a b s t r a c t

cytochrome c, an electron carrier that normally resides in the mitochondrial intermembrane space, may translo- cate to the cytosol under ischemic and Hypoxic conditions and contribute to mitochondrial permeability transi- tion pore opening. In addition, reperfusion of brain tissue following ischemia initiates a cell death cascade that includes cytochrome c-mediated induction of apoptosis. Further studies are needed to determine the contribu- tion of cytochrome c in the regulation of cell death, as well as its value as an in vivo prognostic marker after car- diac arrest and resuscitation.

(C) 2015

Introduction

The cytochrome complex (CYTc) is a small heme protein of the inner mitochondrial membrane. Unlike other cytochromes, CYTc is highly sol- uble and is an essential component of the electron transport chain [1]. Although it undergoes oxidation and reduction, it does not bind oxygen but transfers electrons between complexes III and IV [2,3]. In mitochon- drial electron transport chain, the heme group of CYTc accepts electrons from the b-c1 complex and transfers electrons to the cytochrome oxi- dase complex [3,4].

Immediately after restoration of spontaneous circulation (ROSC), patients enter the post-resuscitation phase during which several factors may affect outcome [5]. Despite the advances in cardiopulmonary re- suscitation (CPR) and post-resuscitation care, prognosis of cardiac arrest victims remains dismal. Systemic biomarkers provide little information, whereas many of the accepted predictors of poor outcome in comatose survivors of cardiac arrest are unreliable [6]. Although research has been focused on the mitochondrial permeability transition pore (mtPTP) and its role in post-resuscitation apoptosis, data from various studies indi- cate that CYTc may play a pivotal role in the pathophysiology of post- cardiac arrest syndrome.

? Source of support: none.

* Corresponding author at: National and Kapodistrian University of Athens, Medical School, MSc “Cardiopulmonary Resuscitation,” Hospital “Henry Dunant,” 107 Mesogion Ave, 11526, Athens, Greece. Tel.: +30 2110121756; fax: +30 2110121758.

E-mail address: [email protected] (A. Chalkias).

Cardiac arrest and Post-cardiac arrest syndrome

Cardiac arrest is a major cause of death, as approximately 300,000 arrested people are treated annually by medical personnel. The annual in- cidence of emergency medical services-treated out-of-hospital ventricular fibrillation arrest is 17 per 100,000, and survival to hospital discharge is 10.7%, whereas the reported incidence of in-hospital cardiac arrest is 1-5 per 1000 admissions, and survival to hospital discharge is 17.6% [5].

Despite the term post-cardiac, the pathophysiological cascade of post-cardiac arrest syndrome is activated by the onset of cardiac arrest and results in 4 key components with common pathophysiological ori- gin [7]. These are the post-cardiac arrest myocardial dysfunction, the post-cardiac arrest brain injury, the systemic ischemia/reperfusion (I/R) response, and the persistent precipitating underlying pathology. The se- verity of these disorders is not uniform and varies in individual patients based on the patient’s prearrest state of health, the severity of the ische- mic insult, the cause and duration of cardiac arrest, the time to CPR, the time to ROSC, and the quality of post-resuscitation care [7,8].

The time frame of post-cardiac arrest syndrome includes 4 distinct time-defined phases. The immediate post-arrest phase is defined as the first 20 minutes after ROSC, the early post-arrest phase is the period be- tween 20 minutes and 6-12 hours after ROSC, the intermediate phase is between 6-12 and 72 hours, whereas the recovery period is the period extending beyond 3 days [6]. Although therapeutic aggressive interven- tions may be more effective during the early and intermediate phases, specific attention should be given to all 4 phases of post-cardiac arrest syndrome [8]. Causes of death in this setting can be divided into neuro- logical causes, resulting from ischemic-anoxic encephalopathy, and he- modynamic causes, leading to multiple organ failure [5,8].

