Curcumin plays a local anti-inflammatory and antioxidant role via the HMGB1/TLR4/NF-ΚB pathway in rat masseter muscle under psychological stress.

BACKGROUND AND OBJECTIVE: Psychological stress causes structural and metabolic dysfunction of masseter muscles. The anti-inflammatory and anti-oxidative polyphenol curcumin plays a local antioxidant role in rat masseter muscles under psychological stress by an as-yet-unknown mechanism. The present study aimed to assess curcumin anti-inflammatory and anti-oxidative effects on masseter muscle and its possible molecular mechanisms. METHODS: We constructed a rat model of chronic unpredictable moderate stress (CUMS). Psychological stress was assessed by determining the levels of adrenocorticotropic hormone (ACTH) and cortisol in serum. Enzyme-linked immunosorbent assays measured inflammatory cytokines and markers of oxidative stress in masseter muscles. Levels of high-mobility group box 1 (HMGB1), interleukin (IL)-1ß, IL-6 and tumour necrosis factor-alpha (TNF-α) were determined using quantitative PCR analyses and immunofluorescent staining. Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) activation were examined using western blotting. RESULTS: The CUMS group showed increased serum cortisol and ACTH levels. Pathological changes in the ultrastructure, oxidative stress and inflammatory cytokines in the masseter muscles were also observed. Curcumin treatment (50, 100 mg/kg) ameliorated these changes significantly by varying degrees. Mechanistically, increased levels of phosphorylated NF-κB, toll-like receptor 4 and HMGB1 were observed, which were also ameliorated by curcumin treatment. CONCLUSION: Curcumin can reduce local pathological changes, levels of oxidative stress and inflammatory factors in masseter muscles. Psychological stress activates HMGB1 expression and increases the expression of downstream TLR4 and p-NF-κB, which could be reduced by curcumin. Thus, curcumin might exert anti-inflammatory and antioxidant effects in masseter muscles via the HMGB1/TLR4/NF-κB pathway.

Expression of myosin heavy chain isoform mRNA transcripts in the masseter and medial pterygoid muscles / Expresión de transcripciones de ARNm de isoforma de cadena pesada de miosina en los músculos masetero y pterigoideo medial

SUMMARY: Both the masseter and medial pterygoid muscles elevate the mandible, raising the lower jaw by acting simultaneously on the lateral and medial surfaces of the mandibular ramus. Nevertheless, electromyographic studies indicate that these muscles, as well as the superficial and deep heads of the masseter, act in a different way during mastication. We have analyzed by real time quantitative polymerase chain reaction (RT-qPCR) the expression of myosin heavy chain (MHC) isoforms in the masseter and medial pterygoid muscles in humans in order to identify possible differences in the expression patterns that may be related to functional differences identified with electromyography. Our findings indicate that the expression pattern of MHC isoforms in the two muscles is characteristic of fast and powerful phasic muscles. We have also observed a high percentage of expression of the MHC-IIx isoform and the expression of the MHC-M isoform at the mRNA level in both muscles, an isoform that does not translate into protein in the masticatory muscles of humans. The high percentage of expression of the MHC-IIx isoform in humans can be related to a high contractile speed of the masseter and medial pterygoid in humans. On the other hand, the low percentage of expression of the MHC-M isoform at the mRNA level in both muscles can be related to the complex evolutionary process that has reduced the size and force of the masticatory muscles in humans.

RESUMEN: Los músculos masetero y pterigoideo medial elevan la mandíbula actuando de forma simultánea sobre las caras lateral y medial de su rama. Sin embargo, los estudios electromiográficos indican que estos dos músculos actúan de forma diferente durante la masticación, de la misma forma que lo hacen las porciones superficial y profunda del músculo masetero. En el presente estudio hemos analizado mediante PCR en tiempo real la expresión de las isoformas de la cadena pesada de la miosina o myosin heavy chain (MHC) en los músculos masetero y pterigoideo medial en humanos, con la finalidad de identificar diferencias en los patrones de expresión que se puedan relacionar con las diferencias funcionales identificadas con la electromiografía. Nuestros resultados indican que el patrón de expresión de las isoformas de la MHC en los dos músculos es la característica de los músculos rápidos y potentes. También hemos observado un elevado porcentaje de expresión de la isoforma MHC-IIx y la expresión a nivel de ARNm de la isoforma MHC-M en los dos músculos, una isoforma que no se detecta a nivel de proteína en los músculos masticadores humanos. El elevado porcentaje de expresión de la isoforma MHC-IIx que hemos observado se puede relacionar con una elevada velocidad de contracción de los músculos masetero y pterigoideo medial en los humanos. Por otro lado, el bajo porcentaje de expresión de la isoforma MHC-M a nivel de ARNm en ambos músculos se puede relacionar con los procesos evolutivos complejos que han reducido el tamaño y la fuerza de los músculos masticadores en los humanos.

