Fig 1.
Experimental procedure, daily consumption of food and water, and body weight.
(A) Dobutamine (DOB) and clenbuterol (CB) was administered once daily for 1 week via intraperitoneal injection (I.P.) at a dose of 2 mg/kg, dissolved in saline. Age-matched control mice (Control) received an identical volume of saline only. (B) In cardiac muscle (CA), the muscle mass per tibial length ratio was significantly increased in the DOB group (**P < 0.01 vs. Control), as well as the CB group (*P < 0.05 vs. Control). (C) In masseter muscle (MA), the muscle mass per tibial length ratio was significantly increased in the CB group (**P < 0.01 vs. Control), but not in the DOB group (D) Typical cross-sections of HE staining of masseter muscle (MA) in the three groups (Control (upper left), DOB (upper right) and CB (lower left)). CSA was significantly increased in the CB group (**P < 0.01 vs. Control), but not in the DOB group (lower right). Scale bars: 50 μm.
Fig 2.
Effects of DOB and CB on fibrosis and apoptosis in cardiac and masseter muscles.
(A-B) Representative images of Masson-trichrome-stained sections of cardiac muscle (A) and masseter muscle (B) in the Control (upper left), DOB (upper right) and CB (lower left) groups. The area of fibrosis was significantly increased in both the DOB-treated cardiac (A) and masseter (B) muscle (*P < 0.05, **P < 0.01 vs. Control), but not in the CB-treated cardiac (A) or masseter (B) muscle (lower right). Scale bars: 50 μm (A) and 100 μm (B). (C-D) Representative images of TUNEL-stained sections of cardiac muscle (C) and masseter muscle (D) in the Control (upper left), DOB (upper right) and CB (lower left) groups. TUNEL-positive nuclei (black arrows) were counted in cardiac muscle (C) and masseter muscle (D) after 1 week of DOB or CB infusion and expressed as percentage of total myocytes (lower right). The number of TUNEL-positive nuclei was significantly increased in both the DOB-treated cardiac muscle (**P < 0.01 vs. Control) and masseter muscle (*P < 0.05 vs. Control), but not in the CB-treated cardiac or masseter muscle. Scale bars: 5 μm (C) and (D). CA; cardiac muscle, MA; masseter muscle.
Fig 3.
Effects of DOB and CB on maximal isometric force and ATPase activity and tension cost.
(A) Maximal isometric force at pCa 4.6 (saturating [Ca2+]) was significantly smaller in the DOB group than in the Control (n = 8 each). (B) Maximal ATPase activity at pCa 4.6 in the DOB group or the CB group showed no significant differences from the Control (n = 8 each). (C) Relationship between ATPase activity and isometric force in the skinned masseter preparations from the Control, DOB, and CB groups. The average ATPase activity values at pCa 6.1, 5.8, 5.5, 5.1 and 4.6 were plotted against the corresponding average force values. The data points of the three groups (open circle, Control; closed circle, DOB; gray circle, CB) were fitted by linear regression as indicated (solid line, Control; dashed line DOB; long dashed line CB). The formulae of the regression lines are y = 4.16x + 74.28, r = 0.91 (Control), y = 4.24x + 84.38, r = 0.91 (DOB), y = 3.56x + 73.37, r = 0.9 (CB), (r: correlation coefficient). Slopes of the regression lines (ATPase activity/isometric force) indicate the tension cost. (D) The average tension cost in the DOB group or the CB group showed no significant difference from the Control (n = 8 each). CA; cardiac muscle, MA; masseter muscle.
Fig 4.
Effects of DOB and CB on Akt and ERK1/2 signaling pathways in cardiac and masseter muscles.
(A) Akt phosphorylation (Ser-473) of cardiac muscle was significantly increased in the CB group (*P < 0.05 vs. Control). It was also increased, though without statistical significance, in the DOB group. (B) Akt phosphorylation (Ser-473) of masseter muscle was significantly increased in the CB group (*P < 0.05 vs. Control), but not in the DOB group. (C) ERK1/2 phosphorylation (Thr-202/Tyr-204) in cardiac muscle was significantly increased in the DOB group (*P < 0.05 vs. Control), but not in the CB group. (D) ERK1/2 phosphorylation (Thr-202/Tyr-204) in masseter muscle was also significantly increased in the DOB group (*P < 0.05 vs. Control), but not in the CB group. CA; cardiac muscle, MA; masseter muscle.
Fig 5.
Effects of DOB and CB on expression of BAX and LC3 in cardiac and masseter muscles.
(A-B) BAX expression was significantly increased in the DOB group, but not in the CB group, in both cardiac (A) and masseter (B) muscles (*P < 0.05 each vs. Control). (C-D) LC3-II expression was significantly increased in the CB group, but not in the DOB group, in both cardiac (C) and masseter muscles (D) (*P < 0.05 or **P < 0.01 each vs. Control). MA; masseter muscle.
Fig 6.
Effects of DOB and CB on mTORC1/C2 and CaMKII signaling pathways in masseter muscle.
(A) In masseter muscle, mTOR phosphorylation on serine 2448, a specific marker of mTORC1 formation, was significantly increased in the CB group (**P < 0.01 vs. Control), but not in the DOB group in masseter muscle. (B) mTOR phosphorylation on serine 2481, a specific marker of mTORC2 formation, was significantly increased in the DOB group (*P < 0.05 vs. Control), but not in the CB group in masseter muscle. (C-D) CaMKII phosphorylation was significantly increased in the DOB group (**P < 0.01 vs. Control), but not in the CB group (C). CaMKII oxidation was also significantly increased in the DOB group (*P < 0.05 vs. Control), but not in the CB group (D). MA; masseter muscle.
Fig 7.
Effects of DOB and CB on Ca2+ homeostasis and PLN phosphorylation in masseter muscle.
(A) NFATc1 phosphorylation on serine 259 was significantly greater in masseter muscle of the DOB group compared with the control group (*P < 0.05 vs. Control), but this was not the case in the CB group. (B) The calculated μ-calpain fractional activation index (i.e., percentage of autolysed calpain) was significantly increased in the DOB group (**P < 0.01 vs. Control), but not in the CB group. (C) PLN phosphorylation (Ser-16) was significantly increased in the DOB group (*P < 0.05 vs. Control), but not in the CB group. (D) PLN phosphorylation (Thr-17) was significantly increased in the DOB group (*P < 0.05 vs. Control), but not in the CB group.
Fig 8.
Schematic illustration of the proposed roles of β-AR signaling in masseter muscle.
This scheme illustrates the proposed relationship of β1-AR and β2-AR signaling in masseter muscle. Chronic β1-AR stimulation with DOB induces muscle fibrosis and apoptosis with the increase of ERK1/2, CaMKII and mTORC2 activation (left). Chronic β1-AR stimulation with CB induces muscle hypertrophy and autophagy with increase of mTORC1 activation and LC3-II expression (right). Solid black lines represent findings in this study and solid grey lines represents findings reported previously (Lynch et al., 2008 [93], Ohnuki et al. 2014 [11], Ohnuki et al 2016 [12], Okumura et al. 2014 [22]).