Cardioprotective Natural Compound Pinocembrin Attenuates Acute Ischemic Myocardial Injury via Enhancing Glycolysis

Purpose Emerging evidence has shown that pinocembrin protects the myocardium from ischemic injury in animals. However, it is unknown whether it has cardioprotection when given at the onset of reperfusion. Also, mechanisms mediating the cardioprotective actions of pinocembrin were largely unknown. Thus, this study is aimed at investigating the effects of pinocembrin postconditioning on ischemia-reperfusion (I/R) injury and the underlying mechanisms. Methods The in vivo mouse model of myocardial I/R injury, ex vivo isolated rat heart with global I/R, and in vitro hypoxia/reoxygenation (H/R) injury model for primary cardiomyocytes were used. Results We found that pinocembrin postconditioning significantly reduced the infarct size and improved cardiac contractile function after acute myocardial I/R. Mechanically, in primary cardiomyocytes, we found that pinocembrin may confer protection in part via direct stimulation of cardiac glycolysis via promoting the expression of the glycolytic enzyme, PFKFB3. Besides, PFKFB3 inhibition abolished pinocembrin-induced glycolysis and protection in cardiomyocytes. More importantly, PFKFB3 knockdown via cardiotropic adeno-associated virus (AAV) abrogated cardioprotective effects of pinocembrin. Moreover, we demonstrated that HIF1α is a key transcription factor driving pinocembrin-induced PFKFB3 expression in cardiomyocytes. Conclusions In conclusion, these results established that the acute cardioprotective benefits of pinocembrin are mediated in part via enhancing PFKFB3-mediated glycolysis via HIF1α, which may provide a new therapeutic target to impede the progression of myocardial I/R injury.


Introduction
Cardiovascular disease remains the main cause of death worldwide [1]. Currently, there are few effective drugs to protect the heart after ischemia/reperfusion (I/R) injury. Therefore, it has become a hot research field to find the molecular mechanism of coronary artery disease progression and development that can protect the myocardium from I/R injury. With this in mind, more and more attention is focused on pharmacological interventions because it can induce cardioprotection and easy to implement [2]. However, for patients with ischemic heart disease, there are still many restrictive drugs available clinically.
Pinocembrin (5,7-dihydroxyflavone) is an abundant flavonoid isolated from propolis and some plants. It has various biological functions such as anti-inflammatory, antioxidation, and antibacterial [3]. Previous studies have confirmed that pinocembrin confers neuroprotective effects during cerebral ischemic I/R [4][5][6]. Recent research has also tried to determine whether pinocembrin is beneficial for cardiac injury. For example, several studies suggest that pinocembrin can improve the cardiac function of myocardial I/R rats, reduce ventricular arrhythmia, and reduce the area of myocardial infarction [7,8]. However, it is still unclear whether pinocembrin given at the onset of reperfusion has cardioprotective effects, which is a more clinically effective method. In addition, the underlying mechanism by which pinocembrin can provide cardioprotection is largely unknown.
Recent findings report that changes in energy metabolism relate to many human diseases, and targeted energy metabolism intervention may have beneficial effects on these diseases [9]. Due to the mechanical function of the heart, it is an organ with high energy requirements. In general, most of the energy (about 70%) of a healthy heart comes from the βoxidation of fatty acids, and the rest of the energy comes from glucose oxidation [10]. Nevertheless, in a pathological environment, substrate utilization may change [11]. In patients with diabetes, circulating blood glucose levels at admission are related to the clinical outcome after acute myocardial infarction (AMI), suggesting that it may be related to myocardial metabolism [12]. A metabolic shift from β-oxidation to glycolysis metabolism will reduce the cell's need for oxygen by 11-13%, and NAD+ precursors have been shown to activate cellular glycolysis to protect the heart from ischemic injury [13]. It is worth noting that redirect energy metabolism to glycolysis can reduce oxidative damage and inhibit apoptosis [14][15][16]. However, few agents that target energy metabolism are clinically safe and useful for patients. At the same time, it is unclear whether and how pinocembrin regulates acute myocardial I/R glycolysis.
In this study, we (i) examine the role of pinocembrin in rat and mouse cardiac I/R injury ex vivo and in vivo, respectively, (ii) clarify the effects of pinocembrin on the glycolytic metabolism during I/R, and (iii) investigate the underlying molecular basis that contributes to the pinocembrininduced cardioprotection. Our results uncover a novel mechanism for pinocembrin-induced cardioprotection and suggest a potential application value of pinocembrin in the protection of hearts against I/R injury.

Animals.
Male Sprague-Dawley rats (aged 6-8 weeks) and C57BL/6 mice (aged 8-10 weeks) were from Shanghai Slac Laboratory Animal Co. Ltd. All animals were housed in a temperature-controlled (24°C) and humidity-controlled (40-70%) barrier system with a 12-hour light/dark cycle. All animal experiments were conducted to the guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (revised in 2011), and all procedures were approved by the Shanghai University of Health and Medicine. Rats and mice were selected for treatment and observed in a randomized, double-blind trial.
2.3. Ex Vivo Global I/R Injury Model. As mentioned before, the heart was rapidly excised at 37°C and perfused at a constant pressure of 80 mmHg with Krebs-Henseleit buffer (KHB) using the Langendorff technique (Xie et al., 2005). The PowerLab system (AD instrument, Australia) was used to monitor the left ventricular imaging pressure (LVDP), left ventricular imaging pressure (LVEDP), maximum rate of pressure changes over time (+dP/dtmax), and pressure decay over time (-dP/dtmax). Pinocembrin's final concentration of 10, 30, and 100 μM perfusate was added with 5 minutes of reperfusion. In order to assess the infarct size, the isolated rat heart was reperfused for 2 hours after 30 minutes of ischemia. The slices were incubated in 1% w/v triphenyltetrazolium chloride (TTC, pH 7.4) for 15 min and then fixed in 10% formaldehyde. The Image-Pro Plus software (Media Cybernetics) was used to calculate the infarct area. The infarct area was expressed as a percentage of the LV area at risk.
2.4. In Vivo Myocardial I/R Model. Surgical ligation of the left coronary artery (LCA) was performed as described previously [17]. Mice were anesthetized with pentobarbital sodium (50 mg/kg) and ketamine (50 mg/kg) by intraperitoneal injection, followed by orally intubating and ventilating. The core body temperature is always maintained at 37°C. An internal sternotomy was then performed with electrocautery, and then, the proximal LCA was displayed and ligated. We investigated the magnitude of protection afforded by two different doses of pinocembrin (5 and 10 mg/kg i.v.). Accordingly, the mice received at random an intravenous injection of either saline, 5 mg/kg of pinocembrin, and 10 mg/kg of pinocembrin. These intravenous injections were performed during the last 5 min of ischemia, prior to reperfusion. After 30 minutes of coronary artery occlusion, the suture was cut and the blood vessels were allowed to reperfuse. After 24 hours of reperfusion, the mice were anesthetized with isoflurane. Transthoracic echocardiography was used to determine the left ventricular ejection fraction. After reperfusion, blood samples were taken followed by centrifugation at 3000 rpm for cardiac troponin T (cTnT) and lactate dehydrogenase (LDH) measurements. Measurement of an area at risk and infarct size was performed as reported previously.
2.5. Isolation and Culture of Primary Cardiomyocytes. Continuous enzymatic digestion and isolation were used to obtain neonatal rat and mouse cardiomyocytes [18,19]. Neonatal rats were decapitated, and hearts were immediately placed in HBSS. The ventricle was taken and digested with trypsin and collagenase several times for 10 min at 37°C. Cardiomyocytes were suspended in sterile DMEM. The cells were preplated twice (37°C for 45 minutes) to reduce fibroblast contamination. Hypoxia/reoxygenation (H/R) was conducted as previously described in cardiomyocytes to simulate MI/R in vivo [20].
2.6. Measurement of LDH Release and cTnT Release. Necrotic cell death was assessed by the activity of the supernatant LDH, just like in previous studies [21]. The plasma cTnT level was used as an indicator of myocardial cell damage and was measured using a mouse cTnT ELISA kit (Wuhan Elabo Biotech Co., Ltd., China) according to the instructions.
2.10. Seahorse Extracellular Flux Analyzer Assays. Cellular bioenergetics was measured using a Seahorse XFe24 extracellular flux analyzer in intact cardiomyocytes. We conducted glycolysis stress testing following the manufacturer's instructions as previously reported [25]. Glycolysis stress test: cells were incubated in glucose-free Seahorse assay media supplemented with 1 mM pyruvate at 37°C in an incubator without CO 2 for 1 h prior to the assay. Injectors were loaded to add 20 mM glucose, 1 μM oligomycin, and 100 mM 2 deoxyglucose (2-DG), and glycolysis, glycolytic capacity, and glycolytic reserves were calculated as an extracellular acidification rate (ECAR).
2.11. Generation of Plasmids and Transfection. p-GL4-basic luciferase expression vectors containing various lengths of 5′-flanking regions from the human PFKFB3 gene promoter and an HIF1-luciferase reporter gene were prepared by gene synthesis by General Biol (Anhui, China). siRNAs for mouse HIF1α were obtained from RiboBio (Guangzhou, China). Transfection was performed with Lipofectamine 3000 (Thermo, MA, USA) following the manufacturer's protocol. Luciferase activities were measured by using a Dual-Luciferase® Reporter Assay System (Promega, USA).
2.12. Statistical Analysis. Statistical analysis was undertaken only for studies where each group size was at least n = 5 using software GraphPad Prism (v7, GraphPad Software, USA). Data were presented as means ± SEM. Comparisons between 2 groups were performed using unpaired, two-tailed t test, and ANOVA with post hoc Dunnett's test was used for among multiple groups. A P value < 0.05 was deemed statistically significant.

