Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul;14(7):e1688.
doi: 10.1002/ctm2.1688.

Salbutamol repurposing ameliorates neuromuscular junction defects and muscle atrophy in Col6a1-/- mouse model of collagen VI-related myopathies

Sonia Calabrò  1   2 , Leonardo Nogara  3   4   5 , Yongzhi Jian  6 , Manuel Valentin  6 , Dario Bizzotto  1 , Paola Braghetta  1 , Loris Russo  1 , Lisa Gambarotto  1 , Bert Blaauw  3   4 , Said Hashemolhosseini  6 , Paolo Bonaldo  1 , Matilde Cescon  1
Affiliations

Salbutamol repurposing ameliorates neuromuscular junction defects and muscle atrophy in Col6a1-/- mouse model of collagen VI-related myopathies

Sonia Calabrò et al. Clin Transl Med. 2024 Jul.
No abstract available

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Salbutamol treatment ameliorates neuromuscular junction (NMJ) fragmentation, promotes NMJ remodelling and induces NMJ functional improvement in Col6a1−/− mice. (A) Whole‐mount hemidiaphragms preparations of vehicle‐treated WT and Col6a1− /− mice, stained with 555‐BTX. White arrowheads indicate AChR cluster fragments. Scale bar, 10 µm. (B) Quantification of the average number of fragments per NMJ in vehicle‐treated WT versus Col6a1− /− mice. Data are shown as mean ± s.e.m. (***p < .001; Mann–Whitney test; n = 546–604 NMJs from 5 to 7 mice, each group). (C) Percentage of total NMJs displaying the number of fragments reported in the x‐axis in vehicle‐treated WT versus Col6a1− /− mice. Data are shown as mean ± s.e.m. (*p < .05; **p < .01; Mann–Whitney test; n = 5–7 mice, each group). (D) Whole‐mount hemidiaphragm preparations of vehicle‐ and salbutamol‐treated Col6a1− /− mice, stained with 555‐BTX to highlight AChR cluster fragmentation (white arrowheads). Scale bar, 10 µm. (E) Quantification of the average number of fragments per NMJ in vehicle‐ versus salbutamol‐treated Col6a1− /− mice. Data are shown as mean ± s.e.m. (***p < .001; Kruskal–Wallis test with Dunn's post hoc test for multiple comparisons; n = 352–604 NMJs from 4 to 7 mice, each group). (F) Percentage of total NMJs displaying the number of fragments reported in the x‐axis in vehicle‐ versus salbutamol‐treated Col6a1− /− mice. Data are shown as mean ± s.e.m. (*p < .05; **p < .01; Kruskal–Wallis test with Dunn's post hoc test for multiple comparisons; n = 4–7 mice, each group). (G–L) Quantitative analysis of postsynaptic parameters in diaphragm muscles of vehicle‐ and salbutamol‐treated Col6a1− /− mice, showing AChR perimeter (G) and area (H), and endplate diameter (I), perimeter (J), area (K) and compactness (L). Data are shown as mean ± s.e.m. (*p < .05, **p < .01; ***p < .001; Kruskal–Wallis test with Dunn's post hoc test for multiple comparison; n = 96–147 NMJs from 4 to 7 mice, each group). (M) Average hanging time of WT and Col6a1− /− mice subjected to the four‐limb hanging test at T0 (before treatment). Data are shown as mean ± s.e.m. (***p < .001; Mann–Whitney test; n = 28–30 mice, each group). (N, O) Individual four‐limb hanging performances of Col6a1− /− mice before (T0) and after (W4) 4‐week‐long treatment with vehicle or salbutamol. The average hanging time (N) and the holding impulse (O) are shown as outcome measures (*p < .05; NS, not significant; two‐tailed paired t‐test for parametric data and Wilcoxon matched pairs signed rank test for non‐parametric data; n = 5–15 mice, each group). (P–W) Quantitative analysis of electrophysiological parameters measured in ex vivo diaphragm preparations of vehicle‐treated WT mice, vehicle‐treated Col6a1− /− mice and 8 mg/kg/die salbutamol‐treated Col6a1− /− mice, showing nerve‐evoked endplate potentials (EPP) (P) and miniature endplate potentials (mEPP) (Q) amplitudes, quantal content (R), input resistance (S), endplate currents (EPC) (T), miniature endplate currents (mEPC) (U), frequency of mEPP (V), and EPP amplitude decrement at 5 Hz (W). Error bars indicate s.e.m. (*p < .05; **p < .01; ***p < .001; unpaired two‐tailed Student's t‐test; n = 8 mice, each group). AChR, acetylcholine receptor; Salb, salbutamol; veh, vehicle; WT, wild type.
FIGURE 2
FIGURE 2
