FIG. 2.

Effect of recombinant Grp94 expression on myofiber CSA of unloaded soleus muscles. Bar: 100 μm. *p<0.01; **p=0.003; ***p=0.001. (A) Visualization of myofibers transfected with Grp94 cDNA (pT94) or with the empty vector (pT) by means of GFP fluorescence (sarcoplasmic fluorescence). Transverse cryosections of ambulatory (A) and unloaded (U) soleus muscles were counterstained with α-sarcoglycan immunolabeling (sarcolemmal fluorescence) to visualize untransfected fibers for comparison of myofiber CSA. (B) Myofibers transfected with pT94 express recombinant Grp94. Serial consecutive transverse cryosections of a representative U soleus muscle were labeled with indirect immunoperoxidase (dark staining) using anti-GFP antibodies, anti-Grp94 mAb 3C4, which recognizes only recombinant Grp94, and anti-ɛMy mAb, which identifies regenerating myofibers (indicated by arrows). Note that recombinant Grp94 is expressed in adult GFP positive myofibers. (C) Recombinant Grp94 expression attenuates CSA decrease of unloaded myofibers. Bars and error bars correspond to mean and SE values of CSA of untransfected, and pT94 and pT transfected myofibers. At least 30 myofibers (GFP+and GFP−, each) were evaluated for a same muscle, which was considered as a single experimental unit. n indicates the number of muscles evaluated in each group. Unloading significantly decreased CSA compared to A myofibers. Asterisk indicates the presence of significant difference versus CSA values of untransfected fibers of the same pT94 transfected muscles and versus those of pT-transfected and untransfected fibers of muscles obtained from U rats matched on body weight (Within-subject ANOVA and Bonferroni post-hoc analysis). (D) Myofiber CSA distribution from seven pT94 U solei. Relative frequency histogram of CSA values of Grp94-transfected fibers (n=662) and of untransfected ones (n=480). (E) Recombinant Grp94 expression attenuates unloading-induced CSA decrease of both slow and fast myofibers. Bars and error bars correspond to mean and SE values of CSA of pT94 transfected and untransfected myofibers of unloaded muscles. At least 50 myofibers (GFP+ and GFP−, each) were evaluated for the slow fiber type in a same muscle, which was considered as a single experimental unit. (Paired Student's t-test). At least 20 myofibers (GFP+ and GFP−, each) were evaluated for the fast fiber type (Paired Student's t-test). ANOVA, analysis of variance; CSA, cross-sectional area; GFP, green fluorescent protein; mAb, monoclonal antibody; My, myosin heavy chain; pT, empty vector codifying for GFP; pT94, bicistronic vector codifying for recombinant Grp94 and GFP. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars