Life Sciences 96 (2014) 53–58

Contents lists available at ScienceDirect

Life Sciences

j ourna l homepage: www.e lsev ie r .com/ locate / l i fesc ie

Citicoline (CDP-choline) protects myocardium from ischemia/reperfusion injury via inhibiting mitochondrial permeability transition

Luz Hernández-Esquivel a, Natalia Pavón b, Mabel Buelna-Chontal c, Héctor González-Pacheco d,Javier Belmont a, Edmundo Chávez a,⁎a Departamento de Bioquímica, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico, D. F. Mexicob Departamento de Farmacología, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico, D. F. Mexicoc Departamento de Biomedicina Cardiovascular, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico, D. F. Mexicod Unidad Coronaria, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico, D. F. Mexico

⁎ Corresponding author at: Departamento de BioqCardiología, Ignacio Chávez, Juan Badiano # 1, Tlalpan, D.F

E-mail address: echavez@salud.gob.mx (E. Chávez).

0024-3205/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.lfs.2013.12.026

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 7 October 2013Accepted 17 December 2013

Keywords:CDP-cholineOxidative stressHeart reperfusionRat heartMitochondriaPermeability transition

Aims: Oxidative stress emerges after reperfusion of an organ following an ischemic period and results in tissuedamage. In the heart, an amplified generation of reactive oxygen species and a significant Ca2+ accumulationcause ventricular arrhythmias and mitochondrial dysfunction. This occurs in consequence of increased non-specific permeability. A number of works have shown that permeability transition is a common substrate thatunderlies the reperfusion-induced heart injury. The aim of this work was to explore the possibility that CDP-choline may circumvent heart damage and mitochondrial permeability transition.Main methods: Rats were injected i.p. with CDP-choline at 20 mg/kg body weight. Heart electric behavior wasfollowed during a closure/opening cycle of the left coronary descendent artery. Heartmitochondriawere isolatedfrom rats treated with CDP-choline, and their function was evaluated by analyzing Ca2+ movements, achieve-ment of a high level of the transmembrane potential, and respiratory control. Oxidative stress was estimated

following the activity of the enzymes cis-aconitase and superoxide dismutase, as well as the disruption of mito-chondrial DNA.Key findings: This study shows that CDP-choline avoided ventricular arrhythmias and drop of blood pressure.Results also show that mitochondria, isolated from CDP-choline-treated rats, maintained selective permeability,retained accumulated Ca2+, an elevated value of transmembrane potential, and a high ratio of respiratory con-trol. Furthermore, activity of cis-aconitase enzyme and mDNA structure were preserved.Significance: This work introduces CDP-choline as a useful tool to preserve heart function from reperfusiondamage by inhibiting mitochondrial permeability transition.

© 2013 Elsevier Inc. All rights reserved.

Introduction

The reduction of blood flow, hence, oxygen deprivation in hearttissue by coronary occlusion results in a severe insult to the structureand function of myocardial cells. The therapy has focused on restoringoxygen supply through the use of proteolytic enzymes, implantationof stents, or coronary bypass. However, when blood flow is abruptlyre-established, heart injury occurs. Reperfusion results in a significantproblem since an increase in reactive oxygen species (ROS), formedthrough dissimilar mechanisms, generates oxidative stress (Petersonet al., 1985; Bobadilla et al., 2001; Sloan et al., 2012). ROS depress theactivity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA 2), a mem-brane Ca2+ pump that plays a crucial role in calcium handling, whichis determinant in myocardial contractility. The resulting cellular Ca2+

overload contributes markedly to the myocardial injury (Luo and

uímica, Instituto Nacional de., México, D. F. 014080, México.

ghts reserved.

Anderson, 2013). The aforementioned leads to contractile dysfunctionand ventricular arrhythmias. There is a generalized consensus thatischemia-reperfusion injury has a common substrate: mitochondrialpermeability transition (Di Lisa et al., 2003; Weiss et al., 2003; Brookeset al., 2004).

In this context, a wide variety of molecules have been introduced asinhibitors of permeability transition and then as protectors againstreperfusion-induced myocardial damage. Among these molecules, cy-closporin A has shown in in vivo and in vitro experiments that it avoidsreperfusion damage in cardiomyocytes (Griffiths et al., 2000) and inwhole rat heart (Arteaga et al., 1992). By the same token, in a previouswork, we showed that octylguanidine, through its interaction withadenine nucleotide translocase, inhibits permeability transition and, inconsequence, protects the myocardium from ischemia reperfusiondamage (Parra et al., 2005; Pavón et al., 2009).

