Kinetic properties of cardiac myosin heavy chain isoforms in rat

The head portion of the myosin heavy chain (MHC) is essential in force generation. As previously shown, Ca2+-activated fibres of mammalian skeletal muscle display a strong correlation between their MHC isoform complement and the kinetics of stretch activation, suggesting isoform-specific differences in kinetic properties of myosin heads. Using the same methodology on muscle strips of atria and ventricles of hyper- and hypothyroid rats, this study showed that the kinetics of cardiac alphaMHC are 3 times faster than those of cardiac betaMHC under isometric conditions and maximal Ca2+ activation. Comparison of rat heart and skeletal muscle fibres revealed that 100% alphaMHC heart muscle strips exhibited faster stretch activation kinetics (time parameter t3: 108+/-18 ms, mean+/-SD) than rat type-IIA fibres ( t3: 157+/-19 ms), but slower than type-IID fibres ( t3: 55+/-10 ms). The kinetics of 100% betaMHC heart muscle strips ( t3: 351+/-44 ms) were faster than that of type-I fibres in rat skeletal muscle ( t3: 901+/-348 ms). This difference between the two muscle types calls in question the generally accepted identity of betaMHC and MHCIbeta.     

Kinetics of cardiac myosin isoforms in mouse myocardium are affected differently by presence of myosin binding protein-C

We tested whether cardiac myosin binding protein-C (cMyBP-C) affects myosin cross-bridge kinetics in the two cardiac myosin heavy chain (MyHC) isoforms. Mice lacking cMyBP-C (t/t) and transgenic controls \(( {\text{WT}}^{\text{t/t}} )\) were fed l-thyroxine (T4) to induce 90/10 % expression of α/β-MyHC. Non-transgenic (NTG) and t/t mice were fed 6-n-propyl-2-thiouracil (PTU) to induce 100 % expression of β-MyHC. Ca²⁺-activated, chemically-skinned myocardium underwent length perturbation analysis with varying [MgATP] to estimate the MgADP release rate \(\left( {k_{ - ADP} } \right)\) and MgATP binding rate \(\left( {k_{ + ATP} } \right)\). Values for \(k_{ - ADP}\) were not significantly different between \({\text{t/t}}_{\text{T4}}\) (102.2 ± 7.0 s^?1) and \({\text{WT}}^{\text{t/t}}_{\text{T4}}\) (91.3 ± 8.9 s^?1), but \(k_{ + ATP}\) was lower in \({\text{t/t}}_{\text{T4}}\) (165.9 ± 12.5 mM^?1 s^?1) compared to \({\text{WT}}^{\text{t/t}}_{\text{T4}}\) (298.6 ± 15.7 mM^?1 s^?1, P < 0.01). In myocardium expressing β-MyHC, values for \(k_{ - ADP}\) were higher in \({\text{t/t}}_{\text{PTU}}\) (24.8 ± 1.0 s^?1) compared to \({\text{NTG}}_{\text{PTU}}\) (15.6 ± 1.3 s^?1, P < 0.01), and \(k_{ + ATP}\) was not different. At saturating [MgATP], myosin detachment rate approximates \(k_{ - ADP}\), and detachment rate decreased as sarcomere length (SL) was increased in both \({\text{t/t}}_{\text{T4}}\) and \({\text{WT}}^{\text{t/t}}_{\text{T4}}\) with similar sensitivities to SL. In myocardium expressing β-MyHC, detachment rate decreased more as SL increased in \({\text{t/t}}_{\text{PTU}}\) (21.5 ± 1.3 s^?1 at 2.2 μm and 13.3 ± 0.9 s^?1 at 3.3 μm) compared to \({\text{NTG}}_{\text{PTU}}\) (15.8 ± 0.3 s^?1 at 2.2 μm and 10.9 ± 0.3 s^?1 at 3.3 μm) as detected by repeated-measures ANOVA (P < 0.01). These findings suggest that cMyBP-C reduces MgADP release rate for β-MyHC, but not for α-MyHC, even as the number of cMyBP-C that overlap with the thin filament is reduced to zero. Therefore, cMyBP-C appears to affect β-MyHC kinetics independent of its interaction with the thin filament.     

Embryonic myosin heavy chain and troponin T isoforms remain in the cremaster muscle of adult rat cryptorchidism induced with flutamide

We examined the immunolocalization of isoforms of muscle proteins, myosin and troponin, in the cremaster muscle of the undescended testis (cryptorchidism). In cryptorchid rats induced by a nonsteroidal androgen antagonist, flutamide, the cremaster muscle contained embryonic myosin and embryonic/cardiac troponin T in both immunofluorescence microscopy and Western blotting using antibodies against myosin and troponin T specific for embryonic, cardiac and fast skeletal muscles. However, in muscles other than the cremaster muscle, i. e., the masseter, pectoral and abdominal muscles, embryonic isoforms of these proteins were undetectable by immunohistochemistry with these antibodies, even in the muscles from cryptorchid rats. Our results showing that high levels of embryonic isoforms of muscle proteins were specifically present in the cremaster muscle of cryptorchid rats induced by flutamide suggest that flutamide treatment of pregnant rats might affect genes controlling the development of the lumbar region of the fetus body resulting in the presence of embryonic protein isoforms in the cremaster muscles which are closely associated with undescended testes.     

Clenbuterol induces expression of multiple myosin heavy chain isoforms in rat soleus fibres

Uterine secretory cells receive a sympathetic cholinergic secremotor innervation. Nitric oxide (NO) has been suggested to be a second messenger of neurogenic modulated glandular secretion of the seminal vesicle. Thus a similar pattern for nervous induced carbohydrate secretion of the endometrium was assumed. The nitric oxide synthase (NOS) activity was estimated via formation of L ‐citrulline from L ‐arginine and histochemically with the nicotinamide‐adenine dinucleotide phosphate diaphorase (NADPH‐d) nitro blue technique. The carbohydrate secretion from everted uterine horns placed in organ baths was estimated. A calcium dependent formation of citrulline was found in the uterine horn suggesting an NOS activity. Strong NADPH staining cells were found in the glandular ducts of the endometrium and in the epithelial linings of the oviduct. Carbachol induced carbohydrate secretion of the endometrium while N‐nitro L ‐arginin (L ‐NNA) and N‐nitro L ‐arginin methyl ester (L ‐NAME) inhibited the carbachol induced secretion. The isomer D ‐NAME had no effect on carbachol induced secretion. When L ‐arginine was administered together with L ‐NNA no inhibitory effect on carbachol induced secretion was seen. L ‐arginine only had no effect on carbohydrate secretion. The NO donor glyceryl tritrate increased carbohydrate secretion but no synergistic effect was seen in combination with carbachol. The results suggest that glandular NO production is a prerequisite for muscarinic carbohydrate secretion of the endometrium.     

Clenbuterol induces expression of multiple myosin heavy chain isoforms in rat soleus fibres

Clenbuterol, a beta2-agonist, administration results in hypertrophy of fast fibres and an increase in the fast myosin heavy chain (MHC) composition of both fast and slow muscles. The present study was designed to determine the phenotypic response at the single fibre level. Clenbuterol was added to the drinking water (30 mg L(-1)) of adult male Wistar rats for 4 weeks. Single fibres from the soleus muscle of control (10 rats; 555 fibres) and clenbuterol-treated (10 rats; 577 fibres) were dissected and their MHC isoform composition was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. Body, heart, and soleus weights were 9, 24, and 27% higher in clenbuterol-treated than control rats. The mean cross-sectional areas of fast and slow/fast hybrid fibres were approximately 64 and approximately 74% larger in the clenbuterol-treated than control rats, whereas the size of the slow fibres were similar in the two groups. Fibres from control soleus showed three MHC patterns: pure type I (84%), pure type IIa (4%), and type I + IIa (12%) MHC. Some fibres from clenbuterol-treated soleus showed a de novo expression of type IIx MHC resulting in the following fibre type proportions: pure type I (62%), pure type IIa (2%), type I + IIa (26%), type I + IIa + IIx (6%), and type IIa + IIx (1%). In those fibres containing multiple MHCs, there was a shift towards the faster MHC isoforms after clenbuterol treatment. These data indicate that clenbuterol results in muscle fibre hypertrophy, stimulates a de novo expression of type IIx MHC and increases the percentage of fibres containing multiple MHC isoforms in the rat soleus muscle. These phenotypic changes at the single fibre level are consistent with a clenbuterol-related shift in the functional properties of the soleus towards those observed in a faster muscle.     

