Histochemical Methods for the Diagnosis of Mitochondrial Diseases

Paepe, Boél De; Bleecker, Jan De; Coster, Rudy Van

doi:10.1002/0471142905.hg1902s63

Cited by 17 publications

(15 citation statements)

References 21 publications

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“…Neighboring sections were stained for fiber-typing using monoclonal antibodies BA-F8 (type I), SC-71 (IIa), and BF-F3 (IIb), obtained from DSHB at the University of Iowa, and mitochondrial complex I/IV activities with NADH tetrazolium reductase and cytochrome C oxidase stainings. All stainings were performed following a previously published protocol (De Paepe et al, 2009). For fiber type quantification, a total of 2–4 tissue sections of 7 mice per genotype were scanned using an automated slide scanner (Zeiss Axio Scan.Z1) and analyzed by either quantitative fluorescence (gastrocnemius) or manual count (plantaris) using Image J.…”

Section: Star Methodsmentioning

confidence: 99%

CRY1/2 Selectively Repress PPARδ and Limit Exercise Capacity

et al. 2017

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SUMMARY Cellular metabolite balance and mitochondrial function are under circadian control, but the pathways connecting the molecular clock to these functions are unclear. Peroxisome proliferator activated receptor delta (PPARδ) enables preferential utilization of lipids as fuel during exercise and is a major driver of exercise endurance. We show here that the circadian repressors CRY1 and CRY2 function as co-repressors for PPARδ. Cry1−/−;Cry2−/− myotubes and muscles exhibit elevated expression of PPARδ target genes, particularly in the context of exercise. Notably, CRY1/2 seem to repress a distinct subset of PPARδ target genes in muscle compared to the co-repressor NCOR1. In vivo, genetic disruption of Cry1 and Cry2 enhances sprint exercise performance in mice. Collectively, our data demonstrate that CRY1 and CRY2 modulate exercise physiology by altering the activity of several transcription factors, including CLOCK/BMAL1 and PPARδ and thereby alter energy storage and substrate selection for energy production.

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Section: Star Methodsmentioning

confidence: 99%

CRY1/2 Selectively Repress PPARδ and Limit Exercise Capacity

et al. 2017

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“…Gastrocnemius muscle samples were embedded in optimal cutting temperature mounting medium (Histolab Products, Gothenburg, Sweden) and frozen in liquid nitrogen followed by cryosectioning and staining with haematoxylin-eosin (H-E; Histolab Products), Nile Red (Sigma-Aldrich, St Louis, MO, USA) or MitoTracker Red (Thermo Fisher Scientific, Waltham, MA, USA). Enzymatic stainings were performed as previously described [ 20 ]. For immunofluorescence, sections were incubated with primary antibodies followed by incubation with secondary antibodies (see ESM Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

Overexpression of protein kinase STK25 in mice exacerbates ectopic lipid accumulation, mitochondrial dysfunction and insulin resistance in skeletal muscle

et al. 2016

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Aims/hypothesis Understanding the molecular networks controlling ectopic lipid deposition and insulin responsiveness in skeletal muscle is essential for developing new strategies to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator of liver steatosis, hepatic lipid metabolism and whole body glucose and insulin homeostasis. Here, we assessed the role of STK25 in control of ectopic fat storage and insulin responsiveness in skeletal muscle.Methods Skeletal muscle morphology was studied by histological examination, exercise performance and insulin sensitivity were assessed by treadmill running and euglycaemichyperinsulinaemic clamp, respectively, and muscle lipid metabolism was analysed by ex vivo assays in Stk25 transgenic and wild-type mice fed a high-fat diet. Lipid accumulation and mitochondrial function were also studied in rodent myoblasts overexpressing STK25. Global quantitative phosphoproteomics was performed in skeletal muscle of Stk25 transgenic and wild-type mice fed a high-fat diet to identify potential downstream mediators of STK25 action. Results We found that overexpression of STK25 in transgenic mice fed a high-fat diet increases intramyocellular lipid accumulation, impairs skeletal muscle mitochondrial function and sarcomeric ultrastructure, and induces perimysial and endomysial fibrosis, thereby reducing endurance exercise capacity and muscle insulin sensitivity. Furthermore, we observed enhanced lipid accumulation and impaired mitochondrial function in rodent myoblasts overexpressing STK25, demonstrating an autonomous action for STK25 within cells. Global phosphoproteomic analysis revealed alterations in the total abundance and phosphorylation status of different target proteins located predominantly to mitochondria and sarcomeric contractile elements in Stk25 transgenic vs wild-type muscle, respectively, providing a possible molecular mechanism for the observed phenotype. Conclusions/interpretation STK25 emerges as a new regulator of the complex interplay between lipid storage, mitochondrial energetics and insulin action in skeletal muscle, highlighting the potential of STK25 antagonists for type 2 diabetes treatment.

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“…For light microscopic studies, muscle segments were snap-frozen in 2-methylbutane, cooled in liquid nitrogen, and sectioned at 10-μm thickness on a Leitz rotary crytome at -20°C. Sequential sections were stained with H&E and using the NADH-tetrazolium reductase procedure with standard methodologies (36). Muscle used in ultrastructural evaluations was fixed isometrically in 3% phosphate-buffered…”

Section: Methodsmentioning

confidence: 99%

A calcium channel mutant mouse model of hypokalemic periodic paralysis

Hernández‐Ochoa

et al. 2012

J. Clin. Invest.

135

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Hypokalemic periodic paralysis (HypoPP) is a familial skeletal muscle disorder that presents with recurrent episodes of severe weakness lasting hours to days associated with reduced serum potassium (K + ). HypoPP is genetically heterogeneous, with missense mutations of a calcium channel (Ca V 1.1) or a sodium channel (Na V 1.4) accounting for 60% and 20% of cases, respectively. The mechanistic link between Ca V 1.1 mutations and the ictal loss of muscle excitability during an attack of weakness in HypoPP is unknown. To address this question, we developed a mouse model for HypoPP with a targeted Ca V 1.1 R528H mutation. The Ca v 1.1 R528H mice had a HypoPP phenotype for which low K + challenge produced a paradoxical depolarization of the resting potential, loss of muscle excitability, and weakness. A vacuolar myopathy with dilated transverse tubules and disruption of the triad junctions impaired Ca 2+ release and likely contributed to the mild permanent weakness. Fibers from the Ca V 1.1 R528H mouse had a small anomalous inward current at the resting potential, similar to our observations in the Na V 1.4 R669H HypoPP mouse model. This "gating pore current" may be a common mechanism for paradoxical depolarization and susceptibility to HypoPP arising from missense mutations in the S4 voltage sensor of either calcium or sodium channels.

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Histochemical Methods for the Diagnosis of Mitochondrial Diseases

Cited by 17 publications

References 21 publications

CRY1/2 Selectively Repress PPARδ and Limit Exercise Capacity

CRY1/2 Selectively Repress PPARδ and Limit Exercise Capacity

Overexpression of protein kinase STK25 in mice exacerbates ectopic lipid accumulation, mitochondrial dysfunction and insulin resistance in skeletal muscle

A calcium channel mutant mouse model of hypokalemic periodic paralysis

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