Jung A. Kim

Gene expression during inactivity-induced muscle atrophy: effects of brief bouts of a forceful contraction countermeasure

Anabolic and catabolic markers of muscle protein metabolism were examined in inactivity-induced atrophying muscles with and without daily short-duration, high-resistance isometric contractions. Inactivity was achieved via spinal cord isolation (SI), which results in near inactivity of the hindlimb musculature without compromising the motoneuron-muscle connectivity. Adult rats were assigned to a control (Con) or SI group in which one limb was stimulated (SI-Stim, 5 consecutive days of brief bouts of high-load isometric contractions) while the other served as a SI control (SI). Both the medial gastrocnemius (MG) and soleus weights (relative to body weight) were approximately 71% of Con in the SI, but maintained at Con in the SI-Stim group. Activity of the IGF-1/phosphatidylinositol 3-kinase (PI3K)/Akt pathway of protein synthesis was similar among all groups in the MG. Expression of atrogin-1 and muscle RING finger-1 (MuRF-1), markers of protein degradation, were higher in the MG and soleus of the SI than Con and maintained at Con in the SI-Stim. Compared with Con, the anti-growth factor myostatin was unaffected in the MG and soleus in the SI but was lower in the MG of the SI-Stim. These results demonstrate that upregulation of specific protein catabolic pathways plays a critical role in SI-induced atrophy, while this response was blunted by 4 min of daily high-resistance electromechanical stimulation and was able to preserve most of the muscle mass. Although the protein anabolic pathway (IGF-1/PI3K/Akt) appears to play a minor role in regulating mass in the SI model, increased translational capacity may have contributed to mass preservation in response to isometric contractions.

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.

Architectural and fiber type distribution properties of selected rhesus leg muscles: feasibility of multiple independent biopsies.

In experiments involving primates, e.g. before and after spaceflight, needle biopsies were thought to be a logical and feasible means of obtaining metabolic and morphological information from skeletal muscles. However, the feasibility of obtaining consistent, repeatable biopsies from individual muscles had to be demonstrated prior to the acceptance of this procedure. To study this approach, the architectural properties and the fiber type distributions at three levels and two regions along the proximo-distal axis of the soleus, medial gastrocnemius and tibialis anterior of adult rhesus monkeys were determined. In each muscle, biopsies were taken from specific regions where the fiber type distribution was determined. Within each region of each muscle, the fiber type populations were similar at the three levels studied. The percentage of fast or oxidative fibers in the biopsies and in the regions of the same muscle were highly correlated, i.e. r = 0.98 for both comparisons. In addition, based on normalized values (z scores), 25/26 and 22/26 biopsies were within the 95% confidence interval, i.e. the biopsies were a representative sample of the mean fiber type population of that region of the muscle. In all muscles, the mean fiber lengths were no more than one third the length of the muscle. Together, these data indicate the feasibility of obtaining independent, repeated biopsies having similar fiber types from each of the muscles studied.

Differential modulation of myosin heavy chain phenotype in an inactive extensor and flexor muscle of adult rats

The effects of chronic neuromuscular inactivity on the phenotype and size of muscle fibres in a fast ankle extensor (medial gastrocnemius, MG) and a fast ankle flexor (tibialis anterior, TA) muscle of the rat hindlimb were determined. Inactivity was produced by spinal cord isolation (SI), i.e. complete spinal cord transections at a mid‐thoracic and high sacral level and bilateral deafferentation between the transection sites. After 90 days of SI, the MG and TA muscle weights were 53 and 45% lower than in age‐matched controls. Overall mean fibre sizes in the deep (close to the bone) and superficial (away from the bone) regions were ∼60 and 65% smaller in the MG and ∼40 and 50% smaller in the TA of SI than control rats, respectively. The myosin heavy chain (MHC) composition shifted towards the faster isoforms after SI: the MG showed an increase in both types IIx (20%) and IIb (23%), whereas the TA showed a marked increase in type IIx (94%) and a decrease in type IIb (18%) MHC. Both muscles in SI rats showed no type IIa and only one MG muscle had ∼5% type I MHC. These results show that prolonged inactivity has a stronger effect on a fast extensor compared with a fast flexor in the rat hindlimb. The larger decrease in mass and fibre size in the MG than the TA most probably reflects the larger impact of chronic inactivity on the normally more highly recruited extensor than flexor muscle. The primary shift to type IIb MHC in the MG and type IIx MHC in the TA indicate a different default mode for an inactive extensor vs. flexor muscle, and may reflect differing activity‐independent neural influences, i.e. neurotrophic factors, on muscle fibre phenotype in extensors vs. flexors.