B2 exon splicing of nonmuscle myosin heavy chain IIB is differently regulated in developing and adult rat brain

Two isoforms of nonmuscle myosin heavy chain IIB (MHC-IIB) are generated by alternative splicing; MHC-IIB(B2) differs from MHC-IIB(DeltaB2) by the insertion of B2 exon cassette near the actin binding region. Here we examined expressions of the two splice variants in developing and adult rat brains by in situ hybridization with isoform-specific oligonucleotide probes. In adult, MHC-IIB(DeltaB2) mRNA was highly expressed in neurons of the cerebral cortex, hippocampus, and cerebellum, whereas MHC-IIB(B2) mRNA was mainly distributed in the brainstem and cerebellum, with the highest level in Purkinje cells. During development, MHC-IIB(DeltaB2) mRNA was predominantly expressed in various regions of embryonic and neonatal brains, whereas MHC-IIB(B2) mRNA was low during embryonic stages. Up-regulation of MHC-IIB(B2) started in the cerebellum during early postnatal stages when dendritogenesis and synaptogenesis occur actively in Purkinje cells. We further employed immunofluorescence using two antibodies (one recognizing both splicing variants and another specific to MHC-IIB(B2)), and found similar and dense localization in cell bodies and dendrites of Purkinje cells. Therefore, splicing of the B2 exon cassette undergoes distinct temporal and spatial regulations in the brain in vivo, and the different exon usage seems unlikely to affect the somato-dendritic localization of MHC-IIB.     

Fine-tuning of cross-bridge kinetics in cardiac muscle of rat and mouse by myosin light chain isoforms

Cross-bridge kinetics underlying stretch-induced force transients was studied in cardiac muscle strips with different myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. The force transients were induced by stepwise stretches of maximally Ca²⁺-activated skinned muscle strips. The MHC and MLC isoforms were analyzed by electrophoreses after the mechanical experiments. Muscle strips of euthyroid rats and mice exclusively containing α-MHC were used. In addition, muscle strips of hyper- and hypothyroid rats containing different combinations of MHC and MLC isoforms were used. The thyroid hormone is known to alter the expression of MHC but not of MLC isoforms. In muscle strips containing exclusively α-MHC, atrial MLC isoforms (all atria of rats and mice) were associated with about 30% faster kinetics than ventricular MLC isoforms (ventricles of hyperthyroid rats and some muscle strips of ventricles of euthyroid rats and mice). On the other hand, in muscle strips containing exclusively ventricular MLC isoforms, α-MHC (ventricles of hyperthyroid rats) was associated with about 2.6 times faster kinetics than β-MHC (ventricles of hypothyroid rats). We conclude that the MLC isoforms fine-tune cross-bridge kinetics, which underlies stretch-induced force transients, whereas the MHC isoforms mainly determine this kinetics. The effect of MLC isoforms on the cross-bridge kinetics may partially contribute to the faster twitch contraction in atria than in ventricles. Furthermore, it may play a role in various cardiomyopathies where atrial MLC isoforms are partially expressed in ventricles or ventricular MLC isoforms are partially expressed in atria.     

