DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model.
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Myotonic dystrophy (DM) is the most common form of muscular dystrophy and is caused by expansion of a CTG trinucleotide repeat on human chromosome 19. Patients with DM develop atrioventricular conduction disturbances, the principal cardiac manifestation of this disease. The etiology of the pathophysiological changes observed in DM has yet to be resolved. Haploinsufficiency of myotonic dystrophy protein kinase (DMPK), DM locus-associated homeodomain protein (DMAHP) and/or titration of RNA-binding proteins by expanded CUG sequences have been hypothesized to underlie the multi-system defects observed in DM. Using an in vivo murine electrophysiology study, we show that cardiac conduction is exquisitely sensitive to DMPK gene dosage. DMPK-/- mice develop cardiac conduction defects which include first-, second-, and third-degree atrioventricular (A-V) block. Our results demonstrate that the A-V node and the His-Purkinje regions of the conduction system are specifically compromised by DMPK loss. Importantly, DMPK+/- mice develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients. These results demonstrate that DMPK dosage is a critical element modulating cardiac conduction integrity and conclusively link haploinsufficiency of DMPK with cardiac disease in myotonic dystrophy. (+info)
(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: implications for congenital myotonic dystrophy.
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Although the mutation for myotonic dystrophy has been identified as a (CTG)n repeat expansion located in the 3'-untranslated region of a gene located on chromosome 19, the mechanism of disease pathogenesis is not understood. The objective of this study was to assess the effect of (CTG)n repeats on the differentiation of myoblasts in cell culture. We report here that C2C12 myoblast cell lines permanently transfected with plasmid expressing 500 bases long CTG repeat sequences, exhibited a drastic reduction in their ability to fuse and differentiate into myotubes. The percentage of cells fused into myotubes in C2 C12 cells (53.4+/-4.4%) was strikingly different from those in the two CTG repeat carrying clones (1.8+/-0.4% and 3.3+/-0. 7%). Control C2C12 cells permanently transfected with vector alone did not show such an effect. This finding may have important implications in understanding the pathogenesis of congenital myotonic dystrophy. (+info)
Relationships among electrophysiological findings and clinical status, heart function, and extent of DNA mutation in myotonic dystrophy.
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BACKGROUND: Impulse-conduction abnormalities and arrhythmias are common in myotonic dystrophy (MD). This study was performed to determine whether a correlation exists between electrophysiological (EP) testing data and clinical status, heart function, or size of the DNA abnormality (cytosine-thymine-guanine sequence repeat). METHODS AND RESULTS: Eighty-three MD patients underwent invasive EP studies prompted primarily by the presence of asymptomatic conduction abnormalities. AV conduction disturbances were common and mainly distal (HV interval, 66.2+/-14 ms). AV conduction observed from the surface ECG was generally concordant with endocardial measurements. However, 11 of 20 patients with normal surface ECGs had abnormal subhisian conduction. Atrial arrhythmias were inducible in 41% of cases and correlated with prolongation of the AH interval (P=0.02) and a shorter atrial refractory period (P=0.04). Induction of ventricular arrhythmias (18%) correlated strongly with age (P=0. 0003). After adjustment for age, the extent of DNA mutation correlated with the Walton score (P=0.0018) but not with conduction abnormalities or induction of arrhythmias. CONCLUSIONS: Prolongation of the HV interval is the most common conduction abnormality in MD and can be reliably recognized only by invasive EP testing. It raises the issue of prophylactic pacing to limit the incidence of sudden death in MD. Atrial and ventricular arrhythmias are often inducible, although their predictive value remains to be determined. Young age emerged as the most powerful predictor of inducible ventricular tachyarrhythmias. Conversely, we found no relationship between ECG or EP abnormalities recorded during invasive testing and the DNA mutation size or severity of peripheral muscle involvement. (+info)
Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.
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1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability. (+info)
Tumor necrosis factor system activity is associated with insulin resistance and dyslipidemia in myotonic dystrophy.
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Myotonic dystrophy (MyD) is a multisystem autosomal dominant disorder associated with progressive muscle wasting and weakness. The striking metabolic abnormality in MyD is insulin resistance. The mechanism by which target tissues are insensitive to insulin action remains uncertain. In a recent study, plasma soluble tumor necrosis factor receptor (sTNFR)2 levels were found to be associated with muscle tissue mass and insulin resistance. Given these associations, we speculated that disorders of the muscle cell membrane could lead simultaneously to insulin insensitivity and sTNFR2 leakage in MyD. To test this hypothesis, we measured the levels of circulating sTNFR1 and sTNFR2 and insulin resistance in MyD patients. We studied 22 MyD patients and 24 age-, BMI-, and fat mass-matched control subjects. Both MyD men and women showed higher plasma insulin levels in the presence of comparable glucose concentrations than did control subjects. sTNFR2, but not sTNFR1, levels were approximately 1.5-fold higher in MyD patients. In parallel with these findings, the fasting insulin resistance index (FIRI) was also higher in MyD patients. In fact, in the whole population, fasting insulin and FIRI strongly correlated with sTNFR2 in both men (r = 0.77 and r = 0.81, P<0.0001, respectively) and women (r = 0.67 and r = 0.64, P = 0.001, respectively). sTNFR2 levels were also associated with the insulin sensitivity index (S(I)), calculated from an oral glucose tolerance test (OGTT) according to the method by Cederholm and Wibell (r = -0.43, P = 0.006). We constructed a multiple linear regression to predict FIRI, with BMI, waist-to-hip ratio, and sTNFR2 as independent variables. In this model, both BMI (P = 0.0014) and sTNFR2 (P = 0.0048) levels contributed independently to 46% of the variance of FIRI. In another model, in which FIRI was substituted for S(I) from the OGTT, both BMI (P = 0.0001) and sTNFR2 (P = 0.04) levels contributed independently to 48% of the variance of S(I) from the OGTT. Plasma cholesterol and triglyceride concentrations were significantly increased in MyD patients. sTNFR1 and sTNFR2 levels were found to be strongly associated with plasma cholesterol, LDL cholesterol, and triglycerides. sTNFR1 and sTNFR2 also correlated with serum creatine kinase activity in MyD patients (r = 0.57, P = 0.006; r = 0.75, P<0.0001, respectively). In conclusion, here we describe, for the first time to our knowledge, a relationship between insulin action and plasma sTNFR2 concentration in MyD patients. We have also found increased concentrations of plasma triglycerides and cholesterol levels in parallel with sTNFR1 and sTNFR2 concentrations in MyD patients. We speculate that the latter associations are dependent on, and secondary to, increased tumor necrosis factor (TNF)-alpha action. Whether TNF action is implicated in the pathogenesis of MyD or is a simple marker of disease activity awaits further studies. (+info)
Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression.
