This work reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a magnetized polycaprolactone... Show moreThis work reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a magnetized polycaprolactone-based composite. In situ iron oxide nanoparticle deposition on oxidized graphene yields homogeneously dispersed magnetic particles with sizes above 0.5 µm and low aspect ratio, preventing cellular internalization and toxicity. With these fillers, homogeneous magnetic composites with high magnetic content (up to 20 weight %) are obtained and processed in a solvent-free manner for the first time. MEW of magnetic composites enabled the creation of skeletal muscle-inspired design of hexagonal scaffolds with tunable fiber diameter, reconfigurable modularity, and zonal distribution of magneto-active and nonactive material, with elastic tensile deformability. External magnetic fields below 300 mT are sufficient to trigger out-of-plane reversible deformation. In vitro culture of C2C12 myoblasts on three-dimensional (3D) Matrigel/collagen/MEW scaffolds showed that microfibers guided the formation of 3D myotube architectures, and the presence of magnetic particles does not significantly affect viability or differentiation rates after 8 days. Centimeter-sized skeletal muscle constructs allowed for reversible, continued, and dynamic magneto-mechanical stimulation. Overall, these innovative microfiber scaffolds provide magnetically deformable platforms suitable for dynamic culture of skeletal muscle, offering potential for in vitro disease modeling. Show less
BackgroundBecker muscular dystrophy (BMD) is an X-linked disorder characterized by slow, progressive muscle damage and muscle weakness. Hallmarks include fibre-size variation and replacement of... Show moreBackgroundBecker muscular dystrophy (BMD) is an X-linked disorder characterized by slow, progressive muscle damage and muscle weakness. Hallmarks include fibre-size variation and replacement of skeletal muscle with fibrous and adipose tissues, after repeated cycles of regeneration. Muscle histology can detect these features, but the required biopsies are invasive, are difficult to repeat and capture only small muscle volumes. Diffusion-tensor magnetic resonance imaging (DT-MRI) is a potential non-invasive alternative that can calculate muscle fibre diameters when applied with the novel random permeable barrier model (RPBM). In this study, we assessed muscle fibre diameters using DT-MRI in BMD patients and healthy controls and compared these with histology. MethodsWe included 13 BMD patients and 9 age-matched controls, who underwent water-fat MRI and DT-MRI at multiple diffusion times, allowing RPBM parameter estimation in the lower leg muscles. Tibialis anterior muscle biopsies were taken from the contralateral leg in 6 BMD patients who underwent DT-MRI and from an additional 32 BMD patients and 15 healthy controls. Laminin and Sirius-red stainings were performed to evaluate muscle fibre morphology and fibrosis. Twelve ambulant patients from the MRI cohort underwent the North Star ambulatory assessment, and 6-min walk, rise-from-floor and 10-m run/walk functional tests. ResultsRPBM fibre diameter was significantly larger in BMD patients (P = 0.015): mean (SD) = 68.0 (25.3) mu m versus 59.4 (19.2) mu m in controls. Inter-muscle differences were also observed (P <= 0.002). Both inter- and intra-individual RPBM fibre diameter variability were similar between groups. Laminin staining agreed with the RPBM, showing larger median fibre diameters in patients than in controls: 72.5 (7.9) versus 63.2 (6.9) mu m, P = 0.006. However, despite showing similar inter-individual variation, patients showed more intra-individual fibre diameter variability than controls-mean variance (SD) = 34.2 (7.9) versus 21.4 (6.9) mu m, P < 0.001-and larger fibrosis areas: median (interquartile range) = 21.7 (5.6)% versus 14.9 (3.4)%, P < 0.001. Despite good overall agreement of RPBM and laminin fibre diameters, they were not associated in patients who underwent DT-MRI and muscle biopsy, perhaps due to lack of colocalization of DT-MRI with biopsy samples. ConclusionsDT-MRI RPBM metrics agree with histology and can quantify changes in muscle fibre size that are associated with regeneration without the need for biopsies. They therefore show promise as imaging biomarkers for muscular dystrophies. Show less