Trans-trigeminal transport of masseter-derived neprilysin to hippocampus.

OBJECTIVE: To show the possible occurrence of exosomal transport of neprilysin from masseter muscle to hippocampus via trigeminal nerve in the living mouse. DESIGN: Mouse C2C12 myotube-derived exosomes were labeled with near-infrared (NIR) dye and injected into the masseter muscle to track their fluorescence from masseter muscle to hippocampus via trigeminal nerve. A plasmid vector encoding green fluorescent protein (GFP)-tagged neprilysin (GFP-neprilysin) was transfected into masseter muscle of C57BL/6 J mice. Expression of mRNA and encoded protein of the transgene was identified in masseter muscle, trigeminal nerve and hippocampus by RT-PCR and Western blot, respectively. RESULTS: Peak of exosomal NIR in masseter muscle at time 0 rapidly reduced at 3 h and 6 h along with the subsequent increases in trigeminal nerve and hippocampus. Expression of GFP-neprilysin mRNA was detected in masseter muscle, but not trigeminal nerve and hippocampus. On the other hand, the corresponding protein of GFP-neprilysin was identified in the three tissues on day 3 after transfection into masseter muscle as a single band on Western blots with anti-GFP and anti-neprilysin antibodies. CONCLUSION: The appearance of GFP-neprilysin protein in trigeminal nerve and hippocampus without a corresponding mRNA expression indicated the protein's origin from the masseter muscle. Concomitant migration of NIR-exosomes from masseter muscle to hippocampus via trigeminal nerve suggested the possible occurrence of exosomal transport of neprilysin.

Mandibular lateral deviation induces alteration in vascular endothelial growth factor expression and oxidative stress/nitric oxide generation in rat condyle, synovial membrane and masseter muscle.

OBJECTIVE: We aimed to investigate alteration in cellular signaling mediated by vascular endothelial growth factor (VEGF) and parameters of oxidative stress/nitric oxide generation, superoxide dismutase (SOD) and neuronal nitric oxide synthase (nNOS), underlying altered functional mechanical loading of TMJ (temporomandibular joint) during lateral mandibular deviation. DESIGN: Thirty-eight 5-week-old male Wistar rats were divided into experimental group, which received acrylic resin appliance that shifted mandible to the left during closure, and control group. Computed tomography and histomorphometry were used for condyle analyses, while samples of condyle, synovial membrane and m. masseter were analyzed with enzyme-linked immunosorbent assay and spectrophotometry to determine VEGF and nNOS protein concentrations, and SOD activity. RESULTS: Experimental group of rats developed smaller and asymmetrical mandibles. Less of new bone and cartilage formation and larger bone marrow cavities area were found in the experimental group. Higher VEGF expression in condyle and m. masseter as well as higher nNOS expression in m. masseter and synovial membrane were found in the experimental compared to the control group. Alteration of SOD activity was found in m. masseter and synovial membrane in the experimental group. CONCLUSIONS: Lateral mandibular deviation induces mandibular and condylar morphological changes as well as significant cellular signaling alterations in condyle, synovial membrane and masticatory muscle. Cellular VEGF protein overexpression and oxidative stress/nitric oxide disbalance could be the mechanisms underlying unbalanced functional TMJ loading due to mandibular deviation.

Retrograde transport of masseter muscle-derived neprilysin to hippocampus.

