Hyperkalemic or Low Potassium Cardioplegia Protects against Reduction of Energy Metabolism by Oxidative Stress

Open-heart surgery is often an unavoidable option for the treatment of cardiovascular disease and prevention of cardiomyopathy. Cardiopulmonary bypass surgery requires manipulating cardiac contractile function via the perfusion of a cardioplegic solution. Procedure-associated ischemia and reperfusion (I/R) injury, a major source of oxidative stress, affects postoperative cardiac performance and long-term outcomes. Using large-scale liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics, we addressed whether cardioplegic solutions affect the baseline cellular metabolism and prevent metabolic reprogramming by oxidative stress. AC16 cardiomyocytes in culture were treated with commonly used cardioplegic solutions, High K+ (HK), Low K+ (LK), Del Nido (DN), histidine–tryptophan–ketoglutarate (HTK), or Celsior (CS). The overall metabolic profile shown by the principal component analysis (PCA) and heatmap revealed that HK or LK had a minimal impact on the baseline 78 metabolites, whereas HTK or CS significantly repressed the levels of multiple amino acids and sugars. H2O2-induced sublethal mild oxidative stress causes decreases in NAD, nicotinamide, or acetylcarnitine, but increases in glucose derivatives, including glucose 6-P, glucose 1-P, fructose, mannose, and mannose 6-P. Additional increases include metabolites of the pentose phosphate pathway, D-ribose-5-P, L-arabitol, adonitol, and xylitol. Pretreatment with HK or LK cardioplegic solution prevented most metabolic changes and increases of reactive oxygen species (ROS) elicited by H2O2. Our data indicate that HK and LK cardioplegic solutions preserve baseline metabolism and protect against metabolic reprogramming by oxidative stress.


Introduction
Cardiovascular disease is the principal cause of mortality worldwide. Most cardiac patients suffering from coronary artery disease have plaque buildup in the wall of the coronary arteries. With advancements in medical technology, an occluded coronary artery can be re-opened by the percutaneous coronary intervention technique. Open-heart surgery remains an option of treatment, especially for multivessel disease, diffuse or complex coronary disease, with or without valvular disease [1][2][3]. The development of cardiopulmonary bypass (CPB) using a heart-lung machine to sustain systematic circulation during operation has revolutionized cardiac surgery and reduced the mortality of patients with coronary artery disease. However, approximately 67% of CPB patients have post-operative complications, including atrial fibrillation, pulmonary dysfunction, or renal failure [4]. One contributing factor to these types of organ injury is procedure-associated ischemia and reperfusion (I/R), which produce oxidative stress. streptomycin, GIBCO) for culture in a 5% CO 2 incubator at 37 • C with weekly subculture. The cells were seeded at a density of 0.3 × 10 6 cells per well in 6-well plates and grown to 90% confluence before experiments.
Cells were treated with cardioplegic solutions according to the ratios used clinically. HK or LK (0.4 mL) was mixed with 1.6 mL DMEM, whereas DN (1.6 mL) was mixed with 0.4 mL DMEM. For HTK or CS, 2 mL pure crystalloid solution was added to AC16 cells. Cells in 6-well plates were serum-starved overnight in 0.5%FBS/DMEM/F12 and treated with a cardioplegic solution for 3 h followed by exposure to 200 µM H 2 O 2 for 1 h.

