Fibre morphology, intramyocellular lipid content and 3D capillary architecture in human postural, respiratory and locomotor muscles in type 2 diabetes mellitus

Fibre morphology, intramyocellular lipid content and 3D capillary architecture in human postural, respiratory and locomotor muscles in type 2 diabetes mellitus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fibre morphology, intramyocellular lipid content and 3D capillary architecture in human postural, respiratory and locomotor muscles in type 2 diabetes mellitus Nataša Pollak, Jiří Janáček, František Saudek, Erika Cvetko, Barbora Radochová, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8618826/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Histochemistry and Cell Biology → Version 1 posted 11 You are reading this latest preprint version Abstract In type 2 diabetes mellitus (T2DM), skeletal muscle is a major site of metabolic and microvascular dysfunction, yet most human data derive from large locomotor muscles, whereas postural and respiratory muscles remain less well characterised. We examined whether T2DM alters fibre morphology, intramyocellular lipid (IMCL) content, and 3D capillary architecture across functionally distinct muscles. Postural (splenius capitis, SC), respiratory (diaphragm, DIA; external intercostal, EXT), and locomotor (vastus lateralis, VL) muscles from adult males (T2DM vs. control, n = 24/group) were sampled < 24h post-mortem. Analysis included myosin-heavy-chain fibre typing, Sudan Black B IMCL quantification, and 3D capillary morphometry (length, tortuosity, anisotropy, branching density). Groups were age-matched (T2DM 70.8 ± 7.4 vs 69.7 ± 11.8 years; p = 0.684), but BMI was higher in T2DM (31.9 ± 4.7 vs 24.8 ± 2.7 kg/m²; p < 0.0001). Fibre-type profiles were similar, except for elevated 2a/2x hybrids in T2DM VL (p = 0.014). Mean fibre diameters were preserved, though type 1 fibres were larger in T2DM SC (p = 0.0238). IMCL was higher in T2DM SC and EXT (p < 0.05), with non-significant differences in VL and DIA. Type 1 and 2a fibres had higher IMCL than glycolytic fibres, with no group-by-fibre-type interaction. BMI strongly predicted VL IMCL (p < 0.0001), while age associated negatively with IMCL in respiratory muscles (p ≤ 0.05). Capillary length per fibre volume was selectively reduced in DIA (p = 0.0115); other indices were preserved, except for higher anisotropy in EXT (p = 0.0495). Overall, these functionally diverse muscles showed subtle, muscle-specific remodelling, with adiposity-linked IMCL accumulation and reduced DIA capillary supply despite largely preserved global architecture, suggesting selective metabolic and microvascular vulnerability. Type 2 diabetes mellitus skeletal muscle myosin heavy chain isoforms intramyocellular lipid 3D capillary morphometry metabolic myopathy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle oxidative metabolism, including reduced expression or abundance of oxidative phosphorylation components and reduced insulin-stimulated non-oxidative glucose disposal, which together contribute to dysglycaemia (Patti et al. 2003 ; Yokoyama et al. 2008 ; Öhman et al. 2021 ). Skeletal muscle is the principal site of insulin-stimulated glucose disposal in humans and comprises roughly 30–40% of adult body mass, underscoring its central role in systemic insulin resistance (Baron et al. 1988 ; Janssen et al. 2000 ). In T2DM, changes in fibre-type composition and oxidative capacity, mitochondrial dysfunction, and impaired microvascular perfusion collectively contribute to reduced aerobic metabolism and insulin responsiveness (Oberbach et al. 2006 ; Park et al. 2020 ; Öhman et al. 2021 ). However, most human data are derived from superficial locomotor muscles such as the vastus lateralis, whereas postural and respiratory muscles that sustain continuous low-intensity activity for posture and breathing remain comparatively understudied. In locomotor muscles, obesity and T2DM have often been associated with a relative shift away from slow oxidative fibres and reduced oxidative enzyme activity, although findings vary across cohorts (He et al. 2001 ; Oberbach et al. 2006 ). Conversely, ageing is often characterised by preferential atrophy and loss of type II fibres, which can increase the relative representation of slow oxidative fibres, although the extent of this remodelling varies by muscle (Lexell et al. 1988 ; Frontera et al. 2000 ). Intermuscular heterogeneity is considerable; fibre-type proportions differ markedly between neck and limb muscles even within the same individual (Vikne et al. 2012 ; Cornwall and Kennedy 2015 ). In obesity and T2DM, chronic metabolic overload further promotes the accumulation of intramyocellular lipid (IMCL), a critical energy reservoir and lipid buffer (da Silva Rosa et al. 2020 ). Seminal ¹H-MRS studies reported an inverse association between IMCL and insulin sensitivity in humans (Krssak et al. 1999 ), and subsequent work implicated lipid-derived intermediates, including diacylglycerols and ceramides and their intracellular localisation, in impaired insulin signalling and mitochondrial function (Szendroedi et al. 2014 ; Perreault et al. 2018 ). The skeletal muscle capillary network is essential for oxygen and substrate delivery and is affected by obesity, ageing, and T2DM, although human findings remain variable across studies and populations (Prior et al. 2015 ; Park et al. 2020 ). Early clamp studies associated reduced capillary density and a lower proportion of slow oxidative (type 1) fibres with insulin resistance (Lillioja et al. 1987 ), whereas later work demonstrated that insulin-mediated microvascular recruitment is blunted in obesity and T2DM (Clerk et al. 2006 ; Park et al. 2020 ). Ageing and T2DM can also impair microvascular function, including endothelial glycocalyx alterations, with effects that depend on muscle type and physical activity level (Groen et al. 2014 ; Prior et al. 2015 ; Bosutti et al. 2015 ). Conventional two-dimensional capillary profile counts are sensitive to section orientation and fibre size, which can bias estimates of capillary supply in architecturally complex muscles. Modern three-dimensional confocal imaging with vector-based reconstruction enables direct quantification of capillary length and network geometry (tortuosity, anisotropy, and branching density) and permits correction for axial shrinkage in thick sections, providing a less biased representation of microvascular architecture (Čebašek et al. 2010 ; Janáček et al. 2011 , 2012 ). Against this background, we analysed autopsy samples from four functionally distinct muscles, (postural, respiratory and locomotor), from individuals with and without T2DM. These muscles differ in metabolic phenotype and habitual functional demands, spanning near-continuous respiratory or postural activity to intermittent locomotion(Mizuno 1991 ; Pollak et al. 2025 ). Using myosin heavy chain (MyHC)-based fibre typing, Sudan Black B histochemistry, and 3D confocal morphometry, we sought to determine whether T2DM is associated with muscle-specific alterations in fibre-type composition, fibre size, IMCL content, and capillary network geometry, and whether the structural hierarchy among postural, respiratory, and locomotor muscles is preserved in T2DM. Methods Study design This comparative study was conducted on human skeletal muscle samples obtained post-mortem from individuals with T2DM and age-matched non-diabetic controls (n = 24 per group). Four functionally distinct skeletal muscles were analysed: the postural muscle splenius capitis (SC), the respiratory muscles external intercostal (EXT) and diaphragm (DIA), and the locomotor muscle vastus lateralis (VL). Muscle samples were collected from each individual within 24 h post-mortem during routine autopsies at the Institute of Forensic Medicine, University of Ljubljana. Medical records were reviewed to confirm diabetes status and to exclude individuals with neuromuscular, other endocrine disorders, malignancy, or chronic systemic conditions likely to affect muscle phenotype, as well as prolonged immobilisation or chronic corticosteroid therapy. Only male individuals were included to eliminate potential confounding arising from sex-specific hormonal variability. Long-term glycaemic control in the T2DM group and normoglycaemia in the control group were evaluated using post-mortem HbA1c. This marker remains stable after death, providing an objective assessment of ante-mortem glycaemic status for all subjects. Medical records indicated a T2DM duration of at least 10 years in all affected individuals, and that glucose-lowering management consisted of oral antihyperglycaemic agents administered as monotherapy or combination therapy (most commonly metformin, sulfonylureas and/or DPP-4 inhibitors). All procedures were reviewed and approved by the National Medical Ethics Committee of the Republic of Slovenia (Permit Nos. : 0120–536/2019/4 and 0120/536/2019/7) and were conducted in accordance with the Declaration of Helsinki. Muscle tissue sampling Muscle samples were obtained from standardised anatomical sites: SC at the mid-portion (C4 level); EXT at the sixth intercostal space (midclavicular line); DIA at the costal portion (midclavicular line); and VL at the distal third of the thigh. Each sample (≈ 1 cm³) was rapidly frozen in liquid nitrogen and stored at − 80°C until further processing. Serial transverse cryosections were prepared using a Leica CM1950 cryostat (Leica Microsystems GmbH, Wetzlar, Germany). Thin 10-µm sections were used for histochemistry and fibre typing, whereas adjacent thick 100-µm sections were prepared for three-dimensional (3D) capillary analysis. Histochemistry: intramyocellular lipids Neutral lipids were visualised using Sudan Black B staining (Sigma-Aldrich Corp, St. Louis, MO, USA) on 10-µm cryosections. Serial sections from the same sample were analysed to align fibre type and lipid measurements. Sections were equilibrated to room temperature, rinsed briefly in 70% ethanol, stained for 60 minutes in saturated Sudan Black B solution prepared in 70% ethanol, rinsed in tap water and mounted in glycerol-gelatin. The IMCL index was calculated as the percentage of fibre area occupied by Sudan Black–positive lipid droplets. Immunohistochemistry: myosin heavy chain isoform expression Serial 10-µm transverse sections were incubated with normal rabbit serum (1:40 in PBS containing 0.5% BSA) to block non-specific binding, followed by incubation with monoclonal antibodies against MyHC isoforms: BA-D5 (MyHC-1), SC-71 (MyHC-2a), and 6H1 (MyHC-2x) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), each diluted 1:100 in PBS (pH 7.4). Immunoreactivity was visualised using a diaminobenzidine (DAB)-based peroxidase detection system according to the manufacturer’s instructions. Fibres were classified as type 1, type 2a, type 2x, and hybrid type 1/2a and type 2a/2x based on established MyHC staining patterns (Schiaffino and Reggiani 2011 ). Light microscopy and image analysis Images of MyHC-stained serial sections were acquired in brightfield on a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) using a 20× Plan Fluor objective (numerical aperture 0.50) equipped with a KERN ODC 841 digital camera (KERN & SOHN GmbH, Balingen, Germany) and VIS Pro KERN OXM 902 software. Illumination, aperture, and exposure settings were standardised within each imaging batch to ensure reproducible conditions. High-resolution fields (5440 × 3648 pixels) were sampled systematically, with at least three randomly selected regions per muscle, yielding a minimum of 100 analysed fibres per muscle (total area: ~8.3 × 10⁵ µm²). Images were saved as 24-bit RGB TIFF files (8 bits per colour channel). Image analysis was performed in Ellipse 2.081 (ViDiTo, Košice, Slovakia). Fibre type classification was conducted using the dedicated software developed by Karen et al. (Karen et al. 2009 ), which semiautomatically aligns serial sections and assigns fibre types according to myosin heavy chain expression profiles. For each muscle, fibre-type proportions (%) and fibre diameter, defined as the minimal Feret diameter, were calculated. The IMCL index was determined as the percentage of the muscle fibre cross-sectional area occupied by Sudan Black B-positive lipid droplets. All imaging and analyses were performed by a single trained evaluator blinded to group. 