Study On Functional Effects And Action Mechanism Of Cardiotrophin-1 On Denervation Atrophy Of Skeletal Muscle

Objective: (1) To investigate the expression patterns of Cardiotrophin-1 (CT-1) and its receptor (gp130/LIFR-β) in skeletal muscle with denervaion time. (2) To evaluate the potential myotrophic effect of CT-1 on skeletal muscle postdenervation. (3) To assess the function of CT-1-treated denervated muscle after reinnervation. (4) To explore the action mechanism of CT-1 on alleviating denervation atrophy of skeletal muscle. (5) To determine the side effects of CT-1 on myocardium, smooth muscle and normal skeletal muscle, and definite the suitable dose administered to protect skeletal muscle against denervation atrophy.Methods: 110 mice were divided into 11 groups randomly, whose right tibial nerves were partly removed. The expression of CT-1 and its receptor (gp130/LIFR-β) in gastrocnemius muscle of mice was assayed by Northern blot analysis 0, 6, 12h and 1, 3, 5, 7, 10, 14, 21, 28d after denervation. Another 30 mice in three groups were intraperitoneally injected with recombinant mouse CT-1 (rmCT-1,100μg/kg/d) after the tibial nerve was transected. Gastrocnemius muscle of the mice were isolated respectively 7, 14, 28d postdenervation. The changes of wet weight, cross-sectional area, total protein, ultrastructure and muscle tension of the denervated muscle were detected. All the results were compared with those of control groups. Thirty mice whose tibial nerve was cut were intraperitoneally injected rmCT-1 for 4 weeks. Then the transected tibial nerve was repaired. The CMAP, muscle tension, wet weight and total protein of gastracnomius, number of myelinated regenerating fiber and latency of action potential were assayed 4, 8 and 12w respectively after nerve repairing. To explore the mechanism by which CT-1 alleviates denervation atrophy, we analyzed the changes of apoptosis, protein metabolism, excitation-contraction couple and the status of muscle satellite cell in denervated skeletal muscle after CT-1-injection. In details, TUNEL labeling was used to measure apoptosis rate of denervated myocytes. Northern blot analysis was employed to assess the expression of mRNA of Fas, Bcl-2, α-actin, MHC IIa, ubiqutin and RC2 in denervated muscle. Western blot analysis was employed to evaluate the contents of HSP70, HSP90 and Ca²⁺-ATPase in CT-1-treated denervated muscle. HPLC was used to assay the release of MeHis fromdenervated muscle. Electron microscope was used to evaluate the morphological changes of muscle satellite cell. To explore the side effects, effective dose and security dose of CT-1 administered to treat denervation atrophy of skeletal muscle, eighty mice were randomly divided into 8 groups and intraperitoneally injected different doses of rmCT-1 for one month after their tibial nerves were partly removed. The doses of rmCT-1 were 40, 60, 80, 100, 120, 140 and 160μg/kg/d respectively. The other group was intraperitoneally the same dose of vehicle. Northern blot analysis was employed to assay the expression of mRNA of α-actin and MHC IIa in contralateral gastrocnemius muscle, and β-MHC of myocardium. Western blot was employed to evaluate the content of α-actin in smooth muscle cells of aorta. At the same time, the wet weight of gastrocnemius muscle and heart, the thickness of smooth muscle of aorta were assayed.Results: CT-1 and its receptor were both expressed in normal skeletal muscle and showed their own expression patterns with denervation time. Expression of CT-1 declined gradually, whereas two components of the receptor, leukemia inhibitory factor-β (LIFR-β) and glycoprotein 130 (gp130), were both augmented in the initial phase postdenervation. Daily administration of exogenous CT-1 for 2 weeks better sustained the wet weight, cross-sectional area, total protein of denervated muscle, alleviated the dilataltion of sarcoplasmic reticulum and improved the muscle tension. Four weeks later, the results were more satisfactory. Furthermore, the CT-1-treated denervted muscles got ameliorated morphological index and improved contractile function after reinnervation, although the number of regenerated myelinated regenerating fiber and latency of action potential of repaired tibial nerves were not improved. Our experiments suggested that rmCT-1 can inhibit the expression of Fas mRNA, up-regulate the expression of Bcl-2 mRNA, reduce the apoptosis rate of denervated muscle, induce the enhanced expression of α-actin and MHC IIa mRNA, stimulate the synthesis of HSP70 and HSP90, increase the content of Ca²⁺-ATPase, and ameliorate the contractile function of denervated muscle. But the administration of rmCT-1 cannot reduce the expression of ubiqutin and RC2 mRNA in ubiquitin-proteasome pathway, suppress the release of MeHis and activate the muscle satellite cell. It was proved in our study that CT-1 alleviated denervation atrophy in a dose-dependent manner. The effective dose was in a range from 60μg/kg/d to 160 μg/kg/d. But when the given dose was beyond 120μg/kg/d, CT-1 may display its side effects on myocardium, smooth muscle and normal skeletal muscle such as inducingoverexpression of β-MHC mRNA in myocardium, α-actin and MHC IIa mRNA in normal skeletal muscle, and increase the content of α-actin in smooth muscle. High dose CT-1 increased the wet weight of heart and normal gastrocnemius muscle, and thickness of smooth muscle in aorta.Conclusion: The finding presented here demonstrated that reduced expression of endogenous CT-1 in denervated skeletal myocytes. Its diminished myotrophic effect contributes to the early atrophy of denervated muscle. While the up-regulated expression of the receptor allowed denervated muscle to be more responsive to exogenous CT-1. When administered in time, exogenous CT-1 would produce remarkable myotrophic and protective effect on denervated muscle by inducing the synthesis of contractile protein and heat shock protein, increasing the content of Ca²⁺-ATPase and inhibiting myocyte apoptosis. Ultimately, exogenous CT-1 alleviated atrophy of denervated skeletal muscle and ameliorated its contractile function after reinnervation. Therefore, CT-1 was proved to be a potent myotrophic factor, and might be employed as a pharmacological agent to protect skeletal muscle against denervation atrophy. The effective dose is 60 μg/kg/d and the security dose is 100μg/kg/d when CT-1 is administered intraperitoneally to treat denervation atrophy.