Altered myogenesis and premature senescence underlie human TRIM32-related myopathy

RESEARCH Open Access Altered myogenesis and premature senescence underlie human TRIM32-related myopathy E. Servián-Morilla 1,2 , M. Cabrera-Serrano 1,2,3 , E. Rivas-Infante 4,3 , A. Carvajal 5 , P. J. Lamont 3 , A. L. Pelayo-Negro 6 , G. Ravenscroft 3 , R. Junckerstorff 7 , J. M. Dyke 7 , S. Fletcher 8,9 , A. M. Adams 8,9 , F. Mavillard 1,2 , M. A. Fernández-García 10 , J. L. Nieto-González 2,11 , N. G. Laing 3 and C. Paradas 1,2* Abstract TRIM32 is a E3 ubiquitin -ligase containing RING, B-box, coiled-coil and six C-terminal NHL domains. Mutations involving NHL and coiled-coil domains result in a pure myopathy (LGMD2H/STM) while the only described mutation in the B-box domain is associated with a multisystemic disorder without myopathy (Bardet-Biedl syndrome type11), suggesting that these domains are involved in distinct processes. Knock-out (T32KO) and knockin mice carrying the c.1465G > A (p.D489N) involving the NHL domain (T32KI) show alterations in muscle regrowth after atrophy and satellite cells senescence. Here, we present phenotypical description and functional characterization of mutations in the RING, coiled-coil and NHL domains of TRIM32 causing a muscle dystrophy. Reduced levels of TRIM32 protein was observed in all patient muscle studied, regardless of the type of mutation (missense, single amino acid deletion, and frameshift) or the mutated domain. The affected patients presented with variable phenotypes but predominantly proximal weakness. Two patients had symptoms of both muscular dystrophy and Bardet-Biedl syndrome. The muscle magnetic resonance imaging (MRI) pattern is highly variable among patients and families. Primary myoblast culture from these patients demonstrated common findings consistent with reduced proliferation and differentiation, diminished satellite cell pool, accelerated senescence of muscle, and signs of autophagy activation. Keywords: Muscle dystrophy, TRIM32, E3 ubiquitin -ligase, Proliferation/differentiation, Autophagy Introduction Tripartite motif-containing protein 32 (TRIM32) is an E3 ubiquitin ligase, whose activity is contained in the RING finger domain of the tripartite TRIM/RBCC motif (RING, B-box, coiled-coil) [20,30]. In addition, TRIM32 has six C-terminal NHL domains. The majority of the mutations described in TRIM32 are clustered in the C-terminal NHL domain, which is defined by amino acid sequence homologies to regions of Ncl-1, HT2A and Lin-41 proteins, supporting its role in protein-protein interactions, critical for the ubiquitination process [13, 29,44]. Mutations involving the NHL and coiled-coil domains are associated with limb-girdle muscular dystrophy 2H (LGMD2H) and sarcotubular myopathy (STM), which are considered as a continuum [15,21]. Until now, only one mutation has been described involving the B-box domain, resulting in a different multisystemic disorder called Bardet-Biedl syndrome (BBS) type 11 with no skeletal muscle involvement, in an only family with four affecting siblings [7]. No mutations in the RING finger domain have been reported. Proximal weakness is the characteristic feature of LGMD2H/STM, although other clinical findings, such as facial, axial or distal weakness, can be associated [5,15, 19,28,33,34,39,42]. Pathologically LGMD2H/STM are characterized by segmental vacuolation of the sarcoplasmic reticulum and transverse tubules [42], however vacuoles containing basophilic material consistent with * Correspondence: [email protected] 1 Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 https://doi.org/10.1186/s40478-019-0683-9 autophagic vacuoles have also been observed in the muscle biopsy of these patients [21,28]. The mutation c.1459G > A/p.D487N in the TRIM32 gene, identified as a founder mutation in Hutterite population, has been the most frequently reported [15], but a recent series of 12 non-Hutterite patients with a TRIM32-related myopathy, with mutations located both in the NHL and coiled-coil domains, has been described [21]. A yeast model has shown that TRIM32 mutations involving the NHL domain introduce conformational changes that impair the interaction properties of the protein, and consequently the ubiquitination process [39]. The most relevant mechanistic studies have been performed in the Trim32 knockout (T32KO) and the knock-in mice carrying the Hutterite mutation (T32KI) [25,26]. TRIM32, as a ubiquitous E3 ubiquitin ligase, has been demonstrated to promote degradation of several targets [1,8,18,22,24,29,31,37], so the absence or abnormal function of TRIM32 due to recessive mutations would lead to loss of ubiquitination and accumulation of the TRIM32 substrates. E3 small ubiquitin-related modifier (SUMO) ligase (PIAS4) [1] and N-myc down-regulated protein 2 (NDRG2) have been previously identified as important TRIM32 substrates. Overexpression of PIAS4 is implicated in regulation of cellular senescence [4] and TRIM32-deficient primary myoblasts from T32KO mice have been demonstrated to undergo premature senescence and impaired myogenesis due to accumulation of PIAS4 [23]. On the other hand, NDRG2 overexpression in C2C12 myoblasts reduces cell proliferation and delayed cell cycle withdrawal during differentiation [14,31]. Altogether, these results coming from cell and animal models, support the hypothesis that TRIM32 is involved in control of myogenesis and that premature senescence underlies myopathy in LGMD2H. T32KI mice demonstrated that the Hutterite mutation (p.D489N) causes TRIM32 protein degradation, but this finding has not been reported in muscle from patients carrying missense mutations [25]. There is evidence that the EI24 autophagy-associated transmembrane protein is involved in the degradation of RING E3 ligases using the autophagy machinery, establishing a connection between two major protein degradation mechanisms, autophagy and the ubiquitin-proteasome system [11]. However, no specific studies about the role of autophagy in the degradation of TRIM32 have been reported. We aimed to establish if patients with muscular dystrophy due to TRIM32 mutations show evidence for the pathogenic mechanism postulated in the mouse models. This would provide a potential set of functional assays. We studied the muscle and myoblasts from three families with mutations in the NHL, coiled-coil and RING domains of TRIM32 resulting in a null-phenotype. We demonstrated common findings consistent with premature senescence, reduced myoblast proliferation and differentiation and, in addition, signs of autophagy activation seemingly involved in the degradation of the mutated TRIM32. Materials and methods Genetic work-up DNA was extracted from blood using standard procedures. Patients were studied using next generation sequencing (NGS): patient IV.3/Family A and patient II.2/ Family C with a panel of 34 genes associated with autosomal recessive limb-girdle muscular dystrophies, and patient II.3/Family B with a panel of 256 neuromuscular disease genes using HighSeq and NextSeq Illumina platforms. Affected and unaffected relatives of the probands were screened for their family mutations by Sanger sequencing. Histological studies Muscle samples were obtained by open muscle biopsies from the biceps brachii muscle of the three patients from Family A (III.2, IV.3 and III.3), two patients from Family B (II.2 and II.3), one patient from Family C (II.2), five aged-matched healthy controls and four age-matched disease controls (LGMD1B, LGMD1C, LGMD2N, NEM6). Frozen muscle sections were stained for standard histological and histochemical techniques including hematoxylin and eosin (H&E) and neonatal myosin heavy chain (MHC-neo). Stained sections were evaluated with an Olympus BX41 (Tokyo, Japan) equipped with a ColorView IIIu camera (Olympus). Primary cell culture Myoblasts from Family A (III.2 and IV.3), Family B (II.2 and II.3) and seven aged-matched healthy controls and three age-matched disease controls (LGMD2B, and X-EDMD) were used for the experiments. Fresh muscle samples were minced and cultured in a monolayer. Briefly, the culture medium for the myoblast stage (proliferation medium) contains 75% Dulbecco’s minimal essential medium (DMEM, Invitrogen) and 25% M199 medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS), 10 μg/ml insulin, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ ml fungizone, 10 ng/ml epidermal growth factor, and 25 ng/ml fibroblast growth factor. To obtain highly purified myoblasts, each 10 7 cells were mixed with 20 μlof CD56-coated microbeads (Milteny Biotec, Bergisch Gladbach, Germany) and incubated at 4 °C for 15 min. Unbound microbeads were removed by washing cells in excess PBS buffer followed by centrifugation at 400×g for 10 min. The cell pellet was resuspended in PBS buffer to a concentration of 2 × 10 8 cells/ml before Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 2 of 16 separation on a midiMACS cell separator (Milteny Biotec, Bergisch Gladbach, Germany). Transmission Electron microscopy (TEM) A piece of muscle from each biopsy and primary myoblast from patients and controls were fixed in 2,5% glutaraldehyde in 0,1 M sodium cacodylate buffer (pH 7,4) for 3 h at 4 °C, washed with the same buffer, postfixed in 1% osmium tetraoxide in 0,1 M cacodylate buffer (pH 