Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions

ARTICLE Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions Montse Olivé et al. # Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O 2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O 2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation. https://doi.org/10.1038/s41467-019-09111-2 OPEN Correspondence and requests for materials should be addressed to M.O. (email: [email protected]t) or (email: [email protected]) or to N.G.L. (email: [email protected]). # A full list of authors and their affiliations appears at the end of the paper. NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications 1 1234567890():,; Myoglobin, the pigment that gives muscle its red color, is a small cytoplasmic globular hemoprotein highly expressed in cardiac and oxidative skeletal muscle fibers1. By reversibly binding O 2 , myoglobin buffers intracellular O 2 concentrations, facilitates intracellular O 2 transport and serves as a reservoir of oxygen during hypoxic and anoxic conditions1–3.In addition, myoglobin is implicated in vivo in the control of redox pathways in skeletal and cardiac myocytes, acting as scavenger of reactive oxygen species (ROS) and nitric oxide4,5. Myoglobin was the first protein for which a three-dimensional structure was determined by X-ray crystallography6. The backbone of myoglobin consists of eight α-helices, assigned the letters A to H, that wrap around a central pocket containing a heme group, a porphyrin ring that contains a central bound iron atom that is normally in the ferrous oxidation state. Its heme active site is responsible for reversible binding to various ligands including oxygen, carbon monoxide and nitric oxide7. Myoglobin is encoded by the myoglobin gene (MB) on human chromosome 22q12.37. Here we describe myoglobinopathy, a disease caused by a recurrent MB mutation found in 14 patients from six unrelated European families suffering from an autosomal dominant myopathy with variable cardiac involvement and characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. We show that mutation of MB alters the kinetics and thermodynamics of O 2 binding and may result in elevated superoxide levels. Results The clinical phenotype of myoglobinopathy. We studied 14 patients from six unrelated European families (F1–F6), (Fig. 1), suffering from an autosomal dominant myopathy with variable cardiac involvement, identified by their highly characteristic features on muscle biopsies. The clinical presentation was homogeneous among all patients and is summarized in Table 1. Age of onset ranged between 33 and 49 years. Initial symptoms were proximal lower limb, pelvic girdle, and axial muscle weakness, manifesting with difficulties climbing stairs, rising from squatting and standing up from the lying position. Two patients had, in addition, weakness and atrophy of thenar muscles from disease onset. Over the following years, the disease slowly progressed to involve distal leg and hand muscles, proximal muscles of upper limbs, and neck muscles. Five patients complained of dysphagia. Facial and extraocular muscles were not involved. Progression was slow; most patients developed respiratory failure requiring nocturnal non-invasive ventilatory support 10 years after disease onset and they became wheelchair dependent 15–20 years after disease onset. Cardiac involvement, revealed by ultrasound, magnetic resonance imaging (MRI) or post mortem myocardium examination, was observed in six individuals. Cardiac MRI in patient F2, II:2 showed a dilated cardiomyopathy with extensive and diffuse areas of late gadolinium enhancement, indicative of fibrosis at the epicardium and mesocardium, but no involvement of the endocardium, (Supplementary Fig. 1). Six patients died between 18 and 30 years after disease onset, from respiratory and/or cardiac failure. I F1 F2 F3 F4 F5 F6 1 1 123456789101112 910 11 12 13 14 15 16 17 23 212 1 12 34 23 1 12 3456 7 234 56 7 12 34 12 12 12 12 34 5678 1234 5 1234 1234 567 123 123 4567 8 II III I II III I II III I II I II III I II III IV Fig. 1 Pedigrees from the six families affected with myoglobinopathy. F1 and F2 Spanish, F3: Swedish, F4 and F5: French, F6: Dutch. Squares represent males; circles, females; filled black symbols indicate affected individuals; filled gray symbol, clinically affected, but not molecularly tested individuals; and slash, deceased ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 2NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications Serum CK levels were normal to mildly increased, except for a single individual who showed CK levels up to sevenfold above the upper normal limit. EMG was consistent with a myopathy with spontaneous activity at rest. Nerve conduction studies were normal. Muscle imaging studies showed a consistent pattern characterized by fatty degenerative changes in paraspinal and gluteal muscles, especially gluteus maximus and medius. At midthigh, there was preferential involvement of the adductor magnus, semimembranosus, long head of biceps femoris and vastus intermedius, except in individual F6, II:4 who showed predominant involvement of the anterior compartment. At the mid-leg, the soleus was always the first and most affected muscle (Fig. 2). Muscle pathology features associated with MB mutation. Muscle biopsies showed characteristic sarcoplasmic bodies in both Type 1 and Type 2 myofibers in all studied patients, including pre-symptomatic individuals, irrespective of the site of the biopsy (Fig. 3and Supplementary Table 1). The sarcoplasmic bodies were rounded or oval, appeared brown on hematoxylin and eosin and red on the modified Gomori’s trichrome stain. They had a glassy appearance and were easily visualized with all stains and even in non-stained sections. They exhibited autofluorescence emission in a wide range of visible laser excitation lines (Supplementary Fig. 2). Besides the sarcoplasmic bodies, the majority of muscle biopsies showed nonspecific myopathic changes with increased myofiber size variation, increased numbers of internal nuclei and type 1 fiber predominance (Supplementary Table 1). Most muscle biopsies, particularly those showing advanced pathological lesions, showed cytoplasmic vacuoles filled with granular basophilic material, displaying strong acid phosphatase activity and lysosome-associated membrane protein 1 (LAMP1) immunoreactivity (Fig. 3), indicating that they contained lysosomes. No major architectural changes were observed on NADH, SDH, and COX reactions apart from the lack of oxidative activity at the site of vacuoles. Myoglobin, p62, and ubiquitin immunoreactivity was observed in abnormal myofiber regions containing vacuoles and in some but not all sarcoplasmic bodies (Fig. 4), demonstrating abnormal protein aggregation (Fig. 4). However, myoglobin immunostaining was not particularly useful for the diagnosis. Under electron microscopy, the inclusions appeared as very dense bodies measuring 0.3–2.5 µm, often located next to myonuclei or dispersed between myofibrils (Fig. 5a–d and Fig. 5e–g). Some were membrane bound, whereas others were not. Some were denser than others, likely reflecting different stages of the pathological process. Autophagic vacuoles with cellular debris, myelin figures, and filaments measuring 8–12 nm were additional findings. Sarcoplasmic bodies were present in respiratory and cardiac muscles obtained post mortem from individual F1, II:7 and in cardiac muscle from Table 1 Phenotypic features of myoglobinopathy Family 1 Family 2 Family 3 Family 4 Family 5 Family 6 Inheritance pattern AD AD AD AD unknown unknown Myoglobin mutation His98Tyr His98Tyr His98Tyr His98Tyr His98Tyr His98Tyr Country of origin Spain Spain Sweden France France Netherlands No. of patients 2 1 6 3 1 1 Mean age of onset (range) 37.5 (36–39) 38 44.5 (39–49) 46 (44–48) 40 33 Gender (female/male) 0/2 1/0 2/4 2/1 0/1 0/1 Initial symptoms Proximal LL and axial weakness (2/2) Proximal LL and axial weakness (1/1) Distal