Shigeo Kawada

Content and localization of myostatin in mouse skeletal muscles during aging, mechanical unloading and reloading

Changes in myostatin content and localization in mouse skeletal muscles were investigated during aging, hindlimb suspension (HS) and reloading after HS. During aging, the content of myostatin among solubilized proteins in gastrocnemius and plantaris muscles (Gast/Plant) was initially low and increased until their wet weight/body weight ratio reached a peak. It remained unchanged with further aging, although gradual atrophy of the muscles was seen to occur. Also, the myostatin content did not change significantly during HS (up to 14 days) in both Gast/Plant and soleus muscles, though the muscles showed morphological signs of atrophy. However, reloading for 2 days after a 14-day HS caused significant decreases in the myostatin content in both of these muscles. Immunohistochemical observations showed the sarcoplasmic existence of myostatin, the amount of which appeared to decrease after reloading. The results suggest that myostatin plays a part in the processes of muscular growth and loading-induced hypertrophy, but is not involved in either aging-related or unloading-induced muscular atrophy.

Influence of icing on muscle regeneration after crush injury to skeletal muscles in rats

The influence of icing on muscle regeneration after crush injury was examined in the rat extensor digitorum longus. After the injury, animals were randomly divided into nonicing and icing groups. In the latter, ice packs were applied for 20 min. Due to the icing, degeneration of the necrotic muscle fibers and differentiation of satellite cells at early stages of regeneration were retarded by ∼1 day. In the icing group, the ratio of regenerating fibers showing central nucleus at 14 days after the injury was higher, and cross-sectional area of the muscle fibers at 28 days was evidently smaller than in the nonicing group. Besides, the ratio of collagen fibers area at 14 and 28 days after the injury in the icing group was higher than in the nonicing group. These findings suggest that icing applied soon after the injury not only considerably retarded muscle regeneration but also induced impairment of muscle regeneration along with excessive collagen deposition. Macrophages were immunohistochemically demonstrated at the injury site during degeneration and early stages of regeneration. Due to icing, chronological changes in the number of macrophages and immunohistochemical expression of transforming growth factor (TGF)-β1 and IGF-I were also retarded by 1 to 2 days. Since it has been said that macrophages play important roles not only for degeneration, but also for muscle regeneration, the influence of icing on macrophage activities might be closely related to a delay in muscle regeneration, impairment of muscle regeneration, and redundant collagen synthesis.

Vitamin C administration attenuates overload‐induced skeletal muscle hypertrophy in rats

This study aimed to investigate the effects of vitamin C administration on skeletal muscle hypertrophy induced by mechanical overload in rats.

Changes in skeletal muscle size, fibre‐type composition and capillary supply after chronic venous occlusion in rats

Aim:  We have previously shown that surgical occlusion of some veins from skeletal muscle results in muscle hypertrophy without mechanical overloading in the rat. The present study investigated the changes in muscle‐fibre composition and capillary supply in hypertrophied muscles after venous occlusion in the rat hindlimb.

Cystine and Theanine Supplementation Restores High-Intensity Resistance Exercise-Induced Attenuation of Natural Killer Cell Activity in Well-Trained Men

Kawada, S, Kobayashi, K, Ohtani, M, and Fukusaki, C. Cystine and theanine supplementation restores high-intensity resistance exercise-induced attenuation of natural killer cell activity in well-trained men. J Strength Cond Res 24(3): 846-851, 2010-We investigated the effects of supplementation with cystine, a dipeptide of cysteine, and theanine (CT), a precursor of glutamate, on immune variables during high-intensity resistance exercise. Cysteine and glutamate are involved in the formation of glutathione, which modulates the activity of natural killer (NK) cells. In this double-blinded clinical trial, 15 well-trained men (aged 22.8 ± 4.0 years) were divided into 2 groups: placebo (n = 7) and CT (n = 8). The placebo group was administered a powder containing cellulose (950 mg) and glutamate (30 mg), whereas the CT group was administered a powder containing cystine (700 mg) and theanine (280 mg), once daily for 2 weeks. The subjects trained according to their normal schedule (3 times per week) in the first week and trained at double the frequency (6 times per week) in the second week. Concentrations of immunoglobulin (Ig)M, interleukin (IL)-6, IL-8, and salivary IgA and the leukocyte count did not change significantly in either group. There was a significant decrease (p ≤ 0.05) in the NK cell activity (NKCA) in the placebo group after the second week compared with that in the CT group (placebo: 69.2 ± 16.1% vs. CT: 101.7 ± 38.7%). Phytohemagglutinin-induced lymphocyte blastoid transformation did not change significantly in either group. These results suggest that NKCA is not affected in a normal training schedule with or without CT supplementation. However, high-intensity and high-frequency resistance exercises cause attenuation of NKCA, which CT supplementation appears to restore. Therefore, in practical application, CT supplementation would be useful for athletes to restore the attenuation of NKCA during high-intensity and high-frequency training.

