Sample collection

Human vastus lateralis biopsies were collected from two cohorts. The collection of muscle biopsies from male control participants and male participants with type 2 diabetes in Denmark was approved by the ethical committee for the Capital Region of Denmark (H-15010122) and was conducted in accordance with the Declaration of Helsinki. All study participants were informed and signed a consent form before enrolment in the study. Characteristics of participants in this study and the biopsy procedure have been described previously [23, 24]. For collection of muscle biopsies from control participants and patients with type 2 diabetes in Canada, all procedures were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Université du Québec à Montréal (CIEREH-2020-3477). HOMA-IR was calculated as previously described [24]. Informed consent was obtained from all participants. Skeletal muscle biopsy samples were obtained from the vastus lateralis muscle under local anaesthesia using a suction modified Bergström needle performed under local anaesthesia. Samples were immediately frozen in liquid nitrogen and stored at −80°C until use. Sex was self-reported in both cohorts.

Single muscle fibre preparation

Cryopreserved muscle samples were dissected into small sections and immersed in a membrane-permeabilising solution, described previously [15] (relaxing solution containing glycerol; 50:50 vol./vol.) for 24 h at −20°C, after which they were transferred to 4°C. The rigor buffer for 2′- (or 3′)-O-(N-methylanthraniloyl)-ATP (Mant-ATP) chase experiments contained 120 mmol/l potassium acetate, 5 mmol/l magnesium acetate, 2.5 mmol/l K2HPO4, 50 mmol/l MOPS, 2 mmol/l DTT at pH 6.8. These bundles were kept in the membrane-permeabilising solution at 4°C for another 24 h. After these steps, bundles were stored in the membrane-permeabilising buffer at −20°C for use within 2 weeks.

Mant-ATP chase assay

Permeabilised skeletal muscle bundles were transferred to the relaxing solution and individual muscle fibres were isolated. Each muscle fibre was first incubated for 300 s with the rigor buffer described above. A solution containing the rigor buffer with added 250 μmol/l Mant-ATP was then added, and kept there for a further 300 s. This incubation time is chosen to ensure full incorporation of Mant-ATP into the sarcomere prior to the washout step. At the end of this step, another solution comprising the rigor buffer with 4 mmol/l ATP was added, with simultaneous acquisition of images.

For fluorescence acquisition, a Zeiss Axio Scope A1 microscope was used with a Plan-Apochromat 20×/0.8 objective and a Zeiss AxioCam ICm 1 camera. Frames were acquired every 5 s with a 20 ms acquisition/exposure time at 385 nm, for 300 s. These data were then fit to an unconstrained double exponential decay using GraphPad Prism version 9.0 (GraphPad.com):

$$\mathrm\;\mathrm\;=\;1\;-\;\mathrm P1\;\lbrack1-\exp(-t/\mathrm T1)\rbrack\;-\;\mathrm P2\;\lbrack1-\exp(-t/\mathrm T2)\rbrack$$

where P1 is the amplitude of the initial rapid decay, which approximates to the DRX, with T1 as the time constant for this decay, and P2 is the slower second decay approximating the proportion of myosin heads in the SRX, with the associated time constant T2. Mant-ATP chase experiments were performed at ambient laboratory temperature (20°C) for all samples.

Each isolated myofibre was then stained using an MYH7 antibody (A4.951, Developmental Studies Hybridoma Bank – DSHB; RRID: AB_528385) to determine whether the fibre was a type I or non-type I fibre, as previously described [25]. A representative image of positive staining in isolated single fibres is shown in electronic supplementary material (ESM) Fig. 1. Due to the relatively low abundance (<1%) of hybrid (type IIX) fibres in vastus lateralis human biopsies, we considered all non-type I fibres to be type II (fast) fibres [26]. Furthermore, our single-fibre proteomics data indicate the presence of zero pure type IIX fibres in this analysis.

Synthesis and purification of MG

MG was purchased from Sigma-Aldrich as a solution at 40% in H2O (M0252). This solution was then further purified by steam distillation. The collected fractions were adjusted to pH 7.0 with NaOH, and aliquots of them were reacted with H2O2 in order to indirectly quantify the pure MG concentration by titration with KMnO4 [27].

Acute glycation Mant-ATP chase assay

Fibres were incubated for 300 s in a modified relaxing buffer solution containing 5.89 mmol/l Na2ATP, 6.48 mmol/l MgCl2, 40.76 mmol/l propionic acid, 100 mmol/l N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid sodium salt, 6.97 mmol/l EGTA, 14.5 mmol/l sodium creatine phosphate dibasic and KOH to adjust the pH to 7.1 [28]. After this incubation, fibres then went through the same protocol as described above (incubation in rigor buffer and then Mant-ATP buffer, followed by fluorescence image acquisition). Then the same fibres were incubated in a modified relaxing buffer that contained 50 mmol/l MG for 30 min to induce acute glycation of the proteins in these muscle fibres. This concentration was selected based on previous literature that used MG synthesised via the same protocol [29]. This buffer contained 5.89 mmol/l Na2ATP, 6.48 mmol/l MgCl2, 40.76 mmol/l propionic acid, 100 mmol/l N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid sodium salt, 6.97 mmol/l EGTA, 43.5 mmol/l sodium chloride, 50 mmol/l MG and KOH to adjust the pH to 7.4.

Following this incubation, these muscle fibres were incubated for 300 s in the modified relaxing buffer described above, and then went through the same process of incubation in rigor buffer, incubation with Mant-ATP buffer, and then fluorescence image acquisition. For analysis purposes, the results for each individual muscle fibre were paired before and after MG incubation to observe the effects of acute glycation upon myosin dynamics.

X-ray diffraction recordings and analysis

Thin muscle bundles were mounted into a specimen chamber in relaxing buffer, and then clamped at a sarcomere length of 2.00 μm. Subsequently, x-ray diffraction patterns were recorded at 15°C using a CMOS camera (model C11440-22CU, Hamamatsu Photonics, Japan; 2048 × 2048 pixels) in combination with a 4-inch image intensifier (model V7739PMOD, Hamamatsu Photonics). The x-ray wavelength was 0.10 nm, and the specimen-to-detector distance was 2.14 m. For each preparation, approximately 20–50 diffraction patterns were recorded at the BL40XU beamline of SPring-8 (Japan Synchrotron Radiation Research Institute, and were analysed as described previously [30]. To minimise radiation damage, the exposure time was kept low (0.5 or 1 s), and the specimen chamber was moved by 100 μm after each exposure. After x-ray recordings, background scattering was subtracted, and the major myosin meridional reflection intensities/spacing were determined as described previously [30].

Single-fibre proteomics

Proteomics measurements were conducted using a previously described workflow [31]. In brief, an Evosep One HPLC system (Evosep), coupled via electrospray ionisation to a timsTOF SCP mass spectrometer (Bruker), was used as the LC-MS system. Peptides were separated using the ‘60 samples per day’ chromatographic method [31] before electrospray ionisation using a CaptiveSpray ion source and a 10 μm emitter directly within the MS instrument. Samples were then measured using a dia-PASEF workflow described elsewhere [31]. Processing of the raw MS spectra was performed using DIA-NN (version 1.8) in library-based mode, based on an in-house muscle fibre-specific MS library comprising 5000 proteins [32]. The DIA-NN settings were as follows: double-pass mode was selected as the neural network mode, ‘robust LC (high accuracy)’ was chosen as the quantification strategy, prototypic peptides were selected for quantification, the ‘match between runs’ option was enabled and the precursor FDR control was set to 1%. Unless specified, the other parameters remained as default settings.