The nucleotide cytidine-5-diphosphocholine, CDP-choline, has beenintroduced as a molecule that protects brain tissue against ischemicstroke (Secades et al., 2006) and provides neuroprotection againstischemic spinal cord injury (Turkkan et al., 2010). Regarding the effect

54 L. Hernández-Esquivel et al. / Life Sciences 96 (2014) 53–58

of CDP-choline on heart injury, Yilmaz et al. (2008)have shown that thismolecule prevents reperfusion-induced heart damage. Nevertheless, itsmechanism of action is not clearly elucidated yet. These authors pro-posed that citicoline acts through the activation of muscarinic receptorsand vagal pathways. Zweifler (2002) discussed that CDP-choline avoidsbrain ischemia by stabilizing cell membranes and reducing free radicalgeneration. Coskun et al. (2010) concluded that the beneficial cardio-vascular effects of the drug are due to its attenuation of oxidative stress.The presentworkwas aimed at exploring the possibility that the protec-tive effect of CDP-choline on heart ischemia/reperfusion would beexerted by avoiding the opening of themitochondrial non-specific pore.

Materials and methods

This investigation was performed following the procedures pub-lished by NIH for the care of laboratory animals and was approved bythe Institutional Bioethics Committee with the number 13-752. CDP-choline was administered intraperitoneally (n = 6) at a dose of125mg/kg body weight, 5 min before starting the experiment. Thetime and the dose were chosen after CDP-choline injection had beenperformed at different times and establishing a dose–response relation-ship (not shown). It should be noted that CDP-choline has an LD50 inrats, when administered intravenously, of 4.15 g/kg (Grau et al.,1983). The untreated group (n = 6) only received NaCl solution(0.9%). Male Wistar rats, weighing between 280 and 300 g, were anes-thetized with sodium pentobarbital (55 mg/kg, i.p.). Rats were main-tained under assisted respiration through tracheotomy. The chest wasopened by left thoracotomy; the left coronary artery was isolated nearits origin by an intramural 6-0 silk loop. Occlusion of the artery wasattained by passing a short tube over the vessel and clamping it firmlywith the thread, the ischemic period lasted 5min. This timewas chosenaccording to previous publishedworks (Arteaga et al., 1992; Parra et al.,2005; Pavón et al., 2009). The removal of the clamp started reperfusion.One lead-II surface electrocardiograph was used to monitor heart rate.The incidence and the time course of the different types of arrhythmiaswere compared between treated and untreated groups. Blood pressurewas measured with a pressure transducer attached to a femoral cannu-la. Mitochondria were isolated from the ischemic/reperfused hearttissue following the standard centrifugation method of the tissuehomogenized in 250 mM sucrose–1 mM EDTA. Protein was measuredby the Lowry method (1951). The infarct size measurement was per-formed from the left ventricle; after having been perfused with isotonicsaline solution, the tissue was frozen at 4 °C and cut into ~1-mm-thicktransverse slices. The slices were incubated in 1% triphenyltetrazoliumchloride (TTC) in sodium phosphate buffer, pH 7.4, at 37 °C for 20min. Then the sliceswere immersed in 12% formalin to enhance the con-trast between stained and unstained tissue. The differentially stainedtissues were scanned. The ischemic area at risk (left ventricle) and theinfracted area (unstained by TTC)weremeasured using the Image J pro-gram, from NIH. Oxygen consumption was analyzed polarographicallywith a Clark-type electrode. Calcium movements were followed bydual spectrometry at 675–685 nm using the metallochromic indicatorArsenazo III. Transmembrane potential was estimated in a spectropho-tometer at 525–575 nm, using the positively charged dye Safranin.Mitochondrial swelling was followed by changes in absorbance at540 nm. Aconitase activity was analyzed according to Hausladen andFridovich (1994) as follows: mitochondrial protein was solubilized byadding 0.05% Triton X-100 containing 25 mM phosphate, pH 7.2,followed by the addition of 0.6 mM manganese sulfate, 1 mM citrate,and 0.1 mM NADP. The formed cis-aconitase was measured spectro-photometrically at 240 nm. Membrane lipid peroxidation was deter-mined in mitochondria as the concentration of TBARS. MitochondrialDNAwas isolated as described byGarcía et al. (2005). The geneticmate-rialwas analyzed in 0.7% agarose gel, visualized by adding ethidiumbro-mide and using a UV lamp. Superoxide dismutase enzymatic activitywas determined in mitochondria by non-denaturating 8% acrylamide

gel electrophoresis and nitro blue tetrazolium staining, as described byPérez-Torres et al. (2009).

