mRNA expression of myosin heavy chain isoforms in the sphenomandibularis portion of the temporalis muscle / Expresión de ARNm de las isoformas de la cadena pesada de la miosina en la porción esfenomandibular del músculo temporal

SUMMARY: The main objective of this study was to analyze by real-time quantitative polymerase chain reaction (RT-qPCR) the expression patterns of the myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, MHC-IIx) in the sphenomandibularis portion of the temporalis muscle. We expected to find differences between the sphenomandibularis and the other portions of the temporalis that could be related to the functional characteristics of the sphenomandibularis identified by electromyography. We dissected the right temporalis muscle of ten adult human individuals (five men and five women). Samples of the anterior and posterior temporalis and of the sphenomandibularis portion were obtained from each dissected muscle. These samples were analyzed by RT-qPCR to determine the percentages of expression of the MHC-I, MHC-IIa and MHC-IIx isoforms. No significant differences were identified between the anterior and the posterior temporalis in the expression patterns of the MHC-I, MHC-IIa and MHC-IIx isoforms. However, there were significant differences between the sphenomandibularis and the anterior temporalis. Specifically, the sphenomandibularis portion had a higher percentage of expression of the MHC-I isoform (P=0.04) and a lower percentage of expression of the MHC-IIx isoform (P=0.003). The pattern of expression that we observed in the sphenomandibularis reflects a greater resistance to fatigue, a lower contraction speed, and a lower capacity of force generation in the sphenomandibularis compared to the anterior temporalis. These characteristics are consistent with electromyographic findings on the functional differences between these two portions.

RESUMEN: El principal objetivo de este estudio fue analizar mediante real-time quantitative polymerase chain reaction (RT-qPCR) los patrones de expresión de las isoformas de la cadena pesada de la miosina (MHC-I, MHC-IIa y MHC-IIx) en la porción esfenomandibular del músculo temporal. Se esperó encontrar diferencias entre el esfenomandibular y las otras porciones del músculo temporal que se pudieran relacionar con las características funcionales del esfenomandibular, identificadas mediante electromiografía. Para obtener estos resultados, se diseccionó el músculo temporal derecho en diez humanos adultos (cinco hombres y cinco mujeres) y se obtuvieron muestras de la porción anterior y posterior del músculo temporal y de su porción esfenomandibular. Estas muestras fueron analizadas mediante RT-qPCR para determinar los porcentajes de expresión de las isoformas MHC-I, MHC- IIa y MHC-IIx. No se identificaron diferencias significativas de los patrones de expresión entre la porción anterior y la porción posterior del músculo temporal, pero sí que se observaron diferencias significativas entre la porción anterior del músculo temporal y su porción esfenomandibular. Concretamente, la porción esfenomandibular presentó un mayor porcentaje de expresión de la isoforma MHC-I (P=0.04) y un menor porcentaje de expresión de la isoforma MHC-IIx (P=0.003). El patrón de expresión que hemos observado en la porción esfenomandibular del músculo temporal refleja una mayor resistencia a la fatiga, una velocidad de contracción más lenta y una menor capacidad de generar fuerza si se compara esta porción con la porción anterior del músclo temporal. Estas características son consistentes con las diferencias funcionales que presentan estas dos porciones, que han sido descritas mediante electromiografía.

Increasing the expression of microRNA-126-5p in the temporal muscle can promote angiogenesis in the chronically ischemic brains of rats subjected to two-vessel occlusion plus encephalo-myo-synangiosis.

BACKGROUND: miR-126-5p plays an important role in promoting endothelial cell (EC) proliferation. We thus explored whether miR-126-5p can promote EC proliferation and angiogenesis in chronically ischemic brains (CIBs). RESULTS: Improved revascularization in moyamoya patients was correlated with upregulated miR-126-5p expression in the TM and DM. In vitro experiments showed that miR-126-5p promoted EC proliferation through the PI3K/Akt pathway. CIBs from the agomir group exhibited significantly higher p-Akt, VEGF, CD31 and eNOS expression compared with the control CIBs. The ICBP and the RCF were significantly better in the agomir compared with the control group. CONCLUSION: Increasing miR-126-5p expression in the TM can promote EC proliferation and angiogenesis in CIBs of 2VO+EMS rats through the PI3K/Akt pathway. METHODS: We assessed the correlation between revascularization and miR-126-5p expression in the temporal muscle (TM) and dura mater (DM) of moyamoya patients. The effect of miR-126-5p on EC proliferation and downstream signaling pathways was explored in vitro. We established an animal model of two-vessel occlusion plus encephalo-myo-synangiosis (2VO+EMS), transfected the TM with miR-126-5p agomir/antagomir, compared the expression of miR-126-5p and relevant downstream cytokines in brain tissue among different groups, and investigated the improvement in cerebral blood perfusion (ICBP) and the recovery of cognitive function (RCF).

Proteomics and immunohistochemistry identify the expression of α-cardiac myosin heavy chain in the jaw-closing muscles of sooty mangabeys (order Primates).

OBJECTIVE: The jaw-closing muscles of humans and nonprimate mammals express alpha-cardiac fibers but MyHC α-cardiac has not been identified in the jaw adductors of nonhuman primates. We determined whether MyHC α-cardiac is expressed in the superficial masseter and temporalis muscles of the sooty mangabey (Cercocebus atys), an African Old World monkey that specializes on hard seeds. DESIGN: LC-MS/MS based proteomics was used to identify the presence of MyHC Iα. Immunohistochemistry was used to analyze the composition and distribution of fiber types in the superficial masseter and temporalis muscles of eight C. atys. Serial sections were stained against MyHC α-cardiac (MYH6), as well as MyHC-1 (NOQ7.5.4D), MyHC-2 (MY-32), and MyHC-M (2F4). RESULTS: Proteomics analysis identified the presence of Myosin-6 (MyHC α-cardiac) in both heart atrium and superficial masseter. MyHC α-cardiac was expressed in abundance in the superficial masseter and temporalis muscles of all eight individuals and hybrid fibers were common. CONCLUSIONS: The identification of MyHC α-cardiac in the jaw adductors of sooty mangabeys is a novel finding for nonhuman primates. The abundance of MyHC α-cardiac indicates a fatigue-resistant fiber population characterized by intermediate speed of contraction between pure MyHC-1 and MyHC-2 isoforms. We suggest that α-cardiac fibers may be advantageous to sooty mangabeys, whose feeding behavior includes frequent crushing of relatively large, hard seeds during the power stroke of ingestion. Additional studies comparing jaw-adductor fiber phenotype of hard-object feeding primates and other mammals are needed to explore this relationship further.

Expression of myosin heavy chain isoforms mRNA transcripts in the temporalis muscle of common chimpanzees (Pan troglodytes).

PURPOSE: The common chimpanzee (Pan troglodytes) is the primate that is phylogenetically most closely related to humans (Homo sapiens). In order to shed light on the anatomy and function of the temporalis muscle in the chimpanzee, we have analyzed the expression patterns of the mRNA transcripts of the myosin heavy chain (MyHC) isoforms in different parts of the muscle. BASIC PROCEDURES: We dissected the superficial, deep and sphenomandibularis portions of the temporalis muscle in five adult P. troglodytes and quantified the expression of the mRNA transcripts of the MyHC isoforms in each portion using real-time quantitative polymerase chain reaction. MAIN FINDINGS: We observed significant differences in the patterns of expression of the mRNA transcripts of the MyHC-IIM isoform between the sphenomandibularis portion and the anterior superficial temporalis (33.6% vs 47.0%; P=0.032) and between the sphenomandibularis portion and the anterior deep temporalis (33.6% vs 43.0; P=0.016). We also observed non-significant differences between the patterns of expression in the anterior and posterior superficial temporalis. PRINCIPAL CONCLUSIONS: The differential expression patterns of the mRNA transcripts of the MyHC isoforms in the temporalis muscle in P. troglodytes may be related to the functional differences that have been observed in electromyographic studies in other species of primates. Our findings can be applicable to the fields of comparative anatomy, evolutionary anatomy, and anthropology.

Regional variation in IIM myosin heavy chain expression in the temporalis muscle of female and male baboons (Papio anubis).

