Ciliary neurotrophic factor is a regulator of muscular strength in aging

Abstract

Ciliary neurotrophic factor (CNTF) participates in the survival of motor neurons and reduces the denervation-induced atrophy of skeletal muscles. Experiments performed in rats show a decrease in peripheral CNTF synthesis during aging, associated with an overexpression of its alpha-binding receptor component by skeletal muscles. Measurement of sciatic nerve CNTF production and of the muscular performance developed by the animals revealed a strong correlation between the two studied parameters (r = 0.8; p < 0.0003). Furthermore, the twitch and tetanic tensions measured in the isolated soleus skeletal muscle in 24-month-old animals increased 2. 5-fold by continuous in vivo administration of CNTF. Analyses of the activation level of leukemia inhibitory factor receptor beta- and signal transducer and activator of transcription 3-signaling molecules in response to exogenous CNTF revealed an increased tyrosine phosphorylation positively correlated with the twitch tension developed by the soleus muscle of the animals.

Fig. 1.

CNTF expression in sciatic nerve of adult (6 months) and aged (24 months) rats. A, ELISA detection of CNTF in sciatic nerve extracts from two different strains of rats. The results are expressed in nanograms per milligram of total protein. Values are mean ± SEM. Left, Adult rats,n = 4; aged rats, n = 3; *p < 0.05; Student’s t test.Right, Adult rats, n = 7; aged rats,n = 8; **p < 0.001; Student’st test. B, CNTF and GAPDH mRNA levels in sciatic nerves from male Sprague Dawley rats were determined by Northern blot and quantified by laser densitometry. Adult rats,n = 3; aged rats, n = 8; *p < 0.05; Student’s t test. Values are mean ± SEM.

Fig. 2.

Regulation of CNTF receptor components in skeletal muscles during aging. A, Expression of CNTFRα mRNA in different hindlimb muscles. Experiments were performed in the soleus, extensor digitorum longus (EDL), gastrocnemius (gastroc.), and tibialis posterior (tib.post.) muscles of male Sprague Dawley rats aged of 3, 6, 12, 18, and 24 months.B, CNTFRα mRNA expression in the soleus muscles from six adult (6 months) and seven aged (24 months) male Sprague Dawley rats. CNTFRα mRNA was also readily detectable in soleus muscle from adult rats after a longer exposure time, as shown in A.C, Expression of CNTF receptor subunits in the soleus muscle from adult (hatched bars) and aged (black bars) male Sprague Dawley rats. CNTFRα expression detected in the soleus muscle by Northern blot analysis was quantified by laser densitometry, and the obtained values were presented as a CNTFRα/GAPDH ratio. Gp130 content was determined in the same muscles by ELISA detection after total protein content determination, and the results were expressed in nanograms per milligram of protein. LIFRβ determination was achieved by immunoprecipitation and Western blotting, and the signals were quantified by laser densitometry. Adult rats,n = 6; aged rats, n = 7; *p < 0.0001; Student’s t test. Values are mean ± SEM.

Fig. 3.

Swimming speed as a function of CNTF content in the sciatic nerve. Swimming speed was determined as described in detail in Materials and Methods and sciatic CNTF content was monitored by ELISA. A positive correlation between the two parameters was observed,r = 0.80; p < 0.0003.

Fig. 4.

Effect of CNTF treatment on rat weight. Control aged rats, n = 4; saline-treated animals,n = 4; CNTF-treated animals, n= 6. No significant variation of rat weight is observed between control or saline-treated and CNTF-treated rats; p = 0.55 and p = 0.12, respectively (Student’st test). Values are mean ± SEM.

Fig. 5.

Effect of CNTF treatment on physiological properties of soleus muscle. Twitch tension (A) and tetanic tension (B) were determined after electrical stimulation of the isolated muscles after treatment, as described in Materials and Methods. The forces are expressed in percentage (tensions in millinewtons per milligram of muscle wet weight), and the control aged group values defined the 100% reference. Control aged animals of 24 months, n = 4; saline-treated aged animals, n = 4; CNTF-treated aged animals, n = 6; control adult rats of 6 months, n = 5. Values are mean ± SEM. *p < 0.001; different from saline-treated animals, Student’s t test. C, Effect of CNTF treatment on cross-sectional area of soleus muscle fibers in aged animals. **p < 0.05; different from saline-treated animals; Student’s t test. Values are mean ± SEM.

Fig. 6.

Activation of STAT3 and LIFRβ after CNTF treatment. A, Equal amounts of tissue extracts from soleus muscle treated with CNTF or vehicle were immunoprecipitated with an anti-phosphotyrosine monoclonal antibody. Samples were then submitted to SDS-PAGE and Western-blotted onto a membrane. Phosphorylation level of two STAT3 isoforms and LIFRβ were determined by staining the membranes with an anti-STAT3 monoclonal antibody and an anti-LIFRβ polyclonal antibody, respectively. B,C, STAT3 and LIFRβ phosphorylation as a function of the twitch tension developed by saline-treated (closed circles) or CNTF-treated (open circles) aged rats. Signals from the blots (A) were quantified by laser densitometry and expressed in arbitrary units. Twitch tension is expressed in newtons per gram of soleus wet muscle. For each panel, a positive correlation between the two parameters was observed;r = 0.852, p < 0.01 andr = 0.776, p < 0.02 for STAT3 and LIFRβ phosphorylation, respectively.