Tetrazolium reduction assays under-report cell death provoked by clinically relevant concentrations of proteasome inhibitors.

High throughput cell viability screening assays often capitalize on the ability of active enzymes or molecules within viable cells to catalyze a quantifiable chemical reaction. The tetrazolium reduction (MTT) assay relies on oxidoreductases to reduce tetrazolium into purple formazan crystals that are solubilized so absorbance reflects viability, while other assays use cellular ATP to catalyze a luminescence-emitting reaction. It is therefore important to know how accurately these assays report cellular responses, as cytotoxic anti-cancer agents promote cell death via a variety of signaling pathways, some of which may alter how these assays work. In this study, we compared the magnitude of cytotoxicity to different cell types provoked by currently used anti-cancer agents, using three different cell viability assays. We found the three assays were consistent in reporting the viability of cells treated with chemotherapy drugs or the BH3 mimetic navitoclax, but the MTT assay underreported the killing capacity of proteasome inhibitors. Additionally, the MTT assay failed to confirm the induction of caspase-mediated cell death by bortezomib at physiologically relevant concentrations, thereby mischaracterizing the mode of cell death. While the cell viability assays used allow for the rapid identification of novel cytotoxic compounds, our study emphasizes the importance for these screening assays to be complemented with a direct measure of cell death or another independent measure of cell viability. We caution researchers against using MTT assays for monitoring cytotoxicity induced by proteasome inhibitors.

Myogenin promotes myocyte fusion to balance fibre number and size.

Each skeletal muscle acquires its unique size before birth, when terminally differentiating myocytes fuse to form a defined number of multinucleated myofibres. Although mice in which the transcription factor Myogenin is mutated lack most myogenesis and die perinatally, a specific cell biological role for Myogenin has remained elusive. Here we report that loss of function of zebrafish myog prevents formation of almost all multinucleated muscle fibres. A second, Myogenin-independent, fusion pathway in the deep myotome requires Hedgehog signalling. Lack of Myogenin does not prevent terminal differentiation; the smaller myotome has a normal number of myocytes forming more mononuclear, thin, albeit functional, fast muscle fibres. Mechanistically, Myogenin binds to the myomaker promoter and is required for expression of myomaker and other genes essential for myocyte fusion. Adult myog mutants display reduced muscle mass, decreased fibre size and nucleation. Adult-derived myog mutant myocytes show persistent defective fusion ex vivo. Myogenin is therefore essential for muscle homeostasis, regulating myocyte fusion to determine both muscle fibre number and size.

An ultrastructural and histochemical study of the flexor tibialis muscle fiber types in male and female stick insects (Eurycantha calcarata, L).

In this study the ultrastructural and histochemical characteristics of the flexor tibialis muscle fibers of the specialized metathoracic legs in the male and those of homologous and unspecialized ones in the female stick insects, Eurycantha calcarata, L, were examined. For the ultrastructural analysis, the muscle was divided longitudinally and vertically to produce a total of 12 sample parts e.g., anterior-dorsal-distal (ADD), posterior-ventral-medial (PVM) and so on. Light and electron microscopes were used to observe the muscle tissue. The methods for myosin adenosine triphosphatase (mATPase) and nicotine adenine dinucleotide- tetrazolium (NADH-TR) staining were modified from the methods of (Stokes et al., '79; Anttila et al., 2009; Anttila and Manttari, 2009). Sections with thickness of 22 µm, were cut from the anterior and the posterior surfaces of the muscle, using a cryostat. The histochemical and ultrastructural results showed that the muscles of both the male and the female were mixtures of physiological fiber types, with predominantly fast fibers. The muscles were composed of fibers with different staining properties for both mATPase and NADH-TR activities. The population of fibers within the muscles was heterogeneous. The differences between the population of the male and that of the female were significant. The means of most criteria e.g., mitochondrial amount and sarcoplasmic reticulum area predicted that the muscle of the male contained more fast fibers than the female. The histochemical examination also showed that the muscle of the male contained more fibers stained darkly for mATPase and lightly for NADH-TR.

Muscle fiber viability, a novel method for the fast detection of ischemic muscle injury in rats.

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury.