3D capillary network labelling Thick (100 µm) transverse sections were washed in cold PBST (PBS with 0.1% Triton X-100) and fixed at 4°C in 7% formaldehyde with 0.1% glutaraldehyde in PBST. After PBST washes, antigen retrieval was performed with 0.2% proteinase K (0.5 M Tris, pH 8.0, with EDTA) for 5 min at 37°C, followed by further PBST washes. The basal lamina was labelled overnight at 4°C with rabbit anti-collagen IV polyclonal antibody (1:200; Abcam, UK), followed by Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:500; Invitrogen, USA). Endothelial cells were labelled overnight at 4°C with fluorescein-labelled Griffonia simplicifolia lectin I (1:300; Vector Laboratories) and mouse monoclonal antibody F8/86 anti-von Willebrand factor (1:1000; Dako, Denmark), followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500; Invitrogen, USA). Sections were mounted in ProLong™ Gold Antifade (Thermo Fisher Scientific, USA). Negative controls omitting primary antibodies and secondary antibodies showed no specific immunofluorescent signal; additional controls omitting lectin confirmed the absence of lectin-related background. Confocal imaging and 3D reconstruction Image stacks were acquired using a Leica STELLARIS 8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a 40×/1.1 water-immersion objective. Z-stacks were obtained with 1-µm optical steps at a resolution of 512 × 512 pixels (pixel size 0.76 µm). A linear laser power Z-compensation was utilized to avoid loss of signal due to sample thickness. Sequential excitation at 488 nm (detection: 498–585 nm) and 570 nm (detection: 590–700 nm) was used to avoid channel crosstalk. Excitation was provided by a White Light Laser and two HyD X detectors, set to operate in photon counting mode, were used for detection. For each muscle, five randomly selected fields of view (387.5 × 387.5 µm) containing at least 100 fibres in total were analysed. Image stacks were processed in Ellipse 2.081 (ViDiTo, Košice, Slovakia). Z-axis deformation was corrected by axial calibration (Janáček et al. 2011 ). After segmentation, binary images were skeletonised using the six-pass Palágyi algorithm and vectorised into 5-µm line segments, followed by manual refinement in Tracer3D (Cvetko et al. 2013 ) using a Phantom Omni haptic device (3D Systems, Rock Hill, USA). Fibre contours were traced on four planes to calculate fibre diameter, surface area and volume. Quantitative parameters included capillary length per fibre length ( LL ), per fibre surface area ( LSf) , and per fibre or muscle volume ( LVf , LVm ) (Janáček et al. 2011 ; Cvetko et al. 2013 ). Mean capillary length ( MeanCap ) was computed as two-thirds of the total capillary length per unit volume divided by the number of branching points per unit volume, i.e. \(\:MeanCap=\frac{2}{3}\frac{{L}_{V}}{{N}_{V}}\) , where L V is capillary length density, and N V is branching-point density (Janáček et al. 2011 ). Tortuosity was expressed as the ratio of sum of exterior angles to the total capillary length anisotropy as the ratio of the principal eigenvalues of the structural tensor, and branching density (Br_dens) as branch points per muscle volume (Cvetko et al. 2013 ; Eržen et al. 2018 ). All analyses were performed by the same evaluator, blinded to group. Statistical analysis All analyses were performed in Python (Statsmodels 0.14; Python Software Foundation, Wilmington, DE, USA) and GraphPad Prism 10 (GraphPad Software, LLC, San Diego, CA, USA). Normality of distributions and model residuals was assessed using the Shapiro–Wilk and Jarque–Bera tests. Between-group differences in demographic variables were evaluated with independent-samples t-tests. Multivariable models were prespecified and adjusted for age and BMI. For capillary parameters ( LVf , LVm , LL , LSf , MeanCap , Br_dens, Tortuosity, Anisotropy), linear mixed-effects models were fitted with subject as a random intercept to account for repeated fields of view within individuals. Models were fitted separately for each muscle, with fixed effects for group, age, and BMI, and the adjusted group effect was reported as β with 95% confidence intervals and two-sided p-values. Sensitivity analyses included quadratic age (age²) and group-by-age terms. For LVf , an additional model included mean fibre diameter as a covariate. For fibre-level outcomes (fibre-type proportions, fibre diameter, and IMCL index), subject-level means were calculated for each fibre type (1, 2a, 2x, 1/2a, and 2a/2x) within each muscle. These means were compared using two-way ANOVA (Group × Fibre type) with Tukey post hoc tests. Because all four muscles were sampled from the same individuals, within-subject comparisons across muscles were assessed using paired t-tests and a repeated-measures mixed model of the form Outcome ~ Group × Muscle + Age + BMI + (1|Subject) to estimate muscle-specific group effects and group-by-muscle interactions. Within the T2DM subgroup, associations between HbA1c and structural outcomes were examined using linear models adjusted for age and BMI. All tests were two-sided with α = 0.05, and results are presented as mean ± SD or adjusted regression coefficients (β) with 95% confidence intervals and corresponding p-values. Results Study groups Age did not differ significantly between groups (T2DM 70.8 ± 7.4 vs controls 69.7 ± 11.8 years; p = 0.684), whereas BMI was higher in T2DM (31.9 ± 4.7 vs 24.8 ± 2.7 kg·m⁻²; p < 0.0001). The mean duration of T2DM was 16.5 ± 5.1 years. In the control group, all subjects had HbA1c within the normal range (< 5.7% [ 8.0%. Muscle fibre morphology Overall, fibre-type composition and fibre diameter were largely similar between groups, with only muscle-specific differences. Fibre-type composition (Fig. 1 a–d) did not differ significantly between groups in SC, DIA, or EXT (all p ≥ 0.10). In the VL, the proportion of hybrid type 2a/2x fibres was higher in T2DM than in controls (p = 0.0141), whereas all other fibre types did not differ significantly between groups. Fibre diameters did not differ significantly between groups at the whole-muscle level in any of the examined muscles, although SC displayed a borderline trend toward larger mean diameters in T2DM (p = 0.0502). In fibre-type–specific analyses (Fig. 1 e–h), the only significant group difference was observed in the SC, where type 1 fibres were larger in T2DM than in controls (p = 0.0238); no other fibre types in the SC, DIA, EXT or VLs differed between groups (all p > 0.10). Across the combined cohort, age was positively associated with fibre diameter in several fast and hybrid fibre types, including type 1/2a, 2a, 2a/2x and 2x fibres in the EXT, and type 2a, 2a/2x and 2x fibres in the VL, as well as type 2x fibres in the DIA (all p < 0.05), whereas no age effect was detected in any fibre type in the SC. BMI showed no independent association with fibre diameter in any muscle. Within the T2DM subgroup, HbA1c was not associated with fibre diameter except for an isolated positive association in type 2x fibres of the EXT (p = 0.0396). Fibre-type proportions ( a–d ) and fibre diameter (e–h) in splenius capitis (SC; a,e ), external intercostal muscle (EXT; b,f ), diaphragm (DIA; c,g ), and vastus lateralis muscle (VL; d,h ) in control (black) and T2DM (red) subjects. Boxes represent the interquartile range, centre lines denote medians, and whiskers indicate 1.5×IQR (Tukey). Individual points represent observed values. CTRL – control; T2DM – type 2 diabetes mellitus. Asterisks above the bars: * denotes P < 0.05 Expression of MyHC isoforms Type 1, Type 2a, Type 2x, and intramyocellular lipid visualised by Sudan Black in successive transverse sections of the splenius capitis (SC), diaphragm (DIA), external intercostal (EXT), and vastus lateralis (VL) muscles. Within each group, panels are arranged left-to-right as: Type 1 fibres (BA-D5 immunoreactivity) → Type 2a fibres and 2a/2x hybrids (SC-71 immunoreactivity; hybrids identified by co-labelling with 6H1) → Type 2x fibres (6H1 immunoreactivity) → intramyocellular lipid (IMCL) staining by Sudan Black B. DIA – diaphragm, SC – splenius capitis, EXT – external intercostal, VL - vastus lateralis muscle; CTRL – control; DM – type 2 diabetes mellitus. Scale bar = 100 µm. Intramyocellular lipids The IMCL index was higher in T2DM, with significant group differences in the SC (p = 0.0128) and EXT (p = 0.0195) muscles and no significant differences in the VL (p = 0.111) and DIA (p = 0.433). Across fibre types, type 1 and 2a fibres contained more IMCL than type 2x fibres, with no group-by-fibre-type interaction. BMI was the strongest independent predictor of IMCL, especially in VL (β = +0.28 percentage points per kg·m⁻², 95% CI + 0.19 to + 0.36, p < 0.0001), while age was modestly negatively associated with IMCL in DIA and EXT (β = −0.017 and − 0.015 percentage points per year, both p ≤ 0.05). Within the T2DM subgroup, HbA1c showed no consistent association with IMCL in pooled models (p = 0.7100), and all muscle–fibre combinations were non-significant (p ≥ 0.10) except for a small isolated negative association in DIA 1/2a fibres (p = 0.0419). ( a–d ) IMCL (%) across fibre types in the splenius capitis (SC; a ), external intercostal (EXT; b ), diaphragm (DIA; c ), and vastus lateralis (VL; d ) muscles. Boxes represent the interquartile range; centre lines denote medians; whiskers indicate 1.5×IQR (Tukey). Individual points represent observed values. CTRL – control (black); T2DM – type 2 diabetes mellitus (red). Three-dimensional capillary architecture Three-dimensional morphometry showed a selective reduction in capillary length per fibre volume ( LVf ) in the DIA in T2DM, with otherwise preserved capillary geometry and minor, muscle-specific differences. Descriptive values for all capillary parameters are presented in Table 1 and representative confocal z-stack images and 3D reconstructions are shown in Fig. 4 . Table 1 Three-dimensional capillary architecture across four human skeletal muscles in non-diabetic controls and individuals with T2DM Parameter Group DIA SC EXT VL LVm (µm ⁻2 ) ×10 ⁻6 CTRL T2DM 598.75 ± 122.01 601.83 ± 193.74 415.32 ± 70.31 418.83 ± 72.34 422.02 ± 81.17 438.00 ± 91.90 368.89 ± 74.80 435.85 ± 143.04 Tortuosity (rad·µm − 1 ×10 − 3 ) CTRL T2DM 23.21 ± 8.20 20.56 ± 4.97 30.78 ± 11.33 33.37 ± 12.48 26.60 ± 10.27 19.94 ± 5.16 42.12 ± 10.30 42.72 ± 10.21 Anisotropy CTRL T2DM 2.75 ± 0.35 2.61 ± 0.62 2.50 ± 0.36 2.34 ± 0.41 2.24 ± 0.32 2.56 ± 0.45 1.66 ± 0.36 1.61 ± 0.24 MeanCap (µm) CTRL T2DM 373.76 ± 151.00 308.61 ± 202.80 469.86 ± 479.55 415.09 ± 264.33 330.18 ± 147.38 370.10 ± 159.52 236.91 ± 62.56 222.15 ± 73.24 Br_dens CTRL T2DM 1.37 ± 0.49 2.04 ± 1.48 0.93 ± 0.32 0.99 ± 0.52 1.11 ± 0.37 1.08 ± 0.55 1.16 ± 0.34 1.51 ± 0.62 LVf (µm − 2 ×10 − 4 ) CTRL T2DM 20.09 ± 6.46 17.06 ± 6.63 13.64 ± 3.86 12.58 ± 4.74 12.57 ± 4.10 13.03 ± 4.24 10.13 ± 2.80 9.72 ± 3.78 LSf (µm − 1 ×10 − 4 ) CTRL T2DM 200.29 ± 59.60 197.39 ± 68.55 126.42 ± 28.81 136.17 ± 50.30 134.78 ± 34.83 135.25 ± 32.39 145.96 ± 37.81 142.45 ± 57.78 LL (µm) CTRL T2DM 3.39 ± 1.10 3.93 ± 1.50 1.94 ± 0.55 2.47 ± 1.28 2.47 ± 0.63 2.52 ± 0.81 3.70 ± 1.61 3.59 ± 1.69 Capillary network characteristics were estimated by the length of capillaries per volume of muscle tissue ( LVm , [µm –2 ] × 10 –6 ), length of capillaries per length of muscle fibers (LL), length of capillaries per fiber surface ( LSf , [µm–1 ] ×10 –4 ), length of capillaries per fiber volume ( LVf , [µm –2 ]×10 –4 ), Tortuosity ([rad µm –1 ] ×10 –3 ), Anisotropy, mean capillary length ( MeanCap , µm); number of branching per muscle volume (Br_dens, [µm –3 ] ×10 –6 ). Values are mean ± SD. SC – splenius capitis; DIA – diaphragm; EXT – external intercostal; VL – vastus lateralis; CTRL – control; T2DM – type 2 diabetes mellitus. LVf was significantly lower in DIA in T2DM than in controls (p = 0.0115), with a non-significant trend toward lower LVf in SC (p = 0.0768) and no group differences in EXT or VL (both p > 0.10). Anisotropy was modestly higher in EXT in T2DM (p = 0.0495), whereas LVm , LL , LSf , MeanCap , tortuosity and Br_dens showed no group differences (all p > 0.10). In covariate analyses, age was associated with lower tortuosity in SC (p = 0.0140) and VL (p = 0.0401) and with shorter LL in VL (p = 0.0142). BMI was positively associated with LVf (p = 0.0236) and LSf (p = 0.0444) in DIA. Within the T2DM subgroup, HbA1c showed DIA-specific associations, being inversely related to LL (p = 0.0367) and positively related to MeanCap (p = 0.0170), with an additional trend for higher LSf (p = 0.0648). For all other muscles and capillary parameters, HbA1c showed no significant associations (all p ≥ 0.13). Inclusion of Group × Age interaction terms or quadratic Age² terms did not improve model fit and did not change the LVf difference between groups, confirming that the lower LVf in DIA in T2DM was not explained by age distribution. ( a–d ) Representative single optical sections from confocal z-stacks showing merged channels from triple immunofluorescence staining: capillaries (yellow-green) and muscle fibre outlines (red). These panels illustrate the spatial distribution of capillaries relative to fibre boundaries within the image volume used for 3D analysis. ( e–h ) Volume renderings generated from the full z-stacks for the same regions, with the capillary network segmented and displayed throughout the reconstructed volume. ( i–l ) 3D renderings combining muscle fibre volumes (semi-transparent grey) with the reconstructed capillary network (red) for the same regions. Columns correspond to muscles as follows: splenius capitis ( a , e , i ), external intercostal (b , f , j ), diaphragm ( c , g , k ), and vastus lateralis ( d , h , l ). Scale bar = 50 µm (applies to a-d ). Within-subject hierarchy among muscles Because all four muscles were obtained from the same individuals, paired comparisons enabled analysis of intrinsic structural hierarchy independent of interindividual variability. Across subjects, LVf followed the order DIA > SC ≈ EXT > VL, while fibre diameter showed a contrasting pattern (VL > DIA > EXT > SC; all paired comparisons p < 0.01). No significant group-by-muscle interactions were detected, indicating that the relative ordering of capillary supply and fibre morphology across these muscles was preserved in T2DM. Discussion This study analysed four functionally distinct skeletal muscles, SC, DIA, EXT, and VL, obtained from the same older men with and without T2DM, using MyHC-based fibre typing, Sudan Black B IMCL staining, and 3D confocal capillary morphometry. The T2DM group had HbA1c values generally within commonly targeted ranges and no documented advanced diabetic complications, supporting interpretation of the findings as early or moderate structural differences associated with diabetes and adiposity rather than overt advanced myopathy. Overall, group differences were modest and muscle-specific, superimposed on a preserved hierarchy across muscles. Fibre-type composition and mean fibre diameter were largely maintained; IMCL was higher in SC and EXT; oxidative fibres (types 1 and 2a) contained more IMCL than type 2x fibres in both groups; and LVf was selectively reduced in the DIA, while other capillary indices were largely similar between groups. Among capillary endpoints, the DIA showed the clearest group difference, a selective reduction in LVf . SC showed the clearest fibre-size difference (larger type 1 fibres), EXT exhibited a small change in anisotropy, and VL showed a modest increase in 2a/2x hybrids together with BMI-associated IMCL variation. Together, the pattern of results is consistent with a dominant influence of muscle-specific functional demands and oxidative phenotype, and with adiposity as an important covariate, rather than a uniform diabetes effect across the musculature, consistent with previous morphological and microvascular studies in diabetic and obese muscle (Umek et al. 2019 ). The preservation of fibre-type composition in the postural (SC) and respiratory (DIA, EXT) muscles, and the modest increase in 2a/2x hybrids in VL, contrasts with reports of a more pronounced shift toward faster, more glycolytic phenotypes in some locomotor muscles in obesity and T2DM (Park et al. 2009 ; Andreassen et al. 2014 ). The larger SC type 1 fibres in T2DM may reflect chronic loading associated with higher body mass and sustained postural activity, although causal inference is limited by the cross-sectional, post-mortem design. Similar preservation or mild hypertrophy of fibres in frequently recruited muscles has been reported in models of obesity-induced insulin resistance, where increased mechanical load supports fibre growth despite systemic metabolic stress (Ato et al. 2019 ; Umek et al. 2021a ). Continuously active postural and respiratory muscles are exposed to sustained mechanical and metabolic demand, which can favour anabolic signalling and mitochondrial maintenance and may oppose atrophy programmes mediated by FoxO transcription factors (Sandri et al. 2006 ; Ogasawara et al. 2013 ). Conversely, intermittently recruited muscles, such as VL, may be more sensitive to reductions in habitual activity, particularly in older individuals. The fibre-type–specific age associations observed in DIA, EXT, and VL should be interpreted as cohort-specific and may reflect selective survival of larger fibres, unmeasured activity differences, or other confounding, rather than a general ageing signature. The minimal influence of BMI and HbA1c on fibre size suggest that, in this cohort of older men, muscle-specific factors, likely related to regional workload and oxygen demand, outweigh systemic factors such as BMI in determining fibre size (Frontera et al. 2000 ; Cameron et al. 2023 ). Collectively, these findings indicate that diabetes does not uniformly impair myofibre morphology; continuously active muscles, such as the postural SC, retain or even modestly increase their fibre calibre, which may reflect functional adaptation to sustained contractile and metabolic demands rather than pathological hypertrophy. IMCL was modestly higher in T2DM, most clearly in SC and EXT, indicating that habitual postural or respiratory activity does not preclude greater lipid storage when adiposity is higher. This is consistent with evidence that IMCL content reflects both oxidative phenotype and lipid availability, and that IMCL per se is not a direct surrogate of insulin sensitivity without information on lipid species, subcellular localisation, and turnover (Krssak et al. 1999 ; Goodpaster et al. 2001 ; Dubé et al. 2008 ; Coen and Goodpaster 2012 ). These data reinforce the context dependence of IMCL, with muscle and fibre type shaping lipid storage patterns and adiposity contributing substantially to between-group differences (Goodpaster et al. 2001 ). The lack of clear group separation in the DIA and VL, together with the small, muscle-specific but fibre-type independent changes, indicates that diabetes does not cause a uniform lipid overload across the musculature, but is superimposed on an existing gradient in which oxidative fibres store more lipid than glycolytic and hybrid fibres. The strong influence of BMI and the overall weak and regionally limited effects of HbA1c (with only a small isolated negative association in DIA type 1/2a fibres) support the view that adiposity and local oxidative propensity are the main drivers of IMCL in this cohort. The slight negative association between age and IMCL in DIA and EXT may reflect reduced storage capacity or a shift towards greater lipid utilisation in chronically active respiratory tissues. The 3D capillary analyses address limitations inherent to two-dimensional capillary indices. Two-dimensional measures derived from thin sections can be sensitive to fibre size, section orientation, and regional sampling, which likely contributes to heterogeneous findings in human obesity and T2DM. Conventional stereological analyses have reported reduced capillarisation, unchanged indices or subtle changes that are difficult to interpret (Groen et al. 2014 ; Mortensen et al. 2019 ). In contrast, our 3D approach, combining confocal imaging, axial calibration, skeletonisation and vector-based reconstruction (Čebašek et al. 2010 ; Janáček et al. 2011 ), showed that microvascular architecture was largely preserved, with similar LVm , LL , LSf , MeanCap , branching density, and tortuosity between groups, and significant differences limited to DIA LVf and EXT anisotropy. Selective alterations in capillary supply metrics have been described in experimental obesity and diabetes, although the underlying driver varies by model and can include changes in fibre size, capillary remodelling, or both (Poole et al. 2013 ; Gomes et al. 2017 ; Umek et al. 2021b ). The DIA-specific LVf reduction in our study, therefore, likely reflects a modest imbalance between capillary length and fibre volume in a chronically loaded, oxidative muscle rather than frank vessel loss or network disorganisation. Covariate analyses further showed age-related simplification of the capillary network (lower tortuosity in SC and VL, shorter LL in VL), a positive association between BMI and LVf / LSf in the DIA, and DIA-specific associations of HbA1c within the T2DM group (shorter LL , higher MeanCap , trend to higher LS). Together, these patterns suggest that age and adiposity modulate quantitative capillary supply, particularly in the DIA, without disrupting overall microvascular topology, and that the DIA LVf deficit is a stable diabetes-related feature across the studied age range. Across both groups, the intrinsic structural hierarchy among muscles was preserved: LVf followed the order DIA > SC ≈ EXT > VL, whereas fibre diameter exhibited the inverse pattern. This reciprocal relationship reflects a consistent design principle in skeletal muscle, whereby smaller oxidative fibres are supplied by proportionally denser capillary networks to optimise oxygen diffusion(Wüst et al. 2009 ) and parallels previous observations in healthy human muscle(Pollak et al. 2025 ) and in experimental obesity and diabetes models (Umek et al. 2021a ). Its persistence suggests that mechanisms linking myofibre structure and capillary architecture remain largely intact in this cohort. Functionally, such structural stability may contribute to the relative preservation of respiratory and postural performance in individuals with T2DM, even though microvascular impairments and endothelial dysfunction are well documented in other tissues (Groen et al. 2014 ; Sörensen et al. 2016 ). Given the chronic nature of diabetic tissue remodeling, the duration of diabetes is an important contextual exposure variable for interpreting the modest, muscle-specific phenotypes observed here. In our cohort, the recorded time since diagnosis was long (mean duration 16.5 ± 5.1 years), supporting that participants had established chronic disease; however, duration derived from clinical diagnosis is an imprecise surrogate for true glycaemic exposure, because T2DM commonly remains clinically unrecognised for years after onset of dysglycaemia (Harris et al. 1992 ). Accordingly, any inference that links “time since diagnosis” to cumulative hyperglycaemic burden (or to the magnitude of structural differences) should be made cautiously. In addition, all individuals with T2DM were managed with oral antihyperglycaemic therapy (monotherapy or combination therapy), which may itself influence skeletal muscle metabolic and microvascular endpoints; for example, metformin has been shown in insulin-resistant humans to improve insulin-mediated skeletal muscle microvascular responsiveness alongside improved muscle glucose disposal, raising the possibility that treatment could have attenuated or modified diabetes-associated microvascular and lipid-related signals in this relatively well-controlled cohort (Jahn et al. 2022 ). The main strengths of this study are its autopsy-based design, enabling simultaneous sampling of four anatomically and functionally distinct muscles from the same individuals, and the combined assessment of fibre type, IMCL, and 3D capillary architecture. This design reduces interindividual variability, a major confounder in biopsy-based research, and provides a structurally coherent view of how T2DM affects the muscular system. However, several limitations should be acknowledged. The cohort consisted solely of older male individuals, limiting generalisability to women and younger age groups, in whom sex hormones and physical activity patterns may influence muscle phenotype and vascularisation. The post-mortem, cross-sectional design precludes assessment of dynamic perfusion, mitochondrial function or muscle performance; therefore, structural preservation cannot be directly equated with preserved functional capacity. Despite controlled post-mortem intervals, subtle autolytic or fixation-related effects cannot be fully excluded. Furthermore, antidiabetic treatment was not included as a covariate and may have influenced muscle lipid content or microvascular architecture; moreover, despite age/BMI adjustment, residual confounding by adiposity cannot be excluded. Finally, molecular markers of angiogenesis, oxidative metabolism and inflammation (e.g. VEGF, PGC-1α, TNFα) were not measured, so mechanistic interpretations remain hypothesis-generating. Conclusions In this cohort of older men, T2DM was associated with muscle-specific, not generalised, structural differences across functionally diverse skeletal muscles. Fibre-type composition and mean fibre diameter were largely preserved, with larger type 1 fibres in SC and a modest increase in 2a/2x hybrid fibres in VL. IMCL was modestly higher in the SC and EXT, whereas VL and DIA showed no significant group differences, and oxidative fibres contained more IMCL than type 2x fibres in both groups. Three-dimensional capillary morphometry showed a selective reduction in DIA LVf and a small increase in anisotropy in EXT, with otherwise preserved capillary geometry and maintenance of the cross-muscle hierarchy linking fibre calibre and capillary supply. Across outcomes, adiposity and age showed stronger and more consistent associations than HbA1c. BMI emerged as the strongest predictor of IMCL and DIA capillary supply, whereas age influenced fibre size and capillary path geometry. Taken together, these findings suggest that local functional demand, oxidative phenotype, adiposity and ageing, rather than systemic metabolic stress alone, govern long-term muscle architecture in relatively well-controlled T2DM. The relative structural preservation of postural and respiratory muscles may explain the absence of pronounced functional deficits in everyday activities despite systemic metabolic dysfunction. Future work integrating 3D microvascular morphometry with lipid species profiling, perfusion