1. Ca2+ activates proteolysis-related enzyme Ca2+ activates mTOR pathway
ABSTRACT
In vivo intracellular Ca2+ dynamics over 7 days following novel eccentric contractions in rat skeletal muscle
In vitro studies have supported an important role for intracellular calcium ion concentration ([Ca2+]i) as an intracellular signal for protein synthesis and degradation. Eccentric contractions (ECC) facilitate Ca2+ influx from the extracellular space via stretch-activated channels and cause high-levels of [Ca2+]i accumulation. Accumulated [Ca2+]i activates proteolysis-related enzymes and induces muscle damage. A few days after ECC, the damaged muscle fiber shifts from this proteolytic to a regenerative phase. Whether there is any temporal and/or spatial correspondence between [Ca2+]i accumulation and structural damage/repair during recovery from ECC is unknown. PURPOSE: Specifically, we tested the hypothesis that there would be a dynamic pattern of [Ca2+]i accumulation post-ECC that would relate temporally to the damage-recovery cycle. METHODS: In anesthetized adult Wistar rats, the tibialis anterior muscles (TA) were subjected to unexercised controls (CONT) and ECC (5 sets of 40 contractions). After 1day (1D), 3 days (3D), and 7 days (7D) of ECC, the TA was loaded with ratiometric dye Fura-2 AM. We used the 340/380 nm ratio to analyze alterations in [Ca2+]i by in vivo fluorescence imaging. After in vivo observations, the TA muscles were dissected to identify the histological features of the damage-to-regeneration cycle. RESULTS: After ECC, there was profound swelling at 1D followed by infiltration at 3D and regeneration at 7D (i.e. appearance of central nucleus). The mean [Ca2+]i was significantly increased after ECC at 1D (1.47 ± 0.01) and 7D (1.35 ± 0.01) but not at 3D (1.25 ± 0.01) compared with CONT (1.22 ± 0.02). Whereas there was a heterogeneous [Ca2+]i accumulation pattern evident among fibers it was interesting that [Ca2+]i oscillated significantly less the 30 minute observation period at 3D and 7D (mean variation range:3D; 0.05 ± 0.003, 7D; 0.07 ± 0.002) than at other time (CONT; 0.09 ± 0.004, 1D; 0.08 ± 0.004). CONCLUSION: We determined that: 1. There were peaks of [Ca2+]i accumulation during both the swelling (1D) and regeneration (7D) phases. 2. The edematous fibers at 1D and regenerative fibers at 7D evidenced an oscillatory [Ca2+]i pattern. These profiles of [Ca2+]i accumulation may be key to controlling the extended pattern of protein synthesis and degradation that characteristically follows novel ECC.
Ayaka Tabuchi1, Hideki Shirakawa1, David C Poole, FACSM2, Yutaka Kano1.
1. Department of Engineering Science, University of Electro-Communications, Chofu, Tokyo, Japan.
2. Departments of Anatomy, Physiology and Kinesiology, Kansas State University, Manhattan, KS.
INTRODUCTION
RESULTS
In vitro studies have supported an important role for intracellular calcium ion concentration ([Ca2+]i) as an intracellular signal for protein synthesis and degradation (Ref.1). Eccentric contractions (ECC) facilitate Ca2+ influx from the extracellular space via stretch-activated channels and cause high-levels of [Ca2+]i accumulation (Ref.2). Accumulated [Ca2+]i activates proteolysis-related enzymes and induces muscle damage (Ref.3). A few days after ECC, the damaged muscle fiber shifts from this proteolytic to a regenerative phase (Ref.4). Whether there is any temporal and/or spatial correspondence between [Ca2+]i accumulation and structural damage/repair during recovery from ECC is unknown.
0day
1day
3days
7days
ECC
100µm
(A)
(B)
(C)
(D)
Ca2+ activates proteolysis-related enzyme
Ca2+ activates mTOR pathway
Fig. 1 The relationship Ca2+ and muscle fiber adaptation after ECC (Hypothesis).
(E)
(F)
(G)
(H)
100µm
Fig.2 Representative data of fluorescence image (A-D) and HE stain image (E-H).
PURPOSE
(B)
(C) 30
(min)
Ratio
mean:(A)
Δ:(B)
(A)
Fiber 1
Using in vivo bioimaging, we tested the hypothesis that there would be a dynamic pattern of [Ca2+]i accumulation post-ECC that would relate temporally to the damage-recovery cycle.
Fiber 2
Fiber 3
METHODS
Animals
Wistar male rats (9 ~ 14 weeks, N = 33), Tibialis anterior (TA) muscles.
Groups
(1) No ECC (CONT, n=5), (2) 1 day after ECC (1D, n=7) (3) 3 days after ECC (3D, n=6) (4) 7 days after ECC (7D, n=7)
Fig.3 [Ca2+]i profile after ECC at 1, 3 and 7 days.
(A) Mean value observing of 30 min.　(B) Ratio alteration range observing 30 min. (C) The relationship of mean and range. Values shown are mean ± S.E. *P<0.05 vs. CONT.
Key Points
Mean [Ca2+]i increased at 1D and 7D (Fig 3-A) each accompanied with edema and central nuclei.
Relationship was found in mean value and fluctuation range after ECC (Fig 3-C) .
[Ca2+]i showed small fluctuation during observation period at 3D and 7D compared to CONT (Fig 3-B) .
CONCLUSION
Eccentric Contractions
Under anesthetized, tibialis right tibialis anterior muscles were subjected to Eccentric Contractions (ECC). The TA muscle was stimulated (5-10V, 100Hz, stimulus duration 700ms) every 3 sec for 2 min (40 times) through surface electrode with electromotor induced plantar flexion.
There were peaks of [Ca2+]i accumulation during both the swelling (1D) and regeneration (7D) phases. The edematous fibers at 3D and regenerative fibers at 7D evidenced a less oscillatory [Ca2+]i pattern. These profiles of [Ca2+]i accumulation may be key to controlling the extended pattern of protein synthesis and degradation that characteristically follows novel ECC.
Muscle preparation, Microscopy & Image analysis
The TA muscle was gently exteriorized without loss of circulating blood flow. After loading Fura2-AM / KHB solution (20 µM, Dojindo) for 60 min to tibialis anterior muscles, the fluorescence image were captured with excitation at 340 and 380 nm and emission at 510 nm. Images were converted to 340/380 ratio image, and the ratio image data indicating [Ca2+]i were observed for 30 min.
REFERENCE
Hematoxylin and Eosin staining:
At the end of the Ca2+ imaging experiments, TA muscles were resected under anesthesia and blocks were frozen rapidly in isopentane cooled in liquid nitrogen. Serial 10-µm sections were made with a at -20°C and were stained for hematoxylin and eosin (HE).
1. Tu et al. Cell Calcium 　　　　　　　　　 2. Sonobe et al. Am J Physiol Regul Integr Comp Physiol 　　
3. Zhang et al. J Appl Physiol Sudo & Kano. J Physiol Sci