Salbutamol remodels skeletal muscle in Col6a1− /− mice. (A) Growth curve showing changes in body weight over the 4 weeks of treatment (W1–W4) in vehicle‐ and salbutamol‐treated Col6a1 −/− mice. The values registered at each time point are expressed as a percentage of the value measured before treatment (T0). Data are shown as mean ± s.e.m. (*p < .05; **p < .01; ***p < .001, compared to T0 within the same group; 2‐way analysis of variance (ANOVA) test with Dunnet's post hoc test for multiple comparisons; n = 4–13 mice, each group). (B, C) Absolute (B) and normalised (C) weight of tibialis anterior (TA) and gastrocnemius muscles of vehicle‐ and salbutamol‐treated Col6a1−/‐ mice. Data are shown as mean ± s.e.m. (*p < .05; **p < .01; ***p < .001; Kruskal–Wallis test with Dunn's post hoc test for multiple comparisons for non‐parametric data or one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons for parametric data; n = 8–32 muscles, each group). (D) Representative fluorescence microscopy images of cross‐sections of TA muscle from vehicle‐ and salbutamol‐treated Col6a1−/‐ mice, stained with fluorophore‐conjugated wheat germ agglutinin (WGA, green) and Hoechst (blue). Arrowheads point at centrally located nuclei. Scale bar, 100 µm. (E, F) Quantification of myofibre cross‐sectional area (CSA) (E) and minimum Feret's diameter (MFD) (F) in vehicle‐ and salbutamol‐treated Col6a1−/− mice. Data are shown as mean ± s.e.m. (*p < .05; one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons; n = 3–8 mice, each group). (G, H) Graphical representation of CSA (G) and MFD (H) distribution among myofibres. The curves were fitted to data using non‐linear regression (Gaussian). (I–L) Comparison of CSA (I, J) and MFD (K, L) distribution among myofibres between vehicle‐treated and low‐dose (I, K) or high‐dose (J, L) salbutamol‐treated Col6a1−/− muscles. Data are shown as mean ± s.e.m. (*p < .05; **p < .01; ***p < .001; multiple unpaired two‐tailed Student's t‐tests; n = 3–8 mice, each group). (M) Quantification of myofibre density, calculated as the number of myofibres per total area, in vehicle‐ and salbutamol‐treated Col6a1−/− mice. Data are shown as mean ± s.e.m. (*p < .05; one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons; n = 3–8 mice, each group). (N) Quantification of the percentage of centrally nucleated fibres (CNF) per muscle section in vehicle‐ and salbutamol‐treated Col6a1−/− mice. Data are shown as mean ± s.e.m. (one‐way ANOVA with Dunnet's post hoc test for multiple comparisons; n = 3–8 mice, each group). (O) Representative confocal micrographs of TA cross‐sections from vehicle‐ and salbutamol‐treated Col6a1−/− mice, stained with Hoechst (blue) and antibodies for embryonic myosin heavy chain (eMHC, red) and laminin (green). White arrowheads indicate eMHC‐positive myofibres. Scale bar, 100 µm. (P) Quantification of eMHC‐positive myofibres per area unit (in mm2), based on confocal micrographs as in (O). Data are shown as mean ± s.e.m. (**p < .01; one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons; n = 3–6 mice, each group). (Q) Real‐time quantitative polymerase chain reaction (RT‐qPCR) analysis of myosin heavy chain 3 (Myh3) transcript levels in TA and diaphragm muscles of vehicle‐ and salbutamol‐treated Col6a1−/− mice. Data are shown as mean ± s.e.m. (**p < .01; one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons; n = 4–9 mice, each group). Salb, salbutamol.
FIGURE 3
FIGURE 3
Salbutamol modifies muscle fibre composition in Col6a1 − / − mice. (A) Representative reconstruction of optical micrographs of TA cross‐sections from vehicle‐ and salbutamol‐treated Col6a1−/ − mice, following histochemical succinate dehydrogenase (SDH) staining. SDH‐positive fibres are stained in dark blue, whereas SDH‐negative fibres are stained in light blue. Scale bar, 500 µm. (B) Quantification of SDH‐positive area, determined by SDH staining as in (A). Data are shown as mean ± s.e.m. (Kruskal–Wallis test with Dunn's post hoc test for multiple comparisons; n = 3–6 mice, each group). (C, D) RT‐qPCR quantification of the levels of transcripts for different myosin heavy chain (MHC) isoforms in the diaphragm (C) and TA (D) muscles of vehicle‐ and salbutamol‐treated Col6a1−/‐ mice. Data are shown as mean ± s.e.m. (**p < .01; one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons; n = 4–9 mice, each group). (E) Representative merged fluorescence microscopy images of whole TA cross‐sections from vehicle‐ and salbutamol‐treated Col6a1−/− mice, stained with antibodies against MHC‐IIA (red), MHC‐IIB (green) and laminin (white). Dotted white squares highlight the areas shown at higher magnification in the bottom panels. Scale bars, 500 µm (top panels) or 100 µm (bottom panels). (F) Quantification of the percentages of type IIA and type IIB fibres, based on immunofluorescent images as in (E). Data are shown as mean ± s.e.m. (*p < .05; **p < .01; one‐way ANOVA test with Dunnett's post hoc test for multiple comparisons; n = 3–5 mice, each group). Salb, salbutamol.