7,4) for 1 h at 4 °C, and then rinsed with distilled water. Samples were immersed in 2% uranyl acetate, dehydrated through a gradient acetone series (50, 70, 90 and 100%), and embedded in Spurr’s resin. Semithin sections (450 nm thickness) were obtained with a glass knife and stained with 1% toluidine blue for muscle fiber/myoblast localization and reorientation using a conventional optic microscope. Once a suitable block face of the selected area was trimmed, ultrathin sections (70 nm) were obtained using an ultramicrotome (Leica UC7) equipped with a diamond knife (Diatome) and collected on 200 mesh copper grids. Sections were viewed with a Zeiss Libra 120 transmission electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany). Scanning Electron microscopy (SEM) Primary myoblasts from patients and controls were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 3 h at 4 °C and postfixed in 1% osmium tetraoxide in 0,1 M cacodylate buffer (pH 7.4) for 1 h at 4 °C. Samples were then dehydrated through a graded series of acetone solutions, critical point dried, gold sputtered, and examined with a Zeiss Auriga Field Emission Scanning Electron Microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany). Immunohistochemical studies Cultured primary myoblasts from patients and controls were fixed with 4% paraformaldehyde for 30 min, permeabilized in 0.2% Triton X-100 for 10 min and incubated in 1% BSA/PBS for 45 min. The following primary antibodies were used: rabbit polyclonal anti-TRIM32 (NBP1–33737) (1:500; Novus Biologicals); rabbit monoclonal anti-Desmin (D93FD5) (1:100; Abcam); mouse monoclonal anti-Ki67 (B56) (1:200; BD Biosciences); mouse monoclonal anti-Myogenin (FD5) (1:100; Abcam) and incubated for 12 h at 4 °C. Frozen muscle samples were fixed with 4% PFA for 15 min and immersed in citrate buffer (pH 6) for 30 min at 80 °C and washed with PBS. Afterward, muscle samples were blocked with 2% non-fat milk + 0.3% Triton X-100 in PBS for 30 min and with 5% BSA + 0.5% Triton X-100 in PBS for 1 h. The following primary antibodies were used: mouse monoclonal anti-Pax7 (1:50; DHSB); mouse monoclonal anti-collagen VI (3C4) (1:1000; Chemicon) and incubated for 3 days at 4 °C. After washing, samples were incubated with the secondary antibodies conjugated with Cy2 and Cy3 (1:500; Jackson ImmunoResearch) for 1 h. Finally, the nuclei were stained for 20 min with To-pro-3-Iodide (Topro 3) at 1/1000 in PBS and the slides were coverslipped with fluorescence mounting medium (Dako). Images were acquired on a Zeiss LSM 710 confocal laser scanning microscope using 20 x objective with a numerical aperture of 1.3. Images from controls and patients were obtained at the same day and under equal conditions (laser intensities and photomultiplier voltages). Maximal projections of Z-stacked images were analyzed with ImageJ software (RRID:SCR_003070). Senescence-associated β-galactosidase (SA-β-gal) staining Cell senescence was assessed measuring SA-β-gal activity at pH 6.0 [12]. Primary patient myoblasts were fixed in 0.2% glutaraldehyde for 15 min at room temperature and stained in solution containing 1 mg/ml bromo-chloro-indolyl-ga- lactopyranoside (X-gal), 40 mM citrid acid/sodium phosphate (pH 6), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, 2 mM magnesium chloride. SA-β-gal + cells were detected by phase contrast microscopy using Olympus Inverted Microscopes Models IX71. Western blot analysis Frozen muscle samples and primary patient myoblasts were homogenized in RIPA buffer (20 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.1% SDS) containing protease inhibitor cocktail (Roche). Western blot analysis of equal-protein loading were performed with the following primary antibodies: rabbit polyclonal anti-TRIM32 (NBP1–33737) (1:500; Novus Biologicals); mouse monoclonal anti-Myogenin (FD5) (1:100; Abcam); mouse monoclonal anti-Myosin Heavy Chain (A4.1025) (1:500; Millipore); rabbit polyclonal anti-LC3B (1:1000; Cell Signaling), rabbit monoclonal anti-LAMP1 (D2D11) (1:1000; Cell Signaling) and rabbit polyclonal anti-GAPDH (1:2000; Sigma-Aldrich). Immunoreactivity was detected with secondary antibodies conjugated to horseradish peroxidase (1:5000; Jackson Immuno Research) and developed with SuperSignal West Femto (Thermo Fisher Scientific) using an ImageQuant LAS 4000 MiniGold System (GE Healthcare Life Sciences). Proliferation and differentiation assays CD56-positive cells were seeded at 15,000 cells/cm 2 using proliferation medium. We examined proliferation over a period of time from 0 to 8 days. The average number of cells for each time point (0, 4, and 8 days) was then calculated and the values were used to plot growth curves. When the myoblasts started to fuse, the medium was substituted with one containing 2% of FBS without growth factor to induce differentiation. We Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 3 of 16 Fig. 1 (See legend on next page.) Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 4 of 16 measured the fusion index 4 days after medium change by calculating the mean percentage of nuclei in myotubes, relative to the total number of nuclei (myoblasts + myotubes). Autophagy blockade experiment Bafilomycin A1 (Baf-A1), an autophagosome-lysosome fusion inhibitor, inhibits autophagy by preventing the fusion of the lysosome to the autophagosome [46]. Myoblasts after 4 days in proliferation medium were incubated with 1 μM Baf-A1 (Cayman) for 6 h, followed by lysis and western blot analysis. Statistical analysis Graphpad Prism software (RRID: SCR_002798) was used to analyze the data using one-way ANOVA. When the ANOVA analysis revealed significant differences, the post hoc Bonferroni’s test was used for pairwise comparisons. When parametric assumptions were not met, Kruskal-Wallis followed by the post hoc Dunn’s test were used. Data were plotted as mean ± SEM. Results Mutations in NHL, coiled-coil and RING domains of TRIM32 are found in patients with a muscular dystrophy We studied three independent families of Spanish and Australian origin with a muscular dystrophy (Fig. 1a), andidentifiedfournovelTRIM32 mutations located in three different domains; NHL, coiled-coil and RING domains. Patients II.2, II.3 and III.3 from family A were homozygous for TRIM32 c.1771G > A (p.V591 M) involving the fourth NHL repeat. None of the six healthy relatives studied from three different generations were homozygous for the mutation, whose frequency in gnomAD was 0.002%. Patients II.1, II.2, II.3 and II.4 from family B were compound heterozygous for c.650 A > G (p.N217S) and c.1701_1703del (p.F568del) mutations, involving the coiled-coil and fourth NHL repeat respectively. The unaffected mother was heterozygous for the c.650 A > G (p.N217S) mutation and the father was not available, thus the mutations were estimated likely to be in trans. Both mutations were very rare in the population (0.002 and 0.0003% in gnomAD respectively). Patients II.2, II.3 and II.4 from Family C were homozygous for a TRIM32 c.115_116insT (p.C39LfsX17) mutation, involving the RING domain (Fig. 1b). Segregation analysis demonstrated that the variant was found in heterozygous state in the rest of the healthy relatives studied from different generations, and was absent from the gnomAD control population. Distinct clinical and radiological features are identified in all families All patients from Family A presented with foot drop as the first manifestation in their teens. This was the only symptom for years. Over further time, they developed remarkable ankle contractions. Later, they also developed proximal and distal weakness in lower and upper limbs. The youngestpatient(IV.3)showedamoresevereprogression,with need of support for gait in his thirties (Fig. 1c). In Family B, there was a remarkable clinical variability. Patient II.3 presented in her twenties with foot drop and proximal upper limb weakness. At age 55, she also had proximal lower limb weakness, and at age 58 she was unable to walk unsupported. Patient II.2, came to our attention at the age of 57, after the diagnosis of her sister and although she did not have any complains, examination showed mild bilateral proximal upper and lower limb weakness. Patient II.4 showed proximal and distal lower limb weakness at age 57 and patient II.1 showed normal physical exam at age of 67. Patients from Family C, presented with limb-girdle muscle weakness with onset between third and fourth decade and later developed distal limb weakness (Fig. 1c). Interestingly, patients II.3 and II.4 presented additional systemic manifestations typically described in BBS including hypogonadism, hearing loss, and behavioral abnormalities (depression and obsessive-compulsive disorder). (See figure on previous page.) Fig. 1 Pedigrees, genetic findings and clinical features of three families with mutations in TRIM32.aPedigree of Spanish family (family A) having the missense c.1771G > A (p.V591 M) mutation that segregates with the disease: homozygous only in affected patients. Pedigree of Australian family (family B) presenting the missense c.650A > G (p.N217S) mutation in combination with the frameshift c.1701_1703del (p.F568del) mutation that segregate with the disease: compound-heterozygosity only in affected patients. Pedigree of Spanish family (family C) possessing