Lack of desmin causes myofiber impairment and a metabolic shift in trunk myofibers.

aTg(mylz2:EGFP);desma^−/−;desmb^−/− and desma^+/-;desmb^+/- larvae exposed to resistance swimming during 12 h from 4 to 5 dpf. Arrowheads: myofiber breaks. b Proportions of injuries caused by resistance swimming. c Force generation of desma^+/-;desmb^+/- and desma^−/−;desmb^−/− trunk myofibers. d Force generated at optimal stretch in desma^+/-;desmb^+/- and desma^−/−;desmb^−/− (p = 0.002). e Representative swimming tracks of desma^−/−;desmb^−/− and desma^+/-;desmb^+/- controls. f Relative swimming distance at low speed (p = 0.024), high speed (p = 0.019) and total distance (p = 0.016). g F310 trunk immunolabeling of desma^−/−;desmb^−/− and WT. Open arrowheads: F310⁺ myofibers inside the slow domain, arrowheads: lack of F310 labeling in the fast domain. h Comparison of number of fast myofibers inside (p = 0.0018) and outside the slow domain. i S58 trunk immunolabeling in desma^−/−;desmb^−/− and WT. Open arrowheads: lack of slow myofibers in the slow domain, arrowheads: S58⁺ myofibers in the fast domain. j Quantification of number of slow myofibers inside (p = 0.002) and outside (p = 0.0012) of the slow domain. k WT and desma^−/−;desmb^−/− trunk immunolabeled for DAPI/Pax7. Arrowheads: Pax7⁺ nuclei. l Quantification of Pax7⁺/DAPI⁺ cells (p = 0.0008). m Cross-sections of WT and desma^−/−;desmb^−/− trunk immunolabeled for DAPI/PCNA. Arrowheads: PCNA⁺ nuclei. n Quantification of PCNA⁺/DAPI⁺ cells (p = 0.0028). o Cross-sections of WT and desma^−/−;desmb^−/− EOMs immunolabeled for F310. p Quantification of F310⁺ myofibers. q Cross-sections of WT and desma^−/−;desmb^−/− EOMs immunolabeled for S58. r Quantification of S58⁺ myofibers. s DAPI/PCNA/laminin/phalloidin immunolabeling of desma^−/−;desmb^−/− and WT EOMs. Arrowheads: DAPI⁺/PCNA⁺ myonuclei. t Quantification of DAPI⁺/PCNA⁺ myonuclei (p = 0.0039). u DAPI/TUNEL/laminin/phalloidin immunolabeling of desma^−/−;desmb^−/− and WT EOMs. Arrowheads: DAPI⁺/TUNEL⁺ myonuclei. v Quantification of DAPI⁺/TUNEL⁺ myonuclei (p = 0.0032). Statistical analysis: Two-sided t-tests with Welch correction. Data in violin plots is presented as median (line) and quartiles (dashed lines). Data in (c) is presented as mean ± SEM. Dashed lines in cross-sections: myosepta separating fast and slow domains. Age of fish, tissue analyzed, viewed area and level of cross-section is illustrated above panels. Scale bars in a, o, q: 100 µm, g, i, k, m: 50 µm, s, u: 25 µm. Schematic images were adapted from https://www.biorender.com.

fhl2b is upregulated in the EOM in response to desmin-related muscular dystrophy.

a Expression of myofiber-related DEGs for the following comparisons: 5 months desma^−/−:desmb^−/−vs WT EOMs (I), 5 months desma^−/−:desmb^−/−vs WT trunk (II), 20 months desma^−/−:desmb^−/−vs WT EOMs (III), 20 months desma^−/−:desmb^−/−vs WT trunk (IV), 5 months WT EOMs vs WT trunk (V), 5 months desma^−/−:desmb^−/− EOMs vs desma^−/−:desmb^−/− trunk (VI), 20 months WT EOMs vs WT trunk (VII) and 20 months desma^−/−:desmb^−/− EOMs vs desma^−/−:desmb^−/− trunk (VIII). bfhl2b expression in the abovementioned comparisons I-VIII. FHL2 antibody labeling of WT and desma^−/−;desmb^−/− cross-sectioned EOMs at c 5 and d 20 months. e FHL2 positive myofibers quantification of desma^−/−;desmb^−/− versus WT control EOMs in 5 (p = 0.017) and 20-monthold zebrafish (p = 3.7e⁻⁷), respectively and 5-months versus 20-months-old WT (p = 0.014) and desma^−/−:desmb^−/− (p = 0.005), respectively. Data is presented as median (line) and quartiles (dashed lines). f WT, plecb^−/− and obscnb^−/− 12-month-old EOM cross sections immunolabeled with FHL2 antibodies. g Human EOM cross section immunolabeled with DAPI/FHL2/laminin antibodies, dashed square indicates area enlarged below. h Western blot on human EOMs showing FHL2. i Mouse EOM cross section immunolabeled using DAPI/FHL2/laminin antibodies, dashed boxes indicate area enlarged below. j Western blot of mouse EOMs showing FHL2. Statistical analysis in b: Two-sided Wald test with B/H-correction, e: Two-sided t-tests with Welch correction. Scale bars in c, d, f, g, i: 50 µm, g, i bottom panel: 25 µm. White dashed lines outline the entire cross-section of the EOMs. Schematic images were adapted from https://www.biorender.com.

