Human XLMTM muscle fibres display myonuclear perturbations
We isolated single muscle fibres from patients with XLMTM (MTM1, N = 3 patients, n = 20 muscle fibres total) and healthy control donors (CTL, N = 4 patients, n = 20 fibres total). Due to the low proportion of type II myosin heavy chain isoforms in these muscle samples, only fibres positive for type I myosin were analyzed. We then analyzed myonuclear organization using 3D confocal microscope reconstructions (Fig. 2a). As expected, XLMTM patients had a large proportion of fibres with centralized nuclei (assessed using individual confocal Z-slices throughout the fibre, Fig. 2a, b). In addition, mean fibre cross sectional area (CSA) was smaller in patients than in healthy controls (compare distributions of CSA in Fig. 2d, f). The total number of myonuclei per mm fibre length was not significantly different between groups (Fig. 2c). However, as reported previously [22, 31,32,33], this parameter was positively and linearly correlated with the CSA, with larger fibres possessing more nuclei (Fig. 2d). Hence, for any given CSA, the number of nuclei per mm fibre length was greater in XLMTM patients than in healthy controls. This demonstrates an abnormally high density of myonuclei in XLMTM fibres. In agreement with this, myonuclear domain (MND) sizes were dramatically smaller in patients compared to controls (Fig. 2e). As for the previous parameter of number of myonuclei per mm fibre length, MND values were positively and linearly correlated with CSA in both groups (Fig. 2f). However, for any given CSA, MND was smaller in patients than controls (Fig. 2f). Although fibres with lower CSAs are known to have smaller MNDs (hence the positive linear relationship in Fig. 2f), the regression lines and data points would suggest that even though XLMTM fibres are small, they have unusually reduced MND sizes compared to healthy controls, suggesting a primary effect of disease, rather than a secondary effect of hypotrophy.
MND measurement provides valuable information on the average volume of cytoplasm controlled by each myonucleus; however, it does not take into account variability between individual domains, nor the overall spatial arrangement/organization of myonuclei. To investigate this, we calculated a distribution or order score (‘g’) as described previously [4, 22, 31,32,33]. g was significantly lower (i.e. myonuclei are more disorganized in their overall distribution) in XLMTM compared to healthy control fibres (Fig. 2g).
Human XLMTM fibres exhibit a disrupted force generating capacity originating at the myofilaments
Optimal MND sizes and nuclear positioning are essential for cellular and contractile function [22, 28]. To measure the force production of myofibres at the contractile level, we measured the absolute steady-state isometric force at saturating [Ca2+] (pCa 4.50) of membrane-permeabilized fibres (20 fibres per group). In this system, externally applied ATP and Ca2+ directly activate contraction at the level of the myofilaments. Thus, upstream events that normally contribute to muscle contraction are bypassed (e.g. electrical stimulation or excitation–contraction coupling), and the properties of myofilament force production are examined in isolation (see Fig. 1 for schematic diagram). In correlation with the nuclear abnormalities, specific force (defined as absolute (maximal) force divided by CSA) was lower in XLMTM patient fibres compared to healthy controls (Fig. 2h).
The Mtm1 knockout (KO) mouse model of XLMTM displays myonuclear perturbations and disrupted force generating capacity
To investigate whether similar disruptions occur in muscle fibres expressing other myosin isoforms, we used a well-known mouse model of the disease, the Mtm1 KO mouse, where skeletal muscles are mainly composed of the fast type IIx and IIb myosin heavy chains [1]. We then tested 109 muscle fibres from 6 mice: 3 healthy wild types (WT, n = 46 fibres total) and 3 Mtm1 KO mice (n = 63 fibres total). Consistent with our human results, muscle fibre CSA was significantly reduced, and central nucleation significantly increased, in Mtm1 KO mice compared to controls (Fig. 3a, b, d). Also in agreement with our human data, Mtm1 KO mice exhibited increased numbers of myonuclei (Fig. 3d) and smaller MND volumes than WT (Fig. 3e, f). However, unlike XLMTM patients, order score (g) was unchanged in Mtm1 KO mice, suggesting no alterations in the regularity of spacing between myonuclei (Fig. 3g). The nuclear changes described above were associated with a low force generating capacity originating specifically at the level of the myofilaments (Fig. 3h), in agreement with the human data.