FIGURE 4
FIGURE 4
One‐month salbutamol treatment ameliorates muscle force in Col6a1−/− mice and leads to ADRB2 downregulation. (A, B) Absolute (A) and normalised (B) in vivo force measurements of gastrocnemius muscle from vehicle‐treated WT and Col6a1− / − mice. Whole curves of stepwise stimulation frequencies are shown. Tetanus was reached at a stimulation frequency of 100 Hz. Data are shown as mean ± s.e.m. (*p < .05; **p < .01; ***p < .001, comparing vehicle treatment in different genotypes at each stimulation frequency; multiple unpaired t‐tests with Holm–Sidak method to correct for multiple comparisons; n = 12–16 limbs, each group). (C, D) Absolute (C) and normalised (D) in vivo force measurements of gastrocnemius muscle from vehicle‐treated and salbutamol‐treated Col6a1− / − mice. Whole curves of stepwise stimulation frequencies are shown. Tetanus was reached at a stimulation frequency of 100 Hz. Data are shown as mean ± s.e.m. (*p < .05; **p < .01; ***p < .001, comparing salbutamol treatment to vehicle treatment within the same genotype, at each stimulation frequency; two‐way ANOVA with Dunnett's post hoc test for pairwise comparisons; n = 8–12 limbs, each group). (E) Representative western blot for ADRB2 and TH in total protein extracts of TA muscle from vehicle‐treated WT mice and vehicle‐ and salbutamol‐treated Col6a1−/− mice. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. (F) Densitometric quantification of ADRB2 levels normalised to GAPDH levels, determined by three independent western blot experiments as in (E). Data are shown as mean ± s.e.m. (*p < .05; **p < .01; one‐way ANOVA with Dunnett's post hoc test for multiple comparisons; n = 4–9 mice, each group). (G) Densitometric quantification of TH levels normalised to GAPDH levels, determined by two independent western blot experiments as in (E). Data are shown as mean ± s.e.m. (one‐way ANOVA with Dunnett's post hoc test for multiple comparisons; n = 4–9 mice, each group). (H) Representative western blot for ADRB2 and TH in total protein extracts of diaphragm muscle from vehicle‐treated WT mice and vehicle‐ and salbutamol‐treated Col6a1− /‐ mice. GAPDH was used as a loading control. (I) Densitometric quantification of ADRB2 levels normalised to GAPDH levels, determined by two independent western blot experiments as in (G). Data are shown as mean ± s.e.m. (**p < .01; one‐way ANOVA with Dunnett's post hoc test for multiple comparisons; n = 4–9 mice, each group). (J) Densitometric quantification of TH levels normalised to GAPDH levels, determined by two independent western blot experiments as in (G). Data are shown as mean ± s.e.m. (one‐way ANOVA with Dunnett's post hoc test for multiple comparisons; n = 4–9 mice, each group). (K) Representative fluorescence microscopy images of longitudinal sections of gastrocnemius muscles from vehicle‐treated WT and vehicle‐ and high‐dose salbutamol‐treated Col6a1−/− mice, stained with an antibody against TH (green), 555‐BTX (red) and Hoechst. Scale bar, 10 µm. (L) Quantification of the percentages of NMJs showing TH staining according to (K). Data are shown as mean ± s.e.m. (Kruskal–Wallis test with Dunn's post hoc test for multiple comparisons; n = 3 mice, each group). More than 50 NMJs were analyzed for each sample. ADRB2, adrenergic receptor β2; Salb, salbutamol; TH, tyrosine hydroxylase; Veh, vehicle; WT, wild type.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Bonaldo P, Braghetta P, Zanetti M, et al. Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy. Hum Mol Genet. 1998;7:2135‐2140. doi:10.1093/hmg/7.13.2135 - DOI - PubMed
    1. Merlini L, Angelin A, Tiepolo T, et al. Cyclosporin A corrects mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies. Proc Natl Acad Sci USA. 2008;105:5225‐5229. doi:10.1073/pnas.0800962105 - DOI - PMC - PubMed
    1. Chrisam M, Pirozzi M, Castagnaro S, et al. Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI‐null mice. Autophagy. 2015;11:2142‐2152. doi:10.1080/15548627.2015.1108508 - DOI - PMC - PubMed
    1. Gambarotto L, Metti S, Corpetti M, et al. Sustained oral spermidine supplementation rescues functional and structural defects in COL6‐deficient myopathic mice. Autophagy. 2023;19(12):3221‐3229. doi:10.1080/15548627.2023.2241125 - DOI - PMC - PubMed
    1. Cescon M, Gregorio I, Eiber N, et al. Collagen VI is required for the structural and functional integrity of the neuromuscular junction. Acta Neuropathol. 2018;136:483‐499. doi:10.1007/s00401-018-1860-9 - DOI - PubMed

Substances

LinkOut - more resources