Effects of Head-down Tilt on Nerve Conduction in Rhesus Monkeys.

BACKGROUND: Few studies have focused on peripheral nerve conduction during exposure to microgravity. The -6° head-down tilt (HDT) comprises an experimental model used to simulate the space flight environment. This study investigated nerve conduction characteristics of rhesus monkeys before and after prolonged exposure to HDT. METHODS: Six rhesus monkeys (3-4 years old) were tilted backward 6° from the horizontal. Nerve conduction studies (NCSs) were performed on the median, ulnar, tibial, and fibular motor nerves. Analysis of variance with a randomized block design was conducted to compare the differences in the NCS before and 7, 21, and 42 days after the -6° HDT. RESULTS: The proximal amplitude of the CMAP of the median nerve was significantly decreased at 21 and 42 days of HDT compared with the amplitude before HDT (4.38 ± 2.83 vs. 8.40 ± 2.66 mV, F = 4.85, P = 0.013 and 3.30 ± 2.70 vs. 8.40 ± 2.66 mV, F = 5.93, P = 0.004, respectively). The distal amplitude of the CMAP of the median nerve was significantly decreased at 7, 21, and 42 days of HDT compared with the amplitude before HDT (7.28 ± 1.27 vs. 10.25 ± 3.40 mV, F = 4.03, P = 0.039; 5.05 ± 2.01 vs. 10.25 ± 3.40 mV, F = 6.25, P = 0.04; and 3.95 ± 2.79 vs. 10.25 ± 3.40 mV, F = 7.35, P = 0.01; respectively). The proximal amplitude of the CMAP of the tibial nerve was significantly decreased at 42 days of HDT compared with the amplitude before HDT (6.14 ± 1.94 vs. 11.87 ± 3.19 mV, F = 5.02, P = 0.039). CONCLUSIONS: This study demonstrates that the compound muscle action potential amplitudes of nerves are decreased under simulated microgravity in rhesus monkeys. Moreover, rhesus monkeys exposed to HDT might be served as an experimental model for the study of NCS under microgravity.

Optimizing Electrical Stimulation for Promoting Satellite Cell Proliferation in Muscle Disuse Atrophy.

OBJECTIVE: The aim of this study was to investigate the optimal electrical stimulation (ES) protocol in attenuating disuse muscle atrophy by influencing satellite cell activity. DESIGN: This study used a pretest-posttest design. Six ES protocols of different duration (3 hrs day or 2 × 3 hrs day) and frequencies (2, 10, or 20 Hz) were applied on the soleus muscle in mice (n = 8 in each group) that were hindlimb-suspended for 14 days. Muscle mass, cross-sectional area and fiber-type composition, and peak tetanic force of the muscles were measured. Immunohistochemical staining was used to evaluate satellite cell content, activation, proliferation, and differentiation. Cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: ES at 2 Hz for 2 × 3 hrs day achieved the best effect in attenuating the loss of muscle fiber cross-sectional area and force. This stimulation parameter led to a 1.2-fold increase in satellite cell proliferation and was effective in rescuing cells from apoptosis. Besides, satellite cells in the atrophic muscles required different stimulation protocols for different cellular activities such as activation, proliferation, and myogenic differentiation. CONCLUSIONS: This study showed that ES at 2 Hz for 2 × 3 hrs day is the optimal protocol for counteracting muscle disuse atrophy.

Expression and association of TRPC1 with TRPC3 during skeletal myogenesis.

INTRODUCTION: TRPC1 and TRPC3 proteins are widely expressed in skeletal muscles in forming calcium-permeable channels. Herein we characterize the expression pattern of TRPC transcripts during skeletal myogenesis in C2C12 myoblasts. METHODS: We used polymerase chain reaction and Western blotting to detect expression levels, immunohistochemistry for subcellular localization, and co-immunoprecipitation techniques to assess interaction. RESULTS: TRPC1 localizes to the cytoplasm and is enriched in the perinuclear region in undifferentiated myoblasts. Expression of TRPC1 increases significantly during myogenesis and resides mainly in differentiated myocytes and myotubes. TRPC3 is absent in undifferentiated myoblasts, is dramatically upregulated in differentiated culture, and is preferentially expressed in myotubes. Physical interaction of TRPC1-TRPC3 was observed, suggesting the possible existence of heteromers. CONCLUSIONS: Expression of TRPC1 and TRPC3 is tightly regulated during myogensis. Evidence of TRPC1-TRPC3 interaction was first demonstrated in a muscle cell line. The functional consequences of this interaction remain to be established.

Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5-azacytidine enhances therapeutic efficacy for myocardial infarction.