Although the effects of neprilysin (NEP), also called CD10, on the clearance of Alzheimer's disease (AD)-associated amyloid-ß (Aß) have been reported, NEP is not made in the brain, and the mechanism for the transport of NEP to the brain has not been investigated. Our hypothesis is that muscle packages NEP in exosomes in response to a neuromuscular signal and sends it to the brain via retrograde axonal transport. The masseter muscle (MM) and the trigeminal nerve (TGN) are good candidates for this mechanism by virtue of their proximity to the brain. The aim of this study was to trace the NEP protein from the MM, through the TGN, and to the hippocampus (HPC) in muscle contraction models in vitro and in vivo. NEP expression in mouse tissue lysates was analyzed by RT-PCR and Western blot. Four-week-old mice were perfused to remove blood NEP contamination. The MM expressed substantial levels of NEP protein and mRNA. On the other hand, a remarkably high level of NEP protein was measured in the TGN in the absence of mRNA. NEP protein, without the corresponding mRNA, was also detected in the HPC. These results suggested that the MM derived NEP was taken up by the TGN, which in turn permitted NEP access to the central nervous system and within it the HPC. When the MM was induced to contract by electric stimulation in freshly euthanized mice, NEP protein decreased in the MM in a stimulus time-dependent manner, while that in the TGN and the HPC increased sequentially. Furthermore, NIR-labeled exosomes tracked along the same route. Finally, carbachol induced secretion of exosomal NEP in C2C12-derived myotube cells. These results support our hypothesis that MM-derived NEP is transported along the TGN to reach the HPC following electrical or cholinergic stimulation.

Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential.

Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2-5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1+ MMSCs as shown by expression of cardiac progenitor genes (including Nkx2-5), skeletal myogenic capacity is maintained in Sca1- MMSCs with Pax7 expression. Sca1+ MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement.

Ultrastructural changes in human masseter muscles after botulinum neurotoxin a injection.

INTRODUCTION: Botulinum neurotoxin A (BoNTA) has long been used as a therapeutic agent and has been widely accepted as a cosmetic agent in recent years. It can inhibit function and induce structural changes in skeletal muscle. METHODS: Specimens of fresh dissected human masseter muscle were used to observe the ultrastructural changes that occurred at 6 and 12 months following BoNTA injection. RESULTS: The findings observed were muscle fiber distortion, sarcomere shortening, mitochondrial vacuolar degeneration, glycogen accumulation, and H and M band disruption in the triad of tubules. At 12 months after injection, there was still evidence of degenerative changes in muscle ultrastructure, whereas most organelles exhibited a normal structure. DISCUSSION: Profound ultrastructural and organelle disfiguring changes were observed after BoNTA injection into human masseter muscle. Most changes were transient, however, and were resolved by 12 months after injection. Muscle Nerve 57: 96-99, 2018.

Tumor Necrosis Factor Alpha Signaling in Trigeminal Ganglion Contributes to Mechanical Hypersensitivity in Masseter Muscle During Temporomandibular Joint Inflammation.

AIMS: To determine the involvement of tumor necrosis factor alpha (TNFα) signaling in the trigeminal ganglion (TG) in the mechanical hypersensitivity of the masseter muscle during temporomandibular joint (TMJ) inflammation. METHODS: A total of 55 male Sprague-Dawley rats were used. Following injection of Complete Freund's Adjuvant into the TMJ, the mechanical sensitivities of the masseter muscle and the overlying facial skin were measured. Satellite glial cell (SGC) activation and TNFα expression in the TG were investigated immunohistochemically, and the effects of their inhibition on the mechanical hypersensitivity of the masseter muscle were also examined. Student t test or two-way repeated-measures analysis of variance followed by Bonferroni multiple comparisons test were used for statistical analyses. P < .05 was considered to reflect statistical significance. RESULTS: Mechanical allodynia in the masseter muscle was induced without any inflammatory cell infiltration in the muscle after TMJ inflammation. SGC activation and an increased number of TNFα-immunoreactive cells were induced in the TG following TMJ inflammation. Intra-TG administration of an inhibitor of SGC activity or of TNFα-neutralizing antibody depressed both the increased number of TG cells encircled by activated SGCs and the mechanical hypersensitivity of the masseter following TMJ inflammation. CONCLUSION: These findings suggest that persistent masseter hypersensitivity associated with TMJ inflammation was mediated by SGC-TG neuron interactions via TNFα signaling in the TG.

