Tamoxifen prolongs survival and alleviates symptoms in mice with fatal X-linked myotubular myopathy

Abstract

X-linked myotubular myopathy (XLMTM, also known as XLCNM) is a severe congenital muscular disorder due to mutations in the myotubularin gene, MTM1. It is characterized by generalized hypotonia, leading to neonatal death of most patients. No specific treatment exists. Here, we show that tamoxifen, a well-known drug used against breast cancer, rescues the phenotype of Mtm1-deficient mice. Tamoxifen increases lifespan several-fold while improving overall motor function and preventing disease progression including lower limb paralysis. Tamoxifen corrects functional, histological and molecular hallmarks of XLMTM, with improved force output, myonuclei positioning, myofibrillar structure, triad number, and excitation-contraction coupling. Tamoxifen normalizes the expression level of the XLMTM disease modifiers DNM2 and PI3KC2B, likely contributing to the phenotypic rescue. Our findings demonstrate that tamoxifen is a promising candidate for clinical evaluation in XLMTM patients.

Conflict of interest statement

J.L. and B.S.C. are scientific advisors for Dynacure. The remaining authors declare no competing interests.

Figures

Fig. 1

Tamoxifen rescues the phenotype of XLMTM mice. Life-long oral tamoxifen delays disease progression in XLMTM mice. Wild type (WT) and Mtm1−/y mice were fed control or tamoxifen (TAM)-supplemented diets from weaning onward. a Photographs of WT and Mtm1−/y mice, illustrating disease severity in young untreated mice and protection conferred by tamoxifen in adult and old mice. b Kaplan–Meier curves showing the effects of treatments on mouse survival. Early death of untreated Mtm1−/y mice contrasts with prolongation of life span in tamoxifen-treated littermates. Data are from 8 to 18 mice per group. *P ≤ 0.05; ****P ≤ 0.0001. Log-rank (Mantel–Cox) test. c Disease progression was assessed three times per week using a 5-grades clinical scale: muscle function was scored as 1 (normal function of hind limbs), 2 (difficulty in spreading toes), 3 (evident weakness in legs), 4 (paralysis of one hind limb), or 5 (complete paralysis of both legs). WT mice had a clinical score of 1 (normal) throughout the study. Untreated Mtm1−/y mice quickly reached a high clinical grade, whereas disease progressed much slower in tamoxifen-treated littermates. d Mouse motor function was assessed weekly via a horizontal grid-hanging test. The score of untreated Mtm1−/y mice declined quickly. Tamoxifen preserved motor function of Mtm1−/y mice close to WT values. e Mouse body weight was recorded 3 times per week. Tamoxifen affected the growth of WT but not of Mtm1−/y mice. Mtm1−/y mice remained smaller than WT mice throughout. b–e Symbols and TAM doses (0.3, 3, and 30 mg kg−1 of diet) are shown on the right-hand side. c, d, e Data represent the mean ± s.e.m. of 8 to 18 mice as defined in a. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns non-significant. One-way ANOVA with Fisher’s least significance difference (LSD) post-test

Fig. 2

Tamoxifen augments the strength of leg muscles of XLMTM mice. Electrically evoked triceps contractions were recorded under isometric conditions and contractile features analyzed in 42, 84, and 210-day-old mice fed placebo or tamoxifen-supplemented (30 mg kg−1) diets. a Average traces showing absolute phasic (twitch) contraction of the triceps at 42 days (D42). Tamoxifen significantly enhanced the force (tension) in Mtm1−/y mice. b Specific phasic force (see Online Methods for details) of D42 Mtm1−/y mice augmented with tamoxifen. c Specific phasic force of Mtm1−/y mice increased with tamoxifen at all examined ages. d Average traces showing triceps absolute force-frequency curves at D42. Tamoxifen more than doubled the tetanic force of Mtm1−/y mice. e Specific force of Mtm1−/y mice markedly augmented with tamoxifen at all stimulation frequencies. f Specific tetanic force of Mtm1−/y mice was considerably improved at all studied ages. In triceps examined after various treatment durations, tamoxifen rescued the impaired time to twitch peak of Mtm1−/y mice (g) but had no impact on the time required for half-relaxation from peak (h). D42 panels: WT wild type; legend in a stands for a, b, d, e; data represent the mean ± s.e.m. of n = 7 triceps per group. c, f, g, h: black triangles illustrate increasing age (42–84 and 210 days) within each treatment group; columns from left to right: data represent the mean ± s.e.m. of 7; 8; 7; 7; 7; 13; 7; 0; 0; 7; 8; 6 triceps, respectively. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns non-significant, Ø no surviving mice. One-way ANOVA followed by Fisher’s LSD post-test

