Distinct Patterns of Fiber Type Adaptation in Rat Hindlimb Muscles 4 Weeks After Hemorrhagic Stroke.

OBJECTIVE: The aim of this study was to evaluate adaptations in soleus and tibialis anterior muscles in a rat model 4 wks after hemorrhagic stroke. DESIGN: Young adult Sprague Dawley rats were randomly assigned to two groups: stroke and control, with eight soleus and eight tibialis anterior muscles per group. Hemorrhagic stroke was induced in the right caudoputamen of the stroke rats. Control rats had no intervention. Neurologic status was evaluated in both groups before stroke and 4 wks after stroke. Muscles were harvested after poststroke neurologic testing. Muscle fiber types and cross-sectional areas were determined in soleus and tibialis anterior using immunohistochemical labeling for myosin heavy chain. RESULTS: No generalized fiber atrophy was found in any of the muscles. Fiber types shifted from faster to slower in the tibialis anterior of the stroke group, but no fiber type shifts occurred in the soleus muscles of stroke animals. CONCLUSIONS: Because slower myosin heavy chain fiber types are associated with weaker contractile force and slower contractile speed, this faster to slower fiber type shift in tibialis anterior muscles may contribute to weaker and slower muscle contraction in this muscle after stroke. This finding may indicate potential therapeutic benefit from treatments known to influence fiber type plasticity.

Skeletal muscle plasticity after hemorrhagic stroke in rats: influence of spontaneous physical activity.

OBJECTIVE: The aim of this study was to determine the contribution of spontaneous post-stroke physical activity to skeletal muscle plasticity after stroke. DESIGN: A randomized controlled study was conducted of 24 young adult male Sprague-Dawley rats assigned to three experimental groups: (1) STR-hemorrhagic stroke in the right caudoputamen; (2) SHAM-procedural control; and (3) CONT-no intervention (n = 8/group). Neurologic testing was performed before and 2 wks after stroke. Spontaneous physical activity was monitored five nights per week for 1 wk preoperatively and 2 wks postoperatively. Two weeks after stroke induction, bilateral soleus and tibialis anterior muscles were harvested. Myofiber cross-sectional areas were determined, and fiber typing was performed with immunohistochemistry. RESULTS: STR animals demonstrated neurologic deficit in the contralesional hindlimb 2 wks after stroke. Quantity of spontaneous physical activity did not differ between groups within each of the week-long study intervals. No significant difference was found in fiber types or cross-sectional areas in the soleus muscle of STR vs. CONT groups. However, the tibialis anterior muscle of the contralesional hindlimbs of the STR animals showed atrophy in 2x and 1 + 2x myofibers, as well as type 1 hypertrophy. CONCLUSIONS: Skeletal muscle adaptation occurs by 2 wks post-stroke in this model. It is muscle specific and appears to be influenced by factors other than spontaneous post-stroke physical activity.

Influence of insulin and muscle fiber type in nepsilon-(carboxymethyl)-lysine accumulation in soleus muscle of rats with streptozotocin-induced diabetes mellitus.

BACKGROUND: Nepsilon-(carboxymethyl)-lysine (CML) is an advanced glycation end product (AGE), the accumulation of which has been implicated in the etiology of diabetes complications. Skeletal muscle in diabetes demonstrates altered function, and increased accumulation of CML has been found in several fast-twitch muscles of diabetic animals. OBJECTIVE: This study aims to explore the accumulation of CML in soleus (a slow muscle) in diabetic animals, with and without insulin therapy. METHODS: Twenty-one rats were randomly divided into control and diabetes groups (DNI: diabetes without insulin; DI: diabetes with insulin; C: control). Diabetes was induced by intravenous administration of streptozotocin. At the end of the 12-week experimental period the soleus muscle was excised and snap frozen in liquid nitrogen. Muscle cross-sections were immunolabeled for CML. The number of CML-labeled muscle fibers was quantified; fibers were also evaluated for fiber types and cross-sectional areas. RESULTS: The percentage of myofibers immunolabeling for CML was highest in the DNI group (13.8 +/- 2.5%), lower in the DI group (5.4 +/- 1.1%) and lowest in the C group (2.1 +/- 0.6%). Statistical analysis revealed that AGE accumulation was significantly greater in the DNI group than in both C and DI groups (p = 0.0002). There was no significant difference between C and DI groups. In the DNI animals, AGE-positive myofibers showed a higher percentage of fast fiber types than did the AGE-negative fibers (49.5 +/- 6.9 vs. 13.7 +/- 1.5%, p = 0.002). No differences existed in cross-sectional areas between AGE-positive and AGE-negative fibers within any group. CONCLUSION: The greatest accumulation of AGE was in the soleus of the DNI group, and was significantly less in the DI group. These findings may be linked to disordered glucose metabolism, increased oxidative stress and/or fiber type transformation in these muscles.

Advanced glycation end-product accumulation and associated protein modification in type II skeletal muscle with aging.

