Effects of acute cervical stretching on arterial wall elastic properties.

Purpose: Acute (immediate) or regular (mid- or long-term) stretching increases arterial compliance and reduces arterial stiffness. Stretching is widely known to induce arterial functional factor changes, but it is unclear whether stretching alters arterial structural factors. Ultrasound shear wave elastography can quantify the distribution of tissue elastic properties as an index of arterial structural factors. This study thus aimed to examine the effects of acute cervical stretching on arterial wall tissue elastic properties. Methods: Seventeen healthy young adults participated in two different trials for 15 min in random order on separate days: a resting and sitting trial (CON) and a supervised cervical stretching trial (CS). In CS, subjects performed 10 different stretches. At each site, the stretch was held for 30 s followed by a 10-s relaxation period. In CON, subjects rested on a chair for 15 min. Results: After the experiment, carotid arterial compliance, assessed by combined ultrasound imaging and applanation tonometry, was significantly increased in CS, but not in CON. However, there was no significant change in tissue elasticity properties of the arterial wall in either trial, as assessed by ultrasound shear wave elastography. Conclusion: Acute cervical stretching significantly increased carotid artery compliance in young participants, but did not reduce elastic tissue properties (i.e., arterial structural factors) of the carotid artery wall. These results strongly suggest that changes in structural factors have little relation to stretching-induced acute increases in arterial compliance.

Aquaporin 4 Expression Level Is Decreased in Skeletal Muscles with Aging.

Skeletal muscle is a tissue that contains abundant water. However, by aging a decrease in muscle water content is induced in skeletal muscles, which is one of major age-related alterations in skeletal muscles as common as muscle atrophy. Selective water channel aquaporin 4 (AQP4) is one of major water transport networks in the skeletal muscles. However, the effects of aging on water transport via AQP4 in skeletal muscles remain unclear. Thus, the current study investigated the change of the expression level of AQP4 in the aged skeletal muscles. Eight-week-old (the young group) and 2-year-old (the old group) female Fischer 344 rats were used in this study (n = 6/group). In skeletal muscles of each group, the expression levels of some target proteins were quantified by Western blot analysis. As a result, the relative muscle weight in the old group was significantly decreased, compared with that in the young group (p < 0.05). The decline in the muscle water content was accompanied by the decrease in expression of AQP4 in the aged skeletal muscles (p < 0.05, respectively). Moreover, the expression of transient receptor potential vanilloid 4, which synergistically regulates the osmolality together with AQP4, was significantly reduced in the aged skeletal muscles (p < 0.05). Therefore, the current study suggested that water transport abilities via AQP4 may decrease in the aged skeletal muscles, and thereby may be involved in age-related loss of muscle water content.

Cyclin D3 Colocalizes with Myogenin and p21 in Skeletal Muscle Satellite Cells during Early-Stage Functional Overload.

Myogenic cell differentiation is modulated by multiple regulatory factors, such as myogenin, p21, and cyclin D3 during myogenesis in vitro. It is also recognized that myogenin and p21 play important roles in regulating muscle satellite cell (SC) differentiation during overload-induced muscle hypertrophy in vivo. However, the expression patterns and functional role of cyclin D3 in the progress of muscle hypertrophy remain unclear. Thus, the present study investigated cyclin D3 expression in skeletal muscles during early-stage functional overload. Plantaris muscles were exposed to functional overload due to ablation of the gastrocnemius and soleus muscles. As a result, cyclin D3 expression was detected in the nuclei of SCs but not in myonuclei on day 1 after surgery. Cyclin D3 expression, after functional overload, gradually increased, reaching a maximum on day 7 along with myogenin expression. Moreover, in response to the functional overload, cyclin D3 was expressed simultaneously with myogenin and p21 in SC nuclei. Therefore, the present study suggests that cyclin D3 with myogenin and p21 may interactively regulate SC differentiation during early-stage functional overload.

Regular exercise ball training reduces arterial stiffness in sedentary middle-aged males.