Pompe disease is an inherited metabolic myopathy caused by deficiency of acid alpha-glucosidase (GAA), resulting in lysosomal glycogen accumulation. Residual GAA enzyme activity affects disease... Show morePompe disease is an inherited metabolic myopathy caused by deficiency of acid alpha-glucosidase (GAA), resulting in lysosomal glycogen accumulation. Residual GAA enzyme activity affects disease onset and severity, although other factors, including dysregulation of cytoplasmic glycogen metabolism, are suspected to modulate the disease course. In this study, performed in mice and patient biopsies, we found elevated protein levels of enzymes involved in glucose uptake and cytoplasmic glycogen synthesis in skeletal muscle from mice with Pompe disease, including glycogenin (GYG1), glycogen synthase (GYS1), glucose transporter 4 (GLUT4), glycogen branching enzyme 1 (GBE1), and UDP-glucose pyrophosphorylase (UGP2). Expression levels were elevated before the loss of muscle mass and function. For first time, quantitative mass spectrometry in skeletal muscle biopsies from five adult patients with Pompe disease showed increased expression of GBE1 protein relative to healthy controls at the group level. Paired analysis of individual patients who responded well to treatment with enzyme replacement therapy (ERT) showed reduction of GYS1, GYG1, and GBE1 in all patients after start of ERT compared to baseline. These results indicate that metabolic changes precede muscle wasting in Pompe disease, and imply a positive feedforward loop in Pompe disease, in which lysosomal glycogen accumulation promotes cytoplasmic glycogen synthesis and glucose uptake, resulting in aggravation of the disease phenotype. Show less
Panhuis, W.I.H.; Tsaalbi-Shtylik, A.; Schonke, M.; Harmelen, V. van; Pronk, A.C.M.; Streefland, T.C.M.; ... ; Kooijman, S. 2022
DNA damage responses compete for cellular resources with metabolic pathways, but little is known about the metabolic consequences of impaired DNA replication, a process called replication stress.... Show moreDNA damage responses compete for cellular resources with metabolic pathways, but little is known about the metabolic consequences of impaired DNA replication, a process called replication stress. Here we characterized the metabolic consequences of DNA replication stress at endogenous DNA lesions by using mice with a disruption of Rev1, a translesion DNA polymerase specialized in the mutagenic replication of damaged DNA. Male and female Rev1 knockout (KO) mice were compared with wild-type (WT) mice and followed over time to study the natural course of body weight gain and glucose tolerance. Follow-up measurements were performed in female mice for in-depth metabolic characterization. Body weight and fat mass were only increased in female KO mice versus WT mice, whereas glucose intolerance and a reduction in lean mass were observed in both sexes. Female KO mice showed reduced locomotor activity while male KO mice showed increased activity as compared with their WT littermates. Further characterization of female mice revealed that lipid handling was unaffected by Rev1 deletion. An increased respiratory exchange ratio, combined with elevated plasma lactate levels and increased hepatic gluconeogenesis indicated problems with aerobic oxidation and increased reliance on anaerobic glycolysis. Supplementation with the NAD (+) precursor nicotinamide riboside to stimulate aerobic respiration failed to restore the metabolic phenotype. In conclusion, replication stress at endogenous DNA lesions induces a complex metabolic phenotype, most likely initiated by muscular metabolic dysfunction and increased dependence on anaerobic glycolysis. Nicotinamide riboside supplementation after the onset of the metabolic impairment did not rescue this phenotype.NEW & NOTEWORTHY An increasing number of DNA lesions interferes with cellular replication leading to metabolic inflexibility. We utilized Rev1 knockout mice as a model for replication stress, and show a sex-dependent metabolic phenotype, with a pronounced reduction of lean mass and glucose tolerance. These data indicate that in obesity, we may end up in an infinite loop where metabolic disturbance promotes the formation of DNA lesions, which in turn interferes with cellular replication causing further metabolic disturbances. Show less
Forsting, J.; Rehmann, R.; Rohm, M.; Guttsches, A.K.; Froeling, M.; Kan, H.E.; ... ; Schlaffke, L. 2022
