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Tooth loss or incorrect positioning causes occlusal disharmony. Furthermore, tooth loss and atrial fibrillation (AF) are both risk factors for ischemic stroke and coronary heart disease. Therefore, we hypothesized that occlusal disharmony-induced stress increases susceptibility to AF, and we designed the present study to test this idea in mice. Bite-opening (BO) was done by cementing a suitable appliance onto the mandibular incisor to cause occlusal disharmony by increasing the vertical height of occlusion by 0.7 mm for a period of 2 weeks. AF susceptibility, evaluated in terms of the duration of AF induced by transesophageal burst pacing, was significantly increased concomitantly with atrial remodeling, including fibrosis, myocyte apoptosis and oxidative DNA damage, in BO mice. The BO-induced atrial remodeling was associated with increased calmodulin kinase II-mediated ryanodine receptor 2 phosphorylation on serine 2814, as well as inhibition of Akt phosphorylation. However, co-treatment with propranolol, a non-selective β-blocker, ameliorated these changes in BO mice. These data suggest that improvement of occlusal disharmony by means of orthodontic treatment might be helpful in the treatment or prevention of AF.

Occlusal disharmony leads to morphological changes in the hippocampus and osteopenia of the lumbar vertebra and long bones in mice, and causes stress. Various types of stress are associated with increased incidence of cardiovascular disease, but the relationship between occlusal disharmony and cardiovascular disease remain poorly understood. Therefore, in this work, we examined the effects of occlusal disharmony on cardiac homeostasis in bite-opening (BO) mice, in which a 0.7 mm space was introduced by cementing a suitable applicance onto the mandibular incisior. We first examined the effects of BO on the level of serum corticosterone, a key biomarker for stress, and on heart rate variability at 14 days after BO treatment, compared with baseline. BO treatment increased serum corticosterone levels by approximately 3.6-fold and the low frequency/high frequency ratio, an index of sympathetic nervous activity, was significantly increased by approximately 4-fold by the BO treatment. We then examined the effects of BO treatment on cardiac homeostasis in mice treated or not treated with the non-selective β-blocker propranolol for 2 weeks. Cardiac function was significantly decreased in the BO group compared to the control group, but propranolol ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but propranolol blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as inhibition of Akt/mTOR signaling and autophagic flux. These data suggest that occlusal disharmony might affect cardiac homeostasis via alteration of the autonomic nervous system.

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

Although multiple factors influence food bite size, the relationship between food bite size per mouthful and mandible or tongue size remains poorly understood. Here, we examined the correlations between food bite size and the lower dental arch size (an indicator of tongue size) in human subjects with good oral and general health, using fish sausage and bread as test foods. Notably, bite size of both foods was significantly positively correlated with the lower dental arch size, whereas masticatory performance (measured in terms of glucose extraction from a gummy jelly) showed no dependence on bite size. Further, bite size was significantly positively correlated with the body mass index. Our findings suggest that larger bite size is associated with larger tongue size, which might be a contributory factor to obesity.

Chronic administration of clenbuterol (CB), a lipophilic β2-adrenoceptor (β2-AR) agonist, induces skeletal muscle hypertrophy and slow-to-fast fiber–type transitions in mammalian species, but the mechanism and pathophysiological roles of these changes have not been explored. Here, we examined the effects of CB not only on masseter muscle mass, fiber diameter, and myosin heavy chain (MHC) composition, but also on daily muscle activity, a factor influencing muscle phenotype, by means of electromyogram analysis in rats. MHC transition towards faster isoforms was induced by 2-week CB treatment. In addition, daily duty time was increased at 1 day, 1 week, and 2 weeks after the start of CB treatment and its increase was greater at high activity level (6-fold) than at low activity level (2-fold). In order to examine whether these effects of CB were mediated through muscle or CNS β2-AR stimulation, we compared these effects of CB with those of salbutamol (SB), a hydrophilic β2-AR agonist. SB treatment induced masseter hypertrophy and MHC transition, like CB, but did not increase daily activity. These results suggest that CB-mediated slow-to-fast MHC transition with hypertrophy was induced through direct muscle β2-AR stimulation, but the increase of daily duty time was mediated through the CNS. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.12271FP] Keywords:: β2-adrenergic signaling, masseter muscle, electromyogram, myosin heavy chain, hypertrophy

Parkinson's disease (PD), a major neurological disease, is characterised by a marked loss of dopaminergic neurons in the substantia nigra. Patients with PD frequently show chewing and swallowing dysfunctions, but little is known about the characteristics of their stomatognathic functions. The purpose of this study was to evaluate the influence of PD on jaw muscle fibre and functions. PD model rats were made by means of the injection of 6-hydroxydopamine (6-OHDA) into the striatum of 8-week-old Sprague-Dawley male rats. Five weeks after the injection, a radio-telemetric device was implanted to record muscle activity continuously from the superficial masseter and anterior belly of digastric muscles. Muscle activity was recorded for 3 days and was evaluated by the total duration of muscle activity per day (duty time). After recording the muscle activities, jaw muscles were isolated for immunohistochemical and PCR analyses. In PD model rats, the following findings of the digastrics muscles verify that compared to the control group: (i) the higher duty time exceeding 5% of the peak activity level, (ii) the higher expression of the mRNA of myosin heavy chain type I, and (iii) the tendency for fast to slow fibre-type transition. With respect to the masseter muscle, there were no significant differences in all analyses. In conclusion, PD leads to the changes in the jaw behaviours, resulting in a PD-specific chewing and swallowing dysfunctions.