OBJECTIVE: The purpose of this study was to determine whether high amounts of fast/type II myosin heavy chain (MyHC) in the superficial as compared to the deep temporalis muscle of adult female and male baboons (Papio anubis) correlates with published data on muscle function during chewing. Electromyographic (EMG) data show a regional specialization in activation from low to high amplitude activity during hard/tough object chewing cycles in the baboon superficial temporalis.(48,49) A positive correlation between fast/type II MyHC amount and EMG activity will support the high occlusal force hypothesis. DESIGN: Deep anterior temporalis (DAT), superficial anterior temporalis (SAT), and superficial posterior temporalis (SPT) muscle samples were analyzed using SDS-PAGE gel electrophoresis to test the prediction that SAT and SPT will show high amounts of fast/type II MyHC compared to DAT. Serial muscle sections were incubated against NOQ7.5.4D and MY32 antibodies to determine the breadth of slow/type I versus fast/type II expression within each section. RESULTS: Type I and type IIM MyHCs comprise nearly 100% of the MyHCs in the temporalis muscle. IIM MyHC was the overwhelmingly predominant fast MyHC, though there was a small amount of type IIA MyHC (≤5%) in DAT in two individuals. SAT and SPT exhibited a fast/type II phenotype and contained large amounts of IIM MyHC whereas DAT exhibited a type I/type II (hybrid) phenotype and contained a significantly greater proportion of MyHC-I. MyHC-I expression in DAT was sexually dimorphic as it was more abundant in females. CONCLUSIONS: The link between the distribution of IIM MyHC and high relative EMG amplitudes in SAT and SPT during hard/tough object chewing cycles is evidence of regional specialization in fibre type to generate high occlusal forces during chewing. The high proportion of MyHC-I in DAT of females may be related to a high frequency of individual fibre recruitment in comparison to males.

Regulation of jaw-specific isoforms of myosin-binding protein-C and tropomyosin in regenerating cat temporalis muscle innervated by limb fast and slow motor nerves.

Cat jaw-closing muscles are a distinct muscle allotype characterized by the expression of masticatory-specific myofibrillar proteins. Transplantation studies showed that expression of masticatory myosin heavy chain (m-MyHC) is promoted by fast motor nerves, but suppressed by slow motor nerves. We investigated whether masticatory myosin-binding protein-C (m-MBP-C) and masticatory tropomyosin (m-Tm) are similarly regulated. Temporalis muscle strips were transplanted into limb muscle beds to allow innervation by fast or slow muscle nerve during regeneration. Regenerated muscles were examined postoperatively up to 168 days by peroxidase IHC using monoclonal antibodies to m-MyHC, m-MBP-C, and m-Tm. Regenerates in both muscle beds expressed fetal and slow MyHCs, m-MyHC, m-MBP-C, and m-Tm during the first 4 weeks. Longer-term regenerates innervated by fast nerve suppressed fetal and slow MyHCs, retaining m-MyHC, m-MBP-C, and m-Tm, whereas fibers innervated by slow nerve suppressed fetal MyHCs and the three masticatory-specific proteins, induced slow MyHC, and showed immunohistochemical characteristics of jaw-slow fibers. We concluded that expression of m-MBP-C and m-Tm is coregulated by m-MyHC and that neural impulses to limb slow muscle are capable of suppressing masticatory-specific proteins and to channel gene expression along the jaw-slow phenotype unique to jaw-closing muscle.

Effect of the use of dry (rigid) or wet (soft) temporal fascia graft on tympanoplasty.

OBJECTIVE: The aim of this study was to research the advantages and disadvantages of using a wet or dry temporalis fascia graft in myringoplasty surgery. STUDY DESIGN: Randomized prospective study. SETTING: Teaching and research hospital. METHODS: Dry graft was used in 210 cases, and wet graft was used in 174 cases in 384 patients who underwent tympanoplasty. OUTCOMES MEASURES: (1) Rate of primary closure of perforation, (2) rate of recurrent and residual perforation, (3) difference in hearing improvement between the two groups, (4) total operation time and graft placement time, and (5) histologic properties of wet and dry fascia grafts. RESULTS: The success rate was 91.4% (159 of 174) in the wet graft group and 88.6% (186 of 210) in the dry graft group. There were no differences between groups regarding recurrent disease, residual disease, and postoperative hearing results (p > .05). The mean operation time and graft placement time were shorter in group 2 (wet graft), and this difference was found to be statistically significant (p < .01). Histologically, the number of fibroblast nuclei was higher in group 2 (wet graft), and this was statistically significant (p < .01). CONCLUSION: The temporal fascia graft has a high success rate regardless of its use, either wet or dry. Using wet grafts can shorten the operation time and result in a high number of fibroblast nuclei histologically.

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.

Enhanced brain angiogenesis in chronic cerebral hypoperfusion after administration of plasmid human vascular endothelial growth factor in combination with indirect vasoreconstructive surgery.

