Isolated Mitochondria State after Myocardial Ischemia-Reperfusion Injury and Cardioprotection: Analysis by Flow Cytometry

Abstract

Rationale: Mitochondria are key organelles involved in cell survival and death during the acute phenomena of myocardial ischemia-reperfusion (i.e., myocardial infarction). To investigate the functions of isolated mitochondria such as calcium retention capacity, oxidative phosphorylation, and reactive oxygen species (ROS) production, already established methods are based on extramitochondrial measurements of the whole mitochondria population.

Objective: The aim of this study was to develop a reliable and well-characterized method for multiparametric analysis of isolated single mitochondrion by flow cytometry (FC) in the context of myocardial infarction. The advantage of FC is the possibility to give a simultaneous analysis of morphological parameters (side and forward scatters: SSC and FSC) for each mitochondrion, combined with intramitochondrial measurements of several biological markers, such as ROS production or membrane potential (Δφm), using specific fluorescent probes.

Methods and results: For this study, a rat model of ischemia-reperfusion and a protective approach of post-conditioning using low reperfusion pressure was used. Thanks to the use of specific probes (NAO, MTR, TMRM, DilC1, and DHR123) combined with flow cytometry, we propose a method: (i) to identify mitochondrial populations of interest based on quality criteria (NAO/TMRM double staining); (ii) to monitor their morphological criteria, especially during swelling due to calcium overload; and (iii) to compare mitochondrial functions (membrane potential and ROS production) in different experimental groups. Applied to mitochondria from ischemic hearts, these measurements revealed that individual mitochondria are altered and that cardioprotection by low-pressure reperfusion reduces damage, as expected.

Conclusions: Our results highlight FC as a reliable and sensitive method to investigate changes in mitochondrial functions and morphology in pathological conditions that disrupts their activity such as the case in ischemia-reperfusion. This methodological approach can be extended to other pathologies involving mitochondrial dysfunctions. Moreover, FC offers the possibility to work with very small amounts of isolated mitochondria, a factor that may limit the use of classical methods.

Keywords: flow cytometry; ischemia reperfusion injury (IRI); mitochondria; postconditioning.

Figure 1

NAO and MTR as specific markers of mitochondria. The probe toxicity was determined by the RCI measurement in isolated mitochondria from rat hearts according to a range of concentrations from 10 to 100 nmol/L for NAO (A) and from 25 to 250 nmol/L for MTR (C). Loading of the isolated mitochondria with the optimal working concentrations (50 nmol/L for NAO and 25 nmol/L for MTR) enabled to properly gate the population of interest (B,D). Quality of mitochondria preparation was evaluated by combination of NAO staining with FSC/SSC dot plot. * p < 0.05 vs. 0 nmol/L. *** p < 0.001 vs. 0 nmol/L (n = 3 per group).

Figure 2

Swelling of mitochondria in time-lapse-dependent manner. Correlated measurements of FSC (A) and SSC (B) reflect the changes in mitochondrial morphology. Histograms show the variation in side or forward scatters per number of events. This figure illustrates an example of the results obtained from 6 different mitochondria preparations.

Figure 3

Validation of DilC1 use to measure Δφm in isolated mitochondria. The probe toxicity was determined by the RCI measurement in isolated mitochondria from rat hearts according to a range of concentrations from 10 to 50 nmol/L for DilC1 and from 15 to 100 nmol/L for TMRM (A,C). As expected, addition of the uncoupling agent FCCP at 10 µmol/L induced a decrease in DilC1 and TMRM fluorescence intensities (B,D), validating the ability of these probes to measure Δφm in our model. *** p < 0.001 vs. + DilC1 alone and vs. + TMRM alone (n = 3 per group).

Figure 4

Validation of DHR 123 used to measure intramitochondrial ROS production. The toxicity was evaluated by RCI from 1 to 15 nmol/L (A). The ROS scavenger: ascorbic acid and hydrogen peroxide (H₂O₂) were used to validate the ability of DHR 123 to detect intramitochondrial ROS production (B). Addition of the uncoupling agent, FCCP, to succinate and ADP, enabled confirmation that the fluorescence of the probe was not dependent on the Δφm (C). *** p < 0.001 vs. + DHR alone (n = 4 per group).

Figure 5

The Δφm is partially maintained after postconditioning. The Δφm was measured by incubating mitochondria from each group with DilC1 at 10 nmol/L (A) or TMRM at 20 nmol/L (B) after stimulation by succinate (3.75 µmol/L), ADP (6.25 µmol/L), and FCCP (10 µmol/L) successively in the same experimental tube. Results were expressed in relative fluorescence intensity. *^,†,‡ p < 0.05 vs. IR and ***^,††† p < 0.001 vs. IR (n = 6 per group).

Figure 6

ROS production is increased by ischemia-reperfusion, which is partially limited by postconditioning. ROS production is detected in mitochondria after labeling with DHR at 10 nmol/L in the three experimental groups after stimulation by succinate (3.75 µmol/L), rotenone (6.25 µmol/L), and antimycin A (1 µmol/L successively in the same experimental tube). ***^{,†††,‡‡‡,+++} p < 0.001 vs. IR and ⁺⁺ p < 0.01 vs. IR (n = 6 per group).