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. 2025 Aug;68(8):1836-1850.
doi: 10.1007/s00125-025-06436-0. Epub 2025 Apr 28.

Dysregulated skeletal muscle myosin super-relaxation and energetics in male participants with type 2 diabetes mellitus

Affiliations

Dysregulated skeletal muscle myosin super-relaxation and energetics in male participants with type 2 diabetes mellitus

Christopher T A Lewis et al. Diabetologia. 2025 Aug.

Abstract

Aims/hypothesis: Disrupted energy balance is critical for the onset and development of type 2 diabetes mellitus. Understanding of the exact underlying metabolic mechanisms remains incomplete, but skeletal muscle is thought to play an important pathogenic role. As the super-relaxed state of its most abundant protein, myosin, regulates cellular energetics, we aimed to investigate whether it is altered in individuals with type 2 diabetes.

Methods: We used vastus lateralis biopsy specimens (obtained from patients with type 2 diabetes and control participants with similar characteristics), and ran a combination of structural and functional assays consisting of loaded 2'- (or 3')-O-(N-methylanthraniloyl)-ATP (Mant-ATP) chase experiments, x-ray diffraction and LC-MS/MS proteomics in isolated muscle fibres.

Results: Our studies revealed a greater muscle myosin super-relaxation and decreased ATP demand in male participants with type 2 diabetes than in control participants. Subsequent proteomic analyses indicated that these (mal)adaptations probably originated from remodelled sarcomeric proteins and greater myosin glycation levels in patients than in control participants.

Conclusions/interpretation: Overall, our findings indicate a complex molecular dysregulation of myosin super-relaxed state and energy consumption in male participants with type 2 diabetes. Ultimately, pharmacological targeting of myosin could benefit skeletal muscle and whole-body metabolic health through enhancement of ATP consumption.

Data availability: The raw MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD053022.

Keywords: Diabetes; Metabolism; Myosin; Skeletal muscle.

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Conflict of interest statement

Data availability: The raw MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD053022 [56]. The rest of the data that support the findings of this study are available from the corresponding author upon reasonable request. Funding: Open access funding provided by Copenhagen University. This work was generously funded by the Lundbeckfonden (R434-2023-311 to JO). The x-ray experiments were performed under approval of the SPring-8 Proposal Review Committee (2022 A1069). MS-based proteomics analyses were performed by the Proteomics Research Infrastructure at the University of Copenhagen, supported by the Novo Nordisk Foundation (grant agreement number NNF19SA0059305). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCIU, MICIU/AEI/10.13039/501100011033) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-zzzzS funded by MCIU). Authors’ relationships and activities: The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work. CTAL is an employee of Novo Nordisk A/S. This position began after the work on this manuscript was complete, and the position had no influence on the results or conclusions drawn. Contribution statement: CTAL, ASD and JO contributed to the study conception and design. Material preparation, data collection and analysis were performed by CTAL, RM-J, LS, EC, AB, JL, RAES, SL, HI, MC, JAM, GG, JA-C, TJH, JV, MA, VM, RH, JG, PG, MA-L, MB, RR, ASD and JO. The first draft of the manuscript was written by CTAL and JO, and all authors commented on all versions of the manuscript and approved the submitted version. JO is the guarantor of this work.

