Bibliografisk note

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A major finding of the present study is that most damaged muscle fibers in McArdle mice were those with higher glycogen and mitochondrial content. In this effect, although a causative association between the amount of glycogen content, on the one hand, and the degree of mitochondrial network disruption, on the other, cannot be inferred, previous studies in McArdle mice have shown that the large muscle glycogen depots that characterize this disease cause myofibrils to tear, change direction, and split from adjacent myofibrils, while T-tubules appear misaligned to myofibrils [21]. These previous results suggest that glycogen accumulation alters the overall ultrastructure of the muscle fibers. In the present study, we have further observed that most damaged fibers presented with a remarkable disruption of intermediate and actin microfilaments and microtubules, along with a disturbance of the mitochondrial network. In this effect, it is well established that the location of mitochondria within the cell as well as the arrangement of the network formed by these organelles is highly dependent on the cytoskeleton structure [30,31,37]. In addition, the cytoskeleton, more specifically βII-tubulin, plays an important role in regulating the local diffusion of ADP through the VDAC pore in the mitochondrial outer membrane in order to control the kinetics of ATP synthesis [38,39]. Furthermore, tubulin has been recognized as an inherent mitochondrial membrane component that acts as a regulator of the mitochondrial permeability transition pore and thereby plays a role in apoptosis, supporting mitochondrial membrane potential and regulating OXPHOS [40]. Given all of the above, the large glycogen deposits inside the skeletal muscle fibers of mice and patients are likely to alter the overall structure of these cells (including the different cytoskeleton components) and subsequently the localization and network arrangement of mitochondria within the cell, thereby potentially affecting, at least partly, the function of these organelles. Additionally, and probably as a consequence of mitochondrial network disruption, we also observed an altered subcellular localization of the different proteins involved in mitochondrial fission (Drp1 and Fis 1) and fusion (Mfn2 and Opa1), as well as lower levels of Mfn2 in the skeletal muscle of McArdle mice, possibly leading to lower mitochondrial fusion. Furthermore, Drp1 frequently co-localized with isolated (and enlarged) mitochondria, thereby suggesting that mitochondrial fission was actively involved in the separation of potentially damaged mitochondria from the network. Besides their negative effect on mitochondrial dynamics, changes in the cellular distribution of the different fission/fusion proteins might also affect mitochondrial integrity, membrane potential and oxidative metabolism. In this regard, it has been observed that Opa1 is involved in maintaining the shape of mitochondrial cristae by stabilizing respiratory chain super-complexes [ 41–43], while loss of Mfn2 function causes metabolic alterations in mitochondria (i.e., lower membrane potential and cellular oxygen consumption, as well as depressed substrate oxidation) with the absence of both mitofusins Opa1 and Mfn2 leading to severe mtDNA depletion [33]. Consistent with this, we observed an impaired mitochondrial content and biogenesis as well as a decrease in OXPHOS complex proteins and activities in the skeletal muscle of McArdle mice. Additionally, we report the presence of c-shaped or “donut” like mitochondria, a phenomenon that has been previously associated with oxidative stress and/or impaired energy production [29,44]; in this regard, the observed increased levels of the Nrf2 protein in the skeletal muscle of McArdle mice might represent a compensatory adaptation aiming at reducing the oxidative stress caused by mitochondrial metabolism impairment

Funding Information:
The present study was funded by grants received from the Fondo de Investigaciones Sanitarias (FIS, PI17/02052, PI18/00139, PI19/01313, and PI20/00645) and cofunded by ‘Fondos FEDER’. Gisela Nogales-Gadea and Carmen Fiuza-Luces are supported by the Miguel Servet research contracts (ISCIII CD14/00032 and CP18/00034, respectively and cofounded by Fondos FEDER′). Research by Pedro L. Valenzuela is funded by a postdoctoral contract granted by Instituto de Salud Carlos III (Sara Borrell, CD21/00138). Monica Villarreal Salazar is supported by the Mexican National Council for Science and Technology (CONACYT).

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