Compound Pinocembrin
Significantly Improved the Cardiac Function and Reduced Infarct Size after I/R Ex Vivo. We perfused isolated rat hearts to explore the cardioprotective effects of pinocembrin against I/R injury. Ten to 100 μM pinocembrin were delivered during the first 5 min of reperfusion (Figure 1 Next, we explore whether pinocembrin improves cell survival during I/R via examining lactate dehydrogenase (LDH) release, an indicator of myocardial injury. Little LDH release was detected in the coronary efflux before ischemia, while LDH release was obviously induced at the end of reperfusion, while pinocembrin significantly inhibited the release of LDH from 10 to 100 μM ( Figure 2(e)). Consistently, pinocembrin significantly reduced the I/R-induced infarction after 2 h of reperfusion at the concentration of 30 μM (Figure 2(f)). These results demonstrated that pinocembrin exhibits protective effects on cardiac I/R injury ex vivo.

Compound Pinocembrin Protects Hearts from Myocardial I/R Injury In Vivo.
To explore the cardioprotective effects of pinocembrin on myocardial I/R injury in vivo, pinocembrin was intravenously injected into wild-type (WT) mice 5 minutes before the end of sustained ischemia (i.v., 5 mg/kg and 10 mg/kg), followed by reperfusion for 24 hours (Figure 1(b)). Evans-blue/TTC dye method was used to determine the infarct size. After reperfusion for 24 hours, no difference of area at risk (AAR) was observed between each group (Figures 3(a) and 3(b)). Nevertheless, compared with the I/R group, pinocembrin i.v. treatment significantly decreased the infarct size by 20% (Figures 3(a) and 3(c)). Besides, plasma levels of cTnI and LDH activity were markedly elevated during myocardial I/R, which were both suppressed with pinocembrin i.v. treatment (Figures 3(d) and 3(e)). Furthermore, the echocardiographic results showed that pinocembrin can significantly improve I/R-suppressed ejection fraction (EF) and fractional shortening (FS) (Figure 3(f)).

Protective Effects of Pinocembrin against H/R Induced Cardiomyocyte Injury.
To determine whether pinocembrin confers cardioprotective effects through its direct action on the cardiomyocytes, isolated neonatal rat and mouse cardiomyocytes were subjected to H/R and applied 30 μM pinocembrin during the onset of reperfusion. In accordance with the effects of pinocembrin on the myocardial I/R injury, simulated I/R-reduced cell viability was significantly improved by pinocembrin treatment (data not shown).

