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

Abstract

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.

Keywords: ciclosporin A-postconditioning–ischemia reperfusion injury (IRI)-delayed reperfusion-mitochondria-permeability transition pore; donation after circulatory death (DCD); heart transplantation.

Figure 1

Functional recovery (rate pressure product or RPP) assessed at reperfusion. Postconditioning (POST) lost its efficacy if delayed beyond 3 min of reperfusion, whereas cyclosporine A (CsA) administration showed higher RPP even after a 10 min delay. N = 6 hearts/group. *** p < 0.001 vs. control.

Figure 2

(a): Myocardial necrosis assessed by LDH release (Panel a) and infarct size measurement (Panel b). Postconditioning (POST) lost its efficacy if delayed beyond 3 min of reperfusion, whereas CsA reperfusion showed higher RPP even after 10 min of reperfusion. N = 6 hearts/group. * p < 0.05, ** p < 0.01 vs. control. (b): Myocardial necrosis assessed by LDH release (Panel a) and infarct size measurement (Panel b). Postconditioning (POST) lost its efficacy if delayed beyond 3 min of reperfusion, whereas CsA reperfusion showed higher RPP even after 10 min of reperfusion. N = 6 hearts/group. ** p < 0.01 vs. control. (c): Example of heart slices of the left ventricle after the sequence of ischemia reperfusion and triphenyltetrazolium chloride staining. The colored areas (brick red) correspond to the viable tissue, while the clear areas (whitish) are the dead areas resulting from the infarction. Infarct size is expressed as % of infarcted areas compared to healthy areas.

Figure 2

(a): Myocardial necrosis assessed by LDH release (Panel a) and infarct size measurement (Panel b). Postconditioning (POST) lost its efficacy if delayed beyond 3 min of reperfusion, whereas CsA reperfusion showed higher RPP even after 10 min of reperfusion. N = 6 hearts/group. * p < 0.05, ** p < 0.01 vs. control. (b): Myocardial necrosis assessed by LDH release (Panel a) and infarct size measurement (Panel b). Postconditioning (POST) lost its efficacy if delayed beyond 3 min of reperfusion, whereas CsA reperfusion showed higher RPP even after 10 min of reperfusion. N = 6 hearts/group. ** p < 0.01 vs. control. (c): Example of heart slices of the left ventricle after the sequence of ischemia reperfusion and triphenyltetrazolium chloride staining. The colored areas (brick red) correspond to the viable tissue, while the clear areas (whitish) are the dead areas resulting from the infarction. Infarct size is expressed as % of infarcted areas compared to healthy areas.

Figure 3

(A) Calcium retention capacity (CRC) is used as an indicator of mitochondrial permeability transition pore (MPTP), with the presence of complex II substrates. Delayed protection maintained higher CRC in all CsA groups, while MPTP inhibition was abolished in delayed POST-10 and -20. The insert shows classical plots obtained on sham, ischemic control, and protected hearts. A schematic diagram is shown in Figure S1 of the Supplementary Data to better understand the principle of CRC measurement. Abbreviations: CsA for cyclosporine A and POST for ischemic postconditioning. N = 6 hearts/group. * p < 0.05 and *** p < 0.001 vs. control. (B): Example of dot plot of CRC measurements obtained from each group of hearts. Pulses of 10 nmol of Ca2+ per mg of protein were added every minute to the populations of mitochondria. The calcium pulse was recorded as a fluorescence peak, and the Ca2+ was then rapidly taken up by the mitochondrial calcium uniporter. When the MPTP was open, the mitochondria and a release of Ca2+ were observed. The number of pulses was used as an indicator of the susceptibility of MPTP to mitochondrial Ca2+ overload.

Figure 4

Extramitochondrial H₂O₂ index assessed in the presence of pyruvate-glutamate-malate- (PGM), substrates of complex I, PGM + rotenone (Rot) and PGM + rotenone + antimycin (AA). 20 min after the onset of reperfusion, the H₂O₂ index remained lower in the delayed CsA group but returned to control values in delayed POST-10 and -20. Abbreviations: CsA for cyclosporine A and POST for ischemic postconditioning. N = 6 hearts/group. * p < 0.05 and *** p < 0.001 vs. control.

Figure 5

The experimental design included 10 groups of 12 animals. Sham underwent no ischemia reperfusion. All other groups experienced 40 min of ischemia followed by 60 min of reperfusion. POST-0 group (postconditioning without delay) consisted of two sequences of ischemia reperfusion at the onset of reperfusion. CsA-0 group (cyclosporin A at reperfusion without delay) involving CsA perfusion at 250 nM during the first 10 min of reperfusion. CsA and POST interventions were deferred for delayed groups by 3, 10, or 20 min.