Mitochondrial dysfunction upon impaired oxygen delivery: cardiac arrest versus hemorrhagic shock

Mitochondrial dysfunction upon impaired oxygen delivery: cardiac arrest versus hemorrhagic shock | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondrial dysfunction upon impaired oxygen delivery: cardiac arrest versus hemorrhagic shock Sergejs Zavadskis, Alexander Franz Szinovatz, Sergiu Dumitrescu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9282225/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Cardiac arrest (CA) and hemorrhagic shock (HS) have similar mortality but differ in oxygen delivery patterns: HS is a progressive low-flow state, while CA causes abrupt no-flow ischemia followed by reperfusion. Whether these distinct conditions lead to similar mitochondrial dysfunction remains unclear. Rats were subjected to either CA (8 min, followed by extracorporeal resuscitation and 24 h recovery, n = 5) or HS (MAP 40 mmHg for 1 h, followed by 24 h recovery, n = 7), with SHAM controls (n = 6). Shock severity was assessed via lactate and base excess. After 24 h, brain and liver tissues were analyzed for mitochondrial respiration and oxoglutarate dehydrogenase complex (OGDHC) activity. Both CA and HS impaired mitochondrial function, but via different mechanisms. CA primarily disrupted the inner mitochondrial membrane, increasing leak respiration. In contrast, HS impaired electron transfer between complexes III and IV, likely due to leak of cytochrome c through outer membrane permeability. This defect was more pronounced with with complex I substrates compared to complex II substrate. In the brain, CA was associated with increased succinate-driven respiration, suggesting activation of compensatory reaction. These findings indicate that CA and HS induce distinct mitochondrial injuries, affecting inner versus outer membranes, respectively, with implications for targeted therapeutic strategies. Health sciences/Cardiology Health sciences/Diseases Health sciences/Medical research Biological sciences/Neuroscience Biological sciences/Physiology Cardiac arrest hemorrhagic shock mitochondria liver brain cortex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Impaired oxygen delivery (DO 2 ) is a critical factor in cardiac arrest (CA) and hemorrhagic shock (HS). Adequate DO 2 is necessary for sufficient mitochondrial function supporting energy demand in tissues. Impaired DO 2 leads to profound anaerobic metabolism, damage to mitochondria which may cause secondary tissue injury during resuscitation. HS and CA represent two clinically relevant impaired DO 2 , low blood flow for a longer time and no flow for the short time, respectively. There is substantial evidence demonstrating impaired mitochondrial function following CA. A decrease in ATP-linked mitochondrial respiration was reported during early return of spontaneous circulation (ROSC) in rats subjected to CA (8 min CA/1 h ROSC; ) 1 . However, it has been suggested that mitochondrial dysfunction may not play a predominant role in the very early phase of post-resuscitation injury but instead becomes more prominent during later resuscitation phase. For example, in a rabbit model of CA (10 min CA/24 h ROSC) 2 , mitochondrial dysfunction was more evident at 24 hours of ROSC. At this time an increase in the leak respiration was observed, suggesting enhanced mitochondrial membrane permeability 2 . A similar impairment of mitochondrial membrane permeability was demonstrated in a pig model of CA (8 min CA/24 h ROSC) 3 , and this defect was ameliorated by therapeutic hypothermia. In general, three major processes may contribute to this phenomenon: (i) oxidative damage, (ii) activation of transmembrane ion channels such as uncoupling proteins (UCPs), and (iii) activation of mitochondrial phospholipases 4 . Ongoing oxidative stress and oxidation of polyunsaturated fatty acids (PUFAs) in mitochondrial membranes have been proposed as mechanisms contributing to increased membrane permeability 5 . In addition, elevated levels of mitochondrial uncoupling protein 2 (UCP2) have been associated with ischemic brain damage 6 . Furthermore, activation of phospholipase C and phospholipase A2 was demonstrated in a model of CA as early as 2 minutes after the onset of brain ischemia 7 . In addition, increased cytochrome c levels were detected in the cerebrospinal fluid of rats subjected to CA (8 min CA/24 h, 48 h, and 7 days ROSC) 8 , with peak concentrations at 24 hours after ROSC. These findings suggest that the release of cytochrome c may be secondary to damage of the outer mitochondrial membrane. It is widely accepted that pathological mitochondrial alterations also occur following hemorrhagic shock (HS) 9 . However, information regarding mitochondrial dysfunction in the brain upon HS remains limited. Although it has been suggested that mitochondrial function is impaired in the brain after HS 10 , this issue has not been comprehensively investigated. Instead, mitochondrial dysfunction following HS and resuscitation was observed in peripheral blood mononuclear cells (PBMCs), and it was associated with subsequent immunosuppression 11 . In rats subjected to HS (40 mmHg for 1 h followed by 1h resuscitation), decreased mitochondrial respiratory capacity across all mitochondrial complexes was reported in the kidney 12 . Similarly, HS (40 mmHg for 2 h/no resuscitation) significantly reduced mitochondrial oxygen consumption rate, ATP production, and mitochondrial membrane potential in rat splenocytes 13 . A decrease in ATP levels in the kidney and liver, along with reduced mitochondrial respiration supported by complex I substrates, was demonstrated following HS (40 mmHg; 60 min resuscitation) 14 . In addition, myocardial ATP and creatine phosphate content were decreased after 60 minutes resuscitation following HS induced 50 mm Hg MAP for 1 h 15 . Only a limited number of studies have shown the markers of mitochondrial dysfunction 24 hours after resuscitation or later 16 , even though that post-hemorrhagic mortality is often related to secondary complications such as multiple organ failure (MOF), which typically develop 24 hours or later after the shock phase. Collectively, comparing mitochondrial responses in HS and CA addresses a fundamental question in shock biology: whether distinct temporal patterns of impaired oxygen delivery activate shared or divergent molecular pathways leading to mitochondrial dysfunction. Elucidating these mechanisms is crucial not only for advancing the conceptual understanding of bioenergetic failure in critical illness but also for guiding the development of targeted therapeutic interventions in HS and CA. This has also a clinical relevance particularly for traumatic CA, where the phase of HS is followed by the CA 17 and the understanding the underlying mechanisms may have an important therapeutic relevance. The existing body of evidence regarding mitochondrial dysfunction in HS and CA has been generated in heterogeneous experimental models that differ substantially in insult duration, severity, and resuscitation protocols. Such variability precludes direct comparison and does not allow definitive conclusions regarding whether HS and CA activate similar or distinct mitochondrial injury pathways. The aim of the present study was to directly compare mitochondrial alterations induced by HS and CA under standardized experimental conditions characterized by comparable mortality in order to address a fundamental mechanistic question: whether distinct modes of impaired oxygen delivery and progressive hypoperfusion in HS versus abrupt circulatory arrest in CA activate convergent or divergent mitochondrial pathophysiological pathways. All analyses were performed 24 hours after resuscitation, a clinically relevant time point associated with the development of secondary organ injury in both brain and liver. As for CA, the brain is especially vulnerable to ischemia because of its high metabolic demand and limited energy reserves. Likewise, during HS a prolonged hypoperfusion can lead to brain tissue damage. Among neural tissues, we focused on the cerebral cortex as it primarily mediates cognition and appropriate social responses, and cortical damage often impairs recovery and return to the previous lifestyle 18,19 . Since ischemia-reperfusion after CA also affects peripheral organs, and the liver, playing a central role in systemic metabolism and inflammatory regulation, its compared analysis will give us insights into organ-specific responses. Similarly in HS, liver is a known key factor in prevention of MOF development 20 . Methods Experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and ARRIVE guidelines 21 and was approved by the relevant ministry for the HS-model (TVA Nr. 1358971-2023) and the CA-model (GZ 2023-0.896.943) respectively. Rat CA model used in this study resulted in 20% mortality by 24 hours of resuscitation, while HS model resulted in 10% mortality. There were no mortalities in the SHAM groups. The experimental animals, male outbred Sprague Dawley rats (400 – 600 g; Charles River Laboratories, Research Models and Services, Germany), were housed in pairs for at least one week for adaptation purposes for the HS experiments, and the minimal adaptation time was doubled for the CA groups, provided with ad libitum access to water and standard lab rodent pellet diet. HS model: On the day of surgery, rats received analgesia with Buprenorphine (0.01 mg/kg s.c., VetViva, Wels, Austria) and Meloxicam (1 mg/kg p.o., Boehringer Ingelheim, Vienna, Austria). After 1 hour, anesthesia was induced with sevoflurane gas (6–7%, 0.8 L/min; AbbVie GmbH, Ludwigshafen am Rhein, Germany) for 3–4 minutes in an induction chamber and maintained via nose cone mask (2–3%, 0.2–0.3 L/min) for the whole duration of the surgical intervention. The abdomen and right inguinal region were shaved, and an ophthalmic ointment was applied. Animals were placed supine on a Small Animal Physiological Monitoring System (Harvard Apparatus, Holliston, MA, USA). Body temperature was continuously monitored with a rectal probe and maintained using a heating pad integrated with the monitoring system. At the right inguinal region, femoral artery was catheterized (0.6 mm inner diameter and a 1.1 mm outer diameter Silclear™ Tubing, Silclear, Hampshire, UK) for blood withdrawal, sampling, and invasive blood pressure monitoring. At the same incision spot, the femoral vein was cathetherized for fluid resuscitation and treatment infusion. After catheterization, animals were allowed to stabilize for 10 minutes. Afterwards, controlled blood withdrawal was performed in 0.5-1 mL steps until mean arterial pressure (MAP) reached 40 ± 5 mmHg. Withdrawn blood was discarded, and total blood loss including samplings did not exceed 50% of the estimated circulating volume. MAP was maintained around the target value of 40 mmHg for 60–75 minutes with additional small withdrawals as needed. Hemorrhagic shock was confirmed by blood gas analysis indicating anaerobic metabolism (lactate > 3.0 mmol/L; base excess < –5.0 mEq/L), and shock duration was adjusted (60–75 minutes) based on these values. At the end of the shock period, the following compounds were administered via femoral vein diluted in a 0.2 mL Ringer’s solution bolus. CA-model: The cardiac arrest model has been established by the experimental resuscitation research group, department of emergency medicine, Medical University of Vienna. Detailed experimental protocol and the efficacy of the model was published previously 22 . In brief, male rats (n=5) were sedated with sevoflurane and received analgesia with piritramide and were mechanically ventilated. Similarly to HS intravenous catheters (Argyle 2.5-french umbilical neonatal vessel catheter) were inserted into the left femoral artery and vein, to determine blood pressure and sampling, respectively. In addition, a venous ECMO drainage cannula (adapted venous cannula, 14GA Venflon BD Luer-Lok, Helsingborg, Sweden) was inserted via the right jugular vein with the tip in the inferior vena cava and used to install a pacing catheter to induce VFCA, as well as an arterial ECMO cannula (custom-made; Dipl.-Ing. Martin Humbs, Valley, Germany) into the right femoral artery. Ventricular fibrillation cardiac arrest (VFCA) was induced by electrical current of 12 V/50 Hz at a max. of 8 mA applied via the pacing catheter lying in the veins next to the right heart. VFCA was defined as VF pattern in the ECG curve and drop of systolic aortic pressure < 20 mmHg with corresponding loss of arterial pulsation in the arterial pressure monitoring. After 8 min of untreated CA, ventilation and ECPR were initiated. After 2 min of ECPR, defibrillation with 2 biphasic shocks à 5 Joule (Heart-Start MRx Defibrillator; Philips, Andover, Mass) were administered every 2 min of resuscitation if shockable rhythm was present; mechanical ventilation (RR 20/min; FiO 2 1.0) was continued and epinephrine (10 μg/kg BW) was administered i.v. every 2 min during ECPR until ROSC was achieved or resuscitation efforts were halted after a maximum of 10 min of ECPR. Upon achieving return of spontaneous resuscitation (ROSC) the extracorporeal membrane oxygenation (ECMO) was halted, and the blood from the ECMO circuit was returned via the arterial in-flow cannula. Prompt removal of the venous ECMO cannula occurred after ROSC, and the wound was closed aseptically. Piritramide (3 mg/kg BW s.c. at 6-h intervals was used to eliminate any pain in the post-operative phase. Animals were kept normothermic by Harvard apparatus homeothermic blanket contril unit for the whole observation period after CA. SHAM group: The animals randomly selected to the SHAM group were handled, prepared and cathetherized identically to the shock group animals with the following notes. No blood was withdrawn besides blood samplings for determination of lactate and base excess. The blood collected for samples was substituted by the Ringer’s solution. Euthanasia and tissue sample collection: After 24 observation period, the animals were put to sleep in a transparent enclosure connected to Sevoflurane (8%, 1.6 L/min) supply. Afterwards, the respiratory rate of the animals was monitored until bradypnea (<30 breaths per minute) was reached. Next, the animal was promptly removed from the box, checked for the absence of the paw pinch reflex to ensure sufficient depth of anaesthesia and decapitated on a rodent DCAP guillotine (Precision Instruments, FL, USA). Next, the cranium of the animal was put on ice, dissected and brain tissue was extracted and cortex was isolated. Cortex and liver were harvested as soon as possible and separately placed in ice-cold CUSTODIOL® (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany) organ preservation solution. All organ extractions and preparations were performed on Petri dishes placed on ice. Next, a piece of liver tissue and cortex were used for mitochondrial respiration analysis and enzymatic activity of OGDHC. Lactate and base excess . The blood gas analysis was carried out on ABL800 FLEX (Radiometer, Krefeld, Germany) machine. Each blood sampling required a 0.3 mL pre-draw to flush the catheter, afterwards a small blood sample (approx. 0.1mL was collected in heparinized 1mL syringe. The machine was set to 95μL sample injection mode and the results were corrected for the animal rectal temperature at the time of sample collection. Mitochondrial function: We extracted cortex and liver and determined mitochondrial function in freshly prepared tissue homogenates using complex I (glutamate/malate) and complex II (succinate) mitochondrial substrates. All reagents were obtained from Sigma-Aldrich (Vienna, Austria), unless stated otherwise. Freshly excised liver or cerebral cortex tissue was cut into ~2 × 2 mm fragments with surgical scissors and washed extensively in ice-cold tissue preparation buffer (250 mM sucrose, 10 mM Tris, 0.5 mM EDTA (Radnor, PA, USA), 0.05% BSA) to remove residual blood and connective tissue. Tissue was homogenized on ice in glass vials at a buffer to tissue ratio of 4:1 (w/v) for cortex samples and 10:1 (w/v) for liver samples. Mitochondrial respiratory activity was measured with high-resolution oxygraphy (Oroboros O2k, Oroboros Instruments, Innsbruck, Austria) and data was recorded using the Oroboros DatLab software (Oroboros Instruments, Innsbruck, Austria). The instrument was calibrated at 37 °C for at least 30 minutes prior to each measurement using incubation buffer (80 mM KCl, 5 mM KH₂PO₄, 20 mM Tris-HCl, 1 mM iron-free diethylenetriaminepentaacetic acid and 0.1% Bovine Serum Albumin; pH adjusted to 7.4 with 6 M KOH at 22 °C). Here we used previously described experimental protocol 23 . To initiate mitochondrial respiration, we added different mitochondrial substrates to the end concentration of 10 mM, glutamate and malate (Fluka, Buchs, Switzerland) mixture to stimulate complex I or succinate to stimulate complex II. This setup allows to determine leak respiration at the inner mitochondrial membrane, characterized by the presence of reducing substrates in the absence of ADP (non-phosphorylating state). Next, we stimulated state III respiration and ATP synthesis by adding ADP (1 mM) to each chamber. Afterwards, maximal respiratory capacity of the ETC was measured after stepwise titration with the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.3 µM).Subsequently, the effect of cyt c (2.5 µM), an electron carrier between complex III and IV, on the respiration was assessed. Finally, mitochondrial respiration was terminated by the addition of complex III inhibitor Myxothiazol (12.5 µM) to confirm mitochondrial specificity. All reagents were obtained from Sigma-Aldrich (Vienna, Austria). From the oxygen uptake kinetics, we determined proton leak at the inner mitochondrial membrane, the respiration linked to ATP synthesis, coupling of oxidation and phosphorylation (respiratory control ratio defined as the ratio of state 3 / leak respiration ), maximal capacity of respiratory chain, and impact of exogenous cytc on the respiration. The data was evaluated by calculating the oxygen consumption rate by the homogenized tissue (nmol/min), corrected by substraction of myxothiazol respiratory value and normalization for 10 mg of tissue wet weight. All parameters were determined in the presence of either substrates of complex I or complex II. Activity of oxoglutarate dehydrogenase complex (OGDHC): OGDHC activity was determined by monitoring the reduction of NAD⁺ during 2-oxoglutarate turnover catalyzed by the enzyme. Frozen rat tissue was kept fully cold (on dry ice/ice) and homogenized in ice-cold homogenization buffer (0.05 % BSA, 0.5 mM EDTA, 1 % protease inhibitor cocktail, 250 mM sucrose, 10 mM TRIS). For each sample, homogenization buffer was added at 5× the wet tissue weight for cerebral cortex and 10× the wet tissue weight for liver tissues. The tissue was homogenized using a glass–Teflon homogenizer coupled to an overhead mixer until no visible clumps remained (around 10x). The homogenate was transferred to cryovials and further lysed by probe sonication (3× 500 J for 15 s, with 15 s pauses between pulses) while kept on ice. Following homogenization/sonication, samples were centrifuged for 10 min and then incubated on ice with RIPA buffer added in a 1:4 ratio for 20 min. A dilution series of the tissue lysate was prepared with homogenization buffer (1:5, 1:10, 1:20) and measured in triplicate. A tissue blank was prepared using incubation buffer and homogenization buffer (no homogenate). For the assay, 211 µL of master mix was dispensed into each well, consisting of 200 µL incubation buffer (1 mM CaCl₂, 0.05 mM CoA, 1 mM DTT, 1 mM MgCl₂·6H₂O, 50 mM MOPS, 2.5 mM NAD⁺; pH adjusted to ~7.22), 8 µL TPP, and 3 µL 2-oxoglutarate. Then, 20 µL of diluted tissue lysate was added to the respective wells (final volume 231 µL). Wells were checked to ensure no bubbles were present, and contents were mixed by inverse pipetting when plating. The plate was then run in a plate reader (POLARstar OMEGA, BMG LABTECH) by monitoring NADH formation photometrically over time. OGDHC activity was calculated from a calibration curve, generated using a dilution series of NADH standards. All reagents obtained from Sigma-Aldrich Austria. Results The pathogenesis of both hemorrhagic shock (HS) and cardiac arrest (CA) is characterized by transient impairment of blood flow, which is reflected by changes in mean arterial pressure (MAP). In the context of HS, the MAP is moderately reduced to approximately 40 mm Hg, but the duration of this reduction is prolonged up to 60 min. In contrast, during CA, the MAP drops significantly to around 5 mm Hg, but the duration of the event is much shorter (8 minutes). A possibility of different mortality rates between these models can not be excluded, out of CA animals (n = 5) one died before the endpoint and was excluded from the study, whereas there were no mortalities in the SHAM (n = 6) or HS (n = 7) animal groups, although previously 10% mortality was determined in this model. To facilitate the comparison of these two experimental models, which exhibit markedly different time courses, we classified the experimental timeline into three distinct phases: the stabilization phase (baseline, BL), the shock phase, and the resuscitation/reanimation phase (Fig. 1 ). The duration of each phase is explicitly represented on the horizontal axis of Fig. 1 A, with time for HS and CA indicated in minutes. The shock phase was subdivided into three subphases: shock onset, mid-shock, and end of shock. Similarly, the resuscitation/reanimation phase was divided into Early Resuscitation/ ROSC (ER) and Late Resuscitation/ ROSC (LR). Figure 1 B demonstrates that both HS and CA result in an increase in lactate levels, with CA inducing a more pronounced elevation. In HS, lactate levels returned to baseline values during the LR phase, whereas in CA, lactate levels remained significantly elevated above control values for up to 24 hours. Figure 1 C highlights a marked decrease in the base excess during both the ER and LR phases, which normalized by 24 hours post-event in both models. The data presented in Fig. 1 strongly suggest that CA-mediated injury is more severe compared to HS-mediated injury. To further investigate the pathological impact of this differential injury, we examined whether these observed differences are reflected in mitochondrial dysfunction. Analyzing mitochondrial respiratory function in animals subjected to CA, we observed a marked increase in the leak respiration rate in the cortex. This was observed both with complex I (glutamate/malate) and complex II (succinate) substrates, as demonstrated in Fig. 2 (A-B). A similar, although more modest, increase in the leak respiration rate was noted in the liver when complex I substrate was used, as shown in Fig. 2 C, and a not significant increase was observed with complex II substrate (Fig. 2 D). These findings suggest a disruption in the permeability of the inner mitochondrial membrane in animals subjected to CA. In contrast, no significant changes in leak respiration were detected in either the liver or the brain of animals subjected to HS, Fig. 2 (A-D). Disruption of inner mitochondrial membrane may impair ATP-synthesis. To address this issue, we next examined ATP-synthesis-linked respiration. Figure 3 reveals that in animals subjected to CA, the ATP-linked respiration rate was significantly increased in the cortex, both with substrates of complex I and II as shown in Fig. 3 (A-B), and only modestly elevated in the liver with succinate (complex II substrate), Fig. 3 D. No significant changes were observed in liver with complex I substrate (Fig. 3 C). No changes were observed in animals subjected to HS. The increase in the ATP-linked respiration may, in theory, compensate for the elevated leak respiration and facilitate the coupling of oxidation and phosphorylation in the cortex. To address this point, we analyzed respiratory control ratio (RCR) characterizing the coupling of oxidation and phosphorylation. To assess the potential impact of altered ATP-synthesis linked respiration on oxidative phosphorylation (OxPhos), we calculated the Respiratory Control Ratio (RCR) based on the data presented in Figs. 2 and 3 . Our analysis shown in Fig. 4 (A-B) reveals that despite an increase in the leak respiration in cortical tissue derived from CA animals, the RCR was elevated, slightly with complex I substrate and much more pronounced with complex II substrate. Notably, in both control and HS brain samples, there was no significant coupling between oxidation and phosphorylation (RCR = 1.25). In contrast in liver samples as exhibited in Fig. 4 (C-D) the RCR was approximately 2.2. This suggests that CA may enhance succinate-driven respiration in cortex, potentially as a compensatory mechanism to counteract the complex I deficiency effected b CA. In contrast, in the liver obtained from CA animals, we observed a decrease in RCR, indicating a disruption in the coupling of oxidation and phosphorylation, which may impair ATP synthesis. This alteration was observed exclusively in the presence of complex I substrates. In contrast to CA animals, no significant changes in RCR were noted in animals subjected to HS. It is important to consider that ATP synthesis may not fully engage the capacity of the mitochondrial respiratory chain, a process that can itself be compromised. Therefore, we next assessed the maximal capacity of the respiratory chain to further characterize the pathological changes in mitochondria. The maximal capacity of the respiratory chain (induced by mitochondrial uncoupler, FCCP) was increased in CA animals, in the cortex (with both complex I and complex II mitochondrial substrates, Fig. 5 (A-B)) and in the liver, but only with succinate, as shown in Fig. 5 D. In contrast to CA animals, under HS, the maximal capacity of the respiratory chain was significantly decreased in the liver in the presence of complex I substrates (Fig. 5 C). Thus, HS induces a distinct impairment of mitochondrial function compared to CA. This impairment occurs at the high electron flow through the mitochondrial electron transport chain (ETC). It may be due to either a slowdown at a specific segment of the ETC or a limitation in the entry of complex I substrates into the TCA cycle. One component often limiting electron flow is cytochrome c shuttling electrons between complex III and IV. To investigate the impact of cytochrome c, we treated mitochondria with exogenous cytochrome c and observed how this impacts the maximal capacity of the respiratory chain. As shown on Fig. 6 (A-D), we did not observe any effect of cytochrome c in the cortex or liver of animals subjected to CA. In contrast, in animals subjected to HS, cytochrome c significantly increased the impaired maximal respiratory chain capacity in both cortex and liver. This effect, however, was observed only in the presence of complex I substrates (glutamate/malate), demonstrated in Fig. 6 (A,C). The selective effect on glutamate-supported respiration suggests that electron transfer complex I substrates to the tricarboxylic acid (TCA) cycle may also be impaired. Previous studies have demonstrated that in numerous neurological disorders, the activity of OGDHC is reduced, often as a consequence of neuroinflammation. OGDHC is a key enzyme regulating glutamate utilization. In light of these findings, we assessed OGDHC activity in the examined tissues. OGDHC activity was assessed retrospectively using samples from a tissue bank collected during previous experiments conducted under the same experimental conditions (Fig. 7 ). This approach was necessary because most of the tissue from the original experiments had been consumed in the mitochondrial analyses. In these samples, we did not observe any decrease in OGDHC activity, as shown in Fig. 7 (B-D). On the contrary, OGDHC activity was increased in liver samples from HS animals as illustrated in Fig. 7 (A) and remained unchanged in the other experimental groups. These findings suggest that impaired OGDHC activity is unlikely to account for the observed decrease in respiratory chain function in HS animals. Discussion Here we demonstrate that both cardiac arrest (CA) and hemorrhagic shock (HS) impair mitochondrial function; however, they appear to affect distinct mitochondrial structures. In CA, the predominant mechanism is an increased proton leak across the inner mitochondrial membrane (IMM), whereas in HS the primary defect is insufficient endogenous cytochrome c to sustain electron transfer within the mitochondrial electron transport chain (ETC), likely due to increased permeability of the outer mitochondrial membrane (OMM). The latter interpretation is supported by the restoration of respiration following the addition of exogenous cytochrome c. Three major processes are known to damage biological membranes: oxidative stress, activation of transmembrane ion channels, and activation of mitochondrial phospholipases 4 . Although all these pathways have been reported in ischemic brain tissue 6 , 7 , the predominant mechanism responsible for mitochondrial membrane damage under our experimental conditions remains to be determined. Cerebral ischemia-reperfusion injury has been shown to induce not only mitochondrial damage but also mitochondrial biogenesis (MB), aiming at restoring mitochondrial function. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is recognized as a central regulator of MB under conditions of impaired DO 2 in brain tissue and has been shown to ameliorate mitochondrial dysfunction induced by cerebral ischemia-reperfusion injury 24 . In our experiments, in addition to increased inner mitochondrial membrane permeability, we observed elevated ATP-linked respiration, suggesting activation of compensatory mitochondrial biogenesis. Interestingly, the increase in ATP-linked respiration in brain tissue was predominantly driven by succinate, a complex II substrate, whereas in control brain tissue and in brain tissue from HS animals, ATP-linked respiration was mainly supported by complex I substrates. This substrate shift may compensate for complex I dysfunction induced by CA. However, such metabolic shift may also have detrimental consequences. Succinate is a metabolic intermediate that accumulates during ischemia-reperfusion 25 . Upon reperfusion, rapid succinate oxidation can drive reverse electron transport to complex I, resulting in a burst of reactive oxygen species (ROS) production and subsequent cellular damage 26 . Therefore, although enhanced complex II–supported ATP synthesis may be beneficial, it may simultaneously promote ROS generation. Further studies are required to clarify the net effect of this metabolic adaptation. In contrast to cortical mitochondria, liver mitochondria from control, HS, and CA animals remained well coupled when energized with both complex I and complex II substrates. We did not observe a significant increase in leak respiration following HS neither in brain nor in liver, suggesting that the IMM is preserved in this model. However, impaired electron transport chain activity was evident in both brain and liver mitochondria isolated from HS animals. This impairment was ameliorated by exogenous cytochrome c, but only in the presence of complex I substrates, and was not observed in CA animals. These findings indicate OMM damage with cytochrome c release in HS. This interpretation is consistent with previous reports demonstrating cytochrome c release and activation of apoptotic pathways in the liver following HS 27,28 . An important unresolved question is why cytochrome c release occurs in HS but not in CA, and conversely, why IMM permeability increases in CA but not in HS. This study does not resolve this issue. Potential mechanisms causing OMM damage include activation of the mitochondrial permeability transition pore (mPTP), matrix swelling with secondary OMM rupture. Alternatively, OMM permeability can be increased via activation of proapoptotic proteins such as Bax and Bak. The mPTP-mediated mechanism appears less likely in HS, as we did not observe increased IMM permeability. In contrast, a proapoptotic shift in Bax/Bak/Bcl-2 protein expression has been described in the liver following HS 29,30 . We speculate that differences in hypoxia duration, oxygen availability, and intracellular ATP levels between CA and HS may underlie these distinct patterns of mitochondrial injury; however, the precise mechanisms remain still unclear. Because complex I appeared more affected than complex II in both models, we assessed the activity of OGDHC, a rate-limiting enzyme in mitochondrial glutamate oxidation 31 , 32 . Given the association between glutamate excitotoxicity and neurological disorders, we hypothesized that reduced OGDHC activity might contribute to impaired electron transfer through complex I. However, no decrease in OGDHC activity was detected; in fact, activity was increased in liver mitochondria from HS animals. These findings suggest that the observed defects are primarily attributable to pathological alterations in mitochondrial membrane integrity rather than impaired substrate oxidation. Our findings are clinically relevant, particularly in traumatic CA, where HS frequently precedes circulatory CA 17 . These data provide a rationale for therapeutic strategies targeting both IMM and OMM permeability, as well as modulation of mitochondrial biogenesis. In conclusion, CA represents a mechanistically distinct mitochondrial insult rather than a simple amplification of HS-induced mitochondrial dysfunction (Fig. 8 ). These findings extend our understanding of mitochondrial pathophysiology in critical illness and have direct translational relevance, particularly in traumatic CA. Further elucidation of these mechanisms is essential for optimizing the timing and selection of mitochondria-targeted therapies in shock and resuscitation. Statistical analysis All data are presented as mean ± standard error of the mean (SEM). Group comparisons were performed using ANOVA followed by Holm–Sidak’s multiple comparisons test, except for Fig. 7 , where a two-tailed t-test was applied. Differences were considered statistically significant when p < 0.05. All calculations were performed using GraphPad software (GraphPad Software, Inc., San Diego, CA). Abbreviations CA, Cardiac arrest DO 2 , oxygen delivery ECMO, the extracorporeal membrane oxygenation ETC, mitochondrial electron transport chain HS, hemorrhagic shock IMM, inner mitochondrial membrane MAP, mean arterial pressure MB, mitochondrial biogenesis MOF, multiple organ failure mPTP, mitochondrial permeability transition pore OGDHC, oxoglutarate dehydrogenase complex OMM, outer mitochondrial membrane PBMCs, peripheral blood mononuclear cells PGC-1α, proliferator-activated receptor gamma coactivator-1α PUFAs, polyunsaturated fatty acids ROS, reactive oxygen species ROSC, return of spontaneous circulation UCPs, uncoupling proteins VFCA, Ventricular fibrillation cardiac arrest Declarations Acknowledgements Authors are thankful to Aniko Gutasi and Sonja Lebel for excellent assistance in the operation facility of L. Boltzmann Institute for Traumatology and to all our students (Jasmin Simon, Katharina Topil, Luka Kovačević, Annamaria Shenoda and Carmen Huber) for their restless support and the team of the Center for Biomedical Research and Translational Surgery, Medical University of Vienna. Funding This project is funded by the Austrian Research Promotion Agency (FFG). https://www.ffg.at/. Grant number #FO999887791. Authors' contributions SZ made substantial contributions to the design of the work and the acquisition and analysis of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work. AFS made substantial contributions to the design of the work and the acquisition and analysis of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work. SD made substantial contributions to the acquisition and analysis of data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. ST made substantial contributions to the acquisition and analysis of data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. MK made substantial contributions to the analysis and interpretation of the data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. CL made substantial contributions to the analysis and interpretation of the data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. JF made substantial contributions to the design, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. MO made substantial contributions to the interpretation, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. JG made substantial contributions to the interpretation, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. MH made substantial contributions to the design, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. WW made substantial contributions to the design, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work. AW made substantial contributions to the conception and interpretation of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work. AVK made substantial contributions to the conception and interpretation of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work. Availability of data and material All data files will be available from the digital data repository for published research of the Ludwig Boltzmann Society (https://creed.lbg.ac.at). Competing interests The authors declare that they have no competing interests. References Ji, X. et al. Cerebral and myocardial mitochondrial injury differ in a rat model of cardiac arrest and cardiopulmonary resuscitation. Biomed. Pharmacother Biomedecine Pharmacother . 140 , 111743 (2021). Kohlhauer, M. et al. Brain and Myocardial Mitochondria Follow Different Patterns of Dysfunction After Cardiac Arrest. Shock 56 , 857–864 (2021). Gong, P. et al. Hypothermia-induced neuroprotection is associated with reduced mitochondrial membrane permeability in a swine model of cardiac arrest. J. Cereb. Blood Flow. Metab. Off J. Int. Soc. Cereb. Blood Flow. Metab. 33 , 928–934 (2013). Kozlov, A. V. & Grillari, J. Pathogenesis of Multiple Organ Failure: The Impact of Systemic Damage to Plasma Membranes. Front. Med. 9 , 806462 (2022). Choudhary, R. C. et al. The Role of Phospholipid Alterations in Mitochondrial and Brain Dysfunction after Cardiac Arrest. Int. J. Mol. Sci. 25 , 4645 (2024). Della-Morte, D. et al. Resveratrol pretreatment protects rat brain from cerebral ischemic damage via a sirtuin 1-uncoupling protein 2 pathway. Neuroscience 159 , 993–1002 (2009). Umemura, A., Mabe, H., Nagai, H. & Sugino, F. Action of phospholipases A2 and C on free fatty acid release during complete ischemia in rat neocortex. Effect of phospholipase C inhibitor and N-methyl-D-aspartate antagonist. J. Neurosurg. 76 , 648–651 (1992). Liu, H. et al. Increased cytochrome c in rat cerebrospinal fluid after cardiac arrest and its effects on hypoxic neuronal survival. Resuscitation 83 , 1491–1496 (2012). Andrianova, N. V. et al. Hemorrhagic Shock and Mitochondria: Pathophysiology and Therapeutic Approaches. Int. J. Mol. Sci. 26 , 1843 (2025). Rao, G., Xie, J., Hedrick, A. & Awasthi, V. Hemorrhagic shock-induced cerebral bioenergetic imbalance is corrected by pharmacologic treatment with EF24 in a rat model. Neuropharmacology 99 , 318–327 (2015). Villarroel, J. P. P. et al. Hemorrhagic shock and resuscitation are associated with peripheral blood mononuclear cell mitochondrial dysfunction and immunosuppression. J. Trauma. Acute Care Surg. 75 , 24–31 (2013). Wang, H. et al. Resveratrol Rescues Kidney Mitochondrial Function Following Hemorrhagic Shock. Shock 44 , 173–180 (2015). Warren, M., Subramani, K., Schwartz, R. & Raju, R. Mitochondrial dysfunction in rat splenocytes following hemorrhagic shock. Biochim. Biophys. Acta Mol. Basis Dis. 1863 , 2526–2533 (2017). Sims, C. A. et al. Nicotinamide mononucleotide preserves mitochondrial function and increases survival in hemorrhagic shock. JCI Insight 3, e120182, 120182 (2018). Masuda, T. et al. Protective effect of urinary trypsin inhibitor on myocardial mitochondria during hemorrhagic shock and reperfusion. Crit. Care Med. 31 , 1987–1992 (2003). Chu, M. M. et al. Effect of high-fat enteral nutrition on hepatocyte injury in response to hemorrhagic shock in the rat. World J. Surg. 31 , 1693–1701 (2007). Carenzo, L. et al. Contemporary management of traumatic cardiac arrest and peri-arrest states: a narrative review. J. Anesth. Analg Crit. Care . 4 , 66 (2024). Nistor, M., Behringer, W., Schmidt, M. & Schiffner, R. A. Systematic Review of Neuroprotective Strategies during Hypovolemia and Hemorrhagic Shock. Int. J. Mol. Sci. 18 , 2247 (2017). Yeo, B. T. T. et al. The organization of the human cerebral cortex estimated by intrinsic functional connectivity. J. Neurophysiol. 106 , 1125–1165 (2011). Veith, N. T., Histing, T., Menger, M. D., Pohlemann, T. & Tschernig, T. Helping prometheus: liver protection in acute hemorrhagic shock. Ann. Transl Med. 5 , 206 (2017). Percie du Sert. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 18 , e3000410 (2020). Stommel, A. M. et al. A ventricular fibrillation cardiac arrest model with extracorporeal cardiopulmonary resuscitation in rats: 8 minutes arrest time leads to increased myocardial damage but does not increase neuronal damage compared to 6 minutes. Front. Vet. Sci. 10 , 1276588 (2023). Zavadskis, S. et al. Interaction of pulsed low frequency electromagnetic field (PEMF) with mitochondria. Sci. Rep. 16 , 6681 (2026). Yuan, Y. et al. Mechanism of PGC-1α-mediated mitochondrial biogenesis in cerebral ischemia-reperfusion injury. Front. Mol. Neurosci. 16 , 1224964 (2023). Tretter, L., Patocs, A. & Chinopoulos, C. Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis. Biochim. Biophys. Acta . 1857 , 1086–1101 (2016). Chouchani, E. T. et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515 , 431–435 (2014). Yang, R. et al. Administration of glutamine after hemorrhagic shock restores cellular energy, reduces cell apoptosis and damage, and increases survival. JPEN J. Parenter. Enter. Nutr. 31 , 94–100 (2007). Childs, E. W., Tharakan, B., Hunter, F. A., Tinsley, J. H. & Cao, X. Apoptotic signaling induces hyperpermeability following hemorrhagic shock. Am. J. Physiol. Heart Circ. Physiol. 292 , H3179–3189 (2007). Zingarelli, B. et al. Liver apoptosis is age dependent and is reduced by activation of peroxisome proliferator-activated receptor-gamma in hemorrhagic shock. Am. J. Physiol. Gastrointest. Liver Physiol. 298 , G133–141 (2010). Yao, H. et al. MKK4 Knockdown Plays a Protective Role in Hemorrhagic Shock-Induced Liver Injury through the JNK Pathway. Oxid. Med. Cell. Longev. 5074153 (2022). (2022). Liu, X., Zhang, G., Yu, D. & Han, J. An Emerging Role for OGDHL: From Mitochondrial Energy Metabolism to Neurodevelopmental Disorders. Biology 14 , 1777 (2025). Weidinger, A. et al. Oxoglutarate dehydrogenase complex controls glutamate-mediated neuronal death. Redox Biol. 62 , 102669 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviewers invited by journal 09 Apr, 2026 Editor invited by journal 07 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9282225","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":621566294,"identity":"516f0a08-5fcb-4d3f-b82b-18f9fe009963","order_by":0,"name":"Sergejs Zavadskis","email":"","orcid":"","institution":"Ludwig Boltzmann Institute for Traumatology","correspondingAuthor":false,"prefix":"","firstName":"Sergejs","middleName":"","lastName":"Zavadskis","suffix":""},{"id":621566296,"identity":"7adb6c03-6f33-4cce-8eab-4802346f9226","order_by":1,"name":"Alexander Franz Szinovatz","email":"","orcid":"","institution":"Medical University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"Franz","lastName":"Szinovatz","suffix":""},{"id":621566300,"identity":"f4d70fae-4c6d-4b0a-9867-b754524f1397","order_by":2,"name":"Sergiu Dumitrescu","email":"","orcid":"","institution":"Ludwig Boltzmann Institute for Traumatology","correspondingAuthor":false,"prefix":"","firstName":"Sergiu","middleName":"","lastName":"Dumitrescu","suffix":""},{"id":621566301,"identity":"6a89294c-037f-4f32-bd4f-2b8c8bb144cb","order_by":3,"name":"Sarolta Takacs","email":"","orcid":"","institution":"Ludwig Boltzmann Institute for Traumatology","correspondingAuthor":false,"prefix":"","firstName":"Sarolta","middleName":"","lastName":"Takacs","suffix":""},{"id":621566302,"identity":"1955ac3f-6552-4a08-86ab-b28deeeb7fa4","order_by":4,"name":"Miriam Karas","email":"","orcid":"","institution":"Ludwig Boltzmann Institute for 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Kozlov","email":"data:image/png;base64,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","orcid":"","institution":"Ludwig Boltzmann Institute for Traumatology","correspondingAuthor":true,"prefix":"","firstName":"Andrey","middleName":"","lastName":"Kozlov","suffix":""}],"badges":[],"createdAt":"2026-03-31 15:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9282225/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9282225/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107483792,"identity":"f4d17b8f-3405-4720-b1f0-602f5f056bd4","added_by":"auto","created_at":"2026-04-22 02:29:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":413595,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental models of hemorrhagic shock (HS) and cardiac arrest (CA). \u003cstrong\u003eA\u003c/strong\u003e. Time course of mean arterial pressure (MAP), \u003cstrong\u003eB.\u003c/strong\u003e The levels of lactate, \u003cstrong\u003eC.\u003c/strong\u003ethe base excess. Both lactate levels and base excess were normalized after 24 h (data not shown) while in CA the levels of lactate were increased even after 24 h. BL – baseline, ER – early resuscitation/ROSC, LR – late resuscitation/ROSC.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/34a691af5be523626d73cf84.png"},{"id":107483716,"identity":"a91e9977-f624-4647-8de4-2ad4c248eb13","added_by":"auto","created_at":"2026-04-22 02:29:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":298512,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on the mitochondrial leak respiration (State 2 respiration) in cortex (A and B) and in liver (C and D) in the presence of glutamate malate (A and C) and succinate (B and D). The data are presented as mean ± SEM. Statistical analyses was performed by ANOVA followed by Holm-Sidak’s multiple comparisons test. * - p \u0026lt; 0.05; **** p\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/913f859c2974f2ab6ce9835f.png"},{"id":107481765,"identity":"b5c6bc09-2bc4-46cb-92de-1113e99e386a","added_by":"auto","created_at":"2026-04-22 02:19:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":350455,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on the ATP-synthesis linked respiration (state 3 respiration) in cortex (A and B) and in liver (C and D) in the presence of glutamate malate (A and C) and succinate (B and D). The data are presented as mean ± SEM. Statistical analyses was performed by ANOVA followed by Holm-Sidak’s multiple comparisons test. * - p \u0026lt; 0.05; **** p\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/015a0d512589104c355008ac.png"},{"id":107198447,"identity":"d29c0c90-267b-4ccb-af11-ebc5c47113a6","added_by":"auto","created_at":"2026-04-18 02:24:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":243199,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on the respiratory control ratio (RCR) in cortex (A and B) and in liver (C and D) in the presence of glutamate malate (A and C) and succinate (B and D). The data are presented as mean ± SEM. Statistical analyses was performed by ANOVA followed by Holm-Sidak’s multiple comparisons test. * p\u0026lt;0.05; **** p\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/e240a00297b970e05e33847f.png"},{"id":107481766,"identity":"75910c23-c239-45c6-9782-fa58f5edcba6","added_by":"auto","created_at":"2026-04-22 02:19:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":351669,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on the maximal capacity of electron transport chain in cortex (A and B) and in liver (C and D) in the presence of glutamate malate (A and C) and succinate (B and D). The data are presented as mean ± SEM. Statistical analyses was performed by ANOVA followed by Holm-Sidak’s multiple comparisons test towards sham group. * p\u0026lt;0.05; *** - p \u0026lt; 0.001; **** p\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/466a5b95f8b64ff1035c861a.png"},{"id":107198449,"identity":"6873a0eb-c1c9-4037-a584-675283e9722f","added_by":"auto","created_at":"2026-04-18 02:24:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":304060,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on the susceptibility of mitochondria to exogenous cytochrome c in cortex (A and B) and in liver (C and D) in the presence of glutamate malate (A and C) and succinate (B and D). The data are presented as mean ± SEM. Statistical analyses was performed by ANOVA followed by Holm-Sidak’s multiple comparisons test towards sham group. * p\u0026lt;0.05; ** - p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/56adaa35360341aca6427aa0.png"},{"id":107483791,"identity":"6297b4b5-e9f8-430e-aea9-91813a69ef6a","added_by":"auto","created_at":"2026-04-22 02:29:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":411942,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on \u0026nbsp;\u0026nbsp;the activity of OGDHC in liver (A and B) and in cortex (C and D). These \u0026nbsp;\u0026nbsp;measurements were performed from the separate groups of animals, which \u0026nbsp;\u0026nbsp;underwent the same surgical procedures. The data are presented as mean ± SEM. \u0026nbsp;\u0026nbsp;Statistical analyses was performed by t-test. ** - p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/0bf8eefd00b64616818923ad.png"},{"id":107481936,"identity":"b3a1e351-d7b7-4781-9afb-2d6c34ad2d45","added_by":"auto","created_at":"2026-04-22 02:21:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":284282,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HS and CA on the mitochondrial function in the brain cortex. HS induced similar changes to the cortex in the liver.