Postconditioning by Delayed Administration of Ciclosporin A: Implication for Donation after Circulatory Death (DCD).

Heart transplantation is facing a shortage of grafts. Donation after Circulatory Death (DCD) would constitute a new potential of available organs. In the present work, we aimed to evaluate whether Postconditioning (ischemic or with ciclosporin-A (CsA)) could reduce ischemia-reperfusion injury in a cardiac arrest model when applied at the start of reperfusion or after a delay. An isolated rat heart model was used as a model of DCD. Hearts were submitted to a cardiac arrest of 40 min of global warm ischemia (37 °C) followed by 3 h of 4 °C-cold preservation, then 60 min reperfusion. Hearts were randomly allocated into the following groups: control, ischemic postconditioning (POST, consisting of two episodes each of 30 s ischemia and 30 s reperfusion at the onset of reperfusion), and CsA group (CsA was perfused at 250 nM for 10 min at reperfusion). In respective subgroups, POST and CsA were applied after a delay of 3, 10, and 20 min. Necrosis was lower in CsA and POST versus controls (p < 0.01) whereas heart functions were improved (p < 0.01). However, while the POST lost its efficacy if delayed beyond 3 min of reperfusion, CsA treatment surprisingly showed a reduction of necrosis even if applied after a delay of 3 and 10 min of reperfusion (p < 0.01). This cardioprotection by delayed CsA application correlated with better functional recovery and higher mitochondrial respiratory index. Furthermore, calcium overload necessary to induce mitochondrial permeability transition pore (MPTP) opening was similar in all cardioprotection groups, suggesting a crucial role of MPTP in this delayed protection of DCD hearts.

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

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.

Quantitative stiffness assessment of cardiac grafts using ultrasound in a porcine model: A tissue biomarker for heart transplantation.

BACKGROUND: Heart transplantation is the definitive treatment for many cardiovascular diseases. However, no ideal approach is established to evaluate heart grafts and it mostly relies on qualitative interpretation of surgeon based on the organ aspect including anatomy, color and manual palpation. In this study we propose to assess quantitatively the Shear Wave Velocity (SWV) using ultrasound as a biomarker of cardiac viability on a porcine model. METHODS: The SWV was assessed quantitatively using a clinical ultrasound elastography device (Aixplorer, Supersonics Imagine, France) linked to a robotic motorized arm (UR3, Universal Robots, Denmark) and the elastic anisotropy was obtained using a custom ultrasound research system. SWV was evaluated as function of time in two porcine heart model during 20h at controlled temperature (4°C). One control group (N = 8) with the heart removed and arrested by cold cardioplegia and immerged in a preservation solution. One ischemic group (N = 6) with the organ harvested after 30 min of in situ warm ischemia, to mimic a donation after cardiac death. Hearts graft were revived at two preservation times, at 4 h (N = 11) and 20 h (N = 10) and the parameters of the cardiac function evaluated. FINDINGS: On control hearts, SWV remained unchanged during the 4h of preservation. SWV increased significantly between 4 and 20h. For the ischemic group, SWV was found higher after 4h (3.04 +/- 0.69 vs 1.69+/-0.19 m/s, p = 0.007) and 20h (4.77+/-1.22 m/s vs 3.40+/-0.75 m/s, p = 0.034) of preservation with significant differences. A good correlation between SWV and cardiac function index was found (r2=0.88) and manual palpation score (r2=0.81). INTERPRETATION: Myocardial stiffness increase was quantified as a function of preservation time and harvesting conditions. The correlation between SWV and cardiac function index suggests that SWV could be used as a marker of graft viability. This technique may be transposed to clinical transplantation for assessing the graft viability during transplantation process. FUNDING: FRM PME20170637799, Agence Biomédecine AOR Greffe 2017, ANR-18-CE18-0015.

Cooling Uncouples Differentially ROS Production from Respiration and Ca2+ Homeostasis Dynamic in Brain and Heart Mitochondria.

Hypothermia provides an effective neuro and cardio-protection in clinical settings implying ischemia/reperfusion injury (I/R). At the onset of reperfusion, succinate-induced reactive oxygen species (ROS) production, impaired oxidative phosphorylation (OXPHOS), and decreased Ca2+ retention capacity (CRC) concur to mitochondrial damages. We explored the effects of temperature from 6 to 37 °C on OXPHOS, ROS production, and CRC, using isolated mitochondria from mouse brain and heart. Oxygen consumption and ROS production was gradually inhibited when cooling from 37 to 6 °C in brain mitochondria (BM) and heart mitochondria (HM). The decrease in ROS production was gradual in BM but steeper between 31 and 20 °C in HM. In respiring mitochondria, the gradual activation of complex II, in addition of complex I, dramatically enhanced ROS production at all temperatures without modifying respiration, likely because of ubiquinone over-reduction. Finally, CRC values were linearly increased by cooling in both BM and HM. In BM, the Ca2+ uptake rate by the mitochondrial calcium uniporter (MCU) decreased by 2.7-fold between 25 and 37 °C, but decreased by 5.7-fold between 25 and 37 °C in HM. In conclusion, mild cold (25-37 °C) exerts differential inhibitory effects by preventing ROS production, by reverse electron transfer (RET) in BM, and by reducing MCU-mediated Ca2+ uptake rate in BM and HM.