The role of voltage-gated calcium channel α2δ-1 in the occurrence and development in myofascial orofacial pain.

Patients who suffer from myofascial orofacial pain could affect their quality of life deeply. The pathogenesis of pain is still unclear. Our objective was to assess Whether Voltage-gated calcium channel α2δ-1(Cavα2δ-1) is related to myofascial orofacial pain. Rats were divided into the masseter tendon ligation group and the sham group. Compared with the sham group, the mechanical pain threshold of the masseter tendon ligation group was reduced on the 4th, 7th, 10th and 14th day after operation(P < 0.05). On the 14th day after operation, Cavα2δ-1 mRNA expression levels in trigeminal ganglion (TG) and the trigeminal spinal subnucleus caudalis and C1-C2 spinal cervical dorsal horn (Vc/C2) of the masseter tendon ligation group were increased (PTG=0.021, PVc/C2=0.012). Rats were divided into three groups. On the 4th day after ligating the superficial tendon of the left masseter muscle of the rats, 10 ul Cavα2δ-1 antisense oligonucleotide, 10 ul Cavα2δ-1 mismatched oligonucleotides and 10 ul normal saline was separately injected into the left masseter muscle of rats in Cavα2δ-1 antisense oligonucleotide group, Cavα2δ-1 mismatched oligonucleotides group and normal saline control group twice a day for 4 days. The mechanical pain threshold of the Cavα2δ-1 antisense oligonucleotides group was higher than Cavα2δ-1 mismatched oligonucleotides group on the 7th and 10th day after operation (P < 0.01). After PC12 cells were treated with lipopolysaccharide, Cavα2δ-1 mRNA expression level increased (P < 0.001). Cavα2δ-1 may be involved in the occurrence and development in myofascial orofacial pain.

Effects of age and diet consistency on the expression of myosin heavy-chain isoforms on jaw-closing and jaw-opening muscles in a rat model.

BACKGROUND: Skeletal craniofacial morphology can be influenced by changes in masticatory muscle function, which may also change the functional profile of the muscles. OBJECTIVES: To investigate the effects of age and functional demands on the expression of Myosin Heavy-Chain (MyHC) isoforms in representative jaw-closing and jaw-opening muscles, namely the masseter and digastric muscles respectively. METHODS: Eighty-four male Wistar rats were divided into four age groups, namely an immature (n = 12; 4-week-old), early adult (n = 24; 16-week-old), adult (n = 24; 26-week-old) and mature adult (n = 24; 38-week-old) group. The three adult groups were divided into two subgroups each based on diet consistency; a control group fed a standard (hard) diet, and an experimental group fed a soft diet. Rats were sacrificed, and masseter and digastric muscles dissected. Real-time quantitative polymerase chain reaction was used to compare the mRNA transcripts of the MyHC isoforms-Myh7 (MyHC-I), Myh2 (MyHC-IIa), Myh4 (MyHC-IIb) and Myh1 (MyHC-IIx)-of deep masseter and digastric muscles. RESULTS: In the masseter muscle, hypofunction increases Myh1 (26, 38 weeks; p < .0001) but decreases Myh4 (26 weeks; p = .046) and Myh2 (26 weeks; p < .0001) expression in adult rats. In the digastric muscle, hypofunction increases Myh1 expression in the mature adult rats (38 weeks; p < .0001), while Myh2 expression decreases in adult rats (26 weeks; p = .021) as does Myh4 (26 weeks; p = .001). Myh7 expression is increased in the digastric muscle of mature adult rats subjected to hypofunction (38 weeks; p = <.0001), while it is very weakly expressed in the masseter. CONCLUSION: In jaw-opening and jaw-closing muscles, differences in myosin expression between hard- and soft-diet-fed rats become evident in adulthood, suggesting that long-term alteration of jaw function is associated with changes in the expression of MyHC isoforms and potential fibre remodelling. This may give insight into the role of function on masticatory muscles and the resultant craniofacial morphology.

Gene expression profiling of the masticatory muscle tendons and Achilles tendons under tensile strain in the Japanese macaque Macaca fuscata.

