D-glucono-1,5-lactone was obtained from MedChem Express (Princeton, NJ, United States). Triphenyltetrazolium chloride (TTC), PI3K inhibitor Wortmannin (Wm; inhibition of PI3K/Akt signaling) and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (inhibition of MEK/ERK signaling) were purchased from Sigma (St. Louis, MO, United States). PKC inhibitors Chelerythrine and Rottlerin were purchased from Topscience (Shanghai, China). PKC inhibitor Gö6976 was purchased from Beyotime (Haimen, China). PKCεV1-2 was obtained from Medbio (Shanghai, China). Dihydroethidium (DHE) was purchased from Beyotime.

Cell Counting Kit-8 (CCK-8) was obtained from Topscience. Lipid Peroxidation Malondialdehyde (MDA) Kit and Total Superoxide Dismutase (SOD) Assay Kit were purchased from Beyotime. In Situ Cell Death Detection Kit was purchased from Roche Co. (Mannheim, Germany). Lactate Dehydrogenase (LDH) Assay Kit, Creatine Kinase MB (CK-MB) isoenzyme Assay Kit and Coenzyme II (NADPH/NADP⁺) Content Test Kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Age-matched male C57BL/6J mice (8- to 10-week-old) were provided by the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). All animals were kept at controlled environmental conditions (22 ± 2°C with a 12 h light/dark cycle), and allowed free access to food and water. All experiments were performed in strict accordance with the recommendations in the Guide for the National Institutes of Health Guidelines for the Use of Laboratory Animals (Publication No. 85–23, revised 1996), and approved by the Animal Care Committee of Northwestern Polytechnical University (Xi’an, China). The mice were allowed to adapt to the new conditions for 1 week before being used in this experiment.

The mouse model of myocardial I/R injury was established by occluding the left anterior descending coronary artery (LAD) for 30 min followed by reperfusion for 120 min. Briefly, mice were anesthetized by pentobarbital sodium (60 mg/kg) after fasted overnight. After endotracheal intubation, mouse heart was exposed between the third and fourth ribs, and LAD was ligated with a 7-0 silk suture. After 30 min of ischemia, the slipknot was released, and myocardium was reperfused for 120 min. Mice in the sham group underwent the same surgical procedures, but without ligation. GDL (5 mg/kg) or its control vehicle (saline) was intraperitoneally administrated at 10 min pre-reperfusion. Surface lead II electrocardiogram (ECG) was continuously recorded during I/R by a physiologic signal-acquisition system (RM6240; Chengdu instrument factory, Chengdu, China), and the arrhythmia incidence and score were assessed according to the Lambeth Conventions (Curtis et al., 2013).

The myocardial infarct size was evaluated by TTC-Evans blue double staining. After 2 h of reperfusion, the LAD was re-tied to stop the blood flow, and 1 mL of 2% Evans blue dye (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was infused into the hearts via the ascending aorta. After that, the heart was sliced into 1 mm sections, and incubated with 1% TTC at 37°C for 20 min. Finally, the slices were store in 4% paraformaldehyde solution before digitally photographed. The area of left ventricle (LV), the area at risk (AAR), and infarct size (INF) were assessed with FIJI (ImageJ) software (National Institutes of Health, United States).

At the end of reperfusion, cardiac apoptosis was evaluated by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s instructions.

Neonatal rat cardiomyocytes (NRCM) were isolated from the hearts of 1- to 3-day-old Sprague–Dawley rats. Briefly, rat hearts were removed, minced and digested with 0.1% Type II collagenase and 0.25% Trypsin. Cardiomyocytes were isolated by differential detachment and verified under the microscope. Isolated NRCM were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution in a humidified 5% CO₂ incubator (Boxun, Shanghai, China) at 37°C.

A cellular model of H/R was established to mimic myocardial I/R injury in vitro. Briefly, hypoxia was induced by incubation of NRCM cells with glucose-free and serum-free DMEM medium in an anaerobic chamber (95% N₂, 5% CO₂) at 37°C for 12 h. To mimic reperfusion, the hypoxic NRCM cells were cultured in serum-free DMEM medium (with 5.5 mM glucose) under normoxic conditions for 2 h at 37°C.

D-glucono-1,5-lactone (1 μM) was administrated before the onset of reoxygenation. To investigate whether blocking PI3K/Akt signaling or MEK/ERK signaling could attenuate GDL-conferred cardioprotection against H/R, ERK inhibitor U0126 (10 μM) or PI3K inhibitor Wm (100 nM) administrated concurrently with GDL before the onset of reoxygenation.