Metabolomics
The samples were collected for LC-MS/MS metabolomics as described [21][22][23][24]. Following a quick rinse with phosphate-buffered saline (PBS), cells were placed in 1 mL/well of 8:2 (v:v) methanol: H 2 O for 30 min incubation on dry ice. This serves to quench metabolism and extract the metabolites. The cells were scraped from the culture plates and transferred to centrifuge tubes. Another 0.7 mL/well of 8:2 (v:v) methanol: H 2 O was added to the plates on the dry ice to scrape and combined with the corresponding centrifuge tubes. Cell-free extracts were collected after 10 min centrifugation at 13,000 rpm under 4 • C. The soluble fractions of the extracts were dried at 4 • C using a speed vacuum. The samples were processed for LC-MS/MS as described [25].
LC-MS/MS analyses were performed using an Agilent 1290 UPLC-6490 QQQ-MS system (Santa Clara, CA, USA). Each sample was injected twice by an auto-sampler set at 4 • C, 10 µL for analysis using a negative ionization mode or 4 µL for analysis using a positive ionization mode. Both chromatographic separations were performed in the hydrophilic interaction chromatography mode on a Waters XBridge BEH Amide column (150 × 2.1 mm, 2.5 µm particle size, Waters Corporation, Milford, MA, USA), with a flow rate of 0.3 mL/min and the column compartment temperature of 40 • C. The mobile phase contained Solvent A (10 mM ammonium acetate and 10 mM ammonium hydroxide in 95% H 2 O/5% ACN) and Solvent B (10 mM ammonium acetate and 10 mM ammonium hydroxide in 5% H 2 O/95% ACN). After an initial 1 min isocratic elution of 90% Solvent B, the percentage of Solvent B decreased to 40% by t = 11 min. Solvent B was maintained at 40% for 4 min (t = 15 min) before gradually returning to 90% in preparation for the next injection. The mass spectrometer was equipped with an electrospray ionization source, and targeted data acquisition was performed in the multiple-reaction monitoring (MRM) mode. Agilent Masshunter Workstation software (Santa Clara, CA, USA) was used to control the operation of the LC-MS/MS system, whereas the extracted MRM peaks were integrated using the Agilent MassHunter Quantitative Data Analysis (Santa Clara, CA, USA).

Cell Morphology
AC16 cells were seeded in 6-well plates for treatments as described in Section 2.2. The cells were fixed in 4% paraformaldehyde for 5 min, washed twice with PBS, and stained with 0.1% Coomassie blue (dissolved in 10% acetic acid and 50% methanol) for 10 min. After washing off the dye, cell morphology was recorded under an inverted microscope with 10× lens (Rebel, Echo, San Diego, CA, USA).

Statistical Analysis
Changes in metabolites or pathways between control versus treatment were analyzed using the web-based software MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/ MetaboAnalyst/ModuleView.xhtml, accessed 3 June 2022). PCA, K-means, heatmap clustering, and pathway analysis overview of altered metabolic profiles were analyzed using MetaboAnalyst 5.0. Bar graphs were presented as means ± standard deviations (SD) of the relative abundance of metabolites and analyzed via one-way ANOVA and corrected by Dunnett's multiple comparisons test with GraphPad Prism 9 software. The data with two grouping variables were analyzed with two-way ANOVA and corrected by Turkey's multiple comparisons test. p value or adjusted p value < 0.05 was set as the threshold for significant difference. Table 1 lists the components of five cardioplegic solutions commonly used in the US and internationally for CPB surgeries (HK, LK, DN, and HTK) or cardiac transplants (CS). To evaluate the impact of these solutions on cellular metabolism, we treated AC16 cardiomyocytes according to their clinical applications, with the final concentration of each component listed in Table 1. The length of treatment was 4 h, mimicking the average CPB surgical time. A total of 94 metabolites were detected among the control (Ctrl) and cells treated with a cardioplegic solution, as shown in the heatmap ( Figure 1A). When compared with the Ctrl, a similar pattern of metabolites was found in HK-and LK-treated groups, but HTK-and CS-treated cells exhibited a clear difference, showing decreases in 12 amino acids and 4 sugars ( Figure 1A). The metabolic profile of DN-treated cells is closer to that of Ctrl, HK, or LK ( Figure 1A,B). The overall metabolic profiles as summed by the principal component analysis (PCA) revealed that HK-or LK-cardioplegia-treated cells showed no significant difference from the Ctrl ( Figure 1B).

Effects of Cardioplegic Solutions on the Metabolome
PCA indicates significant deviations in the profile of HTK-or CS-treated cells ( Figure 1B). The differences in the metabolites are displayed in a volcano plot (Figure 2A,B). The pathway analyses showed that HTK and CS caused significant perturbations of the pathways of: (1) alanine, aspartate, and glutamate metabolism; (2) histidine metabolism; (3) arginine biosynthesis; (4) tricarboxylic acid (TCA) cycle; (5) aminoacyl-tRNA biosynthesis; and (6) valine, leucine, and isoleucine biosynthesis ( Figure 2C,D). CS also affected the glyoxylate and dicarboxylate metabolism pathway ( Figure 2D). These results indicate that unlike HTK or CS, HK or LK cardioplegic solution preserves the basal metabolic profile.

Metabolic Profiles of Oxidative Stress and Impact of Cardioplegic Solutions
H2O2 at the dose of 200 μM was chosen here for metabolomics experiments since it does not cause detectable cytotoxicity, as measured by cell morphology, cell number, or general metabolic activity via CCK-8 assay [24,26]. This dose mimics the range of oxidants in the myocardium during I/R, 10-30 μM, based on the common belief that intracellular oxidants are 10-100-fold lower than extracellular H2O2 concentration [27,28].

Metabolic Profiles of Oxidative Stress and Impact of Cardioplegic Solutions
H 2 O 2 at the dose of 200 µM was chosen here for metabolomics experiments since it does not cause detectable cytotoxicity, as measured by cell morphology, cell number, or general metabolic activity via CCK-8 assay [24,26]. This dose mimics the range of oxidants in the myocardium during I/R, 10-30 µM, based on the common belief that intracellular oxidants are 10-100-fold lower than extracellular H 2 O 2 concentration [27,28].
Compared with the Ctrl or H 2 O 2 -treated cells, pretreatment with HK or LK was able to shift the metabolic profile of H 2 O 2 -treated cells towards that of Ctrl ( Figure 4A). Among the 17 metabolites of 9 significantly changed pathways due to H 2 O 2 treatment, HK or LK pretreatment was able to block H 2 O 2 from inducing increases or decreases in the majority of these metabolites ( Figure 4B). Specifically, the decrease in NAD or acetylcarnitine was attenuated by HK or LK treatment ( Figure 5A,B). H 2 O 2 -induced increases in glucose 6-P, glucose 1-P, mannose 6-P, and oxaloacetic acid were inhibited by HK or LK ( Figure 5B,C). The increases in the metabolites in the pentose phosphate pathway, i.e., xylitol, L-arabitol, adonitol, and D-ribose 5-P, due to H 2 O 2 treatment were prevented by HK or LK ( Figure 5D). These data indicate the capacity of HK or LK for protection against metabolic shifts by oxidative stress. Compared with the Ctrl or H2O2-treated cells, pretreatment with HK or LK was able to shift the metabolic profile of H2O2-treated cells towards that of Ctrl ( Figure 4A). Among the 17 metabolites of 9 significantly changed pathways due to H2O2 treatment, HK or LK pretreatment was able to block H2O2 from inducing increases or decreases in the majority of these metabolites ( Figure 4B). Specifically, the decrease in NAD or acetylcarnitine was attenuated by HK or LK treatment ( Figure 5A,B). H2O2-induced increases in glucose 6-P, glucose 1-P, mannose 6-P, and oxaloacetic acid were inhibited by HK or LK ( Figure 5B,C). The increases in the metabolites in the pentose phosphate pathway, i.e., xylitol, L-arabitol, adonitol, and D-ribose 5-P, due to H2O2 treatment were prevented by HK or LK ( Figure  5D). These data indicate the capacity of HK or LK for protection against metabolic shifts by oxidative stress.

Figure 2.
Metabolites and Metabolic Pathways Altered by HTK and CS. AC16 cells seeded in 6-well plates were used for the collection of aqueous metabolites as described in Figure 1 for LC-MS/MSbased metabolomics. A total of 67 or 65 metabolites showed statistically significant differences as determined by Student's t-test (n = 4, p < 0.05) between control versus HTK or CS and are displayed in the volcano plots (A,B) generated by the MetaboAnalyst 5.0 software. The altered metabolic pathways are displayed with the x-axis representing the pathway impact score computed from pathway topological analysis and the y-axis being the -log10 of the p-value obtained from pathway enrichment analysis (C,D). The metabolic pathways perturbed by the HTK and CS treatment are displayed in the gradient from yellow to red, reflecting the p-value from statistical analysis, as shown in the yaxis, from non-significant (≤1 for −log10, p ≥ 0.1, light yellow) to significant (≥1 but ≤2 for −log10, p ≤ 0.01 to p ≥ 0.001, orange) to highly significant (≥2 for −log10, p ≤ 0.0001, red). The size of the circles reflects the number of metabolites altered in the metabolic pathway (C,D).

Figure 2.
Metabolites and Metabolic Pathways Altered by HTK and CS. AC16 cells seeded in 6-well plates were used for the collection of aqueous metabolites as described in Figure 1 for LC-MS/MSbased metabolomics. A total of 67 or 65 metabolites showed statistically significant differences as determined by Student's t-test (n = 4, p < 0.05) between control versus HTK or CS and are displayed in the volcano plots (A,B) generated by the MetaboAnalyst 5.0 software. The altered metabolic pathways are displayed with the x-axis representing the pathway impact score computed from pathway topological analysis and the y-axis being the -log10 of the p-value obtained from pathway enrichment analysis (C,D). The metabolic pathways perturbed by the HTK and CS treatment are displayed in the gradient from yellow to red, reflecting the p-value from statistical analysis, as shown in the y-axis, from non-significant (≤1 for −log10, p ≥ 0.1, light yellow) to significant (≥1 but ≤2 for −log10, p ≤ 0.01 to p ≥ 0.001, orange) to highly significant (≥2 for −log10, p ≤ 0.0001, red). The size of the circles reflects the number of metabolites altered in the metabolic pathway (C,D).     The metabolites altered by H2O2 treatment belonging to nicotinate and nicotinamide metabolism, TCA cycle, glycolysis/gluconeogenesis, and pentose phosphate pathway were analyzed for the protective effect of HK or LK cardioplegia solution. * indicates significant difference (n = 4, p < 0.05) between control and H2O2-treated cells, whereas # indicates significant difference (n = 4, p < 0.05) between HK or LK with H2O2 treatment in comparison with H2O2 treatment alone. These data were analyzed for the significant differences by one-way ANOVA using GraphPad Prism 9 software.

Protection against ROS Generation
To explore the mechanism of HK or LK for protection against metabolic shift by oxidative stress, we tested whether the cardioplegic solutions affect ROS production by H2O2. AC16 cells were able to tolerate five cardioplegic solutions without morphological evidence of toxicity ( Figure 6A). When the baseline • OH generation was measured by treatment of a cardioplegic solution using DCFH-DA assay, the level of DCF fluorescence in HK-or LK-treated cells was similar to that of the control without any treatment ( Figure  6B). The positive control of 200 μM of H2O2 showed a time-dependent elevation of DCF fluorescence over 240 min ( Figure 6B). DN, HTK, or CS induced elevation of DCF fluorescence, with CS producing nearly half the amount of ROS as H2O2 ( Figure 6B). We then investigated whether any of these cardioplegic solutions are capable of preventing H2O2 from generating • OH. When pretreating cells with a cardioplegic solution, we found an inhibition of H2O2 induced • OH generation by HK or LK, but not DN, HTK, or CS ( Figure  6C). treatment belonging to nicotinate and nicotinamide metabolism, TCA cycle, glycolysis/gluconeogenesis, and pentose phosphate pathway were analyzed for the protective effect of HK or LK cardioplegia solution. * indicates significant difference (n = 4, p < 0.05) between control and H 2 O 2 -treated cells, whereas # indicates significant difference (n = 4, p < 0.05) between HK or LK with H 2 O 2 treatment in comparison with H 2 O 2 treatment alone. These data were analyzed for the significant differences by one-way ANOVA using GraphPad Prism 9 software.

Protection against ROS Generation
To explore the mechanism of HK or LK for protection against metabolic shift by oxidative stress, we tested whether the cardioplegic solutions affect ROS production by H 2 O 2 . AC16 cells were able to tolerate five cardioplegic solutions without morphological evidence of toxicity ( Figure 6A). When the baseline • OH generation was measured by treatment of a cardioplegic solution using DCFH-DA assay, the level of DCF fluorescence in HK-or LK-treated cells was similar to that of the control without any treatment ( Figure 6B). The positive control of 200 µM of H 2 O 2 showed a time-dependent elevation of DCF fluorescence over 240 min ( Figure 6B). DN, HTK, or CS induced elevation of DCF fluorescence, with CS producing nearly half the amount of ROS as H 2 O 2 ( Figure 6B). We then investigated whether any of these cardioplegic solutions are capable of preventing H 2 O 2 from generating • OH. When pretreating cells with a cardioplegic solution, we found an inhibition of H 2 O 2 induced • OH generation by HK or LK, but not DN, HTK, or CS ( Figure 6C). Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 14 indicates significant difference from H2O2-treated cells (C). These data were analyzed for the significant differences by one-way ANOVA using GraphPad Prism 9 software.

Discussion
Using LC-MS/MS-based metabolomics, we evaluated the impact of five cardioplegic solutions on cellular metabolism and metabolic alterations by oxidative stress. Our data indicate that HK and LK cardioplegia solutions did not cause significant changes in the baseline metabolic profile, whereas HTK and CS induced significant metabolic shifts. Induction of oxidative stress by H2O2 treatment resulted in decreases in NAD and acetylcarnitine but increases in glucose 6-P, glucose 1-P, mannose 6-P, oxaloacetic acid, and four sugar alcohols from the pentose phosphate pathway. Our data suggest that HK and LK cardioplegic solutions can provide protection against alterations in energy metabolism due to oxidative stress at the cellular level. Such protection correlates with an inhibition of ROS generation.
The cardioplegic solutions are capable of supporting an extended period of myocardial ischemia with organ preservation during cardiac surgery. The choice of which cardioplegic solution is based on the preference and experience of each surgeon. HK, LK, and DN, the so-called blood cardioplegia, require isogenic mixing of the patient's blood for their clinical application, whereas HTK or CS is perfused into the myocardium as a pure crystalloid. Experimental or clinical evidence suggests that blood cardioplegia is superior to the crystalloids for cardiac protection [29][30][31][32][33][34][35]. Among the many advantages of blood cardioplegia are oxygen-carrying capacity, continuous supply of metabolic substrates, indicates significant difference from H2O2-treated cells (C). These data were analyzed for the significant differences by one-way ANOVA using GraphPad Prism 9 software.

Discussion
Using LC-MS/MS-based metabolomics, we evaluated the impact of five cardioplegic solutions on cellular metabolism and metabolic alterations by oxidative stress. Our data indicate that HK and LK cardioplegia solutions did not cause significant changes in the baseline metabolic profile, whereas HTK and CS induced significant metabolic shifts. Induction of oxidative stress by H2O2 treatment resulted in decreases in NAD and acetylcarnitine but increases in glucose 6-P, glucose 1-P, mannose 6-P, oxaloacetic acid, and four sugar alcohols from the pentose phosphate pathway. Our data suggest that HK and LK cardioplegic solutions can provide protection against alterations in energy metabolism due to oxidative stress at the cellular level. Such protection correlates with an inhibition of ROS generation.
The cardioplegic solutions are capable of supporting an extended period of myocardial ischemia with organ preservation during cardiac surgery. The choice of which cardioplegic solution is based on the preference and experience of each surgeon. HK, LK, and DN, the so-called blood cardioplegia, require isogenic mixing of the patient's blood for their clinical application, whereas HTK or CS is perfused into the myocardium as a pure crystalloid. Experimental or clinical evidence suggests that blood cardioplegia is superior to the crystalloids for cardiac protection [29][30][31][32][33][34][35]. Among the many advantages of blood cardioplegia are oxygen-carrying capacity, continuous supply of metabolic substrates, indicates significant difference from H 2 O 2 -treated cells (C). These data were analyzed for the significant differences by one-way ANOVA using GraphPad Prism 9 software.

Discussion
Using LC-MS/MS-based metabolomics, we evaluated the impact of five cardioplegic solutions on cellular metabolism and metabolic alterations by oxidative stress. Our data indicate that HK and LK cardioplegia solutions did not cause significant changes in the baseline metabolic profile, whereas HTK and CS induced significant metabolic shifts. Induction of oxidative stress by H 2 O 2 treatment resulted in decreases in NAD and acetylcarnitine but increases in glucose 6-P, glucose 1-P, mannose 6-P, oxaloacetic acid, and four sugar alcohols from the pentose phosphate pathway. Our data suggest that HK and LK cardioplegic solutions can provide protection against alterations in energy metabolism due to oxidative stress at the cellular level. Such protection correlates with an inhibition of ROS generation.
The cardioplegic solutions are capable of supporting an extended period of myocardial ischemia with organ preservation during cardiac surgery. The choice of which cardioplegic solution is based on the preference and experience of each surgeon. HK, LK, and DN, the so-called blood cardioplegia, require isogenic mixing of the patient's blood for their clinical application, whereas HTK or CS is perfused into the myocardium as a pure crystalloid. Experimental or clinical evidence suggests that blood cardioplegia is superior to the crystalloids for cardiac protection [29][30][31][32][33][34][35]. Among the many advantages of blood cardioplegia are oxygen-carrying capacity, continuous supply of metabolic substrates, physiologic buffering, and consistent osmotic pressure [36,37]. We mimicked the clinical application by mixing HK, LK, and DN with the culture medium. This approach has maintained the supply of nutrients, including glucose, amino acids, vitamins, and minerals, which were absent in the crystalloid solution HTK or CS. Therefore, the large deviation in the basal metabolic profile by HTK or CS could result from nutrient deprivation in the cell culture experiments. Nevertheless, our finding of HK or LK having a minimal impact on the baseline metabolic profile yet protecting against metabolic switch by oxidants supports the clinical observation of the superiority of the blood cardioplegia.
The metabolic shift detected here by oxidative stress resembles that of surgery-induced ischemia. A decrease in NAD level has been reported in myocardial tissue due to CABG surgery [38,39]. Metabolic reprogramming indeed occurs with CPB since a nuclear magnetic resonance study of the left atrial tissue showed increases in glucose, pyruvate, citrate, and lactate, indicating a reduction of glycolytic energy metabolism [40]. On the other hand, increases in sugar alcohols have been linked to apoptosis of cardiomyocytes during ischemia [41][42][43]. Although xylitol, adonitol, arabitol, and ribose-5-P may not be inducers of apoptosis, the correlation of their elevations with cell death suggests the functionality as a biomarker of cell injury. Inhibition of H 2 O 2 -induced metabolic changes by HK and LK suggest the cytoprotective capacity of HK and LK against ischemic injury or oxidative stress.
Ischemia causes a shift from aerobic to anaerobic metabolism, where ATP production is decreased due to O 2 deprivation for the respiratory chain. Glycolysis, the main ATP source in the ischemic myocardium, is progressively limited by the restraint of glyceraldehyde 3-phosphate dehydrogenase flux due to NADH accumulation [44]. High levels of NADH and FADH 2 inhibit β-oxidation, leading to an accumulation of long-chain acyl CoA, acylcarnitine esters, and free fatty acids [45,46]. These lipophilic compounds can act as "detergents", altering membrane functions, such as Na + /K + ATPase, and sarcoplasmic reticular Ca 2+ -stimulated ATPase [47]. Upon reperfusion, these changes contribute to the elevation of cytosolic Ca 2+ [48]. Oxidative stress can cause Ca 2+ influx and elevation of cytosolic Ca 2+ . Elevation of cytosolic Ca 2+ either by ischemic reperfusion or oxidative stress causes mitochondrial membrane permeability transition and a further increase in oxidant production [5,6].
HK and LK deliver 20 mg/L or 0.085 mM lidocaine, whereas DN introduces 104 mg/L or 0.444 mM lidocaine to the cells in the culture. In contrast, lidocaine is absent in HTK and CS. Lidocaine is a sodium channel blocker that inhibits the entry of Na + into cells through a fast voltage-gated Na + channel (Na V ). A number of publications have reported the cytotoxicity of lidocaine in a variety of cell types in the mM concentration range [49,50]. Cytotoxicity may result from high doses that allow lidocaine to act as a surfactant and disrupt the plasma membrane [50]. This may explain the lack of full protection of DN against the metabolic change of oxidative stress.
In cardiomyocytes, lidocaine appears to serve as a cytoprotective agent. Lidocaine protects H9C2 cardiomyocytes against loss of viability by hypoxia [51]. Protection against myocardial infarction has been reported in mice with lidocaine injection [52]. A similar discovery was reported with regional ischemia in rats [53]. CPB in a canine model showed that lidocaine protected against mortality due to global myocardial ischemia and reperfusion [54]. An early study using a canine model of regional ischemic reperfusion showed a reduction of infarct size by lidocaine in association with decreased lipid peroxidation [55]. The protective effect of lidocaine against myocardial infarction was also observed in a porcine experimental model [56]. These protective effects may result from the inhibition of Na v and the prevention of intracellular Na + increase. Na + efflux is coupled to Ca 2+ influx via the sodium-calcium exchanger (NCX). Inhibition of intracellular Ca 2+ elevation by lidocaine has been demonstrated by pacing isolated rat hearts [57]. In cardiomyocytes, late openings of the sodium channel by H 2 O 2 treatment may contribute to an increase in cytosolic Ca 2+ [58]. Lidocaine may block H 2 O 2 from inducing elevation of cytosolic Ca 2+ and, therefore, production of ROS from the mitochondria. A preliminary experiment indeed showed protection against ROS generation by lidocaine (Figure 7). Early evidence indicates that lidocaine preserved mitochondrial oxidative phosphorylation during ischemic reperfusion [59,60]. Consistent with the protective role of lidocaine, an inhibitor of NCX was capable of preventing intracellular Ca 2+ overload or mitochondrial Ca 2+ increase and enhancing cardiac recovery following ischemia [61,62]. Therefore, the observed protective effect of HK or LK may be related to the presence of lidocaine and the inhibition of ROS generation.
showed protection against ROS generation by lidocaine (Figure 7). Early evidence indicates that lidocaine preserved mitochondrial oxidative phosphorylation during ischemic reperfusion [59,60]. Consistent with the protective role of lidocaine, an inhibitor of NCX was capable of preventing intracellular Ca 2+ overload or mitochondrial Ca 2+ increase and enhancing cardiac recovery following ischemia [61,62]. Therefore, the observed protective effect of HK or LK may be related to the presence of lidocaine and the inhibition of ROS generation.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary Material. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary Material. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.