Calcineurin Mediated Slow Muscle Fiber Program In Hindlimb Suspension And Reloading Conditions

Abstract

Calcineurin (CaN) signaling pathway has been implicated in the transcriptional regulation of slow muscle fiber genes and in muscle hypertrophy. We used the hindlimb suspension (HS) and reloading model with or without administration of FK506, a specific inhibitor of CaN, to elucidate the hypothesis that CaN regulates muscle regrowth and fiber type transformation toward a slow profile during reloading conditions. PURPOSE Our aim was to investigate the functional role of CaN as a regulator of muscle growth and/or muscle fiber type under conditions of recovery from inactivity. METHODS Female ICR mice (8 weeks of age, 28–32 g) were used. To examine the effects of HS and reloading on skeletal muscle fiber size and muscle fiber type, animals were designated to 8 weeks of HS and subsequent reloading for 4 weeks. During reloading, animals were treated with FK506 (Fujisawa, Japan) by intraperitoneal administration (3–5 mg/kg/day). After each experimental period, slow-twitchdominant antigravitational soleus muscle was analyzed. Myosin heavy chain (MHC) isoform composition was analyzed with SDS-PAGE. Fiber cross sectional area was determined with immunohistochemical analysis. Expression level of CaN signaling factors were assessed by Western blotting analyse. RESULTS HS treatment resulted in obvious muscle atrophy and slow-to-fast fiber-type transformation in the mouse soleus muscle (decrease in MHC I compositon and new expression of MHC IIb in HS group). Subsequent reloading for 4 weeks following HS induced muscle regrowth and fiber-type reversion toward a slow profile. FK506 administration prevented this kind of reloading-induced transformation of muscle fiber type. Furthermore, we confirmed that FK506 administration attenuated maintenance of fiber cross-sectional area and reloading-induced fiber regrowth, specifically in slow-type muscle fibers. Unexpectedly, we observed an increase in protein contents of CaN and its downstream effectors after HS, although the muscle fiber type transformed toward a fast profile and obvious muscle atrophy was induced. CONCLUSIONS CaN signaling pathway is required for the slow-type muscle fiber program under suspension and reloading conditions. We also confirmed that CaN signaling factors were activated in the soleus muscle under suspended conditions. These results imply that the CaN signaling pathway may have undefined opposing roles in the regulation of muscle fiber type and size under these conditions. Supported by Grants #15200048 and in part by the Nishihira/Tsukuba Project of COE (Center of Excellence) from the Japan Ministry of Education, Culture, Sports, Science and Technology.