Response to denervation of rabbit soleus and gastrocnemius muscles. Time-course study of postnatal changes in myosin isoforms,fiber types,and contractile properties

Summary— In contrast to general belief, the response of rabbit muscles to denervation is maturation to slow-like type muscles [7]. We report now an investigation by biochemical, morphological, and mechanical studies of the time course effects of muscle denervation on the slow-type soleus and fast-type gastrocnemius to help clucidate the mechanism of maturation of rabbit denervated muscles to slow-like muscles. In both muscles, denervation induced selective progressive atrophy of most fast fibers and hypertrophy of many slow fibers which displayed wide Z-lines; this was accompanied by the appearance of hybrid LC1F- and LC1E-associated slow myosins. The percentage of slow myosins increased with age similarly in the contralateral and denervated soleus. On the other hand, the percentage of slow myosins remained low in the contralateral gastrocnemius, whereas it increased to 95% in the denervated gastrocnemius; in the denervated gastrocnemius, the percentage of slow myosins reached 50% at about 35 days postnatal. At this age, the maximal shortening velocity of the denervated gastrocnemius and its twitch contraction time were already those of a slow-type muscle. This suggests that in addition to myosin, other proteins contributed to the mechanical properties of the denervated gastrocnemius. Transformation of rabbit denervated muscles to slow-like type muscles, which are associated with a lower energy requirement and higher muscle endurance than fast-type muscles, may constitute an adequate model for human neuromuscular pathology.     

The FgfrL1 receptor is required for development of slow muscle fibers

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.     

Strategies of adaptation of small arteries in diaphragm and gastrocnemius muscle to aerobic exercise training

Aerobic exercise training is associated with adaptive changes in skeletal muscles and their vascular bed; such changes in individual muscles may vary depending on their characteristics and recruitment. This study was aimed at comparing the effects of eight-week treadmill training on the locomotor and respiratory muscles in rats. The training course increased the aerobic performance in rats, which was evidenced by an increase in maximum O₂ consumption and a decrease in the blood lactate concentration in ramp test. The succinate dehydrogenase activity was increased in the red portion of the gastrocnemius muscle, but not in the diaphragm of trained rats. Arterial segments were isolated from feed arteries and studied by wire myography. The relaxation in response to acetylcholine in gastrocnemius arteries in trained animals was higher as compared with controls (due to higher NO production), while contractile responses to noradrenaline (in the presence of propranolol) were not changed. On the contrary, the endothelial function of diaphragm arteries was not affected by training, but contractile responses to activation of α-adrenoceptors were markedly increased. Thus, aerobic training may increase the blood supply rate to both locomotor and respiratory muscles, but the underlying regulatory mechanisms are different. The results obtained allow us to reveal the physiological mechanisms that determine the physical performance of the body under conditions of compromised functioning of the respiratory system.     

Diaphragm muscle and its feed artery after chronic respiratory airway obstruction in rats

A chronic respiratory load was produced in Wistar rats by tracheal binding to produce a twofold increase of pleural pressure oscillation amplitude during respiration. Eight weeks after the surgery, a higher proportion of type-I muscle fibers (MFI) in the costal diaphragm along with a greater MFI cross-section area and a higher succinate dehydrogenase activity in MFII in the lumbar diaphragm were observed. During recording the mechanical activity of ring preparations of diaphragm arteries under isometric conditions, an increase in endothelium-dependent relaxation was found, whereas endothelium-independent relaxation and arterial reactivity to noradrenaline did not change. Tracheal binding did not produce any changes of MF in the gastrocnemius muscle, but endothelium-dependent relaxation of gastrocnemius feed arteries was reduced. We conclude that chronic respiratory load affects the endothelial function in diaphragm arteries in a manner favorable for blood flow control in the diaphragm. Functional alterations in gastrocnemius arteries may be associated with the reduced locomotor activity of operated rats.     

Changes in the diaphragm muscle and its feed artery after chronic respiratory airway obstruction in rats

A chronic respiratory load was produced in Wistar rats by tracheal binding to produce a twofold increase of pleural pressure oscillation amplitude during respiration. Eight weeks after the surgery, a higher proportion of type-I muscle fibers (MFI) in the costal diaphragm along with a greater MFI cross-section area and a higher succinate dehydrogenase activity in MFII in the crural diaphragm were observed. During recording the mechanical activity of ring preparations of diaphragm arteries under isometric conditions, an increase in endothelium-dependent relaxation was found, whereas endothelium-independent relaxation and arterial reactivity to noradrenaline did not change. Tracheal binding did not produce any changes of MF in the gastrocnemius muscle, but endothelium-dependent relaxation of gastrocnemius feed arteries was reduced. We conclude that chronic respiratory load affects the endothelial function in diaphragm arteries in a manner favorable for blood flow control in the diaphragm. Functional alterations in gastrocnemius arteries may be associated with the reduced locomotor activity of operated rats.     

