Nogales-Gadea, G. | Search bioRxiv

bioRxiv2026

Patient-derived 3D engineered human muscle model recapitulates CLCN1 mis-splicing and myotonia in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) lacks human in vitro models that directly link RNA toxicity to mature skeletal muscle function, particularly myotonia. Here, we engineer contractile 3D human skeletal muscle tissues from immortalized myoblasts derived from three DM1 patients representing juvenile, adult, and late-onset subtypes. These tissues reproduce key molecular features of DM1, including nuclear RNA foci, MBNL1 sequestration, and widespread mis-splicing. Functionally, DM1 tissues exhibit impaired calcium handling, subtype-dependent weakness, rapid fatigue, and a fiber-type distribution characterized by increased slow type I fibers and pathological MyHC-I/IIx hybrids. Notably, the 3D environment enables expression and complete pathogenic mis-splicing of CLCN1--undetectable in matched 2D cultures--accompanied by myotonia-like delayed relaxation. Using this model, we assessed therapeutic responses of candidate small-molecule modulators. Phenylbutazone reduced RNA foci and MBNL1 sequestration but failed to rescue spliceopathy or function. In contrast, calcitriol induced coordinated transcriptomic remodeling and robustly rescued myotonia-like relaxation despite persistent CLCN1 mis-splicing. These findings establish a functionally mature human DM1 muscle model and highlight compensatory network activation as a strategy to improve muscle function in DM1.

bioengineering↗

bioRxiv2025

Characterisation of DMPK and MBNL1 expression in cell models of Myotonic Dystrophy: A platform for drug screening

Myotonic dystrophy type I (DM1) is caused by CTG repeat expansions in the DMPK gene leading to mRNA toxicity and sequestration of the splicing regulator MBNL1, affecting many tissues. We have developed an in vitro screening platform based on ddPCR and in-cell western to quantify these mRNAs and proteins and characterised more than 20 cell models to define DM1 biomarkers that could be useful for drug screening. DMPK protein levels were reduced in DM1-immortalised myoblasts and myotubes, but not in fibroblasts, while MBNL1 protein was consistently lower in all DM1 myogenic cultures, whether primary or immortalised. Myogenic differentiation of cultures led to an increase in DMPK mRNA expression, which was translated into increased MBNL1 sequestration in foci. We further corroborated the platforms ability to assess therapeutic outcomes, evaluating the effect of a DMPK gapmer ASO and one siRNA: while the gapmer increased MBNL1 protein levels, the siRNA had no significant effect on MBNL1 release. Our platform and the in-depth characterisation of some of the most used models would be of use to the DM1 research community. Significance statementMyotonic dystrophy type I (DM1) is a multisystemic disease with a complex pathogenesis and multiple outcome measures for drug assessment in vitro. In the last years, the increasing number of new potential therapies targeting DM1 in clinical trials has increased the need for robust and rapid evaluation of preclinical candidates, as well as in-depth knowledge of the cell models used. Here, we present a new cell-based platform that enables robust quantification of DMPK and MBNL1 in cell culture for cell model characterisation and drug screening. Indeed, we highlight the differences observed in DMPK and MBNL1 protein quantification in primary fibroblasts and myotubes, immortalised fibroblasts, myoblasts and myotubes. We then, we performed a proof-of-concept drug evaluation of potential therapeutic strategies targeting DMPK, showing the most suitable for targeting the DMPK expanded transcript.

cell biology↗