Regulation of chloride ion conductance during skeletal muscle development and in disease. Focus on “Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1”

Abstract

EDITORIAL FOCUSRegulation of chloride ion conductance during skeletal muscle development and in disease. Focus on “Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1”Thomas A. CooperThomas A. CooperPublished Online:01 Apr 2007https://doi.org/10.1152/ajpcell.00002.2007This is the final version - click for previous versionMoreSectionsPDF (47 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat productive investigations of disease mechanisms that also reveal new information about normal regulation elicit a particularly satisfying sense of a two-for-one deal. In the case of Lueck et al. (Ref. 13; see page 1291 of this issue), a detailed study of the molecular basis for the myotonia (delayed relaxation of muscle contraction) observed in individuals with myotonic dystrophy, type 1 (DM1) has also elucidated a novel mechanism for how the gene encoding the muscle-specific chloride channel (CLCN1) is “turned on” during development. CLCN1 is responsible for establishing ∼90% of the chloride conductance in skeletal muscle (5). During normal contraction stimulation, a single action potential is generated within the muscle fiber and then dampened. Loss of CLCN1 function results in reduced chloride conductance and hyperexcitability of the sarcolemma. The result is myotonia, in which a voluntary contraction initiates a series of continuous action potentials within the muscle fiber, resulting in delayed relaxation. This is perceived as stiffness by affected individuals (3).Mutations in the CLCN1 gene cause a congenital form of myotonia most often due to loss of CLCN1 function. The myotonia in DM results from a loss of CLCN1 function due to a novel mechanism in which the CLCN1 gene is not directly affected but rather CLCN1 expression is altered in trans by expression of a toxic RNA from the mutated gene. DM is dominantly inherited and the most common cause of adult-onset muscular dystrophy. Two mutations are known to cause DM, either a CTG repeat expansion in the 3′-untranslated region of the DMPK gene (DM type 1 or DM1) or a CCTG expansion in intron 1 of the ZNF9 gene (DM2). The main features of the disease are progressive skeletal muscle wasting, cardiac conduction defects, central nervous system dysfunction, cataracts, insulin resistance, and myotonia (1). DM is one of a growing number of microsatellite expansion disorders in which repeating units of 3–10 nucleotides located within the transcribed region of a gene causes disease by expansion. The mechanism by which an expansion causes disease varies among diseases. For example, expanded CAG repeats located within the coding regions of different genes cause Huntington's disease or spinal cerebellar ataxias. The mutation results in insertion of expanded polyglutamine tracts, which bestow a gain- and/or loss-of-function that ultimately results in cellular dysfunction (7).How do expansions within noncoding regions result in DM? Several pieces of evidence indicate that the primary mechanism of pathogenesis involves a toxic gain of function of RNA transcribed from the expanded allele. First, in situ hybridization demonstrated that the expanded alleles are transcribed and the RNA containing long tracts of CUG or CCUG RNA accumulates in nuclear foci. Second, the HSALR mouse model for DM1 expresses a human skeletal α-actin transgene containing 250 CTG repeats and reproduces several DM1 features of the disease, including myotonia and histological changes (14). The HSALR mouse model was used to further examine the basis for myotonia in the study by Lueck et al. (13) in this issue. Third, none of the many DM cases that have been characterized are due to a loss of function mutation in the DMPK or ZNF9 genes, strongly suggesting that the expansion is required to cause disease. Fourth, the fact that expansions within two unrelated genes cause strikingly similar disease phenotypes in DM1 and DM2 argue that the gain of function of the expanded RNA rather than loss of function of the genes is responsible for the common disease features (16).The molecular basis for some primary features of the disease is now understood. Nuclear accumulation of CUG or CCUG repeat-containing RNA disrupts the activities of RNA binding proteins that regulate pre-mRNA alternative splicing. Alternative splicing is the primary mechanism by which 20,000–25,000 genes generates the hundreds of thousands of proteins that are in the human proteome. At least 70% of human genes express multiple mRNAs by alternative splicing, which allows expression of multiple, and in some cases, hundreds of proteins from individual genes. Alternative