Trypanosoma cruzi: Histochemical Characterization of Parasitized Skeletal Muscle Fibers1

C57B1/6 mice were infected with Brasil strain Trypanosoma cruzi trypomastigotes. The leg muscles of the mice were serial-sectioned with a cryostat, and individual fibers were classified histochemically as type I or type II on the basis of succinic dehydrogenase or adenosine triphosphatase activity. Although markedly more type II fibers were present in the leg muscles, the percentage of infected type I fibers was nearly five-fold higher than type II. This is the first demonstration of a preferential in vivo distribution of T. cruzi in muscle fibers based upon muscle type.     

Trypanosoma cruzi: aberrant expression of class II major histocompatibility complex molecules in skeletal and heart muscle cells of chronically infected mice

In isolated skeletal, heart, and smooth muscle cells from BALB/c and C3H/HeJ mice infected with different strains of Trypanosoma cruzi the presence of class II MHC molecules was investigated by immunocytochemical techniques. We employed single muscle fibers instead of conventional cryostat sections to obtain a more accurate antigen localization. Approximately half of the skeletal muscle cells isolated from the rectus femoris expressed Ia antigens on their surface, irrespective of the mouse or parasite strain combination. Ia expression was apparent only at 30 days postinfection and thereafter. The heart muscle cells expressed class II molecules only at 1 and 3 months postinfection. In no case did the smooth muscle cells from infected mice express Ia antigens. Studies of the same molecules in the noninfected animals gave constantly negative results. We conclude that in the course of the chronic infection of mice with T. cruzi, ectopic expression of class II MHC molecules occurs at the surface of skeletal and heart muscle cells, providing a possible mechanism for explaining the anti-striped muscle autoreactivity present in Chagas' disease.     

Analysis of the composition of skeletal muscle fibers in skaters' muscles

Muscle fibers composition was investigated in vastus lateralis muscles of 103 skaters. In the total population of skaters muscle fibers composition was as follows: muscle fibers, type I--in 54 +/- 2%, muscle fibers, type II A--in 35 +/- 1% and muscle fibers, type II B--in 11 +/- 1%. 13 +/- 8% muscle fibers, type I; 1.56 +/- 6% muscle fibers, type II A and 31 +/- 7% muscle fibers, type II B were observed in the muscles of sprinters, while the muscles of long-distance skaters contained 60 +/- 4%, 33 +/- 4% and 7 +/- 2% muscle fibers, respectively.     

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.     

Loss of ovarian function in mice results in abrogated skeletal muscle PPARδ and FoxO1-mediated gene expression

Menopause, the age-related loss of ovarian hormone production, promotes increased adiposity and associated metabolic pathology, but molecular mechanisms remain unclear. We previously reported that estrogen increases skeletal muscle PPARδ expression in vivo, and transgenic mice overexpressing muscle-specific PPARδ are reportedly protected from diet-induced obesity. We thus hypothesized that obesity observed in ovariectomized mice, a model of menopause, may result in part from abrogated expression of muscle PPARδ and/or downstream mediators such as FoxO1. To test this hypothesis, we ovariectomized (OVX) or sham-ovariectomized (SHM) 10-week old female C57Bl/6J mice, and subsequently harvested quadriceps muscles 12 weeks later for gene expression studies. Compared to SHM, muscle from OVX mice displayed significantly decreased expression of PPARδ (3.4-fold), FoxO1 (4.5-fold), PDK-4 (2.3-fold), and UCP-2 (1.8-fold). Consistent with studies indicating PPARδ and FoxO1 regulate muscle fiber type, we observed dramatic OVX-specific decreases in slow isoforms of the contractile proteins myosin light chain (11.1-fold) and troponin C (11.8-fold). In addition, muscles from OVX mice expressed 57% less myogenin (drives type I fiber formation), 2-fold more MyoD (drives type II fiber formation), and 1.6-fold less musclin (produced exclusively by type II fibers) than SHM, collectively suggesting a shift towards less type I oxidative fibers. Finally, and consistent with changes in PPARδ and FoxO1 activity, we observed decreased expression of atrogin-1 (2.3-fold) and MuRF-1 (1.9-fold) in OVX mice. In conclusion, muscles from ovariectomized mice display decreased PPARδ and FoxO1 expression, abrogated expression of downstream targets involved in lipid and protein metabolism, and gene expression profiles indicating less type I oxidative fibers.     

Adaptations of diaphragm and medial gastrocnemius muscles to inactivity.

