Duicu 2012 Rev Med Chir Soc Med Nat Iasi
|Duicu O, Gheorgheosu D, Mirica N, Trancota S, Cristina D, Fira-Mladinescu O, Muntean D (2012) High-resolution respirometry with multiple substrates titration in permeabilized myocardial fibers. Rev Med Chir Soc Med Nat Iasi 116:207-13.|
Abstract: The present study was purported to standardize the high-resolution respirometry technique for the analysis of oxidative phosphorylation (OXPHOS) in saponin-skinned cardiac fibers. Preparation of permeabilized myocardial fibers allows the assessment of respiratory function of the entire population of mitochondria in their normal intracellular position. Adult male rat ventricular bundles were permeabilized with saponin (50 microg/ml) and samples (1-3 mg wet weight) were transferred into the Oxygraph-2k (Oroboros Instr., Austria) chambers containing air saturated incubation buffer in order to measure Complex I (CI) and II (CII) dependent respiration. The following values (expressed in pmol O2 x s(-1) x mg(-1)) were obtained: CI_LEAK, 67.18 +/- 5.12 (CI dependent basal respiration, after glutamate and malate addition); CI_P, 247.37 +/- 49.90 (CI_OXPHOS state after ADP addition); CI_Pc, 252.036 +/- 53.13 (the intactness of the outer mitochondrial membrane assessed after cytochrome c addition); CI+II_P, 342.90 +/- 62.48 (maximum OXPHOS state obtained after succinate addition by activating convergent electron flow from CI+II into the Q junction of the electron transport system (ET-pathway); CII_P, 302.26 +/- 50.16 (CII dependent OXPHOS state obtained after the addition of a CI inhibitor rotenone, with the subsequent entry of electrons from CII only into the Q junction); ET capacity, 331.11 +/- 62.39 (uncoupled respiration). The standardized technique will allow the systematic characterization of mitochondrial respiratory dysfunction associated with several cardiac pathologies in both animals and humans.
• Keywords: Standardized technique, SUIT protocol, Cardiac pathologies
• O2k-Network Lab: RO Timisoara Muntean DM
Labels: MiParea: Respiration
Organism: Rat Tissue;cell: Heart Preparation: Permeabilized tissue
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k