In adult CMs, ryanodine receptor 2 (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction (E-C) coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. In this study, after the knockout of RYR2 gene (RYR2–/–), induced pluripotent stem cells (iPSCs) were able to differentiate into cardiomyocytes (CMs) with an efficiency similar to control iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, significantly reduced frequency and intense abnormalities of Ca2+ transients were observed in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular calcium ([Ca2+]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, the compensatory mechanism for Ca2+ handling in RYR2–/–-iPSC-CMs is partially mediated by the Inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced L-type Ca2+ current (ICaL) density. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmogenic disorder. RYR2 mutations are the genetic cause of CPVT Type 1. So far, the pathogenic mechanism of how RYR2 mutations remodel cardiac rhythm remains controversial, because all existing hypotheses cannot independently and universally represent the mechanism behind CPVT. Patient-specific iPSCs offer a unique opportunity for CPVT modeling and investigation in vitro. In this study, the effects of four different RYR2 mutations (R420W, A2254C, E4076K, and H4742Y) on cardiac Ca2+ handling were examined individually. The R420W mutation in CPVTa-iPSC-CMs showed no effect on the amplitude of paced Ca2+ transients but led to an increased Ca2+ leak and a decreased SR Ca2+ content. Moreover, CPVTa-iPSC-CMs presented an enhanced sensitivity to [Ca2+]o and caffeine but a lower ICaL density. Compared to Ctrl cells, CPVTb-iPSC-CMs carrying the A2254V mutation displayed Ca2+ transients with smaller amplitude and higher frequency. More importantly, CPVTb-iPSC-CMs showed remarkably severe Ca2+ leak and unaltered SR Ca2+ content. The A2254V mutation also enhanced the sensitivity of iPSC-CMs to [Ca2+]o and caffeine. Interestingly, the ICaL density was found higher in CPVTb-iPSC-CMs. As for the E4076K mutation, it caused a reduction in both amplitude and frequency of Ca2+ transients in CPVTc-iPSC-CMs. In addition, the sensitivity to [Ca2+]o was diminished in CPVTc-iPSC-CMs, while the caffeine sensitivity and ICaL density were not changed. Regarding the H4742Y mutation, it led to a reduction of Ca2+ transient amplitude. In addition, CPVTd-iPSC-CMs manifested unique SR Ca2+ leak, which was resistant to tetracaine, suggesting a conformational remodeling of the H4742Y-mutated RYR2. Furthermore, CPVTd-iPSC-CMs also showed enhanced sensitivity to [Ca2+]o and caffeine, although the ICaL density was comparable to Ctrl-iPSC-CMs. In summary, the A2254V variation presented a typical gain-of-function mutation, rendering the RYR2 hyperactive, while the E4076K variation was identified as a loss-of-function mutation, leading to hypoactive RYR2. R420W and H4742Y mutations did not enhance or suppress the activity of RYR2. However, by destabilizing the N-terminal domain (NTD) of RYR2, the R420W mutation caused Ca2+ leak via the mutant channel, which could be blocked by RYR2 inhibitor. As for the H4742Y mutation, it led to a consistent and inhibitor-resistant Ca2+ leak via RYR2, suggesting a structural remodeling of RYR2 that disturbs complete closure of the channel. These results confirmed the importance of RYR2 on the maintenance of Ca2+ handling and gained evidence for the theory that the underlying mechanisms of CPVT caused by mutations in RYR2 should be mutation-specific rather than unified. This study suggests hiPSC-CMs as a suitable platform for modeling cardiac arrhythmogenic disease, interpreting potential molecular and pathophysiological mechanisms, testing new therapeutic compounds, and guiding mechanism-specific therapy.:Abbreviations V List of figures VIII List of tables X 1 Introduction 