Cyclosporin A modulates cellular localization of MEF2C protein and blocks fiber hypertrophy in the overloaded soleus muscle of mice

Abstract

The molecular signaling pathway linked to hypertrophy of the anti-gravity/postural soleus muscle after mechanical overloading has not been identified. Using reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analyses, we investigated whether the amounts of myocyte enhancer factor (MEF)2C, MEF2D, and myogenin change in the mechanically overloaded soleus muscle after treatment with the calcineurin inhibitor cyclosporine A (CsA). Adult male ICR mice were subjected to a surgical ablation of the gastrocnemius muscle and treated with either CsA (25 mg/kg) or vehicle, once daily. They were killed at 2, 4, 7, 10, and 14 days post-injury. Mechanical overloading resulted in a significant increase in the wet weight and the cross-sectional area of slow and fast fibers of the soleus muscle in placebo-treated mice but not CsA-treated mice. RT-PCR analysis did not show a marked difference in MEF2C and MEF2D mRNA levels in the overloaded soleus muscle in placebo- or CsA-administered mice. After 2 days of mechanical overloading, we observed co-localization of MEF2C and myogenin in several mononuclear cells under both conditions. These MEF2C-positive mononuclear cells also possessed immunoreactivity for c-Met, a satellite cell marker. At 4 days, mechanical overloading induced marked expression of MEF2C but not MEF2D in the subsarcolemmal region in a group of myotubes and/or myofibers. Such a MEF2C-positive region emerged less often in the hypertrophied soleus muscle subjected to the treatment with CsA. At 7 days, we observed many mononuclear cells possessing both MEF2C and myogenin protein in mice treated with CsA, but not the placebo. Our results demonstrated that CsA treatment modulates the amount and cellular localization of MEF2C protein. The modulation of MEF2C by CsA treatment may inhibit the hypertrophic process in the soleus muscle after mechanical overloading.

Galactokinase Is a Novel Modifier of Calcineurin-Induced Cardiomyopathy in Drosophila

“…The expression of a dominant-negative Mef2 inhibited CanA actinduced cardiac enlargement and overexpressing Mef2 caused cardiac chamber dilation (Van Oort et al 2006). In skeletal muscle, calcineurin activates Mef2 with exercise (Wu et al 2001;Sakuma et al 2008). Mechanistically, calcineurin was found to co-immunoprecipitate with Mef2 and induce activation of Mef2 through dephosphorylation (Wu et al 2001).…”

Cyclosporin A inhibits the growth of neonatal MHC-expressing myotubes independent of NFATc1 and NFATc3 in the mechanically overloaded soleus muscle of mice

“…This dose given to mice or rats reduces calcineurin activity in skeletal muscles by > 50% [32], although the present study did not examine calcineurin activity. In fact, CsA at 25 mg/Kg once daily in our previous study [18] blocked both fiber transformation and hypertrophy of the soleus muscle of mice after mechanical overloading. Therefore, the amount and frequency of CsA treatment employed here would be appropriate.…”

“…Compensatory enlargement of the soleus muscle was induced in one leg of each mouse by surgical removal of the ipsilateral gastrocnemius muscles as described previously [18]. A sham operation was performed on the contralateral leg after the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital.…”

“…Sodium dodecylsulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) (12.5% acrylamide) and Western blotting were performed next as described previously [18]. Proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes (Hybond-ECL Western, Amersham, Arlington Heights, IL).…”

“…A Ca 2+ /calmodulin-dependent protein serine/threonine phosphatase, calcineurin has been implicated as a molecular decoder modulating the differentiated myoblasts that fuse into myotubes [8] and regenera-tion of skeletal muscle fibers [9][10][11][12][13] or enhancing the slower transformation of myosin heavy chain (MHC) and fiber types [14][15][16]. Although several lines of evidence exclude a functional role for calcineurin in the hypertrophy of muscle fibers in vivo [6,16,17], much evidence obtained using several transgenic mice or pharmacological inhibition of calcineurin [13,18,19], indicates a selective influence of calcineurin on the size of the anti-gravity/postural soleus muscle. However, no downstream modulator of calcineurin signaling has been identified.…”