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, Ca(2+) 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 Ca(2+) challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H(2)O(2) 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.

Figure 1. Simplified schematic representation of the presumed structural effect of preparing isolated mitochondria and permeabilized myofibers from skeletal muscle.

Mitochondria exhibit a three-dimensional reticular morphology in intact myofibers and physically interact with surrounding mitochondria and other structures (e.g., sarcoplasmic reticulum (SR), cytoskeleton, lipid droplets). In isolated mitochondria, only a fraction of total mitochondria are recovered, which are morphologically distinct from that of intact muscle and lack functional interactions with surrounding cellular structures. In permeabilized myofibers, the sarcolemma is selectively and partially dissolved and the cytoplasm is washed out (for specific depiction of membrane permeabilization process, see [22]). This method provides access through diffusion to mitochondria located within the cells, where the intracellular cytoarchitectural environment is preserved and >95% of mitochondria are present.

Figure 2. Altered mitochondrial morphology and increased mPTP sensitivity to Ca²⁺ in isolated mitochondria.

(A) Three-dimensional reconstruction of isolated mitochondria and (B) permeabilized myofiber incubated with MitoTracker Red and imaged using confocal microscopy (see also Video S1 and Video S2). (C) Representative traces of calcium uptake by mitochondria within permeabilized myofibers and isolated mitochondria. Vertical dotted lines indicate time taken as mPTP opening. (D) Quantification of time to mPTP opening in mitochondria from both types of preparations. (E) Quantification of calcium retention capacity (CRC) calculated as the amount of Ca²⁺ taken by mitochondria before opening of the mPTP. N = 8 animals per group, values are means ± s.e.m. ** = p<0.01.

Figure 3. Quantitative and qualitative alterations of mitochondrial respiration in isolated mitochondria.

(A) Schematic diagram of the relevant mitochondrial components involved in mitochondrial respiration and antioxidant defenses. represent the sequence and site of action of each substrate added to the respirometry assay. Bold italicized items are matrix components which may be partially lost during mitochondrial isolation. (B) Mitochondrial oxygen consumption with sequential substrate addition protocol in both preparations, expressed relative to permeabilized myofibers. Baseline – permeabilized myofibers or isolated mitochondria without substrate; GM – Glutamate-Malate; State 3 GM – GM + ADP; State 3 GMS – State 3 GM + Succinate; TMPD – State 3 GMS + Antimycin A (AA) + TMPD + Ascorbate. (C) Respiratory control ratio (RCR) for both preparations. (D) Mitochondrial respiration ratios calculated for both preparations, representing the relative activity of complexes I, II and IV. Abbreviations: I, II, III, IV, IV – electron transport chain complexes I to IV, and ATP synthase (V); NAD⁺ – nicotinamide adenine nucleotide; NADH – reduced NAD⁺; TCA – tricarboxilic acid cycle; MnSOD – manganese superoxide dismutase; GPx – glutathione peroxidase; TPx – thioredoxin peroxidase. N = 8 animals per group, values are means ± s.e.m. * = p<0.05 ** = p<0.01.

Figure 4. Altered stoichiometry of electron transport chain and ATP synthase complexes in isolated mitochondria.

(A) Quantification of Western blots probed for the relative abundance of representative subunits of each of the four mitochondrial electron transport chain complexes (I–IV) and the ATP synthase (V). Mean optical density values are expressed relative to complex I within a given preparation. (B) Representative Western blots from whole muscle and purified isolated mitochondria. N = 8 animals per group, values are means ± s.e.m. * = p<0.05 ** = p<0.01.

Figure 5. Increased mitochondrial reactive oxygen species production in isolated mitochondria.

(A) H₂O₂ release by mitochondria of both preparations during different activation states, normalized to mitochondrial content (COX activity) and expressed relative to permeabilized myofibers values. (B) H₂O₂ release by mitochondria, normalized to oxygen consumption and expressed relative to permeabilized myofibers values. (C) Effect of adding antimycin A (AA) on maximal H₂O₂ production per O₂ flux (JO₂). (D) Effect of adding increasing amount of ADP on H₂O₂ production. N = 8 animals per group, values are means ± s.e.m. * = p<0.05 ** = p<0.01.

Figure 6. Summary of the functional impact of mitochondrial isolation.

Average values in isolated mitochondria are expressed relative to values in permeabilized myofibers. Shown are values under state 2 respiration with glutamate-malate (GM) and normalized for mitochondrial content estimated with COX activity. Relative to permeabilized myofibers, isolated mitochondria exhibit marked sensitivity to mPTP opening, expressed as the inverse of time required for mPTP opening upon Ca²⁺ challenge. Oxygen consumption under state 2 respiration normalized for mitochondrial content is significantly lower in isolated mitochondria than in permeabilized myofibers. Finally, reactive oxygen species production (measured as H2O2 release) normalized to O2 flux is higher than in permeabilized myofibers.