Distribution pattern of muscle fibre types in soft palate of the dog (Canis familiaris, L.).

Distribution pattern of fibre types was studied in the muscles of the soft palate (palatinus, levator veli palatini and tensor veli palatini muscles) in the dog. The fibrillar classification was based on using histochemistry and immunohistochemistry methods: myofibrillar adenosine thriphosphatase (mATPase) to different pH of pre-incubation; nicotine adenine dinucleotide (reduced) tetrazolium reductase (NADH-TR) and finally, application of specific monoclonal antibodies against myosin heavy chain isoforms I, IIa and IIx. In the palatinus and levator veli palatini muscles, pure type I fibres and the hybrid type IIax and IIc were shown, with a checkerboard distribution in the first and a clear predominance of hybrid fibre types (about 98% of the total population) in levator veli palatini muscle. On the other hand, in the tensor veli palatini muscle, type IIx and IIm fibres were identified (fast-twitch fibres related to fast-moving muscles and the powerful jaw muscles of carnivores). The tensor veli palatini muscle had a different distribution and fibrillar composition with predominantly type IIm fibres in its central zone, whilst the peripheral zone was primarily type I and IIx fibres.

Abnormal mitochondria organization and oxidative activity in the palate muscles of long-term snorers with obstructive sleep apnea.

BACKGROUND: Histopathological alterations and a reduced number of capillaries have been observed in the palate muscles of snorers with obstructive sleep apnea syndrome (OSAS). These changes may create a substrate for decreased microcirculation, impaired aerobic metabolism and muscle dysfunction and contribute to upper airway obstruction during sleep. OBJECTIVES: The aim was to analyze mitochondria distribution and oxidative enzyme activity in relation to capillary supply in the palate muscles of patients with a history of long-term snoring and OSAS. METHODS: Palatopharyngeus (PP) and uvula (UV) muscle samples were obtained from 8 patients undergoing uvulopalatopharyngoplasty due to habitual snoring and OSAS. The muscles were analyzed with enzyme- and immunohistochemistry and morphometry. RESULTS: Abnormalities in the internal organization of mitochondria and oxidative activity were observed in 39 ± 15% of the fibers in the PP and 4 ± 3% in the UV, but not in control samples. The majority of these fibers had a lobulated contour and trabecular internal organization of mitochondria. The number of capillaries around abnormal fibers (PP 0.9 ± 0.3, UV 0.4 ± 0.1) was lower than in fibers of a normal appearance in both patients (PP 1.4 ± 0.6, UV 1.2 ± 0.3) and references (PP 2.7 ± 0.7, UV 1.9 ± 0.9) (p < 0.05). CONCLUSIONS: Abnormal mitochondrial distribution, a low capillary supply and signs of impaired oxidative activity suggest that muscle dysfunction of the palate muscles in long-term snorers may contribute to the upper airway obstruction during sleep. The cause of these abnormalities remains unclear, but local muscle and nerve trauma due to vibration and stretch is a possible etiology.

Nicotinamide adenine dinucleotide tetrazolium reductase identifies microvasculature activation in muscle from adult patients with dermatomyositis.

OBJECTIVE: Previous work has suggested involvement of the muscle microvasculature in the pathogenesis of dermatomyositis (DM). Our study evaluates whether standard histochemical reactions can identify microvascular changes in muscle biopsies from patients with DM compared to myopathic and nonmyopathic controls. METHODS: Muscle biopsies were obtained from 111 patients, including 45 patients with DM. Microvascular quantitation was performed on transversely oriented 1-µm toluidine blue-stained plastic sections. Histoenzymatic procedures included alkaline phosphatase (AP), nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), cytochrome C oxidase (COX), and myosin ATPase reactions. RESULTS: Capillary density was significantly lower in DM muscle biopsies compared to biopsies from patients with noninflammatory myopathies (NIM; n = 26) and healthy control muscle (n = 27; mean ± SD: 252 ± 114 vs 402 ± 56 and 325 ± 109 capillaries/mm(2), respectively; p values < 0.05). In contrast, a marked increase in the number of capillaries staining with NADH-TR was noted in DM compared to other idiopathic inflammatory myopathies (IIM; n = 13), NIM, and controls (49.8 ± 50.7 vs 8.0 ± 7.1, 6.7 ± 7.2, and 3.6 ± 2.8 capillaries/mm(2); p < 0.05 compared to DM). DM capillaries also demonstrated mildly increased staining with AP compared to controls; however, no increased SDH or COX reactivity was observed. CONCLUSION: DM muscle capillaries are highly reactive with NADH-TR compared to myopathic and nonmyopathic controls. The lack of staining of DM capillaries with mitochondrial SDH and COX reactions suggests that NADH-TR reactivity may be secondary to activation of the microvascular endoplasmic reticulum, rather than mitochondrial hyperplasia.