Both Achilles and masticatory muscle tendons are large load-bearing structures, and excessive mechanical loading leads to hypertrophic changes in these tendons. In the maxillofacial region, hyperplasia of the masticatory muscle tendons and aponeurosis affect muscle extensibility resulting in limited mouth opening. Although gene expression profiles of Achilles and patellar tendons under mechanical strain are well investigated in rodents, the gene expression profile of the masticatory muscle tendons remains unexplored. Herein, we examined the gene expression pattern of masticatory muscle tendons and compared it with that of Achilles tendons under tensile strain conditions in the Japanese macaque Macaca fuscata. Primary tenocytes isolated from the masticatory muscle tendons (temporal tendon and masseter aponeurosis) and Achilles tendons were mechanically loaded using the tensile force and gene expression was analyzed using the next-generation sequencing. In tendons exposed to tensile strain, we identified 1076 differentially expressed genes with a false discovery rate (FDR) < 10-10. To identify genes that are differentially expressed in temporal tendon and masseter aponeurosis, an FDR of < 10-10 was used, whereas the FDR for Achilles tendons was set at > 0.05. Results showed that 147 genes are differentially expressed between temporal tendons and masseter aponeurosis, out of which, 125 human orthologs were identified using the Ensemble database. Eight of these orthologs were related to tendons and among them the expression of the glycoprotein nmb and sphingosine kinase 1 was increased in temporal tendons and masseter aponeurosis following exposure to tensile strain. Moreover, the expression of tubulin beta 3 class III, which promotes cell cycle progression, and septin 9, which promotes cytoskeletal rearrangements, were decreased in stretched Achilles tendon cells and their expression was increased in stretched masseter aponeurosis and temporal tendon cells. In conclusion, cyclic strain differentially affects gene expression in Achilles tendons and tendons of the masticatory muscles.

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.

Age-related decrease of cholinergic parasympathetic reflex vasodilation in the rat masseter muscle.

Skeletal muscle hemodynamics, including that in jaw muscles, is an important in their functions and is modulated by aging. Marked blood flow increases mediated by parasympathetic vasodilation may be important for blood flow in the masseter muscle (MBF); however, the relationship between parasympathetic vasodilation and aging is unclear. We examined the effect of aging on parasympathetic vasodilation evoked by trigeminal afferent inputs and their mechanisms by investigating the MBF during stimulation of the lingual nerve (LN) in young and old urethane-anesthetized and vago-sympathectomized rats. Electrical stimulation of the central cut end of the LN elicited intensity- and frequency-dependent increases in MBF in young rats, while these increases were significantly reduced in old rats. Increases in the MBF evoked by LN stimulation in the young rats were greatly reduced by hexamethonium and atropine administration. Increases in MBF in young rats were produced by exogenous acetylcholine in a dose-dependent manner, whereas acetylcholine did not influence the MBF in old rats. Significant levels of muscarinic acetylcholine receptor type 1 (MR1) and type 3 (MR3) mRNA were observed in the masseter muscle in young rats, but not in old rats. Our results indicate that cholinergic parasympathetic reflex vasodilation evoked by trigeminal afferent inputs to the masseter muscle is reduced by aging and that this reduction may be mediated by suppression of the expression of MR1 and MR3 in the masseter muscle with age.

Quantification of the Masseter Muscle Hardness of Stroke Patients Using the MyotonPRO Apparatus: Intra- and Inter-Rater Reliability and Its Correlation with Masticatory Performance.

BACKGROUND Chewing dysfunction is one of the most common serious complications after a stroke. It may be influenced by the hardness of the masseter muscle and masticatory performance; however, the association between these 2 factors is not explicit. Thus, it is meaningful to explore the functional status of the masseter muscle among stroke patients. The main objectives of this study were to examine the intra- and inter-rater reliability of the MyotonPRO apparatus in measuring masseter muscle hardness in stroke patients and to investigate the correlation between the bilateral masseter muscle hardness and masticatory performance in these patients. MATERIAL AND METHODS A total of 20 stroke patients participated in our study. The hardness of the masseter muscle was measured by 2 physiotherapists using the MyotonPRO apparatus. Overall, each patient masticated 2 pieces of red-blue bicolor chewing gum for 20 chewing cycles each. The chewing pieces were analyzed using ViewGum software for masticatory performance. RESULTS The intra- and inter-rater reliability of the MyotonPRO apparatus for measuring bilateral masseter hardness of stroke patients was excellent. The correlation analysis showed that the hardness index of the masseter muscle on the affected side was moderately correlated with the masticatory performance of the same side. CONCLUSIONS The MyotonPRO device can be used for measuring the masseter muscle hardness of stroke patients, with excellent reliability. This study established the construct validity between the stiffness of the masseter muscle and masticatory performance.

