Michael J. Horton

Specialized Cranial Muscles: How Different Are They from Limb and Abdominal Muscles?

Mammalian skeletal muscle fibers can be classified into functional types by the heavy chain (MyHC) and light chain (MyLC) isoforms of myosin (the primary motor protein) that they contain. Most human skeletal muscle contains fiber types and myosin isoforms I, IIA and IIX. Some highly specialized muscle fibers in human extraocular and jaw-closing muscles express either novel myosins or unusual combinations of isoforms of unknown functional significance. Extrinsic laryngeal muscles may express the extraocular MyHC isoform for rapid contraction and a tonic MyHC isoform for slow tonic contractions. In jaw-closing muscles, fiber phenotypes and myosin expression have been characterized as highly unusual. The jaw-closing muscles of most carnivores and primates have tissue-specific expression of the type IIM or ‘type II masticatory’ MyHC. Human jaw-closing muscles, however, do not contain IIM myosin. Rather, they express myosins typical of developing or cardiac muscle in addition to type I, IIA and IIX myosins, and many of their fibers are hybrids, expressing two or more isoforms. Fiber morphology is also unusual in that the type II fibers are mostly of smaller diameter than type I. By combining physiological and biochemical techniques it is possible to determine the maximum velocity of unloaded shortening (Vo) of an individual skeletal muscle fiber and subsequently determine the type and amount of myosin isoform. When analyzed, some laryngeal fibers shorten at much faster rates than type II fibers from limb and abdominal muscle. Yet some type I fibers in masseter show an opposite trend towards speeds 10-fold slower than type I fibers of limb muscle. These unusual shortening velocities are most probably regulated by MyHC isoforms in laryngeal fibers and by MyLC isoforms in masseter. For the jaw-closing muscles, this finding represents the first case in human muscle of physiological regulation of kinetics by light chains. Together, these results demonstrate that, compared to other skeletal muscles, cranial muscles have a wider repertoire of contractile protein expression and function. Molecular techniques for reverse transcription of mRNA and amplification by polymerase chain reaction have been applied to typing of single fibers isolated from limb muscles, successfully identifying pure type I, IIA and IIX and hybrid type I/IIA and IIA/IIX fibers. This demonstrates the potential for future studies of the regulation of gene expression in jaw-closing and laryngeal muscles, which have such a variety of complex fiber types fitting them for their roles in vivo.

Abundant expression of myosin heavy-chain IIB RNA in a subset of human masseter muscle fibres

Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is believed this is the first report of the substantial expression of IIB mRNA in man as demonstrated in a subset of masseter fibres, but rarely in limb muscle, and in only a few fibres of the external oblique. These findings provide further evidence for the complexity of myosin gene expression, especially in jaw-closing muscles.

UNLOADED SHORTENING VELOCITY AND MYOSIN HEAVY CHAIN VARIATIONS IN HUMAN LARYNGEAL MUSCLE FIBERS

Myosin description in human laryngeal muscles is incomplete, but evidence suggests the presence of type I, IIA, IIX, and tonic myosin heavy chain (MHC) fibers. This study describes the unloaded shortening velocity (V0) of chemically skinned laryngeal muscle fibers measured by the slack test method in relation to MHC content. Skeletal fibers from human laryngeal and limb muscle biopsy specimens were obtained for determination of V0, and subsequently, glycerol–sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to determine the MHC isoform content. The fibers from human limb muscle had shortening speeds similar to those in previous reports on human skeletal fibers. Type I, IIA, and IIX fibers of laryngeal muscle had shortening speeds similar to those of fibers from limb muscle, but laryngeal fibers with heterogeneous MHC expression had a wide range of shortening speeds, some being nearly twice as fast as limb fibers. In addition, MHC isoform bands from human extraocular muscle comigrated with some bands from laryngeal muscle — a finding suggesting that extraocular myosin may also be expressed.

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

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 staining 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 confirmed 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.

