The role of myostatin and activin receptor IIB in the regulation of unloading-induced myofiber type-specific skeletal muscle atrophy.

Chronic unloading induces decrements in muscle size and strength. This adaptation is governed by a number of molecular factors including myostatin, a potent negative regulator of muscle mass. Myostatin must first be secreted into the circulation and then bind to the membrane-bound activin receptor IIB (actRIIB) to exert its atrophic action. Therefore, we hypothesized that myofiber type-specific atrophy observed after hindlimb suspension (HLS) would be related to myofiber type-specific expression of myostatin and/or actRIIB. Wistar rats underwent HLS for 10 days, after which the tibialis anterior was harvested for frozen cross sectioning. Simultaneous multichannel immunofluorescent staining combined with differential interference contrast imaging was employed to analyze myofiber type-specific expression of myostatin and actRIIB and myofiber type cross-sectional area (CSA) across fiber types, myonuclei, and satellite cells. Hindlimb suspension (HLS) induced significant myofiber type-specific atrophy in myosin heavy chain (MHC) IIx (P < 0.05) and MHC IIb myofibers (P < 0.05). Myostatin staining associated with myonuclei was less in HLS rats compared with controls, while satellite cell staining for myostatin remained unchanged. In contrast, the total number myonuclei and satellite cells per myofiber was reduced in HLS compared with ambulatory control rats (P < 0.01). Sarcoplasmic actRIIB staining differed between myofiber types (I < IIa < IIx < IIb) independent of loading conditions. Myofiber types exhibiting the greatest cytoplasmic staining of actRIIB corresponded to those exhibiting the greatest degree of atrophy following HLS. Our data suggest that differential expression of actRIIB may be responsible for myostatin-induced myofiber type-selective atrophy observed during chronic unloading.

A three-dimensional tissue culture model of bone formation utilizing rotational co-culture of human adult osteoblasts and osteoclasts.

Living bone is a complex, three-dimensional composite material consisting of numerous cell types spatially organized within a mineralized extracellular matrix. To date, mechanistic investigation of the complex cellular level cross-talk between the major bone-forming cells involved in the response of bone to mechanical and biochemical stimuli has been hindered by the lack of a suitable in vitro model that captures the "coupled" nature of this response. Using a novel rotational co-culture approach, we have generated large (>4mm diameter), three-dimensional mineralized tissue constructs from a mixture of normal human primary osteoblast and osteoclast precursor cells without the need for any exogenous osteoconductive scaffolding material that might interfere with such cell-cell interactions. Mature, differentiated bone constructs consist of an outer region inhabited by osteoclasts and osteoblasts and a central region containing osteocytes encased in a self-assembled, porous mineralized extracellular matrix. Bone constructs exhibit morphological, mineral and biochemical features similar to remodeling human trabecular bone, including the expression of mRNA for SOST, BGLAP, ACP5, BMP-2, BMP-4 and BMP-7 within the construct and the secretion of BMP-2 protein into the medium. This "coupled" model of bone formation will allow the future investigation of various stimuli on the process of normal bone formation/remodeling as it relates to the cellular function of osteoblasts, osteoclasts and osteocytes in the generation of human mineralized tissue.

Obese mice incur greater myofiber membrane disruption in response to mechanical load compared with lean mice.

OBJECTIVE: Obesity is associated with modified transmembrane signaling events in skeletal muscle, such as insulin signaling and glucose transport. The underlying cause of these obesity-related effects on transmembrane signaling is still unknown. In general, the function of membrane proteins responsible for transmembrane signaling is modulated by the biochemical makeup of the membrane, such as lipid composition, in which they are embedded. Any obesity-related alterations in membrane composition would also be predicted to modify membrane biomechanical properties and membrane susceptibility to mechanical load-induced damage. The primary objective of this study was to investigate whether obesity influences myofiber membrane susceptibility to mechanical damage in skeletal muscle. DESIGN AND METHODS: Myofiber membrane damage was compared between 12-week-old obese, hypercholesterolemic (B6.V Lep(ob) /J) and isogenic, normocholesterolemic control (C57BL6/J) male mice following either normal cage activity or strenuous eccentric exercise (downhill running). Myofiber membrane damage was quantified in perfusion-fixed frozen sections of the gastrocnemius muscle via sarcoplasmic concentration of either albumin (cage activity experiment) or a fluorescent marker that had been injected immediately before activity (eccentric exercise experiment). RESULTS: Obese mice exhibited evidence of increased myofiber membrane damage compared with lean mice after both normal cage activity and eccentric exercise indicating that myofiber membranes of obese mice are more susceptible to mechanical damage in general and that eccentric exercise exacerbates this effect. CONCLUSIONS: These observations are consistent with the notion that obesity influences the biochemical and biomechanical properties of myofiber membranes.

Laser capture microdissection of metachromatically stained skeletal muscle allows quantification of fiber type specific gene expression.

Skeletal muscle contains various myofiber types closely associated with satellite stem cells, vasculature, and neurons, thus making it difficult to perform genetic or proteomic expression analysis with sufficient cellular specificity to resolve differences at the individual cell or myofiber type level. Here, we describe the combination of a simple histochemical method capable of simultaneously identifying Type I, IIA, IIB, and IIC myofibers followed by laser capture micro-dissection (LCM) to compare the expression profiles of individual fiber types, myonuclear domains, and satellite cells in frozen muscle sections of control and atrophied muscle. Quantitative RT-PCR (qPCR) was used to verify the integrity of the cell-specific RNAs harvested after histologic staining, while qPCR for specific genes of interest was used to quantify atrophy-associated changes in mRNA. Our data demonstrate that the differential myofiber atrophy previously described by histologic means is related to differential expression of atrophy-related genes, such as MuRF1 and MAFbx (a.k.a. Atrogin-1), within different myofiber type populations. This spatially resolved molecular pathology (SRMP) technique allowed quantitation of atrophy-related gene products within individual fiber types that could not be resolved by expression analysis of the whole muscle. The present study demonstrates the importance of fiber type specific expression profiling in understanding skeletal muscle biology especially during muscle atrophy and provides a practical method of performing such research.

Proteomic analysis of skeletal muscle tissue using SELDI-TOF MS: application to disuse atrophy.

Skeletal muscle atrophy in response to disuse/unloading is a complex adaptation that involves many components of the muscle tissue. The underlying mechanisms that initiate and control the loss of muscle tissue during this response, especially contractile proteins located within the myofibers, are as yet unclear. One approach capable of distinguishing protein changes specifically associated with disuse/unloading-induced skeletal muscle atrophy is to compare the proteomic profiles of similar muscles between control, unloaded/atrophied, and unloaded/"atrophy-protected" experimental conditions. By utilizing a subtractive proteomic analysis approach, those proteins specifically modulated during the atrophic response can be identified and discriminated from those associated with disuse in general. We here describe the use of SELDI-TOF MS coupled with micro-scale preparative ion-exchange chromatography to detect proteins potentially specifically associated with the atrophic response in rat skeletal muscle.

Near infrared spectroscopy-derived interstitial hydrogen ion concentration and tissue oxygen saturation during ambulation.

