Muscle Myosin Heavy Chain Isoforms Research Articles

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways.

Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation and remission, and in controls. The expression kinetics of the SMMHC (+)insert was then assessed at multiple time points in two studies: first, in horses with heaves treated for a 1-year period with antigen avoidance alone, inhaled corticosteroids alone or both; second, in horses with heaves before and after a 30-day natural antigen exposure. Gene expression analysis was assessed by quantitative PCR and protein expression was confirmed by targeted mass spectrometry. The (+)insert SMMHC isoform was significantly increased in central and peripheral airways, but not in the trachea of heaves-affected horses in clinical exacerbation when compared horses with heaves in remission and controls. Both corticosteroid administration and antigen avoidance led to a significant reduction of the (+)insert expression in the airways. The (+)insert SMMHC isoform was not significantly increased in airways after 1 month of antigenic re-exposure. The (+)insert SMMHC expression is increased throughout the bronchial tree in horses with heaves and reversible by corticosteroids administration and antigen avoidance.

Electrophoretic separation of reptilian skeletal and cardiac muscle myosin heavy chain isoforms: dependence on gel format.

This report provides a comparison of multiple gel formats to study myosin heavy chain (MHC) isoforms that are expressed in reptilian skeletal and cardiac muscles of five turtle species, water monitor, and prehensile tailed skink. Three gel formats were tested. The results identify one format that is superior, for the overall extent of electrophoretic separation and for the assessment of the number of MHC isoforms in reptilian striated muscles. The same format was shown previously to separate MHC isoforms that are expressed in American alligator. The results also show that another gel format reveals the distinct electrophoretic mobility of MHC isoforms in atrial, ventricular, and jaw adductor samples, compared to those expressed in skeletal muscles in the limbs and elsewhere in the body. In addition, the results reveal that the electrophoretic mobility of specific MHC isoforms, relative to other isoforms, depends on the gel format, as shown previously for mammalian and avian species. The discovery of the expression of masticatory MHC, which is abundantly expressed in jaw adductors of members of Carnivora and several other vertebrate orders, in the homologous muscles of prehensile tailed skink, an herbivore, and the carnivorous water monitor, was made during the course of this study.

CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression

Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T-helper 2 (Th2) inflammatory response. CD4(+) T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4(+) T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen-stimulated CD4(+) T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co-incubation with antigen-stimulated CD4(+) T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4(+) T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co-incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper-responsiveness and asthma.

Chapter 12 - Chronic nitric oxide synthase inhibition does not impair upper airway muscle adaptation to chronic intermittent hypoxia in the rat

Nitric oxide (NO) is an important modulator of striated muscle function. Nitric oxide synthase (NOS) expression and activity is altered by hypoxia and NO is implicated in respiratory muscle remodeling following chronic sustained hypoxia. We sought to determine if NO is implicated in upper airway dilator muscle adaptation to chronic intermittent hypoxia (CIH). Thirty-two adult male Wistar rats (284±13, mean±SD) were exposed to alternating bouts of hypoxia (90 s; 5% O2 at the nadir) and normoxia (210 s; 21% O2) for 12 cycles per hour, 8h/day for 3 weeks. Sham animals were exposed to normoxia in parallel. Half of the animals in both groups received the nNOS inhibitor-L-NNA (2mM) in the drinking water throughout the study (N=8 for all groups). Sternohyoid (pharyngeal dilator) muscle contractile and endurance properties were determined ex vivo. Sternohyoid muscle myosin heavy chain (MHC) isoform composition and cross-sectional area was determined by fluorescence microscopy. Chronic nNOS blockade did not alter sternohyoid muscle peak force or force-frequency relationship in sham or CIH-treated animals. In contrast, chronic nNOS blockade significantly decreased sternohyoid muscle endurance with equivalent effects in sham and CIH-treated rats. Our results suggest that NO is an important modulator of sternohyoid muscle endurance. However, our data provide no evidence to suggest that NO is implicated in upper airway muscle adaptation to CIH.