Muscle diffusion tensor imaging (mDTI)-based tractography is a promising tool with which to detect subclinical changes in muscle injuries and to evaluate pathophysiology in neuromuscular diseases.... Show moreMuscle diffusion tensor imaging (mDTI)-based tractography is a promising tool with which to detect subclinical changes in muscle injuries and to evaluate pathophysiology in neuromuscular diseases. Classic region of interest (ROI)-based tractography is very time-consuming and requires an examiner with extensive experience. (Semi)automatic approaches such as volume-based tractography (VBT) can diminish this problem but its robustness and stability are unknown. The aim of the current study was to assess the performance of VBT in a multicenter setting and to evaluate semiautomatic segmentation approaches in the analysis of VBT-derived data in terms of the comparability of the outcome measures. Five traveling volunteers underwent 3-T mDTI of seven calf muscles of both legs at six different MR sites. Tract properties and diffusion metrics were calculated using VBT. Within-subject coefficients of variance (wsCVs) and intraclass correlation coefficients (ICCs) were calculated to assess the multicenter reproducibility of tract properties such as tract density (TD), mean tract length, volume and tract propagation angle, and diffusion metrics such as fractional anisotropy, mean diffusivity, axial diffusivity (lambda(1)) and radial diffusivity in traveling subjects. Furthermore, 50 individual datasets from five different centers (10 datasets per center) were pooled to assess the feasibility of VBT with manual and semiautomatic segmentation. To assess the differences of tract properties and diffusion metrics between segmentation approaches an ANOVA was performed, and ICC and Bland-Altman plots were analyzed. wsCVs and ICCs showed good reproducibility of the tract properties TD and volume, as well as diffusion metrics. ANOVA showed no significant differences between manual and semiautomatic approaches. ICCs were excellent (>= 0.992) and Bland-Altman analysis did not reveal any systemic bias between the methods. Tract properties and diffusion metrics derived from VBT showed good comparability among centers. Semiautomatic approaches revealed excellent agreement with gold standard of manual segmentation. These findings suggest that pooling data from different centers to construct a reference database for tractography results is feasible using semiautomatic segmentation approaches. Show less
In this acute intervention study, we investigated the potential benefit of ketone supplementation in humans by studying cardiac phosphocreatine to adenosine-triphosphate ratios (PCr/ATP) and... Show moreIn this acute intervention study, we investigated the potential benefit of ketone supplementation in humans by studying cardiac phosphocreatine to adenosine-triphosphate ratios (PCr/ATP) and skeletal muscle PCr recovery using phosphorus magnetic resonance spectroscopy (P-31-MRS) before and after ingestion of a ketone ester drink. We recruited 28 healthy individuals: 12 aged 23-70 years for cardiac P-31-MRS, and 16 aged 60-75 years for skeletal muscle P-31-MRS. Baseline and post-intervention resting cardiac and dynamic skeletal muscle P-31-MRS scans were performed in one visit, where 25 g of the ketone monoester, deltaG(R), was administered after the baseline scan. Administration was timed so that post-intervention P-31-MRS would take place 30 min after deltaG(R) ingestion. The deltaG(R) ketone drink was well-tolerated by all participants. In participants who provided blood samples, post-intervention blood glucose, lactate and non-esterified fatty acid concentrations decreased significantly (-28.8%, p MUCH LESS-THAN 0.001; -28.2%, p = 0.02; and -49.1%, p MUCH LESS-THAN 0.001, respectively), while levels of the ketone body D-beta-hydroxybutyrate significantly increased from mean (standard deviation) 0.7 (0.3) to 4.0 (1.1) mmol/L after 30 min (p MUCH LESS-THAN 0.001). There were no significant changes in cardiac PCr/ATP or skeletal muscle metabolic parameters between baseline and post-intervention. Acute ketone supplementation caused mild ketosis in blood, with drops in glucose, lactate, and free fatty acids; however, such changes were not associated with changes in P-31-MRS measures in the heart or in skeletal muscle. Future work may focus on the effect of longer-term ketone supplementation on tissue energetics in groups with compromised mitochondrial function. Show less
