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".

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 adultonset 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 gainand/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 HSA mouse model for DM1 expresses a human skeletal -actin transgene containing 250 CTG repeats and reproduces several DM1 features of the disease, including myotonia and histological changes (14). The HSA 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 HSA DM1 mouse model contain additional exons (exon 7a) Address for reprint requests and other correspondence: T. A. Cooper, Depts. of Pathology and Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030 (e-mail: [email protected]). Am J Physiol Cell Physiol 292: C1245–C1247, 2007; doi:10.1152/ajpcell.00002.2007.