TRPV4 deficiency increases skeletal muscle metabolic capacity and resistance against diet-induced obesity.

Transient receptor potential channel V4 (TRPV4) functions as a nonselective cation channel in various cells and plays physiological roles in osmotic and thermal sensation. However, the function of TRPV4 in energy metabolism is unknown. Here, we report that TRPV4 deficiency results in increased muscle oxidative capacity and resistance to diet-induced obesity in mice. Although no difference in body weight was observed between wild-type and Trpv4(-/-) mice when fed a standard chow diet, obesity phenotypes induced by a high-fat diet were significantly improved in Trpv4(-/-) mice, without any change in food intake. Quantitative analysis of mRNA revealed the constitutive upregulation of many genes, including those for transcription factors such as peroxisome proliferator-activated receptor α and for metabolic enzymes such as phosphoenolpyruvate carboxykinase. These upregulated genes were especially prominent in oxidative skeletal muscle, in which the activity of Ca(2+)-dependent phosphatase calcineurin was elevated, suggesting that other Ca(2+) channels function in the skeletal muscle of Trpv4(-/-) mice. Indeed, gene expressions for TRPC3 and TRPC6 increased in the muscles of Trpv4(-/-) mice compared with those of wild-type mice. The number of oxidative type I fiber also increased in the mutant muscles following myogenin gene induction. These results strongly suggested that inactivation of Trpv4 induces compensatory increases in TRPC3 and TRPC6 production, and elevation of calcineurin activity, affecting energy metabolism through increased expression of genes involved in fuel oxidation in skeletal muscle and thereby contributing to increased energy expenditure and protection from diet-induced obesity in mice.

An in vitro evaluation of Candida tropicalis infectivity using human cell monolayers.

The aim of this study was to investigate the interaction of Candida tropicalis with three different human cell lines: TCC-SUP (epithelial cells from urinary bladder), HeLa (epithelial cells from cervical carcinoma) and Caco-2 (epithelial cells from colorectal adenocarcinoma). In particular we sought to assess the degree of cell damage and activity reduction induced by C. tropicalis adhesion and the role of secreted aspartyl proteinase (SAP) gene expression in this process. Two C. tropicalis strains were used: the reference strain ATCC 750 and a clinical isolate from urine (U69). The ability of C. tropicalis to adhere to a confluent layer of human cells was determined using an adaptation of the crystal violet staining method; cell damage and cell activity inhibition induced by the adhesion of C. tropicalis were assessed by measuring lactate dehydrogenase and tetrazolium salt (MTS) reduction, respectively. C. tropicalis SAP gene expression was determined by real-time PCR. Both C. tropicalis strains were able to adhere to the different human cells, although in a strain- and cell-line-dependent manner. Concerning the cellular response to C. tropicalis, the highest inhibition of cell activity was obtained for Caco-2, followed by TCC-SUP and HeLa cells. The highest percentage of cell damage (around 14 %) was observed for TCC-SUP cells in contact with the U69 isolate and for Caco-2 in contact with the reference strain. Real-time PCR analysis revealed a wide range of expression profiles of SAP genes for both C. tropicalis strains in contact with the different types of epithelial cells. SAPT3 was the gene expressed at the highest level for both C. tropicalis strains in contact with the three human epithelial cell lines. The results highlight that the response of human cells to C. tropicalis adhesion, as well as production of SAPs, is dependent on both the strain and the epithelial cell line.

Muscle fiber type characterization and myosin heavy chain (MyHC) isoform expression in Mediterranean buffaloes.