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Myotonic dystrophy (DM), the most common inherited muscle disorder, is caused by a CTG expansion in the 3"-untranslated region of a protein kinase gene ( DMPK ). The complex and variable phenotype is most likely caused by a complex molecular pathogenesis, including deficiency of the DMPK protein, a trans -dominant misregulation of RNA homeostasis and haploinsufficiency of a neighboring homeobox gene [DM locus-associated homeodomain protein (DMAHP )]. Here, we study the allele-specific transcriptional activity of the DMAHP/SIX5 gene in DM patient tissues. Using a quantitative fluorescent RT-PCR assay, we tested allele-specific accumulation of DMAHP/SIX5 transcripts in both total and poly(A)+pools. In muscle biopsies, we found that transcript reductions of DMAHP/SIX5 alleles in cis with CTG expansions correlated with the extent of expansion. A patient with approximately 90 CTG repeats in muscle DNA (normal n < 37) showed a 20% reduction of allele-specific transcript levels, while four other DM patients with larger expansions showed 80% reductions. The effects of the CTG expansions on DMAHP transcription were tissue specific: autopsy tissues from a patient with 1500 repeats showed 80% reductions in muscle and liver; however, RNA from other tissues (lung, aorta, heart conduction tissue, cerebellum) showed 0-20% reductions. Our results suggest that the effect of the CTG repeat on the DMAHP/SIX5 promoter is variable and tissue-specific. Our data are consistent with abnormalities of DMAHP/SIX5 probably having a more prominent role in disease pathogenesis in muscle, liver and brain, but being less important in other tissues. (+info)
Simultaneous analysis of expression of the three myotonic dystrophy locus genes in adult skeletal muscle samples: the CTG expansion correlates inversely with DMPK and 59 expression levels, but not DMAHP levels.
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The causative mutation in the majority of cases of myotonic dystrophy has been shown to be the expansion of a CTG trinucleotide repeat, but the mechanism(s) by which this repeat leads to the very complex symptomatology in this disorder remains controversial. We have developed a highly sensitive and quantifiable assay, based on competitive RT-PCR, to test the hypothesis that the expansion disrupts the expression of the genes in its immediate vicinity, DMPK, 59 and DMAHP. In order to avoid cell culture-induced artifacts we performed these experiments using adult skeletal muscle biopsy samples and analysed total cytoplasmic poly(A)+mRNA levels for each gene simultaneously, as this is more physiologically relevant than allele-specific levels. There was considerable overlap between the expression levels of the three genes in myotonic dystrophy patient samples and samples from control individuals. However, in the myotonic dystrophy samples we detected a strong inverse correlation between the repeat size and the levels of expression of DMPK and 59. This is the first report of a possible effect of the CTG expansion on gene 59. Our results indicate that whilst a simple dosage model of gene expression in the presence of the mutation is unlikely to be sufficient in itself to explain the complex molecular pathology in this disease, the repeat expansion may be a significant modifier of the expression of these two genes. (+info)
Myotonic dystrophy is associated with a reduced level of RNA from the DMWD allele adjacent to the expanded repeat.
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Myotonic dystrophy is caused by the expansion of a CTG repeat sequence. The mechanism by which this expanded repeat produces the pathophysiology of myotonic dystrophy is not clear. It has been shown previously that expansion of the repeat produces allele-specific effects on transcripts from two genes, DMPK and SIX5. We have examined the effect of repeat expansion on the level of RNA from a third gene, DMWD. We have identified a polymorphism in this gene and developed a quantitative allele-specific assay for DMWD RNA levels, which we have applied to nuclear and cytoplasmic fractions of RNA from DM cell lines. We have found that the level of the DM-associated allele in the cytoplasm of DM cell lines is reduced by 20-50% compared with the wild-type allele, similar to the level of reduction found for SIX5 in allele-specific analysis. However, no such reduction is observed in RNA from the nuclear fraction of DM cell lines. This may reflect the complex nature of processing transcriptional units at the DM locus. (+info)