Expression of neonatal splice isoforms in adult skeletal muscle has become a classic molecular feature of DM1: more than 21 alternative splicing events have been shown to be misregulated in adult DM1 heart, skeletal muscle, or brain tissues.9, 15
The mechanisms of splicing misregulation in DM1 are directly linked to the effects of the CUGexp
RNA on the functions of MBNL and CELF protein families, RNA binding proteins that normally regulate splicing during development. The expression of CUGexp
RNA causes a loss of MBNL1 function by sequestration and a gain of CELF1 function by stabilizing the protein via protein kinase C-mediated hyperphosphorylation.37
The identification of some of the same splicing abnormalities in mouse models of neuromuscular disorders generated by different mutations prompted us to examine the relationship between degenerating/regenerating skeletal muscle and altered splicing patterns.
In this study, we examined eight alternative splicing events in six independent mouse models of muscular dystrophy and muscle injury including EpA960/HSA-Cre-ERT2
(+ tam), 129P1/ReJ-Lama2dy
, cardiotoxin injury, and notexin injury. The results demonstrated that all of the alternative splicing events that are misregulated in skeletal muscle tissue from one or more DM1 mouse model(s) and human DM1 patients5, 15, 38
are also misregulated in non-DM1 mouse models of skeletal muscle damage and degeneration.
Each of the six mouse models displayed histological evidence of degeneration and regeneration with varying levels of severity. Importantly, the degree of misregulation of the alternative splicing events correlated with the degree of degeneration/regeneration observed histologically. In addition, the severity of the histopathology was consistent with the eMHC expression level by Western blot analyses. These results indicate that while splicing misregulation can reflect a primary disease process, analysis of splicing is complicated by overlapping splicing abnormalities resulting from muscle regeneration. Histological evaluation of the extent of regeneration provides an important parameter when evaluating the basis for abnormalities in splicing regulation or express of splicing regulators.
Regeneration of skeletal muscle requires rapid activation and proliferation of satellite stem cells followed by differentiation to myoblasts and fusion to form myotubes.24
This process resembles that of normal skeletal muscle development24
and it is not surprising that regenerating muscle recapitulates neonatal alternative splicing patterns. Western blot data showed increased protein levels of CELF1, CELF2, and MBNL1 in the non-DM1 mouse models of muscle degeneration. This result is also consistent with reversion to neonatal expression patterns because all three proteins decrease during postnatal skeletal muscle development7, 8
(and data not shown). It is important to note, however, that an increase in nuclear MBNL1 due to nuclear-cytoplasmic shuttling is proposed to be the main mechanism of MBNL1-mediated splicing transitions during postnatal skeletal muscle development.8
Reversion of MBNL1-regulated splicing events to early postnatal patterns is not consistent with increased MBNL1 protein levels observed in the dystrophy models based on Western blot analysis. However, the lack of complete correlation between protein levels and misregulated alternative splicing is likely to reflect an absence of information regarding the nuclear concentration of MBNL1 that would be obtained by immunolocalization studies.
Several results support the contention that CELF1 up-regulation and altered splicing in DM1 is a primary response to the CUGexp
RNA rather than secondary to regeneration. First, the splicing misregulation and increased CELF1 protein levels observed in skeletal muscle tissue from individuals with DM1 are not associated with a robust regenerative response.1, 39–41
This observation strongly suggests that aberrant expression of neonatal splice isoforms in adult DM1 skeletal muscle likely occurs in mature myofibers. Second, misregulated splicing and up-regulation of CELF1 is observed in DM1 cardiac tissue, which does not undergo regeneration.5, 42
Third, a heart-specific and inducible transgenic mouse model for DM1 has shown that misregulated splicing and elevated CELF1 is observed within 6 hours following induced expression of CUGexp
RNA demonstrating these as primary responses to the pathogenic RNA.42
Finally, in the present study, we used immunofluorescent staining for CELF1 combined with in situ
hybridization to detect CUGexp
RNA foci and detected elevated CELF1 in nuclei of non-regenerating muscle fibers and that were coincident with RNA foci.
We conclude that expression of neonatal alternative splicing isoforms in adult skeletal muscle occurs in several diverse models of muscle degeneration and muscle damage as a result of a robust regenerative process. Therefore, it is important to distinguish whether changes in alternative splicing patterns and expression of splicing regulators are primary effects of the pathogenic mechanism or secondary to muscle regeneration. It is important to note that these processes are not mutually exclusive. The EpA960/HSA-Cre-ERT2 (+ tam) DM1 model in skeletal muscle exhibits misregulated splicing, up-regulated CELF1, and diffuse degeneration/regeneration. Our results indicate that assays for splicing misregulation and altered expression of CELF1 are likely to reflect the combined effects of CUGexp RNA in mature skeletal muscle fibers as well as effects secondary to regeneration. The results described here for skeletal muscle tissue from mouse models are likely to be applicable to tissue samples from individuals with degenerative muscle disease, and stress the importance of evaluating cause-effect relationships between disease processes and misregulated splicing.