One mechanism that may influence the quality of skeletal muscle proteins, and explain the age-related decline in contractility, is protein damage. Advanced glycation end-products (AGE) in vivo are useful biomarkers of damage. In this study, comparison of extensor digitorum longus (EDL) muscles from young (8 months), old (33 months), and very old (36 months) Fischer 344 Brown Norway F1 (F344BNF1) hybrid rats shows that muscles from the very old rats have a significantly higher percentage of myofibers that immunolabel intracellularly for AGE-antibody 6D12 compared to the younger age group. The AGE-modified proteins, determined in the semimembranosus muscles from young (9 months) and old (27 months) F344 rats, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry include creatine kinase, carbonic anhydrase III, beta-enolase, actin, and voltage-dependent anion-selective channel 1. Moreover, there is a significant increase in AGE modification of beta-enolase with age. These results identify a common subset of proteins that contain AGE and suggest that metabolic proteins are targets for glycation with aging.

Advanced glycation end product in diabetic rat skeletal muscle in vivo.

BACKGROUND: Advanced glycation end products (AGEs) are implicated in the etiology of diabetic complications in the kidney, nerve and eye. Skeletal muscle contractile parameters have also been found to be altered in diabetes. Glycation has not been extensively studied in skeletal muscle, but AGE-modified proteins may influence contractility. OBJECTIVE AND METHODS: The aim of this study was to use immunohistochemistry to identify distribution patterns of the AGE Nepsilon-(carboxymethyl)-lysine in plantaris muscle of diabetic rats. RESULTS: Results revealed the presence of Nepsilon-(carboxymethyl)-lysine intracellularly and also at sites along the myofiber periphery. The number of myofibers immunolabeling for AGE in animals with diabetes was more than 4-fold greater than in control animals. Additionally, there was a greater proportion of slow + fast myosin heavy chain coexpression in the AGE-positive cells from diabetic animals than in AGE-positive fibers from control animals. No significant difference was present between cross-sectional areas of AGE-positive fibers and AGE-negative fibers within the respective experimental groups. CONCLUSIONS: AGE accumulation is greater in skeletal muscle in vivo from diabetic animals than in control animals. This AGE accumulation appears to be associated with fiber-type transformation rather than with myofiber size. Further study is needed to determine the identity of these AGE-modified proteins and to determine how they influence skeletal muscle function in diabetes.

Effect of endurance exercise on myosin heavy chain isoform expression in diabetic rats with peripheral neuropathy.

OBJECTIVE: This study evaluated the effect of endurance exercise on myosin heavy chain (MHC) isoform expression in soleus muscle of diabetic rats with peripheral neuropathy. DESIGN: Male Sprague Dawley rats were randomly divided into four groups: control sedentary, diabetic sedentary, control exercise, and diabetic exercise. The exercised animals performed treadmill running five times per week. After 12 wks, electrophysiologic testing documented peripheral neuropathy in the diabetic rats. The soleus muscles were then excised and quick-frozen. Cross-sections were immunohistochemically stained for slow, fast, developmental, and neonatal MHCs. Fiber-type composition and fiber cross-sectional areas were then determined. RESULTS: The diabetic groups showed a significantly greater percentage of fast MHC than did the control groups, regardless of exercise status (diabetic sedentary, 22.6%; diabetic exercise, 25.2%; control sedentary, 13.5%; control exercise, 13.1%). The diabetics also showed a significantly lower percentage of slow-only MHC than controls (diabetic sedentary, 77.1%; diabetic exercise, 74.3%; control sedentary, 86.2%; control exercise, 86.1%). No differences in muscle fiber cross-sectional area existed between the groups. The exercised animals showed greater expression of developmental MHC than did the sedentary animals (diabetic sedentary, 1.6%; diabetic exercise, 3.8%; control sedentary, 0.8%; control exercise, 2.0%). CONCLUSION: The altered slow and fast MHC expression in the diabetic muscle is similar to MHC expression in several other conditions, including decreased neuromuscular activity and denervation. Mechanisms of this MHC expression shift are unknown. Chronic endurance training does not alter adult MHC expression in the diabetic animals. The developmental MHC expression is likely a manifestation of uphill treadmill running due to eccentric contractions in the soleus resulting in myofiber injury and regeneration.

Myosin heavy chain isoform immunolabelling in diabetic rats with peripheral neuropathy.

This study evaluated mature and immature myosin heavy chain (MHC) isoform immunolocalisation in soleus muscle of diabetic rats with documented motor neuropathy. Sprague Dawley rats were assigned to one of three groups: control (C), diabetic with insulin (DI), or diabetic without insulin (DNI). Twelve weeks after diabetes induction, soleus muscles were excised and quick-frozen. Cross-sections were labelled immunohistochemically for slow, fast, developmental and neonatal MHC isoforms to determine fiber-type composition. Fiber cross-sectional areas were determined morphometrically. Results revealed that DNI and DI muscles contained greater percentages of myofibers positive for fast MHC compared with controls. DNI animals also showed a lower percentage of myofibers positive for slow MHC compared to the DI group. The number of fibers immunolabelled for developmental MHC isoforms was greater in DNI animals than in the other groups. The differences in slow and fast MHC-labelling appear to indicate a condition of altered neuromuscular activity affecting the diabetic muscles. The increase in developmental MHC-labelling in the DNI muscles could indicate myofiber regeneration or reinnervation that would be more pronounced in the DNI animals in context of their more severe neuropathy. Insulin appeared to influence muscle fiber cross-sectional area and possibly fiber-type grouping frequency; the potential mechanism for these effects was not elucidated.

