Skeletal muscle degeneration and loss of function is at the basis of Duchenne muscular dystrophy (DMD) pathology. Our work focuses on understanding differences between normal, wild-type mouse muscle and the same muscle in the mdx mouse, a spontaneous mutant mouse line that is an animal model for DMD. The differences we study affect muscle microtubule organization. Normal mouse muscles have a periodic grid-like microtubule network, whereas mdx mouse muscles have a disordered, denser network. We still understand little of muscle microtubule organization despite the essential and diverse roles played by microtubules in all mammalian cells and their dramatic reorganization at every stage of muscle development. Furthermore, physiological and pharmacological research on mdx muscle, complemented by RNAseq of human DMD samples, has led others to claim that microtubules underlie mdx and DMD pathology. Previously we showed that microtubules are dynamic, constantly growing, and that their path is aligned with that of the protein dystrophin, which is lacking in mdx and DMD and directly interacts with microtubules. However, a dystrophin construct lacking the microtubule binding site was able to rescue microtubules in a transgenic mdx mouse line, casting doubt on the overall role of dystrophin in microtubule organization. We decided to search for other molecules potentially responsible for the differences in microtubule organization. Microtubules are assembled from linear chains of alpha- and beta-tubulin dimers. Both alpha- and beta-tubulins have 8 to 10 isotypes, allowing for structural and possibly functional diversity. We noticed that the RNA for one of the beta-tubulin isotypes, tubb6 (beta 6 class V), is most increased in DMD compared to normal muscle; furthermore, overexpression of this ubiquitous, minor beta-tubulin isoform modifies the microtubule network of proliferating cells (Bhattacharya et al. 2011, Mol Biol Cell, 22, 1025-34). Tubb6 thus appeared worth investigating. Last year we reported that tubb6 is remarkably increased in mdx compared to wild-type mice (from 3- to 20- fold depending on specific muscle). Additionally, overexpression of a GFP construct of tubb6 causes distortion and density increase of the microtubule network in WT mouse muscle. In comparison, overexpression of the same GFP construct of another beta tubulin, TUBB5, caused no modification of the microtubule network. When tubb6 was decreased or suppressed in mdx muscle by shRNA treatment, the fibers expressing the shRNA --but not a non-specific shRNA or an RNA against TUBB5-- showed a nearly normal microtubule network. Thus, manipulating the level of tubb6 allows us to modify the organization of muscle microtubules, regardless of the presence or absence of dystrophin, suggesting that tubb6 plays a role in muscle microtubule organization and in the microtubule alterations of mdx. But why would tubb6 be increased in mdx and DMD, if it is toxic to muscle? We found some hints by analyzing changes in tubb6 expression during muscle development: we found out that tubb6 increases more than 2-fold during differentiation of the mouse muscle cell line C2. It is therefore possible that tubb6 plays a useful role during the muscle regeneration that takes place in muscle diseases such as DMD and in the mdx mouse, since regeneration replays the events of differentiation. In support of this hypothesis, we found that tubb6 in mdx muscle is not uniformly distributed but is highly concentrated in myotubes and a subset of muscle fibers in regeneration areas. To confirm a link between tubb6 expression and regeneration, we collaborated with the Mammen laboratory in NIAMS and used an experimental model of acute muscle regeneration in which cardiotoxin is injected in the tibialis anterior (TA) mouse muscle. We found that the level of tubb6 RNA increases sharply, with a time-course matching that of other molecules involved in regeneration: the satellite cell marker Pax7, the differentiation marker myogenin, and the perinatal myosin heavy chain MyH3. We also observed that the increase in tubb6 in a number of mouse lines rescued from mdx by transgenic expression of dystrophin constructs, is linked to the degree of muscle regeneration that takes place in that mouse model. The change in microtubule organization observed in mdx muscle fibers is therefore not a simple direct consequence of the lack of dystrophin, but is linked to muscle regeneration. This work is presently under review for publication. This year has also seen the conclusion of a collaboration with the laboratory of Thomas Jensen (Copenhagen) and publication of the results obtained by Thomas's graduate student, Jonas Roland, who spent a few months in our laboratory to measure Fluorescence Recovery after Photobleaching (FRAP) of a GFP-beta-actin construct expressed in mouse muscle fibers. The results showed that muscle stimulation by insulin, contrary to an earlier proposal, does not cause an increase in beta-actin mobility, helping to clarify the mechanism of muscle insulin activation.
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