Altered Behavioral Responses Show GABA Sensitivity in Muscleblind-Like 2-Deficient Mice: Implications for CNS Symptoms in Myotonic Dystrophy.

Considerable evidence from mouse models and human postmortem brain suggests loss of Muscleblind-like protein 2 (MBNL2) function in brain is a major driver of CNS symptoms in Myotonic dystrophy type 1 (DM1). Increased hypersomnia, fatigue, and surgical complications associated with general anesthesia suggest possible sensitivity to GABAergic inhibition in DM1. To test the hypothesis that MBNL2 depletion leads to behavioral sensitivity to GABAA receptor (GABAA-R) modulation, Mbnl2 knock-out (KO) and wild-type (WT) littermates were treated with the anesthetic sevoflurane, the benzodiazepine diazepam, the imidazopyridine zolpidem, and the benzodiazepine rescue agent, flumazenil (Ro 15-1788), and assessed for various behavioral metrics. Mbnl2 KO mice exhibited delayed recovery following sevoflurane, delayed emergence and recovery from zolpidem, and enhanced sleep time at baseline that was modulated by flumazenil. A significantly higher proportion of Mbnl2 KO mice also loss their righting reflex [loss of righting reflex (LORR)] from a standard diazepam dose. We further examined whether MBNL2 depletion affects total GABAA-R mRNA subunit levels and validated RNA-sequencing data of mis-spliced Gabrg2, whose isoform ratios are known to regulate GABA sensitivity and associated behaviors. While no other GABAA-R subunit mRNA levels tested were altered in Mbnl2 KO mouse prefrontal cortex, Gabrg2S/L mRNA ratio levels were significantly altered. Taken together, our findings indicate that loss of MBNL2 function affects GABAergic function in a mouse model of myotonic dystrophy (DM1).

Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation.

BACKGROUND: Skeletal muscle contributes to roughly 40% of lean body mass, and its loss contributes to morbidity and mortality in a variety of pathogenic conditions. Significant insights into muscle function have been made using cultured cells, in particular, the C2C12 myoblast line. However, differentiation of these cells in vitro typically yields immature myotubes relative to skeletal muscles in vivo. While many efforts have attempted to improve the maturity of cultured myotubes, including the use of bioengineered substrates, lack of molecular characterization has precluded their widespread implementation. This study characterizes morphological, molecular, and transcriptional features of C2C12 myotubes cultured on crosslinked, micropatterned gelatin substrates fabricated using previously established methods and compares them to myotubes grown on unpatterned gelatin or traditional plasticware. METHODS: We used immunocytochemistry, SDS-PAGE, and RNAseq to characterize C2C12 myotubes grown on micropatterned gelatin hydrogels, unpatterned gelatin hydrogels, and typical cell culture substrates (i.e., plastic or collagen-coated glass) across a differentiation time course. The ability to form aligned sarcomeres and myofilament protein concentration was assessed. Additionally, the transcriptome was analyzed across the differentiation time course. RESULTS: C2C12 myotubes grown on micropatterned gelatin hydrogels display an increased ability to form aligned sarcomeres as well as increased contractile protein content relative to myotubes cultured on unpatterned gelatin and plastic. Additionally, genes related to sarcomere formation and in vivo muscle maturation are upregulated in myotubes grown on micropatterned gelatin hydrogels relative to control myotubes. CONCLUSIONS: Our results suggest that growing C2C12 myotubes on micropatterned gelatin hydrogels accelerates sarcomere formation and yields a more fully matured myotube culture. Thus, the use of micropatterned hydrogels is a viable and simple approach to better model skeletal muscle biology in vitro.