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/96f78639b266ef44a1b4bab4.png"},{"id":107705285,"identity":"61b7256d-41b8-47ed-851c-20f2efc3052b","added_by":"auto","created_at":"2026-04-24 09:10:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2969453,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9282225/v1/c961dbd8-1f2e-4658-b708-3a0b7e72789d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial dysfunction upon impaired oxygen delivery: cardiac arrest versus hemorrhagic shock","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImpaired oxygen delivery (DO\u003csub\u003e2\u003c/sub\u003e) is a critical factor in cardiac arrest (CA) and hemorrhagic shock (HS). Adequate DO\u003csub\u003e2\u003c/sub\u003e is necessary for sufficient mitochondrial function supporting energy demand in tissues. Impaired DO\u003csub\u003e2\u003c/sub\u003e leads to profound anaerobic metabolism, damage to mitochondria which may cause secondary tissue injury during resuscitation. HS and CA represent two clinically relevant impaired DO\u003csub\u003e2\u003c/sub\u003e, low blood flow for a longer time and no flow for the short time, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is substantial evidence demonstrating impaired mitochondrial function following CA. A decrease in ATP-linked mitochondrial respiration was reported during early return of spontaneous circulation (ROSC) in rats subjected to CA (8 min CA/1 h ROSC; )\u003csup\u003e1\u003c/sup\u003e. However, it has been suggested that mitochondrial dysfunction may not play a predominant role in the very early phase of post-resuscitation injury but instead becomes more prominent during later resuscitation phase. For example, in a rabbit model of CA (10 min CA/24 h ROSC)\u003csup\u003e2\u003c/sup\u003e, mitochondrial dysfunction was more evident at 24 hours of ROSC. At this time an increase in the leak respiration was observed, suggesting enhanced mitochondrial membrane permeability\u003csup\u003e2\u003c/sup\u003e. A similar impairment of mitochondrial membrane permeability was demonstrated in a pig model of CA (8 min CA/24 h ROSC)\u003csup\u003e3\u003c/sup\u003e, and this defect was ameliorated by therapeutic hypothermia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn general, three major processes may contribute to this phenomenon: (i) oxidative damage, (ii) activation of transmembrane ion channels such as uncoupling proteins (UCPs), and (iii) activation of mitochondrial phospholipases\u003csup\u003e4\u003c/sup\u003e. Ongoing oxidative stress and oxidation of polyunsaturated fatty acids (PUFAs) in mitochondrial membranes have been proposed as mechanisms contributing to increased membrane permeability\u003csup\u003e5\u003c/sup\u003e. In addition, elevated levels of mitochondrial uncoupling protein 2 (UCP2) have been associated with ischemic brain damage\u003csup\u003e6\u003c/sup\u003e. Furthermore, activation of phospholipase C and phospholipase A2 was demonstrated in a model of CA as early as 2 minutes after the onset of brain ischemia\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, increased cytochrome c levels were detected in the cerebrospinal fluid of rats subjected to CA (8 min CA/24 h, 48 h, and 7 days ROSC)\u003csup\u003e8\u003c/sup\u003e, with peak concentrations at 24 hours after ROSC. These findings suggest that the release of cytochrome c may be secondary to damage of the outer mitochondrial membrane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is widely accepted that pathological mitochondrial alterations also occur following hemorrhagic shock (HS)\u003csup\u003e9\u003c/sup\u003e. However, information regarding mitochondrial dysfunction in the brain upon HS remains limited. Although it has been suggested that mitochondrial function is impaired in the brain after HS\u003csup\u003e10\u003c/sup\u003e, this issue has not been comprehensively investigated. Instead, mitochondrial dysfunction following HS and resuscitation was observed in peripheral blood mononuclear cells (PBMCs), and it was associated with subsequent immunosuppression\u003csup\u003e11\u003c/sup\u003e. In rats subjected to HS (40 mmHg for 1 h followed by 1h resuscitation), decreased mitochondrial respiratory capacity across all mitochondrial complexes was reported in the kidney\u003csup\u003e12\u003c/sup\u003e. Similarly, HS (40 mmHg for 2 h/no resuscitation) significantly reduced mitochondrial oxygen consumption rate, ATP production, and mitochondrial membrane potential in rat splenocytes\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA decrease in ATP levels in the kidney and liver, along with reduced mitochondrial respiration supported by complex I substrates, was demonstrated following HS (40 mmHg; 60 min resuscitation)\u003csup\u003e14\u003c/sup\u003e. In addition, myocardial ATP and creatine phosphate content were decreased after 60 minutes resuscitation following HS induced 50 mm Hg MAP for 1 h\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOnly a limited number of studies have shown the markers of mitochondrial dysfunction 24 hours after resuscitation or later\u003csup\u003e16\u003c/sup\u003e, even though that post-hemorrhagic mortality is often related to secondary complications such as multiple organ failure (MOF), which typically develop 24 hours or later after the shock phase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, comparing mitochondrial responses in HS and CA addresses a fundamental question in shock biology: whether distinct temporal patterns of impaired oxygen delivery activate shared or divergent molecular pathways leading to mitochondrial dysfunction. Elucidating these mechanisms is crucial not only for advancing the conceptual understanding of bioenergetic failure in critical illness but also for guiding the development of targeted therapeutic interventions in HS and CA. This has also a clinical relevance particularly for traumatic CA, where the phase of HS is followed by the CA\u003csup\u003e17\u003c/sup\u003e and the understanding the underlying mechanisms may have an important therapeutic relevance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe existing body of evidence regarding mitochondrial dysfunction in HS and CA has been generated in heterogeneous experimental models that differ substantially in insult duration, severity, and resuscitation protocols. Such variability precludes direct comparison and does not allow definitive conclusions regarding whether HS and CA activate similar or distinct mitochondrial injury pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe aim\u003c/strong\u003e of the present study was to directly compare mitochondrial alterations induced by HS and CA under standardized experimental conditions characterized by comparable mortality in order to address a fundamental mechanistic question: whether distinct modes of impaired oxygen delivery and progressive hypoperfusion in HS versus abrupt circulatory arrest in CA activate convergent or divergent mitochondrial pathophysiological pathways. All analyses were performed 24 hours after resuscitation, a clinically relevant time point associated with the development of secondary organ injury in both brain and liver.\u003c/p\u003e\n\u003cp\u003eAs for CA, the brain is especially vulnerable to ischemia because of its high metabolic demand and limited energy reserves. Likewise, during HS a prolonged hypoperfusion can lead to brain tissue damage. Among neural tissues, we focused on the cerebral cortex as it primarily mediates cognition and appropriate social responses, and cortical damage often impairs recovery and return to the previous lifestyle\u003csup\u003e18,19\u003c/sup\u003e. Since ischemia-reperfusion after CA also affects peripheral organs, and the liver, playing a central role in systemic metabolism and inflammatory regulation, its compared analysis will give us insights into organ-specific responses. Similarly in HS, liver is a known key factor in prevention of MOF development\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eExperiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and ARRIVE guidelines\u003csup\u003e21\u003c/sup\u003e and was approved by the relevant ministry for the HS-model (TVA Nr. 1358971-2023) and the CA-model (GZ 2023-0.896.943)\u0026nbsp;respectively.\u0026nbsp;Rat CA model used in this study resulted in 20% mortality by 24 hours of resuscitation, while HS model resulted in 10% mortality. There were no mortalities in the SHAM groups. The experimental animals, male outbred Sprague Dawley rats (400 \u0026ndash; 600 g; Charles River Laboratories, Research Models and Services, Germany), were housed in pairs for at least one week for adaptation purposes for the HS experiments, and the minimal adaptation time was doubled for the CA groups, provided with ad libitum access to water and standard lab rodent pellet diet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHS model:\u0026nbsp;\u003c/strong\u003eOn the day of surgery, rats received analgesia with Buprenorphine (0.01 mg/kg s.c., VetViva, Wels, Austria) and Meloxicam (1 mg/kg p.o., Boehringer Ingelheim, Vienna, Austria). After 1 hour, anesthesia was induced with sevoflurane gas (6\u0026ndash;7%, 0.8 L/min; AbbVie GmbH, Ludwigshafen am Rhein, Germany) for 3\u0026ndash;4 minutes in an induction chamber and maintained via nose cone mask (2\u0026ndash;3%, 0.2\u0026ndash;0.3 L/min) for the whole duration of the surgical intervention. The abdomen and right inguinal region were shaved, and an ophthalmic ointment was applied. Animals were placed supine on a Small Animal Physiological Monitoring System (Harvard Apparatus, Holliston, MA, USA). Body temperature was continuously monitored with a rectal probe and maintained using a heating pad integrated with the monitoring system. At the right inguinal region, femoral artery was catheterized (0.6 mm inner diameter and a 1.1 mm outer diameter Silclear\u0026trade; Tubing, Silclear, Hampshire, UK) for blood withdrawal, sampling, and invasive blood pressure monitoring. At the same incision spot, the femoral vein was cathetherized for fluid resuscitation and treatment infusion. After catheterization, animals were allowed to stabilize for 10 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfterwards, controlled blood withdrawal was performed in 0.5-1 mL steps until mean arterial pressure (MAP) reached 40 \u0026plusmn; 5 mmHg. Withdrawn blood was discarded, and total blood loss including samplings did not exceed 50% of the estimated circulating volume. MAP was maintained around the target value of 40 mmHg for 60\u0026ndash;75 minutes with additional small withdrawals as needed. Hemorrhagic shock was confirmed by blood gas analysis indicating anaerobic metabolism (lactate \u0026gt; 3.0 mmol/L; base excess \u0026lt; \u0026ndash;5.0 mEq/L), and shock duration was adjusted (60\u0026ndash;75 minutes) based on these values. At the end of the shock period, the following compounds were administered via femoral vein diluted in a 0.2 mL Ringer\u0026rsquo;s solution bolus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCA-model:\u0026nbsp;\u003c/strong\u003eThe cardiac arrest model has been established by the experimental resuscitation research group, department of emergency medicine, Medical University of Vienna. Detailed experimental protocol and the efficacy of the model was published previously\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn brief, male rats (n=5) were sedated with sevoflurane and received analgesia with piritramide and were mechanically ventilated. Similarly to HS intravenous catheters (Argyle 2.5-french umbilical neonatal vessel catheter) were inserted into the left femoral artery and vein, to determine blood pressure and sampling, respectively. In addition, a venous ECMO drainage cannula (adapted venous cannula, 14GA Venflon BD Luer-Lok, Helsingborg, Sweden) was inserted via the right jugular vein with the tip in the inferior vena cava and used to install a pacing catheter to induce VFCA, as well as an arterial ECMO cannula (custom-made; Dipl.-Ing. Martin Humbs, Valley, Germany) into the right femoral artery. Ventricular fibrillation cardiac arrest (VFCA) was induced by electrical current of 12\u0026thinsp;V/50\u0026thinsp;Hz at a max. of 8\u0026thinsp;mA applied via the pacing catheter lying in the veins next to the right heart. VFCA was defined as VF pattern in the ECG curve and drop of systolic aortic pressure\u0026thinsp;\u0026lt;\u0026thinsp;20\u0026thinsp;mmHg with corresponding loss of arterial pulsation in the arterial pressure monitoring. After 8\u0026thinsp;min of untreated CA, ventilation and ECPR were initiated. After 2\u0026thinsp;min of ECPR, defibrillation with 2 biphasic shocks\u0026nbsp;\u0026agrave;\u0026nbsp;5 Joule (Heart-Start MRx Defibrillator; Philips, Andover, Mass) were administered every 2\u0026thinsp;min of resuscitation if shockable rhythm was present; mechanical ventilation (RR 20/min; FiO\u003csub\u003e2\u003c/sub\u003e 1.0) was continued and epinephrine (10\u0026thinsp;\u0026mu;g/kg BW) was administered i.v. every 2\u0026thinsp;min during ECPR until ROSC was achieved or resuscitation efforts were halted after a maximum of 10\u0026thinsp;min of ECPR. Upon achieving return of spontaneous resuscitation (ROSC) the extracorporeal membrane oxygenation (ECMO) was halted, and the blood from the ECMO circuit was returned via the arterial in-flow cannula. Prompt removal of the venous ECMO cannula occurred after ROSC, and the wound was closed aseptically. Piritramide (3\u0026thinsp;mg/kg BW s.c. at 6-h intervals was used to eliminate any pain in the post-operative phase. Animals were kept normothermic by Harvard apparatus homeothermic blanket contril unit for the whole observation period after CA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSHAM group:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe animals randomly selected to the SHAM group were handled, prepared and cathetherized identically to the shock group animals with the following notes. No blood was withdrawn besides blood samplings for determination of lactate and base excess. The blood collected for samples was substituted by \u003cstrong\u003ethe Ringer\u0026rsquo;s solution.