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.

Interference with SRF expression in skeletal muscles reduces peripheral nerve regeneration in mice.

Traumatic injury of peripheral nerves typically also damages nerve surrounding tissue including muscles. Hence, molecular and cellular interactions of neighboring damaged tissues might be decisive for successful axonal regeneration of injured nerves. So far, the contribution of muscles and muscle-derived molecules to peripheral nerve regeneration has only poorly been studied. Herein, we conditionally ablated SRF (serum response factor), an important myofiber transcription factor, in skeletal muscles of mice. Subsequently, the impact of this myofiber-restricted SRF deletion on peripheral nerve regeneration, i.e. facial nerve injury was analyzed. Quantification of facial nerve regeneration by retrograde tracer transport, inspection of neuromuscular junctions (NMJs) and recovery of whisker movement revealed reduced axonal regeneration upon muscle specific Srf deletion. In contrast, responses in brainstem facial motor neuron cell bodies such as regeneration-associated gene (RAG) induction of Atf3, synaptic stripping and neuroinflammation were not overly affected by SRF deficiency. Mechanistically, SRF in myofibers appears to stimulate nerve regeneration through regulation of muscular satellite cell (SC) proliferation. In summary, our data suggest a role of muscle cells and SRF expression within muscles for regeneration of injured peripheral nerves.

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.

The ultrastructural anatomy of the nuclear envelope in the masseter muscle indicates its role in the metabolism of the intracellular Ca+.

Specific ultrastructural anatomy of masticatory muscles is commonly referred to a general pattern assigned to striated muscles. Junctional feet consisting of calcium channels of the sarcoplasmic reticulum (i.e. the ryanodine receptors, RyRs) physically connected to the calcium channels of the t-tubules build triads within striated muscles. Functional RyRs were demonstrated in the nuclear envelopes of pancreas and of a skeletal muscle derived cell line, but not in muscle in situ. It was hypothesized that ryanodine receptors (RyRs) could also exist in the nuclear envelope in the masseter muscle, thus aiming at studying this by transmission electron microscopy. There were identified paired and consistent subsarcolemmal clusters of mitochondria, appearing as outpockets of the muscle fibers, usually flanking an endomysial microvessel. It was observed on grazing longitudinal cuts that the I-band-limited mitochondria were not strictly located in a single intermyofibrillar space but continued transversally over the I-band to the next intermyofibrillar space. It appeared that the I-band-limited transverse mitochondria participate with the column-forming mitochondria in building a rather incomplete mitochondrial reticulum of the masseter muscle. Subsarcolemmal nuclei presented nuclear envelope-associated RyRs. Moreover, t-tubules were contacting the nuclear envelope and they were seemingly filled from the perinuclear space. This could suggest that nucleoplasmic calcium could contribute to balance the cytosolic concentration via pre-built anatomical routes: (i) indirectly, via the RyRs of the nuclear envelope and (ii) directly via the communication of t-tubules and sarcoplasmic reticulum through the perinuclear space.

Serotonin1B receptor-mediated presynaptic inhibition of proprioceptive sensory inputs to jaw-closing motoneurons.

The proprioceptive sensory inputs from neurons in the mesencephalic trigeminal nucleus (MesV) to masseter motoneurons (MMNs) play an important role in regulating masseter muscle activity during mastication. Several histological studies have shown that serotonin (5-HT) fibers densely innervate both the MesV and the trigeminal motor nucleus. However, the functional roles of 5-HT in the regulation of the excitatory synaptic inputs from MesV afferents to MMNs remain to be clarified. Thus, using the whole-cell recording technique in brainstem slice preparations from juvenile Wistar rats aged between postnatal days 8 and 12, we examined the effects of 5-HT on the excitatory synaptic inputs from MesV afferents to MMNs. Bath application of 5-HT reduced the peak amplitude of excitatory postsynaptic potentials evoked in MMNs by electrical stimulation of the MesV afferents (eEPSPs), and this inhibitory effect of 5-HT on eEPSPs was replicated with the 5-HT1B receptor agonist CP-93129 but not by the 5-HT1A receptor agonist 8-OH-DPAT. Moreover, the 5-HT1B receptor antagonist SB-224289 but not the 5-HT1A receptor antagonist WAY-100635 antagonized the inhibitory effect of 5-HT on eEPSPs. CP-93129 increased the paired-pulse ratio and decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs), while it did not alter the mEPSC amplitude. These results suggest that presynaptic 5-HT1B receptors are involved in the inhibition of the excitatory synaptic inputs from MesV afferents to MMNs. Such inhibition may regulate MesV afferent activity during mastication.