http://dx.doi.org/10.1016/j.ajem.2015.09.006

0735-6757/(C) 2015

The cytochrome c

Cytochrome c is a small, very stable hemoprotein containing covalent- ly bound heme c as a prosthetic group and functions as an electron shuttle between complex III and complex IV of the respiratory chain [3,9]. Cyto- chrome c is synthesized in the cytosol as a single polypeptide chain of 104 amino acid residues (apoprotein), and upon translocation to the mi- tochondria, it is covalently bound to the heme prosthetic group [10]. Ac- cording to crystallographic data, CYTc appears roughly as a sphere with the diameter of 3.4 nm [9]. In mitochondria, at least 15% of CYTc is bound to acidic phospholipids of the inner mitochondrial membrane via both electrostatic and hydrophobic interactions [11], whereas the re- maining CYTc is loosely attached to the inner mitochondrial membrane, as a result of weak electrostatic interactions, and can be readily mobilized [2,12]. Loosely bound CYTc is implicated in electron transport, inhibition of Reactive oxygen species formation, and prevention of oxidative stress, whereas tightly bound CYTc is probably bound to cardiolipin which appears to be necessary for the insertion of CYTc into mitochondri- al membranes [13]. Cardiolipin-bound CYTc does not participate in elec- tron shuttling of the respiratory chain but may account for the peroxidase activity recently attributed to CYTc. Cytochrome c can catalyze several reactions such as hydroxylation and aromatic oxidation and shows peroxidase activity by oxidation of various electron donors [11].

Cytochrome c and programmed cell death

Apoptosis is the process of programmed cell death characterized by specific morphological cellular changes and death. These changes in- clude blebbing, cell shrinkage, nuclear fragmentation, chromatin con- densation, and chromosomal DNA fragmentation [14]. Upon apoptotic stimuli, CYTc is released into the cytosol where, in the presence of aden- osine triphospate (ATP), it mediates the allosteric activation and heptaoligomerization of the adaptor molecule apoptosis-protease activating factor 1 (Apaf-1), generating apoptosomes [15]. Each apoptosome is a complex molecule that can recruit and activate caspase-9, leading to proteolytic self-processing under the regulation of several Heat shock proteins [16]. The aforementioned processes result in the catalytic maturation of caspase-9 and other caspases that eventu- ally mediate the biochemical and morphological features of apoptosis. However, the caspase cascade can be activated by other soluble mito- chondrial proteins upon apoptosis induction, which enhance the neu- tralization of the caspase-inhibitory proteins [9]. Active caspase-9 then initiates apoptosis by cleaving and thereby activating executioner caspases [17]. Although it was shown in 2000 that mammalian cells lacking CYTc could not activate caspases in response to mitochondrial pathway stimulation [18], Hao et al [19] reported that the electron transport function of CYTc is independent of its ability to engage Apaf- 1 and induce apoptosome formation and caspase activation. Of note, ex- cessive caspase-1 activity can cause pyroptosis, a nonapoptotic type of programmed cell death characterized by plasma membrane rupture and the release of proinflammatory intracellular contents [20,21].

Inhibition of caspases protects cells only transiently against cell

death because once mitochondria are irreversibly permeabilized, cell death proceeds regardless of caspase activity [17]. This caspase- independent cell death may result from the loss of essential mitochon- drial functions and/or from the apoptogenic function of the flavoprotein apoptosis-inducing factor (AIF) and endonuclease G [22]. Once in the cytosol, both proteins are able to translocate to the nucleus where they promote DNA fragmentation and apoptotic cell death [23,24].

Antiapoptotic B-cell lymphoma-2 (Bcl-2) family proteins prevent the release of both CYTc and AIF apoptotic protease, whereas in spite of their stabilization effect on the mitochondrial outer membrane, Bcl- 2 proteins may also be involved in the direct binding of apoptotic prote- ase activating molecules as regulatory elements further downstream from the mitochondrial apoptotic signals. Interestingly, acute stress seems to suppress the mitochondrial apoptotic pathway of neutrophils

consequent to downregulation of proapoptotic Bcl-2 proteins [25], whereas the increased plasma levels of CYTc likely originate from or- gans that suffer I/R injury during cardiac arrest and resuscitation [26].

