The Effects of a Calcineurin Inhibitor on Muscle Fibre Type and the Pathology of Centronuclear Myopathy in PlnOE Mice
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The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for pumping calcium from the cytosol back into the lumen of the sarcoplasmic reticulum post contraction. SERCA activity can be reduced by an inhibitory protein, phospholamban (PLN), which binds directly to the pump. Interestingly, overexpressing PLN in transgenic mice (PlnOE) creates a replica of centronuclear myopathy (CNM). CNM is a grouping of rare congenital diseases with three common muscular abnormalities: abundant central nuclei, centrally aggregated oxidative activity and type 1 fibre predominance. Calcineurin (CnA) is a Ca2+-calmodulin dependent serine-threonine phosphatase, which is known to activate a signalling cascade leading to slow-twitch fibre gene expression. Upon activation by excess Ca2+, CnA dephosphorylates nuclear factor of activated T-cells (NFAT) proteins, which then translocate to the nucleus where they act as transcription factors for slow-twitch fibre promotion. Western blotting of muscle lysates from PlnOE mice showed increased CnA and nuclear NFAT content in slow twitch muscle, soleus and gluteus minimus, while no differences were found in the diaphragm which contains much less CNM disease symptoms. Therefore, this study examined the hypothesis that CnA activation may be driving the predominance of type I fibres and may be targeted as a potential therapeutic strategy to alleviate the CNM phenotype in PlnOE mice. Here, developing (4 week old) and adult (4-6 month old) PlnOE mice were treated with the CnA inhibitor, cyclosporin A (CsA), at 25mg/kg body weight twice a day for two weeks. Soleus muscles from vehicle and CsA treated mice were excised for histology and immunofluorescence to assess the CNM phenotype, Western blotting for protein content and whole muscle contractility to assess muscle function. CsA treatment blunted type I fibre predominance and promoted hybrid and type II fibres in both age groups. CsA treated adult PlnOE mice were also found to have increased force, decreased fibrosis, reduced PLN and sarcolipin content. CsA treatment did not eliminate central aggregations of oxidative activity or have an effect on the percentage of fibres displaying central nuclei suggesting these CNM features may not be connected to CnA activity. CnA content was reduced in the CsA treated animals but changes in CnA activity in this study are unclear as western blotting showed increased activity while immunofluorescence displayed decreased activity and less nuclear NFAT. Surprisingly, CsA treatment had minimal effects in developing PlnOE mice with only small difference in fibre type observed between vehicle and CsA animals. In summary, CsA treatment provides modest improvements in fibre type, cellular phenotype and muscle function in adult PlnOE animals however it has little effect on younger mice developing the disease. Targeting fibre type as a potential treatment for CNM merits further research to determine the appropriate timing for treatment, other potential pharmaceuticals that do not disrupt immune signalling or hypertrophy and the underlying mechanism by which the overexpression of PLN causes CNM.
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Paige Chambers (2017). The Effects of a Calcineurin Inhibitor on Muscle Fibre Type and the Pathology of Centronuclear Myopathy in PlnOE Mice. UWSpace. http://hdl.handle.net/10012/11281