imaging, mitochondrial assessments, and functional testing in the same individuals will be required to link these subtle structural differences to physiological performance and to identify mechanisms supporting muscle microvascular resilience in T2DM. Declarations Conflicts of interest Authors declare no conflicts of interest. Funding The authors gratefully acknowledge financial support from the Slovenian Research and Innovation Agency (ARIS), Slovenia, (Grant No. P3-0043 and N3-0256); Czech Science Foundation (GAČR), Czech Republic (Grant No. 22-02756K); and the Ministry of Education, Youth and Sports of the Czech Republic (MEYS) through the Large RI Project LM2023050 Czech-BioImaging as well as the European Regional Development Fund (ERDF) (Grant No. CZ.02.1.01/0.0/0.0/18_046/0016045). Author Contribution NP: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Validation; Visualization; Writing – original draft; Writing – review & editing. JJ: Conceptualization; Methodology; Software; Validation; Resources; Formal analysis; Writing – review & editing; Supervision; Project administration; Funding acquisition. FS: Conceptualization; Methodology; Validation; Writing – review & editing. EC: Conceptualization; Methodology; Validation; Resources; Writing – review & editing; Visualization; Supervision; Project administration; Funding acquisition. BR: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Data curation; Writing – review & editing; Visualization; Project administration. AA: Conceptualization; Methodology; Writing – review & editing; Supervision; Project administration. CKU: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Writing – review & editing; Visualization. DAB: Methodology; Software; Validation; Formal analysis; Investigation; Data curation; Writing – review & editing; Visualization. ŽŠ: Conceptualization; Methodology; Investigation; Writing – review & editing; Visualization. LP: Conceptualization; Methodology; Investigation; Writing – review & editing; Visualization. RTT: Conceptualization; Methodology; Formal analysis; Investigation; Data curation; Writing – review & editing; Visualization. NU: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Data curation; Writing – original draft; Writing – review & editing; Visualization; Supervision; Project administration; Funding acquisition. Acknowledgement The authors thank Majda Črnak Maasarani, Marko Slak, Andreja Vidmar for their technical assistance. Data Availability The datasets used and analysed during the present study are available from the corresponding author upon reasonable request. 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Cite Share Download PDF Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Histochemistry and Cell Biology → Version 1 posted Editorial decision: Revision requested 03 Feb, 2026 Reviews received at journal 03 Feb, 2026 Reviews received at journal 30 Jan, 2026 Reviews received at journal 22 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviewers invited by journal 19 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Submission checks completed at journal 16 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":1618182,"visible":true,"origin":"","legend":"\u003cp\u003eFibre-type proportions and fibre diameter in control and T2DM\u003c/p\u003e\n\u003cp\u003eFibre-type proportions (\u003cstrong\u003ea–d\u003c/strong\u003e) and fibre diameter (e–h) in splenius capitis (SC; \u003cstrong\u003ea,e\u003c/strong\u003e), external intercostal muscle (EXT; \u003cstrong\u003eb,f\u003c/strong\u003e), diaphragm (DIA;\u003cstrong\u003e c,g\u003c/strong\u003e), and vastus lateralis muscle (VL; \u003cstrong\u003ed,h\u003c/strong\u003e) in control (black) and T2DM (red) subjects. Boxes represent the interquartile range, centre lines denote medians, and whiskers indicate 1.5×IQR (Tukey). Individual points represent observed values. CTRL – control; T2DM – type 2 diabetes mellitus. Asterisks above the bars: * denotes \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8618826/v1/9cf8226cffc9218bbcac3b2a.png"},{"id":101256986,"identity":"49b50b22-b011-4d64-ae87-7c330ede3050","added_by":"auto","created_at":"2026-01-27 19:14:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50962227,"visible":true,"origin":"","legend":"\u003cp\u003eFibre type-specific myosin heavy chain expression and intramyocellular lipid content in four human skeletal muscles from control and T2DM subjects\u003c/p\u003e\n\u003cp\u003eExpression of MyHC isoforms Type 1, Type 2a, Type 2x, and intramyocellular lipid visualised by Sudan Black in successive transverse sections of the splenius capitis (SC), diaphragm (DIA), external intercostal (EXT), and vastus lateralis (VL) muscles. Within each group, panels are arranged left-to-right as: Type 1 fibres (BA-D5 immunoreactivity) → Type 2a fibres and 2a/2x hybrids (SC-71 immunoreactivity; hybrids identified by co-labelling with 6H1) → Type 2x fibres (6H1 immunoreactivity) → intramyocellular lipid (IMCL) staining by Sudan Black B. DIA – diaphragm, SC – splenius capitis, EXT – external intercostal, VL - vastus lateralis muscle; CTRL – control; DM – type 2 diabetes mellitus. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8618826/v1/fd0c2077a4be9b2be0414385.png"},{"id":101256984,"identity":"7a08490b-5c28-49f9-81bb-2e93ed6ba0d8","added_by":"auto","created_at":"2026-01-27 19:14:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":846672,"visible":true,"origin":"","legend":"\u003cp\u003eFibre-type–specific intramyocellular lipid content (IMCL) in control and diabetic subjects\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea–d\u003c/strong\u003e) IMCL (%) across fibre types in the splenius capitis (SC; \u003cstrong\u003ea\u003c/strong\u003e), external intercostal (EXT; \u003cstrong\u003eb\u003c/strong\u003e), diaphragm (DIA; \u003cstrong\u003ec\u003c/strong\u003e), and vastus lateralis (VL; \u003cstrong\u003ed\u003c/strong\u003e) muscles. Boxes represent the interquartile range; centre lines denote medians; whiskers indicate 1.5×IQR (Tukey). Individual points represent observed values. CTRL – control (black); T2DM – type 2 diabetes mellitus (red).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8618826/v1/0a70f9ad86a4d22b4392114a.png"},{"id":101256985,"identity":"b5cb86ef-d672-40a3-8675-65d0eabfa5c5","added_by":"auto","created_at":"2026-01-27 19:14:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10410685,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal z-stack images and 3D reconstructions of the capillary network across four human skeletal muscles from the control group.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea–d\u003c/strong\u003e) Representative single optical sections from confocal z-stacks showing merged channels from triple immunofluorescence staining: capillaries (yellow-green) and muscle fibre outlines (red). These panels illustrate the spatial distribution of capillaries relative to fibre boundaries within the image volume used for 3D analysis. (\u003cstrong\u003ee–h\u003c/strong\u003e) Volume renderings generated from the full z-stacks for the same regions, with the capillary network segmented and displayed throughout the reconstructed volume. (\u003cstrong\u003ei–l\u003c/strong\u003e) 3D renderings combining muscle fibre volumes (semi-transparent grey) with the reconstructed capillary network (red) for the same regions. Columns correspond to muscles as follows: splenius capitis (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e), external intercostal \u003cstrong\u003e(b\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e), diaphragm (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e), and vastus lateralis (\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e). Scale bar = 50 μm (applies to \u003cstrong\u003ea-d\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8618826/v1/28431ec66fe3a868be79ad50.png"},{"id":106810265,"identity":"afeb209d-613a-46fc-8a90-3d5df73a956e","added_by":"auto","created_at":"2026-04-13 16:15:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":61491096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8618826/v1/9c74fe4d-dcab-48d1-a781-74d9f7ee4d73.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fibre morphology, intramyocellular lipid content and 3D capillary architecture in human postural, respiratory and locomotor muscles in type 2 diabetes mellitus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eType 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle oxidative metabolism, including reduced expression or abundance of oxidative phosphorylation components and reduced insulin-stimulated non-oxidative glucose disposal, which together contribute to dysglycaemia (Patti et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Yokoyama et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; \u0026Ouml;hman et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Skeletal muscle is the principal site of insulin-stimulated glucose disposal in humans and comprises roughly 30\u0026ndash;40% of adult body mass, underscoring its central role in systemic insulin resistance (Baron et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Janssen et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In T2DM, changes in fibre-type composition and oxidative capacity, mitochondrial dysfunction, and impaired microvascular perfusion collectively contribute to reduced aerobic metabolism and insulin responsiveness (Oberbach et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; \u0026Ouml;hman et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, most human data are derived from superficial locomotor muscles such as the vastus lateralis, whereas postural and respiratory muscles that sustain continuous low-intensity activity for posture and breathing remain comparatively understudied.\u003c/p\u003e \u003cp\u003eIn locomotor muscles, obesity and T2DM have often been associated with a relative shift away from slow oxidative fibres and reduced oxidative enzyme activity, although findings vary across cohorts (He et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Oberbach et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Conversely, ageing is often characterised by preferential atrophy and loss of type II fibres, which can increase the relative representation of slow oxidative fibres, although the extent of this remodelling varies by muscle (Lexell et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Frontera et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Intermuscular heterogeneity is considerable; fibre-type proportions differ markedly between neck and limb muscles even within the same individual (Vikne et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cornwall and Kennedy \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In obesity and T2DM, chronic metabolic overload further promotes the accumulation of intramyocellular lipid (IMCL), a critical energy reservoir and lipid buffer (da Silva Rosa et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Seminal \u0026sup1;H-MRS studies reported an inverse association between IMCL and insulin sensitivity in humans (Krssak et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), and subsequent work implicated lipid-derived intermediates, including diacylglycerols and ceramides and their intracellular localisation, in impaired insulin signalling and mitochondrial function (Szendroedi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Perreault et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe skeletal muscle capillary network is essential for oxygen and substrate delivery and is affected by obesity, ageing, and T2DM, although human findings remain variable across studies and populations (Prior et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Early clamp studies associated reduced capillary density and a lower proportion of slow oxidative (type 1) fibres with insulin resistance (Lillioja et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), whereas later work demonstrated that insulin-mediated microvascular recruitment is blunted in obesity and T2DM (Clerk et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ageing and T2DM can also impair microvascular function, including endothelial glycocalyx alterations, with effects that depend on muscle type and physical activity level (Groen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Prior et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bosutti et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Conventional two-dimensional capillary profile counts are sensitive to section orientation and fibre size, which can bias estimates of capillary supply in architecturally complex muscles. Modern three-dimensional confocal imaging with vector-based reconstruction enables direct quantification of capillary length and network geometry (tortuosity, anisotropy, and branching density) and permits correction for axial shrinkage in thick sections, providing a less biased representation of microvascular architecture (Čebašek et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jan\u0026aacute;ček