Skeletal muscle histology and biochemistry of an elite sprinter, the African cheetah

To establish a skeletal muscle profile for elite sprinters, we obtained muscle biopsy samples from the vastus lateralis, gastrocnemius and soleus of African cheetahs (Acinonyx jubatus). Muscle ultrastructure was characterized by the fiber type composition and mitochondrial volume density of each sample. Maximum enzyme activity, myoglobin content and mixed fiber metabolite content were used to assess the major biochemical pathways. The results demonstrate a preponderance of fast-twitch fibers in the locomotor muscles of cheetahs; 83% of the total number of fibers examined in the vastus lateralis and nearly 61% of the gastrocnemius were comprised of fast-twitch fibers. The total mitochondrial volume density of the limb muscles ranged from 2.0 to 3.9% for two wild cheetahs. Enzyme activities reflected the sprinting capability of the cheetah. Maximum activities for pyruvate kinase and lactate dehydrogenase in the vastus lateralis were 1519.00 ± 203.60 and 1929.25±482.35 μmol min⁻¹ · g wet wt⁻¹, respectively, and indicated a high capacity for glycolysis. This study demonstrates that the locomotor muscles of cheetahs are poised for anaerobically based exercise. Fiber type composition, mitochondrial content and glycolytic enzyme capacities in the locomotor muscles of these sprinting cats are at the extreme range of values for other sprinters bred or trained for this activity including greyhounds, thoroughbred horses and elite human athletes. Accepted: 5 June 1997     

Skeletal muscle fiber composition of the English sparrow (Passer domesticus)

Substrate utilization by English sparrow skeletal muscle has been extensively studied in our lab. However, there are few published studies on the muscle fiber composition of English sparrow wing and gastrocnemius muscles. The objective of the present study was to examine the fiber type composition of a variety of muscles in the English sparrow. The classification of a muscle fiber as fast glycolytic, slow oxidative, or fast oxidative glycolytic provides insight into the physiological function of muscles. Therefore, we completed mATPase and NADH stains on four muscles of the sparrow wing, as well as the gastrocnemius muscle, to characterize these muscle fiber types. Results show that the fibers of extensor digitorum communis, extensor metacarpi ulnaris, and extensor metacarpi radialis are homogeneous fast oxidative. The fibers of the supinator are homogeneous fast oxidative in 62.5% of samples, and heterogeneous (45.2% fast oxidative, 54.8% fast nonoxidative) in 37.5% of samples. Whereas the gastrocnemius muscle fibers are heterogeneous (10% fast oxidative, 64% fast nonoxidative, 26% slow oxidative) in all muscles examined.     

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles—The role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A₈ and A₁₂) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.     

Development of the mitochondrial apparatus and blood supply of skeletal muscle fibers during ontogenesis of domestic fowl

The diameter, length, and numerical density of capillaries, diameter of muscle fibers, size and numerical density of mitochondrial profiles, and relative volume of mitochondria in them were determined in the chicken red oxidative gastrocnemius and white glycolytic pectoral muscle during development from day 10 of embryogenesis to six month of postnatal life. The bulk blood flow was measured in these muscles by hydrogen clearance during postembryonic development. During embryogenesis, the fibers of gastrocnemius muscle develop and grow at a higher rate, while during postembryonic development, those of the pectoral muscle develop faster. The density of mitochondrial profiles increases during embryogenesis and decreases after hatching, while their mean size increases, especially in the oxidative fibers, but it somewhat decreases in 6-month old chicks. Redistribution of mitochondria across the fiber section during development takes place in both muscles: they are localized predominantly in the center in 18-day embryos and in the periphery, especially in the gastrocnemius fibers, in 6-month old chicks. At hatching, the length of capillaries is similar in both muscles, but as chicks grow, the proportion of longer (more than 600 µm) capillaries in the pectoral muscle sharply increases, while their density and bulk blood flow decrease. Ratios were determined between structural parameters of the capillary bed and mitochondria, on the one hand, and oxygen consumption (ml/min per 1 mm fiber and 100 g muscle mass), on the other.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 135–144.Original Russian Text Copyright © 2005 by Belichenko, Korostyshevskaya, Maksimov, Shoshenko.     

Single-fiber myosin heavy chain polymorphism: how many patterns and what proportions?