Adult and developmental myosin heavy chain isoforms in soleus muscle of aging Fischer Brown Norway rat.

Fiber type shifts in aging skeletal muscle have been studied with myofibrillar ATPase histochemistry and gel electrophoresis, but less commonly with immunohistochemistry. Immunohistochemical study of myosin heavy chains (MHCs) in single myofibers yields additional information about aged skeletal muscle. Furthermore, many studies of aging rodent skeletal muscle have been performed on fast-MHC-predominant muscle and in several different strains. The aim of this study was to evaluate immunohistochemically MHC characteristics in the slow-MHC-predominant soleus muscle in the Fischer Brown Norway F1 hybrid aging rat (FBN). Three age groups of FBN rats were studied: 12 months, 30 months, and 36 months. Soleus muscles were excised, quick-frozen, and stained immunohistochemically for slow, fast, developmental, and neonatal MHC isoforms. Cross-sections were evaluated for the number and cross-sectional areas of fibers expressing each isoform. Single myofibers in soleus muscles of the aged rats showed significantly greater amounts of coexpression of slow and fast MHC than did younger animals. This change began by 30 months of age, but did not reach statistical significance until 36 months of age. The soleus from 36-month-old rats also expressed greater amounts of developmental MHC than did the other groups. These developmental MHC-positive myofibers also coexpressed either slow or slow and fast MHC. The age-related increase in MHC coexpression of slow with fast isoforms may indicate a fiber type shift suggestive of denervation that outpaces reinnervation. The developmental MHC-positive fibers provide evidence of ongoing myofiber remodeling in the oldest rats in the midst of the fiber degeneration of aging.

Effects of endurance exercise-training on single-fiber contractile properties of insulin-treated streptozotocin-induced diabetic rats.

The purpose of this study was to characterize the contractile properties of individual skinned muscle fibers from insulin-treated streptozotocin-induced diabetic rats after an endurance exercise training program. We hypothesized that single-fiber contractile function would decrease in the diabetic sedentary rats and that endurance exercise would preserve the function. In the study, 28 rats were assigned to either a nondiabetic sedentary, a nondiabetic exercise, a diabetic sedentary, or a diabetic exercise group. Rats in the diabetic groups received subcutaneous intermediate-lasting insulin daily. The exercise-trained rats ran on a treadmill at a moderate intensity for 60 min, five times per week. After 12 wk, the extensor digitorum longus and soleus muscles were dissected. Single-fiber diameter, Ca(2+)-activated peak force, specific tension, activation threshold, and pCa(50) as well as the myosin heavy chain isoform expression (MHC) were determined. We found that in MHC type II fibers from extensor digitorum longus muscle, diameters were significantly smaller from diabetic sedentary rats compared with nondiabetic sedentary rats (P < 0.001). Among the nondiabetic rats, fiber diameters were smaller with exercise (P = 0.038). The absolute force-generating capacity of single fibers was lower in muscles from diabetic rats. There was greater specific tension (force normalized to cross-sectional area) by fibers from the rats that followed an endurance exercise program compared with sedentary. From the results, we conclude that alterations in the properties of contractile proteins are not implicated in the decrease in strength associated with diabetes and that endurance-exercise training does not prevent or increase muscle weakness in diabetic rats.

Muscle activity and aging affect myosin structural distribution and force generation in rat fibers.

The purpose of this study was to determine whether increased muscle activity could reverse myosin structural alterations that occur in aged rat muscle and whether those alterations could be induced in young rat muscle by decreased activity. Semimembranosus muscle activity was increased by electrical stimulation (200-ms trains, 154 Hz, 5 V) through a nerve cuff on the tibial branch of the ischiatic nerve. The protocol consisted of 5 sets of 6-10 maximal isometric contractions performed twice per week for 4 or 8-10 wk. Decreased muscle activity was induced by denervation of the semimembranosus muscle for 2 or 4 wk. Semimembranosus fibers were then studied for Ca(2+)-activated force generation. Fibers were also spin labeled on the myosin catalytic domain and studied using electron paramagnetic resonance (EPR) spectroscopy to assess myosin structural distribution. Increased muscle activity for 4 and 8-10 wk in approximately 32-mo-old rats resulted in -16 and +4% changes in specific tension, respectively (P < 0.01). EPR spectra showed that the fraction of myosin heads in the strong-binding structural state during contraction was reduced at 4 wk (0.241 +/- 0.020 vs. 0.269 +/- 0.018, P = 0.046) but returned to normal by 8-10 wk (P = 0.67). Decreased muscle activity for 2 and 4 wk in approximately 9-mo-old rats resulted in 23 and 34% reductions, respectively, in specific tension; EPR spectra showed 16 and 35% decreases in strong-binding myosin (P < 0.01). These data support the hypothesis that changes in muscle activity affect muscle strength, at least in part through alterations in myosin structure and function.