Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Regulation of myogenin protein expression in denervated muscles from young and old rats.

Myogenin is a muscle-specific transcription factor participating in denervation-induced increases in nicotinic ACh receptor (nAChR) gene expression. Although myogenin RNA expression in denervated muscle is well documented, surprisingly little is known about myogenin protein expression. Therefore, we assayed myogenin protein and RNA in innervated and denervated muscles from young (4 mo) and old (24-32 mo) rats and compared this expression to that of the nAChR alpha-subunit RNA. These assays revealed increased myogenin protein expression within 1 day of denervation, preceding detectable increases in nAChR RNA. By 3 days of denervation, myogenin and nAChR alpha-subunit RNA were increased 500- and 130-fold, respectively, whereas myogenin protein increased 14-fold. Interestingly, old rats (32 mo) had 6-fold higher myogenin protein and approximately 80-fold higher mRNA levels than young rats. However, after denervation, expression levels were similar for young and old animals. The increased myogenin expression during aging, which tends to localize to small fibers, likely reflects spontaneous denervation and/or regeneration. Our results show that increased myogenin protein in denervated muscles correlates with the upregulation of its mRNA.

Changes in rat muscle with compensatory overload occur in a sequential manner.

The present study was initiated to determine the time course of changes in the profile of selected skeletal muscle myofibril proteins during compensatory overload. Whole muscle isometric contractile properties were measured to assess the physiological consequences of the overload stimulus. Compensatory overload of plantaris muscle of rats was induced by surgical ablation of the synergistic soleus and gastrocnemius muscles. Myosin light chain (LC) and tropomyosin (TM) compositions of control (CP) and overloaded plantaris (OP) muscles were determined by electrophoresis and myofibrillar ATPase assays were performed to assess changes in contractile protein interactions. Within one week of overload decreases in the alpha:beta TM ratio and myofibrillar ATPase activity were observed. Following 30 days of overload, a transition in type II to type I fibres was associated with an increase in slow myosin LC1. Interestingly, after 77 days of overload, the TM subunit ratio returned to one resembling a fast twitch muscle. It is proposed that the early and transitory changes in the TM subunits of OP, as well as the rapid initial depression in maximum tetanic isometric force and myofibrillar ATPase activity may be explained as a result of muscle fibre degeneration-regeneration. We propose that alterations in protein expression induced by compensatory overload reflect both degenerative-regenerative change and increased neuromuscular activity.

Sarcomere dynamics and contraction-induced injury to maximally activated single muscle fibres from soleus muscles of rats.

1. The focal nature of contraction-induced injury to skeletal muscle fibres may arise from heterogeneities in sarcomere length that develop during contractions. We tested the hypothesis that when a maximally activated single permeabilized fibre segment is stretched and a deficit in maximum isometric force (force deficit) is produced, the regions of sarcomeres with the longest lengths of prior to the stretch contain the majority of the damaged sarcomeres when the fibre is returned to optimum length (Lo) after the stretch. 2. Single fibre segments (n = 16) were obtained from soleus muscles of rats. Average sarcomere length at five discrete positions along the length of each fibre was determined by lateral deflection of a diode laser spot. Diffraction patterns were obtained while fibres were relaxed and immediately before, during and after a single stretch of 40% strain relative to Lo. Following the stretch, the regions of each fibre that potentially contained damaged sarcomeres were identified by an increased scatter of the first-order diffraction patterns. The damage was confirmed by light and electron microscopy. 3. While single fibre segments were in relaxing solution, the mean value for all of the average sarcomere lengths sampled (n = 80) was 2.53 +/- 0.01 microns (range, 2.40-2.68 microns). During the maximum isometric contraction before each stretch, the mean sarcomere length decreased to 2.42 +/- 0.02 microns and the range increased to 2.12-3.01 microns. 4. During the stretch of 40% strain, all regions of sarcomeres were stretched onto the descending limb of the length-force curve, but sarcomere lengthening was non-uniform. After the stretch, when the maximally activated fibres were returned to Lo, the force deficit was 10 +/- 1%. Microscopic evaluation confirmed that the regions with the longest sarcomere lengths before the stretch contained the majority of the damaged sarcomeres after the stretch. We conclude that when heterogeneities in sarcomere length develop in single permeabilized fibre segments during a maximum isometric contraction, the sarcomeres in the regions with the longest lengths are the most susceptible to contraction-induced injury.

Contraction-induced injury to single fiber segments from fast and slow muscles of rats by single stretches.

Susceptibility to contraction-induced injury was investigated in single permeabilized muscle fiber segments from fast extensor digitorum longus and slow soleus muscles of rats. We tested the hypotheses that, after single stretches of varying strains and under three conditions of Ca2+ activation (none, submaximum, and maximum), 1) the magnitude of the deficit in maximum isometric force is dependent on the work done to stretch the fiber, and 2) for each condition of activation and strain, fast fibers incur greater force deficits than slow fibers. When all data on force deficits were analyzed together, the best predictors of the overall force deficits for both fast and slow muscle fibers were linear regression models that introduced the simultaneous but independent effects of strain and average force (r2 = 0.52 and 0.63, respectively). Under comparable conditions, greater force deficits were produced in fast than slows fibers. Despite differences in the strain required to produce injury in fast and slow muscle fibers, for a given force deficit, the ultrastructural damage was strikingly similar.