Dominant Negative Inhibition of Human Immunodeficiency Virus Particle Production by the Nonmyristoylated Form of Gag

ABSTRACT Myristoylation of human immunodeficiency virus (HIV) Gag protein is essential for membrane targeting of Gag and production of viral particles. We show here that coexpression of wild-type and nonmyristoylated forms of HIV Gag resulted in severe inhibition of viral particle production, indicating that the nonmyristoylated counterpart had a dominant negative effect on particle release. When coexpressed, the nonmyristoylated Gag partially incorporated into membrane and lipid raft fractions, likely through coassembly with the wild-type Gag. The membrane and raft associations of the wild-type Gag appeared unaffected, and yet particle production was severely impaired. When viral particles produced from the coexpressing cells were analyzed, the wild-type Gag was more abundant than the nonmyristoylated Gag. Confocal microscopy showed that both forms of Gag were diffusely distributed in the cytoplasm of coexpressing cells but that a portion of the wild-type Gag population was accumulated in EEA1- and CD63-positive endosomes. The intracellular accumulation of Gag was more frequently observed at late time points. The Gag accumulation was also observed on the cell surface protrusion. Electron microscopy of the coexpressing cells revealed budding arrest phenotypes, including the occurrence of interconnected virions on the plasma membrane, and intracellular budding. We also show that the inhibition of particle production and the Gag accumulation to endosomes were suppressed when the nucleocapsid (NC) domain was deleted from the nonmyristoylated Gag, although the NC-deleted Gag was still capable of coassembly. Overall, our data indicate that coassembly with the nonmyristoylated Gag impairs HIV particle release, a phenomenon that may involve NC-mediated Gag-Gag interaction.

Increased oxygen tension attenuates acute ultraviolet-B-induced skin angiogenesis and wrinkle formation

Acute ultraviolet (UV)-B irradiation causes skin wrinkle formation associated with hyperplasia of cutaneous blood vessels. This study reports that increased dermal oxygen tension attenuates acute UVB-induced angiogenesis and wrinkle formation. Twenty-four hairless mice (HOS:HR-1) were assigned to 3 groups: 1) control group, 2) UVB-irradiated (UVB) group, and 3) UVB-irradiated and hyperoxia-exposed (UVB+HO) group. The backs of the mice were exposed to UVB irradiation 3 times per week for a 5-wk period. To increase dermal oxygen tension, the mice were exposed to hyperoxia (90% oxygen) for 2 h immediately after each UVB irradiation. Hyperoxic exposure increased dermal oxygen tension by about 10 times compared with the control level. Degree of wrinkle formation and epidermal thickness increased significantly after a 5-wk UVB-irradiation period, whereas hyperoxic exposure attenuated these increases. Tissue adenosine triphosphate concentration and angiogenesis increased significantly only in the UVB group compared with the control group. Although the expression of hypoxia inducible factor-1alpha mRNA, a key molecule for angiogenesis, increased significantly in the UVB and UVB+HO groups compared with the control group, the protein level increased significantly only in the UVB group. The activity of matrix metalloproteinase-2 and -9, critical molecules for angiogenesis, did not increase in the UVB and UVB+HO groups compared with the control group. Active type 1 collagenase activity and soluble collagen content in all of the groups were roughly similar. These results suggest that increased dermal oxygen tension attenuates angiogenesis and wrinkle formation following acute UVB irradiation.