[Purpose] Reports suggest that static stretching, which improves body flexibility, could reduce arterial stiffness. Regular training using an exercise ball would increase flexibility in a different manner, compared to that from static stretching; however, it remains unclear whether such exercise can reduce arterial stiffness. This study aimed to clarify the effect of exercise ball training on arterial stiffness in sedentary middle-aged participants. [Participants and Methods] Fifteen healthy middle-aged males (age, 52 ± 12 years) were divided into a control group (n=7, CON) and an intervention group (n=8, INT). The CON group did not alter physical activity levels throughout the study period, while the INT group participated in supervised training sessions using an exercise ball for 20-30 min, 5 days/week, for a duration of 4 weeks. [Results] Exercise ball training significantly increased the sit-and-reach test score (CON, -3.8 ± 11.1% vs. INT, 33.8 ± 47.5%) and reduced cardio-ankle vascular index (CON, -0.8 ± 4.1% vs. INT, -5.7 ± 4.1%) and heart-ankle pulse wave velocity (CON, 1.6 ± 4.5% vs. INT, -4.2 ± 4.6%), as an index of arterial stiffness. [Conclusion] Four weeks of supervised training using an exercise ball as well as regular static stretching would increase body flexibility and reduce systemic arterial stiffness among sedentary middle-aged males.

Effects of trunk stretching using an exercise ball on central arterial stiffness and carotid arterial compliance.

PURPOSE: Acute or regular stretching exercises reduce arterial stiffness, but whether stretching exercises per se can reduce central arterial stiffness remain controversial. Recent studies have suggested that mechanical stimulation of arteries can directly modulate arterial stiffness, rather than causing systemic effects. Thus, this study aimed to examine the effects of trunk stretching using an exercise ball on central arterial stiffness and carotid arterial compliance. METHODS: Twelve healthy young adults participated in two different trials for 30 min each in random order on separate days: a resting and sitting trial (CON); and supervised passive trunk stretching using the exercise ball (EB). In EB, subjects preformed six types of passive trunk stretching using the exercise ball. At each site, passive stretching was held for 30 s followed by a 30-s relaxation period, repeated 5 times during the 30-min trial. In CON, subjects rested on a comfortable chair for 30 min. RESULTS: After the experiment, carotid-femoral pulse wave velocity was significantly reduced in EB, but not in CON (EB vs. CON: -4.5 ± 1.2% vs. 0.2 ± 0.9%; P < 0.05). Carotid arterial compliance was also significantly increased in EB, but not in CON (EB vs. CON: 38.4 ± 11.4% vs. 4.1 ± 9.4%; P < 0.05). Supplemental experiments also confirmed that stretching of lower extremity did not reduce carotid-femoral pulse wave velocity. CONCLUSION: Our findings indicate that acute, direct trunk stretching using an exercise ball reduces central arterial stiffness and increases carotid arterial compliance in young healthy men.

Decrease in AQP4 expression level in atrophied skeletal muscles with innervation.

Functional interaction between the selective water channel AQP4 and several ion channels, such as TRPV4, NKCC1, and Na+ /K+ -ATPase, closely participate to regulate osmotic homeostasis. In the skeletal muscles, the decrease in APQ4 expression due to denervation was followed by the restoration of AQP4 expression during reinnervation. These findings raised the possibility that innervation status is an essential factor to regulate AQP4 expression in the skeletal muscles. This study investigated this hypothesis using disuse muscle atrophy model with innervation. Adult female Fischer 344 rats (8 weeks of age) were randomly assigned to either control (C) or cast immobilization (IM) groups (n = 6 per group). Two weeks after cast immobilization, the tibialis anterior muscles of each group were removed and the expression levels of some target proteins were quantified by western blot analysis. The expression level of AQP4 significantly decreased at 2 weeks post-immobilization (p < 0.05). Moreover, the expression levels of TRPV4, NKCC1, and Na+ /K+ -ATPase significantly decreased at 2 weeks post-immobilization (p < 0.05). This study suggested that innervation status is not always a key regulatory factor to maintain the expression of AQP4 in the skeletal muscles. Moreover, the transport of water and ions by AQP4 may be changed during immobilization-induced muscle atrophy.

Poor Walking Speed Is Associated With Higher Segment-Specific Arterial Stiffness in Older Adult Japanese Community Dwellers: A Cross-Sectional Study.