Results

Fig. 1 shows the EKG tracings from control heart rats and those treat-ed with CDP-choline subjected to cardiac ischemia/reperfusion. As ob-served, Fig. 1A shows that heart from rat treated with CDP-cholinewas in sinus rhythm. In Fig. 1B, it is observed that the treatment withCDP-choline avoided the appearance of reperfusion-induced arrhyth-mias, and the heart remained in sinus rhythm. In addition, it is illustrat-ed that blood pressure was maintained within normal values (lowertrace). Fig. 1C shows that heart from rat untreated with CDP-cholineremained in sinus rhythm; however, after removal of the occlusion, re-perfusion arrhythmias became apparent (Fig. 1D). These arrhythmiaslasted until the end of the experiment, approximately 10 min later. Itshould be noted that blood pressure was almost abolished in thelower trace.

An objective quantification of the latter results is shown in Fig. 2.Fig. 2A illustrates that, during the occlusion period, the heart rate was380 ± 20 beats per min in rats of the untreated group. In the first min-ute of the reperfusion period, an increase in heart rate occurred, that is,610 ± 20 beats per min. After this time, cardiac frequency in untreatedrats reached up to 650 ± 20 beats per min and remained unchangeduntil the end of the experiment (closed symbols). Remarkably, in CDP-choline-treated rats, ventricular tachycardia remained stable (opensymbols). Fig. 2B shows that, in control rats (closed symbols), bloodpressure diminished from 50 to 10 mmHg during the reperfusionphase. In contrast, in CDP-choline-treated rats, the magnitude of thisvariable remained unchanged (open symbols). This parameter reflectsthe efficiency of heart beats in these rats.

Aimed at knowing whether or not CDP-choline reduces the infarctsize, the experiment shown in Fig. 3 was performed. As observed, inhearts treated with CDP-choline, the infarct size was diminished byaround 50%.

The next experimentswere performed to assess the protective effectof CDP-choline on mitochondrial membrane leakage. To meet such apurpose, mitochondria were isolated from the left ventricle of CDP-choline-treated and -untreated rats, after having been subjected to thereperfusion process. A characteristic of permeability transition is theinability of mitochondria to retain accumulated Ca2+. As observed intrace a of Fig. 4A, indeed, untreated mitochondria, isolated from thereperfused left ventricle, were unable to retain Ca2+ as a consequenceof pore opening. Trace b shows that the opposite occurred in mitochon-dria isolated from CDP-choline-treated mitochondria; as shown, theseorganelles maintain Ca2+ accumulated inside the matrix. As a compar-ison, trace c shows Ca2+ retention by control mitochondria. Mitochon-drial swelling is also an important characteristic of the permeabilitytransition process. The experiment shown in trace a of Fig. 4B indicatesthat the addition of 50 μM Ca2+ to CDP-choline-untreated mitochon-dria from ischemic/reperfused rats induced a fast and large amplitudeswelling. The observed swelling in these mitochondria contrasts withthat obtained when the experiment was performed with mitochondriaisolated from CDP-choline-treated rats or control mitochondria (tracesb and c, respectively). As shown, Ca2+ addition did not induce a changein the volume of these mitochondria. To further investigate the resis-tance conferred by citicoline to permeability transition, the ability tobuild up a transmembrane electrical gradient (Δψ) was analyzed.Fig. 4C reveals that the addition of 50 μM Ca2+ to mitochondria fromreperfused hearts induced a fast collapse of Δψ (trace a). A differentpicture was observed in mitochondria isolated from reperfused heartsfrom rats treated with citicoline. As shown in trace b, the transmem-brane potential was maintained at high levels despite the addition of50 μM Ca2+, similarly to control mitochondria (trace c). It should benoted that the uncoupler CCCP was added in order to attain a total col-lapse of Δψ.

Fig. 1. Electrocardiogram and blood pressure tracings of CDP-choline-treated rats (A and B) and CDP-choline untreated rats (C and D). The bars at the side of blood pressure represent100 mmHg. The tracing represents one of six separate experiments.