Altogether, our findings suggest a novel etiology in XLMTM, associated with MND changes, altered nuclear arrangement and reduced force production related to the myofibrils themselves.
Delivery of wild type MTM1 gene rescues myonuclear density and distribution defects in dogs with XLMTM
We isolated 186 individual myofibres (minimum of 10 fibres per dog) from 15 dogs: 3 healthy control dogs (Healthy), 3 animals expressing the p.N155K mutation in the MTM1 gene which were injected with a saline solution (XLMTM), and 9 dogs also carrying the mutation but where three different doses of rAAV8-cMTM1 were administered (AAVLow, N = 3; AAVMid, N = 3; AAVHigh, N = 3). As expected from previous published data [6, 9, 23], a high proportion of muscle fibres from XLMTM and AAVLow groups possessed central nuclei, but in AAVMid and AAVHigh groups, this was rescued to levels indistinguishable from healthy controls (Fig. 4b). In addition, mean fibre CSA was smaller in XLMTM and AAVLow dogs, but approaching that of the healthy control group following AAVMid and AAVHigh treatments (compare distributions of CSA in Figs. 4d, f). XLMTM and AAVLow dogs exhibited increased numbers of myonuclei (Fig. 4d), smaller MND volumes (Fig. 4e, f), and reduced order score (g), suggesting a higher density of myonuclei, and irregularities in their spacing thoughout the fibre (Fig. 4g). This is in agreement with the findings in XLMTM patients. Treatment with AAVMid and AAVHigh doses was capable of restoring all these parameters to levels that were comparable with healthy controls (Fig. 4a–g).
Myonuclear size scaling is known to happen in response to changes in nuclear number and MND volumes in order to modulate synthetic activity [42]. Here, even though we found higher numbers of nuclei and smaller MND sizes in XLMTM and AAVLow animals, we did not observe any difference in myonuclear size (projected area in the 2D X–Y plane) or aspect ratio between groups (Fig. 4h, i).
These findings partially confirm our initial hypothesis and imply that there is an increased number of myonuclei within myofibres of the canine model of XLMTM, which results in smaller MND sizes. Myonuclei are arranged in both the centre and periphery of myofibres in diseased animals and are inconsistent or more unevenly spaced (i.e. lower order (‘g’) score). AAVMid and AAVHigh doses of the canine MTM1 gene were able to fully restore myofibre CSAs, and fully rescue MND sizes and nuclear distribution parameters to those of healthy animals (Fig. 4d–g).
Delivery of wild type MTM1 gene rescues disrupted histone acetylation in XLMTM dogs
The above nuclear changes might be associated with global gene transcription abnormalities, since nuclear cooperation is known to be affected by nuclear spacing within muscle fibres [37]. To investigate this specific point, we used immunofluorescence to label acetyl histone H3 (Lys9/Lys14) (AcH3). This histone modification is well documented as a marker of actively transcribed regions of DNA, and nuclear abundance/fluorescence intensity of this marker is positively correlated with global nuclear transcription levels [22, 31]. The mean fluorescence intensity of AcH3 within individual myonuclei was significantly lower in XLMTM and AAVLow dogs than in healthy, AAVMid and AAVHigh animals (Fig. 5a, b). Next, we analysed the variability of pixel intensities within each single nucleus (standard deviation of all AcH3 positive pixel values, as a percentage of the mean value). We observed that the AcH3 intensity variability within each nucleus was significantly greater in XLMTM and AAVLow animals than in the other groups (Fig. 5c), suggesting that the distribution of this marker is altered within each nucleus, likely corresponding to altered chromatin organization and/or altered distribution of active domains of DNA. Together with the reduced mean fluorescence intensity of AcH3, this suggests that XLMTM and AAVLow animals display reduced or altered global transcriptional output.
