Cell-Permeable Succinate Improves Platelet Respiration in Patients Undergoing Cardiopulmonary Bypass: A Pilot Study

: Open-heart surgery with cardiopulmonary bypass (CPB) remains the standard approach for complex cardiac pathologies, such as advanced coronary heart disease and severe valvular defects. Platelet dysfunction has been widely reported, with both structural and functional changes being elicited by the CPB circuit. Succinate is a mitochondrial substrate that is metabolized through complex II (CII) but is impermeable to cellular membranes when given exogenously. Cell-permeable succinates are novel prodrugs developed to support mitochondrial electron transport (ET) and prevent energy depletion in various pathologies. The aim of the present pilot study was to investigate the role of NV118 (diacetoxymethyl succinate), a cell-permeable succinate, on platelet respiration in a pilot group of patients undergoing CPB. Blood samples (20 mL) were collected from participants before (prior to heparin administration) and after CPB (within 10 min after protamine sulphate administration). Platelets were isolated through a two-step centrifugation protocol. Mitochondrial respiration was analyzed by means of high-resolution respirometry in the presence of NV118 or its solvent (DMSO). The main respiratory parameters recorded were as follows: respiration, organ protection at the mitochondrial level during CPB are clearly worthy and important areas for future investigation.


. Introduction
One of the most important medical advances of the th century was the development of cardiopulmonary bypass (CPB) which paved the way to open-heart surgery [ ]. While being an irreplaceable part of cardiothoracic surgery, it is well established that extracorporeal circulation systems are associated with the activation of in ammatory and oxidative stress responses [ , ]. Moreover, the exposure of blood to the synthetic surfaces of the heart-lung machine as well as to non-physiological shear stress due to mechanical pumping are responsible for the perioperative hemostatic abnormalities; both bleeding and thrombotic events were reported as major causes of morbidity and mortality in these patients [ , ]. In particular, the CPB circuit determines several structural and functional platelets abnormalities. The activation of platelets that remain in circulation by the bypass circuit will render them hypo-responsive to stimulation with various agonists, which may impair their hemostatic properties [ ].
The last decade has witnessed an increased interest for another pathomechanism in the setting of open-heart surgery, namely mitochondrial dysfunction. During CPB, cardiac mitochondrial dysfunction results from systemic in ammation, surgical trauma, and ischemia/reperfusion injury, and in turn, impairs postoperative myocardial contractility and predisposes one to arrhythmias [ ]. Not only cardiac but also cerebral mitochondrial dysfunction was reported to occur during CPB. A recent study performed in piglets subjected to h of CPB showed that, even in the absence of local markers of ischemia, CPB was associated with decreased mitochondrial respiration and increased mitochondrial ROS formation in brain cortex homogenates [ ].There is one pioneering study in the literature that assessed the mitochondrial respiration of intact platelets isolated from adult patients before and after CPB using the extracellular ux analyzer Seahorse XF ; these authors found neither changes in respiratory parameters nor an association between platelet respiration and aggregation/postoperative bleeding [ ]. Of note, the latter observation has been also recapitulated in chronic conditions by the group of Cezar Watala; in a recent elegant study performed in the experimental model of type diabetes, they reported that elevated platelet mitochondria respiration occurred later in the evolution of the disease and was not related to the earlier observed changes in platelet activation/reactivity [ ].
However, the intraoperative impairment of platelets due to blood exposure to the CPB circuit occurs in cardiac surgery dependent on its duration, and whether their damage can be rescued has not been investigated so far. Nowadays, several pharmacological strategies for mitochondrial protection or recovery after injury are currently under investigation. Cell-permeable succinates are prodrugs able to pass over the cellular membrane, release succinate in the cytosol, and enhance mitochondrial ATP production. They have been developed to , ( ), bypass mitochondrial complex I defects [ ] and have been reported to support electron transport in various pathologies associated with complex I dysfunction/inhibition [ , ].
The present pilot study aimed to assess whether a rst-generation cell-permeable succinate, NV (diacetoxymethyl succinate), is able to modulate platelets bioenergetics in patients undergoing cardiopulmonary bypass. Here, we report that NV elicited a global improvement in the respiratory function of intact platelets isolated both prior to and after cardiopulmonary bypass.