Metabolic and morphological stability of motoneurons in response to chronically elevated neuromuscular activity

The purpose of this study was to determine the plasticity of spinal motoneuron size and succinate dehydrogenase activity in response to increased levels of neuromuscular activation and/or increased target size. The plantaris muscles of adult rats were functionally overloaded for one or 10 weeks via the removal of the soleus and gastrocnemius muscles bilaterally. In addition, one group of functionally overloaded rats at each time period was trained daily (1 h/day) on a treadmill. The plantaris muscle on one side in each rat was injected with the fluorescent tracer Nuclear Yellow two days prior to the end of the study to retrogradely label the associated motor pool. At one week, the plantaris weight was increased compared to control, whereas there was no change in motoneuron size. Succinate dehydrogenase activity was unaffected in either the muscle or motoneurons. At 10 weeks, the plantaris muscle weight was larger and the succinate dehydrogenase activity lower in the functionally overloaded rats compared to age-matched controls. Training further increased the hypertrophic response, whereas the succinate dehydrogenase activity returned to control levels. In contrast, mean motoneuron size and succinate dehydrogenase activity were similar among the three groups. These data indicate that overload of a specific motor pool, involving both an increase in activation and an increase in target size, had a minimal effect on the size or the oxidative potential of the associated motoneurons. Thus, it appears that the spinal motoneurons, unlike the muscle fibers, are highly stable over a wide range of levels of chronic neuromuscular activity.

Selectively reshaping a muscle phenotype: functional overload of cat plantaris.

Introduction: Functional overload (FO) of the fast plantaris muscle was studied in treadmill‐exercised (FO‐Ex) or sedentary (FO‐Sed) adult cats. Methods: Mechanical, phenotype, and kinematics analyses were performed. Results: Plantigrade vs. normal digitigrade posture was observed early post‐FO. Relative plantaris mass was greater in FO‐Sed (10%) and FO‐Ex (60%) cats than in controls 12 weeks post‐FO. Specific tension was similar across groups, indicating functional hypertrophy. Fiber size was greater, percent slow fibers higher, percent IIa myosin heavy chain (MHC) higher, and IIx MHC lower in FO‐Ex than controls. Twitch and half‐relaxation times were longer, and the frequency–tension curve shifted toward that observed in slow muscles. Electromyography (EMG) and tendon force amplitudes during stepping were larger, and the yield (lengthening) phase occurred at a longer muscle length before compared with after FO. Discussion: Reshaping the plantaris phenotype was highly dependent on the overload stimulus, indicating that electrical stimulation paradigms used during rehabilitation should be performed with the muscles under “loaded” conditions. Muscle Nerve, 2011

Electromechanical modulation of catabolic and anabolic pathways in chronically inactive, but neurally intact, muscles

The extent and mechanisms by which neural input regulates skeletal muscle mass remain largely unknown. Adult spinal cord isolated (SI) rats were implanted unilaterally with a microstimulator, whereas the contralateral limb served as SI control (SI‐C). A 100‐HZ, 1‐s stimulus was delivered every 30 s for 5 min, followed by a 5‐min rest. This was repeated six times consecutively (SI‐Stim1) or with a 9‐h interval after the third bout (SI‐Stim2) for 30 days (1 min of daily activity). SI‐Stim1 and SI‐Stim2 paradigms attenuated plantaris atrophy by 20% and 38%, respectively, whereas only SI‐Stim2 blunted soleus atrophy (24%) relative to SI‐C. Muscle mass changes occurred independent of the IGF‐1/PI3K/Akt pathway. No relationships between SI or electromechanical stimulation and expression of several atrophy markers were observed. These data suggest that regulatory mechanisms for maintaining muscle mass previously shown in acute states of atrophy differ substantially from those observed in chronic states. Muscle Nerve, 2010

Spinal neuronal activation during locomotor-like activity enabled by epidural stimulation and 5-hydroxytryptamine agonists in spinal rats.