An important proapoptotic stimulus is the sustained elevation in cal- cium levels [27]. The release of small amounts of CYTc leads to an inter- action with the IP3 receptor on the endoplasmic reticulum (ER), causing ER calcium release. The overall increase in calcium triggers a massive re- lease of CYTc, which then acts in the positive feedback loop to maintain ER calcium release through the IP3 receptors [24]. Of note, these calcium events are linked to the coordinate release of CYTc from all mitochon- dria, identifying a feed-forward mechanism resulting in augmented CYTc release that amplifies the apoptotic signal [24].

oxidative damage to the mitochondria can occur on proteins in-

volved in respiration as well as lipids critical for respiratory protein function. The inner mitochondrial membrane lipid cardiolipin can be- come oxidized, resulting in failure of oxidative phosphorylation [28]. In addition, ROS production during the reperfusion phase and subse- quent Mitochondrial dysfunction activate the intrinsic apoptotic path- way. Cytochrome c is actively targeted by stress signaling during I/R, and phosphorylation of CYTc partially inhibits respiration [29]. In con- trast to phosphorylated CYTc, this dephosphorylated CYTc may have the full capability to bind to Apaf-1 and trigger downstream caspase ac- tivation [30]. In the progression of brain reperfusion injury, mitochon- drial respiration begins to diminish, and mitochondrial dysfunction eventually culminates in cell death possibly due to peroxidation of cardiolipin by CYTc [31-33]. Cytochrome c binds to cardiolipin in the inner mitochondrial membrane, thus anchoring its presence and keep- ing it from releasing out of the mitochondria and initiating apoptosis [29]. Extensive peroxidation of cardiolipin has been demonstrated dur- ing brain reperfusion [29], whereas multiple studies have demonstrated the neuroprotective and antiapoptotic effect of therapies designed to activate cell survival signaling and prevent apoptotic release of CYTc [34,35]. Whereas the initial attraction between cardiolipin and CYTc is electrostatic because of to the extreme positive charge on the latter, the final interaction is hydrophobic, where a hydrophobic tail from cardiolipin inserts itself into the hydrophobic portion of cytochrome.

On the other hand, the data regarding the effects of CYTc on necrosis or paraptosis are scarce. Necrosis is a form of cell injury that results in the pre- mature death of cells in living tissue by autolysis [14]. Necrosis is caused by factors external to the cell or tissue-such as infection, toxins, or trauma-that result in the unregulated digestion of cell components [22]. Cells that die because of necrosis do not follow the apoptotic signal trans- duction pathway, but rather various receptors are activated that result in the loss of cell membrane integrity and an uncontrolled release of cell prod- ucts into the extracellular space [22]. Necrosis may occur because of exter- nal or internal factors, as well as by components of the immune system [23]. Paraptosis was originally identified in 2000 and often exists in paral- lel with apoptosis [36,37]. Paraptosis is a type of programmed cell death, morphologically distinct from apoptosis and necrosis [24]. The defining features of paraptosis are cytoplasmic vacuolation, independent of cas- pase activation and inhibition, and lack of apoptotic morphology [36,37]. Paraptosis lacks several of the hallmark characteristics of apo- ptosis, such as membrane blebbing, chromatin condensation, and nucle- ar fragmentation [38]. Like apoptosis and other types of programmed cell death, the cell is involved in causing its own death, and gene expres- sion is required. This is in contrast to necrosis, which is nonprogrammed cell death that results from injury to the cell. Paraptosis can be triggered by the tumor necrosis factor receptor family member TAJ/TROY, the ap- optotic protein Bcl-2-associated X (Bax), and human insulin-like growth factor I receptor and takes place in neurodegenerative condi-

tions such as the post-cardiac arrest brain injury [5,39,40].

Cytochrome c and cardiac arrest

At the beginning of cardiac arrest, the abrupt loss of effective blood

flow is followed by a temporal increase in blood flow due to the

hypotension-induced baroreflex withdrawal, which increases the vas- cular resistance for 30-60 seconds [8,41]. During this period, the resulting I/R injures mitochondria, increasing the concentration of cir- culating CYTc [42]. The systemic and pulmonary blood flow continues until the pressure gradient between the aorta and the right side of the heart as well as between the pulmonary artery and the left atrium has been completely dissipated. Although even small amounts of CYTc may have a significant effect on initiation of caspase activation and ap- optosis within cardiomyocytes [42], there is evidence that CYTc release may continue even after this time point [43]. Using a murine model of 8- minute untreated cardiac arrest, Han et al [42] identified multiple spe- cific defects in mitochondrial function following cardiac arrest. They re- ported reduced electron flow activity in specific complexes and loss of CYTc, increased ROS generation leading to H₂O₂ production, and oxida- tion of some mitochondrial proteins by tyrosine nitration. Although at the end of eighth minute they observed an increased generation of ROS from complex I and a significant loss of mitochondrial CYTc, H₂O₂ generation from complex I was maximized within 30 minutes following reperfusion, whereas impairments in electron flow at complex IV be- came very pronounced after 60 minutes of reperfusion. In addition, Leung et al [43] submitted isolated neonatal rabbit hearts to 60 minutes of warm crystalloid cardioplegic arrest followed by 120 minutes of re- perfusion and reported that cardioplegic arrest was associated with mi- tochondrial permeability transition pore opening, Bax translocation, cytochrome c release, ROS production, and electron transport chain dysfunction.