55L. Hernández-Esquivel et al. / Life Sciences 96 (2014) 53–58

In the next experiment, the activity of the enzyme aconitase wasmeasured in mitochondria isolated from reperfused rat hearts treatedand untreated with citicoline. Table 1 shows that, in untreated mito-chondria, oxidative stress promoted the inhibition of the enzyme activ-ity by around 45%. In contrast to the above, CDP-choline effectivelyprotected the enzyme from oxidative stress

Mitochondrial DNA is also a target exposed to oxidative damage.As illustrated in Fig. 5, the genetic material isolated from reperfused un-treated hearts was considerably disrupted (track I/R). This contrastswith the intactness observed in mDNA obtained from control hearts

Fig. 2. Time course of cardiac frequency and blood pressure in citicoline-untreated and-treated rats. Panel A shows cardiac frequency. At the indicated time, the left coronaryartery was occluded and the reperfusion was initiated where indicated. Closed symbolsindicate heart frequency in untreated rats. Open symbols indicate heart frequency inciticoline-treated rats. Panel B shows the analysis of blood pressure during ischemia andreperfusion periods. Closed symbols indicate blood pressure from untreated rats. Opensymbols correspond to citicoline-treated rats. The values are expressed as mean ± SEM.P = b0.05.

and mDNA isolated from hearts of CDP-choline-treated rats (track Crtland track I/R + Cit, respectively).

Another important issue, related with an increased oxidative stress,is linked to a diminution in the activity of the oxyradical-scavengingsystem. To assess this, the activity of mitochondrial superoxide dismut-ase (SOD) was analyzed. Fig. 6 indicates that, certainly, a diminution byabout 65% was found in the activity of SOD, as caused by ischemia-reperfusion (I/R); however, citicoline partially avoided inactivation ofthis enzyme (I/R + Cit).

The experimental results shown in Fig. 7 illustrate the protectiongranted by CDP-choline on TBARS generation by membrane lipidsperoxidation. As observed, after reperfusion-induced oxidative stress,an increased amount of TBARS was produced in mitochondria (I/R).This amount was considerably diminished after treatment with CDP-choline (CIT + I/R), a value closely similar to that of the control (Crtl).

The experiment shown in Table 2 was designed to explore furtherthe protective effect of citicoline on mitochondria isolated from the leftventricle of the reperfused heart. As shown, respiratory control frommitochondria not subjected to ischemia/reperfusion attained a highvalue, using succinate or malate glutamate as substrates; however, thevalues decreased in mitochondria subjected to ischemia/reperfusion.Remarkably, mitochondria from citicoline-treated rats maintained ahigh value of respiratory control with glutamate/malate as well aswith succinate.

To explore the possibility that the protection exerted by citicolinewould be due to a direct effect on cardiacmitochondria and not throughcell membrane receptors, in vitro experiments were carried out. Toreach this goal, H2O2 was used to induce oxidative stress, according toCortés-Rojo et al. (2007).Fig. 8A, trace a, shows that after the addition

Fig. 3. The reducing effect of CDP-choline on the infarct size. Infarct sizewas expressed as apercentage of ischemic area at risk. Experimental conditions as described under theMate-rials and Methods section. n = 5. The values represent mean ± S.D.

Fig. 4. The protective effect of CDP-choline onmitochondrial functions. Heartmitochondriawere isolated from control and reperfused ventricles fromuntreated and citicoline-treated rats.Panel A shows mitochondrial Ca2+ movements. Protein (2 mg) was added to the medium containing 125 mM KCl; 10 mM succinate; 2 mM phosphate; 10 mM HEPES, pH 7.3; 2 μgoligomycin; 5 μg rotenone; 100 μMADP; 50 μMCaCl2; and 50 μMArsenazo III. Panel B illustrates the protective effect of CDP-choline onmitochondrial swelling. Themediumwas similarto that described for Panel A, except that Arsenazowas not added.Where indicated, 50 μMCa2+was added. Panel C illustrates the protective effect of citicoline on Ca2+-induced collapseof the transmembrane electrical gradient. Where indicated, 50 μM CaCl2 or 2 μM CCCP was added. The incubation medium was essentially as described for panel A, except that 10 μMSafranin was added instead of Arsenazo III.