Stimulation of β-adrenergic receptors in cardiac myocytes activates cyclic AMP– dependent protein kinase A (PKA). PKA-mediated phosphorylation of myofibrils decreases their longitudinal stiffness, but its effect on transverse stiffness is not fully understood. We thus examined the effects of PKA treatment on the transverse stiffness of cardiac myofibrils by atomic force microscopy and determined the phosphorylation levels of myofibril components by SDS-PAGE. Transverse stiffness was significantly decreased by PKA treatment concomitantly with increased phosphorylation of troponin I, myosin-binding protein C, and titin (also called connectin). Subsequent treatment with protein phosphatase 1 abrogated these PKA-mediated effects. [Supplementary methods: available only at http://dx.doi.org/10.1254/jphs.13110SC] Keywords:: stiffness, catecholamine, contractility

Background and aims The mechanisms of cancer cell growth and metastasis are still not entirely understood, especially from the viewpoint of chemical reactions in tumours. Glycolytic metabolism is markedly accelerated in cancer cells, causing the accumulation of glucose (a reducing sugar) and methionine (an amino acid), which can non-enzymatically react and form carcinogenic substances. There is speculation that this reaction produces gaseous sulfur-containing compounds in tumour tissue. The aims of this study were to clarify the products in tumour and to investigate their effect on tumour proliferation. Methods Products formed in the reaction between glucose and methionine or its metabolites were analysed in vitro using gas chromatography. Flatus samples from patients with colon cancer and exhaled air samples from patients with lung cancer were analysed using near-edge x-ray fine adsorption structure spectroscopy and compared with those from healthy individuals. The tumour proliferation rates of mice into which HT29 human colon cancer cells had been implanted were compared with those of mice in which the cancer cells were surrounded by sodium hyaluronate gel to prevent diffusion of gaseous material into the healthy cells. Results Gaseous sulfur-containing compounds such as methanethiol and hydrogen sulfide were produced when glucose was allowed to react with methionine or its metabolites homocysteine or cysteine. Near-edge x-ray fine adsorption structure spectroscopy showed that the concentrations of sulfur-containing compounds in the samples of flatus from patients with colon cancer and in the samples of exhaled air from patients with lung cancer were significantly higher than in those from healthy individuals. Animal experiments showed that preventing the diffusion of sulfur-containing compounds had a pronounced antitumour effect. Conclusions Gaseous sulfur-containing compounds are the main products in tumours and preventing the diffusion of these compounds reduces the tumour proliferation rate, which suggests the possibility of a new approach to cancer treatment.

Mammalian skeletal muscles change their contractile-protein phenotype in response to mechanical loading and/or chronic electrical stimulation, implying that the phenotypic changes in masticatory muscles might result from new masticatory-loading conditions. To analyze the effects of increased occlusal vertical dimension (OVD) on daily activities and fibre-type compositions in jaw muscles, we measured the total duration of daily activity (duty time) and the myosin heavy chain (MyHC) compositions in the masseter and digastric muscles of freely moving control and bite-opened rats. In the control state, the duty time of the digastric muscle was higher than that of the masseter muscle at activity levels exceeding 5 and 20% of the day's peak activity. The opposite was true at activity levels exceeding 50 and 80% of the day's peak activity. The MyHCs consisted of a mixture of fast and slow types in the digastric muscle. The masseter consisted of mostly fast-type MyHC. The increment of OVD increased not only the duty time at activity levels exceeding 5, 20, 50 and 80% of the day’ peak activity in both muscles but also the proportion of MyHC IIa in the masseter muscle and MyHC I in the digastric muscle at the expense of that of MyHC IIb. These results suggest that the increment of OVD changes masseter and digastric muscles towards slower phenotypes by an increase in their daily activities.

Mandibular movement is achieved by coordinated actions of the jaw muscles. To understand the assigned functional role (e.g., motor or postural role) of each jaw muscle, we characterised not only their contractile and energy-consumption properties but also their compositions of myosin heavy chain (MHC) isoforms. The Ca(2+)-dependent isometric tension development and ATPase activity were simultaneously measured in chemically skinned fibers harvested from rat jaw-closing (masseter and temporalis) and jaw-opening (digastric) muscles. After the measurements of isometric tension development and ATPase activity, the MHC compositions in each preparation were determined by SDS-gel electrophoresis. The Ca(2+)-sensitivity of isometric tension development and ATPase activity was significantly (P

Obesity is well known to be associated with a wide variety of illnesses, and is an increasing problem not only in developed countries but also in developing countries. It is well known that large bite size contributes to excess energy intake and obesity, whereas an increased number of chews before swallowing the food bolus is associated with suppression of obesity. However, the effect of food diameter on bite size per mouthful and on chewing behavior remains poorly understood. Here, we examined the effects of food diameter on bite size and chewing behavior using a masticatory counter during the mastication of stick-type biscuits having the same length (10 cm) and ingredients, but with four different diameters (3.0, 3.5, 4.0, and 8.0 mm). Bite length and bite weight per mouthful were similar among the 3.0, 3.5, and 4.0 mm groups. However, bite length in the 8.0 mm group was significantly smaller, whereas bite weight was significantly greater than in the 3.0/3.5 mm groups. Further, the number of chews gradually increased, whereas the number of chews per bite weight gradually decreased, with an increase of biscuit diameter. These results indicate that a smaller biscuit diameter is associated with a smaller bite weight per mouthful and a greater number of chews per bite weight. This is the first report to quantity the effect of food diameter on bite weight per mouthful and on chewing behavior; these results should be helpful in the design of effective, safe, and low-cost behavioral modification therapy to combat obesity.