At the end of reperfusion or reoxygenation, heart tissue or cell culture was collected, and proteins were isolated using radio-immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Roche), and subsequently quantitated by BCA kit (Pierce Chemical Company, Rockford, IL, United States). Equal amount of proteins were loaded and separated by 10 or 12% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h, and subsequently incubated with primary antibodies against p-ERK (1:1000; Cell Signaling Technology/CST, Danvers, MA, United States), ERK (1:1000; Proteintech, Wuhan, China), p-Akt (1:1000; CST), Akt (1:1000; CST), Bcl2 (1:1000; Proteintech), Bax (1:1000; Proteintech), Caspase3 (1:1000; Proteintech), The nuclear factor erythroid 2 related factor 2 (NRF2; 1:1000; Proteintech), SOD2 (1:1000; Proteintech) and PKCε (1:1000; Proteintech) overnight at 4°C. Subsequently, the membranes were incubated with appropriate secondary antibodies for 1–2 h. Finally, the blots were visualized by Tanon imaging system (Tanon Science & Technology Co., Ltd., China) and quantified by densitometry using Quantity One software (Bio-Rad, Hercules, CA, United States).

Cardiac tissues were fixed overnight with 4% paraformaldehyde, embedded in paraffin, and sectioned with a microtome in 5 μm slices. Myocardial slices were stained with PKCε antibodies (Proteintech), and photographed by an optical microscope (Nikon, Melville, NY, United States). For the colocalization analysis, Manders’ coefficient was calculated using Coloc2 plugin in FIJI (ImageJ) image processing software.¹

All data were presented as means ± SEM and performed using Graph Pad Prism 8.0 software. Student’s t-test or Chi-square test and Fisher’s exact test (for arrhythmia incidence) was used to determine the significant difference among two groups, while one-way ANOVA with Bonferroni’s post hoc test was used among more than two groups. Statistical significance was accepted at P < 0.05.

D-glucono-1,5-lactone, a derivate of glucose, is a ring-shaped molecule characterized by a tetrahydropyran substituted by three hydroxyl groups, one ketone group, and one hydroxymethyl group (its chemical structure is displayed in Figure 1A). The schematic of the experimental design in vivo is shown in Figure 1B. GDL administration reduced myocardial infarction size in IR + GDL group (25 ± 3%) compared with I/R group (40 ± 2%) (Figure 1C). In line with this, serum levels of cardiac injury markers, LDH and CK-MB were increased following I/R injury, while were decreased with GDL pre-treatment (Figures 1D,E).

Imbalance between pro-survival and apoptotic signaling is believed to be central to myocyte dysfunction following I/R injury. Thus, we assessed the expression and (or) activity of key players in pro-survival (Akt and ERK) and apoptotic (Bcl2, Bax, and Caspase3) signaling pathways by Western blot analysis, demonstrating that GDL treatment stimulated ERK phosphorylation, especially p42 ERK, whereas no significant difference in Akt expression and phosphorylation was observed between I/R and I/R + GDL mice (Figure 1F). GDL administration following I/R decreased apoptotic signaling in cardiac tissues as evidenced by increased Bcl2/Bax ratio and decreased Caspase3 activation (Figure 1G). In agreement with this, the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining showed that GDL decreased I/R-induced apoptosis in cardiac tissue (Figures 1H,I).

Reperfusion-induced arrhythmia is a common clinical manifestation of myocardial I/R injury. The surface ECG was recorded in each group, and arrhythmias were analyzed 30 min post-reperfusion according to the Lambeth conventions (Curtis et al., 2013). ECG showed that arrhythmias were commonly seen during the onset of reperfusion, of which, mostly were premature ventricular contraction (PVC), rarely were ventricular tachycardia (VT), and none were ventricular fibrillation (VF). Representative examples of different ventricular arrhythmias (PVC and VT) were illustrated in Figure 2A. GDL decreased reperfusion-induced arrhythmias (Figure 2B), mainly due to a decrease of PVC but not VT occurrence (Figures 2C,D). Of note, we found that QT interval and corrected QT (QTc) interval, a risk factor for the development of ventricular arrhythmia, were decreased in mice with GDL treatment (Figures 2E,F). Heart rate showed no apparent changes during baseline, ischemia or reperfusion in I/R-injured mice with or without GDL treatment (Figure 2G).

Although GDL is known as an antioxidant, its regulatory role in oxidative stress in vivo, especially in the context of I/R, was not investigated. As shown in Figures 3A,B, exposure to I/R displayed an increase of MDA and a decrease of SOD2 activity in either serum or heart tissue. GDL treatment decreased MDA levels both in serum and heart tissue of I/R-injured mice, yet no significant changes of SOD2 activity were observed after GDL treatment (Figures 3A,B).