BACKGROUND AND PURPOSE: The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (BMSCs) after myocardial infarction (MI) is controversial. Our previous results suggested that hypergravity promoted the cardiomyogenic differentiation of BMSCs, and thus we postulated that ex vivo pretreatment of BMSCs using hypergravity and 5-azacytidine (5-Aza) would lead to cardiomyogenic differentiation and result in superior biological and functional effects on cardiac regeneration of infarcted myocardium. METHODS: We used a rat MI model generated by ligation of the coronary artery. Homogeneous rat BMSCs were isolated, culture expanded, and differentiated into a cardiac lineage by adding hypergravity (2G) for 3 days and 5-Aza (50 lmol/L, 24 h). Rats underwent BMSCs (labeled with DAPI) injection after the infarction and were randomized into five groups. Group A rats received the control medium, Group B rats received unmodified BMSCs, Group C rats received BMSCs treated with hypergravity, Group D rats received BMSCs treated with 5-Aza, and Group E rats received BMSCs treated with 5-Aza and hypergravity (n = 6). RESULTS: After hypergravity and 5-Aza treatment, BMSCs showed positive for the early muscle and cardiac markers GATA-4, MEF-2, and Nkx2-5 with RT-PCR. We also found that hypergravity could enhance the activities of MEF-2 via promoting the nuclear export of HDAC5. The frozen section showed that the implanted BMSCs labeled with DAPI survived and angiogenesis was identified at the implantation site. In Groups B, C, D, and E rats, pre-treated BMSCs colocalized with α-actinin, and Group E rats showed a significantly larger increase in left ventricular function. CONCLUSIONS: The biological ex vivo cardiomyogenic differentiation of adult BMSCs with hypergravity and 5-Aza prior to their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a rat model of the infarcted myocardium.

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND: Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells. METHODS AND RESULTS: Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and beta-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes). CONCLUSION: Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.

[Disruption of microfilament cytoskeleton induced by simulated microgravity increases the activity of COL1A1 promoter].

It is well known that cytoskeleton system is the sensor of gravity in cells. Under microgravity condition, cytoskeleton is associated with the changes of cell shape, function, signaling and so on; but the relationship between cytoskeleton and gene expression is not fully understood. In present study, we discussed the effects of cell microfilament on the activity of collagen type I alpha 1 chain gene (COL1A1) promoter under microgravity simulated by clinostat and/or cytochalasin B as microfilament depolymerizer in the established EGFP-ROS cell line using the method of fluorescence semi-quantitative analysis and the fluorescent stain of microfilament. Compared with the normal control, the microfilament of ROS17/2.8 cell tended to disassemble, marginal distribution of fiber stress, and showed reducing stress fibers after spaceflight in Photon-M1 or clinorotation simulated microgravity, which suggested that microgravity destroyed the well-order cell cytoskeleton and induced a rearrangement. Treatment with suitable concentration of cytochalasin B in normal gravity induced disruption of microfilament, increased the activity of COL1A1 promoter and resulted in a dose-dependent increase of EGFP fluorescence. Therefore, a certain extent disruption of the microfilament system was associated with increased activity of the COL1A1 promoter. All above demonstrate that microfilament cytoskeleton system takes part in the regulation of COL1A1 promoter activity and plays an important role in the signaling of microgravity.

[Establishment of COL1A1-EGFP stably transfected ROS17/2.8 cell lines].

OBJECTIVE: To obtain ROS17/2.8 cell lines which were stably expressing EGFP reporter gene drived by COL1A1 promoter. METHOD: A 3.6 Kb COL1A1 promoter from rat was cloned into pMD-18-T vector by PCR. This amplified promoter vector was digested to get several different length fragments which were then fused with EGFP reporter gene to construct eukaryotic expression vectors. ROS17/2.8 cell was stably transfected with these vectors by LipofectAMINE(TM) and selected by G418. RESULT: The COL1A1-EGFP stably transfected cell lines were established. CONCLUSION: The cell lines will be useful for studying the effects of microgravity on the activity of COL1A1 promoter and expression of gene related with bone form.

[Effects of clinorotation-induced weightlessness on growth factor and ERK signal transduction in cultured cardiac fibroblasts].

OBJECTIVE: To study the changes of expression and secretion of some growth factors and ERK1/2 activation in cardiac fibroblasts under clinorotation conditions. METHOD: Primary cultured neonatal rat cardiac fibroblasts were clinorotated to simulate weightlessness. The bFGF and TGF(beta)1 expressions in the cells and Ang II concentration in the medium were detected by western blotting and radioimmunoassay respectively. The expression and phosphorylated level of ERK1/2 were analyzed by western blotting. RESULT: Compared to the control group, bFGF expression and Ang II concentration increased in clinorotation group, but there was no evident change in TGF(beta)1 expression. At the same time, the expression of ERK1/2 increased but their phosphorylated level decreased. CONCLUSION: Different growth factors responds to clinorotation by different ways. As one of the most important reactions of growth factor signal downstream pathways, ERK1/2 activation is inhibited in clinorotation.