An optimized protocol for coupling oxygen consumption rates with β-oxidation in isolated mitochondria from mouse soleus

Sánchez-González, Cristina; Formentini, Laura

doi:10.1016/j.xpro.2021.100735

Cited by 6 publications

(3 citation statements)

References 27 publications

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“…The heart, liver, brain, and soleus and gastrocnemius muscles were dissected after removing the animals’ hair, skin and fascia, as in ref. 63 .…”

Section: Methodsmentioning

confidence: 99%

An ETFDH-driven metabolon supports OXPHOS efficiency in skeletal muscle by regulating coenzyme Q homeostasis

Herrero Martín,

Salegi Ansa,

Álvarez-Rivera

et al. 2024

Nat Metab

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Coenzyme Q (Q) is a key lipid electron transporter, but several aspects of its biosynthesis and redox homeostasis remain undefined. Various flavoproteins reduce ubiquinone (oxidized form of Q) to ubiquinol (QH2); however, in eukaryotes, only oxidative phosphorylation (OXPHOS) complex III (CIII) oxidizes QH2 to Q. The mechanism of action of CIII is still debated. Herein, we show that the Q reductase electron-transfer flavoprotein dehydrogenase (ETFDH) is essential for CIII activity in skeletal muscle. We identify a complex (comprising ETFDH, CIII and the Q-biosynthesis regulator COQ2) that directs electrons from lipid substrates to the respiratory chain, thereby reducing electron leaks and reactive oxygen species production. This metabolon maintains total Q levels, minimizes QH2-reductive stress and improves OXPHOS efficiency. Muscle-specific Etfdh−/− mice develop myopathy due to CIII dysfunction, indicating that ETFDH is a required OXPHOS component and a potential therapeutic target for mitochondrial redox medicine.

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“…The heart, liver, brain, and soleus and gastrocnemius muscles were dissected after removing the animals’ hair, skin and fascia, as in ref. 63 .…”

Section: Methodsmentioning

confidence: 99%

An ETFDH-driven metabolon supports OXPHOS efficiency in skeletal muscle by regulating coenzyme Q homeostasis

Herrero Martín,

Salegi Ansa,

Álvarez-Rivera

et al. 2024

Nat Metab

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show abstract

“…Gastrocnemius muscles were dissected from hind limbs after hair, skin, and surrounding fascia removal. Achilles tendon was cut as close as possible to the knee and muscles separated without WAT deposits [ 64 ]. Rapidly after dissection, muscles were placed in p24 plates with 500 µl of complete DMEM (previously maintained in hypoxia for 24 h) and cultured in hypoxia (0.5% O 2 ).…”

Section: Methodsmentioning

confidence: 99%

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

et al. 2022

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Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.

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“…We rationalized that digestion of the proteins that link mitochondria to LDs could enhance the detachment of PDM from lipid droplets, an approach that would require addressing the potential damage induced by the proteolytic activity. Accordingly, several studies in oxidative tissues, including skeletal and cardiac muscle, used protease treatments to disperse the tissue in the process of mitochondrial isolation without impairing their oxidative function (Kras et al, 2016;Lai et al, 2019;S ánchez-Gonz ález & Formentini, 2021).…”

Section: Introductionmentioning

confidence: 99%

Mitochondria isolated from lipid droplets of white adipose tissue reveal functional differences based on lipid droplet size

Brownstein,

Veliova,

Acin-Perez

et al. 2023

Life Sci. Alliance

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Recent studies in brown adipose tissue (BAT) described a unique subpopulation of mitochondria bound to lipid droplets (LDs), which were termed PeriDroplet Mitochondria (PDM). PDM can be isolated from BAT by differential centrifugation and salt washes. Contrary to BAT, this approach has so far not led to the successful isolation of PDM from white adipose tissue (WAT). Here, we developed a method to isolate PDM from WAT with high yield and purity by an optimized proteolytic treatment that preserves the respiratory function of mitochondria. Using this approach, we show that, contrary to BAT, WAT PDM have lower respiratory and ATP synthesis capacities compared with WAT cytoplasmic mitochondria (CM). Furthermore, by isolating PDM from LDs of different sizes, we found a negative correlation between LD size and the respiratory capacity of their PDM in WAT. Thus, our new isolation method reveals tissue-specific characteristics of PDM and establishes the existence of heterogeneity in PDM function determined by LD size.

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An optimized protocol for coupling oxygen consumption rates with β-oxidation in isolated mitochondria from mouse soleus

Cited by 6 publications

References 27 publications

An ETFDH-driven metabolon supports OXPHOS efficiency in skeletal muscle by regulating coenzyme Q homeostasis

An ETFDH-driven metabolon supports OXPHOS efficiency in skeletal muscle by regulating coenzyme Q homeostasis

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

Mitochondria isolated from lipid droplets of white adipose tissue reveal functional differences based on lipid droplet size

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