Isolation and Purification of Satellite Cells from Young Rats by Percoll Density Gradient Centrifugation.

Satellite cells (SCs) are myogenic stem cells that play an important role in skeletal muscle regeneration and hypertrophy. Primary cultures of SCs are useful to analyze cell functions; however, it is difficult to obtain highly pure SCs from young rats with the conventional procedures. The purpose of this study is to establish a purification method for SC isolation from young rats and quantitatively evaluate the purification procedure employing Percoll, a common research tool to purify cells. We elucidated the purity of SCs collected by Percoll density gradient centrifugation using real-time RT-qPCR and immunocytochemistry for desmin. Percoll treatment increased the purity of SCs isolated from young rats to nearly 90%, which was comparable to that achieved with the conventional method using middle-aged rats.

Fast-to-slow shift of muscle fiber-type composition by dietary apple polyphenols in rats: Impact of the low-dose supplementation.

Our previous studies demonstrated that an 8-week intake of 5% (w/w) apple polyphenol (APP) in the diet improves muscle endurance of young-adult rats. In order to identify a lower limit of the dietary contribution of APP to the effect, the experiments were designed for lower-dose supplementation (8-week feeding of 0.5% APP in AIN-93G diet) to 12-week-old male Sprague-Dawley rats. Results clearly showed that the 0.5% APP diet significantly up-regulates slower myosin-heavy-chain (MyHC) isoform ratios (IIx and IIa relative to total MyHC) and myoglobin expression in lower hind-limb muscles examined (P < 0.05). There was a trend to increased fatigue resistance detected from measurements of relative isometric plantar-flexion force torque generated by a stimulus train delivered to the tibial nerve (F(98, 1372) = 1.246, P = 0.0574). Importantly, there was no significant difference in the animal body-phenotypes or locomotor activity shown as total moving distance in light and dark periods. Therefore, the present study encourages the notion that even low APP-intake may increase the proportions of fatigue-resistant myofibers, and has promise as a strategy for modifying performance in human sports and improving function in age-related muscle atrophy.

Data in support of protocol for rat single muscle-fiber isolation and culture.

This data article contains data related to the research article entitled, "Protocol for rat single muscle-fiber isolation and culture" by Komiya et al. [1]. It has yet to be shown whether adult myosin heavy chain (MyHC) isoforms are expressed at a readily detectable level in cultured myotubes. In this study, we examined whether adult MyHC isoforms could be detected in myotubes differentiated from rat satellite cells using a Western blotting assay and specific antibodies against slow MyHC, fast MyHC and pan-MyHC. Results showed that slow adult MyHC isoforms were faintly detected in myotubes, suggesting that rat myotubes express adult MyHC isoforms although that amount is very low.

Improvement of Endurance Based on Muscle Fiber-Type Composition by Treatment with Dietary Apple Polyphenols in Rats.

A recent study demonstrated a positive effect of apple polyphenol (APP) intake on muscle endurance of young-adult animals. While an enhancement of lipid metabolism may be responsible, in part, for the improvement, the contributing mechanisms still need clarification. Here we show that an 8-week intake of 5% (w/w) APP in the diet, up-regulates two features related to fiber type: the ratio of myosin heavy chain (MyHC) type IIx/IIb and myoglobin protein expression in plantaris muscle of 9-week-old male Fischer F344 rats compared to pair-fed controls (P < 0.05). Results were demonstrated by our SDS-PAGE system specialized for MyHC isoform separation and western blotting of whole muscles. Animal-growth profiles (food intake, body-weight gain, and internal-organ weights) did not differ between the control and 5% APP-fed animals (n = 9/group). Findings may account for the increase in fatigue resistance of lower hind limb muscles, as evidenced by a slower decline in the maximum isometric planter-flexion torque generated by a 100-s train of electrical stimulation of the tibial nerve. Additionally, the fatigue resistance was lower after 8 weeks of a 0.5% APP diet than after 5% APP, supporting an APP-dose dependency of the shift in fiber-type composition. Therefore, the present study highlights a promising contribution of dietary APP intake to increasing endurance based on fiber-type composition in rat muscle. Results may help in developing a novel strategy for application in animal sciences, and human sports and age-related health sciences.

Protocol for rat single muscle fiber isolation and culture.

To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro.