Addition of oh8dG to Cardioplegia Attenuated Myocardial Oxidative Injury through the Inhibition of Sodium Bicarbonate Cotransporter Activity

The biomarker 8-hydroxy-2′-deoxyguanosine (oh8dG) is derived from oxidized nucleic acids or products of oxidant-mediated DNA damage. Enhanced sodium bicarbonate cotransporter (NBC) activity is caused by reactive oxygen species (ROS) production in ventricular myocytes. Thus, we hypothesized that cardioplegia-solution-mediated ROS generation may be involved in the regulation of NBC activity in cardiomyocytes and that oh8dG treatment may modulate ROS and associated NBC activity. Langendorff-free cardioplegia-arrested cardiac strips and cardiomyocytes were isolated to determine the NBC activity and effects of oh8dG on oxidative-stress-mediated cardiac damage markers. We first determined the histidine-tryptophan-ketoglutarate (HTK) solution mediated NBC activity in cardiac strips and cells. The oh8dG treatment attenuated NBC activity in the electroneutral or electrogenic form of NBC. Additionally, exposure to HTK solution induced ROS, whereas co-administration of oh8dG attenuated ROS-mediated NBC activity, reduced ROS levels, and decreased the expression of apoptotic markers and fibrosis-associated proteins in cardiac cells. The oh8dG-administrated cardiac tissues were also protected from enhanced HTK-induced damage markers, heat shock protein 60 and polyADP-ribose. Our results show that oh8dG has a protective role against myocardial oxidative damage and provides a useful treatment strategy for restoring cardiac function.


Introduction
To decrease primary graft failure and postoperative myocardial dysfunction, donor heart preservation is an important process during heart transplantation [1,2]. Extracted donor hearts are exposed to ischemia, which leads to the deletion of the adenosine triphosphate (ATP) stores that initiate anaerobic metabolism and intracellular acidosis [3,4]. Increased intracellular enhanced activity of the sodium-hydrogen exchanger (NHE) leads to sodium influx into myocardial cells [3,4]. After the transplantation procedure, reperfusion of the donor heart rapidly initiates the washout of hydrogen (H + ) to the interstitial space. It creates a huge gradient of H + , which leads to further sodium influx through NHE [5]. The influx of sodium leads to upregulation of the sodium-calcium exchanger (NCX) in reverse mode, which eventually results in the import of calcium into myocardial cells [5]. Intracellular calcium overload enhances myocardial cell death by increased reactive oxygen species (ROS) levels, hyper-contracture, and apoptosis [3][4][5]. To decrease ischemia-reperfusion injury, retrieved organs are generally preserved in cold (4 • C) organpreserving solutions [6]. One of the most frequently used organ-preserving solutions is the histidine-tryptophan-ketoglutarate (HTK) solution [7]. As antioxidants, histidine,

ROS Staining
Isolated cardiomyocytes, cardiac strips, and cardiac tissues were attached to poly-Llysine (P8920, Sigma-Aldrich)-coated coverslips for 15 min at room temperature. Next, 10 µM 2 , 7 -Dichlorofluorescin diacetate (DCFDA) was loaded for 15 min and then rinsed with 1× DPBS. Confocal images were obtained at 488 nm using a Zeiss LSM 700 confocal microscope (Fluo-view, Carl Zeiss, Oberkochen, Germany). Images were collected from five separate preparations of cardiac tissue, and the results are presented as the average from all experiments.

Cell Culture and DNA Transfection
Human HEK293T (H.T), lung fibroblast MRC5, lung adenocarcinoma A549 cells were purchased from ATCC (Washington, DC, USA). Cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS) and 100 U/mL penicillinstreptomycin antibiotics at 37 • C in 5% CO 2 /95% air. Human NBCe1-B (an electrogenic form of sodium bicarbonate cotransporter, encoded by SLC4A4) and NBCn1 (electroneutral form of sodium bicarbonate cotransporter, encoded by SLC4A7) clones were provided by Shmuel Muallem (National Institutes of Health, Bethesda, MD, USA). Plasmid DNAs (1 µg/µL) were mixed with 200 µL Jet Prime Buffer (B200225, Polyplus-transfection, Illkirch-Graffenstaden, France). Transfection reagent (4 mL) (21Y0910L1, Polyplus-transfection) was incubated with mixed Jet prime buffer and DNA for 10 min in the dark. Incubated DNAs were transferred into H.T-cultured plates, and all procedures were performed in accordance with the protocol of the manufacturer (Polyplus-transfection). MRC5 cells were used for determination of cell viability, and A549 cells were used for determination of native NBC activity. S0859 (18497, Cayman, Ann Arbor, MI, USA) was used for the specific inhibition of NBC activity.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from the mouse cardiac tissues using the Hybrid-Ribo Ex extraction system (301-001, GeneAll, Seoul, Korea) in accordance with the instructions of the manufacturer. Isolated RNA was quantitated using the Spectrophotometer ND-1000 (Thermo Fisher Scientific), and cDNA was amplified using the TOPscript RT-PCR kit from Enzynomics (RT410S, Daejeon, Korea). The primers and PCR cycling protocol were as follows: mouse Bcl-2 (forward) GCA TCT TCT CCT TCC AGC CTG and (reverse) GAC GCT CTC CAC ACA CAT GAC; mouse p53 (forward) CAT GGC CAT CTA CAA GAA GTC and (reverse) GAA CAT CTC GAA GCG TTT ACG; denaturation at 95 • C (5 min), followed by 35 cycles of 95 • C (1 min), an annealing step (1 min), and an extension step at 72 • C (1 min). The final extension step was performed at 72 • C (10 min).

Transferase (TdT)-Mediated dUTP Nick End Labeling (TUNEL) Assay
Frozen cardiac tissues of mice were sectioned on a glass slide at a thickness of 10 µm. The TUNEL assay was performed in accordance with the protocol of the manufacturer, using a Transdetect In Situ Fluorescein TUNEL Cell Apoptosis Detection kit (FA201-02, TransGen Biotech Co., LTD, China). The slides were three times washed with 1× Dulbecco's phosphate-buffered saline (LB 001-02, DPBS, Kyungsan, Korea) for 10 min and fixed with 4% paraformaldehyde for 30 min at room temperature. The 1× labeling solution (FA201-02, TransGen Biotech Co., LTD, Beijing, China) was added to the surface of the glass slide with tissue attached and incubated at 37 • C in the dark for 1 h. The glass slides were rinsed with 1× DPBS and DAPI-included fluoromount-G (17984-24, Electron Microscopy Sciences, Hatfield, PA, USA) for 5 min. Confocal images were obtained at 358 nm using a Zeiss LSM 700 confocal microscope (Fluo-view, Carl Zeiss). Images were collected from five separate preparations of the cardiac tissues.

Immunostaining
Frozen cardiac tissues of mice were sectioned on the slide glass at a thickness of 10 µm. Slides were fixed with 4% paraformaldehyde or chilled methanol for 10 min. Heat shock protein 60 (ab46798, HSP60, Abcam) and poly (ADP-Ribose) (SM1398, PAR, ORIGENE, Rockville, MD, USA) antibodies were diluted at 1:100 and used to treat fixed samples for 24 h. Secondary antibodies tagged with FITC (for PAR, 1:200 dilution) and rhodamine (for HSP60, 1:200 dilution) were attached, and confocal images were obtained. Images were obtained using an LSM 700 Zeiss confocal microscope (Fluo-view, Carl Zeiss) at 488 nm (FITC), 530 nm (rhodamine), and 358 nm (DAPI for nucleus staining) and analyzed with ZEN software (ZEN 2009 light edition, Carl Zeiss). Images were collected from five separate preparations of cardiac tissue, and the results were averaged from all experiments.

MTT Assay
MRC5 cells were cultured in 96-well plates for 24 h and various dose of oh8dG was added for 1 h. Tetrazolium bromide (MTT; 298-93-1, Merck, Burlington, MA, USA) dye was mixed with 1 mL of PBS, and the cells were treated with 100 µL of MTT dye and incubated for 2 h in the dark. The medium was carefully aspirated from the plate and added 100 µL of DMSO, and the absorbance of sample was measured at 570 nm using a fluorescence microplate reader (VICTOR X3, PerkinElmer, Waltham, MA, USA).

Statistical Analysis
Experimental results are expressed as the mean ± standard error of the mean (SEM), and standard deviation (SD) was also represented. Experiment was performed more than three times and represented in figure legends. Differences between the mean values of the two samples were analyzed using the Student's t-test. Significance was determined by analysis of variance in each experiment (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Treatment of oh8dG Attenuated HTK-Mediated ROS Level in Cardiac Strips and Isolated Cardiomyocytes
Cardioplegia-induced oxidative injury results in cardiomyocytes apoptosis [41,42].
To determine the anti-oxidative role of exogenous oh8dG, we measured ROS levels using DCFDA fluorescence in cardioplegia solution (HTK)-exposed cardiac strips. DCFDA fluorescence was enhanced in HTK-exposed cardiac strips for 1 h and attenuated by the administration of exogenous oh8dG (Figure 1a,b). The HTK-exposed cardiac strips for 2 h were exhausted of DCFDA fluorescence with or without oh8dG (Figure 1a,b). We also confirmed the anti-oxidative role of oh8dG in cardiomyocytes isolated from the mice. Isolated cardiomyocytes were easily damaged by HTK treatment. Thus, the exposure time of HTK was maintained within 30 min, similar to the experimental condition in cardiac strips, which is available in within 2 h. The improved DCFDA fluorescence in HTK-treated cardiomyocytes was attenuated by treatment with exogenous oh8dG (Figure 1c,d). To verify the toxicity of exogenous oh8dG, we performed an MTT assay (Supplementary Figure S1). There was no effect of oh8dG on cell viability. These results indicate that exogenous oh8dG attenuated HTK-mediated ROS levels in cardiac tissues and cells.

The Treatment of oh8dG Attenuated ROS-Mediated NBC Activities
To verify the role of NBC activity in response to ROS and the anti-oxidative effect of oh8dG, cells were transfected with two types of NBC, NBCe1-B, and NBCn1. NBC-transfected H293T cells were treated with oh8dG for 1 h, and the effective dose of oh8dG was determined. Both 10 and 100 µg/mL of oh8dG attenuated the NBC activities independently of the type of NBC, electrogenic and electroneutral (Figure 2a-d). Then, 100 µg/mL of oh8dG was suggested [28], and we also determined the working dose of oh8dG (100 µg/mL) to evaluate the role of oh8dG. We determined the ROSmodulated NBC activity. NBC-overexpressing cells were stimulated with H 2 O 2 with or without oh8dG. Treatment with H 2 O 2 induced two~three-fold high NBC activity in both NBCs, and the co-administration of oh8dG dramatically attenuated NBC activity (Figure 2e-h). We also confirmed the modulatory role of H 2 O 2 on NBC activity in native NBC-expressed A549 cells. NBC activity was enhanced by the treatment of H 2 O 2 , whereas H 2 O 2 -mediated NBC activity was attenuated by the presence of specific NBC inhibitor S0859 (Supplementary Figure S2). NBC activity was dose-dependently inhibited by the oh8dG treatment (Supplementary Figure S3). These results suggested that NBC activity was directly enhanced by treatment with ROS, and these enhanced activities were attenuated by exogenous oh8dG treatment. cells. NBC activity was enhanced by the treatment of H2O2, whereas H2O2-mediated NBC activity was attenuated by the presence of specific NBC inhibitor S0859 (Supplementary Figure S2). NBC activity was dose-dependently inhibited by the oh8dG treatment (Supplementary Figure S3). These results suggested that NBC activity was directly enhanced by treatment with ROS, and these enhanced activities were attenuated by exogenous oh8dG treatment.  Bars present mean ± SEM (n = 4~6 * p < 0.05, ** p < 0.01, and *** p < 0.001). The table represents mean and SD value. (c) DCFDA staining in isolated cardiomyocytes after cardiac arrest with cardioplegia solution (HTK) using Langendorff-free cardioplegia method for 1 min and 100 µg/mL oh8dG was treated for 10 min and 30 min on ice. The scale bar represents 20 µm. (d) Analysis of normalized intensity (total intensity/measuring area) of DCFDA. Bars present mean ± SEM (n = 10, *** p < 0.001). The table represents mean and SD value. *** p < 0.001). The table represents mean and SD value. (c) DCFDA staining in isolated cardiomyocytes after cardiac arrest with cardioplegia solution (HTK) using Langendorff-free cardioplegia method for 1 min and 100 μg/mL oh8dG was treated for 10 min and 30 min on ice. The scale bar represents 20 μm. (d) Analysis of normalized intensity (total intensity/measuring area) of DCFDA. Bars present mean ± SEM (n = 10, *** p < 0.001). The table represents mean and SD value.   . (g,h) The bars present mean ± SEM (n = 3~5, * p < 0.05, ** p < 0.01, and *** p < 0.001). The table represents mean and SD value.

HTK-Exposed Cardiac Tissues Enhanced NBC Activity, Attenuated by the Treatment of oh8dG
To verify the modulatory role of HTK-induced ROS in NBC activity in cardiac tissues, two types of NBC expression were determined. The left and right ventricles of the cardiac tissues were expressed in both NBCs (Figure 3a). Exposure to the HTK solution stimulated NBC activity in isolated cardiomyocytes (Figure 3b,c). Co-stimulation of HTK with oh8dG attenuated HTK-mediated NBC activity (Figure 3b,c). We then confirmed NBC activity in HTK-exposed isolated cardiac strips. oh8dG also attenuated NBC activity (Figure 3d,e). These results indicate that HTK exposure enhanced ROS production and enhanced NBC activity, which was attenuated by exogenous oh8dG administration.

HTK-Exposed Cardiac Tissues Enhanced NBC Activity, Attenuated by the Treatment of oh8dG
To verify the modulatory role of HTK-induced ROS in NBC activity in cardiac tissues, two types of NBC expression were determined. The left and right ventricles of the cardiac tissues were expressed in both NBCs (Figure 3a). Exposure to the HTK solution stimulated NBC activity in isolated cardiomyocytes (Figure 3b,c). Co-stimulation of HTK with oh8dG attenuated HTK-mediated NBC activity (Figure 3b,c). We then confirmed NBC activity in HTK-exposed isolated cardiac strips. oh8dG also attenuated NBC activity (Figure 3d,e). These results indicate that HTK exposure enhanced ROS production and enhanced NBC activity, which was attenuated by exogenous oh8dG administration. Figure 3. HTK-exposed cardiac tissues enhanced NBC activity, attenuated by the treatment of oh8dG. (a) Protein expression of NBCe1 and NBCn1 in left and right ventricle (LV and RV, respectively) cardiac strips of mice (n = 6). β-actin was used as the loading control. (b) NBC activity was assessed by measuring changes of pH i with or without pre-incubation of 100 µg/mL oh8dG for 1 h in cardiomyocytes on ice. HTK solution was treated for 5 min. (c) The bars present mean ± SEM (n = 3, * p < 0.05, and ** p < 0.01) in cardiomyocytes. The table represents mean and SD value. (d) NBC activity in cardiac strips during cardioplegia (HTK)-induced arrest for 1 min. After cardiac arrest, 100 µg/mL oh8dG was pre-incubated for 1 h. (e) The bars present mean ± SEM (n = 3, *** p < 0.001) in cardiac strips. The table represents mean and SD value.

Treatment of oh8dG Attenuated DNA Fragmentation and Expression of the Apoptotic Marker in HTK-Exposed Cardiac Tissues
Ischemia-reperfusion injury-mediated cellular apoptosis is mediated by caspase-3 activation [43] and, subsequently, induces DNA fragmentation [44]. Figure 4a shows an enhanced DAPI staining area that was observed in the HTK-treated cardiomyocytes. HTKmediated DNA fragmentation was reduced by exogenous oh8dG treatment (Figure 4a,b). The HTK-induced increase in the apoptotic marker Bcl-2, but not p-53, was reduced by oh8dG treatment (Figure 4c,d). The cardiac injury induces the fibrosis factor connective tissue growth factor (CTGF) [45,46]. The oh8dG treatment reduced the expression of CTGF (Figure 4e,f). Isolated cardiac strips were exposed to HTK during cardiac arrest. Thus, the inhibitory effect of oh8dG on CTGF expression was assessed using HTK-exposed cardiac strips as a control. These results indicated that HTK-mediated DNA fragmentation and expression of the apoptotic marker Bcl-2 were attenuated by exogenous oh8dG treatment, with a decrease in CTGF.

Treatment of oh8dG Attenuated HTK-Mediated Cardiac Destructive Signals
The exogenous oh8dG showed anti-oxidative and anti-apoptotic effects in cardiac tissues and cells. To verify the tissue-protective role of exogenous oh8dG in cardiac apoptotic death, we performed a TUNEL assay. The number of TUNEL-positive cardiac tissues was increased by HTK exposure in a time-dependent manner. The co-administration of oh8dG dramatically reduced the number of TUNEL-positive cells (Figure 5a,b). Cellular stress and apoptotic signals induce plasma membrane localization of the mitochondrial protein HSP60 [47]. In addition, PAR production is activated by DNA damage and stress [48]. HTK-exposed cardiac tissue showed enhanced membrane expression of HSP60 (Figure 5c). We verified the role of exogenous oh8dG in stress/injury-associated protein expression. Coadministration of oh8dG dramatically attenuated HTK-mediated HSP60/PAR expression (Figure 5c-e). These data indicate that the co-administration of HTK and exogenous oh8dG has a protective effect against HTK-associated cardiac destructive signals.    (b) Analysis of TUNEL positive cells (%) normalized by assessing total intensity/measuring area of FITC (green). Bars present mean ± SEM (n = 3, * p < 0.05). NS; not significant. The table represents mean and SD value. (c) Immunostaining of HSP60 (red), PAR (green), and nucleus (DAPI, blue) in cardiac strips of mice. I.R.HTK; immediately removed HTK (cardioplegia-induced arrest, 1 min).
The heart was treated with HTK solution for 2 h, and 100 µg/mL oh8dG was treated for 1 h and 2 h, respectively on ice. The scale bars represent 20 µm. Analysis of normalized intensity (total intensity/measuring area) of HSP60 (d) and PAR (e). Bars present mean ± SEM (n = 4, * p < 0.05, ** p < 0.01, and *** p < 0.001). The table represents mean and SD value.