Delivery of wild type MTM1 gene rescues contractile protein content and force generation in XLMTM dogs
Optimal gene transcription and myonuclear domain sizes are thought to be a prerequisite for sufficient production of contractile proteins, the most abundant proteins in muscle fibres. Indeed, aberrant myonuclear domain sizes and altered gene transcription have been associated with reduced contractile protein content and impaired force generation in several animal models [22, 29]. We hypothesized that the same might be true in XLMTM dogs. We assessed contractile protein content via several methods. Using fluorescent labelling, the mean pixel intensity of rhodamine phalloidin (which labels actin) within individual muscle fibres was lower in the XLMTM and AAVLow animals (Fig. 6a, see also Fig. 4a). To further support this, our proteomics analysis on single muscle fibres with known CSA revealed that myosin and actin were decreased at the protein level in XLMTM when compared with results from Healthy animals (Fig. 6b, c). This is in line with our ultrastructural analyses that revealed that the XLMTM and AAVLow animals had a lower density of myofibrils within myofibres (Fig. 6d, g). AAVMid and AAVHigh treatments were capable of restoring rhodamine phalloidin staining intensity and myofibrillar density (Fig. 6a, d, e, see also Fig. 4a).
A reduction in contractile protein content might be expected to affect force generating capacity of muscle fibres. In agreement with this, specific force was significantly lower in XLMTM and AAVLow groups, compared to others. AAVMid and AAVHigh treatments restored specific force to normal levels (Fig. 6e). This force deficit could be due to changes in the total number of contractile proteins and/or myosin molecules available, their recruitment upon Ca2+ activation and/or their intrinsic cycling and mechanical properties in binding to actin. To distinguish between these potential mechanisms, we evaluated rigor force (maximum force divided by CSA in the absence of ATP). This parameter was significantly smaller in XLMTM and AAVLow animals than in healthy, AAVMid and AAVHigh dogs (Fig. 6f). As rigor force and specific force were decreased to a similar extent, and as under rigor conditions, all myosin heads are attached [3], we suggest that the major mechanism underlying the force depression is the availability of myosin molecules and other contractile proteins. Together, these data suggest that global nuclear synthetic activity is altered in XLMTM and AAVLow dogs versus healthy and rescued animals, and that this correlates with decreased content of contractile proteins within myofibres as well as a reduction in contractile force.
In the absence of disease, centrally located myonuclei do not alter myofilament force generating capacity
In addition to the abnormalities in MND size and nuclear spacing in XLMTM patients and animal models (Figs. 2–4), another form of mispositioning exists in this disease: nuclei aberrantly placed in the centre, rather than at the periphery of muscle fibres. The presence of central nuclei might have consequences for various aspects of muscle physiology, e.g. physical interference with contractile function, since central nuclei are buried among myofibrils. To determine whether this latter aspect might affect muscle fibre contraction, we used a mouse model of central nucleation, in the absence of other unrelated pathology. Tibialis anterior muscles of wild type mice were treated with notexin, resulting in muscle degeneration (N = 4 mice). Various studies have found that 3 weeks following notexin injury, muscle regeneration has occurred, resulting in the formation of new mature muscle fibres with normal histological structure, apart from the presence of centralized myonuclei (indicative of a past degeneration/regeneration event) [25]. To further exclude potential confounding effects (such as residual, low expression of developmental isoforms of contractile proteins), we allowed muscles to regenerate for 8 weeks following injury with notexin. Muscle fibres from injured legs were centrally nucleated, but they were normal in various other histological respects, resembling the uninjured, contralateral control muscles (Fig. 7a–i). These parameters included: relative expression of type I, IIa, IIb and IIx myosin heavy chain isoforms (Fig. 7a–d, g; N.B. incidences of type I fibres were < 0.2% for both groups); muscle fibre CSA (Fig. 7e, f, h), and tissue organization and architecture (Fig. 7e, f). Interestingly, the force generating capacity was similar between notexin-injured and control muscles, as assessed using skinned/permeabilized muscle fibres (Fig. 6; n = 15 fibres per condition). This suggests that the presence of central nuclei alone does not markedly interfere with muscle contractile capacity.
These results lend support to our initial hypothesis that, in the context of XLMTM, there is a novel, curable pathophysiological mechanism by which variations in nuclear mispositioning and MND volumes modify transcriptional output, which in turn affects density and/or function of the myofilaments themselves, contributing to overall weakness.