Conversion of rat muscle fiber types

Summary Rats were used in this study to determine the time course of conversion of muscle fiber types. The right or left gastrocnemius muscle was removed thereby causing an overload on the ipsilateral soleus and plantaris muscles. The contralateral limb served as a control. The type II to type I fiber conversion was followed histochemically in the soleus and plantaris muscles for one to six weeks following surgery. Muscle sections were stained for myofibrillar actomyosin ATPase and NADH tetrazolium reductase. The type I population in the soleus muscle was 99.3% six weeks after synergist removal. The plantaris muscle underwent a two fold increase in the percentage of type I fibers after six weeks. Transitional fibers were prominent in the plantaris muscle and reached their peak at 4% (P<0.05) of the total population, four weeks after surgery.This research was funded in part by grants from The Graduate School at Washington State University, and The Society of the Sigma Xi     

Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.     

PGC-1α and FOXO1 mRNA levels and fiber characteristics of the soleus and plantaris muscles in rats after hindlimb unloading

Fifteen-week-old rats were subjected to unloading induced by hindlimb suspension for 3 weeks. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and forkhead box-containing protein O1 (FOXO1) mRNA levels and fiber profiles of the soleus and plantaris muscles in rats subjected to unloading (unloaded group) were determined and compared with those of age-matched control rats (control group). The body weight and both the soleus and plantaris muscle weights were lower in the unloaded group than in the control group. The PGC-1α mRNA was downregulated in the soleus, but not in the plantaris muscle of the unloaded group. The FOXO1 mRNA was upregulated in both the soleus and plantaris muscles of the unloaded group. The oxidative enzyme activity was reduced in the soleus, but not in the plantaris muscle of the unloaded group. The percentage of type I fibers was decreased and the percentages of type IIA and IIC fibers were increased in the soleus muscle of the unloaded group, whereas there was no change in fiber type distribution in the plantaris muscle of the unloaded group. Atrophy of all types of fibers was observed in both the soleus and plantaris muscles of the unloaded group. We conclude that decreased oxidative capacity and fiber atrophy in unloaded skeletal muscles are associated with decreased PGC-1α and increased FOXO1 mRNA levels.     

Motoneuron and muscle fiber succinate dehydrogenase activity in control and overloaded plantaris.

To determine the level of coordination in succinate dehydrogenase (SDH) activity between plantaris motoneurons and muscle fibers, the soleus and gastrocnemius muscles were bilaterally excised in four cats to subject the plantaris to functional overload (FO). Five normal cats served as controls. Twelve weeks after surgery the right plantaris in each cat was injected with horseradish peroxidase to identify plantaris motoneurons. SDH activity then was measured in a population of plantaris motoneurons and muscle fibers in each cat. Control motoneurons and muscle fibers had similar mean SDH activities and a similar relationship between cell size and SDH activity. After FO, muscle fiber size doubled and mean muscle fiber SDH activity halved. Motoneuron mean SDH activity and size were unaffected by FO. Total SDH activity was unchanged in both the motoneurons and muscle fibers after FO. These changes suggest a selective increase in contractile proteins with little or no modulation of mitochondrial proteins in the muscle fibers, because total SDH activity was unchanged in muscle fibers after FO. These data demonstrate that although mean SDH activities were similar in control motoneurons and muscle fibers, mean SDH activities in these two cell types can change independently.     

Fiber-type specific expression of α-actinin isoforms in rat skeletal muscle

α-Actinins are actin-binding proteins, and two isoforms (α-actinin-2 and -3) are major structural components of the sarcomeric Z line in mammalian skeletal muscle. Based on human and knockout mice studies, α-actinin-3 is thought to be associated with muscle force output and high contraction velocities. However, fiber-type specific expression of α-actinin isoforms is not well understood and may vary among species. In this study, we investigated the expression of α-actinin isoforms and the difference between fiber types in rat skeletal muscle and compared it with those of humans and mice from previous reports. Soleus and plantaris muscles were analyzed immunohistochemically to identify muscle fiber types and α-actinin protein expression. α-Actinin-2 was stained in all muscle fibers in both the soleus and plantaris muscles; i.e., all α-actinin-3 co-expressed with α-actinin-2 in rat skeletal muscles. The proportions of α-actinin-3 expression, regardless of fiber type, were significantly higher in the plantaris (75.8 ± 0.6%) than the soleus (8.0 ± 1.7%). No α-actinin-3 expression was observed in type I fibers, whereas all type IIx+b fibers expressed α-actinin-3. α-Actinin-3 was also expressed in type IIa fibers; however, approximately 75% of type IIa fibers were not stained by α-actinin-3, and the proportion varied between muscles. The proportion of α-actinin-3 expression in type IIa fibers was significantly higher in the soleus muscle than the plantaris muscle. Our results showed that fiber-type specific expression of α-actinin isoforms in rats is more similar to that in humans compared to that of the mouse, whereas the proportion of α-actinin-3 protein varied between muscles.     