Influence of glutamate-evoked pain and sustained elevated muscle activity on blood oxygenation in the human masseter muscle.

This study aimed to investigate the effect of glutamate-evoked masseter muscle pain on intramuscular oxygenation during rest and sustained elevated muscle activity (SEMA). Seventeen healthy individuals participated in two sessions in which they were injected with glutamate and saline in random order. Each session was divided into three, 10-min periods. During the first (period 1) and the last (period 3) 10-min periods, participants performed five intercalated 1-min bouts of masseter SEMA with 1-min periods of 'rest'. At onset of the second 10-min period, glutamate (0.5 ml, 1 M; Ajinomoto, Tokyo, Japan) or isotonic saline (0.5 ml; 0.9%) was injected into the masseter muscle and the participants kept the muscle relaxed in a resting position for 10 min (period 2). The hemodynamic characteristics of the masseter muscle were recorded simultaneously during the experiment by a laser blood-oxygenation monitor. The results demonstrated that glutamate injections caused significant levels of self-reported pain in the masseter muscle; however, this nociceptive input did not have robust effects on intramuscular oxygenation during rest or SEMA tasks. Interestingly, these findings suggest an uncoupling between acute nociceptive activity and hemodynamic parameters in both resting and low-level active jaw muscles. Further studies are needed to explore the pathophysiological significance of blood-flow changes for persistent jaw-muscle pain conditions.

Changes in caveolin-1, caveolin-3 and vascular endothelial growth factor expression and protein content after botulinum toxin A injection in the right masseter muscle of dystrophin deficient (mdx-) mice.

Progressive muscle wasting, frequently associated with inflammation, muscle fibre degeneration and fibrosis, is a characteristic of DMD (Duchenne muscular dystrophy). Its most common used animal model, the mdx mouse, however can overcome muscle degeneration by regeneration processes and is for this reason not suitable to answer all scientific questions. The aim of this study was to evaluate the ability of botulinum toxin A (BTX-A) in breaking down muscle regeneration in mdx mice. For this purpose, the right masseter muscle of 100 days old mdx and healthy mice was paralyzed by a single specific intramuscular injection of BTX-A. After 21 days, right and left masseter and temporal muscles as well as tongue muscle were carefully dissected, and gene and protein expression of caveolin-1, caveolin-3 and vascular endothelial growth factor (VEGF) were determined using quantitative RT-PCR and Western blot technique. Statistics were performed using Student's t-test and Mann Whitney U-test (significance level: P ≤ 0.05). After BTX-A injection, in both mice strains and for all three studied genes, no significant differences in mRNA amount could be detected between treated and untreated masseter muscles. A significant increase in caveolin-1, caveolin-3 and VEGF mRNA expression could only be found in the right temporal muscle of control mice compared to the left side. All three investigated proteins were more frequent to be found in dystrophic masseter muscle samples compared to the corresponding control samples, whereas significant decreased caveolin-3 protein levels could only be detected in the treated masseter versus untreated masseter muscle of controls. In contrast to previous conclusions, with this study it was not possible to prove a BTX-A-induced dystrophic phenotype in control animals, in which only the known decreases of caveolin-3 protein expression could be verified due to denervation. At the same time, however, gene and protein expression in dystrophic mice was not changed after BTX-A injection.

JSA guideline for the management of malignant hyperthermia crisis 2016.

Malignant hyperthermia (MH) can be fatal if the crisis is not appropriately treated. It is an inherited disease usually triggered by the administration of volatile inhalational anesthetics and/or succinylcholine, a muscle relaxant. In a patient with suspected MH, the mechanism of calcium release from storage in the sarcoplasmic reticulum in the skeletal muscle is abnormally accelerated. Unexplained hypercarbia representing >55 mmHg of end-tidal carbon dioxide, tachycardia, and muscle rigidity (including masseter muscle rigidity) are early signs of the initiation of MH, because the metabolism is accelerated. The body temperature can rise by >0.5 °C/15 min and may reach ≥40 °C. Respiratory and metabolic acidosis, arrhythmia, cola-colored urine, increased levels of serum potassium, and tented T-waves on electrocardiogram are common and can lead to cardiac arrest. MH should be treated by discontinuation of the triggering agents, administration of intravenous dantrolene (initially 1 mg/kg), and reduction of the body temperature. Early diagnosis and sufficient dantrolene with body temperature reduction are essential to relieve the patient's MH crisis. This guideline in Japanese translation has been posted on the website: http://www.anesth.or.jp/guide/pdf/guideline_akuseikounetsu.pdf .

Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.

OBJECTIVES: To evaluate the cellular and matrix effects of botulinum toxin type A (Botox) on mandibular condylar cartilage (MCC) and subchondral bone. MATERIALS AND METHODS: Botox (0.3 unit) was injected into the right masseter of 5-week-old transgenic mice (Col10a1-RFPcherry) at day 1. Left side masseter was used as intra-animal control. The following bone labels were intraperitoneally injected: calcein at day 7, alizarin red at day 14 and calcein at day 21. In addition, EdU was injected 48 and 24 hours before sacrifice. Mice were sacrificed 30 days after Botox injection. Experimental and control side mandibles were dissected and examined by x-ray imaging and micro-CT. Subsequently, MCC along with the subchondral bone was sectioned and stained with tartrate resistant acid phosphatase (TRAP), EdU, TUNEL, alkaline phosphatase, toluidine blue and safranin O. In addition, we performed immunohistochemistry for pSMAD and VEGF. RESULTS: Bone volume fraction, tissue density and trabecular thickness were significantly decreased on the right side of the subchondral bone and mineralized cartilage (Botox was injected) when compared to the left side. There was no significant difference in the mandibular length and condylar head length; however, the condylar width was significantly decreased after Botox injection. Our histology showed decreased numbers of Col10a1 expressing cells, decreased cell proliferation and increased cell apoptosis in the subchondral bone and mandibular condylar cartilage, decreased TRAP activity and mineralization of Botox injected side cartilage and subchondral bone. Furthermore, we observed reduced proteoglycan and glycosaminoglycan distribution and decreased expression of pSMAD 1/5/8 and VEGF in the MCC of the Botox injected side in comparison to control side. CONCLUSION: Injection of Botox in masseter muscle leads to decreased mineralization and matrix deposition, reduced chondrocyte proliferation and differentiation and increased cell apoptosis in the MCC and subchondral bone.

Increased pain and muscle glutamate concentration after single ingestion of monosodium glutamate by myofascial temporomandibular disorders patients.

BACKGROUND: A randomized, double-blinded, placebo-controlled study was conducted to investigate if single monosodium glutamate (MSG) administration would elevate muscle/serum glutamate concentrations and affect muscle pain sensitivity in myofascial temporomandibular disorders (TMD) patients more than in healthy individuals. METHODS: Twelve myofascial TMD patients and 12 sex- and age-matched healthy controls participated in two sessions. Participants drank MSG (150 mg/kg) or NaCl (24 mg/kg; control) diluted in 400 mL of soda. The concentration of glutamate in the masseter muscle, blood plasma and saliva was determined before and after the ingestion of MSG or control. At baseline and every 15 min after the ingestion, pain intensity was scored on a 0-10 numeric rating scale. Pressure pain threshold, pressure pain tolerance (PPTol) and autonomic parameters were measured. All participants were asked to report adverse effects after the ingestion. RESULTS: In TMD, interstitial glutamate concentration was significantly greater after the MSG ingestion when compared with healthy controls. TMD reported a mean pain intensity of 2.8/10 at baseline, which significantly increased by 40% 30 min post MSG ingestion. At baseline, TMD showed lower PPTols in the masseter and trapezius, and higher diastolic blood pressure and heart rate than healthy controls. The MSG ingestion resulted in reports of headache by half of the TMD and healthy controls, respectively. CONCLUSION: These findings suggest that myofascial TMD patients may be particularly sensitive to the effects of ingested MSG. WHAT DOES THIS STUDY ADD?': Elevation of interstitial glutamate concentration in the masseter muscle caused by monosodium glutamate (MSG) ingestion was significantly greater in myofascial myofascial temporomandibular disorders (TMD) patients than healthy individuals. This elevation of interstitial glutamate concentration in the masseter muscle significantly increased the intensity of spontaneous pain in myofascial TMD patients.