Previous studies have reported the existence of skeletal muscle fibers that coexpress multiple myosin heavy chain isoforms. These surveys have usually been limited to studying the polymorphic profiles of skeletal muscle fibers from a limited number of muscles (i.e., usually <4). Additionally, few studies have considered the functional implications of polymorphism. Hence, the primary objective of this study was to survey a relatively large number of rat skeletal muscle/muscle regions and muscle fibers (n approximately 5,000) to test the hypothesis that polymorphic fibers represent a larger fraction of the total pool of fibers than do so-called monomorphic fibers, which express only one myosin heavy chain isoform. Additionally, we used Hill's statistical model of the force-velocity relationship to differentiate the functional consequences of single-fiber myosin heavy chain isoform distributions found in these muscles. The results demonstrate that most muscles and regions of rodent skeletal muscles contain large proportions of polymorphic fibers, with the exception of muscles such as the slow soleus muscle and white regions of fast muscles. Several muscles were also found to have polymorphic profiles that are not consistent with the I<-->IIA<-->IIX<-->IIB scheme of muscle plasticity. For instance, it was found that the diaphragm muscle normally contains I/IIX fibers. Functionally, the high degree of polymorphism may 1) represent a strategy for producing a spectrum of contractile properties that far exceeds that simply defined by the presence of four myosin heavy chain isoforms and 2) result in relatively small differences in function as defined by the force-velocity relationship.     

Fiber type homogeneity of the flight musculature in small birds

Studies of medium- and large-bodied avian species have suggested that variation in flight muscle composition is related to differences in flight behavior. For example, slow-twitch or tonic fibers are generally found only in the flight muscles of non-volant or soaring/gliding birds. However, we know comparatively little about fiber composition of the muscles of the smallest birds. Here we describe the fiber composition of muscles from the wings, shoulders, and legs of two small avian species, which also display very high wingbeat frequencies: Anna's hummingbirds (Calypte anna) and zebra finches (Taeniopygia guttata). All flight muscles examined in both species contained exclusively fast oxidative glycolytic (FOG) fibers. These unique results suggest that fast oxidative fibers are both necessary and sufficient for the full range of flight behaviors in these small-bodied birds. Like all other studied birds, the zebra finch gastrocnemius, a tarsometatarsal extensor, contained a mixture of FOG (27.1%), slow oxidative (SO, 12.7%), and fast glycolytic (FG, 60.2%) fibers. By contrast, the hummingbird gastrocnemius lacked FG fibers (85.5% FOG, 14.5% SO), which may reflect the reduced role of the hindlimb during take-off. We further hypothesize that thermogenic requirements constrain fiber type heterogeneity in these small endothermic vertebrates.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Development of the mitochondrial apparatus and blood supply of skeletal muscle fibers during ontogenesis of domestic fowl

The diameter, length, and numerical density of capillaries, diameter of muscle fibers, size and numerical density of their profiles, and relative volume of mitochondria in them were determined in the chicken red oxidative gastrocnemius and white glycolytic pectoral muscle during development from day 10 of embryogenesis to six month of postnatal life. The bulk blood flow was measured in these muscles by hydrogen clearance during postembryonic development. During embryogenesis, the fibers of gastrocnemius muscle develop and grow at a higher rate, while during postembryonic development, those of the pectoral muscle develop faster. The density of mitochondrial profiles increases during embryogenesis and decreases after hatching, while their mean size increases, especially in the oxidative fibers, but it somewhat decreases in 6-month old chicks. Redistribution of mitochondria by the fiber section during development takes place in both muscles: they are localized predominantly in the center in 18-day embryos and in the periphery, especially in the gastrocnemius fibers, in 6-month old fowl. At hatching, the lengths of capillaries are similar in both muscles, but as chicks grow, the proportion of longer (more than 600 microm) capillaries in the pectoral muscle sharply increases, while their density and bulk blood flow decrease. Ratios were determined between structural parameters of the capillary bed and mitochondria, on the one hand, and oxygen consumption (ml/min per 1 mm fiber and 100 g muscle mass), on the other.     