Furthermore, the damage of fatty acids of the membrane phospho- lipids by ROS lead to a progressive increase in membrane permeability, whereas the activation of phospholipase leads to phospholipid degrada- tion [44,45]. In addition, water enters mitochondria through the mito- chondrial ATP-sensitive K+ channel, which opens in response to an influx of K+ ions. Although the initial movement of water into mito- chondria is a part of the early protective response to ischemia, counter- ing the effect of matrix shrinkage that increases the intermembrane space and is deleterious to respiration [8], the impaired metabolism of mitochondrial fatty acids results in the accumulation of free fatty acids, long-chain acyl CoA, and acylcarnitine, which together with the products of phospholipid degradation incorporate into membranes and impair their function, further releasing CYTc [46]. Interestingly, there is evidence that the release of CYTc is a self-healing mechanism; when the circulating CYTc is taken up by the injured cells, their meta- bolic capacity improves, enhancing their resistance to injury [47,48]. However, additional studies are necessary to confirm this hypothesis.

The role of Cytochrome C in post-cardiac arrest syndrome

Cytochrome c is an electron carrier that normally resides in the mi- tochondrial intermembrane space and is actively involved in oxidative phosphorylation [49]. Nevertheless, it may translocate to the cytosol under ischemic and hypoxic conditions [50]. Interestingly, the release of CYTc from mitochondria during I/R injury has been confirmed by sev- eral studies; however, only 1 study has demonstrated that CYTc is re- leased into the bloodstream and activates apoptosis after resuscitation from cardiac arrest. In this study, release of CYTc was associated with ac- tivation of executioner caspase-3, impaired left ventricular function, and decreased survival [26]. Although the specific mechanism(s) responsible for CYTc release after ROSC is not yet known, a particular mechanism may relate to the severity of post-cardiac arrest tissue injury [51]. Several studies have shown that CYTc release outside the cell occurs without concomitant release of larger molecules such as lactate dehydrogenase, which is considered a marker of cell necrosis with disruption of cell membrane [52-55]. However, a progressive rise in plasma CYTc during the post-resuscitation period is inversely related to survival outcome re- gardless of the responsible mechanism(s) or the reversal of whole-body ischemia [26].