1 1.1 Human induced pluripotent stem cells 1 1.1.1 Generation and characteristics of human induced pluripotent stem cells 1 1.1.2 Differentiation of hiPSCs into cardiomyocytes 3 1.1.3 Modeling of inherited cardiac disease with hiPSCs 4 1.2 Catecholaminergic polymorphic ventricular tachycardia 7 1.2.1 Clinical characteristics and diagnosis of CPVT 7 1.2.2 Genetic background of CPVT 8 1.2.3 Clinical descriptions of CPVT patients recruited in this study 10 1.2.4 Patient-specific iPSC-CMs recapitulate the phenotypes of CPVT in vitro 10 1.3 Cardiac excitation-contraction coupling 11 1.3.1 Cardiac action potential 12 1.3.2 Ca2+ homeostasis in cardiomyocytes 14 1.3.2.1 Ca2+ influx via L-type Ca2+ channel 14 1.3.2.2 Initiation and termination of SR Ca2+ release 15 1.3.2.3 Ca2+ removal from cytosol 17 1.3.3 Cardiomyocyte contraction 20 1.4 Cardiac ryanodine receptor 21 1.4.1 Distribution and classification of RYRs 22 1.4.2 Regulation of RYR2 23 1.4.2.1 Cytosolic Ca2+ 23 1.4.2.2 Luminal Ca2+ 24 1.4.2.3 Phosphorylation by PKA and CaMKII 25 1.4.2.4 Calmodulin 27 1.4.2.5 Caffeine 27 1.4.3 Pathophysiological mechanisms of CPVT associated with RYR2 mutations 28 2 Aims of this study 33 3 Materials and methods 34 3.1 Materials 34 3.1.1 Cells 34 3.1.2 Laboratory devices and experimental hardware 34 3.1.3 Disposable items 36 3.1.4 Chemicals, solutions, and buffers for physiological and molecular experiment 36 3.1.5 Antibodies 40 3.1.6 Primers 41 3.1.7 Chemicals, media and solutions used for cell culture 42 3.1.8 Software 44 3.2 Methods 44 3.2.1 Cell culture 44 3.2.1.1 Preparation of glass coverslips for cell culture 44 3.2.1.2 Coating of plates and dishes 44 3.2.1.3 Cultivation of iPSCs with feeder-free method 45 3.2.1.4 Cryopreservation and thawing of iPSCs 45 3.2.1.5 Spontaneous differentiation of iPSCs in vitro 45 3.2.1.6 Directed differentiation of iPSCs into cardiomyocytes 46 3.2.1.7 First digestion of iPSC-CMs 46 3.2.1.8 Cryopreservation and thawing of iPSC-CMs 46 3.2.1.9 Time-dependent proliferation analysis of iPSC-CMs 47 3.2.1.10 Second digestion of iPSC-CMs 47 3.2.1.11 Collection of cell pellets for molecular experiment 47 3.2.2 Cell viability assay 48 3.2.3 Gene expression analyses 48 3.2.3.1 RNA isolation 48 3.2.3.2 Reverse transcription reaction 48 3.2.3.3 Polymerase chain reaction 49 3.2.3.4 Agarose gel electrophoresis 49 3.2.4 Protein expression analyses 49 3.2.4.1 Western blot 49 3.2.4.1.1 Lysis of cultured cells 49 3.2.4.1.2 SDS-polyacrylamide gel electrophoresis 50 3.2.4.1.3 Transfer and detection of proteins 50 3.2.4.2 Flow cytometry 51 3.2.4.3 Immunofluorescence staining 51 3.2.5 Calcium imaging 51 3.2.5.1 Measurement of spontaneous Ca2+ transients 52 3.2.5.2 Evaluation of diastolic SR Ca2+ leak and SR Ca2+ content 52 3.2.5.3 Assessment of iPSC-CM sensitivity to [Ca2+]o 53 3.2.5.4 Quantification of iPSC-CM response to caffeine 53 3.2.6 Patch-clamp 53 3.2.6.1 Preparation of agar salt bridge 53 3.2.6.2 Assessment of liquid junction 53 3.2.6.3 Measurement of action potential and L-type calcium current 54 3.2.7 Statistical analysis 54 4 Results 55 4.1 IP3R-mediated SR Ca2+ release partially restores the impaired Ca2+ handling in iPSC-CMs with RYR2 deficiency 55 4.1.1 Loss of RYR2 does not alter the pluripotency of RYR2–/–-iPSCs 55 4.1.2 Loss of RYR2 leads to increased death of RYR2–/–-iPSC-CMs 56 4.1.3 Loss of RYR2 does not affect the expression of IP3R in iPSC-CMs 58 4.1.4 RYR2–/–-iPSC-CMs show abnormal Ca2+ transients 60 4.1.5 The sensitivity of RYR2–/–-iPSC-CMs to [Ca2+]o and caffeine is changed 62 4.1.6 IP3R is critical for the generation Ca2+ transients in RYR2–/–-iPSC-CMs 63 4.1.7 SERCA-mediated SR Ca2+ uptake is crucial for the Ca2+ handling in both Ctrl- and RYR2–/–-iPSC-CMs 65 4.1.8 RYR2–/–-iPSC-CMs display abnormal action potentials 66 4.2 Investigation of the impaired function of RYR2 in CPVTa-iPSC-CMs 