Compensatory adaptations of skeletal muscle fiber types to a long-term functional overload

We investigated selected histochemical and histometrical characteristics of the heterogeneous fiber types of rat skeletal muscle following long-term compensatory muscle growth. Sixty days following surgical removal of the synergistic gastrocnemius muscle, the compensated ipsilateral plantaris and soleus muscles and the corresponding control muscles from the contralateral leg were excised and stained histochemically for myofibrillar ATPase and DPNH-diaphorase activities. The number of fibers per cross-section was determined by a direct count from transverse sections taken from the midportion of the muscles. Fiber area was determined by direct planimetry. The plantaris and soleus muscles hypertrophied 103% and 45%, respectively, within 60 days. Compensatory hypertrophy of the plantaris muscle was accompanied by a significant but disproportionate increase in the cross-sectional areas of the three muscle fiber types. There was an approximate 4-fold increase in the number of slow-twitch-oxidative (SO) fibers observed per transverse section. The hypertrophied plantaris muscle exhibited a significantly greater number of fibers per cross-section (29%) than the respective control muscle. The compensated soleus muscle consisted of nearly 100% SO fibers compared to 83% for the control soleus muscle.     

Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat

To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.     

Endogenous expression and developmental changes of HSP72 in rat skeletal muscles

The purpose of the present study was to determine whether endogenous factor(s) contributes to the expression of heat shock proteins (HSPs) during the early developmental stages of rat skeletal muscles. HSP72 was expressed in both the soleus and plantaris muscles at embryonic day 22 (E22). On the basis of myosin heavy chain (MHC) immunohistochemistry, HSP72 was specifically expressed in slow type I fibers in both muscles. These slow fibers were observed throughout the entire cross section of the soleus muscle and only in the deep region (close to the bone) of the plantaris muscle. These results indicate that the expression of HSP72 is related to endogenous factors associated with type I fibers, because E22 rats have minimal exogenous influences and the soleus and plantaris muscles of E22 rats have similar metabolic and contractile profiles at this stage of development. We then examined the changes in HSP72 and heat shock cognate (HSC) 73 in the same two muscles from E22 to postnatal day 56 via Western blotting. The level of HSP72 in the soleus muscle gradually increased in parallel with the increment in the type I MHC isoform. Compared with the soleus, only a small amount of HSP72 could be detected in the plantaris muscle throughout the developmental period. For both muscles, HSC73 reached levels observed in adult muscles at postnatal day 3, and these levels were unchanged thereafter. These results indicate that the expression of HSP72, but not HSC73, is influenced by both endogenous and exogenous factors during the embryonic and early developmental periods.     

Are region-specific changes in fibre types attributable to nonuniform muscle hypertrophy by overloading?

Muscle fibre composition was compared among the proximal (25%), middle (50%) and distal (75%) regions of the muscle length to investigate whether compensatory overload by removal of synergists induces region-specific changes of fibre types in rat soleus and plantaris muscles. In addition, we evaluated fibre cross-sectional area in each region to examine whether fibre recruitment pattern against functional overload is nonuniform in different regions. Increases in muscle mass and fibre area confirmed a significant hypertrophic response in the overloaded soleus and plantaris muscles. Overloading increased the percentage of type I fibres in both muscles and that of type IIA fibres in the plantaris muscle, with the greater changes being found in the middle and distal regions. The percentage of type I fibres in the proximal region was higher than that of the other regions in the control soleus muscle. In the control plantaris muscle, the percentage of type I and IIA fibres in the middle region were higher than that of the proximal and distal regions. With regard to fibre size, type IIB fibre area of the middle and distal regions in the plantaris increased by 51% and 57%, respectively, with the greater changes than that of the proximal region (37%) after overloading. These findings suggest that compensatory overload promoted transformation of type II fibres into type I fibres in rat soleus and plantaris muscles, with the greater changes being found in the middle and distal regions of the plantaris muscle.     

