MICRORNA AND GENE EXPRESSION CHANGES UNDERLYING THE PATHOLOGY OF NEMALINE MYOPATHY

Nemaline myopathy (NM) is a clinically and genetically heterogeneous neuromuscular disorder characterized by muscle weakness and hypotonia due to sarcomeric disarray and the formation of rod-like "nemaline" bodies in the muscle fibers. The disease affects about 1 in 50.000 and is caused by mutations in nebulin and α-skeletal muscle actin, while mutations in α-tropomyosin, β-tropomyosin, troponin T1, and cofilin-2 are more rare. Currently no therapies or markers exist. Gene expression profiling has provided insights into the gene expression changes associated with NM in human patients. Furthermore, a number of microRNAs (miRs) have been shown to be specifically dysregulated in muscles from NM patients. Based on these findings, we hypothesized that unique regulatory mechanisms regulated by miRs may underlie the pathology of NM irrespective of the causative mutation. To study this, we took advantage of a transgenic knockin mouse model of NM, expressing a mutant form of α-skeletal actin (Acta1(H40Y)KI) (KI) that develops a severe form of the disease. Interestingly, while KI males exhibit a severe phenotype resulting in death from around 10 weeks of age, females are less severely affected and are fertile. The first aim of our study was to perform gene expression profiling on diaphragm (DIA) and tibialis anterior (TA) muscle of KI mouse and compare the results with previously published gene expression analyses of NM patients and the Tmslow(Met9Arg) Tg (Tg) mouse model of a mild form of NM. The second aim was to compare the gene expression profile of male and female KI mice to provide insights into the molecular basis for the different severity of the disease in males and females. The third aim was to investigate the miR expression profile in KI males at an early and late stage of the disease to study miR expression in relation to the development of the disease. The gene expression study was conducted on 3-week-old males and 10-week-old males and females. The highest number of dysregulated genes were found in 3-week-old males. Between muscles (DIA and TA) only few altered genes were in common, possibly due to a peculiar gene expression pattern of the DIA. Interestingly, 10-week-old females showed a gene expression pattern more similar to 3-week-old males than to 10-week-old males, reflecting a delay in the development of the phenotype. However, although the specific dysregulated genes were different, some pathways were commonly dysregulated under different conditions. The same pathways, i.e. metabolic pathways, were found to be dysregulated in KI and human NM patients, but not in Tg mice. Expression levels of selected miRs that are altered in human NM patients were also altered in KI mice, but not in Tg mice. MiR-381 was highly upregulated and two of its predicted targets were validated in vitro by a luciferase assay: the calcium channel protein TRPM7 and the ubiquitin-related protein Trim63/MURF1. The results of this study indicate that Acta1(H40Y)KI is a good model of NM that recapitulates NM disease from both a phenotypic and genetic point of view. It is a better model for NM than the Tg mouse, showing gene and miR expression patterns more similar to NM patients. The biological meaning of the altered gene expression patterns and their possible regulation by miRs remain to be investigated in more detail.