OBJECT: Vascular endothelial growth factor (VEGF) is a secreted mitogen associated with angiogenesis. The conceptual basis for therapeutic angiogenesis after plasmid human VEGF gene (phVEGF) transfer has been established in patients presenting with limb ischemia and myocardial infarction. The authors hypothesized that overexpression of VEGF using a gene transfer method combined with indirect vasoreconstruction might induce effective brain angiogenesis in chronic cerebral hypoperfusion, leading to prevention of ischemic attacks. METHODS: A chronic cerebral hypoperfusion model induced by permanent ligation of both common carotid arteries in rats was used in this investigation. Seven days after induction of cerebral hypoperfusion, encephalomyosynangiosis (EMS) and phVEGF administration in the temporal muscle were performed. Fourteen days after treatment, the VEGF gene therapy group displayed numbers and areas of capillary vessels in temporal muscles that were 2.2 and 2.5 times greater, respectively, in comparison with the control group. In the brain, the number and area of capillary vessels in the group treated with the VEGF gene were 1.5 and 1.8 times greater, respectively, relative to the control group. CONCLUSIONS: In rat models of chronic cerebral hypoperfusion, administration of phVEGF combined with indirect vasoreconstructive surgery significantly increased capillary density in the brain. The authors' results indicate that administration of phVEGF may be an effective therapy in patients with chronic cerebral hypoperfusion, such as those with moyamoya disease.

Differential expression of myosin heavy-chain mRNA in muscles of mastication during functional advancement of the mandible in pigs.

Surgical and orthodontic treatment of retrognathia aims to improve orofacial function by adaptation and training of muscle capacity, which is connected with a change in muscle fibre-type proportions. The aim here was to analyse the proportion of myosin-heavy chain (MyHC) gene expression in type I (slow twitch/ST) and type IIb (fast twitch/FT) fibres during sagittal advancement of the mandible by reverse transcriptase-polymerase chain reaction (RT-PCR). The experiments were carried out on 10-week-old pigs (six test animals, six controls) over a 28-day period. Six pigs were fitted with acrylic bite blocks for sagittal advancement of the mandible. Tissue was taken from seven different regions of the masseter, temporal, medial pterygoid, and geniohyoid muscles. The 84 samples were used for histological fibre differentiation with ATPase staining and for isolation of total RNA. To measure the two MyHC isoforms, RT-PCR (in a single tube reaction with MyHC I, MyHC IIb, and GAPDH primers) was used. A significant increase was registered in the percentage of ST fibres and in mRNA from MyHC I in the anterior region of the masseter and in the posterior region of the temporal muscle of the treated animals. The proportion of ST fibres to FT fibres was increased by up to 12% after functional advancement of the mandible. The histological findings corresponded with the data for fibre mRNA generated by RT-PCR.

Elevated abeta42 in skeletal muscle of Alzheimer disease patients suggests peripheral alterations of AbetaPP metabolism.

The levels of amyloid-beta40 (Abeta40) and Abeta42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Abeta42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Abeta40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Abeta40 and muscle Abeta42 levels were correlated with each other (P < 0.001), 2) muscle Abeta40 levels were positively correlated with age (P = 0. 036), and 3) muscle Abeta42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Abeta peptide were discovered by mass spectrometry, revealing the presence of Abeta starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Abeta and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Abeta in the temporalis muscles of AD patients suggest that alterations in AbetaPP and Abeta metabolism may be manifested in peripheral tissues.

The histochemistry of reactive masticatory muscle hypertrophy.

There are two types of hypertrophy of the muscles of mastication in man: reactive hypertrophy, the more common form; and nonreactive enlargements-myositic, genetic, myopathic, and idiopathic. Reactive hypertrophy develops when the masticatory muscle workload is increased by local bone and dental disorders; such triggers are not powerful but act over long periods, thus demanding increased endurance. Exercise for endurance has a greater effect on the muscles of mastication than it has on the large muscles of the limbs; these react solely by stimulating the oxidative metabolism of type 1 fibers, whereas masticatory muscle reacts structurally by hypertrophy and progressive type 1 fiber predominance. Eventually enzyme instability of type 1 fibers and end stage atrophy of type 2 fibers may appear. Unexpectedly, lesions have also been found in control masticatory muscle, in particular type 2 fiber specific atrophy like that seen in long-standing acquired autoimmune myasthenia gravis. It is suggested that the adverse lesions in hypertrophied and control masticatory muscle are the consequence of post-activation fatigue.

Histochemical analysis of the masseter and temporalis muscles in macaca mulatta after mandibular advancement using rigid or nonrigid fixation.