et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAgainst this background, we analysed autopsy samples from four functionally distinct muscles, (postural, respiratory and locomotor), from individuals with and without T2DM. These muscles differ in metabolic phenotype and habitual functional demands, spanning near-continuous respiratory or postural activity to intermittent locomotion(Mizuno \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Pollak et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Using myosin heavy chain (MyHC)-based fibre typing, Sudan Black B histochemistry, and 3D confocal morphometry, we sought to determine whether T2DM is associated with muscle-specific alterations in fibre-type composition, fibre size, IMCL content, and capillary network geometry, and whether the structural hierarchy among postural, respiratory, and locomotor muscles is preserved in T2DM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis comparative study was conducted on human skeletal muscle samples obtained post-mortem from individuals with T2DM and age-matched non-diabetic controls (n\u0026thinsp;=\u0026thinsp;24 per group). Four functionally distinct skeletal muscles were analysed: the postural muscle splenius capitis (SC), the respiratory muscles external intercostal (EXT) and diaphragm (DIA), and the locomotor muscle vastus lateralis (VL). Muscle samples were collected from each individual within 24 h post-mortem during routine autopsies at the Institute of Forensic Medicine, University of Ljubljana. Medical records were reviewed to confirm diabetes status and to exclude individuals with neuromuscular, other endocrine disorders, malignancy, or chronic systemic conditions likely to affect muscle phenotype, as well as prolonged immobilisation or chronic corticosteroid therapy. Only male individuals were included to eliminate potential confounding arising from sex-specific hormonal variability. Long-term glycaemic control in the T2DM group and normoglycaemia in the control group were evaluated using post-mortem HbA1c. This marker remains stable after death, providing an objective assessment of ante-mortem glycaemic status for all subjects. Medical records indicated a T2DM duration of at least 10 years in all affected individuals, and that glucose-lowering management consisted of oral antihyperglycaemic agents administered as monotherapy or combination therapy (most commonly metformin, sulfonylureas and/or DPP-4 inhibitors). All procedures were reviewed and approved by the National Medical Ethics Committee of the Republic of Slovenia (Permit Nos. : 0120\u0026ndash;536/2019/4 and 0120/536/2019/7) and were conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMuscle tissue sampling\u003c/h3\u003e\n\u003cp\u003eMuscle samples were obtained from standardised anatomical sites: SC at the mid-portion (C4 level); EXT at the sixth intercostal space (midclavicular line); DIA at the costal portion (midclavicular line); and VL at the distal third of the thigh. Each sample (\u0026asymp;\u0026thinsp;1 cm\u0026sup3;) was rapidly frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing. Serial transverse cryosections were prepared using a Leica CM1950 cryostat (Leica Microsystems GmbH, Wetzlar, Germany). Thin 10-\u0026micro;m sections were used for histochemistry and fibre typing, whereas adjacent thick 100-\u0026micro;m sections were prepared for three-dimensional (3D) capillary analysis.\u003c/p\u003e\n\u003ch3\u003eHistochemistry: intramyocellular lipids\u003c/h3\u003e\n\u003cp\u003eNeutral lipids were visualised using Sudan Black B staining (Sigma-Aldrich Corp, St. Louis, MO, USA) on 10-\u0026micro;m cryosections. Serial sections from the same sample were analysed to align fibre type and lipid measurements. Sections were equilibrated to room temperature, rinsed briefly in 70% ethanol, stained for 60 minutes in saturated Sudan Black B solution prepared in 70% ethanol, rinsed in tap water and mounted in glycerol-gelatin. The IMCL index was calculated as the percentage of fibre area occupied by Sudan Black\u0026ndash;positive lipid droplets.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry: myosin heavy chain isoform expression\u003c/h3\u003e\n\u003cp\u003eSerial 10-\u0026micro;m transverse sections were incubated with normal rabbit serum (1:40 in PBS containing 0.5% BSA) to block non-specific binding, followed by incubation with monoclonal antibodies against MyHC isoforms: BA-D5 (MyHC-1), SC-71 (MyHC-2a), and 6H1 (MyHC-2x) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), each diluted 1:100 in PBS (pH 7.4). Immunoreactivity was visualised using a diaminobenzidine (DAB)-based peroxidase detection system according to the manufacturer\u0026rsquo;s instructions. Fibres were classified as type 1, type 2a, type 2x, and hybrid type 1/2a and type 2a/2x based on established MyHC staining patterns (Schiaffino and Reggiani \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eLight microscopy and image analysis\u003c/h3\u003e\n\u003cp\u003eImages of MyHC-stained serial sections were acquired in brightfield on a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) using a 20\u0026times; Plan Fluor objective (numerical aperture 0.50) equipped with a KERN ODC 841 digital camera (KERN \u0026amp; SOHN GmbH, Balingen, Germany) and VIS Pro KERN OXM 902 software. Illumination, aperture, and exposure settings were standardised within each imaging batch to ensure reproducible conditions. High-resolution fields (5440 \u0026times; 3648 pixels) were sampled systematically, with at least three randomly selected regions per muscle, yielding a minimum of 100 analysed fibres per muscle (total area: ~8.3 \u0026times; 10⁵ \u0026micro;m\u0026sup2;). Images were saved as 24-bit RGB TIFF files (8 bits per colour channel). Image analysis was performed in Ellipse 2.081 (ViDiTo, Košice, Slovakia). Fibre type classification was conducted using the dedicated software developed by Karen et al. (Karen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which semiautomatically aligns serial sections and assigns fibre types according to myosin heavy chain expression profiles. For each muscle, fibre-type proportions (%) and fibre diameter, defined as the minimal Feret diameter, were calculated. The IMCL index was determined as the percentage of the muscle fibre cross-sectional area occupied by Sudan Black B-positive lipid droplets. All imaging and analyses were performed by a single trained evaluator blinded to group.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3D capillary network labelling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThick (100 \u0026micro;m) transverse sections were washed in cold PBST (PBS with 0.1% Triton X-100) and fixed at 4\u0026deg;C in 7% formaldehyde with 0.1% glutaraldehyde in PBST. After PBST washes, antigen retrieval was performed with 0.2% proteinase K (0.5 M Tris, pH 8.0, with EDTA) for 5 min at 37\u0026deg;C, followed by further PBST washes. The basal lamina was labelled overnight at 4\u0026deg;C with rabbit anti-collagen IV polyclonal antibody (1:200; Abcam, UK), followed by Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:500; Invitrogen, USA). Endothelial cells were labelled overnight at 4\u0026deg;C with fluorescein-labelled Griffonia simplicifolia lectin I (1:300; Vector Laboratories) and mouse monoclonal antibody F8/86 anti-von Willebrand factor (1:1000; Dako, Denmark), followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500; Invitrogen, USA). Sections were mounted in ProLong\u0026trade; Gold Antifade (Thermo Fisher Scientific, USA). Negative controls omitting primary antibodies and secondary antibodies showed no specific immunofluorescent signal; additional controls omitting lectin confirmed the absence of lectin-related background.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConfocal imaging and 3D reconstruction\u003c/h2\u003e \u003cp\u003eImage stacks were acquired using a Leica STELLARIS 8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a 40\u0026times;/1.1 water-immersion objective. Z-stacks were obtained with 1-\u0026micro;m optical steps at a resolution of 512 \u0026times; 512 pixels (pixel size 0.76 \u0026micro;m). A linear laser power Z-compensation was utilized to avoid loss of signal due to sample thickness. Sequential excitation at 488 nm (detection: 498\u0026ndash;585 nm) and 570 nm (detection: 590\u0026ndash;700 nm) was used to avoid channel crosstalk. Excitation was provided by a White Light Laser and two HyD X detectors, set to operate in photon counting mode, were used for detection. For each muscle, five randomly selected fields of view (387.5 \u0026times; 387.5 \u0026micro;m) containing at least 100 fibres in total were analysed. Image stacks were processed in Ellipse 2.081 (ViDiTo, Košice, Slovakia). Z-axis deformation was corrected by axial calibration (Jan\u0026aacute;ček et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). After segmentation, binary images were skeletonised using the six-pass Pal\u0026aacute;gyi algorithm and vectorised into 5-\u0026micro;m line segments, followed by manual refinement in Tracer3D (Cvetko et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) using a Phantom Omni haptic device (3D Systems, Rock Hill, USA). Fibre contours were traced on four planes to calculate fibre diameter, surface area and volume. Quantitative parameters included capillary length per fibre length (\u003cem\u003eLL\u003c/em\u003e), per fibre surface area (\u003cem\u003eLSf)\u003c/em\u003e, and per fibre or muscle volume (\u003cem\u003eLVf\u003c/em\u003e, \u003cem\u003eLVm\u003c/em\u003e) (Jan\u0026aacute;ček et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Cvetko et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Mean capillary length (\u003cem\u003eMeanCap\u003c/em\u003e) was computed as two-thirds of the total capillary length per unit volume divided by the number of branching points per unit volume, i.e. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:MeanCap=\\frac{2}{3}\\frac{{L}_{V}}{{N}_{V}}\\)\u003c/span\u003e\u003c/span\u003e, where \u003cem\u003eL\u003c/em\u003e\u003csub\u003e\u003cem\u003eV\u003c/em\u003e\u003c/sub\u003e is capillary length density, and \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eV\u003c/em\u003e\u003c/sub\u003e is branching-point density (Jan\u0026aacute;ček et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Tortuosity was expressed as the ratio of sum of exterior angles to the total capillary length anisotropy as the ratio of the principal eigenvalues of the structural tensor, and branching density (Br_dens) as branch points per muscle volume (Cvetko et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Eržen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). All analyses were performed by the same evaluator, blinded to group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed in Python (Statsmodels 0.14; Python Software Foundation, Wilmington, DE, USA) and GraphPad Prism 10 (GraphPad Software, LLC, San Diego, CA, USA). Normality of distributions and model residuals was assessed using the Shapiro\u0026ndash;Wilk and Jarque\u0026ndash;Bera tests. Between-group differences in demographic variables were evaluated with independent-samples t-tests. Multivariable models were prespecified and adjusted for age and BMI. For capillary parameters (\u003cem\u003eLVf\u003c/em\u003e, \u003cem\u003eLVm\u003c/em\u003e, \u003cem\u003eLL\u003c/em\u003e, \u003cem\u003eLSf\u003c/em\u003e, \u003cem\u003eMeanCap\u003c/em\u003e, Br_dens, Tortuosity, Anisotropy), linear mixed-effects models were fitted with subject as a random intercept to account for repeated fields of view within individuals. Models were fitted separately for each muscle, with fixed effects for group, age, and BMI, and the adjusted group effect was reported as β with 95% confidence intervals and two-sided p-values. Sensitivity analyses included quadratic age (age\u0026sup2;) and group-by-age terms. For \u003cem\u003eLVf\u003c/em\u003e, an additional model included mean fibre diameter as a covariate. For fibre-level outcomes (fibre-type proportions, fibre diameter, and IMCL index), subject-level means were calculated for each fibre type (1, 2a, 2x, 1/2a, and 2a/2x) within each muscle. These means were compared using two-way ANOVA (Group \u0026times; Fibre type) with Tukey post hoc tests. Because all four muscles were sampled from the same individuals, within-subject comparisons across muscles were assessed using paired t-tests and a repeated-measures mixed model of the form Outcome\u0026thinsp;~\u0026thinsp;Group \u0026times; Muscle\u0026thinsp;+\u0026thinsp;Age\u0026thinsp;+\u0026thinsp;BMI + (1|Subject) to estimate muscle-specific group effects and group-by-muscle interactions. Within the T2DM subgroup, associations between HbA1c and structural outcomes were examined using linear models adjusted for age and BMI. All tests were two-sided with α\u0026thinsp;=\u0026thinsp;0.05, and results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or adjusted regression coefficients (β) with 95% confidence intervals and corresponding p-values.