This study aimed to evaluate myosin heavy chain (MyHC) isoform expression and muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) in Mediterranean buffaloes and possible fibers muscles modulation according to different slaughter weights. The presence of MyHC IIb isoforms was not found. Only three isoforms of MyHC (IIa, IIx/d and I) were observed and their percentages did not vary significantly among slaughter weights. The confirmation of the presence of hybrid muscles fibers (IIA/X) in LD and ST muscles necessitated classifying the fiber types into fast and slow according to their contractile activity, by m-ATPase assay. For both muscles, the muscle fiber frequency was higher for fast than for slow fibers in all weight groups. There was a difference (P<0.05) in the frequency of LD and ST muscle fiber types according to slaughter weights, which demonstrate that the slaughter weight influences the profile of muscle fibers from buffaloes.

Type II muscle fibers atrophy associated with silent corticotroph adenoma in a dog.

The Silent Corticotroph Adenoma (SCA) is a pituitary adenoma variant characterized by the immunoreactivity for adrenocorticotropic hormone (ACTH) and related peptides, without the clinical signs of Cushing's disease. SCA has been postulated to either secrete structurally abnormal ACTH that is inactive but detectable by immunohistochemistry or radioimmunoassay, or to secrete ACTH intermittently or at low levels continuously. Excess of ACTH has been associated to type II muscle atrophy. We describe a case of type II muscle fibers atrophy associated with silent corticotroph adenoma in a dog. The dog showed moderate to severe proximal muscle wasting and weakness with normal levels of muscle-associated enzymes. In the limb muscle biopsies, type II fibers were uniformly smaller than type I fibers. In temporalis muscles, there were few atrophic fibers, and several irregular areas of loss of enzymatic activity observed in NADH, SDH and COX stains. The tumour showed a trabecular growth pattern and immunohistochemical analysis demonstrated the presence of cytoplasmic immunoreactivity for ACTH. The muscle atrophy was considered to be related to an excess of inactive ACTH. Studying spontaneous occurring rare diseases in animals could help to understand the mechanism of similar diseases in human has well.

Human extraocular muscles in ALS.

PURPOSE. To investigate the general morphology, fiber type content, and myosin heavy chain (MyHC) composition of extraocular muscles (EOMs) from postmortem donors with amyotrophic lateral sclerosis (ALS) and to evaluate whether EOMs are affected or truly spared in this disease. METHODS. EOM and limb muscle samples obtained at autopsy from ALS donors and EOM samples from four control donors were processed for immunohistochemistry with monoclonal antibodies against distinct MyHC isoforms and analyzed by SDS-PAGE. In addition, hematoxylin and eosin staining and nicotinamide tetrazolium reductase (NADH-TR) activity were studied. RESULTS. Wide heterogeneity was observed in the appearance of the different EOMs from each single donor and between donors, irrespective of ALS type or onset. Pathologic morphologic findings in ALS EOMs included presence of atrophic and hypertrophic fibers, either clustered in groups or scattered; increased amounts of connective tissue; and areas of fatty replacement. The population of fibers stained with anti-MyHCslow tonic was smaller than that of MyHCIpositive fibers and was mostly located in the orbital layer in most of the ALS EOM samples, whereas an identical staining pattern for both fiber populations was observed in the control specimens. MyHCembryonic was notably absent from the ALS EOMs. CONCLUSIONS. The EOMs showed signs of involvement with altered fiber type composition, contractile protein content, and cellular architecture. However, when compared to the limb muscles, the EOMs were remarkably preserved. EOMs are a useful model for the study of the pathophysiology of ALS.

The fate of chondrocytes during ageing of human thyroid cartilage.