56 L. Hernández-Esquivel et al. / Life Sciences 96 (2014) 53–58

of 100 μM H2O2 the resulted oxidative stress induced permeabilizationof the inner membrane, in such a way that mitochondria resulted un-able to retain Ca2+. As observed (trace b), Ca2+ was retained insidethe matrix after adding CDP-choline regardless of the addition of100 μMH2O2. Trace c shows control mitochondria without the additionof H2O2. Similarly, citicoline protected fromH2O2-inducedmitochondri-al swelling and collapse of the membrane potential (not shown). Toreinforce the notion that protection by CDP-choline must be exertedby circumventing oxidative damage, the experiment shown in Fig. 8Bwas performed. As observed, citicoline was not able to inhibit perme-ability transition induced by carboxyatractyloside (CAT), a reagentthat induces pore opening through a mechanism that is independentfromoxidative stress. Trace a shows the failure ofmitochondria to retainCa2+ when CAT was present in the incubation mixture. Trace b illus-trates that the addition of CDP-choline had no effect on CAT-inducedmembrane permeabilization. Control mitochondria are illustrated intrace c; trace d indicates that addition of citicoline alone had no effectonmembrane permeability. Trace e shows that cyclosporin A effectivelyinhibited permeability transition as induced by CAT. The above exper-iments would indicate that CDP-choline may have a direct effect onmitochondria through penetrating cell membrane. To this respect,Villa et al. (2012) demonstrated that CDP-choline is able to affectthe activities of Krebs cycle in mitochondria located inside synapticvesicles.

Discussion

Among a number of reperfusion manifestations are severe arrhyth-mias and tissue injury (Chávez et al., 1996; Téllez et al., 1999; Parraet al., 2005; Carvajal et al., 1999) following heart ischemia. At the sub-cellular level, reperfusion stimulates mitochondrial Ca2+ overloadand oxidative stress (Hayashi, 2000). The latter brings about opening

Table 1Cis-aconitase activity.

Condition nmol cis-aconitate/min/mg

Control 360 ± 65Reperfused 200 ± 10Reperfused + citicoline 352 ± 45*

Protection by CDP-choline of the aconitase activity ofmitochondria isolated fromhearts ofcontrol, reperfused, and hearts of citicoline-treated rats. Experimental conditions were asdescribed under the Materials and Methods section. *P b 0.05 vs. reperfused. n = 6.

of the non-specific transmembrane pore, a process that leads to mito-chondrial dysfunction and underlies the pathogenesis of heartreperfusion damage (Brookes et al., 2004; Hayashi, 2000). From theabove, it can be inferred that protection of mitochondria against Ca2+

accumulation and ROS-dependent permeabilization results in a resis-tance to reperfusion-induced injury. To achieve this objective, differentchemicals have been introduced into the field, among them are the im-munosuppressant cyclosporin A (Arteaga et al., 1992; Halestrap et al.,1993), the alkylamine octylguanidine (Pavón et al., 2009), the analgesicketorolac (Chávez et al., 1996), the antagonist of the estrogen receptortamoxifen (Ek et al., 2008), and the pineal hormone melatonin(Lochner et al., 2013); all of them inhibitors of the non-specific poreopening. The results in this work indicate that CDP-choline providesresistance against heart injury derived from ischemia/reperfusion. Asdemonstrated, this drug inhibited the incidence of arrhythmias andreduced ventricular tachycardia in rat hearts undergoing ischemia/reperfusion episodes. Different hypotheses have been proposed toexplain the mechanism of action by which CDP-choline protects fromthe deleterious effect of oxidative stress and Ca2+ overload. Zweifler

Fig. 5. Protection by CDP-choline against oxidative stress-induced mitochondrial DNAdamage. Experimental conditions were as described under the Materials and Methodssection. Mitochondria were isolated from hearts subjected to ischemia/reperfusion, fromrats treated or not with CDP-choline. The lines show molecular weight standards (MW),DNA from control mitochondria (Crtl), DNA from CDP-choline-treated mitochondria(I/R + Cit), and DNA from mitochondria isolated from untreated rats (I/R).

Fig. 6. Superoxide dismutase activity inmitochondria isolated from ischemic/reperfused hearts. As indicated, A shows the activity of the standard enzyme (St), untreated rats (Crt), ische-mic reperfused hearts plus CDP-choline (I/R-Cit), and ischemic reperfused without citicoline (I/R). B illustrates the analysis in pixels of the activity. C shows the activity of the enzyme inunits per 200 μg protein. Experimental conditions were as described under the Materials and Methods section. The values represent the average ± SD of five different determinations.P b 0.001.