Background Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. Methodology We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Results and Conclusion Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

Although microtubules are involved in various pathological conditions of the heart including hypertrophy and congestive heart failure, the mechanical role of microtubules in cardiomyocytes under such conditions is not well understood. In the present study, we measured multiple aspects of the mechanical properties of single cardiomyocytes, including tensile stiffness, transverse (indentation) stiffness, and shear stiffness in both transverse and longitudinal planes using carbon fiber–based systems and compared these parameters under control, microtubule depolymerized (colchicine treated), and microtubule hyperpolymerized (paclitaxel treated) conditions. From all of these measurements, we found that only the stiffness against shear in the longitudinal plane was modulated by the microtubule cytoskeleton. A simulation model of the myocyte in which microtubules serve as compression-resistant elements successfully reproduced the experimental results. In the complex strain field that living myocytes experience in the body, observed changes in shear stiffness may have a significant influence on the diastolic property of the diseased heart.

To gain more insight into the molecular mechanism of muscle growth and fiber-type transformations, we analyzed the effects of beta(2)-adrenergic agonist clenbuterol (CB) and/or cyclosporin A (CsA), a potent inhibitor of calcineurin (CaN), on the muscle mass as well as on the mRNA levels of myosin heavy chains (MHC I, IIa, IId/x, IIb), using a real-time RT-PCR with specific primers in rat masseter. In comparison with control, the CB treatment significantly decreased the MHC I mRNA level (p < 0.01), but increased the MHC IId/x mRNA level (p < 0.01), and the CsA treatment significantly decreased the MHC I mRNA level (p < 0.05) in association with the significant decrease in MHC IIb mRNA level (p < 0.05). The CB+CsA treatment significantly decreased the levels of MHC I (p < 0.01) and IIa mRNAs (p < 0.05), but increased the MHC IId/x mRNA level (p < 0.001) in association with a significant decrease in MHC IIb mRNA level (p < 0.01), in comparison with control. The masseter muscle mass was significantly (p < 0.001) increased by either the CB or the CB + CsA treatment, but decreased with the CsA treatment (p < 0.01). These results suggest that in rat masseter muscle, CB has an anabolic action accompanying MHC mRNA I IIa IId/x sequence transition independently of CaN-signaling pathways, and CaN is involved in the type I fiber gene expression and the muscle mass maintenance of type IIb fiber.

The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by atomic force microscopy. The microscopic images of both skeletal and cardiac myofibrils in a rigor state showed periodical striation patterns separated by Z-bands, which is characteristic of striated muscle fibers. However, sarcomere patterns were hardly distinguishable in the stiffness distributions of the relaxed myofibrils of skeletal and cardiac muscles. Myofibrils in a rigor state were significantly stiff compared with those in a relaxed state, and in each state, cardiac myofibrils were significantly stiffer compared with skeletal myofibrils. By proteolytic digestions of sarcomere components of myofibrils, it was suggested that cardiac myofibrils are laterally stiffer than skeletal myofibrils because Z-bands, connectin (titin) filament networks, and other components of sarcomere structures for the former myofibrils are stronger than those for the latter.

To understand the pathophysiology of hereditary cardiomyopathy, the contractile function of cardiomyopathic hamsters has been studied at the cellular level. However, most of the studies to date have described the cell shortening under the unloaded condition. Using a novel force-length measurement system for single cardiomyocytes, we studied the contractile function of cardiomyopathic hamster myocytes over a wide range of loading conditions. Cardiomyocytes were isolated from the ventricles of eight- to 10-week-old cardiomyopathic (CMP) hamsters (Bio TO-2 strain), as well as control (CTRL) Syrian hamsters. A pair of carbon fibers was attached to both ends of single cardiomyocytes and their contractile characteristics were recorded while changing the after-load by controlling the fiber motion. Under the unloaded condition, the shortening fraction (CMP 9.2+/-0.5% vs. CTRL 10.7+/-0.8%, P=0.06) and maximum shortening velocity (CMP 98.2+/-7.3 microm/s vs. CTRL 147.2+/-6.5 microm/s, P0.05) were decreased in CMP hamster myocytes. The peak force under the isometric condition (CMP 35.8+/-2.2 mN/mm2 vs. CTRL 69.0+/-8.4 mN/mm2, P0.05) and external work (CMP 898+/-130 J/m3 vs. CTRL 3058+/-576 J/m3, P0.05) under physiologically loaded conditions were also decreased, but the differences were more pronounced under the loaded conditions. Calcium transients measured by Indo-1 revealed elevated diastolic level, decreased peak level, and slower diastolic decay in CMP myocytes thus being consistent with the observed contractile dysfunction. These results clearly indicate the importance of the loading conditions in evaluating the contractile function of CMP hamster myocytes, and may provide insights into the mechanism of contractile dysfunction in this disease.

Sarcalumenin (SAR), specifically expressed in striated muscle cells, is a Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) of the intracellular Ca2+ store. By generating SAR-deficient mice, we herein examined its physiological role. The mutant mice were apparently normal in growth, health, and reproduction, indicating that SAR is not essential for fundamental muscle functions. SAR-deficient skeletal muscle carrying irregular SR ultrastructures retained normal force generation but showed slow relaxation phases after contractions. A weakened Ca2+ uptake activity was detected in the SR prepared from mutant muscle, indicating that SAR contributes to Ca2+ buffering in the SR lumen and also to the maintenance of Ca2+ pump proteins. Cardiac myocytes from SAR-deficient mice showed slow contraction and relaxation accompanied by impaired Ca2+ transients, and the mutant mice exhibited a number of impairments in cardiac performance as determined in electrocardiography, ventricular catheterization, and echocardiography. The results obtained demonstrate that SAR plays important roles in improving the Ca2+ handling functions of the SR in striated muscle.