Muscle Fiber Type Composition and Effects of Vocal Fold Immobilization on the Two Compartments of the Human Posterior Cricoarytenoid: A Case Study of Four Patients

The human posterior cricoarytenoid (PCA) muscle is divided into two compartments, the vertical and horizontal bellies, which contain differences in their myosin heavy chain (MyHC) composition. Using immunohistochemical techniques on whole PCA samples, this study provides a more thorough description of the fiber type composition of entire bellies of the PCA. Four patients provided complete PCA samples containing both compartments of their right and left sides; two with unilaterally immobilized vocal folds. The horizontal belly had 80% slow (type I) fibers and 20% fast (type II) fibers. The vertical belly contained equal amounts of slow and fast fibers (approximately 55%:45%); clearly distinguishing between two compartments. Atrophy of muscle fibers and fiber type grouping were also present in both normal and affected subjects; providing no clear confirmation of the clinical findings of vocal fold immobilization. Further study of the PCA muscle from patients with unilaterally immobilized vocal folds is needed.

Maximum Shortening Velocity and Myosin Heavy-chain Isoform Expression in Human Masseter Muscle Fibers

While human masseter muscle is known to have unusual co-expression of myosin heavy-chain proteins, cellular kinetics of individual fibers has not yet been tested. Here we examine if myosin heavy-chain protein content is closely correlated to fiber-shortening speed, as previously reported in other human muscles, or if these proteins do not correlate well to shortening speeds, as has been demonstrated previously in rat muscle. Slack-test recordings of single, skinned human masseter fibers at 15°C revealed maximum shortening velocities generally slower and much more variable than those recorded in human limb muscle. The slowest fiber recorded had a maximum shortening velocity (V0) value of 0.027 muscle lengths ·s-1, several times slower than the slowest type I fibers previously measured in humans. By contrast, human limb muscle controls produced V0 measurements comparable with previously published results. Analysis by gel electrophoresis found 63% of masseter fibers to contain pure type I MyHC and the remainder to co-express mostly type I in various combinations with IIA and IIX isoforms. Vo in masseter fibers forms a continuum in which no clear relationship to MyHC isoform content is apparent.

Human Masseter Muscle Fiber Type Properties, Skeletal Malocclusions, and Muscle Growth Factor Expression

PURPOSE We identified masseter muscle fiber type property differences in subjects with dentofacial deformities. PATIENTS AND METHODS Samples of masseter muscle were collected from 139 young adults during mandibular osteotomy procedures to assess mean fiber areas and percent tissue occupancies for the 4 fiber types that comprise the muscle. Subjects were classified into 1 of 6 malocclusion groups based on the presence of a skeletal Class II or III sagittal dimension malocclusion and either a skeletal open, deep, or normal bite vertical dimension malocclusion. In a subpopulation, relative quantities of the muscle growth factors IGF-I and GDF-8 gene expression were quantified by real-time polymerase chain reaction. RESULTS Fiber properties were not different in the sagittal malocclusion groups, but were very different in the vertical malocclusion groups (P ≤ .0004). There were significant mean fiber area differences for type II (P ≤ .0004) and type neonatal-atrial (P = .001) fiber types and for fiber percent occupancy differences for both type I-II hybrid fibers and type II fibers (P ≤ .0004). Growth factor expression differed by gender for IGF-I (P = .02) and GDF-8 (P < .01). The ratio of IGF-I:GDF-8 expression associates with type I and II mean fiber areas. CONCLUSION Fiber type properties are very closely associated with variations in vertical growth of the face, with statistical significance for overall comparisons at P ≤ .0004. An increase in masseter muscle type II fiber mean fiber areas and percent tissue occupancies is inversely related to increases in vertical facial dimension.

Quantification of Myosin Heavy Chain RNA in Human Laryngeal Muscles: Differential Expression in the Vertical and Horizontal Posterior Cricoarytenoid and Thyroarytenoid

Background: Human laryngeal muscles are composed of fibers that express type I, IIA, and IIX myosin heavy chains (MyHC), but the presence and quantity of atypical myosins such as perinatal, extraocular, IIB, and α (cardiac) remain in question. These characteristics have been determined by biochemical or immunohistologic tissue sampling but with no complementary evidence of gene expression at the molecular level. The distribution of myosin, the main motor protein, in relation to structure‐function relationships in this specialized muscle group will be important for understanding laryngeal function in both health and disease.

Epigenetic influence of KAT6B and HDAC4 in the development of skeletal malocclusion.

INTRODUCTION Genetic influences on the development of malocclusion include heritable effects on both masticatory muscles and jaw skeletal morphology. Beyond genetic variations, however, the characteristics of muscle and bone are also influenced by epigenetic mechanisms that produce differences in gene expression. We studied 2 enzymes known to change gene expressions through histone modifications, chromatin-modifying histone acetyltransferase KAT6B and deacetylase HDAC4, to determine their associations with musculoskeletal variations in jaw deformation malocclusions. METHODS Samples of masseter muscle were obtained from subjects undergoing orthognathic surgery from 6 malocclusion classes based on skeletal sagittal and vertical dysplasia. The muscles were characterized for fiber type properties by immunohistochemistry, and their total RNA was isolated for gene expression studies by microarray analysis and quantitative real-time polymerase chain reaction. RESULTS Gene expressions for fast isoforms of myosins and contractile regulatory proteins and for KAT6B and HDAC4 were severalfold greater in masseter muscles from a patient with a deepbite compared with one with an open bite, and genes related to exercise and activity did not differ substantially. In the total population, expressions of HDAC4 (P = 0.03) and KAT6B (P = 0.004) were significantly greater in subjects with sagittal Class III than in Class II malocclusion, whereas HDAC4 tended to correlate negatively with slow myosin type I and positively with fast myosin gene, especially type IIX. CONCLUSIONS These data support other published reports of epigenetic regulation in the determination of skeletal muscle fiber phenotypes and bone growth. Further investigations are needed to elucidate how this regulatory model might apply to musculoskeletal development and malocclusion.

Molecular motor MYO1C, acetyltransferase KAT6B and osteogenetic transcription factor RUNX2 expression in human masseter muscle contributes to development of malocclusion

OBJECTIVE Type I myosins are molecular motors necessary for glucose transport in the cytoplasm and initiation of transcription in the nucleus. Two of these, MYO1H and MYO1C, are paralogs which may be important in the development of malocclusion. The objective of this study was to investigate their gene expression in the masseter muscle of malocclusion subjects. Two functionally related proteins known to contribute to malocclusion were also investigated: KAT6B (a chromatin remodelling epigenetic enzyme which is activated by MYO1C) and RUNX2 (a transcription factor regulating osteogenesis which is activated by KAT6B). DESIGN Masseter muscle samples and malocclusion classifications were obtained from orthognathic surgery subjects. Muscle was sectioned and immunostained to determine fibre type properties. RNA was isolated from the remaining sample to determine expression levels for the four genes by TaqMan(®) RT-PCR. Fibre type properties, gene expression quantities and malocclusion classification were compared. RESULTS There were very significant associations (P<0.0000001) between MYO1C and KAT6B expressions. There were also significant associations (P<0.005) between RUNX2 expression and masseter muscle type II fibre properties. Very few significant associations were identified between MYO1C and masseter muscle fibre type properties. CONCLUSIONS The relationship between MYO1C and KAT6B suggests that the two are interacting in chromatin remodelling for gene expression. This is the nuclear myosin1 (NM1) function of MYO1C. A surprising finding is the relationship between RUNX2 and type II masseter muscle fibres, since RUNX2 expression in mature muscle was previously unknown. Further investigations are necessary to elucidate the role of RUNX2 in adult masseter muscle.