Quantitative MRI and MRS of muscle are increasingly being used to measure individual pathophysiological processes in Becker muscular dystrophy (BMD). In particular, muscle fat fraction was shown to... Show moreQuantitative MRI and MRS of muscle are increasingly being used to measure individual pathophysiological processes in Becker muscular dystrophy (BMD). In particular, muscle fat fraction was shown to be highly associated with functional tests in BMD. However, the muscle strength per unit of contractile cross-sectional area is lower in patients with BMD compared with healthy controls. This suggests that the quality of the non-fat-replaced (NFR) muscle tissue is lower than in healthy controls. Consequently, a measure that reflects changes in muscle tissue itself is needed. Here, we explore the potential of waterT(2)relaxation times, diffusion parameters and phosphorus metabolic indices as early disease markers in patients with BMD. For this purpose, we examined these measures in fat-replaced (FR) and NFR lower leg muscles in patients with BMD and compared these values with those in healthy controls. Quantitative proton MRI (three-point Dixon, multi-spin-echo and diffusion-weighted spin-echo echo planar imaging) and 2D chemical shift imaging(31)P MRS data were acquired in 24 patients with BMD (age 18.8-66.2 years) and 13 healthy controls (age 21.3-63.6 years). Muscle fat fractions, phosphorus metabolic indices, and averages and standard deviations (SDs) of the waterT(2)relaxation times and diffusion tensor imaging (DTI) parameters were assessed in six individual leg muscles. Phosphodiester levels were increased in the NFR and FR tibialis anterior, FR peroneus and FR gastrocnemius lateralis muscles. No clear pattern was visible for the other metabolic indices. IncreasedT(2)SD was found in the majority of FR muscles compared with NFR and healthy control muscles. No differences in average waterT(2)relaxation times or DTI indices were found between groups. Overall, our results indicate that primarily muscles that are further along in the disease process showed increases inT(2)heterogeneity and changes in some metabolic indices. No clear differences were found for the DTI indices between groups. Show less
Garcia-Rodriguez, R.; Hiller, M.; Jimenez-Gracia, L.; Pal, Z. van der; Balog, J.; Adamzek, K.; ... ; Spitali, P. 2020
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD... Show moreDuchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD gene expression in DMD. Additionally, in situ hybridization showed that DMD mes-senger RNA primarily localizes in the nuclear compartment, con-firming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore DMD transcription dynamics revealed a lower rate of DMD transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase in-hibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcrip-tion dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process. Show less
Background: Huntington's disease (HD) is a neurodegenerative disease with cognitive, motor and psychiatric symptoms. A toxic accumulation of misfolded mutant huntingtin protein (Htt) induces... Show moreBackground: Huntington's disease (HD) is a neurodegenerative disease with cognitive, motor and psychiatric symptoms. A toxic accumulation of misfolded mutant huntingtin protein (Htt) induces mitochondrial dysfunction, leading to a bioenergetic insufficiency in neuronal and muscle cells. Improving mitochondrial function has been proposed as an opportunity to treat HD, but it is not known how mitochondrial function in different tissues relates.Objective: We explored associations between central and peripheral mitochondrial function in a group of mild to moderate staged HD patients.Methods: We used phosphorous magnetic resonance spectroscopy (31P-MRS) to measure mitochondrial function in vivo in the calf muscle (peripheral) and the bio-energetic state in the visual cortex (central). Mitochondrial function was also assessed ex vivo in circulating peripheral blood mononuclear cells (PBMCs). Clinical function was determined by the Unified Huntington's Disease Rating Scale (UHDRS) total motor score. Pearson correlation coefficients were computed to assess the correlation between the different variables.Results: We included 23 manifest HD patients for analysis. There was no significant correlation between central bio-energetics and peripheral mitochondrial function. Central mitochondrial function at rest correlated significantly to the UHDRS total motor score (R = -0.45 and -0.48), which increased in a subgroup with the largest number of CAG repeats.Discussion: We did not observe a correlation between peripheral and central mitochondrial function. Central, but not peripheral, mitochondrial function correlated to clinical function. Muscle mitochondrial function is a promising biomarker to evaluate disease-modifying compounds that improve mitochondrial function, but Huntington researchers should use central mitochondrial function to demonstrate proof-of-pharmacology of disease-modifying compounds. Show less