57L. Hernández-Esquivel et al. / Life Sciences 96 (2014) 53–58

(2002) reported that the efficacy of CDP-choline to protect the brain ofthose patients with ischemic injury due to mild cerebral artery stroke isexerted by stabilizing cell membranes and reducing ROS generation. Inturn, Savci et al. (2002) showed that intracerebroventricular adminis-tration of CDP-choline increases blood pressure. Coskun et al. (2010)discussed that the cardiovascular protection is due to the drug-induced attenuation of oxidative damage. Mitochondrial dysfunctionafter reperfusion is characterized, mainly, by its inability to retain accu-mulated Ca2+ and to maintain the transmembrane potential at a highvalue, as well as by undergoing a wide amplitude swelling (Pavónet al., 2009; Zazueta et al., 2007). This cascade of events must be due,plausibly, to an increase in ROS generation. As evidenced in this work,heart reperfusion caused the inhibition of the cis-aconitase enzyme,the diminution of the superoxide dismutase enzyme activity, and thedisruption of mDNA. All of these processes, found in mitochondria iso-lated from reperfused hearts, are relevant markers of oxidative stressand were inhibited by CDP-choline. Ghosh et al. (2010) reported that,in cerebral ischemia/reperfusion, citicoline diminishes mitochondrialmembrane peroxidation and release of cytochrome c. Yilmaz et al.(2008) and Zweifler (2002) argued that the action of CDP-cholinewould be at the plasma membrane level. To this respect, it is importantto note that our results from in vitro experiments indicate that citicolinemay also act directly on mitochondria because the addition of the

Fig. 7. Evaluation of the protection by CDP-choline on reperfusion-induced TBARS gener-ation. TBARS amount was determined by incubating mitochondria (2 mg protein) in 1 mlbasic medium, as described for Fig. 1, during 30 min. Then 1.0 ml of 20% acetic acid and0.8% 2-thiobarbituric acid was added. The mixture was then heated in boiling water for45 min. After cooling, TBARS were extracted into 2 ml n-butanol. After centrifugation,the butanol layer was measured at 532 nm. A standard curve of MDA was preparedwith 1,3,3,3,-tetraetoxypropane. The values represent the average ± SD of six differentdeterminations (P = 0.05). Analysis was performed with unpaired t-Student.

chemical tomitochondria, undergoing an oxidative stress after H2O2 ad-dition, avoided ROS-induced opening of the non-specific pore.

Conclusions

In conclusion, based on results here presented, it seems challengingto propose that the protection exerted by CDP-choline involves theinhibition of the ROS-induced mitochondrial permeability transition.An important finding must be taken into account, i.e., the fact thatCDP-choline effectively reduced the infarct size, plausibly, through pres-ervation of mitochondrial function. This result is in close agreementwith the reports of Jeong et al. (2013), who demonstrate that the com-pound HS-1793, an analogue of resveratrol, protects rat heart againsthypoxia reoxygenation, reducing infarct size via attenuating mitochon-drial damage. Thus, this work contributes to support this nucleotide as auseful drug to prevent the harmful heartmanifestations that occur afterreperfusion. Its favorable effects in the treatment of intracerebralhemorrhage, even at a long term treatment and at high doses, i.e., 1 gper 12 hours (Secades et al., 2006), attested to the efficacy of the drugand its low toxicity.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commer-cial or financial relationships that could be construed as a potential conflict of interest.

Table 2nAgO/min/mg protein.

Substrate State 4 State 3 RC

Glutamate-MalateControl 40 ± 12.5 200 ± 5.5 5 ± 0.3Ischemia-reperfusion 60.2 ± 2 90 ± 5 1.5 ± 0.2I/R + citicoline 40 ± 2* 195 ± 4.3* 4.8 ± 0.4*

SuccinateControl 75 ± 5 300 ± 22 4 ± 0.3Ischemia-reperfusion 160 ± 14 250 ± 19.3 1.5 ± 0.6I/R + Citicoline 90 ± 9* 350 ± 54* 3.8 ± 0.33*

Respiratory control of mitochondria isolated from hearts of control, untreated, andciticoline-treated rats. Mitochondria (0.6 mg/ml incubation mixture) were added to themedium containing 125 mM KCl, 10 mMHEPES, and 3 mM phosphate; where indicated,10 mM malate, 10 mM glutamate, or 10 mM succinate was used. In addition, 2 μM ADPwas added. Respiratory control was calculated by the relationship between nAg oxygenconsumed by Sate 3 respiration, in the presence of ADP over State 4, in the absence ofADP. *P N 0.05 vs. I/R n = 6.