This study compared the histochemical characteristics of the temporalis and masseter muscles in monkeys who underwent mandibular advancement with and without immobilization of the mandible. The results showed, when compared with controls, that the animals who underwent maxillomandibular fixation (MMF) following advancement of the mandible had significant atrophy in both temporalis and masseter muscles. In contrast, much less myoatrophy was noted in the animals who underwent rigid internal fixation and no MMF following advancement of the mandible.

Expression of fiber type specific proteins during ontogeny of canine temporalis muscle.

The canine masticatory muscles contain a unique adult fiber type composition and different contractile protein isoforms than do adult limb muscles. To determine when these characteristic proteins are expressed during development, samples from canine temporalis (masticatory) and pectineus (limb) muscles were compared between 55 days gestation and 60 days postpartum by histochemical, biochemical, and immunocytochemical analysis. At 55 days gestation and 3 days postpartum, both muscles contained identical histochemical type 2C fibers, native myosin isozymes, and myosin light and heavy chains. By 14 days postpartum, fiber-type expression in these muscles diverged, with resultant formation of type 1 and type 2M fibers in the temporalis muscle and type 1 and 2A fibers in the pectineus muscle. The distinctive myosin isoforms, light chains, and heavy chain of the temporalis muscle were also expressed 2 weeks postpartum. Based on the methods used in this study, we conclude that (1) the temporalis muscle develops from embryonic fibers that initially contain a myosin indistinguishable from embryonic limb muscle fibers, suggesting they have a common precursor, and (2) the myosin light chains and heavy chain unique to the temporalis muscle are initially expressed 2 weeks postpartum.

Influence of monoamine oxidase inhibitors and a dopamine uptake blocker on the distribution of 11C-N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 11C-MPTP, in the head of the rhesus monkey.

N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP, is a neurotoxic substance known to induce a parkinsonian syndrome in primates. The distribution of intravenously injected 11C-labelled MPTP (11C-MPTP) in the head of Rhesus monkeys was studied by means of positron emission tomography, PET. The influence of pretreatment with two monoamine oxidase (MAO) inhibitors, namely pargyline and clorgyline, and a dopamine uptake blocker, nomifensine, on the distribution was also evaluated. The 11C-radioactivity was taken up in all brain regions and maximum radioactivities were found 3-8 min after intravenous administration of MPTP. The 11C-MPTP-derived radioactivity showed a constant value throughout the study period in areas corresponding to the striatum and mesencephalon in monkeys not pretreated and in monkeys pretreated with clorgyline and with nomifensine. Pargyline pretreatment, however, resulted in consecutive elimination of 11C-MPTP-derived radioactivity from the different brain regions with half-lives of 40-60 min. The total radioactivity in blood was also higher after pargyline pretreatment indicating successful inhibition of metabolism. The eyes and temporal muscle each showed radioactivity values of the same order in all monkeys irrespective of pretreatment. The results support findings by other authors that MPTP was rapidly converted in the brain to a reactive metabolite which concentration remained constant in the brain during the PET study. Pargyline in the dosage used is known to be a non-selective MAO inhibitor and it prevented the metabolism of 11C-MPTP to the products retained in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Fiber type-specific autoantibodies in a dog with eosinophilic myositis.

Serum from a 2-year-old male Belgian sheepdog with eosinophilic myositis, which particularly affects the masticatory muscles, was tested for the presence of muscle-specific autoantibodies. Control type 2 temporalis muscle fibers were selectively stained following incubation with the patient's serum and staphylococcal protein A conjugated to horseradish peroxidase (SPA-HRPO). Likewise, type 2 fibers in the patient's temporalis muscle were selectively stained with SPA-HRPO. The same staining procedures applied to limb muscle did not result in fiber staining. Proteins isolated from the temporalis and triceps brachii muscles of a normal dog were separated under denaturing conditions by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto nitrocellulose paper and incubated with either sera from the patient, normal dogs, or neuromuscular disease controls. Subsequent incubation with peroxidase-conjugated goat anti-dog IgG demonstrated antibodies to at least four proteins of the temporalis muscle (myosin heavy chain and three unidentified proteins) when incubated with the patient's serum but not with the controls. Under all conditions, antibodies to the proteins of the triceps brachii were not detected. These findings establish the presence of autoantibodies to specific temporalis muscle proteins that may initiate the myonecrosis and inflammatory response as well as limit the distribution of the response.