Feed restriction delays developmental fast skeletal muscle myosin heavy chain isoforms in turkey poults selected for differential growth

Genetic selection has been very successful at significantly increasing BW and breast muscle proportion in commercial broiler and turkey strains. The mechanisms of breast muscle growth in poultry and the interactive effects of nutritional status and selection are not fully understood. The hypothesis underlying the current study is that feed restriction, simply as a vehicle for controlling early growth, would delay the temporal expression pattern of neonatal (nMyHC) and adult (aMyHC) fast skeletal muscle myosin heavy chain (MyHC) isoforms in the pectoralis major muscle of turkey poults. The poultry growth model used to evaluate this hypothesis consisted of a randombred control turkey line (RBC2) that represents commercial turkeys of the 1960s and a line developed from the RBC2 by selection for BW at 16 wk of age (F line). The F line has significantly heavier breast muscles than the RBC2 concomitant with increased BW, but the proportion of breast muscle relative to BW is similar. A quantitative indirect ELISA using fast skeletal MyHC isoform specific monoclonal antibodies revealed no significant line differences in the temporal expression of posthatch fast skeletal muscle MyHC in ad libitum fed poults. Feed restriction, however, altered the temporal expression patterns of nMyHC and aMyHC in both F line and RBC2 poults compared with the poults fed ad libitum.

Skeletal Muscle Myosin Heavy Chain Isoform Content in Relation to Gonadal Hormones and Anabolic-Catabolic Balance in Trained and Untrained Men

Gonadal hormones and anabolic-catabolic hormone balance have potent influence on skeletal muscle tissue, but little is known about their action with regard to myosin heavy chain (MHC) transformation in humans. We investigated the relationship between skeletal muscle MHC isoform content in the vastus lateralis muscle and basal testosterone (T) concentration in 3 groups of subjects: endurance trained (E), sprint/strength trained (S), and untrained (U) young men. We have also determined basal sex hormone-binding globulin and cortisol (C) concentrations in untrained subjects to examine the relationship between MHC composition and the anabolic-catabolic hormone balance. Moreover, basal free testosterone (fT) and bioavailable testosterone (bio-T) concentrations were calculated for this subgroup. Despite significant differences in MHC isoform content (69.4 ± 2.39%, 61.4 ± 8.04%, and 37.5 ± 13.80% of MHC-2 for groups S, U, and E, respectively, Kruskal-Wallis: H = 18.58, p < 0.001), the T concentration was similar in the three groups of subjects (18.84 ± 5.73 nmol·L(-1), 18.60 ± 5.73 nmol·L(-1), and 20.73 ± 4.06 nmol·L(-1) for U, E, and S groups, respectively, Kruskal-Wallis: H = 1.11, p > 0.5). We have also found that in the U group, type 2 MHC in the vastus lateralis muscle is positively correlated with basal fT:C ratio (r = 0.63, p = 0.01). It is concluded that the differences in the training history and training specificity can be distinguished with regard to the MHC composition but not with regard to the basal T concentration. Simultaneously, it has been shown that MHC isoform content in human vastus lateralis muscle may be related to basal anabolic-catabolic hormone balance, and this hypothesis needs further investigation.

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Adaptações neuromusculares ao treinamento de força e concorrente em homens idosos.

This paper aimed to review the results of studies on neuromuscular adaptations to strength training (ST) and concurrent training (CT) in elderly men. A literature search was conducted using PubMed, Scopus, and SciELO. The search was limited to studies published from 1980 to 2012. A total of 3,390 articles were retrieved. After reading their titles, 127 studies were further evaluated by reading their abstracts. This resulted in 92 papers that were read in full; 25 of these were selected and their results were described in the present review. Several studies showed that, in elderly subjects, ST can produce increases in muscle strength, power, activation and mass. ST-induced strength gain may be explained by neural and morphological adaptations. The main neural adaptations to ST included increased recruitment of motor units and increased motor unit firing rate. Morphological adaptations included increases in the physiological cross-sectional area (CSA) of the muscle, in muscle thickness, in muscle fiber pennation angle, and changes in muscle myosin heavy-chain isoforms, resulting in the conversion of muscle fiber from subtype IIx to IIa. The inclusion of moderate-to-high inten- sity (60-85% of maximum strength) ST in the routine of this population is recommended to improve neuromuscular function. CT can promote significant neuromuscular adaptations, but these gains may be of a lower magnitude than those obtained with ST. Although CT has an interference effect on neuromuscular adaptations, it also promotes improvement in cardiovascular function and is therefore the most frequently recommended intervention for health promotion in the elderly.

