Kouichi Shiozawa

Adrenaline increases the rate of cross-bridge cycling in rat cardiac muscle

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.

Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

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.

Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2‐adrenoceptor stimulation

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. Chronic stimulation of β2‐adrenoceptor (β2‐AR) with clenbuterol (CB), a selective β2‐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. 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. 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. We conclude that Epac1 induces β2‐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.

Effects of Addition of Water on Masticatory Behavior and the Mechanical Properties of the Food Bolus

Abstract To elucidate the effects of water on the mechanical properties of the food bolus, the texture of the bolus was assessed. Thirteen adult subjects were instructed to chew 6 g of two test foods, a biscuit and rice cake, with and without water (2 mL). In the early, middle and late stages of mastication, subjects were instructed to spit the food bolus into a cup. Texture parameters (hardness, adhesiveness and cohesiveness) of the bolus were identified by texture profile analysis. Addition of water significantly ( p >0.001) decreased the number of chewing strokes (NCS) until swallowing for both test foods. Hardness of each of the two test food boluses decreased during mastication regardless of the presence or absence of water. Adhesiveness and cohesiveness of the biscuit bolus increased, whereas those of the rice cake bolus decreased regardless of the presence or absence of water. There were no significant differences in the three texture parameters immediately before swallowing for both food boluses with and without water, although the NCS until swallowing decreased during mastication with water. These results suggest that the presence of water and saliva accelerates bolus formation for swallowing the biscuits and rice cakes studied.

Fragmentation of a Viscoelastic Food by Human Mastication

Fragment-size distributions have been studied experimentally in masticated viscoelastic food (fish sausage). The mastication experiment in seven subjects was examined. We classified the obtained re...

Cardiac overexpression of Epac1 in transgenic mice rescues lipopolysaccharide-induced cardiac dysfunction and inhibits Jak-STAT pathway

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the β-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.

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

Clenbuterol (CB), a selective β2‐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 β2‐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.

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-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)-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.

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.

Effects of Posterior Tooth Occlusion on the Reflex Response of the Human Masseter Muscle.

本研究では, 3種の刺激方法 (a. 上顎右側中切歯へのtapping刺激, b. 上顎右側中切歯唇側歯肉への電気刺激, c. オトガイ部へのtapping刺激) で咬筋に誘発された反射性筋活動が, 臼歯部咬合によって抑制されるか否かを検討した。その結果, 3種の刺激で咬筋に誘発された反射性筋活動は, 臼歯部咬合によっていずれも明らかに抑制された。また, この臼歯部咬合による咬筋筋活動の抑制は, 咬合している上下顎臼歯歯肉の浸潤麻酔後には完全に消失した。したがって, この臼歯部咬合による咬筋筋活動の抑制は, 咬合している臼歯部歯根膜中に存在する機械的受容器からの求心性信号によって生じたことが示唆された。