Hyperbaric Hyperoxia Accelerates Fracture Healing in Mice

Increased oxygen tension influences bone metabolism. This study comprised two main experiments: one aimed to determine the bone mineral apposition and bone formation rates in vivo under hyperbaric hyperoxia (HBO), and the other aimed to evaluate the effects of exposure to HBO on fracture healing. In experiment 1, male mice were exposed to HBO [90 min/day at 90% O2 at 2 atmospheres absolute (ATA) for 5 days]. In experiment 2, an open femur fracture model was created in mice, followed by exposure to HBO 5 times/week (90 min/day at 90% O2 at 2 ATA) for 6 weeks after surgery. In experiment 1, HBO treatment significantly increased the mineral apposition and bone formation rates in the lumbar vertebra and femur and type 1 collagen alpha 1 and alkaline phosphatase mRNA expression in the lumbar vertebra. In experiment 2, at 2 weeks after fracture, the fracture callus was significantly larger in the HBO group than in the non-HBO group. Furthermore, at 4 and 6 weeks after fracture, radiographic findings showed accelerated fracture healing in the HBO group. At 6 weeks after fracture, femur stiffness and maximum load were significantly higher in the HBO group than in the non-HBO group. Urinary 8-hydroxy-2′-deoxyguanosine and plasma calcium concentrations were not significantly different between groups. These results suggest that exposure to HBO enhances bone anabolism and accelerates fracture healing without causing oxidative DNA damage or disruption of plasma calcium homeostasis.

Significant roles of microtubules in mature striated muscle deduced from the correlation between tubulin and its molecular chaperone αB-crystallin in rat muscles

To elucidate the significance of cytoskeletal microtubule networks in striated muscles, we analyzed correlation between the content of tubulin (building block of microtubules) and αB-crystallin (a molecular chaperone for tubulin) in a variety of striated muscles expressing different myosin heavy-chain (MHC) isoforms. The content of both tubulin and αB-crystallin was larger in MHC-I dominant soleus muscle and in MHC-α dominant cardiac (atrium and ventricle) muscles; intermediate in MHC-IId dominant masseter, tongue, and diaphragm muscles; and smaller in MHC-IIb dominant plantaris, gastrocnemius, psoas, extensor digitorum longus, and tibialis anterior muscles. Since the muscles of slow-type MHC (MHC-I/α) show the most economical features in their function and metabolism, which suit for continuous activity required to sustain posture and blood pumping, the present results afforded additional support to our hypothesis that microtubule networks transduce mechanical environmental demands to morphological and biochemical responses that eventually evolve adaptive transformation in the function and metabolism of the mature muscles. The comparison of tubulin/αB-crystalline ratios across the muscles of varied MHC isoforms further suggested that mechanical stress fluctuating at the rhythmic frequency of walking and breathing efficiently activates the hypothesized dynamic function of microtubules.

Effects of pre-exposure to hyperbaric hyperoxia on high-intensity exercise performance

This study comprised 2 main experiments: one was to determine the oxidative DNA damage under hyperbaric hyperoxia (HBO), and the other was to evaluate the effects of pre-exposure to HBO on high-intensity exercise performance. Healthy subjects (n = 8) inspired 100% O2 in an experimental chamber at a pressure of 1.3 atmospheres absolute (ATA) for 50 minutes once per week for 2 weeks. Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured as a marker of DNA oxidative damage on day 0 and on days 1, 3, and 5 after each HBO exposure. To investigate the effects of pre-exposure to HBO on high-intensity exercise performance, subjects (n = 6) performed maximal isometric knee extensor exercise (30 repetitions × 2 sets) with and without HBO pre-exposure (100% O2 at 1.3 ATA for 50 minutes). Urinary 8-OHdG did not show any significant change after HBO exposure. Isometric knee extensor torque was significantly lower during the first half of the first set of exercises after HBO pre-exposure compared with the normobaric normoxia (NBO) trial. The decreased torque was associated with the lower integrated electromyography with respect to time. Changes in the degree of ischemia-reperfusion in the vastus lateralis muscle during exercise were larger in the HBO pre-exposure trial than in the NBO trial. Muscle fatigue index, serum lactate concentration, heart rate, and systolic blood pressure showed no differences between the 2 trials. These results indicated that HBO exposure was harmless to DNA, and HBO pre-exposure did not enhance high-intensity exercise performance. As a practical application, athletes who require maximal muscle strength should not inspire high-concentration of O2 just before their competitions because it might, as the case may be, impair their performance.