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEuthanasia and tissue sample collection:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eAfter 24 observation period, the animals were put to sleep in a transparent enclosure connected to Sevoflurane (8%, 1.6 L/min) supply. Afterwards, the respiratory rate of the animals was monitored until bradypnea (\u0026lt;30 breaths per minute) was reached. Next, the animal was promptly removed from the box, checked for the absence of the paw pinch reflex to ensure sufficient depth of anaesthesia and decapitated on a rodent DCAP guillotine (Precision Instruments, FL, USA). Next, the cranium of the animal was put on ice, dissected and brain tissue was extracted and cortex was isolated. Cortex and liver were harvested as soon as possible and separately placed in ice-cold CUSTODIOL\u0026reg; (Dr. Franz K\u0026ouml;hler Chemie GmbH, Bensheim, Germany) organ preservation solution. All organ extractions and preparations were performed on Petri dishes placed on ice. Next, a piece of liver tissue and cortex were used for mitochondrial respiration analysis and enzymatic activity of OGDHC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLactate and base excess\u003c/strong\u003e. The blood gas analysis was carried out on ABL800 FLEX (Radiometer, Krefeld, Germany) machine. Each blood sampling required a 0.3 mL pre-draw to flush the catheter, afterwards a small blood sample (approx. 0.1mL was collected in heparinized 1mL syringe. The machine was set to 95\u0026mu;L sample injection mode and the results were corrected for the animal rectal temperature at the time of sample collection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial function:\u003c/strong\u003e We extracted cortex and liver and determined mitochondrial function in freshly prepared tissue homogenates using complex I (glutamate/malate) and complex II (succinate) mitochondrial substrates. All reagents were obtained from Sigma-Aldrich (Vienna, Austria), unless stated otherwise.\u0026nbsp;\u003cbr\u003eFreshly excised liver or cerebral cortex tissue was cut into ~2 \u0026times; 2 mm fragments with surgical scissors and washed extensively in ice-cold tissue preparation buffer (250 mM sucrose, 10 mM Tris, 0.5 mM EDTA (Radnor, PA, USA), 0.05% BSA) to remove residual blood and connective tissue. Tissue was homogenized on ice in glass vials at a buffer to tissue ratio of 4:1 (w/v) for cortex samples and 10:1 (w/v) for liver samples. Mitochondrial respiratory activity was measured with high-resolution oxygraphy (Oroboros O2k, Oroboros Instruments, Innsbruck, Austria) and data was recorded using the Oroboros DatLab software (Oroboros Instruments, Innsbruck, Austria). The instrument was calibrated at 37 \u0026deg;C for at least 30 minutes prior to each measurement using incubation buffer (80 mM KCl, 5 mM KH₂PO₄, 20 mM Tris-HCl, 1 mM iron-free diethylenetriaminepentaacetic acid and 0.1% Bovine Serum Albumin; pH adjusted to 7.4 with 6 M KOH at 22 \u0026deg;C). Here we used previously described experimental protocol\u003csup\u003e23\u003c/sup\u003e. To initiate mitochondrial respiration, we added different mitochondrial substrates to the end concentration of 10 mM, glutamate and malate (Fluka, Buchs, Switzerland) mixture to stimulate complex I or succinate to stimulate complex II. This setup allows to determine leak respiration at the inner mitochondrial membrane, characterized by the presence of reducing substrates in the absence of ADP (non-phosphorylating state).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we stimulated state III respiration and ATP synthesis by adding ADP (1 mM) to each chamber. Afterwards, maximal respiratory capacity of the ETC was measured after stepwise titration with the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.3 \u0026micro;M).Subsequently, \u0026nbsp;the effect of cyt c (2.5 \u0026micro;M), an electron carrier between complex III and IV, on the respiration was assessed. Finally, mitochondrial respiration was terminated by the addition of complex III inhibitor Myxothiazol (12.5 \u0026micro;M) to confirm mitochondrial specificity. All reagents were obtained from Sigma-Aldrich (Vienna, Austria). From the oxygen uptake kinetics, we determined proton leak at the inner mitochondrial membrane, the respiration linked to ATP synthesis, coupling of oxidation and phosphorylation (respiratory control ratio defined as the ratio of\u0026nbsp;\u003cstrong\u003estate 3 / leak respiration\u003c/strong\u003e), maximal capacity of respiratory chain, and impact of exogenous cytc on the respiration.\u0026nbsp;\u003cstrong\u003eThe data was evaluated by calculating the oxygen consumption rate by the homogenized tissue (nmol/min), corrected by substraction of myxothiazol respiratory value and normalization for 10 mg of tissue wet weight.\u0026nbsp;\u003c/strong\u003eAll parameters were determined in the presence of either substrates of complex I or complex II.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivity of oxoglutarate dehydrogenase complex (OGDHC):\u003c/strong\u003e OGDHC activity was determined by monitoring the reduction of NAD⁺ during 2-oxoglutarate turnover catalyzed by the enzyme. Frozen rat tissue was kept fully cold (on dry ice/ice) and homogenized in ice-cold homogenization buffer (0.05 % BSA, 0.5 mM EDTA, 1 % protease inhibitor cocktail, 250 mM sucrose, 10 mM TRIS). For each sample, homogenization buffer was added at 5\u0026times; the wet tissue weight for cerebral cortex and 10\u0026times; the wet tissue weight for liver tissues. The tissue was homogenized using a glass\u0026ndash;Teflon homogenizer coupled to an overhead mixer until no visible clumps remained (around 10x). The homogenate was transferred to cryovials and further lysed by probe sonication (3\u0026times; 500 J for 15 s, with 15 s pauses between pulses) while kept on ice. Following homogenization/sonication, samples were centrifuged for 10 min and then incubated on ice with RIPA buffer added in a 1:4 ratio for 20 min. A dilution series of the tissue lysate was prepared with homogenization buffer (1:5, 1:10, 1:20) and measured in triplicate. A tissue blank was prepared using incubation buffer and homogenization buffer (no homogenate). For the assay, 211 \u0026micro;L of master mix was dispensed into each well, consisting of 200 \u0026micro;L incubation buffer (1 mM CaCl₂, 0.05 mM CoA, 1 mM DTT, 1 mM MgCl₂\u0026middot;6H₂O, 50 mM MOPS, 2.5 mM NAD⁺; pH adjusted to ~7.22), 8 \u0026micro;L TPP, and 3 \u0026micro;L 2-oxoglutarate. Then, 20 \u0026micro;L of diluted tissue lysate was added to the respective wells (final volume 231 \u0026micro;L). Wells were checked to ensure no bubbles were present, and contents were mixed by inverse pipetting when plating. The plate was then run in a plate reader (POLARstar OMEGA, BMG LABTECH) by monitoring NADH formation photometrically over time. OGDHC activity was calculated from a calibration curve, generated using a dilution series of NADH standards. All reagents obtained from Sigma-Aldrich Austria.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe pathogenesis of both hemorrhagic shock (HS) and cardiac arrest (CA) is characterized by transient impairment of blood flow, which is reflected by changes in mean arterial pressure (MAP). In the context of HS, the MAP is moderately reduced to approximately 40 mm Hg, but the duration of this reduction is prolonged up to 60 min. In contrast, during CA, the MAP drops significantly to around 5 mm Hg, but the duration of the event is much shorter (8 minutes). A possibility of different mortality rates between these models can not be excluded, out of CA animals (n\u0026thinsp;=\u0026thinsp;5) one died before the endpoint and was excluded from the study, whereas there were no mortalities in the SHAM (n\u0026thinsp;=\u0026thinsp;6) or HS (n\u0026thinsp;=\u0026thinsp;7) animal groups, although previously 10% mortality was determined in this model.\u003c/p\u003e \u003cp\u003eTo facilitate the comparison of these two experimental models, which exhibit markedly different time courses, we classified the experimental timeline into three distinct phases: the stabilization phase (baseline, BL), the shock phase, and the resuscitation/reanimation phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The duration of each phase is explicitly represented on the horizontal axis of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, with time for HS and CA indicated in minutes. The shock phase was subdivided into three subphases: shock onset, mid-shock, and end of shock. Similarly, the resuscitation/reanimation phase was divided into Early Resuscitation/ ROSC (ER) and Late Resuscitation/ ROSC (LR).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB demonstrates that both HS and CA result in an increase in lactate levels, with CA inducing a more pronounced elevation. In HS, lactate levels returned to baseline values during the LR phase, whereas in CA, lactate levels remained significantly elevated above control values for up to 24 hours. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC highlights a marked decrease in the base excess during both the ER and LR phases, which normalized by 24 hours post-event in both models.\u003c/p\u003e \u003cp\u003eThe data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e strongly suggest that CA-mediated injury is more severe compared to HS-mediated injury. To further investigate the pathological impact of this differential injury, we examined whether these observed differences are reflected in mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalyzing mitochondrial respiratory function in animals subjected to CA, we observed a marked increase in the leak respiration rate in the cortex. This was observed both with complex I (glutamate/malate) and complex II (succinate) substrates, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A-B). A similar, although more modest, increase in the leak respiration rate was noted in the liver when complex I substrate was used, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and a not significant increase was observed with complex II substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings suggest a disruption in the permeability of the inner mitochondrial membrane in animals subjected to CA. In contrast, no significant changes in leak respiration were detected in either the liver or the brain of animals subjected to HS, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A-D). Disruption of inner mitochondrial membrane may impair ATP-synthesis. To address this issue, we next examined ATP-synthesis-linked respiration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e reveals that in animals subjected to CA, the ATP-linked respiration rate was significantly increased in the cortex, both with substrates of complex I and II as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(A-B), and only modestly elevated in the liver with succinate (complex II substrate), Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. No significant changes were observed in liver with complex I substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). No changes were observed in animals subjected to HS. The increase in the ATP-linked respiration may, in theory, compensate for the elevated leak respiration and facilitate the coupling of oxidation and phosphorylation in the cortex. To address this point, we analyzed respiratory control ratio (RCR) characterizing the coupling of oxidation and phosphorylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the potential impact of altered ATP-synthesis linked respiration on oxidative phosphorylation (OxPhos), we calculated the Respiratory Control Ratio (RCR) based on the data presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Our analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(A-B) reveals that despite an increase in the leak respiration in cortical tissue derived from CA animals, the RCR was elevated, slightly with complex I substrate and much more pronounced with complex II substrate. Notably, in both control and HS brain samples, there was no significant coupling between oxidation and phosphorylation (RCR\u0026thinsp;=\u0026thinsp;1.25). In contrast in liver samples as exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(C-D) the RCR was approximately 2.2. This suggests that CA may enhance succinate-driven respiration in cortex, potentially as a compensatory mechanism to counteract the complex I deficiency effected b CA. In contrast, in the liver obtained from CA animals, we observed a decrease in RCR, indicating a disruption in the coupling of oxidation and phosphorylation, which may impair ATP synthesis. This alteration was observed exclusively in the presence of complex I substrates. In contrast to CA animals, no significant changes in RCR were noted in animals subjected to HS.