Figures

Fig. 1
Fig. 1
Myosin relaxed states are altered in type 2 diabetes (T2DM). (a) Representative fluorescence Mant-ATP decays from single muscle fibres isolated from skeletal muscle biopsies from control participants and participants with T2DM, measured over a 300 s period. (b, c) Percentage of myosin heads in P1/DRX (b) or P2/SRX (c) from single muscle fibres obtained from control participants and participants with T2DM. Values are separated based on fibre type: MyHC type I or MyHC type II. (d) T1 value indicating the ATP turnover lifetime for the DRX. (e) T2 value indicating the ATP turnover lifetime for the SRX. Grey circles represent the values from each individual muscle fibre analysed; coloured triangles represent the mean value for an individual participant; n=9–11 participants per group; 8–12 fibres were analysed per participant. An unpaired Student’s t test was used to calculate statistical significance of the differences among the mean values. (f) Representative x-ray diffraction recordings from permeabilised skeletal muscle bundles from control participants and those with T2DM. The M3 and M6 meridional reflections are indicated. (g) Normalised intensity (AU) of the M3 meridional reflection. (h) M3 meridional spacing, measured in nanometres (nm). (i) Normalised intensity (AU) of the M6 meridional reflection. (j) M6 meridional spacing, measured in nanometres (nm). Values are means ± SEM. All differences were tested for significance using a bilateral Student’s t test (n=6 participants per group): *p<0.05
Fig. 2
Fig. 2
Glycation of type I myosin heavy chain (MYH7) in type 2 diabetes (T2DM). (a) Schematic of the process of glycolysis from which the intracellular MG is formed, and the chemical formulae of some of the MG-derived AGEs that have been detected on various protein residues. The figure was created using BioRender. (b) Quantification of several glycated peptides from MYH7, showing increased glycation in participants with T2DM when compared with control participants. The Mann–Whitney U test was used to calculate the statistical significance of the differences between groups: *p<0.05, **p<0.01 (n=5 participants per group). We detected several peptide fragments modified through formation of CEL, MG-Hs or Nε-(carboxymethyl)lysine. (c) Scheme of MYH7 protein regions, with the location of the glycated residues indicated in red. ELC, essential light chain; MOLD, methylglyoxal-lysine dimer; MODIC, (2-ammonio-6-([2-[(4-ammonio-5-oxido-5-oxopentyl)amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene]amino)hexanoate. RLC, regulatory light chain. The figure was created using BioRender
Fig. 3
Fig. 3
Acute glycation increased the percentage of myosin heads in the SRX in control participants. (a) Workflow for loaded Mant-ATP chase experiments in which single muscle fibres were incubated with 50 mmol/l MG in between matched experiments. The figure was created using BioRender. (b) Percentage of myosin heads in P1/DRX before (black circles) and after (red circles) treatment with MG. (c) Percentage of myosin heads in P2/SRX before (black circles) and after (red circles) treatment with MG. (d) T1 value indicating the ATP turnover lifetime of the DRX before (black circles) and after (red circles) treatment with MG. (e) T2 value indicating the ATP turnover lifetime of the SRX before (black circles) and after (red circles) treatment with MG. Values were separated based on fibre type: MyHC type I or MyHC type II. A paired Student’s t test was used to calculate statistical significance between fibres before and after MG incubation: *p<0.05. Two-way ANOVA with Šídák’s multiple comparisons test was used to calculate the significance between groups (between control and T2DM and between type I and type II fibres): †††p<0.001 (n=4 participants per group)
Fig. 4
Fig. 4
Single-fibre proteomics shows that type I muscle fibres from participants with type 2 diabetes (T2DM) have differential expression of sarcomeric proteins. (a) Workflow for isolation of single skeletal muscle fibres from vastus lateralis muscle biopsies, and downstream processing of these single muscle fibres using MS and a subsequent bioinformatics pipeline. The figure was created using BioRender. (b) Volcano plot showing differentially expressed proteins between type I and type II muscle fibres from both participant groups. (c) Volcano plot showing proteins that are differentially expressed in type I fibres between control and T2DM groups. (d) Volcano plot showing proteins that are differentially expressed in type II fibres between control and T2DM groups. Detailed information on statistical analysis of single-fibre proteomics is provided in Methods (n=5 participants per group). FC, fold change. ACTN3, α-actinin 3; ACTR1A, actin-related protein 1A; ATP2A1, sarcoplasmic/endoplasmic reticulum calcium ATPase 1; DPYSL3, dihydropyrimidinase-like 3; DYNC1H1, dynein cytoplasmic 1 heavy chain 1; ECH1, enoyl-CoA hydratase 1; FABP4, fatty acid binding protein 4; GLO1, glyoxalase I; GOLGA4, golgin subfamily A member 4; MPC2, mitochondrial pyruvate carrier 2; MYBPC2, myosin binding protein C2; MYH2, myosin 2; MYL3, myosin light chain 3; MYL4, myosin light chain 4; MYL6B, myosin light chain 6B; MYLK2, myosin light chain kinase 2; MYOM2, myomesin 2; MYOZ2, myozenin-2; NDUFA10, NADH:ubiquinone oxidoreductase subunit A10; NDUFA12, NADH:ubiquinone oxidoreductase subunit A12; NDUFS5, NADH:ubiquinone oxidoreductase subunit S5; NDUFS8, NADH:ubiquinone oxidoreductase core subunit S8; NUDT5, nudix hydrolase 5; PEPD, peptidase D; PGK2, phosphoglycerate kinase 2; PSMD11, 26S proteasome non-ATPase regulatory subunit 11; SLC25 A11, solute carrier family 25 member 11; SYNPO2, synaptopodin 2; TNNC1, troponin C1; TNNC2, troponin C2; TNNI1, troponin I1; TNNT1, troponin T1; TNNT3, troponin T3; TPM3, tropomyosin 3; ZC3H8, zinc finger CCCH-type containing 8

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