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy groups\u003c/h2\u003e \u003cp\u003eAge did not differ significantly between groups (T2DM 70.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4 vs controls 69.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8 years; p\u0026thinsp;=\u0026thinsp;0.684), whereas BMI was higher in T2DM (31.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 vs 24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 kg\u0026middot;m⁻\u0026sup2;; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The mean duration of T2DM was 16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 years. In the control group, all subjects had HbA1c within the normal range (\u0026lt;\u0026thinsp;5.7% [\u0026lt;\u0026thinsp;39 mmol/mol]), while in the T2DM group, mean HbA1c was 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1% (51.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4 mmol/mol); 4 of 24 individuals (17%) had HbA1c\u0026thinsp;\u0026gt;\u0026thinsp;8.0%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMuscle fibre morphology\u003c/h2\u003e \u003cp\u003eOverall, fibre-type composition and fibre diameter were largely similar between groups, with only muscle-specific differences. Fibre-type composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u0026ndash;d) did not differ significantly between groups in SC, DIA, or EXT (all p\u0026thinsp;\u0026ge;\u0026thinsp;0.10). In the VL, the proportion of hybrid type 2a/2x fibres was higher in T2DM than in controls (p\u0026thinsp;=\u0026thinsp;0.0141), whereas all other fibre types did not differ significantly between groups. Fibre diameters did not differ significantly between groups at the whole-muscle level in any of the examined muscles, although SC displayed a borderline trend toward larger mean diameters in T2DM (p\u0026thinsp;=\u0026thinsp;0.0502). In fibre-type\u0026ndash;specific analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u0026ndash;h), the only significant group difference was observed in the SC, where type 1 fibres were larger in T2DM than in controls (p\u0026thinsp;=\u0026thinsp;0.0238); no other fibre types in the SC, DIA, EXT or VLs differed between groups (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.10).\u003c/p\u003e \u003cp\u003eAcross the combined cohort, age was positively associated with fibre diameter in several fast and hybrid fibre types, including type 1/2a, 2a, 2a/2x and 2x fibres in the EXT, and type 2a, 2a/2x and 2x fibres in the VL, as well as type 2x fibres in the DIA (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no age effect was detected in any fibre type in the SC. BMI showed no independent association with fibre diameter in any muscle. Within the T2DM subgroup, HbA1c was not associated with fibre diameter except for an isolated positive association in type 2x fibres of the EXT (p\u0026thinsp;=\u0026thinsp;0.0396).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFibre-type proportions (\u003cb\u003ea\u0026ndash;d\u003c/b\u003e) and fibre diameter (e\u0026ndash;h) in splenius capitis (SC; \u003cb\u003ea,e\u003c/b\u003e), external intercostal muscle (EXT; \u003cb\u003eb,f\u003c/b\u003e), diaphragm (DIA; \u003cb\u003ec,g\u003c/b\u003e), and vastus lateralis muscle (VL; \u003cb\u003ed,h\u003c/b\u003e) in control (black) and T2DM (red) subjects. Boxes represent the interquartile range, centre lines denote medians, and whiskers indicate 1.5\u0026times;IQR (Tukey). Individual points represent observed values. CTRL \u0026ndash; control; T2DM \u0026ndash; type 2 diabetes mellitus. Asterisks above the bars: * denotes \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExpression of MyHC isoforms Type 1, Type 2a, Type 2x, and intramyocellular lipid visualised by Sudan Black in successive transverse sections of the splenius capitis (SC), diaphragm (DIA), external intercostal (EXT), and vastus lateralis (VL) muscles. Within each group, panels are arranged left-to-right as: Type 1 fibres (BA-D5 immunoreactivity) \u0026rarr; Type 2a fibres and 2a/2x hybrids (SC-71 immunoreactivity; hybrids identified by co-labelling with 6H1) \u0026rarr; Type 2x fibres (6H1 immunoreactivity) \u0026rarr; intramyocellular lipid (IMCL) staining by Sudan Black B. DIA \u0026ndash; diaphragm, SC \u0026ndash; splenius capitis, EXT \u0026ndash; external intercostal, VL - vastus lateralis muscle; CTRL \u0026ndash; control; DM \u0026ndash; type 2 diabetes mellitus. Scale bar =\u0026thinsp;100 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIntramyocellular lipids\u003c/h2\u003e \u003cp\u003eThe IMCL index was higher in T2DM, with significant group differences in the SC (p\u0026thinsp;=\u0026thinsp;0.0128) and EXT (p\u0026thinsp;=\u0026thinsp;0.0195) muscles and no significant differences in the VL (p\u0026thinsp;=\u0026thinsp;0.111) and DIA (p\u0026thinsp;=\u0026thinsp;0.433). Across fibre types, type 1 and 2a fibres contained more IMCL than type 2x fibres, with no group-by-fibre-type interaction.\u003c/p\u003e \u003cp\u003eBMI was the strongest independent predictor of IMCL, especially in VL (β = +0.28 percentage points per kg\u0026middot;m⁻\u0026sup2;, 95% CI\u0026thinsp;+\u0026thinsp;0.19 to +\u0026thinsp;0.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while age was modestly negatively associated with IMCL in DIA and EXT (β = \u0026minus;0.017 and \u0026minus;\u0026thinsp;0.015 percentage points per year, both p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Within the T2DM subgroup, HbA1c showed no consistent association with IMCL in pooled models (p\u0026thinsp;=\u0026thinsp;0.7100), and all muscle\u0026ndash;fibre combinations were non-significant (p\u0026thinsp;\u0026ge;\u0026thinsp;0.10) except for a small isolated negative association in DIA 1/2a fibres (p\u0026thinsp;=\u0026thinsp;0.0419).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(\u003cb\u003ea\u0026ndash;d\u003c/b\u003e) IMCL (%) across fibre types in the splenius capitis (SC; \u003cb\u003ea\u003c/b\u003e), external intercostal (EXT; \u003cb\u003eb\u003c/b\u003e), diaphragm (DIA; \u003cb\u003ec\u003c/b\u003e), and vastus lateralis (VL; \u003cb\u003ed\u003c/b\u003e) muscles. Boxes represent the interquartile range; centre lines denote medians; whiskers indicate 1.5\u0026times;IQR (Tukey). Individual points represent observed values. CTRL \u0026ndash; control (black); T2DM \u0026ndash; type 2 diabetes mellitus (red).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThree-dimensional capillary architecture\u003c/h2\u003e \u003cp\u003eThree-dimensional morphometry showed a selective reduction in capillary length per fibre volume (\u003cem\u003eLVf\u003c/em\u003e) in the DIA in T2DM, with otherwise preserved capillary geometry and minor, muscle-specific differences. Descriptive values for all capillary parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and representative confocal z-stack images and 3D reconstructions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThree-dimensional capillary architecture across four human skeletal muscles in non-diabetic controls and individuals with T2DM\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDIA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEXT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLVm\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;m\u003csup\u003e⁻2\u003c/sup\u003e) \u0026times;10\u003csup\u003e⁻6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e598.75\u0026thinsp;\u0026plusmn;\u0026thinsp;122.01 601.83\u0026thinsp;\u0026plusmn;\u0026thinsp;193.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e415.32\u0026thinsp;\u0026plusmn;\u0026thinsp;70.31 418.83\u0026thinsp;\u0026plusmn;\u0026thinsp;72.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e422.02\u0026thinsp;\u0026plusmn;\u0026thinsp;81.17 438.00\u0026thinsp;\u0026plusmn;\u0026thinsp;91.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e368.89\u0026thinsp;\u0026plusmn;\u0026thinsp;74.80 435.85\u0026thinsp;\u0026plusmn;\u0026thinsp;143.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTortuosity (rad\u0026middot;\u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.21\u0026thinsp;\u0026plusmn;\u0026thinsp;8.20\u003c/p\u003e \u003cp\u003e20.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e30.78\u0026thinsp;\u0026plusmn;\u0026thinsp;11.33\u003c/p\u003e \u003cp\u003e33.37\u0026thinsp;\u0026plusmn;\u0026thinsp;12.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e26.60\u0026thinsp;\u0026plusmn;\u0026thinsp;10.27\u003c/p\u003e \u003cp\u003e19.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e42.12\u0026thinsp;\u0026plusmn;\u0026thinsp;10.30\u003c/p\u003e \u003cp\u003e42.72\u0026thinsp;\u0026plusmn;\u0026thinsp;10.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnisotropy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMeanCap\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e373.76\u0026thinsp;\u0026plusmn;\u0026thinsp;151.00 308.61\u0026thinsp;\u0026plusmn;\u0026thinsp;202.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e469.86\u0026thinsp;\u0026plusmn;\u0026thinsp;479.55 415.09\u0026thinsp;\u0026plusmn;\u0026thinsp;264.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e330.18\u0026thinsp;\u0026plusmn;\u0026thinsp;147.38 370.10\u0026thinsp;\u0026plusmn;\u0026thinsp;159.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e236.91\u0026thinsp;\u0026plusmn;\u0026thinsp;62.56 222.15\u0026thinsp;\u0026plusmn;\u0026thinsp;73.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBr_dens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLVf\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.09\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46\u003c/p\u003e \u003cp\u003e17.06\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86\u003c/p\u003e \u003cp\u003e12.58\u0026thinsp;\u0026plusmn;\u0026thinsp;4.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.10\u003c/p\u003e \u003cp\u003e13.03\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e \u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLSf\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e200.29\u0026thinsp;\u0026plusmn;\u0026thinsp;59.60 197.39\u0026thinsp;\u0026plusmn;\u0026thinsp;68.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e126.42\u0026thinsp;\u0026plusmn;\u0026thinsp;28.81 136.17\u0026thinsp;\u0026plusmn;\u0026thinsp;50.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e134.78\u0026thinsp;\u0026plusmn;\u0026thinsp;34.83 135.25\u0026thinsp;\u0026plusmn;\u0026thinsp;32.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e145.96\u0026thinsp;\u0026plusmn;\u0026thinsp;37.81 142.45\u0026thinsp;\u0026plusmn;\u0026thinsp;57.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLL\u003c/p\u003e \u003cp\u003e(\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003cp\u003eT2DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003cp\u003e3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003cp\u003e2.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e \u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCapillary network characteristics were estimated by the length of capillaries per volume of muscle tissue (\u003cem\u003eLVm\u003c/em\u003e, [\u0026micro;m \u003csup\u003e\u0026ndash;2\u003c/sup\u003e ] \u0026times; 10 \u003csup\u003e\u0026ndash;6\u003c/sup\u003e ), length of capillaries per length of muscle fibers (LL), length of capillaries per fiber surface (\u003cem\u003eLSf\u003c/em\u003e, [\u0026micro;m\u0026ndash;1 ] \u0026times;10 \u003csup\u003e\u0026ndash;4\u003c/sup\u003e ), length of capillaries per fiber volume (\u003cem\u003eLVf\u003c/em\u003e, [\u0026micro;m \u003csup\u003e\u0026ndash;2\u003c/sup\u003e ]\u0026times;10 \u003csup\u003e\u0026ndash;4\u003c/sup\u003e), Tortuosity ([rad \u0026micro;m\u003csup\u003e\u0026ndash;1\u003c/sup\u003e ] \u0026times;10 \u003csup\u003e\u0026ndash;3\u003c/sup\u003e), Anisotropy, mean capillary length (\u003cem\u003eMeanCap\u003c/em\u003e, \u0026micro;m); number of branching per muscle volume (Br_dens, [\u0026micro;m\u003csup\u003e\u0026ndash;3\u003c/sup\u003e] \u0026times;10\u003csup\u003e\u0026ndash;6\u003c/sup\u003e). Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. SC \u0026ndash; splenius capitis; DIA \u0026ndash; diaphragm; EXT \u0026ndash; external intercostal; VL \u0026ndash; vastus lateralis; CTRL \u0026ndash; control; T2DM \u0026ndash; type 2 diabetes mellitus.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLVf\u003c/em\u003e was significantly lower in DIA in T2DM than in controls (p\u0026thinsp;=\u0026thinsp;0.0115), with a non-significant trend toward lower \u003cem\u003eLVf\u003c/em\u003e in SC (p\u0026thinsp;=\u0026thinsp;0.0768) and no group differences in EXT or VL (both p\u0026thinsp;\u0026gt;\u0026thinsp;0.10). Anisotropy was modestly higher in EXT in T2DM (p\u0026thinsp;=\u0026thinsp;0.0495), whereas \u003cem\u003eLVm\u003c/em\u003e, \u003cem\u003eLL\u003c/em\u003e, \u003cem\u003eLSf\u003c/em\u003e, \u003cem\u003eMeanCap\u003c/em\u003e, tortuosity and Br_dens showed no group differences (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.10).