Rita Rani Roy

A new technique for the sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of rat skeletal muscle myosin heavy-chain (MHC) isoforms is presented. This technique allows for the separation of the four identified MHC isoforms known to be present in adult rat skeletal muscle. These types of MHC are commonly called I, IIa, IIx or IId, and IIb. The procedure can be performed using minigel electrophoresis systems and does not involve preparation of gradient-separating gels or the use of special cooling devices. The procedure accommodates both silver and Coomasie Blue staining. Thus the procedure is simple to perform and highly repeatable, providing high-resolution separation of MHC protein isoforms. The percent composition of the four adult MHCs in rat soleus, medial gastrocnemius, diaphragm, and levator ani muscles by use of this procedure and Coomasie Blue staining is similar to that previously reported. This new technique provides a novel and easy-to-perform method for the separation of rat skeletal muscle MHC isoforms.

The role of apoptosis in the elimination of myonuclei during hindlimb unloading-induced atrophy and the inhibition of apoptosis in the prevention of muscle atrophy were examined. The number of nuclei demonstrating double-stranded DNA fragmentation seen by terminal deoxynucleotidyl transferase (TDT) histochemical staining, an indicator of apoptosis, was significantly increased after 14 days of suspension. Double staining with TDT and antilaminin immunohistochemistry revealed that some TDT-positive nuclei were within the fiber lamina and were most likely myonuclei. The number of fibers containing morphologically abnormal nuclei was also significantly greater in suspended compared with control rats. Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance exercise attenuated the increase in TDT-positive nuclei (approximately 26%, P > 0.05) and significantly decreased the number of fibers with morphologically abnormal nuclei. The data suggest that 1) "programmed nuclear death" contributes to the elimination of myonuclei and/or satellite cells from atrophying fibers, and 2) GH/IGF-I administration plus muscle loading ameliorates the apoptosis associated with hindlimb unloading.

The objectives of the present study were to determine the size and enzyme properties of soleus fibers of rats subjected to a 4-day spaceflight (National Aeronautics and Space Administration, STS-41) and the effects of exogenous growth hormone (GH) on the atrophic response of the muscle. Four groups of rats were studied: 1) control (Con), 2) Con plus GH treated (Con + GH), 3) flight (Fl), and 4) F1 plus GH treated (Fl + GH). Cross-sectional area and the activities of succinate dehydrogenase and myofibrillar adenosinetriphosphatase (ATPase) were determined in fibers identified in frozen serial cross sections. Fibers were categorized immunohistochemically as slow, fast, or slow-fast on the basis of their reaction with slow and fast myosin heavy-chain (MHC) monoclonal antibodies. Fibers also were categorized as light or dark on the basis of their staining for ATPase at pH 8.6. After the 4-day flight, mean body weight was significantly decreased compared with control. The absolute and relative (muscle wt/body wt) soleus weights were significantly smaller in the Fl and Fl + GH rats compared with their respective ground-based controls. In both flight groups, the cross-sectional area of the light ATPase fibers was significantly smaller (approximately 30%) than control. Three of 11 flight rats had a higher proportion of fibers expressing both slow and fast MHCs than expected on the basis of the fiber type distribution in the 11 control rats. Mean fiber succinate dehydrogenase and ATPase activities were similar among the four groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic muscle inactivity with spinal cord isolation (SI) decreases expression of slow type I myosin heavy chain (MHC) while increasing expression of the faster MHC isoforms, primarily IIx. The purpose of this study was to determine whether type I MHC downregulation in the soleus muscle of SI rats is regulated transcriptionally and to identify cis-acting elements or regions of the rat type I MHC gene promoter involved in this response. One week of SI significantly decreased in vivo activity of the -3500-, -408-, -299-, -215-, and -171-bp type I MHC promoters. The activity of all tested deletions of the type I MHC promoter, relative to the human skeletal alpha-actin promoter, were significantly reduced in the SI soleus, except activity of the -171-bp promoter, which increased. Mutation of the betae3 element (-214/-190 bp) in the -215- and -408-bp promoters and deletion of this element (-171-bp promoter) attenuated type I downregulation with SI. Gel mobility shift assays demonstrated a decrease in transcription enhancer factor-1 binding to the betae3 element with SI, despite an increase in total binding to this region. These results demonstrate that type I MHC downregulation with SI is transcriptionally regulated and suggest that interactions between transcription enhancer factor-1 and the betae3 element are likely involved in this response.

Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 +/- 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)