Human laryngeal cartilages, especially thyroid cartilage, exhibit gender-specific ageing. In contrast to male thyroid cartilages, the ventral half of the female thyroid cartilage plate remains unmineralized until advanced age. In cartilage specimens from laryngectomies and autopsies, apoptosis was studied immunohistochemically and the oxidative mitochondrial enzyme nicotinamide adenine dinucleotide hydride tetrazolium reductase (NADH-TR) was localized histochemically. In addition, very fresh specimens from laryngectomies were fixed under addition of ruthenium hexamine trichloride or tannin to fixation solution to study cell organelles of chondrocytes by electron microscopic methods. In general, apoptotic chondrocytes decreased in thyroid cartilages of both genders, especially after the second decade. In the age group 41-60 years, thyroid cartilage from male specimens revealed a significantly higher percentage of apoptotic cells than did thyroid cartilage from women (P = 0.004), whereas in the age groups 0-20 years and 61-79 years no statistically significant gender difference was determined. In general, thyroid cartilage from women contained more living chondrocytes into advanced age than men. Chondrocytes adjacent to mineralized cartilage were partly positive for apoptosis and NADH-TR and partly negative. Apoptotic chondrocytes often were localized in areas of asbestoid fibres where vascularization and mineralization took place first. Electron microscopy revealed remnants of chondrocytes in asbestoid fibres. Taken together, it can be assumed that some chondrocytes in thyroid cartilage die by apoptosis and that these chondrocytes are characterized by absent reactivity for the mitochondrial enzyme NADH-TR. A possible influence of sexual hormones on apoptotic death of thyroid cartilage cells requires further elucidation.

Muscle histopathology in myasthenia gravis with antibodies against MuSK and AChR.

AIMS: We compared myopathological features in myasthenia gravis (MG) patients with antibodies against AChR (seropositive) and muscle-specific tyrosin-kinase (MuSK). While the immunopathogenesis of seropositive MG is well known, there is a lack of pathological studies in anti-MuSK antibody-positive (MuSK+) MG. METHODS: We analysed skeletal muscle biopsy features of 13 MG patients: 6 MuSK+ (all women) and 7 anti-AchR antibody-positive (AChR+) (2 women and 5 men). In our histopathological examination, we quantified the atrophy factor of both fibre types, and the extent of minicores, myofibrillar disarray, cytochrome c oxidase (COX)-negative fibres, mitochondrial aggregates and fibre type grouping. RESULTS: Mean muscle fibre atrophy factor was higher in AChR+ MG than MuSK+ MG, both in type I fibres (494 vs. 210) and particularly in type II fibres (1023 vs. 300). Fibre type grouping was observed in AChR+ MG whereas COX-negative fibres were common in MuSK+ MG. Bulbar muscles were more severely affected in MuSK+ MG and the disease was more severe: the onset was usually earlier (39 years) with Myasthenia Gravis Foundation of America score III in MuSK+ MG, and score II was found in AChR+ MG (62 years). CONCLUSIONS: Muscle biopsies of MuSK+ MG show myopathic signs with prominent mitochondrial abnormalities, whereas neurogenic features and atrophy are more frequently found in AChR+ MG. The mitochondrial impairment could explain the oculo-bulbar involvement in MuSK+ MG.

Histological and histochemical effects after occlusion alteration in suprahyoid muscles.

This study verified the effect of unilateral teeth extraction on the suprahyoid muscles in gerbils (Meriones unguiculatus). Ten adult male gerbils weighing about 50g had induced occlusal alterations by upper molar teeth extraction on the left side while the other ten animals were only subjected to surgical stress, control group. After 60 days, animals of both groups, experimental and control had the suprahyoid muscles removed and processed for histological and histochemical (adenosine triphosphatase (ATPase), nicotine adenine dinucleotide tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH)) purposes. The fiber type area was estimated in % according to Weibel method (point-counting method) using a test-system. The myosinic ATPase pH 4.7 activity in the control group of the digastric, milohyoid and geniohyoid muscles presented a small area of type I fiber and a larger area of type IIa fibers; in the experimental group, significant contractile capacity alteration was not observed. Samples of the digastric, milohyoid and geniohyoid muscles, after SDH activity, showed a small area with high metabolic activity fibers, and a large area with intermediary and low metabolic activity fibers in the control group. The milohyoid muscle of the experimental group presented low metabolic fibers in a reduced area, in both sides, however without significant difference. In the experimental group, high metabolic fibers were observed on the left side in a reduced area in the geniohyoid muscle, but without statistical significance. Thus, the geniohyoid muscle did not change the metabolic activity after occlusal alteration. In conclusion, 60 days of unilateral malocclusion induced was able to alter the fibers oxidative activity of the suprahyoid muscles, however, it does not affect the contractile property of the fibers. The digastric muscle has adequate fibers to produce fast contraction and able to resist to fatigue in intermediate degrees, but became more fatigable after unilateral exodontia.