hand weakness (2/6) Proximal LL and axial weakness (6/6) Proximal LL and axial weakness (3/3) Proximal LL weakness (1/1) Proximal LL and axial weakness (1/1) Symptoms at advanced disease Distribution of weakness Proximal and axial > distal 4 EE Proximal and axial > distal 4 EE Proximal and axial > distal 4 EE Proximal and distal 4 EE > axial Proximal and axial > distal 4 EE Proximal and axial > distal legs Involvement of hand muscles 2/2 1/1 6/6 2/2** 1/1 0/1 Facial weakness 0/2 0/1 0/6 0/2 0/1 0/1 Muscle atrophy 2/2 1/1 6/6 2/2 0/1 1/1 Dysphagia 2/2 0/1 2/4 0/2 1/1 0/1 Respiratory insufficiency 2/2 1/1 2/6 1/2 1/1 1/1 Cardiac involvement* 2/2 1/1 2/6 0/2 1/1 0/1 Clinical outcome Mean age at wheelchair dependency 54 65 56 (4/6 patients wheelchair dependent, 2/6 ambulant) 66 (1/3 patients wheelchair dependent, 2/3 ambulant) 56 47 Mean age at death (range) 60.5 (54–67) –64 (58–71) 72 –– Laboratory studies CK fold elevation above normal levels 1.5–2 2 1.5–4 2.3–3.7 4 3.5–7 EMG Myogenic with spontaneous activity at rest Myogenic with spontaneous activity at rest Myogenic with spontaneous activity at rest Myogenic with spontaneous activity at rest Myogenic with spontaneous activity at rest Myogenic with spontaneous activity at rest *As revealed by cardiac ultrasound, cardiac MRI, or by post mortem examination of cardiac muscle. ** Individual III:2 from Fam 4 is in the early stages of the disease and therefore not included in symptoms at advanced disease items. EE: extremities; LL: lower limbs NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 ARTICLE NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications 3 individual F3, III:5 (Fig. 5d and Supplementary Fig. 3), thus indicating that cardiomyopathy was a primary consequence of the disease and not secondary to respiratory failure. Identification of p.His98Tyr MB substitution in six families. To identify the molecular cause of this myopathy two independent groups used two different strategies. The Australian group used whole-exome sequencing of three affected individuals from two Spanish families (F1, II:5 and II:7 and F2, II:2), and gene prioritization of shared variants using skeletal muscle expression enrichment data from FANTOM58. This identified the same heterozygous missense variant (c.292C>T, p.His98Tyr) in a b c d e f g h i Fig. 2 Muscle imaging in myoglobinopathy. Muscle CT scan from individuals F1, II: 7 (a–c); F4, II: 4 (d–f); and F5, II: 6 (g–i) at the pelvic (a,dand g), midthigh (b,e, and h), and mid-leg (c,f, and i). At the pelvis, there is involvement of the gluteus maximus, medius, and minimus. At mid-thigh, there is preferential involvement of the posterior compartment, specially, of the adductor magnus, biceps femoris, and semimembranosus. At the mid-leg, the soleus is the first and most-affected muscle Fig. 3 Histochemical features of myoglobinopathy. aAnterior tibialis muscle biopsy from individual F3, III: 15, 10 years prior to the onset of symptoms, stained with hematoxylin and eosin, showing several rounded brown inclusions (arrows) (sarcoplasmic bodies) in the majority of myofibers and very small vacuoles in some myofibers (arrowhead in a). b,cBiceps brachii from individual F1, II: 7, 15 years after disease onset. bNote the presence of collections of sarcoplasmic bodies within the rimmed vacuoles. d,eSarcoplasmic bodies appear red on modified Gomori trichrome stain. eIn muscle biopsies with more advanced pathological lesions, large numbers of rimmed vacuoles are observed. fNo major architectural changes are seen on NADH reaction, apart from lack of oxidative activity at the site of vacuoles. gFast myosin immunohistochemistry demonstrate the presence of sarcoplasmic bodies in both type 1 (slow) and 2 (fast) myofibers. hMyofiber regions containing vacuoles display strong phosphatase activity, and LAMP1 iimmunoreactivity. Scale bar in a,e, f,g, and h=50 µm; scale bar in b,c,d, and i=20 µm ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 4NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications the myoglobin gene (MB) in all three affected individuals. The Swedish group, studying family F3, identified one region on chromosome 22 through genome-wide linkage analysis, with a statistically significant LOD score. Refinement of the peak with further markers and clarification of the disease state in family members, resulted in a maximum multipoint LOD score of 5.8 at D22S685 (Supplementary Fig. 4). Targeted capture and sequencing of genes within the linkage region in eight individuals, six affected and two unaffected, revealed in all the affected family members, the presence of the same MB (c.292C>T, p.His98Tyr) variant. Sanger sequencing confirmed segregation of the variant with the disease in all available family members and in three additional families (F4, F5, and F6). The variant involves a highly conserved residue in the proximity of the oxygen-binding heme group (Supplementary Fig. 5), it is absent in all unaffected relatives tested, is not present in the 1000genomes, ExAC (http://exac.broadinstitute.org/) or gnomAD ((http://gnomad. broadinstitute.org) data sets and was present on different haplotypes. Moreover, all in silico predictors suggested the observed substitution in MB is deleterious: MutationAssessor (medium functional impact, FI score 3.175), MutationTaster (diseasecausing, p=0.999), PolyPhen-2 (probably damaging, score 1.000), Provean (deleterious, score −4.093) and SIFT (damaging, score 0.001). Thus, we have compelling evidence across six families that the same MB (c.292C>T, p.His98Tyr) variant is responsible for the observed myopathy. Haplotype analysis using microsatellites within 3 Mbp of the MB gene indicated at least three different haplotypes within the six families, suggesting that the c.292C>T variant is a recurrent variant originating on different ancestral backgrounds (Supplementary Table 2). Furthermore, the analysis of haplotypes in family F5 showed that the affected and two unaffected siblings had identical alleles, suggesting that in the affected individual the variant probably arose de novo (Supplementary Table 2). No samples from the parents in family F5 were available for confirmation. Myoglobin knockout mice have a binary phenotype. Twothirds die in utero at ~ E9.5-E10.59, but those that survive live to adulthood with little sign of functional effects, because of multiple compensatory mechanisms10. We therefore hypothesized a dominant gain of function as the cause of the disease in the families and used multiple methods to investigate the pathomechanism. NanoSIMS analysis of the sarcoplasmic bodies. Correlative electron microscopy and NanoSIMS (Nanoscale Secondary Ion Mass Spectrometry) analysis revealed high sulfur content and small iron Fe signals in the sarcoplasmic bodies (Fig. 5h–j). Of note, sulfur is a component of antioxidant systems of cells and reactive sulfur species are formed under conditions of oxidative stress11,12. Iron signals could be related to degradation of myoglobin and other metalloproteins inside the lysosomes13. Comparative NanoSIMS analysis of myoglobinopathy samples with one sample from a patient with Pompe disease containing electron-dense inclusions, two samples showing abundant lipofuscin and two muscle samples from patients affected with distal myopathies with rimmed vacuoles, revealed that the amount of Fe is higher in the myoglobinopathy sarcoplasmic bodies than in other similar inclusions. µFTIR analysis of muscle biopsy samples. Fourier transform infrared microscopy (µFTIR) analysis of muscle samples demonstrated increased carbonyl (ester) groups in the sarcoplasmic bodies indicating lipid oxidation (Fig. 5k–m). Moreover, some sarcoplasmic bodies showed a band at 1627 cm−1, which is characteristic of intermolecular β-sheet structures14 (Fig. 5n) and a feature typical of amyloid formation15,16. However, in vitro, only small amounts of non-fibrillar amyloid β-structure were detected (see Supplementary Note 1 and Supplementary Fig. 