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal.     

大鼠与家兔生后各年龄阶段跖肌纤维的比较

应用建立在肌球蛋白重链异构体基础上的标准肌动球蛋白ATP酶和琥珀酸脱氢酶组织化学方法,分析大鼠和家兔出生后发育各年龄阶段跖肌纤维型分布。在生后2周至24周龄的大鼠和家兔Ⅰ、ⅡX型肌纤维百分比例减少,而ⅡA、ⅡB型纤维则增加。进行大量单肌纤维的组织化学特征的比较和相关性探讨。结果显示动物平均体重与跖肌的平均湿重随生后发育逐渐增加,Ⅰ、ⅡX、ⅡA及ⅡB型纤维均在生后各年龄组的全部肌肉内被发现,但出生后2日龄组是个例外。在生后发育期间,雄性大鼠和家兔ⅡB型纤维的平均肌纤维型构成要大于雌性大鼠和家兔,而雄性大鼠和家兔Ⅰ、ⅡX、ⅡA型三种氧化组织化学分类的肌纤维型构成均小于雌性大鼠和家兔。大鼠Ⅰ、ⅡX、ⅡA和ⅡB型纤维的平均横切面积显然要比家兔的同类型肌纤维要小。在大鼠和家兔可见明显的性别差异。大鼠和家兔的ⅡX型纤维横切面积是最小的,Ⅰ、ⅡA型纤维呈中等大小,ⅡB型纤维最大。该重要的测试有助于我们深入研究啮齿类动物快肌纤维生理特征的适应。     

Contractile properties and myosin expression in rats born and reared in hypergravity

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.     

Multiple stimulations for muscle–nerve–blood vessel unit in compensatory hypertrophied skeletal muscle of rat surgical ablation model

Tissue inflammation and multiple cellular responses in the compensatory enlarged plantaris (OP Plt) muscle induced by surgical ablation of synergistic muscles (soleus and gastrocnemius) were followed over 10 weeks after surgery. Contralateral surgery was performed in adult Wistar male rats. Cellular responses in muscle fibers, blood vessels and nerve fibers were analyzed by immunohistochemistry and electron microscopy. Severe muscle fiber damage and disappearance of capillaries associated with apparent tissue edema were observed in the peripheral portion of OP Plt muscles during the first week, whereas central portions were relatively preserved. Marked cell activation/proliferation was also mainly observed in peripheral portions. Similarly, activated myogenic cells were seen not only inside but also outside of muscle fibers. The former were likely satellite cells and the latter may be interstitial myogenic cells. One week after surgery, small muscle fibers, small arteries and capillaries and several branched-muscle fibers were evident in the periphery, thus indicating new muscle fiber and blood vessel formation. Proliferating cells were also detected in the nerve bundles in the Schwann cell position. These results indicate that the compensatory stimulated/enlarged muscle is a suitable model for analyzing multiple physiological cellular responses in muscle–nerve–blood vessel units under continuous stretch stimulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fiber type changes in rat skeletal muscle after intense interval training

Female Sprague-Dawley rats were subjected to a ten week training program to determine the influence of intense interval running on the fiber type composition of selected hindlimb muscles; soleus (S), plantaris (P), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL). The muscles of one hindlimb were used for histochemical ATPase analysis to determine the distribution of fiber types and those of the contralateral hindlimb were assayed biochemically for citrate synthase activity (an aerobic marker). Training induced a significant increase in citrate synthase activity in each muscle section. The largest absolute increase occurred in the DVL and the largest relative increase occurred in the SVL. The distribution of fiber types within the S (85% slow-twitch) and SVL (100% fast-twitch) remained unchanged with training. However, significant increases in the percentage of type I (slow-twitch) fibers in both the P (2-fold) and DVL (3-fold) were observed with concomitant decreases in the type II (fast-twitch) population. In addition, training induced significant changes in the fast-twitch subtype populations of the DVL (IIB----IIA). These data suggest exercise-induced fiber type transformations occurring both within the fast-twitch population and between fast-twitch and slow-twitch fibers in certain hindlimb muscles of the rat following a high intensity interval training program.     

