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. 2025 Dec 17;16:2409. doi: 10.1038/s41598-025-32385-0

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Structural analysis of Control and FSHD skeletal myotubes with and without EPS. (A) EPS enhances contractility in control myotubes but not in FSHD myotubes. Beat amplitude (%) was quantified using multielectrode array (MEA) analysis in control and FSHD myotubes, with or without 12-hour electrical pulse stimulation (EPS). Left: Representative heat maps showing beat amplitude (%) in CTRL1 and FSHD2 myotubes under the indicated conditions. Right: Each dot represents the beat amplitude (%) recorded from a single electrode (n = 60 electrodes per group). Data are visualized as dot-box plots, presenting the median ± interquartile range (IQR), with mean values indicated by red bars. **p < 0.01, unpaired Student’s t-test. Immunofluorescent images of myotubes from (B) Control and (C) FSHD1 cell lines stained for (i) nuclei, (ii) actin, and (iii) ɑ-actinin, which are blue, green, and red in (iv) merged image, respectively. (D) Immunofluorescent images of myotubes from Control and FSHD2 cell lines under non-paced (-EPS) and paced (+ EPS) conditions. Scale bar = 20 μm. (E-G) Quantitative analysis of images with bars representing mean and error bars of standard deviation. Significance bars were generated using two-way ANOVA and post hoc Tukey’s Honest Significant Difference (HSD) test, with ***p < 0.001, ** p < 0.01, and * p < 0.05; (E) Percent of image area positive for actin stain; (F) Z-line density within cells; (G) Z-line orientational order parameter.