Study On Effects Of Three Supplementations On The Morphology And Function Of Soleus Muscle In Hindlimb-Unloading Rats

BackgroundA great deal of scientific data and experiments have indicated weightlessness and simulated microgravity can lead to apparent muscle atrophy, as not only affects work efficiency of astronauts and their adaptation to the earth when they are back from space, but also sharply threatens their lifesaving ability. Therefore, seeking effective drugs for prevention and treatment of muscle atrophy is one of the subjects waiting for solution in space medical field.Previous studies on weightlessness or simulated weightlessness mostly are focused on changes in extrafusal fiber, and corresponding study on preventive drugs are relevant to their influence on extrafusal fiber. It has been proved that under weightlessness or simulated weightlessness, declined afferent impulse of muscle spindle evokes or aggravates muscle atrophy. On the contrary, increased afferent impulse of muscle spindle alleviates or prevents muscle atrophy. In addition, under weightlessness or simulated weightlessness, skeletal muscle is less driven by motor nerve. The degradation of neuromuscular junction may be one of the reasons for muscle atrophy and lessened muscle strength caused by weightlessness. Experiments have validated choline can increase spread of acetylcholine, endplate current, and prevent patients of Alzheimer's disease from degradation of neuromuscular junction. Based on this we deem theoretically choline that can excite muscle spindle and increase afferent impulse of muscle spindle are probably ideal drugs for prevention and treatment of muscle atrophy. Starting from Choline Chloride and Citicoline, two types of choline that can excite muscle spindle, this experiment compounded three supplementations and set up a simulated weightlessness model by unloading hindlimb of female rats. We observed the effects of three supplementations on morphology and functions of soleus (SOL) muscle in hindlimb-unloading rat in order to provide basic experiment data for new drug development in preventive muscle atrophy. PurposeThe study assumed that weightlessness or simulated weightlessness can result in changes in the morphology of muscle fibers, decrease functions of skeletal muscle and apparent muscle atrophy. The application of some supplementations such as Choline Chloride can alleviate or prevent the occurrence and development of muscle atrophy. The experiment focused on the effects of three supplementations including Choline Chloride, the compound of Choline Chloride and Vitamin and Citicoline on morphology and functions of soleus in hindlimb-unloading rats so as to discover effective drugs to prevent muscle atrophy caused by weightlessness.MethodsSet up hindlimb-unloading rat model, and conduct intragastric water instillation of three supplementations and then, (1) Observe changes in the shape of skeletal muscle after HE staining. Meanwhile, measure and analyze muscle wet weight (MWW) and muscle wet weight to body weight ratio (MWW/BW). (2) Adopt histochemistry method to observe effects on activity of mATPase of SOL. Analyze effects on isoform of myosin heavy chain in skeletal muscle by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (3) Measure function parameters of skeletal muscle and analyze effects on muscle strength and contractile time.Result1. Effects on morphology and function of skeletal muscle in hindlimb-unloading ratsCompared to contrast (CON) group, after 14 days hindlimb-unloading (HU) and hindlimb-unloading plus intragastric water instillation (HU-W) group had apparently shrunk SOL, loose structure, and clear changes in shape. Although, it has no significant difference in body weight (BW), both MWW and MWW/BW dropped indicating obvious atrophy. Cross sectional area (CSA) of SOL fiber I and fiber II were significantly lower than CON group, CSA of single muscle fiber dropped notably. Meanwhile, the percentages of SOL fiber I decreased and the percentages of fiber II increased in HU and HU-W group (P<0.001), and there is a transformation trend of muscle fiber from slow-twitch fiber to fast-twitch fiber. The analysis of myosin isoform by SDS-PAGE represented such changes. Moreover, isometric twitch force (P_t) and tetanic tension (P_o) in HU-W group both declined clearly(P<0.01; P<0.05) while time to peak tension (TPT) and half relaxation time (HRT) significantly declined, indicating that simulated weightlessness resulted in decline in contractile tension, accelerated atrophy and there was a trend from slow-twitch to fast-twitch in SOL fibers.2. Effects of three supplementations on morphology and function in soleus muscle of hindlimb-unloading rats(1) During hindlimb unloading period, intragastric three supplementations instillation were administered. Compared to HU-W group, muscle fiber was compacter, deteriorated shape was improved, skeletal muscle MWW and MWW/BW apparently climbed, especially HU-CC group increased 24.5% (P<0.01), 25.0% (P<0.01) and HU-CS group rose by 18.5% (P<0.05), 16.7% (P<0.05), an evident trend suggesting that certain dosages of Choline Chloride and Citicoline can resist weight loss of muscle and changes in structure of muscle fiber under simulated weightlessness.(2) Compared to HU-W group, after intragastric three supplementations instillation were administered in hindlimb unloading period, fiber I and fiber II of SOL in all groups show evident expansion in fiber CSA, increased proportions in fiber I and decreased proportions in fiber II and in three groups MHC I of SOL increased but MHC IIa declined, representing that certain dosage of supplementations can to some extent delay occurrence and evolution of weightlessness caused muscle atrophy.(3) Compared to HU-W group, after intragastric three supplementations instillation were administered in hindlimb unloading period, P_o and TPT in HU-CC group increased 21.85% and 23.90% respectively with significant difference (P<0.05). P_t and HRT in HU-CS group apparently increased (53.72%, P<0.01; 18.87%, P<0.05), suggesting that both Choline Chloride and Citicoline can to some extent alleviate drop in muscle strength and atrophy caused by simulated weightlessness and prevent weakened function of skeletal muscle and the transformation from slow-twitch to fast-twitch fiber.ConclusionHindlimb unloading can result in apparent disuse atrophy, change in fiber type and decrease in functions of soleus muscles. And some supplementations can alleviate the occurrence and evolution of such muscle atrophy. Especially Choline Chloride has a prominent effect. It perhaps because that Choline Chloride can excite muscle spindle and nourish neuromuscular junction. Further study should set foot in selection of dosage and protein anabolism metabolism.