6). Non-fibrillar β-sheet aggregates have been previously described for other peptides and proteins including β-amyloid peptides related to Alzheimer’s disease16. Both NanoSIMS and µFTIR, therefore, suggest lipid oxidation in patients’muscles contribute to sarcoplasmic body formation. Biochemical characterization. At the molecular level, the His98Tyr variant might impact on different MB functional Fig. 4 Immunohistochemical features of myoglobinopathy. a,bSmall myoglobin aggregates are observed in some myofiber regions and in some sarcoplasmic bodies (double arrow in b), but not in others (arrow in b). cp62 and ubiquitin dimmunoreactivity is observed in some myofiber regions, and in some, but not in all sarcoplasmic bodies indicating protein aggregates. Scale bars =20 µm NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 ARTICLE NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications 5 properties, crucial to the physiological role: these include the interaction of MB with dioxygen, the affinity of MB protein scaffold to hemin, as well as the ability to keep the heme in the reduced state (Fe2+). In vivo, ferrous MB (Fe2+), which is the only myoglobin form that can bind and store O 2 , is prone to autoxidation17, leading to functionally inactive MetMB (Fe3+), which is eventually reduced back to ferrous MB by cytochrome b 5 18,19. First, we evaluated whether the variant affects the affinity of the prosthetic group for the MB polypeptide matrix, as it has been demonstrated that mutations in the vicinity of the porphyrin ring can increase heme dissociation rates (k -H ) by two-orders of CH 3 3000 0.6 ** 0.5 0.4 0.3 A2925 cm –1 /A1654 cm –1 A1740 cm –1 /A2925 cm –1 0.2 0.1 0.0 0.0 0.1 0.2 0.3 0.4 0.5 Sarcoplasmic bodies Surrounding muscle tissue Sarcoplasmic bodies Surrounding muscle tissue 2900 2800 Wavenumber (cm–1) 1700 1600 1500 1680 1660 α-helix + unordered  -sheet 0.005 d2A/d(υ)2 1640 Wavenumber (cm–1) 1620 1600 CH 2 Amide C=O k lm n ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 6NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications magnitude20–22. At pH 7.0, k -H of H98Y was five times higher compared with WT (Table 2and Supplementary Fig. 7). Mutations of the corresponding residue (His97) in sperm whale myoglobin into small and hydrophobic (e.g., Ala, Val) or acidic (e.g., Glu, Asp) amino acids enhanced k -H 21,22 owing to loss of electrostatic interaction between the side chain of His97 and the negatively charged heme propionate-721. His98 of human MB contributes to modulating the solvent accessibility to the heme pocket. Upon exchange of His98 with Tyr, the interaction with the propionate is weakened; Tyr98 can still form H-bonds with propionate-7, nevertheless, k -H is higher for p.His98Tyr compared with WT MB. Notably, this change in k -H is in line with the His97Phe mutant of sperm whale myoglobin21,22,reflecting the similarity between Tyr and Phe in terms of steric hindrance. Our interpretation is further supported by the fact that at pH 5.0 the k -H values for WT and p.His98Tyr are increased (1.44 ± 0.09 h−1and 1.86 ± 0.05 h−1, respectively) and the difference between the two species becomes smaller, because of protonation of the heme propionates, resulting in diminished interaction of the heme group with the protein. Next, we investigated the impact of the mutation on the oxidation state of the heme iron, both from a thermodynamic and kinetic point of view. Oxidized and reduced MB coexist in solution, and the ratio of the two forms is regulated thermodynamically by the reduction potential E°’and kinetically by the protein autoxidation rate17, which in turn are influenced by pH, oxygen concentration, and temperature23. The autoxidation process is kinetically controlled under physiological conditions. We observed nearly identical autoxidation rate constants k ox for WT and p.His98Tyr (see Table 2). Moreover, the spectral features of deoxy-, oxy-, and metmyoglobin forms for both WT and mutant are identical (Supplementary Fig. 8). On the contrary, WT and p.His98Tyr feature slightly different reduction potential (E°’) values. Spectroelectrochemical investigations yielded a E°’of (+0.040 ± 0.005) V vs SHE for WT MB, in excellent agreement with previous findings24, whereas the reduction potential of the p.His98Tyr myoglobin is shifted toward lower values (Fig. 6). E°’ determines the tendency of electron transfer from the Fe (II) center to the bound dioxygen ligand25, a critical step of the autoxidation process yielding inactive MetMB. From a purely thermodynamic point of view, lower E°’values would favor formation of auto-oxidized MetMB. Another important difference between WT and mutant MB concerns the affinity for molecular oxygen. Oxygen-binding studies to both WT and mutant deoxymyoglobins showed formation of oxymyglobin states (Soret band at 418 nm and two distinct bands at 545 and 580 nm) (Fig. 7). Calculation of rate constants, k on ,ofO 2 binding from the slope of the linear plots of k obs values versus O 2 concentration (Fig. 7b) yielded apparent bimolecular binding rates of (1.16 ± 0.7) × 107M−1s−1(WT MB) and (5.6 ± 0.2) × 106M−1s−1(p.His98Tyr), respectively. From the intercept, k off , of these plots, the dissociation constants, K D (=k off /k on ), were calculated to be (1.2 ± 1.1) µMand (8.4 ± 2.5) µM, respectively (see Table 2), demonstrating a reduced affinity of the mutant MB for molecular oxygen. To shed light onto the structural and dynamic changes caused by the His-to-Tyr substitution underlying the complex biochemical picture described above, we performed molecular dynamics (MD) simulations of both WT and mutant species. The analysis of our sampling suggests that the substitution does not lead to major global structural rearrangements. The global fold of the protein appears to be maintained (Fig. 6), and the two proteins feature almost superimposable fluctuation patterns (Supplementary Note 2 and Supplementary Figs. 9 and 10). Nevertheless, MD simulations indicated that the heme group in p.His98Tyr is more solvent exposed than in the WT protein. This may explain the shift of E°’, which is highly (though not solely) influenced by the extent of exposure of the heme to solvent26,27; the latter might also contribute to the increment of K D for O 2 binding to p.His98Tyr, owing to the more polar environment resulting from enhanced accessibility to water. Initial studies using a reporter cell assay, suggest that mutant myoglobin may result in elevated intracellular superoxide levels. Fig. 5 Characterization of sarcoplasmic bodies, the morphological hallmark of myoglobinopathy. Electron micrographs a–dshowing collections of highly electron-dense bodies with some less dense material at their periphery. The sarcoplasmic bodies are seen under the sarcolemma (a) and often next to the nuclei. Some sarcoplasmic bodies are surrounded by a membrane (b). Sarcoplasmic bodies of different electron densities near several vesicular structures (c). Sarcoplasmic bodies observed in the cardiac muscle obtained post mortem from individuals F1, II:7 (d) and F3, III:5. Electron micrographs (e–g) and the corresponding NanoSIMS images (h–j). Blue indicates sulfur (32S), red phosphorus (31P), and green (56Fe), respectively. Sarcoplasmic bodies interspersed between the myofibrils (e), next to nuclei (f), or inside an autophagic vacuole (g). Note the high-sulfur signal in the sarcoplasmic bodies (h–j), and the iron signal (green dots within the sarcoplasmic bodies in i,j). Scale bar in a=2µm, b=5µm, c=0.5 µm, d=1µm, h–j=4µm. kTypical µFTIR spectra and their second derivative of the muscle tissue where the lipid region has been highlighted in orange and the protein region in blue; the inset shows the lipid/ protein ratio (calculated from the Infrared spectra) on an optical image of a tissue section with sarcoplasmic bodies. The color bar represents intensity of the ratio: blue and red mean low and high lipid content, respectively. The scale bar is four microns. lBox