Walking speed as one index of gait ability is an important component of physical fitness among older adults. Walking speed-arterial stiffness relationships have been studied, but whether poor walking speed is associated with higher segment-specific arterial stiffness in older adults is unclear. We thus aimed to examine the relationship between walking speed and segmental arterial stiffness among older community dwellers. This study was a cross-sectional study of 492 older Japanese community dwellers (age range, 65 to 96 years). Heart-brachial PWV (hbPWV), brachial-ankle PWV (baPWV), heart-ankle PWV (haPWV), and cardio-ankle vascular index (CAVI) were used as arterial stiffness indices. Walking speed, strength, flexibility, and cognitive function were also assessed. The participants were categorized into low (Slow), middle (Middle), and high (Fast) tertiles according to walking speed. The CAVI and baPWV were significantly lower in Fast than in Slow. Significant decreasing trends in CAVI and baPWV and a tendency toward decreasing trend in haPWV were observed from Slow to Fast, whereas hbPWV did not significantly differ among tertiles and no trend was evident. The results remained significant after normalizing CAVI and PWVs for multicollinearity of arterial stiffness indices and major confounding factors, such as age, gender, body mass index, blood pressure, cognitive function, and each physical fitness. Therefore, these findings suggest that poor walking speed is associated with higher segment-specific arterial stiffness of the central and lower limbs, but not of upper, in older adult community dwellers.

Time course changes in AQP4 expression patterns in progressive skeletal muscle atrophy during the early stage of denervation.

OBJECTIVES: In the skeletal muscles, water metabolism is mainly regulated by water channel aquaporin 4 (AQP4). Although the expression level of AQP4 was reduced by long-term denervation, during denervation the relationship between muscle atrophy initiation and AQP4 expression decrease initiation remains unknown. The present study examined the relationship between the timing of muscle atrophy initiation and that of AQP4 expression decrease initiation, during the early stage of denervation. METHODS: Female 344 rats (8 weeks of age) were randomly assigned to control (C), day 1 post-sciatic denervation (D1), day 4 post- sciatic denervation (D4) and day 7 post- sciatic denervation (D7) groups (n=6 per group). In the tibialis anterior (TA) muscles of each group, the expression levels of some target proteins were quantified by Western blot analysis. RESULTS: The expression level of AQP4 significantly decreased on day 4 post-denervation (p<0.05). Moreover, the beginning of the decrease in AQP4 expression level was concurrent with the timing of muscle atrophy in the skeletal muscles during the early stage of denervation. CONCLUSIONS: The present study suggested that the progression of the decrease in the AQP4 expression level is partly related to the progression of muscle atrophy during the early stage of denervation.

Negative Mood States Are Related to the Characteristics of Facial Expression Drawing: A Cross-Sectional Study.

An assessment of mood or emotion is important in developing mental health measures, and facial expressions are strongly related to mood or emotion. This study thus aimed to examine the relationship between levels of negative mood and characteristics of mouth parts when moods are drawn as facial expressions on a common platform. A cross-sectional study of Japanese college freshmen was conducted, and 1,068 valid responses were analyzed. The questionnaire survey consisted of participants' characteristics, the Profile of Mood States (POMS), and a sheet of facial expression drawing (FACED), and the sheet was digitized and analyzed using an image-analysis software. Based on the total POMS score as an index of negative mood, the participants were divided into four groups: low (L), normal (N), high (H), and very high (VH). Lengths of drawn lines and between both mouth corners were significantly longer, and circularity and roundness were significantly higher in the L group. With increasing levels of negative mood, significant decreasing trends were observed in these lengths. Convex downward and enclosed figures were significantly predominant in the L group, while convex upward figures were significantly predominant and a tendency toward predominance of no drawn mouths or line figures was found in the H and VH groups. Our results suggest that mood states can be significantly related to the size and figure characteristics of drawn mouths of FACED on a non-verbal common platform. That is, these findings mean that subjects with low negative mood may draw a greater and rounder mouth and figures that may be enclosed and downward convex, while subjects with a high negative mood may not draw the line, or if any, may draw the line shorter and upward convex.

The expression of aquaporin-4 is regulated based on innervation in skeletal muscles.

Aquaporin-4 (AQP4) is a selective water channel, which expresses on the plasma membrane of myofibers and regulates the osmotic pressure, energy metabolism and morphological changes in myofibers by modulating water transport across sarcolemma in skeletal muscles. Although the physiological roles of AQP4 have been gradually clarified in skeletal muscles, the regulatory mechanisms of AQP4 expression have been poorly understood in skeletal muscles. Recently, it was reported that the expression of AQP4 decreased in atrophied skeletal muscles following sciatic nerve transection, but not tail-suspension. Therefore, expecting that the nerve supply to myofibers would be one of the major regulatory factors regulating AQP4 expression in skeletal muscles, we investigated whether the expression patterns of AQP4 were changed in skeletal muscles by denervation and subsequent reinnervation. As a result, while the APQ4 expression levels were significantly decreased by sciatic nerve freezing-induced denervation, subsequently the expression levels of AQP4 were fully restored during reinnervation in skeletal muscles (p < 0.05, respectively). On the other hand, the expression levels of α1-syntrophin and AQP1, which are respectively structural and functional related AQP4 factors, were stably maintained during the denervation and subsequent reinnervation. Therefore, the present study demonstrated that the expression of AQP4 may be regulated depending on the innervation to skeletal muscles. Moreover, AQP4 regulatory mechanisms may be fundamentally different to those of AQP1 in skeletal muscles.