Oxidative stress refers to the pathological process of tissue damage caused by excessive ROS production and/or reduced ROS scavenging capacity. We first evaluated the expression of antioxidant enzymes including NRF2 and SOD2. In line with the results of our studies regarding to SOD activities, no apparent changes of NRF2 and SOD2 expression were observed among groups (Figure 3C). NADPH, a key component in the cellular anti-oxidative defense system, was decreased in the heart of I/R mice, an effect which could be reversed by GDL treatment (Figure 3D). Next, we measured total ROS levels using DHE staining, showing that GDL treatment decreased I/R-induced ROS levels (Figure 3E).

Next, we evaluated the protective effect of GDL and the underlying mechanism in vitro. Our results demonstrated that high concentration of GDL (>100 μM) had a cytotoxic effect on NRCM cells, whereas GDL concentrations at 1 μM and 10 μM did not show this adverse effect (Figure 4A). In the context of H/R injury, GDL concentrations at 1 μM, 10 μM, 100 μM and 1 mM showed beneficial effects against H/R injury, an effect that was diminished when GDL concentration was at 10 mM (Figure 4B). Therefore, GDL concentration in the following experiments was set to be 1 μM when GDL exerted cardioprotective effects against H/R injury and had less cytotoxic effect. As expected, GDL (1 μM) treatment decreased apoptotic signaling in H/R-injured NRCM cells, as evidenced by decreased Caspase3 activation and increased Bcl2/Bax ratio (Figure 4C). In agreement with this result, GDL treatment activated pro-survival signaling including ERK and Akt (Figure 4D).

Both in vivo and in vitro studies have shown that GDL-conferred cardioprotection was associated with increased pro-survival signaling (ERK or Akt). CCK-8 assay showed that blocking ERK signaling by its inhibitor U0126 attenuated GDL-induced cell survival, whereas inhibiting PI3K/Akt signaling via Wortmannin did not show this effect (Figure 5A). Supportively, GDL treatment activated ERK signaling, reaching a peak at 2 h (Figure 5B). In addition, either low or high concentration of GDL strongly activated ERK signaling after treating cardiomyocytes for 2 h (Figure 5C). Western blot analysis showed that blocking ERK signaling attenuated GDL-induced inhibition of Caspase3, yet had no significant effect on Bcl2/Bax ratio (Figures 5D–G).

To further understand how GDL regulates ERK signaling, we used Swiss Target Prediction databases² to sort out the potential targets of GDL. Species were selected as “Homo sapiens,” and a total of 23 targets were obtained under the condition of probability greater than 0.1 (Supplementary Table 1). Among them, Protein kinase C (PKC) isoforms, including PKCδ, PKCα, PKCγ, PKCε, PKCη, PKCθ, stood out as the most possible target for GDL.

Next, Autodock Vina was used to predict the binding modes of GDL with PKC (Trott and Olson, 2009). The crystal structure of PKC (PDB code 1XJD) was obtained from the Protein Data Bank (PDB), and GDL was docked into PKC structure by Autodock Vina. Among twenty conformation generated, the best one has been nominated based on lowest binding energy (-5.0 Kcal/mol). Protein-ligand interaction of docked complex was visualized by PyMOL software³ (Figure 6A). The hydrogen bonds were analyzed by PyMOL software, showing GDL interacts with PKC by forming hydrogen bonds with Q688, F691, and K413 (Figure 6B). Electrostatic surface of PKC was generated by APBS tool, showing GDL buried in a positive charged pocket of PKC (Figure 6C).

The NMR structure of PKC binding with Phorbol-13-acetate (PDB code 1PTR) has been revealed before, thus, we try to compare the binding poses of GDL with Phorbol-13-acetate. GDL was docked into the crystal structure of PKCδ C1 domain by Autodock Vina (binding energy -5.0 Kcal/mol), and we can see GDL occupied the same position in PKC structure as Phorbol-13-acetate after structural alignment (Figure 6D).

Protein kinase C family falls into three groups: the classical PKC (cPKC; α, βI, βII, and γ), atypical PKC (aPKC; ζ and λ/ι) or novel PKC (nPKC; δ, ε, η, θ, μ, and υ). To investigate whether or which PKC isoform contributes to GDL-induced ERK phosphorylation and cardioprotection, we used different PKC inhibitors, including Chelerythrine (a general PKC inhibitor), Gö6976 (an inhibitor of cPKC), Rottlerin (an inhibitor of PKCδ), and PKCεV1-2 (a specific inhibitor of PKCε). Western blot analysis showed that Chelerythrine (5 μM), Gö6976 (1 μM), and Rottlerin (25 μM) had no effect on GDL-induced ERK phosphorylation, whereas PKCε specific inhibitor PKCεV1-2 (20 μM) attenuated this effect (Figures 7A,B). Immunofluorescence staining showed that i.p. administration of GDL in mice increased PKCε translocation into nuclear (Figures 7C,D). Cell viability assay showed that PKCεV1-2 attenuated GDL-conferred cardioprotection against H/R injury (Figure 7E).