To gain more insight into the mechanism of muscle plasticity in response to mechanical overload, we analyzed the effects of bite -opening (BO, 3 mm increase in the vertical dimension for 2 weeks) and/or a calcineurin (CaN) inhibitor, cyclosporin A (CsA, 10 mg/kg body weight, once daily for 2 weeks, ip) treatment on the myosin heavy chain (MHC I, IIa, IId/x, IIb) mRNA levels, using real-time RT-PCR with specific primers and on the muscle mass in rat masseter. As compared with normal control (n = 6), the BO treatment (n = 6) significantly increased the MHC I (p < 0.05) and the IIa mRNA levels (p < 0.01), and the CsA treatment (n = 6) significantly decreased the MHC I mRNA level (p < 0.01) in association with the significant decrease in the MHC IIb mRNA level (p < 0.05). The BO + CsA treatment (n = 6) significantly increased the MHC IIa mRNA level (p < 0.01) in association with the significant decrease in the MHC IIb mRNA level (p < 0.01), as compared with control. The masseter muscle mass was significantly decreased by either the CsA (p < 0.05) or the BO + CsA treatment (p < 0.001), but slightly increased by the BO treatment. These results suggest that in rat masseter the BO treatment produces not only the up-regulation of MHC IIa mRNA independently of CaN-signaling pathways, but also the MHC mRNA transition from IIa to I and the muscle mass maintenance mainly of type IIb fiber through the CaN-signaling pathways.

To clarify the roles of Ca2+ and crossbridge kinetics in determining the cardiac contraction profile, we analyzed the rate of tension development following nitrophenyl-EGTA photolysis and the rate of relaxation following diazo-2 photolysis in the absence and presence of phosphate (Pi, 5 mM) in rat skinned ventricular trabeculae. The rate of tension development was fitted with a single exponential function. The rate constant (kc) increased not only with an increase in prephotolysis tension (initial activation level) under the same postphotolysis tension (final Ca2+ level), but also with an increase in postphotolysis tension under the same prephotolysis tension. Pi increased kc, though decreased both the prephotolysis and postphotolysis tension greatly. The rate of relaxation was fitted with a double-exponential function. The rate constants of both initial rapid phase (kr1), which was higher than kc, and subsequent slow (kr2) relaxation were almost independent of either the prephotolysis tension or the postphotolysis tension (i.e. the extent of relaxation). Pi increased both kr1 and kr2 by about twofold. These results apparently contradicting both the “steric blocking model” and the “kinetic model”, and can be explained in terms of the changes in number of tension-generating crossbridges through the Ca2+-dependent cooperative thin filament activation/inactivation associated with the Pi-modulated changes in number of tension-generating crossbridges. The thin filament activation kinetics seems to be slower than its inactivation.

Objective: To investigate the functional role of myosin light chain (MLC) isoforms in cardiac muscles, we examined the motor function of two different myosins the structure of which differed only in the MLC. Methods: We purified myosin from atria (A-myosin) and ventricles (V-myosin) of young rats, which contained atrial-type and ventricular-type MLCs, respectively, but having identical α-heavy chain isoform. Actin filament velocity (Vel) was determined in the in vitro motility assay. Average force of myosin molecules ( F ) was estimated and single events of actin–myosin interaction were recorded with the laser trap technique. Results: Vel was slightly higher in A-myosin than in V-myosin, while actin-activated ATPase activity was not different. F , determined from force versus actin filament length relation, was ∼60% higher in V-myosin (3.3 vs. 2.1 pN/μm). The mean duration of isometric force events was longer in V-myosin than in A-myosin (323±13 vs. 294±30 ms, p

The mammalian heart is known to contain only two isoformic myosin heavy chain (MHC) genes, α and β. A previously uncharacterized MHC gene was isolated in Syrian hamster hearts (McCully et al., J Mol Biol 1991). We identified the novel MHC gene as a hamster embryonic skeletal MHC gene based on the developmental stage- and tissue-specific expression pattern: the restricted expression of mRNA to striated muscles was highest in embryonic skeletal muscle and was developmentally down-regulated. We confirmed that the embryonic skeletal MHC gene exhibited higher expression in cardiomyopathic than in normal hamster hearts, and was up-regulated during the development of cardiomyopathy. The sporadic expression was highly localized in the endocardium. The present study identified that a very small number of undifferentiated myogenic cells existed in adult hamster endocardium. Moreover, using RT-PCR, a homologue of embryonic skeletal MHC mRNA was also expressed in human embryonic, but not adult ventricles. Our data provide a new insight into the regulatory mechanisms of MHCs in the cardiomyopathic hamster heart.

An X-ray diffraction experiment on ferret heart muscle was made to examine the relationship between tension and mass transfer from the thick to the thin filament associated with the interaction of cross-bridges with actin. A ferret papillary muscle was electrically stimulated for 8 s in the presence of 5 microM ryanodine to give a tetanus. At different extracellular Ca2+ concentrations (2-20 mM), a linear relationship was found between the tension and the mass transfer. It was concluded that a change in tension caused by altering the extracellular Ca2+ concentration is due to a change in the number of myosin heads bound to actin. This is in contrast to the results obtained on skinned preparations, which showed a markedly nonlinear relationship between the number of heads in the vicinity of the thin filaments and force. It was found that the tension per myosin head is similar in twitch and tetanus of cardiac muscle. Also, a similar fraction of myosin heads is recruited in tetanus of cardiac and skeletal muscles.

To study the effects of diet consistency on the fiber phenotypes of rat masseter (1-70 days of age), the mRNAs of myosin heavy chain isoforms (MHC embryonic, neonatal, I, IIa, IId/x and IIb) were measured in total RNA preparations from masseters of hard-diet group (HDG) and soft-diet group (SDG) by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). With respect to the time course of the transition of each MHC mRNA expressed as a percentage relative to the maximum mean, the soft diet facilitated early (9 days after weaning) expression of IId/x and IIb isoforms, and also a decline in the expression of neonatal and IIa isoforms. The expression of neonatal, IIa and IId/x isoforms at 70 days of age was significantly (P

To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.