Involvement of IL-1 in the Maintenance of Masseter Muscle Activity and Glucose Homeostasis.

Physical exercise reportedly stimulates IL-1 production within working skeletal muscles, but its physiological significance remains unknown due to the existence of two distinct IL-1 isoforms, IL-1α and IL-1ß. The regulatory complexities of these two isoforms, in terms of which cells in muscles produce them and their distinct/redundant biological actions, have yet to be elucidated. Taking advantage of our masticatory behavior (Restrained/Gnawing) model, we herein show that IL-1α/1ß-double-knockout (IL-1-KO) mice exhibit compromised masseter muscle (MM) activity which is at least partially attributable to abnormalities of glucose handling (rapid glycogen depletion along with impaired glucose uptake) and dysfunction of IL-6 upregulation in working MMs. In wild-type mice, masticatory behavior clearly increased IL-1ß mRNA expression but no incremental protein abundance was detectable in whole MM homogenates, whereas immunohistochemical staining analysis revealed that both IL-1α- and IL-1ß-immunopositive cells were recruited around blood vessels in the perimysium of MMs after masticatory behavior. In addition to the aforementioned phenotype of IL-1-KO mice, we found the IL-6 mRNA and protein levels in MMs after masticatory behavior to be significantly lower in IL-1-KO than in WT. Thus, our findings confirm that the locally-increased IL-1 elicited by masticatory behavior, although present small in amounts, contributes to supporting MM activity by maintaining normal glucose homeostasis in these muscles. Our data also underscore the importance of IL-1-mediated local interplay between autocrine myokines including IL-6 and paracrine cytokines in active skeletal muscles. This interplay is directly involved in MM performance and fatigability, perhaps mediated through maintaining muscular glucose homeostasis.

Functions of miR-1 and miR-133a during the postnatal development of masseter and gastrocnemius muscles.

The present study investigated the function of miR-1 and miR-133a during the postnatal development of mouse skeletal muscles. The amounts of miR-1 and miR-133a were measured in mouse masseter and gastrocnemius muscles between 1 and 12 weeks after birth with real-time polymerase chain reaction and those of HDACs, MEF2, MyoD family, MCK, SRF, and Cyclin D1 were measured at 2 and 12 weeks with Western blotting. In both the masseter and gastrocnemius muscles, the amount of miR-1 increased between 1 and 12 weeks, whereas the amount of HADC4 decreased between 2 and 12 weeks. In the masseter muscle, those of MEF2, MyoD, Myogenin, and MCK increased between 2 and 12 weeks, whereas, in the gastrocnemius muscle, only those of MRF4 and MCK increased. The extent of these changes in the masseter muscle was greater than that in the gastrocnemius muscle. The amounts of miR-133a, SRF, and Cyclin D1 did not change significantly in the masseter muscle between 1 and 12 weeks after birth. By contrast, in the gastrocnemius muscle, the amounts of miR-133a and Cyclin D1 increased, whereas that of SRF decreased. Our findings suggest that the regulatory pathway of miR-1 via HDAC4 and MEF2 plays a more prominent role during postnatal development in the masseter muscle than in the gastrocnemius muscle, whereas that of miR-133a via SRF plays a more prominent role in the gastrocnemius muscle than in the masseter muscle.

Expression of myostatin in early postnatal mouse masseter and rectus femoris muscles.