Marked decrease of aquaporin-4 protein is independent of the changes in α1-syntrophin and TRPV4 levels in response to denervation-induced muscle atrophy in vivo.

Aquaporin-4 (AQP4) is a selective water channel mediating water transport across cell membranes in skeletal muscles. Recently, it was noted that AQP4 is one of the key molecules regulating muscle morphology. Indeed, the AQP4 accumulation level was stably maintained in hypertrophied skeletal muscles. On the other hand, whether the AQP4 accumulation level is stably maintained in atrophied muscles remains poorly understood. The present study investigated the changes in the AQP4 accumulation level in the atrophied muscles at 2 weeks after denervation. As a result, the accumulation level of AQP4 in the atrophied muscle was significantly decreased compared with that in the control muscle (p < 0.05). Interestingly, the accumulation level of α1-syntrophin, which is an essential factor in regulating the stable accumulation level of AQP4, was stably maintained in the atrophied muscles. On the other hand, the accumulation level of the transient receptor potential vanilloid 4 (TRPV4), which contributes to cell volume control via interaction with AQP4, was significantly increased in the atrophied muscles compared with that in the control muscle (p < 0.05). Therefore, the present study suggested that the imbalance between the AQP4 accumulation level and skeletal muscle volume may be induced in the atrophied muscles by denervation, and the decrease in the accumulation level of AQP4 may be accompanied by defects in the functional and structural relationships with α1-syntrophin and TRPV4.

Aquaporin-4 Protein Is Stably Maintained in the Hypertrophied Muscles by Functional Overload.

Aquaporin-4 (AQP4) is a selective water channel that is located on the plasma membrane of myofibers in skeletal muscle and is bound to α1-syntrophin. It is considered that AQP4 is involved in the modulation of homeostasis in myofibers through the regulation of water transport and osmotic pressure. However, it remains unclear whether AQP4 expression is altered by skeletal muscle hypertrophy to modulate water homeostasis in myofibers. The present study investigated the effect of muscle hypertrophy on the changes in AQP4 and α1-syntrophin expression patterns in myofibers. Novel findings indicated in the present study were as follows: 1) Expression levels of AQP4 and α1-syntrophin were stably maintained in hypertrophied muscles, and 2) AQP4 was not expressed in the myofibers containing the slow-type myosin heavy chain isoform (MHC) with or without the presence of fast-type MHC. The present study suggests that AQP4 may regulate the efficiency of water transport in hypertrophied myofibers through its interaction with α1-syntrophin. In addition, this study suggests that AQP4 expression may be inhibited by a regulatory mechanism activated under physiological conditions that induces the expression of slow-type MHC in skeletal muscles.

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo.

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.

In Vivo Real-Time Imaging of Exogenous HGF-Triggered Cell Migration in Rat Intact Soleus Muscles.

The transplantation of myogenic cells is a potentially effective therapy for muscular dystrophy. However, this therapy has achieved little success because the diffusion of transplanted myogenic cells is limited. Hepatocyte growth factor (HGF) is one of the primary triggers to induce myogenic cell migration in vitro. However, to our knowledge, whether exogenous HGF can trigger the migration of myogenic cells (i.e. satellite cells) in intact skeletal muscles in vivo has not been reported. We previously reported a novel in vivo real-time imaging method in rat skeletal muscles. Therefore, the present study examined the relationship between exogenous HGF treatment and cell migration in rat intact soleus muscles using this imaging method. As a result, it was indicated that the cell migration velocity was enhanced in response to increasing exogenous HGF concentration in skeletal muscles. Furthermore, the expression of MyoD was induced in satellite cells in response to HGF treatment. We first demonstrated in vivo real-time imaging of cell migration triggered by exogenous HGF in intact soleus muscles. The experimental method used in the present study will be a useful tool to understand further the regulatory mechanism of HGF-induced satellite cell migration in skeletal muscles in vivo.

In situ real-time imaging of the satellite cells in rat intact and injured soleus muscles using quantum dots.

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.

Features and a possible role of Mash1-immunoreactive cells in the dentate gyrus of the hippocampus in the adult rat.

