Elena J. Grossman

Effects of inactivity on myosin heavy chain composition and size of rat soleus fibers

Myosin heavy chain (MHC) and fiber size properties of the adult rat soleus were determined after 4–60 days of complete inactivity, i.e., lumbar spinal cord isolation. Soleus atrophy was rapid and progressive, i.e., 25% and 64% decrease in weight and 33% and 75% decrease in fiber size after 4 and 60 days of inactivity, respectively. Changes in MHC occurred at a slower rate than the atrophic response. After 15 days there was de novo expression of type IIx MHC (∼10%). By 60 days, type IIx MHC accounted for 33% of the total MHC content, and 7% of the fibers contained only type IIx MHC. The relative amount of type I MHC was reduced from 93% in control to 49% after 60 days of inactivity. Therefore, the effects of 60 days of inactivity suggest that during this time period at least 75% of fiber size and ∼40% of type I MHC composition of the adult rat soleus can be attributed to activation‐related events.

Myosin heavy chain composition of adult feline (Felis catus) limb and diaphragm muscles

The myosin heavy chain (MHC) compositions of adult feline limb and diaphragm muscles were determined. Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) were able to separate three different MHC isoforms. This was in contrast to rat muscles, in which four MHC isoforms were separated by SDS-PAGE. The fastest migrating cat MHC migrated similar to rat type I MHC and labeled in Western blots with a monoclonal antibody (mAb) specific for slow MHC and was categorized as type I. The other two MHC isoforms labeled in Western blots with a mAb specific for fast MHC and were categorized as type II. The slowest migrating fast isoform migrated similar to rat type IIa MHC and labeled with mAb N2.261, specific for types I and IIa; therefore, this MHC was categorized as type IIa. The intermediate migrating cat MHC did not migrate similar to either rat type IIx or type IIb and was not reactive with mAbs N2.261, 35 (specific for rat I, IIa, and IIb MHCs), or F3 (specific for rat IIb MHC). In tissue sections, type IIB fibers (based on myofibrillar ATPase histochemistry) were also unstained with mAbs N2.261 and 35. Therefore, the intermediate migrating cat MHC was categorized as type IIx. Consequently, feline limb and diaphragm muscles were composed of fibers containing type I, IIa, or IIx MHCs. The observations that type I and IIa isoforms, but not IIx, had similar electrophoretic mobilities in the cat and rat and that type IIb was absent from cat limb muscles suggest that there is greater diversity in MHC isoforms IIb and IIx compared to I and IIa in cats compared to rats.

Persistence of myosin heavy chain-based fiber types in innervated but silenced rat fast muscle

Myosin heavy chain (MHC) profile and size of fibers in deep and superficial regions of the adult rat medial gastrocnemius (MG) were determined after 4, 15, 30, and 60 days of inactivity induced by spinal cord isolation (SI). After 4 days, fiber size decreased by 33 to 50% and 36 to 46% in deep and superficial regions, whereas MHC composition was unaffected. By 15 days, these values were 45 to 78% and 51 to 69%, and MHC composition was shifting toward faster isoforms. By 60 days, there were no pure type I MHC fibers and increases from 1 to 18% and 78 to 93% in pure type IIb fibers in deep and superficial regions. The percentage of type I MHC (gel electrophoresis) was∼10 and ∼3%, and of type IIb ∼40 and ∼60% in control and 60‐day SI rats. Thus, adaptations in the MHC molecule occurred at a slower rate and for a longer duration than the atrophic response.

Does daily activity level determine muscle phenotype

SUMMARY The activation level of a muscle is presumed to be a major determinant of many mechanical and phenotypic properties of its muscle fibers. However, the relationship between the daily activation levels of a muscle and these properties has not been well defined, largely because of the lack of accurate and sustained assessments of the spontaneous activity levels of the muscle. Therefore, we determined the daily activity levels of selected rat hindlimb muscles using intramuscular EMG recordings. To allow comparisons across muscles having varying activity levels and/or muscle fiber type compositions, we recorded EMG activity in a predominantly slow plantarflexor (soleus), a predominantly fast plantarflexor (medial gastrocnemius, MG), a predominantly fast ankle dorsiflexor (tibialis anterior, TA) and a predominantly fast knee extensor (vastus lateralis, VL) in six unanesthetized rats for periods of 24 h. EMG activity levels were correlated with the light:dark cycle, with peak activity levels occurring during the dark period. The soleus was the most active and the TA the least active muscle in all rats. Daily EMG durations were highest for soleus (11–15 h), intermediate for MG (5–9 h) and VL (3–14 h) and lowest for TA (2–3 h). Daily mean EMG amplitudes and integrated EMG levels in the soleus were two- to threefold higher than in the MG and VL and seven- to eightfold higher than in the TA. Despite the three- to fourfold difference in activation levels of the MG and VL vs the TA, all three predominantly fast muscles have been reported to have a similar, very low percentage of slow fibers. Comparing these relative EMG levels to the published fiber type profiles of these muscles yields a very poor relationship between daily activity level and fiber type composition in the same muscles across several species. Although it is clear that changing the levels of activity can modulate the expression of the myosin phenotype, these results indicate that factors other than activation must play critical roles in determining and maintaining normal phenotypic properties of skeletal muscle fibers.

