Myosin Composition Research Articles

Influence of Chronic Lead Intoxication on Functional Characteristics and Isoform Composition of Left Ventricular Myosin in the Rat Heart

Influence of Chronic Lead Intoxication on Functional Characteristics and Isoform Composition of Left Ventricular Myosin in the Rat Heart

Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart

Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition.

Thermal acclimation to cold alters myosin content and contractile properties of rainbow smelt, Osmerus mordax, red muscle

Rainbow smelt (Osmerus mordax), a eurythermal fish, live in environments from −1.8 to 20°C, with some populations facing substantial annual variation in environmental temperature. These different temperature regimes pose distinct challenges to locomotion by smelt. Steady swimming performance, red muscle function and muscle myosin content were examined to assess the prediction that cold acclimation by smelt will lead to improved steady swimming performance and that any performance shift will be associated with changes in red muscle function and in its myosin heavy chain composition. Cold acclimated (4°C) smelt had a faster maximum steady swimming speed and swam with a higher tailbeat frequency than warm acclimated (10°C) smelt when tested at the same temperature (10°C). Muscle mechanics experiments demonstrated faster contractile properties in the cold acclimated fish when tested at 10°C. The red muscle of cold acclimated smelt had a shorter twitch times, a shorter relaxation times and a higher maximum shortening velocity. In addition, red muscle from cold acclimated fish displayed reduced thermal sensitivity to cold, maintaining higher force levels at 4°C compared to red muscle from warm acclimated fish. Immunohistochemistry suggests shifts in muscle myosin composition and a decrease in muscle cross-sectional area with cold acclimation. Dot blot analysis confirmed a shift in myosin content. Rainbow smelt do show a significant thermal acclimation response to cold. An examination of published values of maximum muscle shortening velocity in fishes suggests that smelt are particularly well suited to high levels of activity in very cold water.

Dietary HMB and β-alanine co-supplementation does not improve in situ muscle function in sedentary, aged male rats.

This study evaluated the effects of dietary β-hydroxy-β-methylbutyrate (HMB) combined with β-alanine (β-Ala) in sedentary, aged male rats. It has been suggested that dietary HMB or β-Ala supplementation may mitigate age-related declines in muscle strength and fatigue resistance. A total of 20 aged Sprague-Dawley rats were studied. At age 20 months, 10 rats were administered a control, purified diet and 10 rats were administered a purified diet supplemented with both HMB and β-Ala (HMB+β-Ala) for 8 weeks (approximately equivalent to 3 and 2.4 g per day human dose). We measured medial gastrocnemius (MG) size, force, fatigability, and myosin composition. We also evaluated an array of protein markers related to muscle mitochondria, protein synthesis and breakdown, and autophagy. HMB+β-Ala had no significant effects on body weight, MG mass, force or fatigability, myosin composition, or muscle quality. Compared with control rats, those fed HMB+β-Ala exhibited a reduced (41%, P = 0.039) expression of muscle RING-finger protein 1 (MURF1), a common marker of protein degradation. Muscle from rats fed HMB+β-Ala also exhibited a 45% reduction (P = 0.023) in p70s6K phosphorylation following fatiguing stimulation. These data suggest that HMB+β-Ala at the dose studied may reduce muscle protein breakdown by reducing MURF1 expression, but has minimal effects on muscle function in this model of uncomplicated aging. They do not, however, rule out potential benefits of HMB+β-Ala co-supplementation at other doses or durations of supplementation in combination with exercise or in situations where extreme muscle protein breakdown and loss of mass occur (e.g., bedrest, cachexia, failure-to-thrive).

Adaptability of the Immature Ocular Motor Control System: Unilateral IGF-1 Medial Rectus Treatment.