69 4.2.1 The R420W mutation leads to increased SR Ca2+ leak and decreased SR Ca2+ content 69 4.2.2 The R420W mutation leads to an enhanced sensitivity of iPSC-CMs to [Ca2+]o 70 4.2.3 CPVTa-iPSC-CMs shows increased sensitivity to caffeine 71 4.2.4 CPVTa-iPSC-CMs show reduced ICaL density 72 4.3 Investigation of the impaired function of RYR2 in CPVTb-iPSC-CMs 74 4.3.1 CPVTb-iPSC-CMs show abnormal Ca2+ transients 74 4.3.2 The A2254V mutation intensifies the SR Ca2+ leak in iPSC-CMs 75 4.3.3 The A2254V mutation enhances the sensitivity of iPSC-CMs to [Ca2+]o 76 4.3.4 The A2254V mutation increases the sensitivity of iPSC-CMs to caffeine 78 4.3.5 CPVTb-iPSC-CMs show increased ICaL density 78 4.4 Investigation of the impaired function of RYR2 in CPVTc-iPSC-CMs 79 4.4.1 CPVTc-iPSC-CMs show abnormal Ca2+ transients 79 4.4.2 The E4076K mutation shows no effect on the SR Ca2+ leak and content 80 4.4.3 The E4076K mutation diminishes the sensitivity of iPSC-CMs to [Ca2+]o 81 4.4.4 The E4076K mutation shows almost no effect on the response of iPSC-CMs to caffeine 82 4.4.5 The E4076K mutation does not alter the ICaL density in iPSC-CMs 83 4.5 Investigation of the impaired function of RYR2 in CPVTd-iPSC-CMs 84 4.5.1 CPVTd-iPSC-CMs show abnormal Ca2+ transients 84 4.5.2 The H4742Y mutation leads to a tetracaine-resistant Ca2+ leak in iPSC-CMs 84 4.5.3 The H4742Y mutation improves the sensitivity of iPSC-CMs to [Ca2+]o 86 4.5.4 The H4742Y mutation enhances the response of iPSC-CMs to caffeine 87 4.5.5 The H4742Y mutation alters the gating properties of LTCC in iPSC-CMs 88 5 Discussion 90 5.1 IP3R-mediated compensatory mechanism for Ca2+ handling in iPSC-CMs with RYR2 deficiency 90 5.2 Pathophysiological mechanisms of RYR2 mutation-related CPVT are mutation-specific 93 5.2.1 Dysfunctional Ca2+ handling caused by RYR2-R420W mutation 94 5.2.2 Dysfunctional Ca2+ handling caused by RYR2-A2254V mutation 96 5.2.3 Dysfunctional Ca2+ handling caused by RYR2-E4076K mutation 99 5.2.4 Dysfunctional Ca2+ handling caused by RYR2-H4742Y mutation 101 5.3 Conclusions and future perspectives 104 6 Summary 106 7 Zusammenfassung 108 8 References 111 9 Acknowledgements 131 10 Declaration 132

Background and aims: Leptin resistance or leptin signalling deficiency are associated with increased risk of diabetic cardiomyopathy and heart failure, which is a leading cause of obesity- and diabetes type 2 (T2DM)-related morbidity and mortality. Various metabolic disturbances are involved in this pathogenesis, such as elevated glucose and fatty acid levels, insulin resistance and altered myocardial substrate utilization. Rodent models provided useful insights into the underlying molecular mechanisms of obese- and T2DM-associated cardiometabolic diseases, however, they cannot fully recapitulate the disease phenotype of obese or T2DM patients. The aims of this study were to study the effect of leptin receptor (LEPR) mutations on the leptin-mediated signalling pathways in human cardiomyocytes, and to investigate glucose and fatty acid metabolism in the heart under (patho)physiological conditions. Methods and results: To study the role of LEPR in human cardiomyocytes (CMs), human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were used as a model. In the first part of this study, LEPR expression and function was investigated in wild type (WT)-iPSC-CMs by PCR and Western Blot. LEPR protein expression was almost not detectable in iPSCs and during early cardiac differentiation stages, however mRNA LEPR expression was comparable in the different steps of cardiac development. Importantly, LEPR protein expression was observed in WT-iPSC-CMs at the maturation stages, indicating that LEPR plays an important role in matured CMs. Thanks to CRISPR/Cas9 technology, LEPR mutations were introduced into iPSCs. Among the several clones obtained, 1B2 LEPRΔ/Δ-iPSC line