Stretch activation and isoforms of myosin heavy chain and troponin-T of rat skeletal muscle fibres

Recent studies on single mammalian skeletal muscle fibres revealed a correlation between the kinetics of stretch-induced delayed force increase (stretch activation) and the isoforms of the myosin heavy chain. This observation suggests a causal relation between stretch activation and myosin heavy chain. However, the assumption is weakened by the fact that isoforms of other myofibrillar proteins tend to be coexpressed with myosin heavy chain isoforms. The relation between the isoforms of the tropomyosin-binding troponin subunit and myosin heavy chain is unknown. For a variety of reasons, tropomyosin-binding troponin subunit is a possible candidate for being involved in stretch activation. Therefore, we measured stretch activation of single, maximally Ca2+-activated skinned rat skeletal muscle fibres and characterized them by their myosin heavy chain composition, as well as by the isoform species of tropomyosin-binding troponin subunit. Four myosin heavy chain isoforms (I, IIa, IId or IIx and IIb) and six tropomyosin-binding troponin subunit isoforms (TnT1s, TnT2s, TnT1f, TnT2f, TnT3f, TnT4f) were distinguis hed. The following preferential coexpression patterns of the myosin heavy chain and tropomyosin-binding troponin subunit isoforms were observed: MHCI-TnT1s, MHCIIa-TnT3f, MHCIId-TnT1f, and MHCIIb-TnT4f. Stretch activation kinetics was found to be correlated with the myosin heavy chain isoform complement also in fibres not displaying one of the preferential MHC-TnTf isoform coexpression patterns. This corroborates the assumption of a causal relation between myosin heavy chain and stretch activation This revised version was published online in July 2006 with corrections to the Cover Date.     

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres

Summary Mammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.     

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.     

Stretch activation and myosin heavy chain isoforms of rat,rabbit and human skeletal muscle fibres

The underlying mechanism of stretch-induced delayed force increase (stretch activation) of activated muscles is unknown. To assess the molecular correlate of this phenomenon, we measured stretch activation of single, Ca2+-activated skinned muscle fibres from rat, rabbit and the human and analysed their myosin heavy chain complement by SDS gradient gel electrophoresis. Stretch activation kinetics was found to be closely correlated with the myosin heavy chain isoform complement (I, IIa, IId/x and IIb). In hybrid fibres containing two myosin heavy chain isoforms (especially IId and IIb), the kinetics of stretch activation depended on the percentage distribution of the two isoforms. Muscle fibres of the same type but originating from different mammalian species exhibited similar kinetics of stretch activation. Considering the differing unloaded shortening velocities of these fibres, the time-limiting factors for stretch activation and unloaded shortening velocity appear not to be the same. The stretch activation kinetics of the fibre types IIB, IID and IIA more likely seemed to follow a Normal Gaussian distribution than that of type I fibres. Several type I fibres had extraordinarily slow kinetics. This observation corroborates biochemical data indicating the possible existence of more than one slow myosin heavy chain isoform     

Whole-muscle and single-fibre contractile properties and myosin heavy chain isoforms in humans

 The contractile characteristics of three human muscle groups (triceps surae, quadriceps femoris and triceps brachii) of seven young male subjects were examined. The contractile properties were determined from electrically evoked isometric responses and compared with fibre type composition determined from needle biopsy samples. Fibre types were identified using myosin heavy chain (MHC) isoforms as molecular markers with gel electrophoresis (SDS-PAGE) and histochemical ATPase staining. Four contractile parameters (twitch time to peak torque, the maximal rate of torque development, frequency response and fatiguability) were found to be related to fibre type composition. From the biopsy samples, single muscle fibres were isolated and chemically skinned. Isometric tension (P _o) unloaded shortening velocity (V _o) and rate of tension rise (dP/dt) were determined. Each fibre was classified on the basis of its MHC isoform composition determined by SDS-PAGE. Fibres belonging to the same type showed identical contractile parameters regardless of the muscle of origin, except minor differences in P _o of the fast fibres and dP/dt of slow fibres. The results are in favour of the conclusion that fibre type composition, determined using MHC isoforms as markers, is the major determinant of the diversity of contractile properties among human muscle groups. Received: 26 December 1995 / Received after revision: 26 March 1996 / Accepted: 29 April 1996     

Forced expression and assembly of rat cardiac troponin T isoforms in cultured muscle and nonmuscle cells