Lack of Fhl2 causes EOM myofiber hypertrophy.

Cross-sections of 12 months old WT, desma^−/−;desmb^−/−, desma^−/−;desmb^−/−;fhl2a^−/−, desma^−/−;desmb^−/−;fhl2b^−/− and desma^−/−;desmb^−/−;fhl2a^−/−; fhl2b^−/− zebrafish EOMs. a F-actin labeling (phalloidin) and b average F-actin positive myofiber area quantification in WT, desma^−/−;desmb^−/−, desma^−/−;desmb^−/−;fhl2a^−/−, desma^−/−;desmb^−/−;fhl2b^−/− (p = 0.0002) and desma^−/−;desmb^−/−;fhl2a^−/−;fhl2b^−/− (p = 0.007) mutant zebrafish. c DAPI and TUNEL labeling of myonuclei. Dashed boxes indicate magnified areas in d). d TUNEL (top) and DAPI (bottom) labeling of myonuclei inside the myofiber laminin sheet. Phalloidin labels F-actin in the myofibers. White arrowheads indicate TUNEL positive myonuclei. e Quantification of DAPI⁺/TUNEL⁺ myonuclei in WT, desma^−/−;desmb^−/− (p = 0.021), desma^−/−;desmb^−/−;fhl2a^−/−, desma^−/−;desmb^−/−;fhl2b^−/− and desma^−/−;desmb^−/−;fhl2a^−/−;fhl2b^−/− (p = 2.2e⁻⁷) mutant zebrafish. f DAPI/PCNA labeling of myonuclei. Dashed boxes indicate magnified areas in g). g DAPI/PCNA labeling of myonuclei inside the myofiber laminin sheet. Phalloidin labels F-actin. White arrowheads indicate double positive myonuclei. h Quantification of PCNA⁺ myonuclei in WT, desma^−/−;desmb^−/−, desma^−/−;desmb^−/−;fhl2a^−/− (p = 8.8e⁻⁵), desma^−/−;desmb^−/−;fhl2b^−/− (p = 2.3e⁻⁵), and desma^−/−;desmb^−/−;fhl2a^−/−;fhl2b^−/− (p = 4.6e⁻⁷) mutant zebrafish. Dashed lines outline the entire cross-section of the EOMs. Statistical analysis: Two-sided t-tests with Welch correction. Data in all violin plots is presented as median (line) and quartiles (dashed line). Avg average. Scale bar in a, c, f: 25 µm, d, g: 10 µm.

Muscle specific overexpression of fhl2b significantly prolongs life-span and improves motor function and muscle integrity in dmd^−/− zebrafish larvae.