Immunohistochemical Analysis of Laryngeal Muscles in Normal Horses and Horses With Subclinical Recurrent Laryngeal Neuropathy

We used immunohistochemistry to examine myosin heavy-chain (MyHC)-based fiber-type profiles of the right and left cricoarytenoideus dorsalis (CAD) and arytenoideus transversus (TrA) muscles of six horses without laryngoscopic evidence of recurrent laryngeal neuropathy (RLN). Results showed that CAD and TrA muscles have the same slow, 2a, and 2x fibers as equine limb muscles, but not the faster contracting fibers expressing extraocular and 2B MyHCs found in laryngeal muscles of small mammals. Muscles from three horses showed fiber-type grouping bilaterally in the TrA muscles, but only in the left CAD. Fiber-type grouping suggests that denervation and reinnervation of fibers had occurred, and that these horses had subclinical RLN. There was a virtual elimination of 2x fibers in these muscles, accompanied by a significant increase in the percentage of 2a and slow fibers, and hypertrophy of these fiber types. The results suggest that multiple pathophysiological mechanisms are at work in early RLN, including selective denervation and reinnervation of 2x muscle fibers, corruption of neural impulse traffic that regulates 2x and slow muscle fiber types, and compensatory hypertrophy of remaining fibers. We conclude that horses afflicted with mild RLN are able to remain subclinical by compensatory hypertrophy of surviving muscle fibers. (J Histochem Cytochem 57:787–800, 2009)     

Diaphragm muscle development in bottlenose dolphins (Tursiops truncatus)

Being born directly into the aquatic environment creates unique challenges for the breathing muscles of neonatal cetaceans. Not only must these muscles be active at the instant of birth to ventilate the lungs, but their activities must also be coordinated with those of the locomotor muscles such that breathing takes place only at the water's surface. At least one major locomotory muscle of bottlenose dolphins (Tursiops truncatus) has been demonstrated to be well developed and, therefore, able to power the neonatal dolphin's early movements (Dearolf et al. [2000] J Morphol 244:203-215). Thus, because of the demands for coordinated behavior with the locomotor muscles, it is hypothesized that the breathing muscles of bottlenose dolphins, represented in this study by the diaphragm, will also demonstrate adult morphology at birth. However, histochemical and biochemical analyses demonstrate that neonatal dolphins have immature diaphragms, with only 52% of the adult slow fiber-type profile (neonates: 34% slow-twitch fibers; adults: 66% slow-twitch fibers). The developmental state of the dolphin diaphragm is compared to those of other neonatal mammals, using a muscle development index (% slow-twitch fibers in neonatal muscle / % slow-twitch fibers in adult muscle). Fiber-type profiles reported in the literature are used to calculate index values for the diaphragms of altricial rats, rabbits, and cats, intermediate baboons and humans, and precocial sheep and horses. The dolphin is not unique in having an immature diaphragm at birth; however, there is a positive relationship between the developmental state of the diaphragm and the overall developmental state of the neonate. The presence of type IIc ("undifferentiated") fibers in the diaphragms of altricial developers (e.g., rats, rabbits, and cats) is correlated with the slow contraction speeds recorded from their diaphragms. The diaphragms of neonatal horses and dolphins express little to no type IIc fibers and, thus, may have the ability to contract at the speeds required for their increased ventilation rates. These results lead to the modification of the criterion for evaluating the developmental state of a muscle at birth. Thus, the developmental state of a neonatal muscle should be based on both its value of Dearolf et al.'s (2000) developmental index, as well as the percentage of type IIc fibers found in that muscle.     

The significance of lipoprotein lipase in rat skeletal muscles.

Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.     

A histochemical and enzymatic study of the muscle fiber types in the water monitor, Varanus salvator

Histochemical analysis of five muscles from the water monitor, Varanus salvator, identified three major classes of fibers based on histochemical activities of the enzymes myosin ATPase (mATPase), succinic dehydrogenase (SDH), and alpha-glycerophosphate dehydrogenase (alpha GPDH). Fast-twitch, glycolytic (FG) fibers were the most abundant fiber type and exhibited the following reaction product intensities: mATPase, dark; SDH, light; alpha GPDH, moderate to dark. Fast-twitch, oxidative, glycolytic (FOG) fibers were characteristically mATPase, dark; SDH, light; alpha GPDH, moderate to dark. The third class of fibers had the following histochemical characteristics: mATPase, light; SDH, moderate to dark; alpha GPDH, light. These fibers were considered to be either slow twitch, or tonic, and oxidative (S/O). Pyruvate kinase (PK), alpha GPDH, and citrate synthase (CS) activities were measured in homogenates of the same muscles studied histochemically. There was a positive relationship between both PK and alpha GPDH activities and the percentage of glycolytic fiber types within a muscle. Likewise, CS activities were greater in muscles high in FOG and S/O content. Based on CS activities, Varanus S/O fibers were eight-fold more oxidative than FG fibers within the same muscle. PK/CS ratios suggested that FG fibers possess high anaerobic capacity, similar to the iguanid lizard Dipsosaurus. The fiber type composition of the gastrocnemius muscle, relative to that of other lizard species, suggests that varanid lizards may possess a greater proportion of FOG and S/O fibers than other lizards.