Summary Cardiac troponin T (cTnT), a tropomyosin (TM)-binding subunit of the troponin complex, undergoes a developmentally regulated isoform switch from embryonic form to adult form in the rat heart. To investigate the in vivo assembly of cTnT isoforms, we transiently transfected cDNA clones of either rat cTnT isoform into nonmuscle CHO cells and chick embryo myogenic (CEM) cells. As determined by Western blotting, both isoforms can be expressed in CHO and CEM cells. The expressed proteins had the same mobility as native rat cTnT proteins on SDS polyacrylamide gels and were recognized by anti-TnT antibodies. Conventional and confocal microscopy of transfected cells, double-labelled with antibodies against cTnT and against TM, revealed that neither isoform appears to associate with the nonmuscle TM in CHO cells, although both are able to colocalize with muscle TM-containing microfilament bundles in the myogenic CEM cells. There was no appreciable cTnT isoform-related difference in association with TM, suggesting that the functional significance of isoform variability in rat cTnT does not correspond to an assembly advantage for the maturing cardiac thin filament. To help determine whether cTnT nonassembly in CHO environment is primarily due to the nonmuscle nature of the endogenous TM, or if it involves the absence of other factors specific to muscle, we have isolated several stably-transfected clones of skeletal ßTM-expressing CHO cells which incorporate this muscle TM onto stress fibres. When either isoform of cTnT was transiently expressed in these ßTM-CHO cells, the strictly filamentous ßTM staining pattern was no longer observed. Instead, ßTM codistributed with cTnT in brightly staining aggregates not associated with the intact stress fibres. This suggests that both isoforms of cTnT are interacting with the ßTM in the nonmuscle environment and that other muscle-specific proteins may indeed be required for stable assembly of cTnT onto microfilaments. It also suggests that the interaction between cTnT and muscle TM is stronger than that between muscle TM and nonmuscle microfilaments.     

Heterogeneous postnatal transitions in myosin heavy chain isoforms within the rabbit temporalis muscle

Postnatal changes in the fiber type composition and fiber cross-sectional area were investigated in the superficial (TEM1) and deep (TEM23) temporalis of male rabbits. It was hypothesized that, due to the transition from suckling to chewing during early postnatal development, the proportion of fast fiber types would decrease, while the proportion of fibers positive for myosin heavy chain (MyHC) cardiac alpha would increase, and that, due to the influence of testosterone during late postnatal development, the proportion of these alpha fibers would decrease again. Classification of the fibers types was performed by immunohistochemistry according to their MyHC content. The proportion of alpha fiber types significantly increased in both muscle portions from 2% and 8% for TEM1 and TEM23 at week 1 to 29% and 54% at week 8, respectively,. While in TEM1 the proportion of this fiber type did not change thereafter, it decreased again to 27% in TEM23 at week 20. The change for the fast fiber types was opposite to that of the alpha fiber types. Significantly more MyHC IIX fibers were found in TEM1 than in TEM23 in adult rabbits. In the first 8 weeks, the cross-sectional areas of all fibers increased. After this period, only MyHC cardiac alpha + I fibers continued to increase significantly. It was concluded that there are developmental differences in the myosin heavy chain transitions of the two portions of the temporalis muscle.     

Maximum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibres of rat skeletal muscle