There are no published studies on synaptogenesis focusing on the elimination of the superfluous nicotinic acetylcholine receptor (nAChR) outside the neuromuscular junction and the nAChR subunit switch from the embryonic-type (α2βγδ subunits) to the adult-type (α2β∈δ subunits) in mouse tongues. To identify the time course of nAChR subunit elimination and switch, we analyzed the expression levels of a, e, and γ subunit mRNAs, and the immunolocalization of the 8 subunit protein in the mouse tongue and corresponding hind limb. The analysis included the period from embryonic day (E) 11 to the newborn stage. The nAChR elimination and subunit switch began at E15 in the tongue and at E17 in the hind limb. They were nearly complete at birth in the tongue, but not in the hind limb. The early completion of synaptogenesis in the tongue at birth may be related to the early functional demands placed on the tongue, such as suckling and swallowing, immediately after birth.

X-ray diffraction studies were made using synchrotron radiation on ferret right ventricular papillary muscle under three different thyroid states: euthyroidism, hyperthyroidism, and hypothyroidism. The latter two states were induced by treatment with L-thyroxine and methimazole, respectively. The X-ray equatorial reflections were recorded at a time resolution of 10 ms to study the mass movement of myosin cross-bridges from thick to thin filaments. The myosin isomer content was measured by gel electrophoresis which showed that V3 isomer was predominant in euthyroid muscle and 27% of myosin was V1 isomer in hyperthyroid muscle. The intracellular free Ca concentration was measured by using the aequorin method. The peak Ca concentration was similar in all three states, but in the hypothyroid state the time-to-peak was longer and the decay was slower. The time-to-peak of twitch tension was shorter in hyperthyroidism and longer in hypothyroidism than in euthyroidism. The different time courses of twitch tension in different thyroid states accompanied a cross-bridge movement which closely followed the tension development. In hyperthyroidism, the cross-bridge movement significantly preceded tension development, suggesting that hyperthyroid myosin (V1) has a longer latency period between the shift to the vicinity of the thin filament and force development.

To study the effects of mechanical constraints on the Ca2+ affinity of cardiac troponin C, we analyzed the tension and aequorin light (AL) responses to sinusoidal length changes (5–10% of the initial muscle length) in aequorin-injected, tetanized cardiac muscles. The amplitude of the quasi-sinusoidal tension and AL responses decreased with increasing length-perturbation frequency from 0.5 to 1 Hz at 24°C and from 1 to 3 Hz at 30°C. The increase in AL corresponded well to the decrease in tension; likewise, the decrease in AL to the increase in tension and the tension response lagged behind the length change. A further increase in frequency (>1 Hz at 24°C and >3 Hz at 30°C) markedly increased the amplitude of the tension responses but decreased the amplitude of the AL responses. The increase in AL lagged behind the decrease in tension; likewise, the decrease in AL lagged behind the increase in tension, and the tension response led the length change. From previous mechanistic interpretations of the frequency dependence of the amplitude of tension response, we argue that the Ca2+affinity of cardiac troponin C changes in parallel with the active tension (i.e., the number of active cross bridges) but not with the passive tension produced by the length perturbation-induced cross-bridge strain.

To study the effects of increased occlusal vertical dimension on these kinetics, the actin-filament sliding velocity on masseter myosins in an in vitro motility assay and the ATPase activity of masseter myosins from normal (control) and bite-opened (5.6 mm increase in the vertical dimension for 1 week) guinea-pigs were measured. In control myosin preparations, the average value (mean +/- SD, n = 5) for the actin-filament sliding velocity at 25 degrees C was 4.0 +/- 0.3 microns/sec. In bite-opened myosin preparations (n = 5), it was 3.4 +/- 0.3 microns/sec, a significant (p0.01) decrease. Myosin ATPase activity was also decreased significantly (p0.01) from 1.0 +/- 0.1 to 0.7 +/- 0.1 mumol Pi mg per min (mean +/- SD, n = 5) after the bite opening. These results strongly suggest that in guinea-pigs an increase in occlusal vertical dimension for 1 week decreases the turnover rate of actin-myosin interaction in the masseter through changes in the myosin isozyme. These changes may result in a slowing of the rate of detachment of myosin cross-bridges from actin filaments.

To address controversies concerning the effect of beta-adrenergic stimulation on the rate of cross-bridge cycling in cardiac muscle, we measured ca(2+)-induced isometric tension development, unloaded shortening velocity (Vmax) and ATPase activity of demembranated (Triton X-100 skinned) rat right ventricular trabeculae before and after treatment with the catalytic subunit of protein kinase A (PKA), which is known to mimic the action of beta-adrenergic agonists in demembranated preparations. PKA treatment (1 U/microliter, 40 min) shifted the pCa-tension relation to the right from 5.41 to 5.26 at pCa50 (the [Ca2+] required for half maximal steady state tension) without changing the steepness of the pCa-tension relation and the maximum Ca(2+)-activated tension; Vmax, as determined by the slack test, was increased for a given pCa value, despite the reduced level of isometric tension. PKA treatment also shifted the pCa-ATPase activity to the right slightly from 5.47 to 5.40 at pCa50 (the [Ca2+] required for half maximal ATPase activity), but increased the ATPase activity during a given level of steady isometric tension generation, resulting in a 33% increase of the tension cost (ATPase activity/tension). All the results obtained strongly suggest that, in rat right ventricular trabeculae, beta-adrenergic stimulation may increase the rate of cross-bridge cycling by increasing the rate of cross-bridge detachment from actin through a PKA-mediated mechanism, although PKA reduces the Ca(2+)-sensitivity of the contractile system.

