMSO group (P < 0.05; Fig. 10E).

Fig. 10

Fig. 10

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DISCUSSION

We initially detected that sepsis-induced myopathy was characterized with abnormal emergence of fetal γ-nAChR and neuronal α7-nAChR, which could lead to poor skeletal muscle excitability (7). However, the underlying mechanism remained unclear. In the present study, we further explored the mechanism about sepsis-induced myopathy and the potential role of autophagy in this disease. After establishment of CLP in rat model, neuromuscular dysfunction was observed in anterior tibial muscle at 24 h. Expression of γ- and α7- nAChR increased gradually within the first 24 h. Levels of NRG-1 initially increased, then decreased to a level lower than that observed in the sham group. Autophagy initially increased but then decreased too. This phenomenon could be explained as decompensation of protective factors, leading to expression of γ- and α7-nAChR. Thus, it was reasonable to speculate that there existed an important association between autophagy and sepsis-induced qualitative changes to nAChRs.

We also sought to verify the role of autophagy in regulating NRG-1, γ- and α7-nAChR. To begin with, we found that enhanced autophagy with Rapa led to increased survival rates, alleviated neuromuscular dysfunction, and attenuation of the systemic inflammatory response but no effect on local inflammation in anterior tibialis. Furthermore, we also found that enhancing autophagy with Rapa improved the level of NRG-1, which then suppressed the expression of γ- and α7-nAChR; and limiting autophagy with 3-MA decreased NRG-1 expression, which in turn promoted the expression of γ- and α7-nAChR. These findings indicated that enhanced autophagy protected against neuromuscular dysfunction via increased expression of NRG-1 and decreased expression of γ- and α7-nAChR but without participation of local anti-inflammatory pathways. Thus, we concluded that impaired autophagy regulated NRG-1, and further affected γ- and α7-nAChR, resulting in neuromuscular dysfunction. Enhancing autophagy in the early stages after CLP could prevent NRG-1 form decreasing, and decrease γ- and α7-nAChR production, which would significantly alleviate neuromuscular dysfunction.

We used a septic neuromyopathy model that is considered to be appropriate for studying the pathophysiology of neuromuscular dysfunction (22, 23). CLP-induced sepsis is characterized by a hyper-dynamic state in the early phase (first 5 days after CLP), followed by a hypo-dynamic state during the chronic phase (6–28 days after CLP) (24, 25). We focused on the first 24 h after CLP, aiming to nip the neuromuscular dysfunction in the bud. The results showed that intervention with Rapa or 3-MA could make effects on neuromuscular function not only in septic early phase (24 h), but also in septic chronic phase (7d). Rapa and 3-MA was chosen as pharmacological intervention. Rapa has been widely used to experimentally induce autophagy because of its ability to block the inhibitory action of mammalian target of Rapa on Atg1 (autophagy-related gene 1) (26). 3-MA is a PtdIns3K inhibitor that effectively blocks an early stage of autophagy by inhibiting the class III PtdIns3K (26, 27). We choose the dose of 3-MA as 15 mg/kg, referring to the literature (28). It could inhibit autophagy without acute toxicity. Our results revealed that 3-MA treatment exacerbated myopathy, enhanced local inflammatory factor IL-6, without regulating NRG-1, γ- and α7-nAChR, which may be secondary to a different mechanism of action of Rapa. According to the literature, 3-MA has off-target effects, especially on immune cells. Several studies have demonstrated that inhibition of autophagy with 3-MA can have effects on cytokine transcription, processing, and secretion, including IL-6 family members (29, 30). IL-6 has been suggested as a potential mediator of muscle weakness. It directly increases muscle proteolysis, indirectly interferes with the growth hormone/insulin-like growth factor-1 pathway, contributing to muscle atrophy (31). Thus, 3-MA increased muscle IL-6, deteriorated neuromuscular dysfunction.

LC3 II and p62 were selected as markers for the start-up phase and degradation phase, respectively, of autophagy (26, 27). After establishment of CLP, LC3 II levels decreased after a brief increase, but remained higher than levels observed in the sham group. These findings indicated that the start-up phase of autophagy was enhanced until 24 h after CLP. Levels of p62 decreased at 4 h, which indicated increased activity during the degradation phase. This could be a compensatory response against CLP. Levels of p62 increased significantly from 8 to 24 h, compared with those observed in the sham group, indicating decreased degradation phase activity, which in turn suggests decompensation. Thus, at 24 h after CLP, start-up phase activity increased, while degradation phase activity decreased. We refer to this phenomenon as “incomplete” or “impaired” autophagy (32).

NRG-1 plays a major role in maintaining the number of acetylcholine receptors at the neuromuscular junction, regulating expression, differentiation, and stabilization of postsynaptic acetylcholine receptors (13, 33). Levels of NRG-1 may also reflect myelin sheath thickness, which reflects neuronal functionality (34). In this study, levels of NRG-1 decreased after a brief increase in septic activity. Enhancing autophagy thus increased levels of NRG-1 and reduced levels of γ- and α7-nAChR. Synthesis of γ- and α7-nAChR is initiated within hours of burn, immobilization, or sepsis, which is commonly regarded as the reason for neuromuscular dysfunction, as measured by electromyography (7, 11, 12). In this study, enhancing autophagy with Rapa mediated the expression of NRG-1 and γ- and α7-nACh, facilitating neuromuscular function in young male CLP rats.

However, the male and female rats respond differently to sepsis, and we only studied the male rats. Female rodents have shown an enhanced immunological response with respect to male animals, resulting in better survival after injury, mainly due to differing hormonal milieu of each sex (35). Moreover, autosomal, genetically identical individuals carrying differences in their sex alleles (e.g., female and male mice from the same strain) present with different inflammatory responses, suggesting that factors in addition to sex steroids are involved (35). Thus, sex-based differences should be taken into consideration in further studies. Besides, CLP-induced myopathy in rats with cancer, elderly rats or rats with some chronic organ failure should also be studied as these are phenotypes that get sepsis and their response to infection is much different. The difference might include neuropathology, autophagy, and myelopathy.

In conclusion, our findings suggest that impaired autophagy could worsen septic inflammation and neuromuscular dysfunction in young male rats. Enhancing autophagy during the early stages of sepsis may improve neuromuscular function in septic early phase (24 h) as well as in septic chronic phase (7d), through upregulation of NRG-1 and downregulation of γ- and α7-nAChR. Thus, enhancing or restoring autophagy soon after the onset of sepsis may be an effective adjuvant therapy for treatment of sepsis-induced myopathy.

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Keywords:

Acetylcholine receptors; autophagy; cecal ligation and puncture (CLP); myopathy; neuromuscular dysfunction; sepsis; Abbreviations; 3-MA; 3-methyladenine; Atg1; (autophagy-related gene 1); CLP; cecal ligation and puncture; CMAP; Compound muscle action potential; CRP; C-reactive protein; DMSO; dimethyl sulfoxide; ELISA; Enzyme-linked immunosorbent assay; EMG; Electromyography; IL-6; Interleukin 6; LC3-II; Microtubule-associated protein light chain 3-II; nAChR; nicotinic acetylcholine receptor; NCV; nerve conduction velocity; NRG-1; neuregulin-1; p62; Sequestosome1; Rapa; rapamycin

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