Unilateral treatment with sustained release IGF-1 to one medial rectus muscle in infant monkeys was performed to test the hypothesis that strabismus would develop as a result of changes in extraocular muscles during the critical period of development of binocularity. Sustained release IGF-1 pellets were implanted unilaterally on one medial rectus muscle in normal infant monkeys during the first 2 weeks of life. Eye position was monitored using standard photographic methods. After 3 months of treatment, myofiber and neuromuscular size, myosin composition, and innervation density were quantified in all rectus muscles and compared to those in age-matched controls. Sustained unilateral IGF-1 treatments resulted in strabismus for all treated subjects; 3 of the 4 subjects had a clinically significant strabismus of more than 10°. Both the treated medial rectus and the untreated ipsilateral antagonist lateral rectus muscles had significantly larger myofibers. No adaptation in myofiber size occurred in the contralateral functionally yoked lateral rectus or in myosin composition, neuromuscular junction size, or nerve density. Sustained unilateral IGF-1 treatment to extraocular muscles during the sensitive period of development of orthotropic eye alignment and binocularity was sufficient to disturb ocular motor development, resulting in strabismus in infant monkeys. This could be due to altering fusion of gaze during the early sensitive period. Serial measurements of eye alignment suggested the IGF-1-treated infants received insufficient coordinated binocular experience, preventing the establishment of normal eye alignment. Our results uniquely suggest that abnormal signaling by the extraocular muscles may be a cause of strabismus.

Origin, development, function, and evolution of cypriniform trophic novelties

Cypriniform fishes are characterized by a number of evolutionary trophic novelties. These feeding novelties have allowed cypriniforms to take full advantage of a number of unique trophic niches. One such feeding innovation, the palatal organ, is a complex muscular structure located on the pharyngeal roof of cypriniform fishes. The palatal organ is a dorsal mass of complexly arranged muscle fibers within the buccopharyngeal cavity innervated by the vagal lobe and covered by a taste bud coated epithelium. Here we use developmental data from zebrafish, functional data from carp and goldfish, and histological data and modeling approaches from assorted cypriniform species to address how the palatal organ developed and explain some of its functional evolution. Using zebrafish we first describe the origin and ontogenetic development of the palatal organ. Comparative data on histological structure and myosin composition of the palatal organ across a wide phylogenetic sampling of cypriniforms is then used to determine the variation in structure and potential function of the palatal organ across Cypriniformes. The general assumption has been that the function of the palatal organ is conserved across cypriniforms, and requires the careful sensory control made possible by a hypertrophied vagal lobe to function properly. Few have considered the possibility that the palatal organ may have become adapted for different functions during the course of cypriniform evolution. Its possible role in other feeding modes has been largely overlooked as it has been presumed that the palatal organ's only function is in sorting during benthic feeding events. Using electromyography and high‐speed video to examine palatal organ activity in the common carp and goldfish, muscle activation patterns demonstrate that the palatal organ is likely employed just prior to suction generation, during suction feeding behaviors, during prey processing events, and may even play a role in respiration during stressful conditions. Furthermore, the palatal organ shows behavioral modulation when different food types and positions are used. These data suggest that the palatal organ is a multi‐functional structure with a much wider range of functional repertoires than previously shown. While sorting during benthic feeding may have been the primitive function of the palatal organ, it has likely been secondarily adapted for increasing dietary breadth during the course of cypriniform evolution. This muscular pad has become secondarily modified for different forms of feeding but may even play a more generalized role in suction feeding and prey processing within cypriniforms.

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Acute effects of sex-specific sex hormones on heat shock proteins in fast muscle of male and female rats

Heat shock protein (HSP) expression and sex hormone levels have been shown to influence several aspects of skeletal muscle physiology (e.g., hypertrophy, resistance to oxidative stress), suggesting that sex hormone levels can effect HSP expression. This study evaluated the effects of differing levels of sex-specific sex hormones (i.e., testosterone in males and estrogen in females) on the expression of 4: HSP70, HSC70, HSP25, and αB-crystallin in the quadriceps muscles of male and female rats. Animals were assigned to 1 of 3 groups (n=5M and F/group). The first group (Ctl) consisted of typically cage-housed animals that served as controls. The second group (H) was gonadectomized and received either testosterone (males) or estradiol (females) via injection for 12 consecutive days. The third group (Gx) was gonadectomized and injected as above, but with vehicle only, rather than hormones. Significant sex by condition interactions (P<0.05 by two-way MANOVA) were found for all 4 proteins studied, except for HSP70, which exhibited a significant effect of condition only. The expression of all HSPs was greater (1.9-2.5-fold) in males vs. females in the Ctl group, except for HSP70, which was no different. Generally, gonadectomy appeared to have greater effects in males than females, but administration of the exogenous sex hormones tended to produce more robust relative changes in females than males. There were no differences in myosin composition in any of the groups, suggesting that changes in fiber type were not a factor in the differential protein expression. These data may have implications for sex-related differences in muscular responses to exercise, disuse, and injury.