Transgenic Expression and Purification of Myosin Isoforms Using the Drosophila melanogaster Indirect Flight Muscle System

We report a novel method for the generation of extremely pure transgenic myosin to carry out crystallization screening for x‐ray crystallography. We are optimizing a novel method for the use of genetically engineered Drosophila melanogaster to express histidine‐tagged recombinant muscle myosin isoforms. This method relies on several facts. First, that the Drosophila genome contains only one gene encoding all skeletal muscle myosin heavy chain isoforms. Second, that wild‐type or mutagenized versions of the gene can then be inserted into a specific location in the Drosophila genome. Third, that the Mhc10 fly line has endogenous myosin knocked out in the indirect flight muscles. Fourth, that the Actin88F promoter facilitates high‐level expression of accessible myosin in the thoracic indirect flight muscles. Finally, that cost of fruit fly colony propagation is relatively low. Using these advantages we devised a method for expressing and purifying two isoforms of recombinant histidine‐tagged myosin from an engineered fly strain. The myosin shows ATPase activity and is of sufficient purity and homogeneity for crystallization. This technique may prove useful for the expression and isolation of mutant myosins linked with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization. Supported by NIH R01 GM032443 to SIB.

Nitric oxide synthase inhibition prevents activity‐induced calcineurin–NFATc1 signalling and fast‐to‐slow skeletal muscle fibre type conversions

The calcineurin–NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin–NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml(−1)) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day(−1)) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3β phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3β-induced facilitation of calcineurin–NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling.

Characteristics of Muscle Fibers in Rats with Limb Movements during Sleep after Spinal Cord Injury

Background/Aims: Previous studies have demonstrated that spinal cord injury (SCI) results in changes in sleep patterns through increased arousals and limb movements during sleep. Dramatic changes in muscle myosin heavy-chain isoforms have also been reported. The aim of this study was to investigate the characteristics of muscle fibers after SCI in rats with limb movements during sleep. Methods: Forty male Wistar rats were divided into four groups: SHAM, SCI 3, 7 and 15 days. Animals were subjected to electrode insertion surgery, 24-hour baseline sleep recording, SCI, and subsequent sleep recording for 3, 7, or 15 consecutive days. In addition, the gastrocnemius muscle and spinal cord were collected for histopathological/histochemical analyses. Results: Our results indicate a rapid and progressive decrease in the cross-sectional area of type I fibers in the gastrocnemius muscle (35.76–24.74 µm²) after SCI. Additionally, we found SCI-induced changes in sleep patterns. Following SCI, we also observed limb movements in sleeping rats, as well as significant negative moderate correlations between type I fibers and limb movement. Conclusion: Our study strengthened the hypothesis by correlation between changes in types of muscle fiber (decline in type I fibers) and an increase in limb movements during sleep after SCI.

Comparison of New ELISA Method With Established SDS-PAGE Method for Determination of Muscle Myosin Heavy Chain Isoforms

We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare analysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a "semi-quantitative" or "comparative" measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact nor faster compared to SDS-PAGE.

Effect of season on contractile and metabolic properties of desert camel muscle (Camelus dromedarius)

Thirty fattened one humped desert camels were used to examine the effect of season on contractile and metabolic properties of Longissimus thoracis (LT) muscle. Ten camels were slaughtered according to seasons of the year (winter, summer and autumn). Season significantly influenced muscle chemical composition, ultimate pH (pHu) and color. Activities of metabolic enzymes were higher during autumn season compared to summer and winter for phosphofructokinase (+64% compared to both seasons) and for isocitrate dehydrogenase (+35% and +145% in autumn vs. summer and winter, respectively). Quantification of muscle myosin heavy chain isoforms by SDS-PAGE electrophoresis showed only presence of type I and type IIa MyHC in camel muscle and indicated high proportion in winter for type I and in autumn for type IIa with respect to other seasons. Several correlations between different MyHC proportions and enzyme activities were reported. These findings indicated that muscle characteristics in camels are influenced by season.

Muscle fiber type characterization and myosin heavy chain (MyHC) isoform expression in Mediterranean buffaloes

This study aimed to evaluate myosin heavy chain (MyHC) isoform expression and muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) in Mediterranean buffaloes and possible fibers muscles modulation according to different slaughter weights. The presence of MyHC IIb isoforms was not found. Only three isoforms of MyHC (IIa, IIx/d and I) were observed and their percentages did not vary significantly among slaughter weights. The confirmation of the presence of hybrid muscles fibers (IIA/X) in LD and ST muscles necessitated classifying the fiber types into fast and slow according to their contractile activity, by m-ATPase assay. For both muscles, the muscle fiber frequency was higher for fast than for slow fibers in all weight groups. There was a difference (P<0.05) in the frequency of LD and ST muscle fiber types according to slaughter weights, which demonstrate that the slaughter weight influences the profile of muscle fibers from buffaloes.