Clenbuterol (CB), a selective β 2‐adrenergic receptor (AR) agonist, induces muscle hypertrophy and counteracts muscle atrophy. However, it is paradoxically less effective in slow‐twitch muscle than in fast‐twitch muscle, though slow‐twitch muscle has a greater density of β‐AR. We recently demonstrated that Epac1 (exchange protein activated by cyclic AMP [cAMP]1) plays a pivotal role in β 2‐AR‐mediated masseter muscle hypertrophy through activation of the Akt and calmodulin kinase II (CaMKII)/histone deacetylase 4 (HDAC4) signaling pathways. Here, we investigated the role of Epac1 in the differential hypertrophic effect of CB using tibialis anterior muscle (TA; typical fast‐twitch muscle) and soleus muscle (SOL; typical slow‐twitch muscle) of wild‐type (WT) and Epac1‐null mice (Epac1KO). The TA mass to tibial length (TL) ratio was similar in WT and Epac1KO at baseline and was significantly increased after CB infusion in WT, but not in Epac1KO. The SOL mass to TL ratio was also similar in WT and Epac1KO at baseline, but CB‐induced hypertrophy was suppressed in both mice. In order to understand the mechanism involved, we measured the protein expression levels of β‐AR signaling‐related molecules, and found that phosphodiesterase 4 (PDE4) expression was 12‐fold greater in SOL than in TA. These results are consistent with the idea that increased PDE4‐mediated cAMP hydrolysis occurs in SOL compared to TA, resulting in a reduced cAMP concentration that is insufficient to activate Epac1 and its downstream Akt and CaMKII/HDAC4 hypertrophic signaling pathways in SOL of WT. This scenario can account for the differential effects of CB on fast‐ and slow‐twitch muscles.

As is evident in this review, significant advances have been made in the analysis of crossbridge cycling in cardiac muscle. It becomes apparent that the myocardial crossbridge cycling rate is altered by various factors such as: 1) changes in the intracellular constituents (Ca2+, MgATP, MgADP, Pi, and H+), 2) difference in the type of myosin isozyme, and 3) probably, troponin I, C-protein, and MLC2 phosphorylation. The physiological consequences of an altered crossbridge cycling rate may give rise to the changes in the contraction profile during twitch, tension cost (the ratio of ATPase activity to tension), and the thermal economy (the ratio of heat liberated to tension) of cardiac muscle. These findings and implications should be kept in mind when interpreting cardiac performance under different inotropic states.

This study compares the crossbridge kinetics of intact and skinned preparations from ferret cardiac muscles at 20 degrees C to determine whether skinning causes any alteration in the crossbridge response to an imposed length change. A papillary or trabecular muscle was isolated from the right ventricle, the muscle length adjusted to give the maximum twitch tension (Lmax), and the preparation was subjected to Ba2+ contracture. When steady tension developed, the length of the preparation was perturbed sinusoidally in 19 discrete frequencies, ranging from 0.13 to 135 Hz, and at a small peak-to-peak amplitude (0.25% Lmax). We identified three exponential processes in the sinusodial force-response to the imposed length oscillation, and these were labeled processes B, C, and D in order of increasing speed. A slow process, A, normally present in fast-twitch skeletal muscles, is very small or absent in cardiac muscles. Process B is an exponential delay, and the muscle produces oscillatory work on the forcing apparatus; processes C and D are exponential advances in which the muscle absorbs work. The preparation was chemically skinned and activated in the presence of (mM) CaEGTA 6 (pCa 4.55), MgATP 5, magnesium propionate 1, and phosphate 1, pH 7.0, with ionic strength adjusted to 200 mM with potassium propionate. We found that the crossbridge kinetics were not altered by the skinning procedure. The apparent rate constants extracted from the sinusoidal analysis were nearly identical in Ba2+ contracture (intact preparation) and in Ca2+ activation (skinned preparation), and the Nyquist plots were similar. Because the rate constants changed sensitively with the substrate (MgATP) concentrations, we concluded that the substrate is adequately supplied during Ba2+ contracture in the intact preparation. Our study demonstrates the compatibility of results obtained from an intact and from a skinned preparation.

To characterize the myocardial cross-bridge dynamics in catecholamine-induced positive inotropic state, we studied the effects of adrenaline (6 X 10(-6) M) on the transient central segment length (SL) response to step decrease in tension in rat right ventricular papillary muscle in barium contracture. The time course of this response is thought to reflect the kinetics of actin-myosin interaction. The muscle was released stepwise from the steady contracture tension (Tc) to new steady tension levels (Tr) of varying magnitudes at 22 degrees C. When the tension decrease was less than 0.7 Tc, the SL transient responses comprised, in most cases, four phases. The first phase was a rapid and minute shortening during tension reduction; the second was a slow further shortening; the third, a slow lengthening; and the fourth, an extremely slow shortening toward a new steady length under the new tension. Adrenaline showed almost no effect on Tc and the amplitude of SL transients, but markedly reduced the duration of the second (D2) and third (D3) phases of SL transient regardless of the amplitude of tension reduction. The reduction of duration was 14 +/- 3% in D2 and 26 +/- 5% in D3 at Tr/Tc of 0.84 +/- 0.03 on the average (mean +/- S.D.) in nine preparations. The velocity measured from the quasi-steady SL shortening in the second phase increased with the addition of adrenaline, regardless of the amplitude of tension reduction. The increase in the shortening velocity was 16 +/- 6% (mean +/- S.D., n = 9) at Tr/Tc of 0.18 +/- 0.04. These results suggest that adrenaline increases the rate of cross-bridge cycling in cardiac muscle independent of activation level.