Summary Myosin heavy chain composition of a large number (288) of single fibres from slow (soleus), and fast (superficial part of tibialis anterior, and plantaris) muscles of adult (3–5-month-old) Wistar rats was determined. A combination of SDS-PAGE and monoclonal antibodies against myosin heavy chains allowed to identify four myosin heavy chain isoforms (1, 2A, 2X, and 2B) and to detect myosin heavy chain coexistence. Four groups of fibres containing only one myosin heavy chain (1 myosin heavy chain, 2A myosin heavy chain, 2X myosin heavy chain, and 2B myosin heavy chain), and five groups containing more than one myosin heavy chain 1 and 2A myosin heavy chains, 2A and 2X myosin heavy chains, 2X and minor amounts of 2B (2X-2B fibres), 2B and minor amounts of 2X (2B-2X fibres), and 2A, 2X, and 2B myosin heavy chain were identified and their relative percentages were assessed. Coexistence of fast myosin heavy chain isoforms was found to be very frequent (50% of the fibres in plantaris, and 30% in tibialis anterior), whereas coexistence of slow and fast (2A) myosin heavy chain was very rare. Maximum shortening velocity (V₀) was determined using the slack-test procedure in a subset of 109 fast fibres from the above population. The values of V₀ formed a continuum extending from 2A to 2X to 2X-2B to 2B-2X to 2B fibres. 2A fibres had the lowest value of V₀ and 2B fibres the highest. Only the differences between 2A and 2B and 2A and 2B-2X fibres were statistically significant. Importantly, the variability of V₀ in fibres containing only one myosin heavy chain and in fibres containing a variable proportion of two myosin heavy chain isoforms was similar and, in some case (e.g. 2B fibres), such to encompass the whole range of variation of fast fibres shortening velocities. The results of this study demonstrate that the large variability in maximum shortening velocity of fast fibres is not due to myosin heavy chain coexistence, and therefore suggest that it cannot be explained on the basis of myosin heavy chain composition.     

Hindlimb suspension induces the expression of multiple myosin heavy chain isoforms in single fibres of the rat soleus muscle

The aim of our experiment was to test the hypothesis that the performance of maximal isometric exercise every 20 s would reduce the intermediate frequency force, i.e. the force that appears while stimulating the muscle at 15 and 20 Hz, and would produce less decrease the force at 10 and 50 Hz, while Pt would increase. Such changes in stimulated force should demonstrate the coexistence of potentiation, low frequency fatigue (LFF) and `post-contractile depression' (PCD). The quadriceps muscle of 14 healthy men (aged 19–37 years) was studied. The results have shown, that during isometric exercise of maximal intensity there was significant (P < 0.05) decrease in P15 and P20, increase in Pt, however, MVC and P10 and P50 was unchanged (P > 0.05). LFF manifested itself most significantly which is evident from decrease in P20/P50. During recovery after work there was significant increase in LFF and decrease in P50 which is indicative of the manifestation of PCD. Besides, there was significant (P < 0.05) decrease immediately after exercise in RTP20 and RTP50, while no changes in T50 and RT. There were no significant changes (P > 0.05) however, either in RTP20 and RTP50 or in T50 and RT 20 min after exercise if compared to the initial and immediately post-exercise values.     

Effect of artificial rearing on the contractile properties and myosin heavy chain isoforms of developing rat tongue musculature

This study's purpose was to examine the influence of an altered activity level, via artificial rearing, on the contractile properties, myosin heavy chain phenotypes (MHC), and muscle fiber sizes of the developing rat tongue retractor musculature. Artificially reared rat pups were fed through a gastric cannula, eliminating nutritive suckling from postnatal day 4 to postnatal day 14. Rat pups were observed immediately following artificial rearing (postnatal day 14) and after a 1-mo resumption of function (postnatal day 42). The contractile characteristics of the tongue retractor musculature were measured in response to stimulation of the hypoglossal nerve. At postnatal day 14, artificially reared rat pups demonstrated significantly longer twitch half-decay times, lower fusion frequencies, and a marked decrease in fatigue resistance. These contractile speed and fatigue characteristics were fully recovered following a 1-mo resumption of function. MHC phenotypes of the styloglossus muscle (a tongue retractor) were determined by gel electrophoresis. At postnatal day 14, artificial rearing had not altered the MHC phenotype or muscle fiber sizes of the styloglossus muscle. However, following a 1-mo resumption of function artificially reared rat pups demonstrated a small but significant increase in MHCIIa expression and decrease in MHCIIb expression compared with dam-reared rats. These results support artificial rearing as a useful model for altering the activity level of the tongue and suggest that normal suckling behavior is necessary for the normal postnatal development of the tongue retractor musculature. This may also be the case for premature infants necessarily fed artificially.