AIMS: Myostatin (Mstn) is a member of the transforming growth factor-ß (TGF-ß) family that inhibits muscle differentiation. In this study, we aimed to identify the relationships between Mstn, thyroid hormone receptor alpha (TRα), and myosin heavy chain (MyHC) isoform expression during early postnatal development. METHODS: We investigated the expression of Mstn, TRα, and MyHCs (embryonic, slow, IIa, IIb, and IIx) using quantitative real-time RT-PCR and ELISA (Mstn) in postnatal mouse muscles between day 0 and day 10. We also examined the correlations between Mstn, TR and MyHCs during the early development of mouse masseter muscle (MM) and rectus femoris muscle (RFM). RESULTS: Distinct Mstn mRNA expression patterns were observed in the two muscles despite nearly non-significant changes in the Mstn protein abundance in MM. The expression pattern of the TRα mRNA in the MM differed from that observed in the RFM. The expression of MyHC IIa, IIb and IIx mRNAs increased in the MM and decreased in the RFM from day 0 to day 10, whereas embryonic fiber MyHC mRNA expression was similar in both muscle types. Principal component analysis showed the existence of a correlation between: (1) TRα and MyHC, (2) Mstn and MyHC, and (3) TRα and Mstn in MM. The correlations were different in RFM and MM. Cluster analyses identified the distinct clusters: cluster 1, days 0-4 for the MM and day 0 for the RFM; cluster 2, day 6 for the MM and day 2 for the RFM; and cluster 3, days 8-10 for the MM and days 4-10 for the RFM. CONCLUSIONS: These data suggest that TRα influences MyHC expression in both muscle types. In addition, Mstn has a limited effect in the MM related to the expression of individual MyHCs, as opposed to its role in the RFM, at early postnatal developmental stages. TRα could be involved in regulating both the temporal expression of MyHCs and Mstn at the early postnatal stages in the MM and RFM.

Effects of experimental tooth clenching on pain and intramuscular release of 5-HT and glutamate in patients with myofascial TMD.

OBJECTIVES: It has been suggested that tooth clenching may be associated with local metabolic changes, and is a risk factor for myofascial temporomandibular disorders (M-TMD). This study investigated the effects of experimental tooth clenching on the levels of 5-HT, glutamate, pyruvate, and lactate, as well as on blood flow and pain intensity, in the masseter muscles of M-TMD patients. METHODS: Fifteen patients with M-TMD and 15 pain-free controls participated. Intramuscular microdialysis was performed to collect 5-HT, glutamate, pyruvate, and lactate and to assess blood flow. Two hours after the insertion of a microdialysis catheter, participants performed a 20-minute repetitive tooth clenching task (50% of maximal voluntary contraction). Pain intensity was measured throughout. RESULTS: A significant effect of group (P<0.01), but not of time, was observed on 5-HT levels and blood flow. No significant effects of time or group occurred on glutamate, pyruvate, or lactate levels. Time and group had significant main effects on pain intensity (P<0.05 and <0.001). No significant correlations were identified between: (1) 5-HT, glutamate, and pain intensity; or between (2) pyruvate, lactate, and blood flow. DISCUSSION: This experimental tooth clenching model increased jaw muscle pain levels in M-TMD patients and evoked low levels of jaw muscle pain in controls. M-TMD patients had significantly higher levels of 5-HT than controls and significantly lower blood flow. These 2 factors may facilitate the release of other algesic substances that may cause pain.

ACTN3 R577X genotypes associate with Class II and deepbite malocclusions.

INTRODUCTION: α-Actinins are myofibril anchor proteins that influence the contractile properties of skeletal muscles. ACTN2 is expressed in slow type I and fast type II fibers, whereas ACTN3 is expressed only in fast fibers. ACTN3 homozygosity for the 577X stop codon (ie, changing 577RR to 577XX, the R577X polymorphism) results in the absence of α-actinin-3 in about 18% of Europeans, diminishes fast contractile ability, enhances endurance performance, and reduces bone mass or bone mineral density. We have examined ACTN3 expression and genetic variation in the masseter muscle of orthognathic surgery patients to determine the genotype associations with malocclusion. METHODS: Clinical information, masseter muscle biopsies, and saliva samples were obtained from 60 subjects. Genotyping for ACTN3 single nucleotide polymorphisms, real-time polymerase chain reaction quantitation of muscle gene message, and muscle morphometric fiber type properties were compared to determine statistical differences between genotype and phenotype. RESULTS: Muscle mRNA expression level was significantly different for ACTN3 single nucleotide polymorphism genotypes (P <0.01). The frequency of ACTN3 genotypes was significantly different for the sagittal and vertical classifications of malocclusion, with the clearest association being elevated 577XX genotype in skeletal Class II malocclusion (P = 0.003). This genotype also resulted in significantly smaller diameters of fast type II fibers in masseter muscles (P = 0.002). CONCLUSION: ACTN3 577XX is overrepresented in subjects with skeletal Class II malocclusion, suggesting a biologic influence during bone growth. ACTN3 577XX is underrepresented in subjects with deepbite malocclusion, suggesting that muscle differences contribute to variations in vertical facial dimensions.

