Effects of occlusal disharmony on susceptibility to atrial fibrillation in mice.

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 ß-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.

Effects of occlusal disharmony on cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage in mice.

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 ß-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.

Role of ß-adrenergic signaling in masseter muscle.

In skeletal muscle, the major isoform of ß-adrenergic receptor (ß-AR) is ß2-AR and the minor isoform is ß1-AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the ß-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic ß1- or ß2-AR activation with a specific ß1-AR agonist, dobutamine (DOB), or a specific ß2-AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic ß1-AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic ß2-AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic ß1-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 ß2-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 ß1-AR pathway is deleterious and the ß2-AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness.

Effects of chronic Porphyromonas gingivalis lipopolysaccharide infusion on skeletal muscles in mice.

Periodontitis, which is caused by various oral organisms, predominantly affects adults, and is one of the main causes of tooth loss, as well as leading to progression of numerous systemic diseases. However, its relationship to sarcopenia (aging-associated degenerative loss of skeletal muscle mass and function) remains unclear. The aim of this study was to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on skeletal muscle in mice, and to establish the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) for 4 weeks. This treatment significantly decreased the weight of fast-twitch skeletal muscles (masseter and tibialis anterior muscles), but not that of slow-twitch skeletal muscle (soleus muscle). The area of fibrosis was significantly increased in masseter muscle, but remained unchanged in the other two muscles. The number of apoptotic myocytes was significantly increased (approximately eightfold) in masseter muscle. These data suggest that persistent subclinical exposure to PG-LPS might reduce the size of fast-twitch skeletal muscle, but not slow-twitch skeletal muscle. Masseter muscle appears to be especially susceptible to the adverse effects of PG-LPS, because muscle remodeling (muscle fibrosis and myocyte apoptosis) was induced solely in masseter muscle. Thus, periodontitis might be one of the major causes of oral sarcopenia.

Relationship between bite size per mouthful and dental arch size in healthy subjects.

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.

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in ß-arrestin 2-Akt signaling and dopaminergic behaviors.

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)-ß-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 ß-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with ß-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 ß-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-ß-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 ß-arrestin-dependent, G protein-independent signaling.

Role of phosphodiesterase 4 expression in the Epac1 signaling-dependent skeletal muscle hypertrophic action of clenbuterol.

Clenbuterol (CB), a selective ß2-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 ß-AR We recently demonstrated that Epac1 (exchange protein activated by cyclic AMP [cAMP]1) plays a pivotal role in ß2-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 ß-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.