. . Study Population
The study was conducted in accordance with the statements of the Declaration of Helsinki, and the protocol was approved by the Committee of Research Ethics of "Victor Babes , " University of Medicine and Pharmacy, Timis , oara, Romania (no. / . . and no. p/ / / ). The study protocol was explained, and all participants provided written informed consent.
Thirteen patients (four M, nine F) diagnosed with multivessel coronary artery disease, valvular pathology, or both were admitted for open-heart surgery with CPB in a single center (Institute for Cardiovascular Diseases of Timis , oara) and included in the study. The exclusion criteria were: heart failure > NYHA III and ejection fraction < %, age < or > years, platelet count < . /mm , hemoglobin < g/dL, congenital or acquired platelets disorders, congenital coagulation disorders, disseminated intravascular coagulation, and surgical emergencies. The clinical and laboratory characteristics of the study group are presented in Table . Table . Characteristics of the study group.
Regarding the type of surgery, ( %) patients underwent aortic valve replacement, ( . %) patient had mitral valve replacement, ( . %) patients underwent aorto-coronary bypass graft, and ( . %) patients , ( ), had aortic valve replacement and aorto-coronary bypass surgery. The mean duration of CBP was min. All patients presented preoperative hypertension as a comorbidity for which antihypertensive medication (ACE inhibitors/sartans ± calcium channel blockers) was administered.

. . Platelet Isolation
Twenty ml of peripheral blood was collected before (prior to heparin administration) and after the CPB (within min after protamine sulphate administration) using K (EDTA) as an anticoagulant. Blood samples were prepared and analyzed within -h after collection. The platelet suspension dissolved in mL of homologous plasma was obtained through two-step centrifugation at • C ( min at × g, min at × g) according to a protocol adapted from ref.
[ ] and further used for high-resolution respirometry studies.

. . High-Resolution Respirometry
The mitochondrial respiration of isolated intact (non-permeabilized) platelets was assessed at C through high-resolution respirometry (HRR) using an oxygraph-k (Oroboros Instr., Innsbruck, AT, USA). Platelets ( million/chamber) were suspended in a mitochondrial respiration medium (MiR ): sucrose mM, HEPES mM, taurine mM, K-lactobionate mM, MgCl mM, KH PO mM, EGTA . mM, BSA g/L, and pH . , as described in refs. [ , ]. Prior to each experiment, calibration at air saturation was performed. A Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol was adapted from ref. [ ] to measure complex I (CI) and complex II (CII)-dependent respiration in intact platelets. In brief, the platelet oxygen consumption rate (OCR) was allowed to reach a steady state corresponding to the ROUTINE respiration (respiration in the physiological coupling state). A cell-permeable succinate NV ( µM) or an equivalent volume of dimethyl sulfoxide (DMSO) as a control was added in the xygraphy chambers. ATP-synthase was inhibited using oligomycin ( µg/mL) in order to assess LEAK respiration (the non-phosphorylating respiration), after which, a stepwise titration of FCCP (a protonophore) was performed in order to assess the ET CAPACITY (maximal uncoupled respiration as an indicator of the maximal activity of the electron transport system-ETS). Last but not least, in order to investigate the direct e ects of NV on complex II-supported respiration, complex I was inhibited with rotenone ( µM), allowing the measurement of ETII capacity (the residual succinate-supported respiration). In the end, complex III was inhibited with antimycin A ( µg/mL), allowing the estimation of residual oxygen consumption (ROX). The OCR was expressed in pmol × s − × × − platelets, and all the values were ROX-corrected.

. . Chemicals
All chemicals were obtained from Sigma-Aldrich-Merck. The cell-permeable succinate was generously provided by Abliva AB (Lund, Sweden) and is available in the MitoKit-CII (Oroboros Instr., AT).

. . Data Analysis
DatLab software (Oroboros Instr.) was used to record the oxygen concentration and oxygen consumption rate ( ux) displayed in real time and also for data analysis. Mitochondrial respiration was also corrected for oxygen ux due to instrumental background.
Statistical analysis was performed using GraphPad software (version . ). All data are expressed as mean ± SEM of ROX corrected respiration. , ( ),

. Results
DMSO was used as the solvent for NV , and its e ect on ROUTINE respiration was rstly assessed. As depicted in Figure , no di erences in platelet physiological OCR were found prior to ( . ± . vs. . ± . pmol × s − × × − cells- Figure   Furthermore, the e ect of NV (as compared to DMSO) on intact platelets, isolated both prior to and after CPB, was assessed by recording the above-mentioned respiratory parameters: ROUTINE respiration, LEAK respiration, ET capacity, and ETII.