Genioglossal response to mechanical vibrations of the mandible and the submandibular muscles.

The extrinsic tongue muscles are activated in coordination with pharyngeal muscles to dilate the airways as needed during breathing. The genioglossus (GG) activity is known to be modulated by several reflexes evoked via the mechanoreceptors of the upper airways. The primary objective of this paper was to investigate the effectiveness of activating these reflex pathways using mechanical stimulation of the mandible or the submandibular muscles. In eight healthy subjects, 3-s long, 5-mm vertical mechanical vibrations were delivered at 8 and 12 Hz to the lower jaw in a seated position, while the GG EMG was recorded using a custom-made sublingual electrode, along with the activity of the masseter (MS) and mylohyoid (MH). All three muscle activities were significantly higher during stimulation compared with the baseline (P < 0.02), and the increase was larger at 12 Hz versus 8 Hz (P < 0.02). All three muscle responses had components that synchronized with the mechanical stimuli, but those of MS were much more strongly phase-locked to the vibrational cycle. In 10 healthy subjects, we also applied mechanical vibrations to the submandibular muscles at three different stimulation intensities, while subjects were lying in a supine position. The GG activity increased significantly above the baseline (P = 0.026) in 9 out of 10 subjects, and the elevated activity persisted after termination of the stimulus for a few seconds. The results demonstrate that GG muscle responses can be evoked with mechanical vibrations applied to the lower jaw or the submandibular muscles in healthy subjects during wakefulness. NEW & NOTEWORTHY The evoked responses observed in the genioglossus (GG) activity during mechanical vibrations of the lower jaw or the submandibular muscles may lead to therapeutic applications for improving the patency of airways during sleep. The presence of these GG reflexes may also explain a mechanism by which the vibrations produced during snoring can help the airways stay open in individuals who may otherwise have obstructed airways in sleep.

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.

[Mitochondrial calcium overload in the masseter muscle of rats with occlusal interference: ionic changes and regulation by calmodulin kinase II].

OBJECTIVE: To investigate the changes in mitochondrial calcium and extracellular sodium concentrations in the masseter muscle of rats with occlusal interference and the regulatory mechanism of mitochondrial Ca2+ overload by calmodulin kinase II (CaMK II). METHODS: SD rat models of occlusal interference were established by placing a stainless steel segments (0.8 mm in diameter) to raise the occlusal surface of the upper right first molar. At 3, 7, 14, and 21 days after occlusal interference and at 3 days after removal of occlusal interference, HE staining was used to observe the histomorphological changes of the masseter muscle. Mitochondrial calcium concentration in the masseter muscle was detected using fluorescence spectrophotometry, and direct turbidimetry with potassium pyroantimonate was used to detect the extracellular sodium concentration; the expression levels of masseter muscle p-CaMK II (Thr287) and CaMK II were detected using Western blotting. RESULTS: Compared with those in the corresponding control groups, mitochondrial Ca2+ concentration in the masseter muscle on occlusal interference side increased significantly at 3, 7, 14 and 21 days after occlusal interference (P<0.05), but was significantly lowered at 3 days after removal of the interference (P<0.05). The concentration of extracellular Na+ increased progressively with time at 3, 7, 14 and 21 days after occlusal interference (P<0.05), and was significantly decreased at 3 days after interference removal (P<0.05). Occlusal interference for 3, 7 and 14 days resulted in significantly increased expressions of p-CaMK II (Thr287) and CaMK II (P<0.05), which was significantly decreased at 21 days compared with those in the control groups (P<0.05) and further decreased after removal of occlusal interference (P<0.05). Similar changes were also observed on the side without interference, but the changes on the interference side were more obvious (P<0.05). CONCLUSION: Occlusal interference causes elevated mitochondrial Ca2+ and extracellular Na+ concentrations in the masseter muscle of rats to lead to calcium overload; the increase in mitochondrial Ca2+ concentration is correlated with the phosphorylation level of CaMK II signaling pathway, suggesting a negative feedback regulation mechanism by the CaMK II signal pathway.