SM2+/- male mice are predisposed to develop urinary tract obstruction and hyper contractility of the bladder smooth muscle upon ageing

We previously showed that complete loss of smooth muscle myosin heavy chain isoform 2 (SM2) resulted in postnatal lethality, but in het mice a partial loss of SM2 (SM2(+/-)) was accompanied by down-regulation of SM1 with unaltered SM2:SM1 ratio. To determine whether a normal bladder function would be maintained throughout its lifespan, we aged WT and SM2(+/-) mice up to 18 months and analyzed a) SM2:SM1 ratio b) bladder smooth muscle structure and c) function in SM2(+/-) het mice. A notable finding was that ~50% of 15-18 months old male SM2(+/-) mice exhibited urinary retention in bladder with the distention of upper urethra. In SM2(+/-) mouse bladder with urinary retention, the SM2:SM1 ratio was decreased but not in SM2(+/-) mouse bladder that did not develop urinary retention. Interestingly in the distended bladder the expression levels of α-actin and tropomyosin remained unaltered despite a reduction in the number of myosin thick filaments. These distended bladders showed hypersensitivity to submaximal K(+) depolarization and M3-receptor stimulation, without a significant increase in myosin light chain phosphorylation. We therefore suggest that a partial loss of SM2 may predispose male mice to develop lower urinary tract obstruction during ageing. In addition our data suggest that bladder obstruction can cause a further reduction in SM2 expression and SM2:SM1 ratio, and a hyper-contractility of the bladder smooth muscle.

Effects of Creatine Supplementation During Resistance Training on Myosin Heavy Chain (MHC) Expression in Rat Skeletal Muscle Fibers

The purpose of this study was to utilize a rodent model to test the hypothesis that creatine (Cr) supplementation during resistance training would influence the pattern of slow-twitch muscle myosin heavy chain (MHC) isoforms expression. Male Wistar rats (2-3 months old, 250-300 g) were divided into 4 groups: Nontrained without creatine supplementation (CO), nontrained with creatine supplementation (CR), trained without creatine supplementation (TR), and trained with creatine supplementation (TRCR). TR and TRCR groups were submitted to a resistance training program for 5 weeks (5 days/week) for morphological and biochemical analysis of the soleus muscle. Weightlifting exercise involved jump sessions into water, carrying progressive overload equivalent to percentage of body weight. CR and TRCR groups were given creatine at 0.5 g/kg(-1)/d(-1). Both Cr supplementation and resistance training alone or associated did not result in significant alterations (p > 0.05) in body weight gain, food intake, and muscle weight in the CR, TR and TRCR groups compared to the CO group. Also compared to the CO group, the CR group showed a significant (p < 0.02) increase in MHCI content and a reduction in MHCII; inversely, the TR group increased the MHCII content and reduced MHCI (p < 0.02). When combined, both creatine and resistance training did not promote significant (p > 0.05) changes in MHC content of the TRCR group compared to the CO group. The data show that Cr supplementation provides a potential action to abolish the exercise-induced MHC isoform transitions from slow to fast in slow-twitch muscle. Thus, Cr supplementation might be a suitable strategy to maintaining a slow phenotype in slow muscle during resistance training, which may be favorable to maintenance of muscle oxidative capacity of endurance athletes.

Stength, Endurance or Combined Training Elicit Diverse Skeletal Muscle Myosin Heavy Chain Isoform Proportion but Unaltered Androgen Receptor Concentration in Older Men

We investigated whether the myosin heavy chain (MyHC) proportion and androgen receptor (AR) concentration in skeletal muscle differ following 21 weeks of strength, endurance and combined training in untrained older men. Strength (S) and endurance (E) groups trained twice per week and combined (S+E) group trained four times per week (two strength and two endurance). Muscle biopsies were obtained before and after the training period from m. vastus lateralis (VL) and AR mRNA and protein concentration and MyHC proportion were determined. 1RM increased during the training period in S, S+E and E but the changes were greater in S and S+E than in E. Statistically significant increases were observed only in S and S+E in maximal isometric force as well as in VL thickness. VO (2max) increased significantly only in E. MyHCIIa proportion increased in S, while MyHCIIa proportion decreased and MyHCI increased (p<0.05) in E. No statistically significant changes were observed in serum testosterone and in AR mRNA or protein concentrations. The present results indicate that 21 weeks of strength, endurance or combined training changed MyHC proportion according to the training method but did not have an effect on AR mRNA or protein expression in skeletal muscle at rest.

