Key pointsr Epac (exchange protein directly activated by cyclic AMP (cAMP)), a PKA-independent cAMP sensor, plays important roles in multiple cellular processes, but its role in the pathogenesis of skeletal muscle hypertrophy and myosin heavy chain (MHC) transition is poorly understood.r Chronic stimulation of β 2 -adrenoceptor (β 2 -AR) with clenbuterol (CB), a selective β 2 -AR agonist, induced masseter muscle hypertrophy in wild-type (WT) mice, but not in Epac1-null mice (Epac1KO), even if slow-to-fast MHC isoform transition was similarly induced by CB treatment in both WT and Epac1KO.r Disruption of Epac1 inhibited development of masseter muscle hypertrophy concomitantly with decreased phosphorylation of Akt and its downstream molecules 70 kDa ribosomal S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1, and also, in parallel, glycogen synthase kinase-3β.r Disruption of Epac1 decreased histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy.r We conclude that Epac1 induces β 2 -AR-mediated masseter muscle hypertrophy without influencing slow-to-fast MHC isoform transition, probably via activation of Akt and its downstream molecules and increase of CaMKII-mediated HDAC4 phosphorylation.Abstract The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β 2 -AR and that in the cardiac muscle is β 1 -AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β 2 -AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β 2 -AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.

In skeletal muscle, the major isoform of β-adrenergic receptor (β-AR) is β 2 -AR and the minor isoform is β 1 -AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the β-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic β 1 - or β 2 -AR activation with a specific β 1 -AR agonist, dobutamine (DOB), or a specific β 2 -AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic β 1 -AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic β 2 -AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic β 1 -AR stimulation did not induce muscle hypertrophy, but did induce fibrosis and apoptosis concomitantly with increased levels of p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), calmodulin kinase II (Thr-286) and mammalian target of rapamycin (mTOR) (Ser-2481) phosphorylation. On the other hand, chronic β 2 -AR stimulation in masseter muscle induced muscle hypertrophy without histological abnormalities, as in the case of cardiac muscle, concomitantly with phosphorylation of Akt (Ser-473) and mTOR (Ser-2448) and increased expression of microtubule-associated protein light chain 3-II, an autophagosome marker. These results suggest that the β 1 -AR pathway is deleterious and the β 2 -AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness.

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.

BackgroundGlucocorticoid 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.MethodologyWe 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 ConclusionMasseter 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.

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. Abbreviations AF Atrial fibrillation BO Bite-opening β-AR Beta-adrenergic receptor AC Adenylyl cyclase ATP Adenosine triphosphate cAMP Cyclic adenosine monophosphate Epac Exchange protein activated by cAMP Control Control group Pro + BO BO plus propranolol treatment group NS Not significant BW Body weight ECG Electrocardiogram NE Norepinephrine TUNEL Terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-deoxyuridine triphosphate (dUTP) nick-end labeling 8-OHdG 8-Hydroxy-2′-deoxyguanosine CaMKII Calmodulin-dependent protein kinase II Ca 2+ Calcium oxidized-CaMKII Ox-CaMKII RyR2 Ryanodine receptor 2