plot graphic of lipid/protein ratio (2925 cm−1/ 1654 cm−1). mBox plot graphics representing COOH/CH 2 ratio indicating lipid oxidation (1739 cm−1/2925 cm−1). Ratios were calculated from the second derivative of the spectra of three different samples from three different patients (at least 10 sarcoplasmic bodies per patient). Boxplots denote the median (center line), interquartile range (box), whiskers that represents the most extreme data that are not >1.5x IQR from the edge of the box and outliers that are the points outside this range. T-tests were used to compare the sarcoplasmic body ratios with the surrounding tissue ratios and determine the pvalue (*p<0.005). nInfrared second derivative spectrum of the amide region of one sarcoplasmic body (green) showing an increase of β-sheet structures, indicating protein aggregation. Second derivative of the amide region corresponding to the tissue surrounding the sarcoplasmic bodies (black) Table 2 Effect of mutation on biochemical properties of MB K D, O2 (µM) k -H at pH 7.0 (h−1)ak -H at pH 5.0 (h−1)bE°’by spectroelectr. (V vs SHE) cE°’by SWV (V vs SHE) ck OX at pH 7.0 (min−1) Heme SASA (nm2)d WT 1.2 ± 1.1 0.22 ± 0.06 1.44 ± 0.09 +0.040 ± 0.005 −0.056 ± 0.015 1.96 ± 0.39 1.45 ± 0.04 His98Tyr 8.4 ± 2.5 1.19 ± 0.05 1.86 ± 0.05 +0.021 ± 0.005 −0.105 ± 0.015 1.97 ± 0.43 1.80 ± 0.05 Conditions: a0.2 Mphosphate buffer, pH 7.0, and 0.45 sucrose (37 °C); b0.2 Macetate, pH 5.0, and 0.45 Msucrose (37 °C); c0.2 Mphosphate buffer, pH 7.0 (25 °C). dValues obtained from MD simulations NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 ARTICLE NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications 7 HEK cells transfected with MBp.His98Tyr-EGFP had significantly elevated levels (1.45-fold, p=0.007) of intracellular superoxide compared with those transfected with wild-type MB-EGFP (Fig. 7c). Further experimental investigations of structural and functional changes caused by the substitution will shed more light on the altered relationship between structure, dynamics, and function in the p.His98Tyr variant. Discussion Myoglobin belongs to the globin superfamily. Mutations in hemoglobin have been recognized for a long time and result in hemoglobinopathies28. Two other globin proteins (cytoglobin and neuroglobin) have not yet been associated with disease29. Our findings allow us to describe the first disorder caused by a mutation in MB. We propose the disorder should be named Myoglobinopathy. Family 3, first described by Edstrom et al.30,31in 1980, became a classic of the muscle disease literature but its cause remained unsolved until now. Myoglobinopathy has a recognizable clinical phenotype, characterized by disease onset during the fourth or fifth decade of life, manifesting with proximal weakness in the lower extremities and axial muscles, progressing to involve proximal and distal muscles of all limbs with later generalized weakness. Although initially described as a distal myopathy30 our study demonstrates that all patients exhibit proximal weakness as the initial symptom. Respiratory insufficiency is a frequent complication and is the cause of death in the majority of patients. Indeed, most advanced patients will require non-invasive respiratory support. Cardiomyopathy was demonstrated in less than half of the patients. It is uncertain whether myocardial involvement is an inconstant feature of the disease, or it remains subclinical for a long time. In support of this last hypothesis, post mortem examination of cardiac tissue in two individuals who did not manifest symptoms of cardiac failure showed large numbers of sarcoplasmic bodies. The combination of proximal and axial weakness and respiratory insufficiency share some similarities with the phenotype of patients suffering from late-onset Pompe disease. However, cardiomyopathy is usually not a feature of late onset Pompe disease; moreover, distal muscle involvement as revealed by clinical examination and muscle imaging studies does not occur in late