Single-fibre proteomics data analysis/statistical testing

Data analysis was performed using R software (version 4.3.2) and multiple packages from the R environment. The protein groups matrix was loaded into the software, and proteins were log2-transformed before being filtered for at least 70% valid values in at least one of the tested groups (type 2 diabetes or control participants). Next, the filtered protein matrix followed the limma (version 3.54.2) workflow, which included quantile normalisation of all samples and differential expression testing between groups in a pseudobulk manner [33]. To adjust p values for multiple comparison testing, we applied the Benjamini–Hochberg correction for the type I–type II comparison, and the Xiao significance score for the fibre type-specific comparisons of control participants vs those with type 2 diabetes, which takes into consideration both expression fold change and statistical significance. Proteins with a Xiao score under 0.05 were considered as differentially expressed between groups [34].

Myosin heavy chain band isolation for post-translational identifications

Muscle biopsy samples were cut into 15 mg sections, and immersed in a sample buffer (0.5 M Tris buffer at pH 6.8, 0.5 mg/ml bromophenol blue, 10% SDS, 10% glycerol, 1.25% mercaptoethanol) at 4°C. Samples were then homogenised and centrifuged (1200 g), allowing the supernatant to be extracted and used for SDS–PAGE gels using a stacking gel made with acrylamide/bis-acrylamide 37.5:1 and separation gel made with acrylamide/bis-acrylamide 100:1). Proteins were separated, and the individual MYH7 band was excised manually [35].

MS-based glycation peptide mapping

SDS–PAGE bands corresponding to MYH7 from the control or diabetic groups were subjected to in-gel digestion. After reduction with DTT (10 mmol/l) and alkylation of Cys groups with iodoacetamide (50 mmol/l), modified porcine trypsin (Promega) was added at a final ratio of 1:20 (trypsin:protein). Digestion proceeded overnight at 37°C in 100 mmol/l ammonium bicarbonate at pH 7.8. The resulting tryptic peptides were loaded and washed using Evotips, and separated on an Endurance EV1106 column (15 cm × 150 µm internal diameter; 1.9 µm beads) using an Evosep One HPLC system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher) and a 30 SPD preprogrammed gradient.

MS analysis was performed using the data-independent scanning method as described previously [36], with some modifications. Each sample was analysed in a single chromatographic run covering a mass range from 390 to 1000 m/z. The cycle consisted of 255 sequential HCD MS/MS fragmentation events with 2.5 m/z windows from 390–900 m/z, and with 4 m/z windows from 900–1000 m/z. HCD fragmentation was performed using a 33 normalised collision energy. MS/MS scans were performed using a 70 ms injection time, a AGC target setting of 3 × 105 ions and 17,500 resolutions. The whole cycle lasted a maximum of 18 s, depending on the ion intensity during chromatography. The narrow windows used for fragmentation allowed peptide identification using conventional DDA searching algorithms. The SEQUEST HT search program was used, as implemented in Proteome Discoverer 2.5 (Thermo Scientific), against a database containing human myosins and using the following parameters: two maximum missed trypsin cleavage sites, a precursor mass tolerance of 3 Da, and a fragment mass tolerance of 30 ppm. Oxidation on Met (+15.995 Da), CEL (+72.021 Da) and carboxymethyl (+58.005 Da) on Lys, and MG-Hs (+54.011 Da) on Arg were set as variable modifications. Carbamidomethylation (+57.021 Da) on Cys was set as a fixed modification [37].

Statistical analysis

Data are presented as means ± SD. Graphs were prepared and analysed using GraphPad Prism version 9.0. Statistical significance was set to p<0.05 unless otherwise stated. Where data are unevenly distributed, a Mann–Whitney test was applied to assess differences between two groups. Where data are evenly distributed, a bilateral Student’s t test was used to assess differences between two groups; a paired Student’s t test was used in paired experiments. For analyses involving multiple groups, two-way ANOVAs with Šídák’s multiple comparisons test were used. The statistical test applied in each analysis is listed in each figure legend. The number of samples used in each experiment is listed in each individual figure legend, and each sample refers to experimental work on tissue from separate individual participants. Where replicates have been performed upon the same tissue from one individual participant, this is also stated in the figure legend. Throughout the study, experiments were performed unblinded.