was fully characterized and showed normal capacity to differentiate into spontaneously beating CMs. Although the B27 medium represents a well-established medium to cultivate iPSC-CMs, it has limitations for studying CM metabolism due to its high concentration of insulin and glucose, but low concentration of fatty acids. Physiological medium condition (F2) including physiological range of glucose, insulin and fatty acids was found to be fundamental to study LEPR signalling pathway in iPSC-CMs. Western blot analysis showed functional LEPR downstream pathway activation in WT-iPSC-CMs, while the absence of LEPR function was demonstrated in LEPRΔ/Δ-iPSC-CMs cultured in F2 medium. Moreover, improved medium condition, offered by the F2 medium, ameliorates insulin sensitivity as result of increased insulin-dependent AKT phosphorylation in WT-iPSC-CMs, while loss of LEPR function was associated with downregulation of insulin pathway activation. Additionally, leptin direct effect was observed on the regulation of glucose metabolism in WT-iPSC-CMs by reducing glycolytic fluxes, which was not observed in LEPRΔ/Δ-iPSC-CMs, as measured by 13C-isotope-assisted glucose metabolic flux. These data indicate that the signalling interaction between insulin and leptin is important in regulation of glucose metabolism and is abolished in LEPRΔ/Δ-iPSC-CMs. The matured WT-iPSC-CMs in F2 medium display adult CM-like metabolic phenotype such as enhanced mitochondrial respiration and glycolytic function, as measured by Seahorse analyser, compared to the same group cultured in the B27 medium. The mutation generated in LEPRΔ/Δ-iPSC-CMs caused an “energy starvation” status which led to increased AMPK phosphorylation compared to the WT group in B27 medium, which was associated with lower mitochondrial oxygen consumption rate (OCR) linked basal respiration and ATP production. In the next part of this study, the long-term leptin treatment of iPSC-CMs under physiological medium conditions in the presence of physiological range of insulin, glucose, and fatty acids (F2+) influenced LEPR downstream pathway activation such as JAK2 and AMPK suggesting a leptin-dependent role in fatty acid uptake and oxidation in WT-iPSC-CMs. On the contrary, leptin did not affect JAK2 and AMPK activation in LEPRΔ/Δ-iPSC-CMs. Culturing of (WT)-iPSC-CMs in F2+ medium demonstrated no significant difference in mitochondrial oxygen consumption, while slightly lower glycolysis and glycolytic capacity was observed. However, a leptin effect on fatty acid and glucose metabolism was observed in LEPR∆/∆-iPSC-CMs, which is independent from LEPR downstream regulation. To study the effect of high leptin levels, a medium mimicking some of the diabetic hallmarks, such as high glucose, high insulin, and high leptin levels, was used. Metabolic flexibility was observed in WT-iPSC-CMs in F3+ medium as showed by no difference in mitochondrial function in WT-iPSC-CMs in the presence or absence of high leptin. In contrast, LEPRΔ/Δ-iPSC-CMs in F3+ medium demostrated higher OCR compared to F2 medium, which is accompanied by lower glycolysis and glycolytic capacity, indicating the incapability of LEPRΔ/Δ-iPSC-CMs to use glucose as energy source, as measured by Seahorse analysis. Conclusion and outlook: Taken together, this study demonstrates the importance of leptin and LEPR at the late stage of CM maturation and the fundamental role of metabolic medium condition including physiological range of glucose and fatty acid to study the role of leptin in iPSC-CMs. In addition, LEPRΔ/Δ-iPSC-CMs in diabetic condition (F3+) represent a suitable model to investigate leptin-dependent cardiac metabolism, resulting in increased mitochondrial oxygen consumption and decreased glycolytic function, resembling the condition known in obesity-related T2DM patients. Further studies should focus on the regulation of the metabolic switch between glucose and fatty acid utilization in the absence of a functional LEPR. Understanding the contribution of leptin/LEPR signalling in human CM metabolism will shed light on novel therapeutic approaches to treat diabetic cardiomyopathy.