After ROSC, I/R cause intracellular calcium overload, generation of ROS, and mitochondrial injury causing detachment of CYTc from the mi- tochondrial membrane [8]. Cytochrome c release occurs very rapidly and can further progress during reperfusion, suggesting that mitochon- dria can initiate cascades that amplify injury in reperfusion [42]. How- ever, there may be prosurvival signals that promote healing and repair as a response to injury coincident with the injury amplification path- ways. Ayoub et al [56] measured CYTc serially in rats successfully resus- citated from an 8-minute interval of untreated ventricular fibrillation after 8 minutes of closed-chest resuscitation and found that, in survivors, plasma CYTc gradually increased to levels that did not exceed 2 g/mL, returning to baseline within 48-96 hours, while in nonsurvivor rats, CYTc rapidly increased to levels that substantially exceeded those observed in survivor rats, without reversal before demise from cardio- vascular dysfunction. Huttemann et al [29] have shown that both the amount of CYTc in the mitochondria and its redox state may account for the increased ROS production in I/R. The release of CYTc in cytosol and its subsequent entrance in the bloodstream, when cell rupture oc- curs, may be a key step in reperfusion-induced apoptosis/necrosis and Myocardial stunning due to a lower ATP production and an increased ROS production [26]. Indeed, ATP, ROS, and intracellular calcium over- load are strongly implicated in post-resuscitation myocardial dysfunc- tion [9]. In addition, the cytosolic calcium increases and activates various enzymes, causing alterations in contractile proteins and phos- pholipid degradation [45,57]. Loss of phospholipid asymmetry in the plasma membrane with the externalization of phosphatidylserine fur- ther facilitates the recognition of dying cells by macrophages, whereas when phagocytic cells are normally absent, apoptotic cells and their fragments lyse in a process similar to necrosis, that is, “secondary necro- sis” or “post-apoptotic necrosis” [58]. In apoptotic cells, part of the phosphatidylserine pool is translocated to the cell surface, whereas the distribution of the other major phospholipids is not affected [59,60]. Phospholipid scrambling is stimulated by calcium, and recent findings suggest that transmembrane protein 16 F is the calcium responsive component in the plasma membrane and of critical impor- tance for phosphatidylserine exposure by apoptotic cells [61]. Distur- bances in calcium homeostasis and other stresses, such as inhibition of glycosylation and oxidative stress, trigger the ER response, which in- volves the release of signaling proteins and consists of multiple parallel events, such as the apoptotic pathways of stress-activated protein ki- nases (SAPKs) 1 and 2 [58]. SAPKs are closely related to the mitogen- activated protein kinases that regulate cell survival as SAPK1 stimulates the mitochondrial pathway of apoptosis, which is mediated by an aden- osine monophosphate-dependent activation of SAPK2 [62].

CYTc translocation is directly related with the post-resuscitation for- mation of mtPTP. The mtPTP is defined as an increase in the permeability of the mitochondrial membranes to molecules. It is the opening of a pro- tein pore that is formed in the inner membrane of the mitochondria under certain pathological conditions and can lead to mitochondrial swelling and cell death [63]. The mtPTP appears to play a key role in damage caused by ischemia, as occurs in cardiac arrest, although it re- mains closed during ischemia but opens during reperfusion [64,65]. Un- fortunately, little is known about the structure of mtPTP. The only mtPTP components that have been identified so far are the translocation protein located in the mitochondrial outer membrane and cyclophilin-D in the mitochondrial matrix [66,67].

The combined effect of ATP depletion, sarcolemmal calcium in- crease, and increased oxidative stress occurring after the loss of CYTc contributes to mtPTP opening and further mitochondrial depolarization, swelling, and rupture of the external mitochondrial membrane, resulting in efflux of CYTc either through the mtPTP or by selective per- meabilization of the outer mitochondrial membrane without mtPTP opening [26,29,49]. Opening of the mPTP allows molecules of up to 1500 Da to enter the mitochondrial matrix along with water and sol- utes, leading to mitochondrial swelling, unfolding of inner mitochondri- al membrane cristae, and disruption of the outer mitochondrial

membrane, ultimately causing CYTc release to the cytosol [68]. Initially, the influx of water in mitochondria counters the effect of matrix shrink- age which is deleterious to respiration [68], although mtPTP opening can lead to necrosis through ATP depletion, to apoptosis via CYTc re- lease, or to autophagy [69].

Permeabilization of the outer mitochondrial membrane is regulated by the Bcl-2 family proteins, which interact with the outer mitochondri- al membrane and disrupt the normal mitochondrial respiratory func- tion [70]. This effect is modulated by Bcl-2 through oligomerization and formation of channel-like structures in the outer mitochondrial membrane. Among the various family members, Bax, Bcl-2 homologous antagonist/killer, and truncated BH3 interacting domain death agonist are considered proapoptotic and favor formation of the channel-like structures in the outer mitochondrial membrane. Release of CYTc is fur- ther facilitated during I/R by peroxidation of cardiolipin consequent to mitochondrial calcium overload and ROS production [49]. The perme- abilized mitochondria, in turn, release the AIF and endonuclease G fac- tors that translocate to the nucleus and initiate apoptosis through chromatin condensation and deoxyribonucleic acid fragmentation [71]. Considering that CYTc is released in a progressive as well as prom- inent manner, it might represent a potential peripheral biomarker of post-resuscitation reperfusion-associated apoptosis. Besides apoptosis, caspases have been associated with paraptosis, which facilitate the phos- phorylation of caspase-9, enhancing I/R-induced cell death [58]. All these form a vicious cycle that may contribute to the emergence of post- cardiac arrest syndrome and to low survival of cardiac arrest victims.