Cardiac myosin heavy chain isoform exchange alters the phenotype of cTnT-related cardiomyopathies in mouse hearts

Familial hypertrophic cardiomyopathy, FHC, is a clinically heterogeneous, autosomal-dominant disease of the cardiac sarcomere leading to extensive remodeling at both the whole heart and molecular levels. The remodeling patterns are mutation-specific, a finding that extends to the level of single amino acid substitutions at the same peptide residue. Here we utilize two well-characterized transgenic FHC mouse models carrying independent amino acid substitutions in the TM-binding region of cardiac troponin T (cTnT) at residue 92. R92Q and R92L cTnT domains have mutation-specific average peptide conformation and dynamics sufficient to alter thin filament flexibility and cross-bridge formation and R92 mutant myocytes demonstrate mutation-specific temporal molecular remodeling of Ca2+ kinetics and impaired cardiac contractility and relaxation. To determine if a greater economy of contraction at the crossbridge level would rescue the mechanical defects caused by the R92 cTnT mutations, we replaced the endogenous murine α-myosin heavy chain (MyHC) with the β-MyHC isoform. While β-MyHC replacement rescued the systolic dysfunction in R92Q mice, it failed to rescue the defects in diastolic function common to FHC-associated R92 mutations. Surprisingly, a significant component of the whole heart and molecular contractile improvement in the R92Q mice was due to improvements in Ca2+ homeostasis including SR uptake, [Ca2+]i amplitude and phospholamban phosphorylation. Our data demonstrate that while genetically altering the myosin composition of the heart bearing a thin filament FHC mutation is sufficient to improve contractility, diastolic performance is refractory despite improved Ca2+ kinetics. These data reveal a previously unrecognized role for MyHC isoforms with respect to Ca2+ homeostasis in the setting of cardiomyopathic remodeling and demonstrate the overall dominance of the thin filament mutation in determining the degree of diastolic impairment at the myofilament level.

Age-related changes in contraction and relaxation of rat diaphragm

Age-related changes of physiological and biochemical properties were examined in the diaphragm muscle, which has particularly high activation compared to that of other skeletal muscles. The diaphragm from 10-week-, 50-week- and 100-week-old male Wistar rats were used to measure in vitro isometric contractile properties, sarcoplasmic reticulum (SR) Ca2+-ATPase activity, and myosin heavy chain (MHC) isoform composition. Although there were no significant differences in specific twitch tension of the diaphragm among the groups, there was significant reduction in specific tetanic tension in the 50-week to 100-week groups. The contraction time and 1/2 relaxation time of twitch contraction extended with aging, and significant differences were found between 10-week-old and 100-week-old diaphragms. Regarding the activity of SR Ca2+-ATPase, the pattern of age-related change was similar to that in the 1/2 relaxation time and there was a significant difference between 10-week-old and 100-week-old diaphragms. There was a significant increase in the relative composition of the MHC I isoform in 100-week-diaphragms compared to that in 10-week-old diaphragms and a concomitant decrease in the relative composition of fast myosin was noted. These findings demonstrated that older diaphragms have slower contraction and relaxation speeds, and these alterations were attributed to changes in SR Ca2+-ATPase activity and MHC isoform composition.

Stapedius muscle fibre characterization in the noise exposed and auditory deprived rat

In skeletal muscle, interventions that unload the muscle cause slow-to-fast myosin heavy chain (MHC) conversions, whereas fast-to-slow conversions are seen when the muscles are engaged in resistance training and endurance exercise. The stapedius muscle (SM) is reported to prevent cochlear damage by noise. This theory may be supported by showing comparable changes of muscle fibre composition when ears are exposed to longstanding noise (SM training). Comparable changes after sound deprivation (SM unloading) would suggest that the SM needs a certain degree of daily activity evoked by environmental sound to sustain its normal composition. We investigated the difference in myosin composition of SM fibres from rats exposed to noise, from auditory deprived rats and from rats exposed to low level ambient noise (control group). Consecutive complete SM cross-sections were processed by enzymehistochemistry to determine acid/alkali lability of myofibrillar adenosine triphosphatase (mATPase) and by immunohistochemistry using MHC antibodies. Fibres were assigned to mATPase type I, IIA, IIX or ‘Miscellaneous’ categories. Per mATPase category, the fibres were attributed to groups with specific MHC isoform compositions. Auditory deprivation lasting nine weeks was accomplished by closure of the external meatus at the age of three weeks. A slow-to-fast shift was seen in these rats when compared to the control group. The noise exposed group was exposed to 65–90 dB sound pressure level during a period lasting nine weeks from the age of three weeks onwards. A shift from an overwhelming presence of type mATPase IIX, as seen in the control group, to type mATPase IIA occurred in the noise exposed group. Also, more MHC IIA/IIX hybrid fibres were found in the mATPase IIX category. An adaptive response to the acoustic environment in the characteristics of the fibres of the SM, comparable to the response in skeletal muscles on unloading and training activity, can be ascertained. This supports the theory that the SM plays an active role in modulating external acoustic energy on entry to the cochlea. Our results are also in favour of another postulated function of the SM, the unmasking of high-frequency signals in low-frequency background noise.

