Picard 2008 Am J Physiol Regul Integr Comp Physiol

Picard M, Csukly K, Robillard ME, Godin R, Ascah A, Bourcier-Lucas C, Burelle Y (2008) Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles. Am J Physiol Regul Integr Comp Physiol 295:R659-68. doi: 10.1152/ajpregu.90357.2008

» PMID: 18495829 Open Access

Picard M, Csukly K, Robillard ME, Godin R, Ascah A, Bourcier-Lucas C, Burelle Y (2008) Am J Physiol Regul Integr Comp Physiol

Abstract: This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca²⁺-induced mitochondrial Ca²⁺ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124±47 vs. 30.4±6.8 pmol Ca²⁺/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121±60 vs. 40±10 nmol Ca²⁺/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca²⁺ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56±4 vs. 111±11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca²⁺-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca²⁺ dynamics between fiber types.

• Bioblast editor: Gnaiger E

Selected quotes and comments

Communicated by Gnaiger E and Cecatto C (2022-12-23) 

• After the addition of mitochondria, Ca²⁺-pulses (83 nmol/mg protein) were added at 2-min intervals until mitochondrial Ca²⁺-release caused by opening of the PTP was observed. CRC was calculated as the cumulative amount of Ca²⁺ taken by mitochondria before Ca²⁺-release.

Comment: Calcium retention capacity CRC is in this approach not 'the cumulative amount of Ca²⁺ taken by mitochondria', if no correction is made for an increase of Ca²⁺ concentration in the medium before opening of the PTP.

• After the addition of fibers and respiratory substrates, a single pulse of 20 nmol of Ca²⁺ was added. CRC was taken as the total amount of Ca²⁺ accumulated by mitochondria before Ca²⁺ release caused by PTP opening. CRC values were expressed per milligram of dry fiber weight and by unit of CS. [Ca²⁺] in the cuvette was calculated from a standard curve relating [Ca²⁺] to the fluorescence of Ca-green.

Comment: CRC obtain in this approach is actually calcium uptake capacity. 20 nmol Ca²⁺ added to a 600 µL cuvette yields 33 µmol/L Ca²⁺. When [Ca²⁺] subtracted from this initial concentration is subtracted, then actual calcium uptake capacity is obtained. Even when expressed by a mitochondrial marker (CS), these results cannot be directly compared with the results reported for isolated mitochondria.

• Mitochondrial H₂O₂ production was measured in permeabilized fiber bundles and in isolated mitochondria with the fluorescent probe Amplex red (20 µM; excitation-emission, 563–587 nm). Fiber bundles (0.3–1.0 mg dry weight) were incubated at 37 °C in a quartz microcuvette with continuous magnetic stirring in 600 µL of buffer Z (in mM: 110 K-MES, 35 KCl, 1 EGTA, 5 K₂HPO₄, 3 MgCl₂6H₂O, and 0.5 mg/mL BSA, pH 7.3 at 4 °C) supplemented with 1.2 U/mL horseradish peroxidase as described previously (1). Isolated mitochondria were incubated at a final concentration of 0.1 mg/mL in 2 mL of CRC buffer. The rate of H₂O₂ production was monitored before and after the addition of glutamate-malate (5:2.5 mM) or succinate (5 mM) and rotenone (1 µM). Rate of H₂O₂ production was calculated from a standard curve established in the same experimental conditions, except that fibers or isolated mitochondria were absent.

Comment: The incubation media differed between fibers and imt (buffer Z and CRC buffer, respectively; CRC buffer [in mM]: 250 sucrose, 10 MOPS, 0.005 EGTA, 10 Pi-Tris, pH 7.3). The quality of incubation media exerts an influence on H₂O₂ production in mt-preparations, which must be assessed for proper comparison of pfi and imt. For comparison, results must be expressed in comparable units, i.e. per CS marker in both preparations. Oxygen concentration was not monitored or controlled in the cuvette during these measurements.

• .. the respiratory response of ghost fibers {extraction of contractile filaments} to the sequential addition of substrates and inhibitors was similar to that observed in permeabilized bundles, suggesting that disruption of myofilaments had little effect on mitochondria.

• the production of H2O2 per unit of the mitochondrial marker enzyme CS was two- to threefold higher in fibers from WG compared with those of the Sol (Fig. 6A), despite the fact that the WG displayed the lowest sensitivity to PTP opening. A similar difference between the two muscles was also observed in isolated mitochondrial preparations (Fig. 6B).

Comment: There is no indication of 'strikingly different dynamics' emphasized in a later publication (Picard 2011 PLoS One).

• Extraction of myosin by incubation in presence of high concentrations of KCl has been used previously to study mitochondrial function. Electron microscopy and confocal imaging experiments have shown that this procedure effectively removes myosin and entirely disrupts contractile proteins, while mitochondria remain attached to the cytoskeleton, retain their original localization within the myocytes, and maintain normal respiratory parameters (2, 42, 43).

• Preparation of permeabilized muscle fibers: After this permeabilization procedure, fiber bundles were washed three times for 10 min in buffer B (in mM: 2.77 CaK₂EGTA, 7.23 K₂EGTA, 1.38 MgCl₂, 3.0 K₂HPO₄, 0.5 dithiothreitol, 20 imidazole, 100 K-MES, 20 taurine, pH 7.3 at 4°C) supplemented with BSA (2mg/ml). Fiber bundles were kept on ice in the same solution until respirometry analysis. Preparation of permeabilized “ghost” muscle fibers: Fibers were [...] washed three times in low-EGTA sucrose buffer (in mM: 250 sucrose, 10 Tris base, and 0.1 EGTA, pH 7.4), and kept on ice until use for Ca²⁺-induced PTP opening assays.

Comment: The different EGTA concentrations to maintain the fibers could cause differences in the behavior shown in figure 3a.

Labels: MiParea: Respiration, Comparative MiP;environmental MiP 

Stress:Permeability transition  Organism: Rat  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue, Isolated mitochondria 

Regulation: Calcium