The neural networks that generate stepping in complete spinal adult rats remain poorly defined. To address this problem, we used c‐fos (an activity‐dependent marker) to identify active interneurons and motoneurons in the lumbar spinal cord of adult spinal rats during a 30‐min bout of bipedal stepping. Spinal rats were either step trained (30 min/day, 3 days/week, for 7.5 weeks) or not step trained. Stepping was enabled by epidural stimulation and the administration of the serotonergic agonists quipazine and 8‐OHDPAT. A third group of spinal rats served as untreated (no stimulation, drugs, or stepping) controls. The numbers of activated cholinergic central canal cluster cells and partition neurons were higher in both step‐trained and nontrained rats than in untreated rats and were higher in nontrained than in step‐trained rats. The latter finding suggests that daily treatment with epidural stimulation plus serotonergic agonist treatment without step training enhances the excitability of a broader cholinergic interneuronal population than does step training. The numbers of activated interneurons in laminae II–VI of lumbar cross‐sections were higher in both step‐trained and nontrained rats than in untreated rats, and they were highest in step‐trained rats. This finding suggests that this population of interneurons is responsive to epidural stimulation plus serotonergic treatment and that load‐bearing induced when stepping has an additive effect. The numbers of activated motoneurons of all size categories were higher in the step‐trained group than in the other two groups, reflecting a strong effect of loading on motoneuron recruitment. In general, these results indicate that the spinal networks for locomotion are similar with and without brain input.

PPARδ preserves a high resistance to fatigue in the mouse medial gastrocnemius after spinal cord transection.

Introduction: Skeletal muscle oxidative capacity decreases and fatigability increases after spinal cord injury. Transcription factor peroxisome proliferator‐activated receptor δ (PPARδ) promotes a more oxidative phenotype. Methods: We asked whether PPARδ overexpression could ameliorate these deficits in the medial gastrocnemius of spinal cord transected (ST) adult mice. Results: Time‐to‐peak tension and half‐relaxation times were longer in PPARδ‐Con and PPARδ‐ST compared with littermate wild‐type (WT) controls. Fatigue index was 50% higher in PPARδ‐Con than WT‐Con and 70% higher in the PPARδ‐ST than WT‐ST. There was an overall higher percent of darkly stained fibers for succinate dehydrogenase in both PPARδ groups. Conclusions: The results indicate a conversion toward slower, more oxidative, and less fatigable muscle properties with overexpression of PPARδ. Importantly, the elevated fatigue resistance was maintained after ST, suggesting that enhanced PPARδ expression, and possibly small molecule agonists, could ameliorate the increased fatigability routinely observed in chronically paralyzed muscles. Muscle Nerve 53: 287–296, 2016

Spinal neuronal activation during locomotor-like activity enabled by epidural stimulation and 5-hydroxytryptamine agonists in spinal rats: Epidural Stimulation and 5-HT Agonists in Spinal Rats

The neural networks that generate stepping in complete spinal adult rats remain poorly defined. To address this problem, we used c‐fos (an activity‐dependent marker) to identify active interneurons and motoneurons in the lumbar spinal cord of adult spinal rats during a 30‐min bout of bipedal stepping. Spinal rats were either step trained (30 min/day, 3 days/week, for 7.5 weeks) or not step trained. Stepping was enabled by epidural stimulation and the administration of the serotonergic agonists quipazine and 8‐OHDPAT. A third group of spinal rats served as untreated (no stimulation, drugs, or stepping) controls. The numbers of activated cholinergic central canal cluster cells and partition neurons were higher in both step‐trained and nontrained rats than in untreated rats and were higher in nontrained than in step‐trained rats. The latter finding suggests that daily treatment with epidural stimulation plus serotonergic agonist treatment without step training enhances the excitability of a broader cholinergic interneuronal population than does step training. The numbers of activated interneurons in laminae II–VI of lumbar cross‐sections were higher in both step‐trained and nontrained rats than in untreated rats, and they were highest in step‐trained rats. This finding suggests that this population of interneurons is responsive to epidural stimulation plus serotonergic treatment and that load‐bearing induced when stepping has an additive effect. The numbers of activated motoneurons of all size categories were higher in the step‐trained group than in the other two groups, reflecting a strong effect of loading on motoneuron recruitment. In general, these results indicate that the spinal networks for locomotion are similar with and without brain input.