\u003c/p\u003e \u003cp\u003eIn covariate analyses, age was associated with lower tortuosity in SC (p\u0026thinsp;=\u0026thinsp;0.0140) and VL (p\u0026thinsp;=\u0026thinsp;0.0401) and with shorter \u003cem\u003eLL\u003c/em\u003e in VL (p\u0026thinsp;=\u0026thinsp;0.0142). BMI was positively associated with \u003cem\u003eLVf\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0236) and \u003cem\u003eLSf\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0444) in DIA. Within the T2DM subgroup, HbA1c showed DIA-specific associations, being inversely related to \u003cem\u003eLL\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0367) and positively related to \u003cem\u003eMeanCap\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0170), with an additional trend for higher \u003cem\u003eLSf\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0648). For all other muscles and capillary parameters, HbA1c showed no significant associations (all p\u0026thinsp;\u0026ge;\u0026thinsp;0.13). Inclusion of Group \u0026times; Age interaction terms or quadratic Age\u0026sup2; terms did not improve model fit and did not change the \u003cem\u003eLVf\u003c/em\u003e difference between groups, confirming that the lower \u003cem\u003eLVf\u003c/em\u003e in DIA in T2DM was not explained by age distribution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(\u003cb\u003ea\u0026ndash;d\u003c/b\u003e) Representative single optical sections from confocal z-stacks showing merged channels from triple immunofluorescence staining: capillaries (yellow-green) and muscle fibre outlines (red). These panels illustrate the spatial distribution of capillaries relative to fibre boundaries within the image volume used for 3D analysis. (\u003cb\u003ee\u0026ndash;h\u003c/b\u003e) Volume renderings generated from the full z-stacks for the same regions, with the capillary network segmented and displayed throughout the reconstructed volume. (\u003cb\u003ei\u0026ndash;l\u003c/b\u003e) 3D renderings combining muscle fibre volumes (semi-transparent grey) with the reconstructed capillary network (red) for the same regions. Columns correspond to muscles as follows: splenius capitis (\u003cb\u003ea\u003c/b\u003e, \u003cb\u003ee\u003c/b\u003e, \u003cb\u003ei\u003c/b\u003e), external intercostal \u003cb\u003e(b\u003c/b\u003e, \u003cb\u003ef\u003c/b\u003e, \u003cb\u003ej\u003c/b\u003e), diaphragm (\u003cb\u003ec\u003c/b\u003e, \u003cb\u003eg\u003c/b\u003e, \u003cb\u003ek\u003c/b\u003e), and vastus lateralis (\u003cb\u003ed\u003c/b\u003e, \u003cb\u003eh\u003c/b\u003e, \u003cb\u003el\u003c/b\u003e). Scale bar =\u0026thinsp;50 \u0026micro;m (applies to \u003cb\u003ea-d\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWithin-subject hierarchy among muscles\u003c/h2\u003e \u003cp\u003eBecause all four muscles were obtained from the same individuals, paired comparisons enabled analysis of intrinsic structural hierarchy independent of interindividual variability. Across subjects, \u003cem\u003eLVf\u003c/em\u003e followed the order DIA\u0026thinsp;\u0026gt;\u0026thinsp;SC\u0026thinsp;\u0026asymp;\u0026thinsp;EXT\u0026thinsp;\u0026gt;\u0026thinsp;VL, while fibre diameter showed a contrasting pattern (VL\u0026thinsp;\u0026gt;\u0026thinsp;DIA\u0026thinsp;\u0026gt;\u0026thinsp;EXT\u0026thinsp;\u0026gt;\u0026thinsp;SC; all paired comparisons p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). No significant group-by-muscle interactions were detected, indicating that the relative ordering of capillary supply and fibre morphology across these muscles was preserved in T2DM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study analysed four functionally distinct skeletal muscles, SC, DIA, EXT, and VL, obtained from the same older men with and without T2DM, using MyHC-based fibre typing, Sudan Black B IMCL staining, and 3D confocal capillary morphometry. The T2DM group had HbA1c values generally within commonly targeted ranges and no documented advanced diabetic complications, supporting interpretation of the findings as early or moderate structural differences associated with diabetes and adiposity rather than overt advanced myopathy.\u003c/p\u003e \u003cp\u003eOverall, group differences were modest and muscle-specific, superimposed on a preserved hierarchy across muscles. Fibre-type composition and mean fibre diameter were largely maintained; IMCL was higher in SC and EXT; oxidative fibres (types 1 and 2a) contained more IMCL than type 2x fibres in both groups; and \u003cem\u003eLVf\u003c/em\u003e was selectively reduced in the DIA, while other capillary indices were largely similar between groups. Among capillary endpoints, the DIA showed the clearest group difference, a selective reduction in \u003cem\u003eLVf\u003c/em\u003e. SC showed the clearest fibre-size difference (larger type 1 fibres), EXT exhibited a small change in anisotropy, and VL showed a modest increase in 2a/2x hybrids together with BMI-associated IMCL variation. Together, the pattern of results is consistent with a dominant influence of muscle-specific functional demands and oxidative phenotype, and with adiposity as an important covariate, rather than a uniform diabetes effect across the musculature, consistent with previous morphological and microvascular studies in diabetic and obese muscle (Umek et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe preservation of fibre-type composition in the postural (SC) and respiratory (DIA, EXT) muscles, and the modest increase in 2a/2x hybrids in VL, contrasts with reports of a more pronounced shift toward faster, more glycolytic phenotypes in some locomotor muscles in obesity and T2DM (Park et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Andreassen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The larger SC type 1 fibres in T2DM may reflect chronic loading associated with higher body mass and sustained postural activity, although causal inference is limited by the cross-sectional, post-mortem design. Similar preservation or mild hypertrophy of fibres in frequently recruited muscles has been reported in models of obesity-induced insulin resistance, where increased mechanical load supports fibre growth despite systemic metabolic stress (Ato et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Umek et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eContinuously active postural and respiratory muscles are exposed to sustained mechanical and metabolic demand, which can favour anabolic signalling and mitochondrial maintenance and may oppose atrophy programmes mediated by FoxO transcription factors (Sandri et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ogasawara et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Conversely, intermittently recruited muscles, such as VL, may be more sensitive to reductions in habitual activity, particularly in older individuals. The fibre-type\u0026ndash;specific age associations observed in DIA, EXT, and VL should be interpreted as cohort-specific and may reflect selective survival of larger fibres, unmeasured activity differences, or other confounding, rather than a general ageing signature. The minimal influence of BMI and HbA1c on fibre size suggest that, in this cohort of older men, muscle-specific factors, likely related to regional workload and oxygen demand, outweigh systemic factors such as BMI in determining fibre size (Frontera et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Cameron et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Collectively, these findings indicate that diabetes does not uniformly impair myofibre morphology; continuously active muscles, such as the postural SC, retain or even modestly increase their fibre calibre, which may reflect functional adaptation to sustained contractile and metabolic demands rather than pathological hypertrophy.\u003c/p\u003e \u003cp\u003eIMCL was modestly higher in T2DM, most clearly in SC and EXT, indicating that habitual postural or respiratory activity does not preclude greater lipid storage when adiposity is higher. This is consistent with evidence that IMCL content reflects both oxidative phenotype and lipid availability, and that IMCL per se is not a direct surrogate of insulin sensitivity without information on lipid species, subcellular localisation, and turnover (Krssak et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Goodpaster et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Dub\u0026eacute; et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Coen and Goodpaster \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These data reinforce the context dependence of IMCL, with muscle and fibre type shaping lipid storage patterns and adiposity contributing substantially to between-group differences (Goodpaster et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The lack of clear group separation in the DIA and VL, together with the small, muscle-specific but fibre-type independent changes, indicates that diabetes does not cause a uniform lipid overload across the musculature, but is superimposed on an existing gradient in which oxidative fibres store more lipid than glycolytic and hybrid fibres. The strong influence of BMI and the overall weak and regionally limited effects of HbA1c (with only a small isolated negative association in DIA type 1/2a fibres) support the view that adiposity and local oxidative propensity are the main drivers of IMCL in this cohort. The slight negative association between age and IMCL in DIA and EXT may reflect reduced storage capacity or a shift towards greater lipid utilisation in chronically active respiratory tissues.\u003c/p\u003e \u003cp\u003eThe 3D capillary analyses address limitations inherent to two-dimensional capillary indices. Two-dimensional measures derived from thin sections can be sensitive to fibre size, section orientation, and regional sampling, which likely contributes to heterogeneous findings in human obesity and T2DM. Conventional stereological analyses have reported reduced capillarisation, unchanged indices or subtle changes that are difficult to interpret (Groen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mortensen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, our 3D approach, combining confocal imaging, axial calibration, skeletonisation and vector-based reconstruction (Čebašek et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jan\u0026aacute;ček et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), showed that microvascular architecture was largely preserved, with similar \u003cem\u003eLVm\u003c/em\u003e, \u003cem\u003eLL\u003c/em\u003e, \u003cem\u003eLSf\u003c/em\u003e, \u003cem\u003eMeanCap\u003c/em\u003e, branching density, and tortuosity between groups, and significant differences limited to DIA \u003cem\u003eLVf\u003c/em\u003e and EXT anisotropy.\u003c/p\u003e \u003cp\u003eSelective alterations in capillary supply metrics have been described in experimental obesity and diabetes, although the underlying driver varies by model and can include changes in fibre size, capillary remodelling, or both (Poole et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gomes et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Umek et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). The DIA-specific \u003cem\u003eLVf\u003c/em\u003e reduction in our study, therefore, likely reflects a modest imbalance between capillary length and fibre volume in a chronically loaded, oxidative muscle rather than frank vessel loss or network disorganisation. Covariate analyses further showed age-related simplification of the capillary network (lower tortuosity in SC and VL, shorter \u003cem\u003eLL\u003c/em\u003e in VL), a positive association between BMI and \u003cem\u003eLVf\u003c/em\u003e/\u003cem\u003eLSf\u003c/em\u003e in the DIA, and DIA-specific associations of HbA1c within the T2DM group (shorter \u003cem\u003eLL\u003c/em\u003e, higher \u003cem\u003eMeanCap\u003c/em\u003e, trend to higher LS). Together, these patterns suggest that age and adiposity modulate quantitative capillary supply, particularly in the DIA, without disrupting overall microvascular topology, and that the DIA \u003cem\u003eLVf\u003c/em\u003e deficit is a stable diabetes-related feature across the studied age range.