splicing is often regulated such that individual genes express functionally diverse protein isoforms that are tissue specific, expressed at specific developmental stages, or in response to specific cues (2). Not all alternative splicing results in expression of different protein isoforms. A surprisingly high fraction of alternative splicing events (about one-third) introduce premature termination codons (PTCs), which render the out-of-frame mRNAs susceptible to degradation by the nonsense-mediated decay (NMD) pathway (11). This provides a mechanism by which regulation of alternative splicing can determine “on/off” regulation of gene expression and this is particularly relevant to regulation of CLCN1 gene expression.At least 20 alternative splicing events are disrupted in DM heart, skeletal muscle, or brain tissues. Interestingly, most, if not all of these are normally developmentally regulated transitions which occur within the first few weeks after birth (12, 16). In DM, the embryonic splicing patterns are aberrantly expressed in adult tissues. For two genes, expression of the embryonic splicing patterns has been shown to cause features of the disease due to a failure to fulfill the functional requirements of the adult tissue. Failure to express the muscle-specific and high-signaling isoform of the insulin receptor directly correlates with insulin resistance in DM (17, 18). In the second case, CLCN1 mRNAs from individuals with DM as well as the HSALR DM1 mouse model contain additional exons (exon 7a) or introns (intron 2) that introduce PTCs and make these mRNAs likely targets for NMD. Consistent with the NMD hypothesis, there is a loss of CLCN1 mRNA and protein that is sufficient to explain the myotonia observed in individuals with DM and in HSALR mice (4, 15). In addition to providing a mechanism for myotonia in DM, these results suggested that CLCN1 mRNA levels were upregulated during normal muscle development by an alternative splicing switch from out of frame and degraded mRNAs to in frame and relatively stable mRNAs.The paper by Lueck at al. presents a systematic and detailed analysis of CLCN1 mRNA and pre-mRNA expression during normal development and in the HSALR mouse model. The first part of the two-for-one deal is the understanding of how CLCN1 expression is regulated during normal skeletal muscle development. It has been known for some time that chloride conductance increases during postnatal skeletal muscle development and that the change in conductance correlates with a striking increase in the steady-state levels of CLCN1 mRNA (19). The results from Lueck et al. strongly suggest that the primary mechanism of this regulation is an alternative splicing switch rather than upregulation of transcription. The results of RT-PCR analyses demonstrated no difference in the level of gene transcription at embryonic day 18 compared with adult skeletal muscle because comparable amounts of CLCN1 pre-mRNA were detected. In contrast, CLCN1 mRNA undergoes a threefold increase during early postnatal development. The reason for this is that at early postnatal timepoints, at least 40% of CLCN1 mRNAs retain exon 7a that renders the mRNA subject to NMD. Forty percent is likely to be an underestimate because of the propensity for these mRNAs to be degraded. In addition, splicing events not assayed in this study (such as inclusion of intron 2) also induce NMD so an even larger fraction of CLCN1 spliced mRNAs are degraded (15). During the first three postnatal weeks, there is a gradual shift in splicing that removes the PTCs and produces the full length open reading frame. Whole cell patch-clamp analysis on isolated skeletal muscle fibers demonstrated a correlation between CLCN1 channel activity, this alternative splicing transition, and accumulation of CLCN1 mRNA (13).The second component of the two-for-one deal is the elucidation of the mechanism of myotonia in DM based on results from the mouse model. A previous study by Ebralidze et al. (6) proposed an alternative to the NMD hypothesis in which CLCN1 mRNA levels are decreased in DM due to sequestration of transcription factors. The authors proposed that a defect in transcription rather than alternative splicing is determinative for the CLCN1 deficiency (6). In HSALR mice, RT-PCR analysis demonstrated that expression of CLCN1 pre-mRNA was not lower than in control mice as would be expected if transcription was reduced while CLCN1 mRNA levels were significantly reduced as shown previously (15). Overall, the evidence indicates that the predominant pathway for loss of CLCN1 mRNA and myotonia in DM is expression of the embryonic alternative splicing pattern and loss of mRNA by the NMD pathway.The mechanism by which the splicing transitions that are affected