The objective of this study was to determine whether walking and running at different treadmill speeds resulted in different metabolic and cardiovascular responses in the vastus lateralis (VL) and lateral gastrocnemius (LG) by examining metabolite accumulation and tissue oxygen saturation. Ten healthy subjects (6 males, 4 females) completed a submaximal treadmill exercise test, beginning at 3.2 km h(-1) and increasing by 1.6 km h(-1) increments every 3 min until reaching 85% of age-predicted maximal heart rate. Muscle tissue oxygenation (SO(2)), total hemoglobin (HbT) and interstitial hydrogen ion concentration ([H(+)]) were calculated from near infrared spectra collected from VL and LG. The [H(+)] threshold for each muscle was determined using a simultaneous bilinear regression. Muscle and treadmill speed effects were analyzed using a linear mixed model analysis. Paired t-tests were used to test for differences between muscles in the [H(+)] threshold. SO(2) decreased (P = 0.001) during running in the VL and LG, but the SO(2) response across treadmill speeds was different between muscles (P = 0.047). In both muscles, HbT and [H(+)] increased as treadmill speed increased (P < 0.001), but the response to exercise was not different between muscles. The [H(+)] threshold occurred at a lower whole-body VO(2) in the LG (1.22 ± 0.63 L min(-1)) than in the VL (1.46 ± 0.58 L min(-1), P = 0.01). In conclusion, interstitial [H(+)] and SO(2) are aggregate measures of local metabolite production and the cardiovascular response. Inferred from simultaneous SO(2) and [H(+)] measures in the VL and LG muscles, muscle perfusion is well matched to VL and LG work during walking, but not running.

Capillary and venous samples of total creatine kinase are similar after eccentric exercise.

Circulating creatine kinase (CK) levels are often monitored as an indirect biomarker of muscle damage after resistive exercise. The purpose of the present investigation was to evaluate whether capillary whole-blood sampling, a simpler and less invasive method for obtaining a venous blood sample, would allow for a reliable measurement of total CK compared to venipuncture. Fifteen untrained subjects performed 50 maximal eccentric elbow extensions to induce muscle damage of the biceps brachii. Capillary (fingerstick) and venous whole-blood samples were collected contemporaneously at baseline and again at 24, 48, 72, and 96 hours post-exercise. Using a commercial CK analysis kit with a protocol modification to account for a reduced sample size, total CK activity of the capillary and venous samples was analyzed concurrently via spectrophotometry. Results indicated a 0.997 correlation between sampling sites for total CK, with disagreement between the venous and capillary samples estimated at <12% across the range of CK values. These findings indicate capillary sampling for total CK activity provides a valid alternative to venipuncture and should be considered by researchers, clinicians, and strength and conditioning specialists as an alternate sampling technique when indirectly evaluating muscle damage after exercise.

Ionizing radiation-induced bioeffects in space and strategies to reduce cellular injury and carcinogenesis.

BACKGROUND: The bioeffects of space radiation on organisms outside of the environment of Earth's magnetosphere are a concern for long-duration exploration spaceflights. Potential mutagenic effects from space radiation exposure result from direct DNA damage or indirectly from the production of reactive oxygen species (ROS). HYPOTHESES: 1) Transepithelial electrical resistance (TER) measurements in cell culture monolayers may be used as a model system for detecting cell damage produced by exposure to simulated space radiation and for testing potential chemoprotective agents; 2) biomarkers of exposure that quantitate indirect radiation effects may allow prediction of cellular DNA damage; and 3) a multiple agent, chemoprevention cocktail may reduce the bioeffects of simulated space radiation. METHODS: Normal human and canine lung, breast, and renal epithelial cells were assayed in vitro and exposed to escalating doses of gamma or heavy-ion carbon (290 MeV/u), ceon (400 MeV/u), or iron (600 MeV/u) irradiation. Post-exposure measurements of TER, lipid peroxidation (LP) via measurement of 4-hydroxy-nonenal (4-HNE), and malonaldehyde (MDA) and assessment of chromosome damage via fluorescence in situ hybridization with tandem labeling of chromosome 1 were performed. RESULTS: Cells exposed to intermediate or high doses of radiation (5, 10, and 25 Gy) showed characteristic diminution in TER, thought to be secondary to dysfunction of tight junctions, and associated with membrane LP and other mechanisms. The cells also showed increases in 4-HNE + MDA measurements and increased frequency of chromosomal aberrations. Preliminary studies of cells incubated with media containing a combination of chemoprotective agents at the time of radiation exposure showed a 15-50% reduction in the radiation-induced changes in membrane resistance, levels of LP, and chromosomal aberrations relative to their unprotected cellular counterparts. CONCLUSION: TER measurement, in conjunction with measures of LP, may provide a useful model for determination of physiological changes caused by radiation exposure and the efficacy of chemoprotective agents. A multi-agent mixture of chemoprotective agents may be more effective than previously evaluated single agents alone.

