Cardioprotective effect of ivabradine via the AMPK/SIRT1/PGC-1α
signaling pathway in myocardial ischemia/reperfusion injury
induced in H9c2 cell

Post-resuscitation myocardial dysfunction (PRMD) is the most severe myocardial ischemia-reperfusion injury (MIRI) and is characterized by difficult treatment and poor prognosis. Research has shown the protective effects of the rational use of ivabradine (IVA) against PRMD; however, the molecular mechanisms of IVA remain unknown. In this study, an ischemia-reperfusion injury (IRI) model was established using hypoxic chambers. The results demonstrated that pretreatment with IVA reduced IRI-induced cytotoxicity and apoptosis. IVA attenuated mitochondrial damage, eliminated excess reactive oxygen species (ROS), suppressed IRI-induced ATP and NAD, and increased the AMP/ATP ratio. We further found that IVA increased the mRNA levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and upregulated the expression levels of phosphorylated AMP-activated protein kinase (p-AMPK)/AMPK, SIRT1, and PGC-1α proteins. Interestingly, no change in AMPK mRNA levels was observed. Cardiomyocyte energy metabolism significantly changed after IRI. The aim of this study was to demonstrate the cardioprotective effect of Ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway in myocardial ischemia/reperfusion injury-induced in H9c2 cell.


Introduction
Post-resuscitation myocardial dysfunction (PRMD) is the most severe myocardial ischemia-reperfusion injury (MIRI) and is closely associated with early death after resuscitation, especially within the first 24 h after the restoration of spontaneous circulation (ROSC) (Jentzer et al., 2015). Studies have shown that the severity of PRMD is closely related to the duration of myocardial ischemia and the dose of epinephrine (Palmer et al., 2004;Yamaguchi et al., 2002). In the resuscitation process of cardiac arrest (CA) patients, the use of epinephrine (especially large doses) can significantly increase the heart rate after resuscitation and the recurrence rate of arrhythmia, and even ventricular fibrillation after resuscitation and those factors would aggravate PRMD. Therefore, controlling the heart rate is one of the clinical methods used to treat cardiovascular diseases (Yip et al., 2016). Many previous studies have reported the therapeutic value of β-blockers for PRMD in animal models and clinical studies Ji et al., 2012;Link et al., 2015;Yang et al., 2015). The limited use of beta-blockers to treat PRMD patients is probably due to their negative inotropic, negative chronotropic, and negative dromotropic effects (Link et al., 2015). Therefore, it is necessary to find new safe and effective drugs for controlling heart rate.
IVA is the only hyperpolarization-activated cyclic nucleotide-gated (HCN) channel inhibitor that has been demonstrated to control heart rate and protect heart function to date (Kuwabara et al., 2013;Xia et al., 2010;Yu et al., 2015). In fact, the IVA treatment was compared with the effect of beta-blockers. Evidence provides that IVA treatment is associated with an increase in the ejection fraction (EF) and reductions in end-diastolic volumes (EDVs) and end-systolic volumes (ESV)s (Fasullo et al., 2009). Our previous results also indicated that IVA could alleviate myocardial dysfunction and myocardial injury in a porcine cardiopulmonary resuscitation (CPR) model, suggesting that IVA may be beneficial for the treatment of PRMD patients (Yang et al., 2020).
In clinical practice, patients with acute coronary syndrome (ACS) have got beneficial effects with the administration of IVA (Oliphant et al., 2016;Trivi et al., 2014). A further study found that IVA has multi-effects such as anti-inflammatory, antioxidant, and endothelial cell function improvements Marazia et al., 2015;Custodis et al., 2008). Furthermore, IVA could reduce myocardial oxygen consumption, without affecting myocardial contraction or inducing heart arrhythmias (Treptau et al., 2015). Under heart rate control conditions, IVA protects against MIRI primarily by reducing the range of myocardial infarction and the production of reactive oxygen species (ROS), and by increasing the activity of ventricular myocytes and adenosine triphosphate (ATP) in mitochondria (Kleinbongard et al., 2015). However, the molecular mechanism underlying these protective effects of IVA remains unknown.
The MIRI effect may be controlled by decreasing ATP production and oxidative stress levels, avoiding the increase of apoptosis (Yellon and Hausenloy, 2007;Ovize et al., 2010). This study investigated the cytoprotective effects of IVA against ischemia-anoxia-induced heart injury, especially the role of AMP-activated protein kinase (AMPK). AMPK is a well-known intracellular energy receptor that protects against metabolic stress conditions, such as ischemia/reperfusion (I/R) (Hong et al., 2003). Some studies have shown that the AMPK/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) signaling pathway plays a crucial role in the protection of cardiac function by participating in energy metabolism, oxidative stress, and mitochondrial function (Chau et al., 2010;Tian et al., 2019). The aim of this study is to demonstrate that IVA may regulate mitochondrial energy metabolism and oxidative stress through the AMPK/SIRT1/PGC-1α signaling pathway in an I/R model of H9c2 cardiomyocytes.