The effects of 2 wk of inactivity on in vitro contractile properties of diaphragm and medial gastrocnemius (MG) muscles were examined in adult hamsters. In addition, inactivity effects on fiber-type proportions and cross-sectional areas were studied. Inactivity of the right hemidiaphragm or MG muscle was induced by either tetrodotoxin (TTX) blockade of nerve impulses or denervation (DNV). Inactivity effects on diaphragm or MG were compared with corresponding sham (saline-treated or untreated control) muscles. After both TTX- and DNV-induced inactivity, isometric twitch contraction and half-relaxation times were prolonged, maximum tetanic force decreased, and fatigue resistance improved. Proportions of type I and II fibers in both diaphragm and MG were unaffected by TTX- and DNV-induced inactivity. However, in both muscles, type I fibers hypertrophied, whereas type II fibers atrophied. In diaphragm, contractile and morphometric adaptations after DNV were generally more pronounced than those induced by TTX. In addition, compared with corresponding untreated or saline-treated control groups, inactivity effects (both TTX and DNV) on MG were generally greater than those induced in diaphragm, with the exception of hypertrophy of type I fibers. We conclude that inactivity exerts differential effects on type I and II fibers in both diaphragm and MG. Yet, these morphometric adaptations cannot completely account for the adaptations in muscle contractile and fatigue properties after inactivity.     

Distribution of fiber types in locomotory muscles of dogs

The distribution of Type I and Type II fibers, as determined from histochemical estimation of myofibrillar ATPase activity, was studied within and among the locomotory muscles of the forelimb, trunk, and hindlimb of three mongrel dogs. All Type II fibers had high oxidative capacities as estimated from the histochemical assay for reduced nicotinamide adenine dinucleotide tetrazolium reductase, so they were not further divided into subpopulations. Furthermore, Type I and Type II fibers had similar oxidative potentials as indicated by both histochemistry and biochemistry. Type I fiber populations ranged between 14% and 100% in the muscles sampled. The highest percentages of Type I fibers were found in deep muscles of physiological extensor groups in the arm and thigh that serve to resist gravity (antigravity muscles) when the dog is in the quadrupedal standing position. More superficial muscles in these same groups had fewer Type I fibers. The patterns of Type I fiber distribution among muscles in the antigravity groups of the forearm and leg were the opposite of those in the arm and thigh, with the more superficial muscles of the distal limb segments having more Type I fibers than the deeper muscles. In all limb segments, muscle groups that do not serve to resist gravity did not show as much intermuscular variation. Type I fiber populations in these muscles did not exceed 50%. A stratification of fiber types also existed within muscles, both in extensor and flexor groups, with the deeper portions of the muscles having more Type I fibers than the more superficial portions.     

Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

Summary The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar.Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar. Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

The development of the pattern of innervation in chicken hindlimb muscles: evidence for specification of nerve-muscle connections

The pattern of innervation in 13 chicken hindlimb muscles was studied at various stages of development in order to examine the mechanisms which regulate its formation. The pattern of innervation was visualized by examining the distribution of fiber types within each muscle. It was found that the fiber type which a myotube acquired was influenced by both its time of formation and its position within a muscle. The earliest generation of myotubes (primary) had a marked tendency to become type I fibers, whereas, in contrast, the later generation of myotubes (secondary) tended to differentiate into type II fibers. There were regions of muscle, however, in which primary myotubes differentiated into type II fibers and other regions in which secondary myotubes acquired type I characteristics. During the development of some muscles the pattern of fiber types changed as a result of either a selective loss of type I fibers or, in other cases, a rearrangement of some of the initial neuromuscular contacts. These observations are consistent with the pattern of innervation of a muscle being established as a result of differential projection patterns of fast and slow motoneurons and the existence of some type of chemoaffinity where particular myotubes are preferentially innervated by particular motoneurons.     

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.     

Effect of hypothyroidism on myosin heavy chain expression in rat pharyngeal dilator muscles.

Although the association between hypothyroidism and obstructive sleep apnea is well established, the effect of thyroid hormone deficiency on contractile proteins in pharyngeal dilator muscles responsible for maintaining upper airway patency is unknown. In the present study, the effects of hypothyroidism on myosin heavy chain (MHC) expression were examined in the sternohyoid, geniohyoid, and genioglossus muscles of adult rats (n = 20). The relative proportions of MHC isoforms present were determined using MHC-specific monoclonal antibodies and oligonucleotide probes. All control muscles showed a paucity of type I MHC fibers, with greater than 90% of fibers containing fast-twitch type II MHCs. In the genioglossus muscle, a population of non-IIa non-IIb fast-twitch type II fibers (putatively identified as type IIx MHC fibers) were detected. Hypothyroidism induced significant changes in MHC expression in all muscles studied. In the sternohyoid, type I fibers increased from 6.2 to 16.9%, whereas type IIa fibers increased from 25.9 to 30.7%. Type I fibers in the geniohyoid increased from 1.2 to 12.8%, whereas type IIa fibers increased from 34.1 to 42.7%. The genioglossus showed the smallest relative increase in type I expression but the greatest induction of type IIa MHC. None of the muscles examined demonstrated reinduction of embryonic or neonatal MHC in response to thyroid hormone deficiency. In summary, hypothyroidism alters the MHC profile of pharyngeal dilators in a muscle-specific manner. These changes may play a role in the pathogenesis of obstructive apnea in hypothyroid patients.     

