Diaphragm Assessment in Mice Overexpressing Phospholamban in Slow‐Twitch Type I Muscle Fibers

Abstract

Phospholamban (PLN) is a small transmembrane protein that regulates the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump through direct physical interactions. Recent work published by our laboratory revealed that when PLN was transgenetically overexpressed in mice (PlnOE), the postural hind limb muscles presented a phenotype for human Centronuclear Myopathy (CNM). Specifically, PlnOE soleus and gluteus minimus muscles displayed: centrally located oxidative activity, a predominance of hypotrophied type I fibers, and an abundance of central nuclei. To further assess this novel model for CNM, we examined the diaphragm muscles of PlnOE mice, since studies assessing respiratory or diaphragm function in animal models of CNM were limited. Interestingly, our unpublished results suggest that unlike the soleus and gluteus minimus, diaphragm muscles did not display the main characteristics of CNM. Rather than exhibiting type I fiber predominance, the diaphragm muscles exhibited lower type I and greater type II fiber counts, which is of importance since the PlnOE mice were designed to overexpress PLN specifically in their type I fibers. Here, we sought to determine why the PlnOE diaphragm failed to exhibit type I fiber predominance similar to that seen in the soleus and gluteus minimus muscles. Specifically, we focused on calciuneurin (CnA), a Ca2+ ‐dependent phosphatase, and its downstream effector target, nuclear factor of activating T‐cell (NFAT), that upon dephosphorylation by CnA, translocates to the nucleus and acts as a transcription factor for myosin heavy chain I (MHCI). We found that CnA content was significantly higher in both soleus (p=0.02) and gluteus minimus (p=0.05) muscles of PlnOE mice compared to wild type animals. Corresponding with this, nuclear NFAT levels were significantly increased in the gluteus minimus (p=0.01) and trending towards a significant increase in soleus muscle (p=0.07). In contrast, there were no differences between WT and PlnOE in CnA (p=0.23) or nuclear NFAT (p=0.42) content in diaphragm, which suggests the differences in fiber type transition and the CNM phenotype across muscles may be associated with the CnA‐NFAT pathway and calcium signaling. Together these data suggest that the diaphragm of the PlnOE mice may have some undetermined protective mechanism for better regulating cytosolic calcium levels and fiber type expression. Uncovering the mechanisms behind this resistance within the diaphragm of PLNOE mice may aid in exploring new therapeutic strategies for CNM.