Fig. 3

Tamoxifen mitigates muscle structure and improves sarcomeric organization in XLMTM mice. Muscle structure and sarcomere ultrastructure were examined by histology and transmission electron microscopy, respectively, in 42-day-old (D42) mice either untreated (control; Ctrl) or treated with tamoxifen (TAM; 30 mg kg−1 of diet). a Representative pictures of hematoxylin–eosin stained sections from the tibialis anterior (TA) of D42 mice. Note the small size of the Mtm1−/y myofibres and the presence of mislocalized nuclei, a hallmark of Mtm1−/y mice and XLMTM patients (arrows). b Representative pictures of succinate dehydrogenase (SDH) activity in TA sections of D42 mice, demonstrating abnormal distribution of oxidative staining. The intense staining forming a ring at the periphery of many Mtm1−/y myofibres (examples shown by asterisks) is due to accumulated mitochondria and other organelles and is a hallmark of the pathology. c The percentage of nuclei abnormally positioned (either internally or centrally located) in TA myofibres of D42 mice were counted from hematoxylin–eosin-stained sections. That feature was reduced by 53.3% with TAM. Data represent the mean ± s.e.m. of n = 2–4 TA per group. ****P ≤ 0.0001. One-way ANOVA followed by Fisher’s LSD post-test. d Sarcomere ultrastructure revealed by transmission electron microscopy in the TA from untreated (control) and tamoxifen-treated Mtm1−/y mice at D42. Note overall disorganization of the sarcomeres in untreated TA with disruption of the Z-lines (structures running perpendicular to the sarcomeres and holding myofibrils together; arrowheads) and shifted sarcomeres (dashed arrow). Tamoxifen ameliorated the organization of the sarcomeres as demonstrated by in-register Z-lines in adjacent myofibrils (arrowheads). The bar represents 50 µm in a and b, and 2 µm in d

Fig. 4

Tamoxifen reduces the expression of XLMTM disease modifiers and alters the expression of estrogen receptors. The mRNA and protein levels of known XLMTM disease modifiers and of estrogen receptors, which mediate most tamoxifen actions, were determined in gastrocnemius muscle of 42-days old (D42) wild type (WT) and Mtm1−/y mice, either untreated (control; Ctrl) or tamoxifen (TAM)-treated (30 mg kg−1 of diet). a mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). From left to right, relative expression (as percentage of WT Ctrl) of mRNA encoding amphiphysin 2/BIN1 (Bin1; all transcripts), muscle-specific BIN1 (Bin1; exon 11-containing transcript), dynamin-2 (Dnm2), PI3KC2B (Pik3c2b), estrogen receptor (ER) α (Esr1), and ERβ (Esr2). b Representative immunoblots of proteins of interest, as indicated. DNM2: dynamin 2; MyHC: myosin heavy chains; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. Position of molecular weight markers (kDa) is shown. c–f Levels (normalized to GAPDH and expressed as percentage of WT Ctrl) of proteins selected for their role in XLMTM and tamoxifen signaling. c Proteins involved in the “MAD”-pathway. From top to bottom: myotubularin (MTM1), amphiphysin 2/BIN1 (pan-isoforms), amphiphysin 2/BIN1 (muscle-specific isoform), dynamin-2 (DNM2). d Other disease modifiers and protein deregulated in absence of MTM1. From top to bottom: desmin, dysferlin, PI3KC2B. e Estrogen receptors. Top: ERα; bottom: ERβ. f Myosin heavy chains, a major constituent of sarcomeres. Data represent the mean ± s.e.m. of n = 6–8 muscles per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns non-significant. One-way ANOVA followed by Fisher’s LSD post-test

