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 (2022-12-18)

• After the addition of mitochondria, Ca²⁺-pulses (83 nmol/mg protein) were addedat 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 Ca^{2+ added to a 600 µL cuvette yields 30 mmol/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; RC 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. Oxygen concentration was not monitored or controlled in the cuvette during these measurements.

Labels: MiParea: Respiration, Comparative MiP;environmental MiP 

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

Regulation: Calcium