Soleus muscle force following downhill running in ovariectomized rats treated with estrogen.

The ovariectomized (OVX) rat model was used to investigate the effects of estrogen treatment on soleus muscle functionality in situ following muscle injury induced by downhill running. Fifty immature, 24- to 26-d-old, OVX rats were randomly assigned to 5 separate experimental groups: sedentary controls (OVX-Sed), placebo-treated and studied immediately after exercise (OVX-Pb0), placebo-treated and studied 72 h after exercise (OVX-Pb72), estradiol-treated and studied immediately after exercise (OVX-Ed0), and estradiol-treated and studied 72 h after exercise (OVX-Ed72). At the age of 9 weeks, under ketamine and xylazine anesthesia i.p., the rats were subcutaneously implanted with either placebo or 17beta-estradiol-impregnated pellets (0.05 mg/pellet, 3 week release). Treatment with 17beta-estradiol increased the estradiol plasma levels in OVX animals to those normally seen during the proestrous cycle of normal animals. Three weeks after the implantation the rats were subjected to a 90 min intermittent downhill running protocol. Our results indicate that the exercise protocol used in the study induced injury in the soleus muscle, as it was detected by the significant reduction in unfused (stimulation at 10, 20, and 40 Hz) and maximal (Po) tetanic force, as well as the decreased ability of the soleus muscle to maintain tension (stimulation at 40 Hz for 3 min) in OVX-Pb0 and OVX-Pb72 placebo-treated animals subjected to downhill running (injured muscles) as compared with OVX-Sed control rats (uninjured muscle). Estradiol replacement in OVX rats partially protected the soleus muscle from the injury normally induced by downhill running. As compared with the OVX-Pb0 and OVX-Pb72 placebo-treated groups, the soleus muscles of OVX-Ed0 and OVX-Ed72 estradiol-treated rats were capable of producing significantly greater unfused tetanic force and had an increased ability to maintain tension after fatigue. However, estrogen at the dose administered did not prevent the decrease in maximal tetanic force. We postulate that the protective effect of estrogens on muscle strength may be related to the ability of estrogen hormones to attenuate the E--C coupling failure and (or) the disorganization of the contractile apparatus associated with eccentric exercise through a mechanism or mechanisms yet to be fully understood.

Mechanical stimulation of the plantar foot surface attenuates soleus muscle atrophy induced by hindlimb unloading in rats.

Unloading-induced muscle atrophy occurs in the aging population, bed-ridden patients, and astronauts. This study was designed to determine whether dynamic foot stimulation (DFS) applied to the plantar surface of the rat foot can serve as a countermeasure to soleus muscle atrophy normally observed in hindlimb unloaded (HU) rats. Forty-four mature (6 mo old), male Wistar rats were randomly assigned to ambulatory control, HU alone, HU with active DFS (i.e., plantar contact with active inflation), HU with passive DFS (i.e., plantar contact without active inflation), and HU while wearing a DFS boot with no plantar contact groups. Application of active DFS during HU significantly counteracted the atrophic response by preventing approximately 85% of the reduction in type I myofiber cross-sectional area (CSA) in the soleus while preventing approximately 57% of the reduction in type I myofiber CSA and 43% of the reduction in type IIA myofiber CSA of the medial gastrocnemius muscle. Wearing of a DFS boot without active inflation prevented myofiber atrophy in the soleus of HU animals in a fashion similar to that observed in HU animals that wore an actively inflated DFS boot. However, when a DFS boot without plantar surface contact was worn during HU, no significant protection from HU-induced myofiber atrophy was observed. These results illustrate that the application of mechanical foot stimulation to the plantar surface of the rat foot is an effective countermeasure to muscle atrophy induced by HU.