Fig. 5

Tamoxifen improves triad density and excitation-contraction coupling, Triads and excitation–contraction coupling were examined in 42 days old (D42) mice either untreated (control; Ctrl) or treated with tamoxifen (TAM; 30 mg kg−1 in diet). a Triads—specialized membrane structures made of a t-tubule (tub) flanked by terminal cisternae (cis) arising from the sarcoplasmic reticulum and controlling Ca2+ release—were visualized by transmission electron microscopy (TEM) in the tibialis anterior (TA) of mice at D42. Note the abnormal shape of the remaining triads in the Mtm1−/y mouse and recovery with tamoxifen treatment. The bar represents 100 nm. b The number of well-positioned triads per sarcomere unit was determined from TEM pictures in TA from wild type (WT) and Mtm1−/y mice at D42. TAM partly rescued the much decreased triad density in Mtm1−/y mice. Mean ± s.e.m. of n = 2–3 mice. c–e Excitation–contraction coupling assessed via live imaging of Ca2+ fluxes induced by KCl depolarization in single FDB myofibers (see Online Methods for details). c Average traces of the responses of FDB fibers to KCl, an experimental setting that mimics muscle depolarization (n = 17–29 fibers). d Representative images (pseudo-colored) illustrating cytosolic Ca2+ levels in FDB fibers at baseline (−; before KCl pulse) and at the peak of KCl-induced response (+). The bar represents 100 μm. e Quantification of cytosolic Ca2+ at the peak response. TAM enhanced Mtm1−/y cytosolic Ca2+ to levels found in WT. Data shown in e represent, from left to right, the mean ± s.e.m. of n = 29, 53, 53, and 31 fibers. f Representative western blots of DHPR and RyR1. g, h Quantification of DHPR and RyR1, respectively. Mean ± s.e.m. of n = 7 muscles. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns non-significant. One-way ANOVA followed by Fisher’s LSD post-test

Fig. 6

Myopathic features after long-term tamoxifen treatment, Muscle structure and triads were examined by histology and transmission electron microscopy, respectively, in wild type (WT) and Mtm1−/y mice during long-term treatment with tamoxifen (30 mg kg−1 in diet). a Representative pictures of tibialis anterior (TA) sections from untreated WT and Mtm1−/y mice at D42, and tamoxifen-treated Mtm1−/y mice at D42, D84, and D210 stained with hematoxylin–eosin (HE) or for succinate dehydrogenase (SDH) activity. Muscle structure of Mtm1−/y mice was stable over the duration of tamoxifen treatment. The bar represents 50 µm. b Scatter plots showing the distribution of TA myofiber diameter in mice at D42, D84, and D210 (black triangles illustrate increasing age). Fiber size remained almost constant from D42 to D210. N = 600 myofibers per group. c The percentage of abnormally positioned nuclei (either internally or centrally located) in TA myofibres of mice at D42, D84, and D210 were counted from hematoxylin–eosin-stained sections. The density of abnormally positioned nuclei increased significantly with ageing. Mean ± s.e.m. of n = 2–5 TA per group. d The number of well-positioned triads relative to sarcomere number, determined from TEM pictures in TA from wild type and Mtm1−/y mice, showed no significant change from D42 to D84. Black triangles illustrate increasing age (42–84 and 210 days). Mean ± s.e.m. of n = 2–3 TA per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ##P ≤ 0.01; Mtm1−/y control vs tamoxifen at D42. ns non-significant; Ø no surviving mice. One-way ANOVA followed by Fisher’s LSD post-test