Discussion
In the present study, we addressed that exposure to HTK solution in cardiac cells induced ROS and co-administration of exogenous oh8dG attenuated ROS-mediated NBC activity and reduced expression of apoptotic markers and fibrosis-associated protein CTGF. The exogenous oh8dG-administrated cardiac tissues were protected from the HTK-induced damage markers HSP60 and PAR. Our results addressed exogenous oh8dG as a protective supplement against potential HTK-solution-mediated oxidative damage. Thus, our observations in cardiac tissues are consistent with previous studies in various experimental systems showing the protective role of exogenous oh8dG against oxidative or inflammatory signals [21][22][23][24][25][26][27][28].
NBC activity is modulated by various cellular stimulants, such as ROS [18], acidic [49], hypoxia [50], and inflammatory cytokines [49]. Our previous study showed that HTKinduced arrest mediated more acidic intracellular circumstances through the involvement of sodium-potassium-chloride cotransporter Nkcc1 and chloride-bicarbonate exchanger Slc26a6 in the db/db diabetic mouse model's heart than in wild-type mice during reperfusion [38].
Although NBC is considered an intracellular pH regulator, it has not been revealed whether HTK is involved in the activity of NBCs. The present study showed that NBC activity was increased by HTK-mediated ROS generation. Enhanced NBC activity may mediate sodium and bicarbonate uptake and, subsequently, coordinate ion transport, such as calcium and chloride. We speculate that the accumulation of sodium and bicarbonate through enhanced NBC may mediate the activation of the chloride/bicarbonate exchanger (chloride influx and bicarbonate efflux) and NCX exchanger (calcium influx and sodium efflux). In addition, enhanced sodium and chloride may facilitate water influx to maintain cellular osmotic power ( Figure 6). Therefore, calcium overload, enhanced sodium/chloride level, and subsequent water intake occur in the cytosol of cardiomyocytes and, subsequently, induced cytotoxic edema and impaired cardiomyocytes' function. Similarly, it has been addressed that the angiotensin II-mediated modulation of NBC and pathological output of associated ion transporters in cardiopathies [51]. Although experimental limitations were revealed, our results provide new potential targets to maintain cardiac function after cardiac surgery or heart transplantation using the HTK solution. Modulation of NBC is essential, to protect the heart during the reperfusion period of cardiac surgery or transplantation, by decreasing ROS generation. idants 2022, 11, x FOR PEER REVIEW 13 of 17 Figure 6. Schematic model of enhanced NBC and its coordinated channels and transporters in HTKexposed cardiomyocytes and preserving effect of exogenous oh8dG. HTK-solution-exposed cardiac tissues produce oxidative signals. Oxidants enhance cardiac NBC activity through the involvement of ERK [51] and, subsequently, induce convergent involvement of NCX, CBE (SLC26A6), and aquaporin (AQP), which cause calcium overload and cyto-edema during reperfusion [16]. Cardiac calcium overload also mediates ROS generation [52]. Thus, the modulation of NBC activity may be a potential strategy against cardiac damage such as arrhythmic events or cardiac sudden death. The application of exogenous oh8dG may attenuate HTK-mediated NBC hyperactivity and the associated damage markers.
ROS-mediated NBC activities, including NBCn and NBCe, were attenuated by treatment with exogenous oh8dG. The signaling of oh8dG has been known in the inactivation of Ras in cancer cells [53]. Ras signaling is involved in migration in various cellular systems, such as vascular smooth muscle cells, endothelial cells, and several cancers [54][55][56][57]. In addition, NBCn is known to provide a migratory module in various cell types, such as vascular smooth muscle cells, breast cancers, and synoviocytes [49,58,59]. Currently, there is no direct evidence for an association between Ras and NBCn. In this regard, the proteinprotein network between NBCn and Ras signaling is an attractive and changeable issue for future studies.
The protective role of exogenous oh8dG in various inflammatory and oxidative stress-mediated diseases has been highlighted in different studies [21,23,[29][30][31][32][33]. Our results indicate that long-term exposure to HTK solution mediated the enhancement of tissue damage markers. Cardiac tissues should be maintained and protected from damage signals during cardioplegia exposure. The static hypothermic preservation method is a low-cost and standard method for heart transplantation [60]. HTK has been suggested as an effective cardiac preservation solution to regenerate cardiac function [61,62]; however, HTK could not fully protect against reperfusion injury during hypothermic organ preservation [63]. Since ROS generation is the main initiator of reperfusion injury, the addition of various ROS scavengers has been attempted in order to decrease reperfusion injury during heart transplantation [64][65][66]. However, the cardio-protective effect of decreasing ROS with ROS scavengers is insufficient. Since the generation of ROS and various ion transporters are involved in modulating pH during reperfusion, we thought that modulation of both ROS and ion channels is essential to decrease reperfusion injury. Therefore, Figure 6. Schematic model of enhanced NBC and its coordinated channels and transporters in HTKexposed cardiomyocytes and preserving effect of exogenous oh8dG. HTK-solution-exposed cardiac tissues produce oxidative signals. Oxidants enhance cardiac NBC activity through the involvement of ERK [51] and, subsequently, induce convergent involvement of NCX, CBE (SLC26A6), and aquaporin (AQP), which cause calcium overload and cyto-edema during reperfusion [16]. Cardiac calcium overload also mediates ROS generation [52]. Thus, the modulation of NBC activity may be a potential strategy against cardiac damage such as arrhythmic events or cardiac sudden death. The application of exogenous oh8dG may attenuate HTK-mediated NBC hyperactivity and the associated damage markers.
ROS-mediated NBC activities, including NBCn and NBCe, were attenuated by treatment with exogenous oh8dG. The signaling of oh8dG has been known in the inactivation of Ras in cancer cells [53]. Ras signaling is involved in migration in various cellular systems, such as vascular smooth muscle cells, endothelial cells, and several cancers [54][55][56][57]. In addition, NBCn is known to provide a migratory module in various cell types, such as vascular smooth muscle cells, breast cancers, and synoviocytes [49,58,59]. Currently, there is no direct evidence for an association between Ras and NBCn. In this regard, the protein-protein network between NBCn and Ras signaling is an attractive and changeable issue for future studies.
The protective role of exogenous oh8dG in various inflammatory and oxidative stressmediated diseases has been highlighted in different studies [21,23,[29][30][31][32][33]. Our results indicate that long-term exposure to HTK solution mediated the enhancement of tissue damage markers. Cardiac tissues should be maintained and protected from damage signals during cardioplegia exposure. The static hypothermic preservation method is a low-cost and standard method for heart transplantation [60]. HTK has been suggested as an effective cardiac preservation solution to regenerate cardiac function [61,62]; however, HTK could not fully protect against reperfusion injury during hypothermic organ preservation [63]. Since ROS generation is the main initiator of reperfusion injury, the addition of various ROS scavengers has been attempted in order to decrease reperfusion injury during heart transplantation [64][65][66]. However, the cardio-protective effect of decreasing ROS with ROS scavengers is insufficient. Since the generation of ROS and various ion transporters are involved in modulating pH during reperfusion, we thought that modulation of both ROS and ion channels is essential to decrease reperfusion injury. Therefore, the possibility of adding exogenous oh8dG, which leads to decreased ROS and modulation of NBC activity, to the preservation solution could be considered.
Our study has some experimental limitations. Cardiac function after reperfusion could not be evaluated because the Langendorff perfusion system was not used. We evaluated various cardiac injury markers to determine whether HTK leads to apoptosis during organ preservation. Although co-administration of exogenous oh8dG with HTK requires additional experiments on organ preservation for long-term exposure, the cardioprotective effect of exogenous oh8dG might provide improved beneficial effects to restore functional cardiac recovery during reperfusion.