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.