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Contexte : Au cours de la dernière décennie, le nombre de personnes en attente d’une transplantation cardiaque a augmenté d’environ 25%, tandis que le nombre de greffes effectuées chaque année est resté stable. Le taux de décès des patients en attente d’une greffe cardiaque est d’environ 15-20%. Le don d’organe suite à un décès cardiocirculatoire (DDC) est une alternative au don après décès neurologique (DDN) qui a permis d’augmenter le nombre d’organes disponibles comme les poumons, les reins et les foies. Compte tenu de la survenue d’une mort cardiovasculaire dans les protocoles DDC, le cœur est rarement greffé à cause des dommages infligés durant la période d’ischémie chaude. Notre équipe a précédemment démontré que l’utilisation du Celastrol, ainsi que notre analogue synthétique inhibiteur de la HSP90 ont des effets cardioprotecteurs, quand administrés au moment de la reperfusion dans des modèles in vitro de culture cellulaire et ex vivo dans des cœurs de rats montés sur le système de perfusion Langendorff. L’objectif est d’évaluer les mécanismes cardioprotecteurs rapides d’une nouvelle formulation de l’inhibiteur HSP90, et de comprendre l’efficacité de ce nouveau composé synthétique sur deux lignées de cellules : les cardiomyoblastes H9c2 issus de rats et les cardiomyocytes dérivés de cellules souches pluripotentes humaines (iPSC-CMs). Méthodes/Résultats : Les cellules H9c2 et iPSC-CMs ont été cultivées. La signalisation cellulaire a été analysée par western blot pour évaluer le niveau d’activation de ces différentes voies. Suite à l’optimisation des conditions pour les cellules iPSC-CMs, les deux lignées cellulaires ont été mises en condition ischémique (sans glucose, 95% N2, 5% CO2) durant la nuit, puis reperfusées, en conditions normales, avec différentes concentrations de l’inhibiteur HSP90. La viabilité cellulaire ainsi que l’ouverture des pores mitochondriaux (mPTP) ont été évaluées à l’aide de kits d’analyses, la production de radicaux libres d’oxygène à l’aide de kits de fluorescence et l’expression des ARN messagers de gènes antioxydants à l’aide de la réaction en chaîne par polymérase (PCR). Les résultats ont montré une augmentation de l’activation des voies cytoprotectrices quand les deux lignées cellulaires étaient traitées à la concentration 10-6M du composé sans stress 4 ischémique : augmentation de HO-1 and HSP-70 dans les 30 premières minutes et AKT et ERK après 1 heure de traitement et 3 heures de récupération. Contrairement à nos attentes, le traitement au moment de la reperfusion à la concentration 10-6M a montré une diminution de la viabilité des cellules, alors que la concentration 10-7M l’a augmenté. À une concentration de 10- 7M, il y a eu diminution de la production de radicaux libres comparativement au groupe témoin. Comme attendu, cette concentration a aussi démontré une diminution de l’ouverture des mPTP. Tous ces résultats ont été observés, autant dans les cellules humaines que celles de rats. Une évaluation préliminaire de l’expression des gènes antioxydants dans les cellules H9c2 a seulement montré une augmentation de l’expression des gènes CAT et HO-1. Conclusion : Notre groupe de recherche a précédemment démontré l’efficacité des composés issus du Celastrol sur la réduction des dommages myocardiques dus à la reperfusion dans les modèles d’ischémie, incluant l’infarctus du myocarde et la donation après décès cardiocirculatoire. Ces expériences ont montré les effets bénéfiques du nouveau composé synthétique sur l’expression des gènes antioxydants, et sur l’activation