a Experimental setup to generate dmd^−/− zebrafish overexpressing fhl2b. One cell-stage eggs from in-crossed dmd^+/- zebrafish were injected with a 503unc:fhl2b-T2A-EGFP plasmid, raised and crossed into dmd^+/- to generate stable lines. b Survival tests over 30 days for three different 503unc:fhl2b-T2A-EGFP lines. Kaplan-Meier log rank test was used to calculate significance between dmd^−/− and dmd^−/−:Tg(503unc: fhl2b-T2A-EGFP) for each of the three founder lines (Founder 1: p = 0.0228, Founder 2: p = 0.0534, Founder 3: p = 0.0188 and all founder lines combined: p < 0.0001). c Spontaneous swimming tests showed significant increases in low speed (p = 0.0459) and total distance (p = 0.0385) in dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) compared to dmd^−/− larvae. d Representative swimming tracks of sibling control, dmd^−/− and dmd^−/−:Tg(503unc: fhl2b-T2A-EGFP) larvae. e F-actin (phalloidin) labeling of trunk muscle in sibling control, dmd^−/−, dmd^−/−:Tg(503unc:EGFP) and dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) larvae. f Magnification of dashed boxes in e) showing detached myofibers and empty areas in dmd^−/− and dmd^−/−:Tg(503unc:EGFP) larvae (arrowheads) whereas dmd^−/−:Tg(503unc: fhl2b-T2A-EGFP) larvae display g small diameter intensely EGFP positive myofibers (green) in corresponding areas (open arrowheads). DAPI in blue. h Quantification of detached F-actin⁺ myofibers in somite segments at 3, 5, 6 and 7 dpf. i Number of myofiber breaks per somite. In total, dmd^−/−:Tg(503unc: fhl2b-T2A-EGFP) larvae show more small (1–5 breaks) myofiber detachments per somite (p = 0.0441) and less large (> 10 breaks) myofiber detachments per somite (p = 0.001) as compared to dmd^−/− larvae. Statistical analysis in c, i: Two-sided t-tests with Welch correction. Data in violin plots (c, i) are presented as median (line) and quartiles (dashed line). Data in all survival graphs (b) are presented as mean ± SEM. Scale bar in e: 100 µm, f: 25 µm. Schematic images were adapted from https://www.biorender.com.

Muscle specific overexpression of fhl2b ameliorates axon and neuromuscular junction integrity in dmd^−/− zebrafish larvae.

a Upset plot showing intersection of DEGs between dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) vs sibling controls (dmd^+/+, dmd^+/-) and dmd^−/− vs sibling controls (Gene set A: disease-related DEGs shared between dmd^−/− and dmd^−/−;Tg(503unc:fhl2b-T2A-EGFP); Gene set B: DEGs unique to dmd^−/−;Tg(503unc: fhl2b-T2A-EGFP) larvae; Gene set C: disease-related DEGs specific to dmd^−/− larvae). b Heatmap displaying the expression of 1054 DEGs in Gene set C. c GO terms enriched in genes from gene set C. Yellow boxes: GO terms related to axon and neuron projection guidance; BP: Biological Process. d Heatmap displaying the expression of axon and neuron guidance related DEGs, red boxes: semaphorin and ephrin DEGs. Normalized counts for edre-mir-206 (p.adj=0.00005, p.adj=0.009) and fhdac4 (p.adj=0.02). Comparisons were made between dmd^−/− vs sibling controls and dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) vs sibling controls, respectively. g Sibling control, dmd^−/− and dmd^−/−;Tg(503unc:fhl2b-T2A-EGFP) larvae immunolabeled for acetylated tubulin and h α-bungarotoxin. i Magnification of dashed boxes in g), arrowheads: axons, open arrowheads: axon/NMJ overlap and arrows: NMJs lacking axon overlap. j Sibling control, dmd^−/− and dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) larvae immunolabeled for SV2 and α-bungarotoxin. Arrowheads: SV2⁺/α-bungarotoxin⁺ NMJs. k Quantifications of α-bungarotoxin⁺ NMJs where p < 0.0001 for dmd^−/− vs sibling control and p = 0.0008 for dmd^−/− vs dmd^-/-:Tg(503unc:fhl2b-T2A-EGFP). l Quantifications of SV2⁺/α-bungarotoxin⁺ NMJs where p = 4.6e⁻⁵ for dmd^−/− vs sibling control, p = 0.044 for dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) vs sibling control and p = 0.024 for dmd^−/− vs dmd^-/-:Tg(503unc:fhl2b-T2A-EGFP). m Filament reconstruction of acetylated tubulin labeled sibling control, dmd^−/− and dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) larvae. n) Volume reconstruction of acetylated tubulin/α-bungarotoxin labeled sibling control, dmd^−/− and dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP). o Quantification of total axon filament length based on reconstructions in (m) where p = 0.014 for dmd^−/− vs sibling control and p = 9.7e⁻⁵ for dmd^−/− vs dmd^-/-:Tg(503unc:fhl2b-T2A-EGFP). p Quantification of total axon volume based on reconstructions in (n) where p = 0.0005 for dmd^−/− vs sibling control, p = 0.043 for dmd^−/−;Tg(503unc:fhl2b-T2A-EGFP) vs sibling control and p = 0.002 for dmd^−/− vs dmd^-/-;Tg(503unc:fhl2b-T2A-EGFP). Statistical analysis: Two-sided t-tests with Welch correction. Trunk region viewed is indicated in illustration above. Scale bar in g, h, m, n: 50 µm, i, j: 25 µm. Schematic images were adapted from https://www.biorender.com.