Effects of undernutrition on diaphragm fiber size, SDH activity, and fatigue resistance

The influence of prolonged nutritional deprivation on the succinate dehydrogenase (SDH) activity and cross-sectional areas of individual fibers in the rat diaphragm and deep portion of the medial gastrocnemius (MGr) muscles was determined. Fatigue resistance of the diaphragm was measured by means of an in vitro nerve-muscle strip preparation. Fiber SDH activity and cross-sectional area were quantified by means of an image processing system. Diaphragm fatigue resistance was significantly improved in the nutritionally deprived (ND) group. In both muscles, nutritional deprivation resulted in a significant decrease in fiber cross-sectional area (both type I and II), type II fibers showing greater atrophy. The SDH activities of type I and II fibers in the diaphragm were not affected by nutritional deprivation. This contrasted with a significant decrease in the SDH activity of both type I and II fibers in the MGr of ND animals. An assessment of the interrelationships between fiber atrophy and fiber SDH activity revealed a greater effect of malnutrition on those diaphragm type II fibers that had the lowest relative SDH activities and the largest cross-sectional areas. By comparison, the effect of malnutrition on type I and II fibers in the MGr was nonselective with regard to fiber SDH activity. We conclude that the enhanced diaphragm fatigue resistance in the ND animals does not result from an increase in the oxidative capacity of muscle fibers and is best explained by the pattern of diaphragm muscle fiber atrophy.     

Conversion of rat muscle fiber types. A time course study

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 less than 0.05) of the total population, four weeks after surgery.     

Long-term activity in upper- and lower-limb muscles of humans.

Despite limited data on humans, previous studies suggest that there is an association between the duration of daily muscle activity and the proportion of type I muscle fibers. We quantified the activity of limb muscles in healthy men and women during normal use and compared these measurements with published reports on fiber-type proportions. Seven men (age range = 21-28 yr) and seven women (age range = 18-26 yr) participated in two 10-h recording sessions. Electromyogram (EMG) activity of four muscles in nondominant upper (first dorsal interosseus and biceps brachii) and lower limbs (vastus medialis and vastus lateralis) was recorded with surface electrodes. Hand and arm muscles were active for 18% of the recording time, whereas leg muscles were active for only 10% of the recording time. On average, upper-limb muscles were activated 67% more often than lower-limb muscles. When lower-limb muscles were activated, however, the mean amplitude of each burst was greater in leg muscles [18 and 17% maximum voluntary contraction (MVC)] compared with hand (8% MVC) and arm (6% MVC) muscles. Temporal association in activity between pairs of muscles was high for the two lower-limb muscles (r2 = 0.7) and relatively weak for the two upper-limb muscles (r2 = 0.09). Long-term muscle activity was only different between men and women for the biceps brachii muscle. We found no relation between duration of muscle activity in 10-h recordings and the reported values of type I fibers in men and women.     

Influence of the sagittal advancement of mandibulae on myofibrillar ATPase activity and myosin heavy chain content in the masticatory muscles of pigs

Endurance muscle stress leads to polymorphic expression of myosin heavy chains (MyHC). Histochemical and electrophoretic analyses were performed on different masticatory muscles (masseter, temporal, geniohyoid and medial pterygoid) of 10 weeks old pigs after 28 days of chronic sagittal advancement of the mandibulae. The differentiation between fiber types was investigated histochemically with the myofibrillar ATPase (mATPase) method and by immunohistochemistry. Expression of different MyHC isoforms was also assessed by means of immunoblotting with monoclonal antibodies. The results of both methods were compared. Chronic sagittal advancement of the mandibulae led to an increase in the cross-sectional area of type I fibers and type I MyHC in the anterior part of the masseter, the distal part of the temporal and the medial pterygoid muscle. In the present study, clear differentiation between type I and type II muscle fibers in all histological analyses was possible. However, mATPase classification of subtypes of type II fibers may lead to misinterpretations. Additionally, a direct correlation between the type I MyHC concentration and the type I fibers was seen in enzyme histochemical and immunohistochemical staining. The defined cross section of fibers is important for the histological investigation in small muscles. The immunoblot method seems to be more sensitive and less subjective for measurement of muscle changes. It can be concluded that the immunoblot method used for measuring the MyHC content is a valid alternative for fiber typing in small muscles as it is less time-consuming and more sensitive than qualitative histochemistry.     

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.