. . Cell-Permeable Succinate Improved Mitochondrial Respiration Prior to CPB
As depicted in Figure  ). Finally, the residual succinate-supported respiration was measured after rotenone addition to inhibit CI. As shown in Figure D, the ET capacity dependent on CII signi cantly increased from . ± . to .

. . Cell-Permeable Succinate Improved Mitochondrial Respiration after CPB
Platelets isolated after CPB were exposed to NV vs. DMSO, with a comparable increase for almost all respiratory parameters (pmol × s − × × − cells), as previously described prior to CPB. Thus, ROUTINE respiration ( Figure

. Discussion
The main nding of this pilot study is the improvement (via NV ) of mitochondrial bioenergetics in intact platelets isolated from patients prior to and after cardiopulmonary bypass. To our knowledge, this is the rst study which investigated the e ect of cell-permeable succinate on platelets exposed to extracorporeal circulation. Here, we report a signi cant increase in all respiratory parameters, in the presence of NV , in isolated platelets, regardless of the moment of their harvesting.
In particular, routine respiration is dependent on the existent mitochondrial matrix succinate that platelets retain while ex vivo. Since the addition of a membrane-permeable succinate increased oxygen consumption both pre-and post-CPB, this indicates that platelet oxidative metabolism is limited by the concentration of succinate as an energetic substrate. The addition of a cell-permeable succinate would correct this in any condition. From a translational perspective, this observation may have signi cant clinical implications. For example, the administration of permeable succinates in extracorporeal circulation will correct the substrate de cit and improve platelet bioenergetics.
As previously reported by the Gary Fiskum group in their pioneering work that analyzed platelet respiration in the setting of CPB using the Seahorse extracellular ux analyzer [ ], we did not nd signi cant di erences in platelet respiration prior to or after CPB using the HRR technique in this pilot study (data not shown). The mean duration of CBP was . h in this group. We cannot exclude the possibility that longer periods of extracorporeal circulation and/or the presence of metabolic comorbidities (reported to be associated with mitochondrial dysfunction) in patients undergoing open-heart surgery might elicit platelet mitochondrial respiratory dysfunction. In recent years, the use of peripheral platelets as an easy obtainable source of viable mitochondria has been brought to light, which allows the analysis of mitochondrial respiratory dys/ Additionally, the past few decades witnessed an increasing interest in the development of drugs capable of supporting mitochondrial function, and cell-permeable succinates have emerged as such compounds. Succinate is the substrate of succinate dehydrogenase, which is complex II (embedded in the inner mitochondrial membrane), being crucial for oxidative phosphorylation as it couples the tricarboxylic cycle with the electron transport system activity. Succinate causes the ETS to run sequentially from complex II (i.e., it bypasses CI) all the way down to complex IV, where oxygen is converted to water [ ]. Thus, it is particularly useful in pathological conditions associated with dysfunctional CI either due to genetic mutations or damage due to increased oxidative stress or drug toxicity (more frequent in practice). However, succinate is not cell-permeable (being an anion in living organisms), so it has limited intracellular uptake when exogenously administered. To date, cell-permeable succinate prodrugs have emerged as alternative fuel sources in order to prevent energetic dysfunction in various pathological conditions. Accordingly, succinate prodrugs have been reported to improve mitochondrial respiration in cellular models of drug toxicity [ , , ], peripheral blood mononuclear cells from acute poisoning with carbon monoxide [ ], and broblasts cultured from a patient with Leigh syndrome (mitochondrial disease with CI defect) [ ].
Whether cardiac and cerebral mitochondrial dysfunction reported to occur in some of the patients undergoing CPB can be alleviated by cell-permeable succinates awaits future investigation. , ( ), We acknowledge the small number of subjects investigated, and thus, the limited statistical power as an important limitation of this pilot study.

. Conclusions
NV , a cell-permeable succinate prodrug, improved platelet bioenergetics in the setting of cardiopulmonary bypass. Whether this e ect can support platelet function during CPB is yet another area of enquiry for future investigation. This nding may also be a voice in the discussion on new pharmacological approaches of organ protection during cardiac surgery.