Roles of TRPV1 and TRPA1 in Spontaneous Pain from Inflamed Masseter Muscle.

Craniofacial muscle pain, such as spontaneous pain and bite-evoked pain, are major symptoms in patients with temporomandibular disorders and infection. However, the underlying mechanisms of muscle pain, especially mechanisms of highly prevalent spontaneous pain, are poorly understood. Recently, we reported that transient receptor potential vanilloid 1 (TRPV1) contributes to spontaneous pain but only marginally contributes to bite-evoked pain during masseter inflammation. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1) in spontaneous and bite-evoked pain during masseter inflammation, and dissected the relative contributions of TRPA1 and TRPV1. Masseter inflammation increased mouse grimace scale (MGS) scores and face wiping behaviors. Pharmacological or genetic inhibition of TRPA1 significantly attenuated MGS but not face wiping behaviors. MGS scores were also attenuated by scavenging putative endogenous ligands for TRPV1 or TRPA1. Simultaneous inhibition of TRPA1 by AP18 and TRPV1 by AMG9810 in masseter muscle resulted in robust inhibition of both MGS and face wiping behaviors. Administration of AP18 or AMG9810 to masseter muscle induced conditioned place preference (CPP). The extent of CPP following simultaneous administration of AP18 and AMG9810 was greater than that induced by the individual antagonists. In contrast, inflammation-induced reduction of bite force was not affected by the inhibition of TRPA1 alone or in combination with TRPV1. These results suggest that simultaneous inhibition of TRPV1 and TRPA1 produces additive relief of spontaneous pain, but does not ameliorate bite-evoked pain during masseter inflammation. Our results provide further evidence that distinct mechanisms underlie spontaneous and bite-evoked pain from inflamed masseter muscle.

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.

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.

Early molecular response and microanatomical changes in the masseter muscle and mandibular head after botulinum toxin intervention in adult mice.

BACKGROUND: Masseter muscle paralysis induced by botulinum toxin type A (BoNTA) evokes subchondral bone loss in mandibular heads of adult rats and growing mice after 4 weeks. However, the primary cellular and molecular events leading to altered bone remodeling remain unexplored. Thus, the aim of the current work has been to assess the molecular response that precedes the early microanatomical changes in the masseter muscle and subchondral bone of the mandibular head in adult mice after BoNTA intervention. METHODS: A pre-clinical in vivo study was performed by a single intramuscular injection of 0.2 U BoNTA in the right masseter (experimental) of adult BALB/c mice. The contralateral masseter was injected with vehicle (control). Changes in mRNA levels of molecular markers of bone loss or muscle atrophy/regeneration were addressed by qPCR at day 2 or 7, respectively. mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) was assessed in mandibular heads, whilst mRNA levels of Atrogin-1/MAFbx, MuRF-1 and Myogenin were addressed in masseter muscles. In order to identify the early microanatomical changes at day 14, fiber diameters in transversal sections of masseter muscles were quantified, and histomorphometric analysis was used to determine the bone per tissue area and the trabecular thickness of subchondral bone of the mandibular heads. RESULTS: An increase of up to 4-fold in RANKL mRNA levels were detected in mandibular heads of the BoNTA-injected sides as early as 2 days after intervention. Moreover, a 4-6 fold increase in Atrogin-1/MAFbx and MuRF-1 and an up to 25 fold increase in Myogenin mRNA level were detected in masseter muscles 7 days after BoNTA injections. Masseter muscle mass, as well as individual muscle fiber diameter, were significantly reduced in BoNTA-injected side after 14 days post-intervention. At the same time, in the mandibular heads from the treated side, the subchondral bone loss was evinced by a significant reduction in bone per tissue area (-40%) and trabecular thickness (-55%). CONCLUSIONS: Our results show that masseter muscle paralysis induced by BoNTA leads to significant microanatomical changes by day 14, preceded by molecular changes as early as 2 days in bone, and 7 days in muscle. Therefore, masseter muscle atrophy and subchondral bone loss detected at 14 days are preceded by molecular responses that occur during the first week after BoNTA intervention.

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.