Biology subjects

Doig, C. L.

Publications and source records attributed to Doig, C. L..

3 recordsLinked to original sources

bioRxiv2023

Spatial proteomics reveals heterogeneity in neural markers underpinning high-fat diet-induced myopathy in male mice.

Metabolic dysfunction in skeletal muscle disturbs its contractile response as well as its innervation and vascular networks. The molecular drivers responsible for affecting decline in function remain poorly defined. To provide insight and locate these, we mapped changes in the spatial proteome occurring as a result of impaired metabolic health. We exposed male mice (C57/BL6J) to diet induced obesity to investigate the impairment of muscle metabolic function and myopathy. We conducted digital spatial profiling using the NanoString GeoMx(R) platform on recovered skeletal muscle (tibialis anterior) comparing it to standard fed controls. Digital spatial profiling revealed areas with shifts in the contractile protein desmin and CD31 expression, a marker of tissue stress and cellular maladaptation. We find increased expression of proinflammatory markers were identified in areas of elevated Desmin in obese samples compared to controls. Our data suggest a dietary-driven relationship between the spatial abundance of the sarcomere protein desmin and the influx of neural and inflammatory mediators to muscle. This supports the concept of pro-inflammatory events underpinning the muscle metabolic dysfunction associated with chronic non-communicative diseases such as type 2 diabetes, metabolic syndrome, and chronic obstructive pulmonary disorder.

physiology↗

bioRxiv2022

Rapid isolation of respiring skeletal muscle mitochondria using nitrogen cavitation.

BackgroundMethods of isolating mitochondria commonly utilize mechanical force and shear stress to homogenize tissue followed by purification by multiple rounds of ultracentrifugation. Existing protocols can be time-consuming with some physically impairing integrity of the sensitive mitochondrial double membrane. MethodsHere, we describe a method for the recovery of intact, respiring mitochondria from murine skeletal muscle tissue and cell lines using nitrogen cavitation in combination with differential centrifugation. ResultsThis protocol results in high yield, pure and respiring mitochondria without the need for purification gradients or ultracentrifugation. The protocol takes under an hour and requires limited specialised equipment. Our methodology is successful in extracting mitochondria of both cell extracts and skeletal muscle tissue. This represents an improved yield in comparison to many of the existing methods. Western blotting and electron microscopy demonstrate an enrichment of mitochondria with their ultrastructure well-preserved and an absence of contamination from cytoplasmic or nuclear fractions. Using respirometry analysis we show that mitochondria extracted from the murine skeletal muscle cell lines and tibialis anterior have an appropriate respiratory control ratio. These measures are indicative of healthy coupled mitochondria. ConclusionOur method successfully demonstrates the rapid isolation of functional mitochondria and will benefit researchers studying mitochondrial bioenergetics as well as providing greater throughput and application for time-sensitive assays.

molecular biology↗

bioRxiv2019

Induction of the nicotinamide riboside kinase NAD+ salvage pathway in skeletal muscle of H6PDH KO mice

BackgroundHexose-6-Phosphate Dehydrogenase (H6PDH) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PDH with 11{beta}-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PDH in ER/SR NAD(P)(H) homeostasis is incomplete. Muscle specific lack of H6PDH results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PDH deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation.\n\nMethodsWe analysed skeletal muscle from H6PDH knockout (H6PDKO), H6PDH and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subject to biochemical analysis including NAD(H) measurement, LC/MS based metabolomics, Western immunoblotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO.\n\nResultsH6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or Acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability.\n\nConclusionsThese findings suggest a complex metabolic response to changes to muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PDH. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.

physiology↗