Mitochondrial Structure and Function Are Disrupted by Standard Isolation Methods

Abstract

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca2+ handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca2+ challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H2O2 production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.

Alterations in intrinsic mitochondrial function with aging are fiber type‐specific and do not explain differential atrophy between muscles

“…One of the primary benefits of using the permeabilized myofiber technique is the preservation of mitochondrial structure and content with this method (Saks et al, 1998;Picard et al, 2011). In addition, we have also found that routine mitochondrial isolation methods markedly exaggerate the degree of mitochondrial dysfunction in severely atrophied aging muscle (Picard et al, 2010).…”

Proline as a fuel for insect flight: enhancing carbohydrate oxidation in hymenopterans

“…However, the interpretation of the results obtained by Campbell et al [24] presents some difficulties. First, it is possible that isolated mitochondria and permeabilized muscle fibre preparations show different functional properties as reported in vertebrates [61]. A recent study on mosquitoes does not support this possibility as the two preparations responded similarly to various substrates, including proline [48].…”

Metformin-treated patients with type 2 diabetes have normal mitochondrial complex I respiration

“…Studies carried out on rat muscle (permeabilised fibres and isolated mitochondria) or liver or in cell lines may not be representative of skeletal muscle from human patients with type 2 diabetes. Recently, it has also been reported that fundamental differences exist in the respiratory responses of isolated mitochondria and permeabilised fibres [23].…”