Nodal pathway genes are down-regulated in facial asymmetry.

PURPOSE: Facial asymmetry is a common comorbid condition in patients with jaw deformation malocclusion. Heritability of malocclusion is advancing rapidly, but very little is known regarding genetic contributions to asymmetry. This study identifies differences in expression of key asymmetry-producing genes that are down-regulated in patients with facial asymmetry. METHODS: Masseter muscle samples were collected during bilateral sagittal split osteotomy orthognathic surgery to correct skeletal-based malocclusion. Patients were classified as class II or III and open or deep bite malocclusion with or without facial asymmetry. Muscle samples were analyzed for gene expression differences on Affymetrix HT2.0 microarray global expression chips. RESULTS: Overall gene expression was different for asymmetric patients compared with other malocclusion classifications by principal component analysis (P < 0.05). We identified differences in the nodal signaling pathway, which promotes development of mesoderm and endoderm and left-right patterning during embryogenesis. Nodal and Lefty expression was 1.39- to 1.84-fold greater (P < 3.41 × 10), whereas integral membrane Nodal modulators Nomo1,2,3 were -5.63 to -5.81 (P < 3.05 × 10) less in asymmetry subjects. Fold differences among intracellular pathway members were negative in the range of -7.02 to -2.47 (P < 0.003). Finally Pitx2, an upstream effector of Nodal known to influence the size of type II skeletal muscle fibers was also significantly decreased in facial asymmetry (P < 0.05). CONCLUSIONS: When facial asymmetry is part of skeletal malocclusion, there are decreases in nodal signaling pathway genes in masseter muscle. This data suggest that the nodal signaling pathway is down-regulated to help promote development of asymmetry. Pitx2 expression differences also contributed to both skeletal and muscle development in this condition.

Involvement of trigeminal transition zone and laminated subnucleus caudalis in masseter muscle hypersensitivity associated with tooth inflammation.

A rat model of pulpitis/periapical periodontitis was used to study mechanisms underlying extraterritorial enhancement of masseter response associated with tooth inflammation. Periapical bone loss gradually increased and peaked at 6 weeks after complete Freund's adjuvant (CFA) application to the upper molar tooth pulp (M1). On day 3, the number of Fos-immunoreactive (IR) cells was significantly larger in M1 CFA rats compared with M1 vehicle (veh) rats in the trigeminal subnucleus interpolaris/caudalis transition zone (Vi/Vc). The number of Fos-IR cells was significantly larger in M1 CFA and masseter (Mass) capsaicin applied (M1 CFA/Mass cap) rats compared with M1 veh/Mass veh rats in the contralateral Vc and Vi/Vc. The number of phosphorylated extracellular signal-regulated kinase (pERK)-IR cells was significantly larger in M1 CFA/Mass cap and M1 veh/Mass cap rats compared to Mass-vehicle applied rats with M1 vehicle or CFA in the Vi/Vc. Pulpal CFA application caused significant increase in the number of Fos-IR cells in the Vi/Vc but not Vc on week 6. The number of pERK-IR cells was significantly lager in the rats with capsaicin application to the Mass compared to Mass-vehicle treated rats after pulpal CFA- or vehicle-application. However, capsaicin application to the Mass did not further affect the number of Fos-IR cells in the Vi/Vc in pulpal CFA-applied rats. The digastric electromyographic (d-EMG) activity after Mass-capsaicin application was significantly increased on day 3 and lasted longer at 6 weeks after pulpal CFA application, and these increase and duration were significantly attenuated by i.t. PD98059, a MEK1 inhibitor. These findings suggest that Vi/Vc and Vc neuronal excitation is involved in the facilitation of extraterritorial hyperalgesia for Mass primed with periapical periodontitis or acute pulpal-inflammation. Furthermore, phosphorylation of ERK in the Vi/Vc and Vc play pivotal roles in masseter hyperalgesia after pulpitis or periapical periodontitis.