Co-expression of myosin II regulatory light chain and the NMDAR1 subunit in neonatal and adult mouse brain

Movement of glutamate receptors in neurons likely involves direct and indirect association of receptor subunits with microtubule- and actin-based motor proteins. We have previously shown that myosin II regulatory light chain (RLC) binds directly to subunits of the NMDA-type glutamate receptor (NR), suggesting that NMDA receptors are closely associated with a myosin II motor complex. Using a polyclonal antibody predicted to recognize all RLC isoforms previously described in rodent brain, we report the expression of RLC and the NR1 subunit in cortex, hippocampus and cerebellum of postnatal day 0 (P0) and adult mouse. Although myosin RLC was not exclusively localized with NR1 by immunohistochemistry, co-staining was striking in the neuronal soma of deep cortical neurons and Purkinje neurons of the cerebellum which showed a punctate, perinuclear pattern of immunoreactivity. These neuronal populations were identified using a monoclonal antibody directed against a nuclear-specific, transcriptional repressor, chicken ovalbumin upstream promoter-transcription factor (COUP-TF)-interacting protein 2 (CTIP2). Co-expression of NR1 and a myosin II motor was validated using an isoform specific anti-nonmuscle myosin II-B heavy chain (NMHC II-B) antibody. Our findings support the idea that there is regional heterogeneity in the molecular composition of the NMDA receptor-associated cytoskeleton, and suggest that NR subunits may be associated with an actin-based, myosin II-B motor within the endomembrane system of some neuronal populations. Differential staining patterns observed with light and heavy chain antibodies, however, suggest that there is also heterogeneity in the composition of myosin II complexes in brain.

Quantitative Proteomics Profiling of Sarcomere Associated Proteins in Limb and Extraocular Muscle Allotypes

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

Stapedius muscle fiber characterization during postnatal development in the rat

The stapedius muscle (SM) is reported to prevent cochlear damage by noise. Functional demands are then the ability of fast contraction with long endurance. At the end of the third postnatal week, the middle ear of the rat is completely pneumatized and according to electrophysiological data, the auditory function starts to match the adult. We investigated the developmental changes in myosin composition of SM fibres using consecutive complete SM cross-sections (taken from rats on post natal day (PND) 7, 14, 16, 21, 28, 42 and 84) which were processed by enzymehistochemistry to determine acid/alkali lability of myofibrillar adenosine triphosphatase (mATPase) and by immunohistochemistry using myosin heavy chain (MHC) antibodies (mAb). Fibres were assigned to mATPase type I, IIA, IIB, IIX or ‘Miscellaneous’ categories. Per mATPase category, the fibres were attributed to groups with specific MHC isoform compositions. Neonatal MHC expression could not be documented with the mAb used. However, embryonal (Emb) MHC was expressed at PND 7, very little at PND 14; at later PND fibres did not show Emb MHC. In general, the mATPase-based classification did not show large alterations after PND 21. Expression of MHC IIB, which was present in almost 50% of the fibres at PND 7 and 14, diminished to 3% at PND 84. A decrease in number of fibres expressing more than one MHC isoform was found. These results show that the SM is a precociously developing muscle compared to limb muscles and even to the diaphragm. Moreover, it is shown that the expression of the adult MHC isoform phenotype coincides with the onset of auditory function in the third postnatal week.