\u003c/p\u003e \u003cp\u003eIt is important to consider that ATP synthesis may not fully engage the capacity of the mitochondrial respiratory chain, a process that can itself be compromised. Therefore, we next assessed the maximal capacity of the respiratory chain to further characterize the pathological changes in mitochondria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe maximal capacity of the respiratory chain (induced by mitochondrial uncoupler, FCCP) was increased in CA animals, in the cortex (with both complex I and complex II mitochondrial substrates, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A-B)) and in the liver, but only with succinate, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. In contrast to CA animals, under HS, the maximal capacity of the respiratory chain was significantly decreased in the liver in the presence of complex I substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Thus, HS induces a distinct impairment of mitochondrial function compared to CA. This impairment occurs at the high electron flow through the mitochondrial electron transport chain (ETC). It may be due to either a slowdown at a specific segment of the ETC or a limitation in the entry of complex I substrates into the TCA cycle. One component often limiting electron flow is cytochrome c shuttling electrons between complex III and IV. To investigate the impact of cytochrome c, we treated mitochondria with exogenous cytochrome c and observed how this impacts the maximal capacity of the respiratory chain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(A-D), we did not observe any effect of cytochrome \u003cem\u003ec\u003c/em\u003e in the cortex or liver of animals subjected to CA. In contrast, in animals subjected to HS, cytochrome \u003cem\u003ec\u003c/em\u003e significantly increased the impaired maximal respiratory chain capacity in both cortex and liver. This effect, however, was observed only in the presence of complex I substrates (glutamate/malate), demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(A,C).\u003c/p\u003e \u003cp\u003eThe selective effect on glutamate-supported respiration suggests that electron transfer complex I substrates to the tricarboxylic acid (TCA) cycle may also be impaired. Previous studies have demonstrated that in numerous neurological disorders, the activity of OGDHC is reduced, often as a consequence of neuroinflammation. OGDHC is a key enzyme regulating glutamate utilization. In light of these findings, we assessed OGDHC activity in the examined tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOGDHC activity was assessed retrospectively using samples from a tissue bank collected during previous experiments conducted under the same experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This approach was necessary because most of the tissue from the original experiments had been consumed in the mitochondrial analyses. In these samples, we did not observe any decrease in OGDHC activity, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(B-D). On the contrary, OGDHC activity was increased in liver samples from HS animals as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(A) and remained unchanged in the other experimental groups. These findings suggest that impaired OGDHC activity is unlikely to account for the observed decrease in respiratory chain function in HS animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we demonstrate that both cardiac arrest (CA) and hemorrhagic shock (HS) impair mitochondrial function; however, they appear to affect distinct mitochondrial structures. In CA, the predominant mechanism is an increased proton leak across the inner mitochondrial membrane (IMM), whereas in HS the primary defect is insufficient endogenous cytochrome c to sustain electron transfer within the mitochondrial electron transport chain (ETC), likely due to increased permeability of the outer mitochondrial membrane (OMM). The latter interpretation is supported by the restoration of respiration following the addition of exogenous cytochrome c.\u003c/p\u003e \u003cp\u003eThree major processes are known to damage biological membranes: oxidative stress, activation of transmembrane ion channels, and activation of mitochondrial phospholipases\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Although all these pathways have been reported in ischemic brain tissue\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, the predominant mechanism responsible for mitochondrial membrane damage under our experimental conditions remains to be determined.\u003c/p\u003e \u003cp\u003eCerebral ischemia-reperfusion injury has been shown to induce not only mitochondrial damage but also mitochondrial biogenesis (MB), aiming at restoring mitochondrial function. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is recognized as a central regulator of MB under conditions of impaired DO\u003csub\u003e2\u003c/sub\u003e in brain tissue and has been shown to ameliorate mitochondrial dysfunction induced by cerebral ischemia-reperfusion injury\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In our experiments, in addition to increased inner mitochondrial membrane permeability, we observed elevated ATP-linked respiration, suggesting activation of compensatory mitochondrial biogenesis.\u003c/p\u003e \u003cp\u003eInterestingly, the increase in ATP-linked respiration in brain tissue was predominantly driven by succinate, a complex II substrate, whereas in control brain tissue and in brain tissue from HS animals, ATP-linked respiration was mainly supported by complex I substrates. This substrate shift may compensate for complex I dysfunction induced by CA.\u003c/p\u003e \u003cp\u003eHowever, such metabolic shift may also have detrimental consequences. Succinate is a metabolic intermediate that accumulates during ischemia-reperfusion\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Upon reperfusion, rapid succinate oxidation can drive reverse electron transport to complex I, resulting in a burst of reactive oxygen species (ROS) production and subsequent cellular damage\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Therefore, although enhanced complex II\u0026ndash;supported ATP synthesis may be beneficial, it may simultaneously promote ROS generation. Further studies are required to clarify the net effect of this metabolic adaptation.\u003c/p\u003e \u003cp\u003eIn contrast to cortical mitochondria, liver mitochondria from control, HS, and CA animals remained well coupled when energized with both complex I and complex II substrates. We did not observe a significant increase in leak respiration following HS neither in brain nor in liver, suggesting that the IMM is preserved in this model. However, impaired electron transport chain activity was evident in both brain and liver mitochondria isolated from HS animals. This impairment was ameliorated by exogenous cytochrome c, but only in the presence of complex I substrates, and was not observed in CA animals. These findings indicate OMM damage with cytochrome c release in HS. This interpretation is consistent with previous reports demonstrating cytochrome c release and activation of apoptotic pathways in the liver following HS\u003csup\u003e27,28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn important unresolved question is why cytochrome c release occurs in HS but not in CA, and conversely, why IMM permeability increases in CA but not in HS. This study does not resolve this issue. Potential mechanisms causing OMM damage include activation of the mitochondrial permeability transition pore (mPTP), matrix swelling with secondary OMM rupture. Alternatively, OMM permeability can be increased via activation of proapoptotic proteins such as Bax and Bak. The mPTP-mediated mechanism appears less likely in HS, as we did not observe increased IMM permeability. In contrast, a proapoptotic shift in Bax/Bak/Bcl-2 protein expression has been described in the liver following HS\u003csup\u003e29,30\u003c/sup\u003e. We speculate that differences in hypoxia duration, oxygen availability, and intracellular ATP levels between CA and HS may underlie these distinct patterns of mitochondrial injury; however, the precise mechanisms remain still unclear.\u003c/p\u003e \u003cp\u003eBecause complex I appeared more affected than complex II in both models, we assessed the activity of OGDHC, a rate-limiting enzyme in mitochondrial glutamate oxidation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Given the association between glutamate excitotoxicity and neurological disorders, we hypothesized that reduced OGDHC activity might contribute to impaired electron transfer through complex I. However, no decrease in OGDHC activity was detected; in fact, activity was increased in liver mitochondria from HS animals. These findings suggest that the observed defects are primarily attributable to pathological alterations in mitochondrial membrane integrity rather than impaired substrate oxidation.\u003c/p\u003e \u003cp\u003eOur findings are clinically relevant, particularly in traumatic CA, where HS frequently precedes circulatory CA\u003csup\u003e17\u003c/sup\u003e. These data provide a rationale for therapeutic strategies targeting both IMM and OMM permeability, as well as modulation of mitochondrial biogenesis.\u003c/p\u003e \u003cp\u003eIn conclusion, CA represents a mechanistically distinct mitochondrial insult rather than a simple amplification of HS-induced mitochondrial dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings extend our understanding of mitochondrial pathophysiology in critical illness and have direct translational relevance, particularly in traumatic CA. Further elucidation of these mechanisms is essential for optimizing the timing and selection of mitochondria-targeted therapies in shock and resuscitation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Group comparisons were performed using ANOVA followed by Holm\u0026ndash;Sidak\u0026rsquo;s multiple comparisons test, except for Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, where a two-tailed t-test was applied. Differences were considered statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All calculations were performed using GraphPad software (GraphPad Software, Inc., San Diego, CA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCA, Cardiac arrest\u003c/p\u003e\n\u003cp\u003eDO\u003csub\u003e2\u003c/sub\u003e, oxygen delivery\u003c/p\u003e\n\u003cp\u003eECMO, the extracorporeal membrane oxygenation\u003c/p\u003e\n\u003cp\u003eETC, mitochondrial electron transport chain\u003c/p\u003e\n\u003cp\u003eHS, hemorrhagic shock\u003c/p\u003e\n\u003cp\u003eIMM, inner mitochondrial membrane\u003c/p\u003e\n\u003cp\u003eMAP, mean arterial pressure\u003c/p\u003e\n\u003cp\u003eMB, mitochondrial biogenesis\u003c/p\u003e\n\u003cp\u003eMOF, multiple organ failure\u003c/p\u003e\n\u003cp\u003emPTP, mitochondrial permeability transition pore\u003c/p\u003e\n\u003cp\u003eOGDHC, oxoglutarate dehydrogenase complex\u003c/p\u003e\n\u003cp\u003eOMM, outer mitochondrial membrane\u003c/p\u003e\n\u003cp\u003ePBMCs, peripheral blood mononuclear cells\u003c/p\u003e\n\u003cp\u003ePGC-1\u0026alpha;, proliferator-activated receptor gamma coactivator-1\u0026alpha;\u003c/p\u003e\n\u003cp\u003ePUFAs, polyunsaturated fatty acids\u003c/p\u003e\n\u003cp\u003eROS, reactive oxygen species\u003c/p\u003e\n\u003cp\u003eROSC, return of spontaneous circulation\u003c/p\u003e\n\u003cp\u003eUCPs, uncoupling proteins\u003c/p\u003e\n\u003cp\u003eVFCA, Ventricular fibrillation cardiac arrest\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are thankful to Aniko Gutasi and Sonja Lebel for excellent assistance in the operation facility of L. Boltzmann Institute for Traumatology and to all our students (Jasmin Simon, Katharina Topil, Luka Kovačević, Annamaria Shenoda and Carmen Huber) for their restless support and the team of the Center for Biomedical Research and Translational Surgery, Medical University of Vienna. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eFunding \u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project is funded by the Austrian Research Promotion Agency (FFG). https://www.ffg.at/. Grant number #FO999887791.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eAuthors\u0026apos; contributions\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSZ\u003c/strong\u003e made substantial contributions to the design of the work and the acquisition and analysis of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAFS\u003c/strong\u003e made substantial contributions to the design of the work and the acquisition and analysis of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e made substantial contributions to the acquisition and analysis of data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eST\u003c/strong\u003e made substantial contributions to the acquisition and analysis of data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMK\u003c/strong\u003e made substantial contributions to the analysis and interpretation of the data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCL\u003c/strong\u003e made substantial contributions to the analysis and interpretation of the data, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJF\u003c/strong\u003e made substantial contributions to the design, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMO\u003c/strong\u003e made substantial contributions to the interpretation, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJG\u003c/strong\u003e made substantial contributions to the interpretation, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMH\u003c/strong\u003e made substantial contributions to the design, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWW\u003c/strong\u003e made substantial contributions to the design, revised it critically for important intellectual content, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAW\u003c/strong\u003e made substantial contributions to the conception and interpretation of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVK\u003c/strong\u003e made substantial contributions to the conception and interpretation of data, drafted the work, approved the version to be published and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eAvailability of data and material\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data files will be available from the digital data repository for published research of the Ludwig Boltzmann Society (https://creed.lbg.ac.at).