\u003c/p\u003e \u003cp\u003eAcross both groups, the intrinsic structural hierarchy among muscles was preserved: \u003cem\u003eLVf\u003c/em\u003e followed the order DIA\u0026thinsp;\u0026gt;\u0026thinsp;SC\u0026thinsp;\u0026asymp;\u0026thinsp;EXT\u0026thinsp;\u0026gt;\u0026thinsp;VL, whereas fibre diameter exhibited the inverse pattern. This reciprocal relationship reflects a consistent design principle in skeletal muscle, whereby smaller oxidative fibres are supplied by proportionally denser capillary networks to optimise oxygen diffusion(W\u0026uuml;st et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and parallels previous observations in healthy human muscle(Pollak et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and in experimental obesity and diabetes models (Umek et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Its persistence suggests that mechanisms linking myofibre structure and capillary architecture remain largely intact in this cohort. Functionally, such structural stability may contribute to the relative preservation of respiratory and postural performance in individuals with T2DM, even though microvascular impairments and endothelial dysfunction are well documented in other tissues (Groen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; S\u0026ouml;rensen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the chronic nature of diabetic tissue remodeling, the duration of diabetes is an important contextual exposure variable for interpreting the modest, muscle-specific phenotypes observed here. In our cohort, the recorded time since diagnosis was long (mean duration 16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 years), supporting that participants had established chronic disease; however, duration derived from clinical diagnosis is an imprecise surrogate for true glycaemic exposure, because T2DM commonly remains clinically unrecognised for years after onset of dysglycaemia (Harris et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Accordingly, any inference that links \u0026ldquo;time since diagnosis\u0026rdquo; to cumulative hyperglycaemic burden (or to the magnitude of structural differences) should be made cautiously. In addition, all individuals with T2DM were managed with oral antihyperglycaemic therapy (monotherapy or combination therapy), which may itself influence skeletal muscle metabolic and microvascular endpoints; for example, metformin has been shown in insulin-resistant humans to improve insulin-mediated skeletal muscle microvascular responsiveness alongside improved muscle glucose disposal, raising the possibility that treatment could have attenuated or modified diabetes-associated microvascular and lipid-related signals in this relatively well-controlled cohort (Jahn et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main strengths of this study are its autopsy-based design, enabling simultaneous sampling of four anatomically and functionally distinct muscles from the same individuals, and the combined assessment of fibre type, IMCL, and 3D capillary architecture. This design reduces interindividual variability, a major confounder in biopsy-based research, and provides a structurally coherent view of how T2DM affects the muscular system. However, several limitations should be acknowledged. The cohort consisted solely of older male individuals, limiting generalisability to women and younger age groups, in whom sex hormones and physical activity patterns may influence muscle phenotype and vascularisation. The post-mortem, cross-sectional design precludes assessment of dynamic perfusion, mitochondrial function or muscle performance; therefore, structural preservation cannot be directly equated with preserved functional capacity. Despite controlled post-mortem intervals, subtle autolytic or fixation-related effects cannot be fully excluded. Furthermore, antidiabetic treatment was not included as a covariate and may have influenced muscle lipid content or microvascular architecture; moreover, despite age/BMI adjustment, residual confounding by adiposity cannot be excluded. Finally, molecular markers of angiogenesis, oxidative metabolism and inflammation (e.g. VEGF, PGC-1α, TNFα) were not measured, so mechanistic interpretations remain hypothesis-generating.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this cohort of older men, T2DM was associated with muscle-specific, not generalised, structural differences across functionally diverse skeletal muscles. Fibre-type composition and mean fibre diameter were largely preserved, with larger type 1 fibres in SC and a modest increase in 2a/2x hybrid fibres in VL. IMCL was modestly higher in the SC and EXT, whereas VL and DIA showed no significant group differences, and oxidative fibres contained more IMCL than type 2x fibres in both groups. Three-dimensional capillary morphometry showed a selective reduction in DIA \u003cem\u003eLVf\u003c/em\u003e and a small increase in anisotropy in EXT, with otherwise preserved capillary geometry and maintenance of the cross-muscle hierarchy linking fibre calibre and capillary supply. Across outcomes, adiposity and age showed stronger and more consistent associations than HbA1c. BMI emerged as the strongest predictor of IMCL and DIA capillary supply, whereas age influenced fibre size and capillary path geometry. Taken together, these findings suggest that local functional demand, oxidative phenotype, adiposity and ageing, rather than systemic metabolic stress alone, govern long-term muscle architecture in relatively well-controlled T2DM. The relative structural preservation of postural and respiratory muscles may explain the absence of pronounced functional deficits in everyday activities despite systemic metabolic dysfunction. Future work integrating 3D microvascular morphometry with lipid species profiling, perfusion imaging, mitochondrial assessments, and functional testing in the same individuals will be required to link these subtle structural differences to physiological performance and to identify mechanisms supporting muscle microvascular resilience in T2DM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eAuthors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge financial support from the Slovenian Research and Innovation Agency (ARIS), Slovenia, (Grant No. P3-0043 and N3-0256); Czech Science Foundation (GAČR), Czech Republic (Grant No. 22-02756K); and the Ministry of Education, Youth and Sports of the Czech Republic (MEYS) through the Large RI Project LM2023050 Czech-BioImaging as well as the European Regional Development Fund (ERDF) (Grant No.\u003c/p\u003e \u003cp\u003eCZ.02.1.01/0.0/0.0/18_046/0016045).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNP: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Validation; Visualization; Writing \u0026ndash; original draft; Writing \u0026ndash; review \u0026amp; editing. JJ: Conceptualization; Methodology; Software; Validation; Resources; Formal analysis; Writing \u0026ndash; review \u0026amp; editing; Supervision; Project administration; Funding acquisition. FS: Conceptualization; Methodology; Validation; Writing \u0026ndash; review \u0026amp; editing. EC: Conceptualization; Methodology; Validation; Resources; Writing \u0026ndash; review \u0026amp; editing; Visualization; Supervision; Project administration; Funding acquisition. BR: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Data curation; Writing \u0026ndash; review \u0026amp; editing; Visualization; Project administration. AA: Conceptualization; Methodology; Writing \u0026ndash; review \u0026amp; editing; Supervision; Project administration. CKU: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Writing \u0026ndash; review \u0026amp; editing; Visualization. DAB: Methodology; Software; Validation; Formal analysis; Investigation; Data curation; Writing \u0026ndash; review \u0026amp; editing; Visualization. ŽŠ: Conceptualization; Methodology; Investigation; Writing \u0026ndash; review \u0026amp; editing; Visualization. LP: Conceptualization; Methodology; Investigation; Writing \u0026ndash; review \u0026amp; editing; Visualization. RTT: Conceptualization; Methodology; Formal analysis; Investigation; Data curation; Writing \u0026ndash; review \u0026amp; editing; Visualization. NU: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Data curation; Writing \u0026ndash; original draft; Writing \u0026ndash; review \u0026amp; editing; Visualization; Supervision; Project administration; Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Majda Črnak Maasarani, Marko Slak, Andreja Vidmar for their technical assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analysed during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndreassen CS, Jensen JM, Jakobsen J, et al (2014) Striated muscle fiber size, composition, and capillary density in diabetes in relation to neuropathy and muscle strength. 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Diabetes Obes Metab 10:400\u0026ndash;407. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/J.1463-1326.2007.00716.X\u003c/span\u003e\u003cspan address=\"10.1111/J.1463-1326.2007.00716.X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Type 2 diabetes mellitus, skeletal muscle, myosin heavy chain isoforms, intramyocellular lipid, 3D capillary morphometry, metabolic myopathy.","lastPublishedDoi":"10.21203/rs.3.rs-8618826/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8618826/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn type 2 diabetes mellitus (T2DM), skeletal muscle is a major site of metabolic and microvascular dysfunction, yet most human data derive from large locomotor muscles, whereas postural and respiratory muscles remain less well characterised. We examined whether T2DM alters fibre morphology, intramyocellular lipid (IMCL) content, and 3D capillary architecture across functionally distinct muscles. Postural (splenius capitis, SC), respiratory (diaphragm, DIA; external intercostal, EXT), and locomotor (vastus lateralis, VL) muscles from adult males (T2DM vs. control, n\u0026thinsp;=\u0026thinsp;24/group) were sampled\u0026thinsp;\u0026lt;\u0026thinsp;24h post-mortem. Analysis included myosin-heavy-chain fibre typing, Sudan Black B IMCL quantification, and 3D capillary morphometry (length, tortuosity, anisotropy, branching density). Groups were age-matched (T2DM 70.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4 vs 69.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8 years; p\u0026thinsp;=\u0026thinsp;0.684), but BMI was higher in T2DM (31.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 vs 24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 kg/m\u0026sup2;; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Fibre-type profiles were similar, except for elevated 2a/2x hybrids in T2DM VL (p\u0026thinsp;=\u0026thinsp;0.014). Mean fibre diameters were preserved, though type 1 fibres were larger in T2DM SC (p\u0026thinsp;=\u0026thinsp;0.0238). IMCL was higher in T2DM SC and EXT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with non-significant differences in VL and DIA. Type 1 and 2a fibres had higher IMCL than glycolytic fibres, with no group-by-fibre-type interaction. BMI strongly predicted VL IMCL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while age associated negatively with IMCL in respiratory muscles (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Capillary length per fibre volume was selectively reduced in DIA (p\u0026thinsp;=\u0026thinsp;0.0115); other indices were preserved, except for higher anisotropy in EXT (p\u0026thinsp;=\u0026thinsp;0.0495). Overall, these functionally diverse muscles showed subtle, muscle-specific remodelling, with adiposity-linked IMCL accumulation and reduced DIA capillary supply despite largely preserved global architecture, suggesting selective metabolic and microvascular vulnerability.\u003c/p\u003e","manuscriptTitle":"Fibre morphology, intramyocellular lipid content and 3D capillary architecture in human postural, respiratory and locomotor muscles in type 2 diabetes mellitus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 19:14:13","doi":"10.21203/rs.3.rs-8618826/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-03T10:12:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-03T09:09:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T14:40:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-22T15:58:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202447505120100744202203802549174367396","date":"2026-01-22T08:46:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144817257377561152194073007094253290725","date":"2026-01-19T14:22:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283604296693342118575255293152404280774","date":"2026-01-19T12:29:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T11:54:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T10:52:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-17T04:54:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Histochemistry and Cell Biology","date":"2026-01-16T11:54:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a91d54d9-b7c8-45cf-b9a1-61c14e3156fa","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:10:39+00:00","versionOfRecord":{"articleIdentity":"rs-8618826","link":"https://doi.org/10.1007/s00418-026-02477-7","journal":{"identity":"histochemistry-and-cell-biology","isVorOnly":false,"title":"Histochemistry and Cell Biology"},"publishedOn":"2026-04-06 15:57:52","publishedOnDateReadable":"April 6th, 2026"},"versionCreatedAt":"2026-01-27 19:14:13","video":"","vorDoi":"10.1007/s00418-026-02477-7","vorDoiUrl":"https://doi.org/10.1007/s00418-026-02477-7","workflowStages":[]},"version":"v1","identity":"rs-8618826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8618826","identity":"rs-8618826","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}