the homozygous frameshift mutation c.115_116insT (p.C39LfsX17) that segregates with the disease: homozygous only in affected patients. In the non-affected siblings (red symbols) of the three pedigrees we found only one or no mutated alleles. Circles denote female members and squares male members. Double line denotes a consanguineous marriage. Solid black symbols denote affected patients. b. Chromatograms display the homozygous missense c.650A > G (p.N217S), heterozygous missense c.650A > G (p.N217S), heterozygous frameshift c.1701_1703del (p.F568del) and homozygous frameshift mutations in the TRIM32 gene. Arrows indicate the location of the base change. The TRIM32 functional domains RING finger domain, B-box type 1 domain, coiled-coil region, and six NHL repeats are represented in the scheme, where arrows indicate the novel mutations location in the TRIM32 protein structure. c. Distal muscle involvement led to distal atrophy and ankle contractions. Patient II.3 from Family C showed mild paravertebral muscle atrophy with no scapular winging. d. Muscle MRI T1-weighted axial images at gluteus, thigh and calf levels showed different patterns of muscle involvement in the three families Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 5 of 16 No cardiac or respiratory involvement was found in any of the examined patients. Muscle MRI revealed different patterns of muscle involvement in the three families (Fig. 1d). Family A showed diffused fatty degeneration in the thighs and lower legs. Family B displayed a more focal pattern of fatty replacement mainly involving the biceps femoris and semimembranosus muscle in the thigh and mildly Fig. 2 Reduced TRIM32 protein level associated with p.V591 M, p.N217S/F568del and p.C39LfsX17 mutations. a,bWestern blot analysis of amuscle derived from biopsies of family A patients, family C patient and healthy controls, and bprimary myoblasts from family A patients, family B patient, and healthy controls. Anti-TRIM32 antibody revealed a remarkable reduction of TRIM32 protein level in TRIM32 V591M ,TRIM32 N217S/F568del and TRIM32 C39LfsX17 samples compared with controls. An anti-GAPDH blot is shown as a loading control. Arrows indicate the correct band at 72 kDa. c. Immunofluorescence staining and quantification of the percentage of TRIM32 + cells (arrowheads) in primary myoblasts at 8 days growing in proliferation medium from family A patients (n= 2), family B patients (n= 2) and healthy controls (n= 7). Images show TRIM32 fluorescence (red). Cells were counterstained with Topro 3 (blue) to visualize the nuclei. The percentage of TRIM32 + cells was significantly lower in TRIM32 V591M and TRIM32 N217S/F568del myoblasts than in controls. Data from 9 to 31 independent fields were analyzed per condition. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparison test. Scale bar, 50 μm Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 6 of 16 Fig. 3 (See legend on next page.) Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 7 of 16 in the soleus muscle in the lower legs. Interestingly, the asymptomatic patient II.1 did show moderate fat replacement of muscles. Family C showed predominant degeneration of the gluteus muscles, the posterior compartment in the thighs, and the soleus and gastrocnemius muscles in the lower legs. TRIM32 gene mutations lead to a reduced TRIM32 protein level At present, most TRIM32 reported mutations are clustered in the highly conserved C-terminal NHL domain of TRIM32 and may cause conformational changes in the protein that lead to a substantial decrease in its stability. This hypothesis is supported by the reduced level of TRIM32 found in human fibroblasts isolated from LGMD2H patients carrying the homozygous p.D487N substitution and in the muscle from the mouse model T32KI (harboring the p.D489N substitution) [1,25]. Based on these observations, we analyzed the effect of the novel mutations on the presence of TRIM32 in muscle samples from patients. Western blot revealed almost undetectable TRIM32 protein level in TRIM32 C39LfsX17 muscle, which was an expected result because it is a frame-shift mutation resulting in a premature stop codon, which in turn should result in a severely truncated TRIM32 protein. However, we also found a remarkable reduction of TRIM32 level in TRIM32 V591M and TRIM32 N217S/F568del muscles or primary myoblasts, compared to controls (Fig. 2a-b). Nicklas et al. found that proliferating mouse myoblasts in culture displayed nuclear TRIM32 signal and a shift of TRIM32 to the cytoplasm during differentiation [35]. In human samples, immunostaining assay showed a reduced amount of proliferating myoblasts with