Soleus muscle atrophy was induced by hindlimb unloading of male Sprague-Dawley rats (305 +/- 15 g) for 4, 7, and 10-14 days. Controls (291 +/- 14 g) were housed in vivarium cages. Soleus electromyogram (EMG) activity was recorded before and during tail suspension. Unloading caused progressive reduction in the muscle-to-body weight ratio. After 14 days, type I and IIa fibers decreased in area 63 and 47%, respectively. Subsarcolemmal mitochondria and myofibrils were degraded more rapidly than intermyofibrillar mitochondria and the cell membrane. After 10 days, 3% of the fibers exhibited segmental necrosis; affected fibers were all high-oxidative type IIa fibers. This suggested ischemic injury. By 13 days, 30% of the fibers possessed central corelike lesions involving primarily type I fibers. Video monitoring revealed abnormal plantar flexion of the hindfeet by 4 days; this posture shortened the soleus working range. Corelike lesions indicated adaptation to the shortened length. No morphological signs of denervation were detected. EMG activity shifted from tonic to phasic, and aggregate activity was 13% of normal after 7 days. These findings indicate that the atrophy and pathological changes result from unloaded contractions, reduced use, compromised blood flow, and shortened working length.

Spaceflight (flight) and tail suspension-hindlimb unloading (unloaded) produced significant decreases in fiber cross-sectional areas of the adductor longus (AL), a slow-twitch antigravity muscle. However, the mean wet weight of the flight AL muscles was near normal, whereas that of the suspension unloaded AL muscles was significantly reduced. Interstitial edema within the flight AL, but not in the unloaded AL, appeared to account for this apparent disagreement. In both experimental conditions, the slow-twitch oxidative fibers atrophied more than the fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers. Immunostaining showed that slow-twitch oxidative fibers expressed fast myosin, producing hybrid fibers containing slow and fast myosin isoforms. Two-dimensional gel electrophoresis of flight AL muscles revealed increased content of fast myosin light chains and decreased amounts of slow myosin light chains and fatty acid-binding protein. In the flight AL, absolute mitochondrial content decreased, but the relatively greater breakdown of myofibrillar proteins maintained mitochondrial concentration near normal in the central intermyofibrillar regions of fibers. Subsarcolemmal mitochondria were preferentially lost and reduced below normal concentration. Elevated fiber immunostaining for ubiquitin conjugates was suggestive of ubiquitin-mediated breakdown of myofibrillar proteins. On return to weight bearing for 8-11 h, the weakened atrophic muscles exhibited eccentric contraction-like lesions (hyperextension of sarcomeres with A-band filaments pulled apart and fragmented), tearing of the supporting connective tissue, and thrombosis of the microcirculation. Segmental necrosis of muscle fibers, denervation of neuromuscular junctions, and extravasation of red blood cells were minimal. Lymphocyte antibody markers did not indicate a significant immune reaction. The flight AL exhibited threefold more eccentric-like lesions than the unloaded AL; the high reentry G forces experienced by the flight animals, but not the unloaded group, possibly accounted for this difference. Muscle atrophy appears to increase the susceptibility to form eccentric contraction-like lesions after reloading; this may reflect weakening of the myofibrils and extracellular matrix. Microcirculation was also compromised by spaceflight, such that there was increased formation of thrombi in the post-capillary venules and capillaries. This blockage led to edema by 8-11 h after resumption of weight bearing by the COSMOS 2044 rats. The present findings indicate that defective microcirculation most likely accounted for the extensive tissue necrosis and microhemorrhages observed for COSMOS 1887 rats killed 2 days after landing.

Spacelab Life Sciences-1 and -2 provided skeletal muscles from rats dissected in flight for the first time and 2 h to 14 days postflight. The muscles permitted the distinguishing of primary adaptations to microgravity from secondary reloading-induced alterations. In microgravity, rats adopted bipedal forelimb locomotion with the hindlimbs relegated to grasping activities. On landing day, body posture was abnormally low and walking was stilted at a rate one-third of normal. The adductor longus (AL) and soleus muscles exhibited decreased myofiber areas that did not recover 14 days postflight. Doubling of the nonmyofiber area indicated interstitial edema in AL muscles 2.3 h postflight. Solei did not manifest edema postflight, and neither muscle showed edema in flight. Sarcomere eccentric contraction-like lesions were detected in 2.6% of AL myofibers 4.5 h postflight; lesions were absent earlier postflight and in flight. At 9 days postflight, these lesions were repaired but regenerating AL myofibers were present, which suggests that myofiber necrosis occurred 1-2 days postflight. These studies demonstrate that muscle atrophy occurs in microgravity, whereas interstitial edema and sarcomere lesions are postflight phenomena.

Histochemical and ultrastructural analyses were performed postflight on hind limb skeletal muscles of rats orbited for 12.5 days aboard the unmanned Cosmos 1887 biosatellite and returned to Earth 2 days before sacrifice. The antigravity adductor longus (AL), soleus, and plantaris muscles atrophied more than the non-weight-bearing extensor digitorum longus, and slow muscle fibers were more atrophic than fast fibers. Muscle fiber segmental necrosis occurred selectively in the AL and soleus muscles; primarily, macrophages and neutrophils infiltrated and phagocytosed cellular debris. Granule-rich mast cells were diminished in flight AL muscles compared with controls, indicating the mast cell secretion contributed to interstitial tissue edema. Increased ubiquitination of disrupted myofibrils implicated ubiquitin in myofilament degradation. Mitochondrial content and succinic dehydrogenase activity were normal, except for subsarcolemmal decreases. Myofibrillar ATPase activity of flight AL muscle fibers shifted toward the fast type. Absence of capillaries and extravasation of red blood cells indicated failed microcirculation. Muscle fiber regeneration from activated satellite cells was detected. About 17% of the flight AL end plates exhibited total or partial denervation. Thus, skeletal muscle weakness associated with spaceflight can result from muscle fiber atrophy and segmental necrosis, partial motor denervation, and disruption of the microcirculation.

Substitution of chromosome 13 from Brown Norway BN/SsNHsd/Mcw (BN/Mcw) rats into the Dahl salt-sensitive SS/JrHsd/Mcw (SS/Mcw) rats resulted in substantial reduction of blood pressure salt sensitivity in this consomic rat strain designated SSBN13. In the present study, we attempted to identify genes associated with salt-sensitive hypertension by utilizing a custom, known-gene cDNA microarray to compare the mRNA expression profiles in the renal medulla (a tissue playing a pivotal role in long-term blood pressure regulation) of SS/Mcw and SSBN13 rats on either low-salt (0.4% NaCl) or high-salt (4% NaCl, 2 wk) diets. To increase the reliability of microarray data, we designed a four-way comparison experiment incorporating several levels of replication and developed a conservative yet robust data analysis method. Using this approach, from the 1,751 genes examined (representing more than 80% of all currently known rat genes), we identified 80 as being differentially expressed in at least 1 of the 4 comparisons. Substantial agreements were found between the microarray results and the results predicted on the basis of the four-way comparison as well as the results of Northern blots of 20 randomly selected genes. Analysis of the four-way comparison further indicated that approximately 75% of the 80 differentially expressed genes were likely related to salt-sensitive hypertension. Many of these genes had not previously been recognized to be important in hypertension, whereas several genes/pathways known to be involved in hypertension were confirmed. These results should provide an informative source for designing future functional studies in salt-sensitive hypertension.