d’une série de voies cytoprotectrices permettant la stabilisation de la membrane mitochondriale, réduisant aussi la production de radicaux libres, et améliorant ultimement la survie cellulaire. Des études supplémentaires sont en cours afin d’améliorer la compréhension des modes d’action, des mécanismes et des dosages optimaux du médicament, ce qui nous permettra de commencer les essais sur animaux dans le but d’introduire cette molécule en clinique dans le contexte de don d’organes. / Background: During the last decade, the number of people waiting for a cardiac transplantation has increased by about 25%, while the number of yearly transplant surgeries performed has remained steady. The death rate of patients awaiting heart transplant is about 15-20%. Organ donation after circulatory death (DCD) is an alternative to donation after neurological death (DND) that has allowed to increase the number of available organs like lungs, livers, and kidneys. However, because of the cardiac death in DCD protocols, the heart is rarely used because of the injuries suffered by the warm ischemia period. Our group has previously shown that Celastrol, along with a synthetic HSP90 inhibitor analog, have cardioprotective effects when given as postconditioning agents at the moment of reperfusion in an in vitro model on cellular cultures and an ex vivo model on rat hearts mounted on a Langendorff perfusion system. The objective is to evaluate the rapid cardioprotective mechanisms of a novel formulation of the HSP90 inhibitor compound, and to understand the efficacy of this new synthetic compound on two cell lines: rat H9c2 cardiomyoblasts and human induced pluripotent stem cell-derived cardiomyocytes (iPSCCMs). Methods/Results: H9c2 rat cardiomyoblasts and human iPSC-CMs were cultured. Cell signaling was analyzed by western blot to evaluate pathway activations. Both cell lines were put in ischemic conditions (no glucose, 95% N2, 5% CO2) overnight, then reperfused (normal conditions) with different concentrations of HSP90i after optimizing the human iPSC-CMs’ stress experiment. Cell viability and mitochondrial permeability transition pore (mPTP) opening were evaluated using assays, oxygen-free radical production by fluorescence assay and antioxidant gene messenger RNA expression via polymerase chain reaction (PCR). Results showed an increase in cytoprotective pathway activation when both cell lines were treated with 10-6M of the compound without any stress: HO-1 and HSP-70 in the first 30 minutes while AKT and ERK after 1 hour of treatment and 3 hours of recuperation. Interestingly, treatment with the compound at 10-6M at the moment of reperfusion showed decreased viability of the cells while 10-7M improved it. Free radical production was also decreased at a concentration of 10-7M 6 when compared to the baseline, and as expected, the compound also decreased mPTP opening. These results were seen in both human and rat cell lines. Preliminary evaluation of antioxidant gene expression in H9c2 cells only showed an increase in the expression of the cytoprotective CAT and HO-1 genes. Conclusion: Our research group has previously demonstrated the efficacy of Celastrol compounds in reducing reperfusion damage in myocardial ischemia models, including myocardial infarction and donation after circulatory death. These experiments have shown that the beneficial effects of this new synthetic compound include the expression of antioxidant genes and the launching of a series of cytoprotective pathways that stabilize the mitochondrial membrane, reduce free radical production, and improve cell survival. Additional studies to fully understand the mode of action, the mechanisms and the optimal dosages are underway to allow us to move to animal trials in order to ultimately introduce the molecule in the clinical field in the context of organ donation.