positive signal of TRIM32 in the nuclei of TRIM32 V591M and TRIM32 N217S/F568del myoblasts (Fig. 2c). These data support that not only the frameshift in theRINGdomainbutalsothesingleaminoaciddeletion and missense TRIM32 mutations identified in the NHL and coiled-coil domains resulted in reduced levels of TRIM32 protein. Mutations in TRIM32 impair proliferation and myogenic differentiation in primary myoblast Data from T32KO mice shows that TRIM32 is necessary for proliferation and differentiation of satellite cells through the regulation of the transcription factor c-Myc [35], and that the loss of TRIM32 function deregulates these processes due to the accumulation of TRIM32 substrates involved in myoblast proliferation and myogenesis [31]. We studied whether loss of protein due to TRIM32 human mutations alter myoblast proliferation in vitro. To address this point, primary myoblasts from patients carrying p.V591 M and p.N217S/p.F568del mutations were cultured. The cell proliferation rate (Fig. 3a) and percentage of Ki67 + cells (Fig. 3b) were significantly lower in mutant myoblasts compared with controls. Next, we investigated if muscle differentiation was also altered when TRIM32 is reduced. The confluent myoblasts were cultured in differentiation medium, in which myoblasts withdraw from the cell cycle, cease to divide, and start to elongate and fuse to form multinucleated myotubes. Myogenin expression (Fig. 4a-b) and fusion index (Fig. 4c) were reduced in mutant myoblasts compared with controls. These results were further supported by western blot in which we found a reduction of the differentiation markers myosin heavy chain and myogenin in TRIM32 V591M muscle compared with controls (Fig. 4d). TRIM32 C39LfsX17 myoblasts showed an extremely slow growth due to the reduced proliferation that precluded the production of enough cells for these experiments. Taken together, our data suggests that the loss of TRIM32 protein due to different mutations reduced myoblast proliferation and delayed myogenic differentiation. TRIM32 mutations in humans lead to premature senescence of myoblasts As mentioned, altered myogenesis and premature senescence are postulated to underlie myopathy in LGMD2H. Aging of muscle is characterized by functional impairment, loss of quiescence and reduction of the pool of satellite cells leading to altered regenerative capacity of the muscle [6,45]. Additional features of senescent cells include overexpression of SA-β-gal [12], and altered morphology [36]. To study whether the reduced cellular growth and myotube formation observed in TRIM32 mutant myoblasts were associated with premature senescence, we analyzed the satellite cells pool, the degree of muscle regeneration and the existence of morphological and metabolic changes in muscle from patients with (See figure on previous page.) Fig. 3 TRIM32 mutant myoblasts exhibit a decrease in cell proliferation. aQuantification of number of primary myoblast (proliferation rate) at 1, 4 and 8 days growing in proliferation medium from family A patients (n= 2), family B patients (n= 2), healthy controls (n= 6) and disease controls (2 LGMD2B, X-EDMD) (n= 3). The proliferation rate is reduced in TRIM32 V591M and TRIM32 N217S/F568del myoblasts compared with controls. Data from 12 to 45 independent fields were analyzed per time point. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. b. Analysis of primary myoblast proliferation at 8 days growing in proliferation medium using Ki67 as a marker of dividing cells from family A patients (n= 2), family B patients (n= 2) and healthy controls (n= 6). Immunofluorescence showing double staining, desmin (red) and Ki67 (green). Nuclei were counterstained with Topro 3 (blue). Quantification of Ki67 + cells revealed a progressive decrease in the percentage of proliferating TRIM32 V591M and TRIM32 N217S/F568del myoblasts compared with controls. Data from 14 to 41 independent fields were analyzed per condition. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Scale bar, 50 μm Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 8 of 16 Fig. 4 (See legend on next page.) Servián-Morilla et al. Acta Neuropathologica Communications (2019) 7:30 Page 9 of 16 with mutation in TRIM32, a putative E3-ubiquitin-ligase gene. Am J Hum Genet 70:663–672. https://doi.org/10.1086/339083 16. Garcia-Prat L, Martinez-Vicente M, Perdiguero E, Ortet L, Rodriguez-Ubreva J, Rebollo E, Ruiz-Bonilla V, Gutarra S, Ballestar E, Serrano AL et al (2016) Autophagy maintains stemness by preventing senescence. Nature 529, 37:– 42. https://doi.org/10.1038/nature16187 17. 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