Pinocembrin Increases Glycolysis in Cardiomyocyte.
During myocardial ischemia, enhanced glycolytic metabolism is essential for maintaining homeostasis of cardiomyocytes. In addition, previous studies reported that pinocembrin was involved in regulating glucose uptake in cancer cells [26]. Subsequently, we explored the effects of pinocembrin on cellular bioenergetics with the Seahorse extracellular flux analyzer and performed glycolysis stress tests to measure glycolysis and glycolytic capacity both in intact rat and mouse cardiomyocytes. Our results indicate that delivery with pinocembrin versus the control increased glycolysis by 21.4% (extracellular acidification rate (ECAR)) after H/R. Pinocembrin also increased glycolytic capacity in cardiomyocyte by 23.7% (Figures 5(a) and 5(b)).
To figure out the underlying mechanism of pinocembrin promoting myocardial glycolysis, mRNA expressions of glycolysis-related genes in cardiomyocytes after H/R were determined with qRT-PCR. As shown in Figure 5(c), pinocembrin significantly increased the expression of glycolysisrelated genes, especially the PFKFB3 gene.

PFKFB3 Inhibition Alters Glycolysis and Abolished
Pinocembrin-Induced Cardioprotection in Cardiomyocytes. Next, we explored whether blockade of PFKFB3 inhibits cardiomyocyte glycolysis and abolishes pinocembrin-afforded cardioprotective effects. As shown in Figures 6(a)-6(d), exposure of cardiomyocyte to 10 μM PFKFB3 inhibitor, 3PO remarkably reversed pinocembrin-enhanced glycolysis. What is more, inhibition of PFKFB3 resulted in a significant increase of cTnI release and LDH release post-H/R in vitro (Figures 6(e)-6(h)), suggesting that disruption of glucose metabolism leads to impaired cardioprotection of pinocembrin during H/R.

PFKFB3 Deficiency in Normal Mice Using AAV9
Abolished Pinocembrin-Afforded Cardioprotective Effects after MI/R. Since pinocembrin alleviated H/R-induced cardiomyocyte death by upregulating glycolysis via PFKFB3, we therefore evaluated the roles of PFKFB3 on myocardial I/R injury in vivo. To determine whether PFKFB3 regulation is directly involved in pinocembrin-related MI/R injury improvement, the WT mice were injected with AAV9 encoding PFKFB3 shRNA to knockdown endogenous PFKFB3. Intravenous injection of AAV9-shPFKFB3 on mice has successfully reduced the protein level of PFKFB3 in the heart (Supplementary Figure 2). Subsequently, mice were insulted with in situ myocardial ischemia-reperfusion to assess the functional role of myocardial-specific PFKFB3 in pinocembrin-afforded cardioprotection. Myocardial injury was measured by infarct size area, serum cTnT levels, and LDH activity. Exposure of mice infected with AAV9-shPFKFB3 to MI/R presented larger infarct sizes and enhanced release of troponin I and LDH (Figures 7(a)-7(c)). More importantly, the deletion of myocardial PFKFB3 abolished pinocembrin-conferred protective effects on those indexes. Furthermore, the pinocembrin-improved EF and FS were also abolished by PFKFB3 deficiency with  Oxidative Medicine and Cellular Longevity AAV9-shPFKFB3 (Figure 7(d)). Collectively, these data confirm that pinocembrin confers cardioprotective effects by enhancing cardiomyocyte glycolysis through activation of PFKFB3.

HIF1α Is a Key Transcription Factor Driving
Pinocembrin-Induced PFKFB3 Expression. Pinocembrin notably promoted PFKFB3 gene expression in cardiomyocytes exposed to H/R (Figure 8(a)). To explore the underlying mechanisms responsible for the increased expression of PFKFB3 in cardiomyocytes induced by pinocembrin, we examined the activity of the mouse PFKFB3 promoter both in the absence or in the presence of pinocembrin. We transfected cardiomyocytes with a construct in which luciferase expression was driven by a 3,308 bp fragment from the mouse PFKFB3 promoter. As shown in Figure 8(b), pinocembrin by itself exerted little effect on PFKFB3 promoter activity, but it cooperated with H/R to increase luciferase activity (Figure 8(b)). Furthermore, we studied the DNA regions required for pinocembrin to increase PFKFB3 5 Oxidative Medicine and Cellular Longevity promoter activity by using a series of promoter constructs containing successive deletions from the 5 ′ end. As shown in Figure 8(c), 5′ deletion mutants revealed that sequences between -50 and -200 and -1108 and -2108 from the transcription start site are essential for the induction of the PFKFB3 gene promoter by pinocembrin.
HIF1α is considered to be an important regulator of glycolysis, and its target genes include PFKFB3 and glucose transporter-1 (GLUT1) [27,28]. Also, pinocembrin increased the expression of HIF1α, which are key transcription factors for PFKFB3 expression (data not shown). To identify transcription factor HIF1α that could modulate PFKFB3 induction by pinocembrin, reporter constructs carrying binding sites for HIF1 were transfected into cardiomyocytes, which were stimulated afterward with pino-cembrin. The results showed that the activity of HIF1 reporter constructs increased upon activation with H/R, and this activity was enhanced by the pinocembrin (Figure 8(d)). Moreover, we observed that reduced expression of HIF1α with specific siRNA greatly decreased PFKFB3 induction by pinocembrin during H/R, corroborating its key role in pinocembrin-induced PFKFB3 expression (Figures 8(e) and 8(f)). Together, our data demonstrates that HIF1α is a key transcription factor driving pinocembrin-induced PFKFB3 expression during H/R.