Endurance Training: Volume-Dependent Adaptational Changes in Myosin

The purpose of this study was to find the effect of different endurance training volumes on the composition and turnover of myosin. Sixteen-week-old male rats of the Wistar strain were divided into three different volume-based training groups. Changes in myosin heavy chain (MyHC), myosin light chain (MyLC) isoforms' composition, their synthesis rate, as well as myosin binding C-protein synthesis rate, and muscle protein degradation rate were measured. In slow-twitch (ST) soleus (Sol) muscle MyHC I isoform relative content increased and MyHC IIa isoform decreased during excessive increase in the volume of endurance training (ET). In plantaris (Pla) muscle excessive increase in ET volume decreased MyHC I and IIb isoforms, and increased MyHC IIa and IId relative content. In extensor digitorum longus (EDL) muscle the relative content of MyHC IId isoform increased during ET, but excessive increase in training volume decreased it. In Pla muscle the relative content of MyLC 1 (slow) isoform decreased during ET, but excessive increase in ET volume decreased the relative content of MyLC 3 (fast) isoform in both fast-twitch (FT) muscles. Decrease in MyHC and myosin binding C-protein synthesis rate in Pla muscle had significant correlation with ET volume (r = - 0.537, p < 0.05 and r = - 0.727, p < 0.001 subsequently). MyHC I and IIb isoforms and MyLC 3 (fast) isoform in Pla muscle and MyHC IIb, IId and MyLC 3 (fast) isoforms in EDL muscle are the most sensitive to the increase in ET volume. Excessive increase in ET volume leads to a decrease in physical working capacity. The degradation of muscle protein increased during ET in all groups.

Isoenergetic Dietary Protein Restriction Decreases Myosin Heavy Chain IIx Fraction and Myosin Heavy Chain Production in Humans

The synthesis of muscle protein is restrained during dietary protein restriction. This is widely understood to vary quantitatively with the degree of nutritional deprivation, but there has been little discussion of qualitative changes in muscle protein deriving from dietary protein restriction. We studied 14 healthy subjects in a 2-sample study. Subjects were randomly assigned to a diet providing an ample, American-style protein intake (1.67 g. kg fat-free mass(-1). d(-1)) or a diet approximating the mean minimum adult protein requirement (0.71 g. kg fat-free mass(-1). d(-1)). We found that consumption of an isoenergetic diet at the mean adult minimum protein requirement for 4 wk produced an 81% lower fractional synthesis rate of myosin heavy chain (MHC) proteins in vastus lateralis muscle than did consumption of an ample protein diet (P = 0.05). Protein deprivation altered the skeletal muscle myosin composition such that the proportion of the total myosin content represented by fast-twitch MHC IIx was 51% lower than with ample intake (P = 0.013). The steady state content of MHC IIx messenger RNA (mRNA) did not differ in subjects consuming the minimum requirement of protein, suggesting that the reduced proportion of MHC IIx arises from posttranscriptional events. A 68% lower rate of 3-methylhistidine excretion with protein restriction (P < 0.01) suggests that myofibrillar protein degradation was lower. We conclude that dietary amino acid scarcity produces a change in myosin isoform distribution via posttranscriptional mechanisms. The relative contribution of inhibited myosin synthesis and inhibited degradation to the altered myosin isoform composition remains unknown. This has implications for the mechanisms by which amino acids govern muscle protein composition in vivo, and further exploration is required.

Staining of Human Thyroarytenoid Muscle with Myosin Antibodies Reveals Some Unique Extrafusal Fibers, but no Muscle Spindles

Summary: This study describes the myosin composition of extrafusal and intrafusal muscle fibers found in the human thyroarytenoid (TA) and sternohyoid (control) muscles. We sought to determine the presence of muscle spindles in the TA muscle, and to identify unusual extrafusal fiber types, using the commonly accepted approach of tissue stainng with myosin isoform specific antibodies. Extrafusal fibers are organized into motor units, which subsequently produce muscle movement, whereas intrafusal fibers compose muscle spindles, the primary stretch receptor that provides afferent (feed back) information to the nervous system for regulation of motor unit length and tonicity. Immunohistochemical identification of muscle spindles was con-firmed in sternohyoid, but not in TA samples; however, some extrafusal fibers contained tonic myosin. These results indicate that human TA muscle functions similar to some mammalian extraocular muscle, performing unloaded (non-weight bearing) contractions without afferent information from native muscle spindles.

Effects of thyroxine on myosin isoform expression and mechanical properties in guinea-pig smooth muscle.