fhl2b overexpression leads to upregulation of regenerative markers in dmd^−/− zebrafish larvae.

a Upset plot showing the intersection of DEGs between Tg(503unc:fhl2b-T2A-EGFP) vs sibling controls and dmd^−/−:Tg(503unc:fhl2b-T2A-EGFP) vs dmd^−/− larvae (Gene set D: DEGs caused by fhl2b overexpression in both comparisons; Gene set E: DEGs caused by fhl2b overexpression in healthy sibling control conditions alone; Gene set F: DEGs caused by fhl2b overexpression in the dmd^−/− disease condition). b Heatmap displaying the expression of DEGs in Gene set F. Red boxes indicate DEGs related to muscle regeneration. c Timelapse images of dmd^−/−;Tg(503unc:EGFP) and dmd^−/−;Tg(503unc:fhl2b-T2A-EGFP) larvae at 3.5, 4 and 4.5 dpf, respectively. Asterisks (*) indicate damaged somites. Area viewed is indicated in zebrafish illustration above. d Birefringence images following the same dmd^−/−;Tg(503unc:EGFP) and dmd^−/−;Tg(503unc:fhl2b-T2A-EGFP larvae at 3, 4 and 5 dpf. Asterisks (*) indicate damaged somites. Trunk region viewed is indicated in zebrafish illustration above. E Quantification of birefringence in damaged somites over time. Birefringence at 4 and 5 dpf was compared to 3 dpf in order to evaluate muscle regeneration progression over time. p = 0.012 at 4 dpf and p = 0.051 at 5 dpf. Statistical analysis in e, f: Two-sided Wald test with B/H-correction, k, l, o, p: Two-sided t-test with Welsh correction. Scale bar in c: 50 µm and d: 200 µm. Schematic images were adapted from https://www.biorender.com.

Muscle wound healing is enhanced in fhl2b overexpressing larvae.

a Normalized counts for mmp13a (p.adj=0.012, p.adj=0.023), mpeg1.2 (p.adj=0.015, p.adj=0.0009), cxcl8b.3 (p.adj=7.4e⁻⁸, p.adj=0.0007) and cxcl8b.1 (p.adj=0.0005, p.adj=0.0001). Comparisons were made between dmd^−/−:Tg(503unc: fhl2b-T2A-EGFP) vs sibling controls (dmd^+/+, dmd^+/-) and Tg(503unc: fhl2b-T2A-EGFP) vs sibling controls, respectively. b Wound healing assay experimental setup indicating area of needle-stick injury and timepoints for the different analyzes presented below. c Lateral view of injured somites in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) zebrafish embryos immunolabeled with neutrophil specific Mpx antibody between 2–18 hpi. Dashed lines indicate wounded areas. Quantifications of Mpx⁺ neutrophils at wound size is presented in d. e Lateral view of injured somites in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) zebrafish embryos immunolabeled with macrophage specific antibody Mfap4 between 6–72 hpi. Dashed lines indicate wounded areas. f Quantification of Mfap4⁺/DAPI⁺ cells in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) at 6 (p = 0.0019), 24 (p = 0.0374), 48 and 72 hpi. g Lateral view of injured somites in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) zebrafish embryos immunolabeled with satellite cell specific Pax7antibody at 24–96 hpi. h Quantification of Pax7⁺/DAPI⁺ cells in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) at 24 (p = 0.0445), 48 (p = 0.0198), 72 (p = 0.0002) and 96 hpi (p = 0.0246). i Lateral view of injured somites in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) zebrafish embryos labeled with phalloidin (F-actin) at 24–96 hpi. j Quantification of F-actin intensity at wound site in sibling controls and Tg(503unc:fhl2b-T2A-EGFP) at 24, 48, 72 (p = 0.0045) and 96 hpi. Statistical analysis in a: Two-sided Wald test with B/H-correction, d, f, h, j: Two-sided t-tests with Welch correction. Data in graphs is presented as mean ± SEM. Scale bar: 25 µm. Schematic images were adapted from https://www.biorender.com.

Model for fhl2b mediated rescue of muscular dystrophy.

fhl2b is upregulated and protects EOMs from muscular dystrophy conditions. dmd^−/− larvae overexpressing fhl2b survive for extended amounts of time due to improved muscle integrity with fewer myofiber detachments and breaks, improved axon and NMJ stability, and accelerated myofiber regeneration. Schematic images were adapted from https://www.biorender.com.