Discussion
In the present study, we observed that glycolysis functionally mediated the cardioprotective responses of pinocembrin. We It would be an attractive treatment principle to reduce the infarct size through pharmaceutical intervention to assist classic reperfusion intervention [29]. Pinocembrin is a potential cardiovascular drug with potential neuroprotective effects on transient and long-term ischemic stroke in rats [30]. Also, a previous study indicated that the administration of pinocembrin before myocardial ischemia improved LV function [7]. Pharmacological postconditioning is easier to implement and has therapeutically potential in both clinical and experimental settings, which avoids the potential injury induced by ischemic conditioning, and therefore has good clinical application prospects [31]. Therefore, our study aims to explore the cardioprotective effects of pinocembrin postconditioning. The ex vivo results showed that pinocembrin from 10 to 100 μM delivered at the first 5 minutes of reperfusion remarkably improved postischemic myocardial function and attenuates cell death in a concentration-dependent manner. Furthermore, the in vivo mouse myocardial I/R injury model was prepared and pinocembrin postconditioning was fulfilled by an intraperitoneal injection of pinocembrin (5 mg/kg and 10 mg/kg body weight) 5 min before reperfusion. Cardiac function, serum LDH activity and cTnT content, and infarct size were significantly improved with pinocembrin treatment. The cardioprotective effects of pinocembrin are consistent with other reports. This suggests that pinocembrin could be a candidate compound that may be used in the clinical treatment of myocardial infarction.
Various metabolic abnormalities occur during myocardial I/R, such as increased fatty acid oxidation and decreased glucose oxidation [32]. This phenomenon is related to the uncoupling of mitochondrial respiration, increased proton leakage, ROS formation, and, more importantly, increased myocardial oxygen consumption [33]. During myocardial I/R, the metabolic shift aimed at   Oxidative Medicine and Cellular Longevity increasing glucose oxidation has proved to be beneficial. Although some therapeutic strategies have tried to reverse this metabolic imbalance, there is still no approved treatment regimen so far [34][35][36]. In addition, strategies aimed at increasing clinical glucose consumption have different results and have not yet reached routine clinical practice. Looking for drugs that can safely induce the transfer of cellular energy metabolism and a better understanding of the protective mechanisms of increased glucose oxidation may facilitate transfer to the clinic. Our results show that increasing glycolysis is responsible for pinocembrin-induced protective effects. This is consistent with previous studies that metabolic shift towards increased glycolysis protects the heart from I/R injury   Oxidative Medicine and Cellular Longevity [13]. Moreover, PFKFB3 expression is notably upregulated during I/R, which directs cellular glucose metabolism from PPP to aerobic glycolysis [37]. Our data have shown that PFKFB3 was significantly upregulated with pinocembrin treatment and specific inhibitor of PFKFB3 abolished pinocembrin-afforded protective effects in cardiomyocytes. Most importantly, knockdown of PFKFB3 in the myocardium using AAV9 reversed the cardioprotection of pinocembrin postconditioning. Previous studies have shown that the main reason responsible for the protection offered by the metabolic shift is the fact that glycolysis is able to produce two molecules of ATP without the need for oxygen, so that uncoupling between mitochondrial full glucose oxidation and glycolysis leads to increased cardiac efficiency [38]. However, potential protective mechanisms of the metabolic shift are largely unknown. Further studies are needed to investigate how pinocembrin regulate the glycolysis and search for its putative downstream targets.

Conclusions
In summary, our findings demonstrate that compound pinocembrin exhibits significant protective effects on