Information on the effects of thyroid hormone on smooth muscle contractile protein expression and mechanical properties is sparse. We have addressed the following questions. (1) Can thyroxine hormone alter myosin isoform composition in smooth muscle? (2) Can a change in myosin isoform composition lead to altered mechanical properties in smooth muscle? (3) Are alterations, if occurring, equal in fast and slow smooth muscle types? Guinea-pigs were treated with thyroxine (T(4)) for 12 days. Control animals were given physiological saline solution. Maximal unloaded shortening velocity (V(max)) was measured in chemically skinned, maximally activated muscle preparations from the aorta and the taenia coli. V(max) increased following thyroxine treatment, by approximately 20 % in the taenia coli. In the aorta, no significant increase in V(max) could be detected. The sensitivity of isometric force to inorganic phosphate (P(i)) was increased in the taenia coli following thyroxine treatment. The expression of mRNA (determined with RT-PCR) for the myosin heavy chain with the seven amino acid insert increased by approximately 70 % in the aorta and about 25 % in the taenia coli following thyroxine treatment. Western blot analysis showed an increase in the inserted myosin heavy chain form in the taenia coli. Expression of mRNA for the myosin essential light chains and the corresponding proteins did not change significantly in either muscle type. No alterations in non-muscle myosin heavy chain isoforms could be detected after thyroxine treatment. In conclusion, thyroxine treatment alters the isoform composition of myosin in fast and slow smooth muscles in vivo. This change is sufficient to increase shortening velocity and sensitivity of isometric force to P(i) in the fast, but not in the slow, smooth muscle type.

Muscle LIM protein is upregulated in fast skeletal muscle during transition toward slower phenotypes.

Muscle LIM protein (MLP) is constitutively expressed in slow, but undetectable in fast, muscles of the rat. Here we show that MLP was upregulated at both the mRNA and protein levels under experimental conditions leading to transitions from fast to slower phenotypes. Chronic low-frequency stimulation and mechanical overloading by synergist removal both induced fast-to-slow shifts in myosin heavy chain (MHC) isoforms and expression of MLP in fast muscles. High amounts of MLP mRNA and protein were also present in fast muscles of the myotonic, hyperactive ADR mouse. Hypothyroidism evoked shifts in myosin composition toward slower isoforms and increased the MLP protein content of soleus (SOL) muscle but failed to induce MLP in fast muscles. Unweighting by hindlimb suspension elicited slow-to-fast transitions in MHC expression without altering MLP levels in SOL muscle. Hyperthyroidism shifted the MHC pattern toward faster isoforms but did not affect MLP content in SOL muscle. We conclude that alterations in MLP expression are associated with transitions from fast to slower phenotypes but not with slow-to-fast muscle fiber transitions.

Stapedius muscle fibre composition in the rat

The stapedius muscle (SM) is supposed to prevent cochlear damage by noise. Consequently functional demands are the ability of fast contraction with long endurance. This implies the presence of a large fraction of myosin type II fibres with an appreciable oxidative capacity. We determined the myosin composition of SM fibres using consecutive complete SM cross-sections (6 week old rats) which were processed by enzyme histochemistry (EHC) to determine acid/alkali lability of myofibrillar adenosine triphosphatase (mATPase) or by immunohistochemistry (IHC) using myosin heavy chain (MyHC) antibodies. Method accuracy was determined in co-processed extensor digitorum longus (EDL). Four hundred SM and 200 EDL fibres were assigned to mATPase type I, IIA, IIB, IIX or ‘miscellaneous’ (‘Misc’) categories. Per mATPase category the fibres were attributed to groups with specific MyHC composition. In the EDL, mATPase type I and IIB fibres expressed only MyHC I and IIB respectively, whereas about 10% of the type IIA and 40% of the type IIX fibres expressed more than one MyHC. Thus IHC detects amounts of myosin isoforms which are not detected by EHC. The mATPase IIX category criterion leaves the possibility that this category contains fibres with myosin type IIA and/or IIB in larger amounts. The criteria of the mATPase categories type I, IIA or IIB preclude assignment to these categories of fibres which also contain other myosin isoforms in larger amounts. Such fibres were classified in one of the mATPase ‘Misc’ categories. Thus in the EDL the capability of the EHC criteria to select ‘pure’ fibres in terms of myosin differs per mATPase category. None of the SM fibres were assigned to the mATPase type I or IIB categories, about 25% to the type IIA, 60% to type IIX and 15% (including most fibres which expressed MyHC I) to a ‘Misc’ category. All SM fibres expressed two or more MyHC isoforms, MyHC IIB occurring in all fibres and substantial amounts of MyHC IIA and/or IIX in most. These findings confirm the hypothesis that such fibres have the capacity to contract fast and have the better fatigue resistance.