Talk:Zuccolotto-dos-Reis 2022 Abstract Bioblast

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Zuccolotto-dos-Reis Felippe H
Zuccolotto-dos-Reis Felippe H, Escarso SHA, Araujo JS, Espreafico EM, Alberici LC, Sobreira CFR (2022) Acetyl CoA driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease. Bioblast 2022: BEC Inaugural Conference.

Link: Bioblast 2022: BEC Inaugural Conference

Zuccolotto-dos-Reis Felippe H, Escarso SHA, Araujo JS, Espreafico EM, Alberici Luciane Carla, Sobreira CFR (2022)

Event: Bioblast 2022

The procedure of freezing human biopsies is common in clinical practice as a form of storage [1,2,3]. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function [4]. To contribute to the laboratorial diagnosis of mitochondrial diseases, this study sought to develop an O2 consumption protocol to measure the whole electron transfer system (ETS) activity in homogenates of frozen skeletal muscle biopsies.

We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; and five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment.

Transmission electron microscopy confirmed that most mitochondria presented areas of membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate, and antimycin. This protocol of acetyl-CoA driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETS abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found a significant correlation between O2 consumption rates and enzyme activity assays of individual ETS complexes.

Here we present a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to the diagnosis of mitochondrial diseases in humans.

Keywords: frozen skeletal muscle biopsy; acetyl-CoA driven respiration (ACoAR); high-resolution respirometry; electron transfer system; mitochondrial diseases

  1. Gorman GS, Chinnery PF, DiMauro S, Hirano M, Koga Y, McFarland R, Suomalainen A, Thorburn DR, Zeviani M, Turnbull DM (2012) Mitochondrial diseases. https://doi.org/10.1038/nrdp.2016.80
  2. Kirby DM, Thorburn DR, Turnbull DM, Taylor RW (2007) Biochemical assays of respiratory chain Complex activity. https://doi.org/10.1016/S0091-679X(06)80004-X
  3. Thorburn DR, Chow CW, Kirby DM (2004) Respiratory chain enzyme analysis in muscle and liver. https://doi.org/10.1016/j.mito.2004.07.003
  4. Larsen S, Wright-Paradis C, Gnaiger E, Helge JW, Boushel R (2012) Cryopreservation of human skeletal muscle impairs mitochondrial function. https://pubmed.ncbi.nlm.nih.gov/22825783/


Affiliations

Zuccolotto-dos-Reis FH1*, Escarso SHA1, Araujo JS2, Espreafico EM2, Alberici LC3, Sobreira CFR1
  1. Department of Neurosciences, Division of Neurology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil, Av. Bandeirantes, 3900, Monte Alegre, Ribeirão Preto, SP, 14049-900 - [email protected]
  2. Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil, Av. Bandeirantes, 3900, Monte Alegre, Ribeirão Preto, SP, 14049-900
  3. Department of BioMolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil, Av. do Café s/n, Monte Alegre, Ribeirão Preto, SP, 14040-900


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Comment by the MitoFit Preprints Editors: This abstract is in the most part a copy from the following reference:
  • Zuccolotto-Dos-Reis FH, Escarso SHA, Araujo JS, Espreafico EM, Alberici LC, Sobreira CFDR. Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease. https://doi.org/10.1111/eci.13574
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