The release of CYTc outside the cell occurs without concomitant re-

lease of larger molecules, such as lactate dehydrogenase, which is considered a marker of cell necrosis, with disruption of the cell mem- brane [54]. As CYTc levels increase after ROSC, despite reversal of whole-body ischemia, it can be concluded that I/R trigger processes leading to a progressive rise in plasma CYTc after return of metabolically adequate blood flow. Huda et al [72] demonstrated no signs that apo- ptosis is activated in neutrophils after tourniquet-induced ischemia in human skeletal muscle 4 hours after reperfusion.

After ROSC, the management and prognosis of post-cardiac arrest brain injury remain suboptimal, and two-thirds of those dying after ad- mission to intensive care unit following out-of-hospital cardiac arrest die of neurological injury. Again, mitochondria play multiple roles in the setting of cerebral I/R injury both as a cause of neuronal damage and as a site of intracellular injury. Reperfusion of brain tissue following ischemia initiates a cell death cascade that includes CYTc-mediated in- duction of apoptosis [29]. However, considering that the blood-brain barrier of various cerebral regions breaks down in a selective and specif- ic manner after ROSC, allowing serum proteins to enter the brain microfluid environment of certain areas [35], the apoptotic process of neuronal cells may not be associated only with the locally released CYTc but also with its circulating blood levels. Therefore, cerebrospinal fluid’s (CSF) CYTc may be a potential biomarker of post-cardiac arrest brain injury, whereas its measuring levels in the bloodstream could also serve as a general marker of organ damage. Liu et al [73] reported that CYTc concentrations increased in CSF following hypoxic-ischemic brain injury in a pediatric rat model. In addition, Ahlemeyer et al [74] found that addition of CYTc to culture media was toxic to both untreated and staurosporine-treated mixed-neuronal cultures derived from chicks or rats. Also, Piel et al. [48] reported that intravenous doses of CYTc can cross the cardiac cell membrane, migrate into mitochondria, and improve cardiac function, which may decrease the severity of post-cardiac arrest brain injury. After intravenous injection, mitochon- drial levels of CYTc remained persistently elevated for 24 hours, and only supranormal levels of mitochondrial CYtc completely overcame the inhibition of myocardial cytochrome oxidase. The authors conclud- ed that the effective half-life of exogenous CYTc is less than 48 hours when injected at 24 hours after sepsis induction [48]. Although overall survival improved after injection of CYTc, the results of Piel et al indicate that increasing the frequency of injection of exogenous CYTc to every 24

hours may improve cardiac function and hemodynamics, thus improv- ing neurologic outcome.

On the other hand, Boiarinov et al [75] studied the effect of intracarotid injection of CYTc on the course of the early post- resuscitation period in a rat hemorrhagic model and reported that CYTc normalized the disturbed metabolic processes in the brain, re- duced the structural changes consequent upon total ischemia, stabilized the activity of the cardiovascular system, and contributed to restoration of functional activity of the central nervous system in the early post-re- suscitation period. Collectively, these studies show that CYTc, when taken up by injured cells, improves metabolic capacity and allows the cells to better withstand an injury. However, additional studies are nec- essary to confirm this hypothesis while it remains unknown whether the increased CYTc in CSF can exacerbate or attenuate cell death. Of note, hypothermia, the only clinically proven effective neuroprotective therapy, inhibits late CYTc translocation in rodents following global is- chemia [76].

Conclusion

There is evidence that CYTc might serve as a potential prognostic and therapeutic biomarker after cardiac arrest. However, there are cru- cial gaps in our knowledge, and further research is needed for the full clarification of its role. In particular, the release of CYTc after ROSC has to be evaluated in both experimental and clinical studies because it re- mains unknown whether and to what extent CYTc increases after ROSC. Furthermore, the role of CYTc in the pathophysiology and severity of post-cardiac arrest syndrome has to be elucidated. CYTc may also serve in the evaluation of CPR and therapeutic interventions during the post-resuscitation period. Research on these issues might further help identify its role during and after cardiac arrest.

Conflicting interests

None.

Acknowledgments

Nothing to acknowledge.

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