in DM are coordinately regulated during normal postnatal developmental is understood to some degree, as is the mechanism by which expression of expanded repeat RNA disrupts this regulation in DM. On the basis of the hypothesis that CUG repeat RNA is pathogenic because it binds and disrupts the functions of RNA binding proteins, two CUG repeat RNA binding proteins were identified: CUG-binding protein 1 (CUG-BP1) and muscleblind-like 1 (MBNL1). Both proteins were subsequently shown to directly regulate alternative splicing by binding to specific sequence motifs near the regulated alternative exons. In addition, MBNL1 and CUG-BP1 are antagonistic regulators of the developmentally regulated splicing transitions that are disrupted in DM1 (16). How does expression of CUG repeat RNA disrupt the functions of these proteins? Intriguingly, it causes both a loss of function for MBNL1 and a gain of function for CUG-BP1. MBNL1 is sequestered on the nuclear foci of CUG repeat-containing mRNA, and the loss of nuclear MBNL1 causes a switch in splicing to the embryonic splicing pattern. Support for this mechanism came from an MBNL1 knockout mouse model which reproduces the embryonic splicing patterns observed in DM1 and HSALR mice, as well as myotonia and other DM-like features (9, 12). Interestingly, the mechanism by which MBNL1 normally regulates these splicing transitions during postnatal skeletal muscle development appears to be by a change in nuclear:cytoplasmic distribution, such that MBNL1 switches from being predominantly cytoplasmic to predominantly nuclear (12). The increased nuclear concentration during postnatal development is thought to drive the developmentally regulated splicing changes that are affected in DM, such as in CLCN1, whereas loss of nuclear MBNL1 due to sequestration is thought to reproduce the low nuclear concentration observed in embryonic muscle and to lead to embryonic splicing patterns (12).CUG-BP1 expression is also regulated during development, such that protein levels are high in embryonic and early postnatal development but low in adult skeletal muscle (10, 12). This loss of CUG-BP1 activity is also thought to control developmental splicing transitions, since forced expression of CUG-BP1 in transgenic mice induces the embryonic (and DM-like) splicing patterns (8). Consistent with expression of embryonic splicing patterns in adult DM1 skeletal muscle, CUG-BP1 steady-state levels are increased in DM1 skeletal muscle. The mechanism by which expression of CUG repeat RNA induces higher CUG-BP1 protein levels remains to be elucidated.Overall, investigations into the aberrant regulation of alternative splicing in DM have identified a coordinated network of developmentally regulated splicing transitions that are part of an postnatal regulatory program, identified the proteins that normally regulate these transitions, and led to an understanding of the mechanisms by which the activities of these proteins are disrupted in the disease. The results by Lueck et al. have firmly established the role of alternative splicing and NMD in the myotonia of DM and have revealed the role for alternative splicing in developmental control of CLCN1 expression. CLCN1 is one of few genes for which mRNA steady-state levels are known to be determined by an alternative splicing decision. However, there are likely to be many more.REFERENCES1 Ashizawa T, Harper PS. Myotonic Dystrophies: An Overview. New York: Elsevier-Academic, 2006.Google Scholar2 Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 27: 27–48, 2003.Google Scholar3 Cannon SC. Pathomechanisms in channelopathies of skeletal muscle and brain. Annu Rev Neurosci 29: 387–415, 2006.Crossref | PubMed | ISI | Google Scholar4 Charlet-B N, Savkur RS, Singh G, Philips AV, Grice EA, and Cooper TA. Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. 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Cooper, Depts. of Pathology and Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030 (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationCited ByChanges in Expression and Cellular Localization of Rat Skeletal Muscle ClC-1 Chloride Channel in Relation to Age, Myofiber Phenotype and PKC Modulation15 May 2020 | Frontiers in Pharmacology, Vol. 11Crowded Charges in Ion Channels29 November 2011Detection and Measurement of Cardiac Ion Channels13 August 2010Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue4 September 2007 | Proceedings of the National Academy of Sciences, Vol. 104, No. 36 More from this issue > Volume 292Issue 4April 2007Pages C1245-C1247 Copyright & PermissionsCopyright © 2007 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.00002.2007PubMed17215330History Published online 1 April 2007 Published in print 1 April 2007 Metrics