Muscle growth and fiber type composition in hind limb muscles during postnatal development in pigs.

Rapid postnatal development in pigs is reflected by differentiation in skeletal muscle. This process depends on muscle function and demands, but a comprehensive overview of individual developmental characteristics of quickly growing leg muscles in pigs is still missing. This study focused on the development of 10 hind limb muscles in pigs. To determine these changes in mass, fiber type patterns and fiber diameters were analyzed 0, 2, 4, 7, 14, 28, 42, 56 and 400 days after birth. Generally, the proportion of slow fibers increased from birth to 8 weeks. Thereafter, only minor changes in muscle fiber type composition were observed. The majority of the muscles contained less then 10% slow-twitch fibers at birth, increasing to between 12 (Musculus vastus lateralis) and 38% (M. gastrocnemius medialis) in adult pigs. By contrast, postural muscles already had 20-30% slow fibers at birth, and this contribution increased up to 65% in adults (i.e. M. vastus intermedius). From birth to the 2nd week, only in slow fibers could activity of oxidative enzymes be detected. A differentiation of fast-twitch fibers into subtypes with high (comparable to type IIA) and low oxidative metabolism (equivalent to type IIB) occurred between the 2nd and 4th week of life. The ratio between type II fibers with high and low oxidative enzyme activity did not change markedly through development in any muscle, although there was a trend towards an increasing proportion of type IIA fibers in the soleus. In the majority of the muscles investigated, the fast-twitch fibers with low oxidative metabolism (IIB) obtained the largest cross-sectional area. In contrast, at birth no remarkable differences in the diameter of fast and slow fibers were found. The rapid increase in muscle mass compared to body mass reflects the high performance in meat production of the cross pig investigated.

With the help of giants.

A childhood fascination with animals, plants, and insects was aided and abetted by many giants, beginning with my parents. The Bronx High School of Science and the City College of New York (CCNY) made a solid and priceless grounding in chemistry and biology available free of charge. Abe Mazur at CCNY revealed the wonders of biochemistry and illustrated that it was possible to pursue these wonders while being paid to do so. He also directed me to Duke University Medical School for PhD work under the tutelage of Phil Handler. With the exception of a sabbatical year at Harvard with Frank Westheimer, my entire career has been spent at Duke serving under three fine and supportive chairmen: Handler, Hill, and Raetz. The premier discoveries to emanate from my laboratory have been the sulfite oxidase, the several superoxide dismutases, the manganese catalase, and the catalase/peroxidase. Many other topics piqued my interest and resulted in ~ 400 publications. Herein I have recounted some of the circumstances surrounding that work and named a few of the people involved. The first 20 years I worked happily at the bench and the next 35 years just as happily facilitating the work of younger people. It has been so rewarding that I wish for nothing more than to be allowed to keep at it.

Fibre composition of human intrinsic tongue muscles.

The muscle fibre composition of three human intrinsic tongue muscles, the longitudinalis, verticalis and transversus, was investigated in four anterior to posterior regions of the tongue using morphological and enzyme- and immunohistochemical techniques. All three muscles typically contained type I, IIA and IM/IIC fibres. Type I fibres expressed slow myosin heavy chain (MyHC), type II fibres fast MyHC, mainly fast A MyHC, whereas type IM/IIC coexpressed slow and fast MyHCs. Type II fibres were in the majority (60%), but regional differences in proportion and diameter of fibre types were obvious. The anterior region of the tongue contained a predominance of relatively small type II fibres (71%), in contrast to the posterior region which instead showed a majority of larger type I and type IM/IIC fibres (66%). In general, the fibre diameter was larger in the posterior region. This muscle fibre composition of the tongue differs from those of limb, orofacial and masticatory muscles, probably reflecting genotypic as well as phenotypic functional specialization in oral function. The predominance of type II fibres and the regional differences in fibre composition, together with intricate muscle structure, suggest generally fast and flexible actions in positioning and shaping the tongue, during vital tasks such as mastication, swallowing, respiration and speech.