Influences of electromechanical events in defining skeletal muscle properties

Inactivity of the cat soleus muscle was induced via spinal cord isolation (SI), and the cats were maintained for 4 months. The soleus was electrically stimulated while lengthening (SI‐L) or shortening (SI‐S) during a simulated step cycle or during isometric (SI‐I) contractions. For the SI, SI‐S, SI‐L, and SI‐I groups, the soleus weights were 33, 55, 55, and 64% of the control, respectively, and the maximum tetanic tensions were 15, 30, 36, and 44% of the control, respectively. The specific tension was lower in all SI groups than in the control. Absolute forces at stimulation frequencies of 5–200 Hz were smaller in all SI groups than in the control. The SI‐I group tended to have higher values for all force‐related parameters than the other SI groups. Fatigue resistance was similar among all groups. The isometric twitch time‐to‐peak tension was shorter, and the frequency of the stimulation–tension response was shifted to the right in all SI groups with respect to the control. Maximum shortening velocities were 70, 59, and 73% faster for the SI, SI‐S, and SI‐L groups and similar to the control for the SI‐I group. Inactivity resulted in an increased percentage of faster myosin heavy chains (MHCs) that was blunted in the SI‐I and SI‐L groups but not in the SI‐S group. Pure type I MHC fibers atrophied by 80, 59, 58, and 47% in the SI, SI‐S, SI‐L, and SI‐I groups. The data from the SI group quantify the contribution of activity‐independent factors in maintaining the mechanical and phenotypic properties of the cat soleus. Relative to a fast‐fatigable muscle, these results suggest that only 25% of the slowness (type I MHC) and none of the resistance to fatigue of the soleus muscle are dependent on activity‐related factors. Short, daily bouts of electromechanical activation ameliorated several of these adaptations, with the isometric contractions being the most effective countermeasure. The clinical implications of these findings for rehabilitation strategies are discussed.

Is spinal cord isolation a good model of muscle disuse

The patterns of normal daily activity that are required to maintain normal skeletal muscle properties remain unknown. The present study was designed to determine whether spinal cord isolation can be used as a reliable experimental model of neuromuscular inactivity, that is, as a baseline for the absence of activity. Electromyograms (EMGs) were recorded from selected hindlimb muscles of unanesthetized rats over 24‐hour periods before and 7, 30, 60, and 90 days after surgical isolation of the lumbar spinal cord. Our data indicate that some rat slow muscle fibers pre‐surgery were activated for less than 3 hours per day. Spinal cord isolation (SI) reduced the mean daily integrated EMG (IEMG) and daily EMG duration in the primary slow extensor muscle (soleus) to <1% of control, and in the primary fast extensor muscles [medial gastrocnemius (MG) and vastus lateralis (VL)] to <2% of control. These parameters were decreased to <8% and 3% of control, respectively, in a primary fast flexor muscle, the tibialis anterior (TA). From 30 to 90 days post‐SI, the mean amplitudes of the spontaneous EMG bursts were relatively normal in the soleus, increased ∼2‐fold in the MG and VL, and increased ∼4‐fold in the TA. Some evidence of the normal antagonistic flexor–extensor relationship was apparent in the brief periods of recorded activity post‐SI. These results indicate that SI eliminates nearly all of the normal EMG activity in the hindlimb muscles in the presence of relatively normal muscle innervation and functional intraspinal neural circuitry. Muscle Nerve, 2006

Activity‐independent neural influences on cat soleus motor unit phenotypes

The physiological and phenotypic properties of motor units in the cat soleus muscle were studied after 4 months of inactivity induced by spinal cord isolation (SI). The soleus of some SI cats were stimulated for 30 min/day during an isometric (SI‐I), shortening (SI‐S), or lengthening (SI‐L) phase of a simulated step cycle. Mean maximum tetanic tensions were approximately 15, 26, 32, and 51% of the control in the SI, SI‐S, SI‐L, and SI‐I groups. Mean time‐to‐peak tension was approximately 50% shorter than the control in all SI groups. One motor unit was glycogen‐depleted in each muscle via repetitive stimulation. Eighteen physiologically slow and 9 fast motor units from the spinal cord–isolated groups consisted of fibers that contained only slow myosin heavy chain (MHC) and sarco(endo)plasmic reticulum calcium‐adenotriphosphatase (SERCA) isoforms. Two motor units (physiologically fast) consisted primarily of fibers that contained both fast and slow MHC and SERCA. These data reflect a dissociation between isometric speed‐related properties and MHC and SERCA isoforms following inactivity. The predominance of fibers containing both fast and slow MHC and SERCA isoforms in 2 motor units demonstrates a strong motoneuronal influence on the muscle‐fiber phenotype even when the motoneurons are silent.

Effect of Altered Thyroid State on the in situ Mechanical Properties of Adult Cat Soleus

To determine the responsiveness of cat hindlimb muscles to thyroid manipulation, adult female cats were made hypothyroid (thyroidectomy plus tapazole treatment), hyperthyroid (synthroid pellets), or maintained euthyroid. After 4 months, the hypothyroid soleus had slower time-to-peak (TPT, 80%) and half-relaxation (HRT) times, whereas the hyperthyroid soleus had faster TPT (20%) and HRT than euthyroid cats. The tension at low stimulation frequencies (5–15 Hz) was higher in hypothyroid and lower in hyperthyroid cats compared to euthyroid cats. Muscle weight, maximum twitch and tetanic (Po) tensions, and maximum rates of shortening (Vmax) were similar across groups. The soleus of hypothyroid cats was more fatigable than normal. The myosin heavy chain (MHC) composition, based on gel electrophoresis, was unaffected by thyroid hormone manipulation. Based on the reaction of monoclonal antibodies for specific MHCs, some fast fibers in the hypothyroid cats coexpressed developmental MHC. These data indicate that 4 months of an altered thyroid state result in changes in the isometric twitch speed properties of the cat soleus, but not the tension-related or isotonic properties. Further, a chronic decrease in thyroid hormone had a greater impact than a chronic increase in thyroid hormone on the mechanical properties of the adult cat soleus.