In order to investigate the reflex regulation of the infrahyoid muscle activities induced by sensory signals (chemical and mechanical) from the tongue in the frog, excitatory and inhibitory reflex responses in the infrahyoid muscles (sternohyoid muscle-SH muscle, omohyoid muscle-OH muscle) by both chemical and mechanical stimulation of the tongue were studied using electromyography (EMG) recordings in bullfrogs, Rana catesbeiana. The chemical stimulations (1 mM quinine-HCl, 0.5-1.5 M NaCl) evoked no reflex response in either the SH or the OH muscles, and this result suggested that the infrahyoid muscle activities may not be reflexly regulated by chemical signals from the tongue in the bullfrog. Both the mechanical stimulation of the tongue and the electrical stimulation of the glossopharyngeal nerve evoked excitatory responses in the SH and the OH muscles, bilaterally and contralaterally, respectively. The electrical stimulation of the glossopharyngeal nerve evoked no inhibitory response in the SH, while the stimulation evoked the inhibitory response in the ipsilateral OH. These differences may be related to a difference in the role of these muscles during tongue movement in the bullfrog.

Quick release of the right ventricular papillary muscle of ferrets, injected with aequorin, from Lmax (initial length) to 92% Lmax during twitch response produced an extra-light signal of aequorin. 2,3-Butanedione monoxime (BDM) at 10 mM decreased the peak tension to less than 10% of that in control and the light signal to 70%. In the BDM-treated muscle, the extra-light in response to the quick release did not occur. A quick stretch from Lmax to 103% Lmax, in the presence and absence of BDM, did not cause any changes in the light signal. The results indicate that the extra-light signal in the quick release is tension dependent. The tension reduction by the quick release decreases the affinity of troponin-C (Tn-C) for Ca2+ without affecting Ca2+ handling system in intact cardiac muscle.

To understand the pathophysiology of hereditary cardiomyopathy, we measured the phosphorylation status of regulatory proteins, troponin I (TnI), troponin T (TnT), myosin light chain 2 (MLC2), and myosin-binding protein C (MyBP-C), and the Ca2+-dependence of tension development and ATPase activity in skinned right ventricular trabeculae obtained from cardiomyopathic (TO-2 strain, n = 8) and control (F1B strain, n = 8) hamsters. The Ca2+ sensitivities of tension development and ATPase activity (mean +/- SD) were significantly (P0.0001) higher in the TO-2 strain (pCa50 5.64 +/- 0.04 in tension and 5.65 +/- 0.04 in ATPase activity) than in the F1B strain (pCa50 5.48 +/- 0.03 in tension and 5.51 +/- 0.03 in ATPase activity). No significant differences in their maximum values were observed between TO-2 (40.8 +/- 7.4 mN/mm2 in tension and 0.52 +/- 0.15 micromol/l/s in ATP consumption) and F1B (42.3 +/- 8.5 mN/mm2 in tension and 0.58 +/- 0.41 micromol/l/s in ATP consumption) preparations, indicating that the tension cost (ATPase activity/tension development) in TO-2 was quite similar to that in F1B. The phosphorylation levels of MLC2 and TnI were significantly (P0.01) lower in TO-2 than in F1B. These results suggest that the increase in the Ca2+ sensitivities of tension development and the ATPase activity in TO-2 hearts result from the decreased basal level of TnI phosphorylation, and these features can be considered to produce the incomplete diastolic relaxation and partly improve the systolic function in TO-2 hearts.

Green fluorescent protein (GFP) is widely used as a biologically inert expression marker for studying the effects of transgene expression in heart tissue, but its influence on the contractile function of cardiomyocytes has not yet been fully evaluated. We measured the contractile function of isolated rat ventricular myocytes before and after infection with a recombinant adenovirus expressing GFP (Adv-GFP). Myocytes infected with a non-transgene-containing adenovirus (Adv-Null) or uninfected myocytes (UI) served as controls. Using a carbon-fiber-based force-length measurement system for single cardiomyocytes, we evaluated the contractile function over a wide range of loading conditions including the shortening fraction (%FS) and maximal shortening velocity (Vmax) under the unloaded condition, and isometric force. At 24 hours after infection, nearly 80% of the Adv-GFP-infected myocytes expressed GFP. We found that the %FS and Vmax did not differ among the three groups, however, the isometric force showed a mild, but significant, decrease only in Adv-GFP myocytes (Adv-GFP: 29.1 +/- 4.0 mN/mm2; Adv-Null: 42.8 +/- 6.2 mN/mm2; UI: 47.1 +/- 4.8 mN/mm2; p = 0.03). An evaluation of the contractile function of isolated cardiomyocytes under high load conditions revealed impaired isometric contractility by GFP expression. Adv-GFP expression may not be an ideal control for specific gene expression experiments in myocardial tissue.

One of the most salient characteristics of the heart is its ability to adjust work output to external load. To examine whether a single cardiomyocyte preparation retains this property, we measured the contractile function of a single rat cardiomyocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control. A pair of carbon fibers was used to clamp the cardiomyocyte, attached to each end under a microscope. One fiber was stiff, serving as a mechanical anchor, while the bending motion of the compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezoelectric translator based on adaptive control, we could change load dynamically during contractions. Under unloaded conditions, maximal shortening velocity was 106 ± 8.9 μm/s ( n = 13 cells), and, under isometric conditions, peak developed force reached 5,720 nN (41.6 ± 5.6 mN/mm2; n = 17 cells). When we simulated physiological working conditions consisting of an isometric contraction, followed by shortening and relaxation, the average work output was 828 ± 123 J/m3( n = 20 cells). The top left corners of tension-length loops obtained under all of these conditions approximate a line, analogous to the end-systolic pressure-volume relation of the ventricle. All of the functional characteristics described were analogous to those established by studies using papillary muscle or trabeculae preparations. In conclusion, the present results confirmed the fact that each myocyte forms the functional basis for ventricular function and that single cell mechanics can be a link between subcellular events and ventricular mechanics.