Neurogenesis occurs throughout life in both the subventricular zone (SVZ) and subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus in the adult brain. In the SVZ, it has been demonstrated that transit-amplifying neural progenitor cells, which appear between neural stem/progenitor cells (NSPCs) and neuroblasts during the neuronal differentiation process, express mammalian achaete-scute homolog 1 (Mash1), which regulates differentiation during neurogenesis. Although Mash1-positive cells (Mash1+ cells) are observed in the SGZ, the importance of Mash1 in hippocampal neurogenesis is not sufficiently understood. In the present study, using immunohistochemical techniques, we examined whether Mash1+ cells in the SGZ act as transit-amplifying neural progenitor cells, and whether chronic treadmill running can induce alterations of the Mash1+ cells in the SGZ of the DG. The present results indicated that Mash1 immunoreactivity is detected in proliferative cells, and that astrocytes or NSPCs and neuroblasts express Mash1. A quantitative analysis of Mash1-positive astrocytes or NSPCs and Mash1-positive neuroblasts indicated that approximately 90% of Mash1+ cells did not belong to astrocytic and neuronal cells. Furthermore, chronic treadmill running induced an increase in the number of proliferating Mash1+ cells. The present study suggests that the majority of the Mash1+ cells in the SGZ may be transit-amplifying neural progenitor cells. In addition, the proliferation of Mash1-positive transit-amplifying neural progenitor cells may contribute to the exercise-induced neurogenesis that is associated with the improvement of learning and memory function.

Effects of chronic treadmill running on neurogenesis in the dentate gyrus of the hippocampus of adult rat.

Proliferating astrocytes and proliferating neuroblasts have been observed in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus of adult rats under normal conditions. However, whether these proliferating cells are stimulated by running has not been determined. Using immunohistochemical techniques, we examined the effects of chronic treadmill running on proliferating astrocytes (PCNA+/GFAP+ cells), proliferating neuroblasts (PCNA+/DCX+ cells) and newly generated postmitotic neurons (DCX+/NeuN+ cells) in the DG of the hippocampus of adult rats and also characterized the morphological features of PCNA+/GFAP+ cells and PCNA+/DCX+ cells. PCNA+/GFAP+ cells with few processes and PCNA+/DCX+ cells without long processes were detected in the SGZ, and we determined that these are morphological features of the astrocytes and neuroblasts with proliferative ability. Chronic treadmill running (at a speed of 22 m/min, 30 min/days for 7 days) significantly increased the numbers of PCNA+/GFAP+ cells and DCX+/NeuN+ cells, and the number of PCNA+/DCX+ cells tended to increase by chronic treadmill running. These results indicate that chronic treadmill running stimulates the proliferation of astrocytes in the SGZ. Furthermore, the present study indicates that chronic treadmill running increases DCX+/NeuN+ cells that are detected in a transient stage during the neuronal maturation process. These events may be the cellular basis mediating both running-induced increases of new neurons in the DG of the hippocampus and running-induced improvement of learning and memory functions of adult rats.

In vivo expression patterns of MyoD, p21, and Rb proteins in myonuclei and satellite cells of denervated rat skeletal muscle.

MyoD, a myogenic regulatory factor, is rapidly expressed in adult skeletal muscles in response to denervation. However, the function(s) of MyoD expressed in denervated muscle has not been adequately elucidated. In vitro, it directly transactivates cyclin-dependent kinase inhibitor p21 (p21) and retinoblastoma protein (Rb), a downstream target of p21. These factors then act to regulate cell cycle withdrawal and antiapoptotic cell death. Using immunohistochemical approaches, we characterized cell types expressing MyoD, p21, and Rb and the relationship among these factors in the myonucleus of denervated muscles. In addition, we quantitatively examined the time course changes and expression patterns among distinct myofiber types of MyoD, p21, and Rb during denervation. Denervation induced MyoD expression in myonuclei and satellite cell nuclei, whereas p21 and Rb were found only in myonuclei. Furthermore, coexpression of MyoD, p21, and Rb was induced in the myonucleus, and quantitative analysis of these factors determined that there was no difference among the three myofiber types. These observations suggest that MyoD may function in myonuclei in response to denervation to protect against denervation-induced apoptosis via perhaps the activation of p21 and Rb, and function of MyoD expressed in satellite cell nuclei may be negatively regulated. The present study provides a molecular basis to further understand the function of MyoD expressed in the myonuclei and satellite cell nuclei of denervated skeletal muscle.