Fig. 8. The in vitro effect of CDP-choline added tomitochondria isolated from control heartrats. Oxidative stress was achieved after adding 100 μM H2O2. Mitochondrial protein(2 mg) was added to the incubation medium. Panel A illustrates calcium movement. Theadditions were as follows: in trace a, 50 μM CaCl2 plus 100 μM H2O2; trace b contained,in addition to Ca2+ and H2O2, 20 μM CDP-choline; in trace c, only Ca2+ was added.Panel B shows the failure of CDP-choline to inhibit CAT-induced permeability transition.The additions were essentially as described for Panel A, except that, in trace a, 0.5 μMcarboxyatractyloside (CAT) was added. In trace b, 0.5 μM CAT plus 20 μM CDP-cholinewas added. Control mitochondria are indicated in trace c. In trace d, 20 μM citicolinewas added, and in trace e, 0.5 μM cyclosporin A plus CAT was added. The incubationmedia were similar to those described for Fig. 3.

58 L. Hernández-Esquivel et al. / Life Sciences 96 (2014) 53–58

References

Arteaga D, Odor A, López RM, Contreras G, Pichardo J, García E. Impairment by cyclosporinA of reperfusion-induced arrhythmias. Life Sci 1992;51(14):1127–34.

Bobadilla I, Franco M, Cruz D, Zamora J, Robles SG, Chávez E. Hypothyroidism providesresistance to reperfusion injury following myocardium ischemia. Int J Biochem CellBiol 2001;33(5):499–506.

Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: alove-hate triangle. Am. J. Physiol. Cell Physiol. 2004;287(4):C817–33.

Carvajal K, El Hafidi M, Baños G. Myocardial damage due to ischemia and reperfusion inhypertriglyceridemic and hypertensive rats: participation of free radicals and calciumoverload. J Hypertens 1999;17(11):1607–16.

Chávez E, Téllez F, Pichardo J, Milán R, Cuellar A, Carvajal K, et al. On the protection byketorolac of reperfusion-induced heart damage. Comp Biochem Physiol C PharmacolToxicol Endocrinol 1996;115(1):95–100.

Cortés-Rojo C, Calderón-Cortés E, Clemente-Guerrero M, Manzo-Avalos S, Uribe S,Boldogh I, et al. Electron transport chain of Saccharomyces cerevisiae mitochondriais inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipidperoxidation involvement. Free Radic Res 2007;41(11):1212–23.

Coskun C, Avci B, Ocak N, Yalcin M, Dirican M, Savci V. Effect of repeatedly givenCDP-choline on cardiovascular and tissue injury in spinal shock conditions: investiga-tion on the acute phase. J Pharm Pharmacol 2010;62(3):497–506.

Di Lisa F, Canton M, Menabó R, Dodoni G, Bernardi P. Mitochondria and reperfusion inju-ry. The role of permeability transition. Basic Res Cardiol 2003;98(4):235–41.

Ek RO, Yildiz Y, Cecen S, Yenisey C, Kavak T. Effects of tamoxifen on ischemia-reperfusioninjury model in ovariectomized rats. Mol Cell Biochem 2008;308(1-2):227–35.

García N, García JJ, Correa F, Chávez E. The permeability transition pore as a pathway forthe release of mitochondrial DNA. Life Sci 2005;76(24):2873–80.

Ghosh S, Das N, Mandal AK, Dunadung SR, Sarkar S. Mannosylated liposomalcytidine 5′diphosphocholine prevented age related global moderate cerebral ischemia reperfu-sion induced mitochondrial cytochrome c release in aged brain. Neuroscience2010;171(4):1287–99.

Grau T, Romero A, Sacristán A, Ortiz JA. CDP-choline: Acute toxicity study.Arzneimittelforschung 1983;33(7A):1033–4.

Griffiths EJ, Ocampo CJ, Savage JS, Stern MD, Silverman HS. Protective effects of lowand high doses of cyclosporin A against reoxygenation injury in isolated ratcardiomyocytes are associated with differential effects on mitochondrial calciumlevels. Cell Calcium 2000;27(2):87–95.

Halestrap AP, Connern CP, Griffiths EI, Kerr PM. Cyclosporin A binding in mitochondrialcyclophilin inhibits the permeability transition pore and protects heart fromischemia/reperfusion injury. Mol Cell Biochem 1993;174(1–2):167–72.