onset Pompe diseases until very late in the course of the illness32. The sarcoplasmic bodies are the pathological hallmark of the disease and are the principal aid to diagnosis. They have some similarities with lipofuscin33 and also with the globular dense inclusions found in Pompe disease33,34 but they are much denser and homogeneous, and usually lack the vacuolar lipid droplets seen in these two conditions. They are also highly reminiscent of the inclusions found in the skeletal muscles from patients with chronic vitamin E deficiency, which are known to be the result of lipid oxidation as a consequence of oxidative stress35. MB accomplishes its physiological tasks thanks to an extremely delicate balance of efficient oxygen binding, resistance to autoxidation, and high heme affinity. The full interplay between the molecular properties affected by the pathogenic mutation is obviously complex. However, it is apparent from our biochemical 800 a b c His98Tyr WT 600 400 I (nA) 200 0 –200 –400 350 0.0 0.1 0.2 Abs (a.u.) 0.3 80 70 60 50 E (mV vs SHE) 40 30 20 –0.2 0.0 0.2 Log x 0.4 0.6 0.8 400 450 500 λ (nm) 550 600 650 –200 0 E (V vs SHE) 200 400 Fig. 6 In vitro and in silico studies of WT and mutant MB. aTypical square wave voltammograms recorded for WT (black) and p.His98Tyr mutant (red) human myoglobin immobilized on a Au electrode in 20 mMphosphate buffer, pH 7.0, 25 °C. bElectronic spectra of p.His98Tyr mutant of human myoglobin obtained at various applied potentials E in spectroelectrochemical experiments carried out with an optical thin-layer electrochemistry cell at pH 7.0, 25 °C. The corresponding Nernst plot is shown in the inset, where x =[(Amax λred −A λred )−(Amax λox −A λox ). cOverlay of cartoon representations of representative structures sampled within the MD simulations for native (red) and p.His98Tyr mutant (black) human myoglobin. His98 is also shown as stick with the same color coding. The heme group and Fe-ligand residue His94 is shown only for WT for sake of clarity ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 8NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications 0.14 0.12 418 434 1.0 0.8 0.6 0.4 Relative absorbance at 418 nm 0.2 0.0 02040 Time (ms) 60 Experimental time trace/wild-type Single exponential fit/wild-type Experimental time trace/H98Y Single exponential fit/H98Y 555 545 580 0.10 0.08 Absorbance 0.06 0.04 0.02 0.00 1400 Human myoglobin wild-type kon = 11.6 (± 0.7) × 106 M–1 s–1 koff = 13.6 (± 11.1) s–1 KD = 1.2 (± 1.1) μM Human myoglobin H98Y kon = 5.6 (± 0.2) × 106 M–1 s–1 koff = 47.5 (± 12.5) s–1 KD = 8.4 (± 2.5) μM 1200 1000 800 600 kobs (s–1) 400 200 0 0.0 WT (26) His98Tyr (23) 0.2 0.4 0.6 DHE fluorescence (au, normalised to t = 0 min) 0.8 1.0 c b a 050 * 100 O2 (μM) 150 200 400 500 Wavelength (nm) 600 700 10 μM O2 Fig. 7 Kinetics of dioxygen binding to wild-type human myoglobin and the variant His98Tyr. aSpectral changes upon reaction of 1 µMferrous wild-type hMb (black line) with 10 µMO 2 . The final spectrum represents oxymyoglobin (red line, 68 ms after mixing). Gray lines represent spectra obtained at 0.68, 2.72, 4.08, 6.12, 8.84, 12.24, 34.00, and 51.00 ms after mixing. The inset depicts experimental time traces at 418 nm of wild-type hMb (solid black line) and p.His98Tyr MB (dashed black line) mixed with 10 µMO 2 and corresponding single-exponential fits (solid red line, wild-type MB; dashed red line, His98Tyr MB). bLinear dependence of k obs values from the O 2 concentration for wild-type MM (gray circles, solid line) and p.His98Tyr MB (white squares, dashed line). cBasal intracellular superoxide levels in HEK293FT cells expressing WT or mutant MB-EGFP. Data presented as individual data points and the mean ± SEM, numbers in parenthesis represent n. *indicates p=0.007 (Mann–Whitney test, two-tailed) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09111-2 ARTICLE NATURE COMMUNICATIONS | (2019) 10:1396 | https://doi.org/10.1038/s41467-019-09111-2 | www.nature.com/naturecommunications 9