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Aims: Myotonic dystrophy type 1 (DM1) predisposes to the development of life-threatening arrhythmias and sudden cardiac death. Our study aimed to evaluate the prognostic value of programmed ventricular stimulation (PVS) in DM1 patients with conduction system disease., Methods and Results: Arrhythmic CArdiac DEath in MYotonic dystrophy type 1 patients (ACADEMY 1) is a double-arm non-randomized interventional prospective study. Myotonic dystrophy type 1 patients with permanent cardiac pacing indication were eligible for the inclusion. The study population underwent to pacemaker (PM) or implantable cardioverter-defibrillator (ICD) implantation according to the inducibility of ventricular tachyarrhythmias at PVS. Primary endpoint of the study was a composite of appropriate ICD therapy and cardiac arrhythmic death. The secondary study endpoint was all-cause mortality. Seventy-two adult-onset DM1 patients (51 ± 12 years; 39 male) were enrolled in the study. A ventricular tachyarrhythmia was induced in 25 patients (34.7%) at PVS (PVS+) who underwent dual chambers ICD implantation. The remaining 47 patients (65.3%) without inducible ventricular tachyarrhythmia (PVS-) were treated with dual-chamber PM. During an average observation period of 44.7 ± 10.2 months, nine patients (12.5%) met the primary endpoint, four in the ICD group (16%) and five (10.6%) in the PM group. Thirteen patients died (18.5%), 2 in the ICD group (8%) and 11 in PM group (23.4%). The Kaplan-Meier analysis did not show a significantly different risk of both primary and secondary endpoint event rates between the two groups., Conclusions: The inducibility of ventricular tachyarrhythmias has shown a limited value in the arrhythmic risk stratification among DM1 patients., (© Crown copyright 2021.)

Biomarker-driven trials hold promise for therapeutic development in chronic diseases, such as muscular dystrophy. Myotonic dystrophy type 1 (DM1) involves RNA toxicity, where transcripts containing expanded CUG-repeats (CUGexp) accumulate in nuclear foci and sequester splicing factors in the Muscleblind-like (Mbnl) family. Oligonucleotide therapies to mitigate RNA toxicity have emerged but reliable measures of target engagement are needed. Here we examined muscle transcriptomes in mouse models of DM1 and found that CUGexp expression or Mbnl gene deletion cause similar dysregulation of alternative splicing. We selected 35 dysregulated exons for further study by targeted RNA sequencing. Across a spectrum of mouse models, the individual splice events and a composite index derived from all events showed a graded response to decrements of Mbnl or increments of CUGexp. Antisense oligonucleotides caused prompt reduction of CUGexp RNA and parallel correction of the splicing index, followed by subsequent elimination of myotonia. These results suggest that targeted splice sequencing may provide a sensitive and reliable way to assess therapeutic impact in DM1., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Expansion of an unstable CTG repeat in the 3'UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUGexp) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUGexp, we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUGexp from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUGexp. Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs' chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1-2 and ZF3-4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1-2 or ZF3-4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1-2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3-4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.

PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA-PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA-PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA-RNA heteroduplexes reveals PNA's intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson-Crick pairing, the structures contain interesting interactions between the RNA's phosphate groups and the Π electrons of the peptide bonds in PNA., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3'-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUG(exp)) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUG(exp)/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUG(exp) foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUG(exp)-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aims: Atrial Preference Pacing (APP) is a pacemaker (PM) algorithm that supports a continuous atrial stimulation instead of a spontaneous atrial rhythm to prevent supraventricular tachyarrhythmias. The role of the APP in the prevention of atrial fibrillation (AF) is still controversial. The aim of our study was to evaluate the effect of preventive atrial pacing on AF incidence in myotonic dystrophy type I patients during a 12-month follow-up period., Methods and Results: We studied 40 patients with myotonic dystrophy type 1 (MD1) who underwent dual-chamber PM implantation for first- and second-degree atrioventricular block. After a 1-month stabilization period, they were randomized to APP algorithm programmed OFF or ON for 6 months each, using a cross-over design. The number of AF episodes during active treatment (APP ON phases) was lower than those registered during no treatment (APP OFF phases). No statistically significant difference was found in AF episodes duration between the two phases. During the APP OFF phases and APP ON phases, the atrial pacing percentage was 0 and 98%, respectively, while the ventricular pacing percentage did not show statistically significant difference (10 vs. 8%, P =0.2). Atrial premature beats count was significantly greater during APP OFF phases than during APP ON phases. Lead parameters remained stable over time and there were no lead-related complications., Conclusions: Based on these 12-month follow-up data, it is concluded that APP is an efficacy algorithm for preventing paroxysmal AF in MD1 patients who underwent dual-chamber PM implantation for atrioventricular conduction disorders.

Aims: Paroxysmal atrial arrhythmias occur in myotonic dystrophy type 1 (MD1) patients frequently. Pacemaker (PM) including detailed diagnostic functions may facilitate the diagnosis and management of frequent paroxysmal atrial tachyarrhythmias that may remain undetected during conventional clinical follow-up. Aim of our study was to evaluate the preventive effects of interatrial septum pacing in the Bachmann's Bundle region on atrial fibrillation (AF) in MD1 patients during 12 months follow up period., Methods and Results: Thirty MD1 patients (age 50.3 +/- 7.3; 11 F) who underwent dual chamber PM implantation were randomized at implantation to receive right atrial appendage pacing (16 patients) or Bachmann's bundle pacing (14 patients). No statistically significant difference in the electrical parameters (P wave amplitude, pacing threshold and lead impedance) was found between the two groups at implantation. Patients were followed at 1 month, 3 months, and every 6 months thereafter. They underwent clinical assessment, a standard 12-lead ECG and assessment of device performance at every visit. We counted the number of episodes of atrial arrhythmia occurred during the collection period and the duration of each episode. At 12 months of follow-up, no statistically significant differences in the number of AF episodes or in AF duration were found. Lead parameters remained stable over time and there were no displacements of the electrodes after implantation., Conclusion: Implantation of an atrial-active fixation lead on the atrial septum is safe and feasible. However, this study showed no significant difference between septal pacing and high atrial pacing, using the endpoints of AF duration and number of AF episodes.

Cardiac involvement in myotonic dystrophy type 1 (DM1) is frequent with increased incidence of conduction disturbances and sudden cardiac death when compared with general population. We describe a 38-year-old man in whom the diagnosis of DM1 was made 8 years after occurrence of cardiac arrest owing to ventricular fibrillation and discuss management of DM1 patients at risk for sudden cardiac death.