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eCompeting interests\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJi, X. et al. Cerebral and myocardial mitochondrial injury differ in a rat model of cardiac arrest and cardiopulmonary resuscitation. \u003cem\u003eBiomed. Pharmacother Biomedecine Pharmacother\u003c/em\u003e. \u003cb\u003e140\u003c/b\u003e, 111743 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohlhauer, M. et al. Brain and Myocardial Mitochondria Follow Different Patterns of Dysfunction After Cardiac Arrest. \u003cem\u003eShock\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e, 857\u0026ndash;864 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong, P. et al. Hypothermia-induced neuroprotection is associated with reduced mitochondrial membrane permeability in a swine model of cardiac arrest. \u003cem\u003eJ. Cereb. Blood Flow. Metab. Off J. Int. Soc. Cereb. Blood Flow. Metab.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 928\u0026ndash;934 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozlov, A. V. \u0026amp; Grillari, J. Pathogenesis of Multiple Organ Failure: The Impact of Systemic Damage to Plasma Membranes. \u003cem\u003eFront. Med.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 806462 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoudhary, R. C. et al. The Role of Phospholipid Alterations in Mitochondrial and Brain Dysfunction after Cardiac Arrest. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 4645 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDella-Morte, D. et al. Resveratrol pretreatment protects rat brain from cerebral ischemic damage via a sirtuin 1-uncoupling protein 2 pathway. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cb\u003e159\u003c/b\u003e, 993\u0026ndash;1002 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmemura, A., Mabe, H., Nagai, H. \u0026amp; Sugino, F. Action of phospholipases A2 and C on free fatty acid release during complete ischemia in rat neocortex. Effect of phospholipase C inhibitor and N-methyl-D-aspartate antagonist. \u003cem\u003eJ. Neurosurg.\u003c/em\u003e \u003cb\u003e76\u003c/b\u003e, 648\u0026ndash;651 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, H. et al. Increased cytochrome c in rat cerebrospinal fluid after cardiac arrest and its effects on hypoxic neuronal survival. \u003cem\u003eResuscitation\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e, 1491\u0026ndash;1496 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrianova, N. V. et al. Hemorrhagic Shock and Mitochondria: Pathophysiology and Therapeutic Approaches. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 1843 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao, G., Xie, J., Hedrick, A. \u0026amp; Awasthi, V. Hemorrhagic shock-induced cerebral bioenergetic imbalance is corrected by pharmacologic treatment with EF24 in a rat model. \u003cem\u003eNeuropharmacology\u003c/em\u003e \u003cb\u003e99\u003c/b\u003e, 318\u0026ndash;327 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillarroel, J. P. P. et al. Hemorrhagic shock and resuscitation are associated with peripheral blood mononuclear cell mitochondrial dysfunction and immunosuppression. \u003cem\u003eJ. Trauma. Acute Care Surg.\u003c/em\u003e \u003cb\u003e75\u003c/b\u003e, 24\u0026ndash;31 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. et al. Resveratrol Rescues Kidney Mitochondrial Function Following Hemorrhagic Shock. \u003cem\u003eShock\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, 173\u0026ndash;180 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarren, M., Subramani, K., Schwartz, R. \u0026amp; Raju, R. Mitochondrial dysfunction in rat splenocytes following hemorrhagic shock. \u003cem\u003eBiochim. Biophys. Acta Mol. Basis Dis.\u003c/em\u003e \u003cb\u003e1863\u003c/b\u003e, 2526\u0026ndash;2533 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSims, C. A. et al. Nicotinamide mononucleotide preserves mitochondrial function and increases survival in hemorrhagic shock. \u003cem\u003eJCI Insight\u003c/em\u003e 3, e120182, 120182 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasuda, T. et al. Protective effect of urinary trypsin inhibitor on myocardial mitochondria during hemorrhagic shock and reperfusion. \u003cem\u003eCrit. Care Med.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 1987\u0026ndash;1992 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu, M. M. et al. Effect of high-fat enteral nutrition on hepatocyte injury in response to hemorrhagic shock in the rat. \u003cem\u003eWorld J. Surg.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 1693\u0026ndash;1701 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarenzo, L. et al. Contemporary management of traumatic cardiac arrest and peri-arrest states: a narrative review. \u003cem\u003eJ. Anesth. Analg Crit. Care\u003c/em\u003e. \u003cb\u003e4\u003c/b\u003e, 66 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNistor, M., Behringer, W., Schmidt, M. \u0026amp; Schiffner, R. A. Systematic Review of Neuroprotective Strategies during Hypovolemia and Hemorrhagic Shock. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 2247 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeo, B. T. T. et al. The organization of the human cerebral cortex estimated by intrinsic functional connectivity. \u003cem\u003eJ. Neurophysiol.\u003c/em\u003e \u003cb\u003e106\u003c/b\u003e, 1125\u0026ndash;1165 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeith, N. T., Histing, T., Menger, M. D., Pohlemann, T. \u0026amp; Tschernig, T. Helping prometheus: liver protection in acute hemorrhagic shock. \u003cem\u003eAnn. Transl Med.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 206 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePercie du Sert. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. \u003cem\u003ePLoS Biol.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, e3000410 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStommel, A. M. et al. A ventricular fibrillation cardiac arrest model with extracorporeal cardiopulmonary resuscitation in rats: 8 minutes arrest time leads to increased myocardial damage but does not increase neuronal damage compared to 6 minutes. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1276588 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZavadskis, S. et al. Interaction of pulsed low frequency electromagnetic field (PEMF) with mitochondria. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 6681 (2026).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan, Y. et al. Mechanism of PGC-1α-mediated mitochondrial biogenesis in cerebral ischemia-reperfusion injury. \u003cem\u003eFront. Mol. Neurosci.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 1224964 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTretter, L., Patocs, A. \u0026amp; Chinopoulos, C. Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e. \u003cb\u003e1857\u003c/b\u003e, 1086\u0026ndash;1101 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChouchani, E. T. et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e515\u003c/b\u003e, 431\u0026ndash;435 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, R. et al. Administration of glutamine after hemorrhagic shock restores cellular energy, reduces cell apoptosis and damage, and increases survival. \u003cem\u003eJPEN J. Parenter. Enter. Nutr.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 94\u0026ndash;100 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChilds, E. W., Tharakan, B., Hunter, F. A., Tinsley, J. H. \u0026amp; Cao, X. Apoptotic signaling induces hyperpermeability following hemorrhagic shock. \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e \u003cb\u003e292\u003c/b\u003e, H3179\u0026ndash;3189 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZingarelli, B. et al. Liver apoptosis is age dependent and is reduced by activation of peroxisome proliferator-activated receptor-gamma in hemorrhagic shock. \u003cem\u003eAm. J. Physiol. Gastrointest. Liver Physiol.\u003c/em\u003e \u003cb\u003e298\u003c/b\u003e, G133\u0026ndash;141 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao, H. et al. MKK4 Knockdown Plays a Protective Role in Hemorrhagic Shock-Induced Liver Injury through the JNK Pathway. \u003cem\u003eOxid. Med. Cell. Longev.\u003c/em\u003e 5074153 (2022). (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, X., Zhang, G., Yu, D. \u0026amp; Han, J. An Emerging Role for OGDHL: From Mitochondrial Energy Metabolism to Neurodevelopmental Disorders. \u003cem\u003eBiology\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 1777 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeidinger, A. et al. Oxoglutarate dehydrogenase complex controls glutamate-mediated neuronal death. \u003cem\u003eRedox Biol.\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 102669 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cardiac arrest, hemorrhagic shock, mitochondria, liver, brain cortex","lastPublishedDoi":"10.21203/rs.3.rs-9282225/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9282225/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCardiac arrest (CA) and hemorrhagic shock (HS) have similar mortality but differ in oxygen delivery patterns: HS is a progressive low-flow state, while CA causes abrupt no-flow ischemia followed by reperfusion. Whether these distinct conditions lead to similar mitochondrial dysfunction remains unclear. Rats were subjected to either CA (8 min, followed by extracorporeal resuscitation and 24 h recovery, n\u0026thinsp;=\u0026thinsp;5) or HS (MAP 40 mmHg for 1 h, followed by 24 h recovery, n\u0026thinsp;=\u0026thinsp;7), with SHAM controls (n\u0026thinsp;=\u0026thinsp;6). Shock severity was assessed via lactate and base excess. After 24 h, brain and liver tissues were analyzed for mitochondrial respiration and oxoglutarate dehydrogenase complex (OGDHC) activity. Both CA and HS impaired mitochondrial function, but via different mechanisms. CA primarily disrupted the inner mitochondrial membrane, increasing leak respiration. In contrast, HS impaired electron transfer between complexes III and IV, likely due to leak of cytochrome c through outer membrane permeability. This defect was more pronounced with with complex I substrates compared to complex II substrate. In the brain, CA was associated with increased succinate-driven respiration, suggesting activation of compensatory reaction. These findings indicate that CA and HS induce distinct mitochondrial injuries, affecting inner versus outer membranes, respectively, with implications for targeted therapeutic strategies.\u003c/p\u003e","manuscriptTitle":"Mitochondrial dysfunction upon impaired oxygen delivery: cardiac arrest versus hemorrhagic shock","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-18 02:24:52","doi":"10.21203/rs.3.rs-9282225/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"286503421472817241382698666131241862954","date":"2026-05-18T06:20:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-10T00:40:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-07T12:18:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T02:39:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-02T02:38:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-31T15:36:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"312f941b-9020-4466-bf69-f99712fde81e","owner":[],"postedDate":"April 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"286503421472817241382698666131241862954","date":"2026-05-18T06:20:52+00:00","index":47,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66147118,"name":"Health sciences/Cardiology"},{"id":66147119,"name":"Health sciences/Diseases"},{"id":66147120,"name":"Health sciences/Medical research"},{"id":66147121,"name":"Biological sciences/Neuroscience"},{"id":66147122,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-18T02:24:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-18 02:24:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9282225","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9282225","identity":"rs-9282225","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}