In mammalian hearts, there are two myosin heavy chain (MHC) isoforms, fasttype α-MHC and slow-type β-MHC, and the isoform composition changes in response to mechanical load. In human atria and rodent ventricles, isoform composition shifts from α-to β-MHC under pressure overload. Since α-MHC is related with higher ATPase activity and higher velocity of muscle shortening compared to β-MHC, the redistribution towards β-MHC could be adaptive response to increased mechanical load for better economy. To dissect the molecular mechanism underlying the higher economy of β-MHC, we purified V1 (homodimer of α-MHC) and V3 (homodimer of β-MHC) myosins from rat ventricles and characterized the functional differences in vitro. Both Ca2+-activated ATPase activity and actin filament velocity (VEL) in the in vitro motility assay were twice higher in V1 compared to V3. Ensemble force generated by several myosin molecules was measured with a laser trap. Isometric force per unit length of actin filament, an index of average force of several myosin molecules and time-averaged force of an individual molecule (Fave), was similar between V1 and V3. Displacement under low load and isometric force generated by a single (or a very small number of) myosin molecule(s) were determined by reducing the number of myosin molecules interacting with actin filaments. Both unitary displacement and unitary force were equal between these myosins. Notably, however, the event duration was significantly longer in V3 under both low load and isometric condition compared to V1. Assuming the two-state model of crossbridge, these results suggest that V3 may have proportionally slower kinetics in both attachment and detachment of the crossbridge, which can explain the similar Fave. Importantly, however, to maintain equal level of isometric force, V3 may hydrolyze less number of ATP molecules and thus have better economy than V1.

The mammalian heart is known to contain only two isoformic myosin heavy chain (MHC) genes, alpha and beta. A previously uncharacterized MHC gene was isolated in Syrian hamster hearts (McCully et al., JMol Biol 1991). We identified the novel MHC gene as a hamster embryonic skeletal MHC gene based on the developmental stage- and tissue-specific expression pattern: the restricted expression ofmRNA to striated muscles was highest in embryonic skeletal muscle and was developmentally down-regulated. We confirmed that the embryonic skeletal MHC gene exhibited higher expression in cardiomyopathic than in normal hamster hearts, and was up-regulated during the development of cardiomyopathy. The sporadic expression was highly localized in the endocardium. The present study identified that a very small number of undifferentiated myogenic cells existed in adult hamster endocardium. Moreover, using RT-PCR, a homologue of embryonic skeletal MHC mRNA was also expressed in human embryonic, but not adult ventricles. Our data provide a new insight into the regulatory mechanisms of MHCs in the cardiomyopathic hamster heart.

It is of paramount importance to investigate the relation between the time-dependent change in intracellular Ca2+concentration ([Ca2+]i) (Ca2+transients) and the mechanical activity of isolated single myocytes to understand the regulatory mechanisms of heart function. However, because of technical difficulties in performing mechanical measurements with single myocytes, the simultaneous recording of Ca2+transients and mechanical activity has mainly been performed with multicellular cardiac preparations that give conflicting results concerning Ca2+transients during isometric twitches and during twitches with unloaded shortening. In the present study, we coupled intracellular Ca2+measurement optics with a force measurement system using carbon fibers to examine the relation between Ca2+transients and the mechanical activity of rat single ventricular myocytes over a wide range of load. To minimize the possible load dependence of sarcoplasmic reticulum Ca2+loading, contraction mode was switched at every twitch from unloaded shortening to isometric contraction. During a twitch with unloaded shortening, the Ca2+transients exhibited a higher peak and a higher rate of decay than transients during an isometric twitch. Similarly, when we changed the contraction mode in every pair of twitches, Ca2+transients were dependent only on the mode of contraction. Mechanical uncoupling with 2,3-butanedione monoxime abolished this dependence on the mode of contraction. Our results suggest that Ca2+transients reflect the affinity of troponin C for Ca2+, which is influenced by the change in strain on the thin filament but not by the length change per se.

In order to study the mechanical activity of a single cardiac myocyte under a wide range of load, we have developed a novel force measurement system using carbon fibers. Newly fabricated Graphite Reinforced by Carbon (GRC) fibers greatly facilitate the firm attachment of cell membrane to the fibers. A pair of fibers was attached to both ends of the cell; the rigid fiber as a mechanical ground and the compliant fiber for the strain gauge. By connecting the compliant fiber to the piezoelectric translator and applying the position signal to the driver, we could make the myocyte contract under isometric condition. Feedback control of the system also enabled us to study the relation between work output and the load. This system can be a useful tool in studying the mechanical activity of the cardiac myocyte under genetic as well as pharmacological interventions.

No published study on synaptogenesis in masseter muscle has focused on the shift of nicotinic acetylcholine receptors (nAChRs) from the embryonic type (alpha(2)-, beta-, gamma- and delta-subunits) to the adult-type (alpha(2)-, beta-, epsilon- and delta-subunits) and the elimination of nAChRs outside the neuromuscular junction. To identify the time course of the nAChR transitions in rat masseter muscle between 1 and 63 days of age, the expression of delta-, epsilon- and gamma-subunit mRNAs was analysed by competitive polymerase chain reaction in combination with reverse transcription. The expression of the delta-subunit was high between 1 and 7 days of age, then decreased by 95% (P0.0001) between 7 and 28 days, suggesting that the nAChR elimination occurs during this period. The quantity of the epsilon-subunit increased by approximately 600% (P0.0001) between 1 and 21 days of age, whereas the quantity of the gamma-subunit decreased by 85% (P0.0001) during the same period. This result indicates that the nAChR type shift is terminated at 21 days of age. The feeding behaviour of the rats inevitably changed from suckling to biting after 19 days of age, because they were weaned at that age. As the nAChR type shift was terminated soon after weaning, the termination could be related to the change in feeding behaviour. However, it might also be the case that nAChR elimination is not directly related to the change in feeding behaviour, as the elimination continued at the same rate for 9 days after weaning (from 19 to 28 days of age).