Hausladen A, Fridovich I. Superoxide and peroxynitrite inactivate aconitase but nitricoxide does not. J Biol Chem 1994;269(47):29405–8.

Hayashi H. Pathogenesis and the role of Ca2+ overload during myocardial ischemia/reperfusion. Nagoya J Med Sci 2000;63(3–4):91–8.

Jeong SH, Hanh TM, Lee SR, Song IS, Noh SJ, Song S, et al. HS-1793, a recently develop-ment resveratrol analogue protects rat heart against hypoxia/reoxygenationinjury via attenuating mitochondrial damage. Bioorg Med Chem Lett 2013;23(14):4225–9.

Lochner A, Huisamen B, Nduhirabandi E. Cardioprotective effect of melatonin againstischemia/reperfusion damage. Front Biosci 2013;5:305–15.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the folin phenolreagent. 1951;193(1):265–75.

Luo M, Anderson ME. Mechanisms of altered Ca2+ handling in heart failure. Circ Res2013;113(6):690–708.

Parra E, Cruz D, García G, Zazueta C, Correa F, García N, et al. Myocardial protective effectof octylguanidine against the damage induced by ischemia reperfusion in rat heart.Mol Cell Biochem 2005;269(1–2):19–26.

Pavón N, Aranda A, García N, Hernández-Esquivel L, Chávez E. In hyperthyroid ratsoctylguanidine protects the heart from reperfusion damage. Endocrine 2009;35(2):158–65.

Pérez-Torres I, Roque P, El HaffidiM, Díaz-Díaz E, Baños E. Association of membrane dam-age and oxidative stress in a rat model of metabolic syndrome. Influence of gender.Free Radic Res 2009;43(8):761–71.

PetersonDA, Asinger RW, Elsperger KJ,HomansDC, Eaton JW. Reactive oxygen speciesmaycause myocardial reperfusion injury. Biochim Biophys Res Commun 1985;127(1):87–93.

Savci V, Cavun S, Goklalay G, Ulus IH. Cardiovascular effects of intracerebroventricularlyinjected CDP-choline in normotensive and hypotensive animals: the involvementof cholinergic system. Naunyn Schmiedebergs Arch Pharmacol 2002;365:388–98.

Secades JJ, Alvarez-Sabin J, Rubio E, Lozano R, Dávalos A, Castillo J, et al. Citicoline in intra-cerebral haemorrage: a double-blind, randomized, placebo-controlled, multi-centrepilot study. Cerebrovasc Dis 2006;21(5-6):380–5.

Sloan RC, Moukdar E, Frasier CR, Patel HD, Bostian PA, Lust RM, et al. Mitochondrial per-meability transition in the diabetic heart: contributions of thiol redox state and mito-chondrial calcium to augmented reperfusion injury. J Mol Cell Cardiol 2012;52(5):1009–18.

Téllez F, Carbajal K, Cruz D, Cárabez A, Chávez E. Effect of perezone on arrhythmias andmarkers of cell injury during reperfusion in the anesthetized rat. Life Sci 1999;65(16):1615–23.

Turkkan A, Alkan T, Goren B, Kocaeli H, Akar E, Korfali E. Citicoline and postconditioningprovides neuroprotection in a rat model of ischemic spinal cord injury. ActaNeurochir (Wien) 2010;152(6):1033–42.

Villa RF, Ferrari F, Gorini A. Effect of CDP-choline on age-dependent modifications ofenergy- and glutamate-linked enzyme activities in synaptic and non synaptic mito-chondria from rat cerebral cortex. Neurochem Int 2012;61(6):1424–32.

Weiss JN, Korge P, Honda HM, Ping P. Role of the mitochondrial permeability transition inmyocardial disease. Circ Res 2003;93(4):292–301.

Yilmaz MS, Coskun C, Yalcin M, Savci V. CDP-choline prevents cardiac arrhythmias andlethality induced by short-term myocardial ischemia-reperfusion injury in rat:involvement of central muscarinic cholinergic mechanisms. Naunyn SchmiedebergsArch Pharmacol 2008;378(3):293–301.

Zazueta C, Franco M, Correa F, García N, Santamaría J, Martínez-Abundis E, et al. Hypothy-roidism provides resistance to kidney mitochondria against the injury induced byischemia-reperfusion. Life Sci 2007;80(14):1252–8.

Zweifler RM. Membrane stabilizer: citicoline. Curr Med Res 2002;18(Suppl. 2):s14–7.