Evolutionary changes in myosin isoform expression in the tongue by quantitative PCR

Tongue and cranial muscles experience unique mechanical demands, particularly in the complex speech of humans, and may require specialized contractile properties. Cranial muscles, derived from somitomeres, display uncommon myosin heavy chain (MHC) isoforms, including cardiac‐α (Ca) and extraocular (EOM). Tongue muscles derive from somites and do not appear to express these isoforms by protein analysis. mRNA analysis may reveal expression of Ca and EOM. We hypothesize that MHC isoform expression will reflect the functional demands on the tongue, including expression of Ca and EOM. Expression of 8 MHC isoforms in intrinsic muscles of the tongues of human, monkey, and rat were determined by QPCR. Rats express mostly 2x (67 ± 4%) with substantial 2b (31 ± 4%) and no slow MHC. Humans and monkeys express primarily conventional MHCs: 2x (20 ± 6% human, 28± 6% monkey), 2a (50 ± 7%, 58 ± 7%) and slow MHC (24 ± 6%, 10 ± 2%). The expression of faster MHC isoforms in rat than primate tongue is similar to limb MHC protein expression patterns. Of the uncommon isoforms, Ca constitutes 5 ± 3% of total MHC in humans, and individual monkey samples contained up to 7.5% EOM MHC. Neonatal and embryonic isoforms contribute less than 1% of MHC in all 3 species. We conclude that the properties of conventional MHC isoforms may not provide sufficient functional diversity for tongue action. Supported by DC05017

Deficiency of smooth muscle myosin heavy chain isoform 2 increases muscle contractility and causes premature postnatal death in mice

Deficiency of smooth muscle myosin heavy chain isoform 2 increases muscle contractility and causes premature postnatal death in mice

Myogenic Growth Factors Can Decrease Extraocular Muscle Force Generation: A Potential Biological Approach to the Treatment of Strabismus

Future pharmacologic treatment of strabismus may be optimized if drugs that are less potentially toxic to patients can be developed. Prior studies have shown that direct injection of extraocular muscles (EOMs) with insulin growth factor or fibroblast growth factor results in significant increases in the generation of EOM force. The purpose of this study was to examine the morphometric and physiological effects of direct EOM injection with the growth factors BMP4, TGFbeta1, Shh, and Wnt3A. One superior rectus muscle of normal adult rabbits was injected with BMP4, TGFbeta1, Shh, or Wnt3A. The contralateral muscle was injected with an equal volume of saline to serve as a control. After 1 week, the animals were euthanatized, and both superior rectus muscles were removed and assayed physiologically. The muscles were stimulated at increasing frequencies to determine force generation. A separate group of treated and control superior rectus muscles were examined histologically for alterations in total muscle cross-sectional area and myosin heavy chain isoform (MyHC) composition. One week after a single injection of BMP4, TGFbeta1, Shh, or Wnt3A, all treated muscles showed significant decreases in generation of force compared with control muscles. BMP4, TGFbeta1, Shh, and Wnt3A significantly decreased the mean myofiber cross-sectional area of fast MyHC-positive myofibers. BMP4 resulted in a conversion of fast-to-slow myofibers and a significant decrease in the percentage of developmental and neonatal MyHC-positive myofibers. Alterations in mean cross-sectional area and proportion of MyHCs were seen after injection with TGFbeta1, Shh, and Wnt3A. TGFbeta1 and BMP4 injections resulted in increased Pax7-positive satellite cells, whereas BMP4, TGFbeta1, and Wnt3A resulted in a decrease in MyoD-positive satellite cells. These results suggest that, rather than using toxins or immunotoxins, a more biological approach to decrease muscle strength is possible and demonstrate the potential utility of myogenic signaling factors for decreasing EOM strength. Ongoing drug-delivery studies will elucidate means of extending treatment effect to make such agents clinically useful.