Regeneration of reinnervated rat soleus muscle is accompanied by fiber transition toward a faster phenotype.

The functional recovery of skeletal muscles after peripheral nerve transection and microsurgical repair is generally incomplete. Several reinnervation abnormalities have been described even after nerve reconstruction surgery. Less is known, however, about the regenerative capacity of reinnervated muscles. Previously, we detected remarkable morphological and motor endplate alterations after inducing muscle necrosis and subsequent regeneration in the reinnervated rat soleus muscle. In the present study, we comparatively analyzed the morphometric properties of different fiber populations, as well as the expression pattern of myosin heavy chain isoforms at both immunohistochemical and mRNA levels in reinnervated versus reinnervated-regenerated muscles. A dramatic slow-to-fast fiber type transition was found in reinnervated soleus, and a further change toward the fast phenotype was observed in reinnervated-regenerated muscles. These findings suggest that the (fast) pattern of reinnervation plays a dominant role in the specification of fiber phenotype during regeneration, which can contribute to the long-lasting functional impairment of the reinnervated muscle. Moreover, because the fast II fibers (and selectively, a certain population of the fast IIB fibers) showed better recovery than did the slow type I fibers, the faster phenotype of the reinnervated-regenerated muscle seems to be actively maintained by selective yet undefined cues.     

Competitive control of myosin expression: hypertrophy vs. hyperthyroidism

The competition between two opposing influences on the phenotypic expression of skeletal muscle myosin were studied to determine which was the dominant regulator. Experimental hyperthyroidism, which induces fast myosin expression, was produced by subcutaneous implantation of a 40-day constant-time-release triiodothyronine pellet. Compensatory hypertrophy, which induces slow myosin expression, was produced by surgical removal of a synergistic hindlimb muscle. Hyperthyroidism increased the percentage of type II fibers and the fast myosin isoforms in both the plantaris and soleus muscles. Hypertrophy significantly increased the percentage of type I fibers and the slow myosin type in the plantaris and soleus muscles. However, with the simultaneous introduction of hyperthyroidism and hypertrophy, only the hyperthyroid effects were observed. Hyperthyroidism and not physiological demand was found to be the dominant regulator of skeletal muscle myosin expression.     

Fiber-type-specific alphaB-crystallin distribution and its shifts with T(3) and PTU treatments in rat hindlimb muscles.

Changes in alphaB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5, 3'-triiodothyronine (T(3)) and propylthiouracil (PTU) treatments. Cellular distributions of alphaB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-alphaB-crystallin antibody. alphaB-crystallin content in both soleus and EDL muscles was significantly decreased after T(3), and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. alphaB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx > or = IIa in soleus muscle. The relation was basically unchanged in both muscles after T(3) treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean alphaB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein alphaB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.     

Skeletal muscle fiber, nerve, and blood vessel breakdown in space-flown rats

Histochemical and ultrastructural analyses were performed postflight on hind limb skeletal muscles of rats orbited for 12.5 days aboard the unmanned Cosmos 1887 biosatellite and returned to Earth 2 days before sacrifice. The antigravity adductor longus (AL), soleus, and plantaris muscles atrophied more than the non-weight-bearing extensor digitorum longus, and slow muscle fibers were more atrophic than fast fibers. Muscle fiber segmental necrosis occurred selectively in the AL and soleus muscles; primarily, macrophages and neutrophils infiltrated and phagocytosed cellular debris. Granule-rich mast cells were diminished in flight AL muscles compared with controls, indicating the mast cell secretion contributed to interstitial tissue edema. Increased ubiquitination of disrupted myofibrils implicated ubiquitin in myofilament degradation. Mitochondrial content and succinic dehydrogenase activity were normal, except for subsarcolemmal decreases. Myofibrillar ATPase activity of flight AL muscle fibers shifted toward the fast type. Absence of capillaries and extravasation of red blood cells indicated failed microcirculation. Muscle fiber regeneration from activated satellite cells was detected. About 17% of the flight AL end plates exhibited total or partial denervation. Thus, skeletal muscle weakness associated with spaceflight can result from muscle fiber atrophy and segmental necrosis, partial motor denervation, and disruption of the microcirculation.     

Effects of mechanical over-loading on the properties of soleus muscle fibers, with or without damage, in wild type and mdx mice

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (～51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.