Materials and Methods
Reagents and antibodies IVA was purchased from Target Mol (Shanghai, China). The primary antibodies used for western blots were as follows: antibodies against total and phosphorylated AMPKα (1:1000) and SIRT1 (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against PGC-1α (1:1000) was purchased from Abcam (Abcam Technology, Cambridge, UK). Tubulin-β was used as a loading control and detected using a monoclonal Tubulin-β antibody (1:500, Affinity Biosciences, USA). NAD + , ATP disodium salt, ADP sodium salt, and 5'-AMP-Na 2 were all purchased from Sigma (Sigma Aldrich, St Louis, MO, USA).

Cell culture
H9c2 embryonic rat cardiomyocytes, acquired from the Chinese Academy of Sciences Shanghai Cell Bank, were cultured in complete high-glucose Dulbecco's Modified Eagle Medium (DMEM), containing 10% fetal bovine serum (FBS), in an atmosphere of 95% humidified air and 5% CO 2 , at 37°C. Cells in the logarithmic growth phase were selected for all experiments. After reaching 80-90% confluence, cells were digested for 1-2 min with 0.25% trypsin and passaged at 1:3 every 2-3 days.
Establishment of a myocardial I/R damage model and cell grouping H9c2 cells were selected for the establishment of an I/R model, as described previously by Kuznetsov et al. (2015). According to the method designated previously by Yang et al. (2015), experimental groups were switch from high-glucose DMEM to serum-and glucose-free anoxic media (139 mmol/L NaCl, 4.7 mmol/L KCl, 0.5 mmol/L MgCl 2 , 10 mmol/L CaCl 2 , and 5 mmol/L HEPES, pH 7.4) and were maintained in anaerobic conditions, at 37°C, for 8 h. Subsequently, the medium was replaced with DMEM containing 10% fetal bovine serum, and cells were cultured in an incubator containing 95% air and 5% CO 2 , at 37°C, for 24 h, to mimic re-oxygenation (reperfusion) conditions. The control group consisted of cells that were constantly maintained in normal medium, without the hypoxic stimulus. Each IVA group was pretreated previously with IVA before being subjected to IRI model treatment.
Cell counting kit-8 (CCK-8) assay Cell viability was determined using CCK-8 (Beyotime, Shanghai, China), according to the manufacturer's protocol. Briefly, the H9c2 cell suspension density was adjusted to 5 × 10 3 cells/well and seeded in 96-well plates for 24 h. Different concentrations (1, 10, 20, 100, and 200 µM) of IVA were added to the wells and incubated for the indicated times (2, 12, and 24 h). Cells were then subjected to the I/R injury (IRI) model. After inducing IRI in H9C2c cells, 10 µL of CCK-8 reagent was added to each well, and incubation was continued for 2 h, at 37°C, in a 5% CO 2 incubator. The absorbance value was measured at 450 nm using a Microplate Reader (Tecan M 1000, Swiss Confederation, Tecan company, German), and the experiment was repeated three times.

Apoptosis assay
To investigate the effects of IVA on H9c2 cell survival after I/R, we used flow cytometry combined with Annexin V and propidium iodide (PI) staining to evaluate the apoptosis rate of IRI model H9c2 cells treated with IVA. Flow cytometry analysis, using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, USA), was performed to quantify and identify the percentage of apoptotic H9c2 cells. The H9c2 cell suspension density was adjusted to 1 × 10 6 cells/well and seeded in 6 well-plates. After IRI treatment, cells were collected, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 500 µL 1× binding buffer. Next, 5 µL Annexin-V and 5 µL PI were added to each group of cells for 15 min, under shading conditions, at room temperature. Subsequently, the treated cells were measured using a flow cytometer (Beckman Coulter, USA) to differentiate necrotic cells from apoptotic cells, according to the manufacturer's instructions, and the experiment was repeated three times.

Morphological examination of mitochondria by transmission electron microscopy
Cells were harvested and fixed with 2.5% glutaraldehyde (TED PELLA INC) for 12 h at 4°C. Change the 2.5% glutaraldehyde to 0.01 M PBS-phosphate buffer (pH7.4) and process for 2 h. Cells were post-fixed with 0.1% osmium acid for 1 h (TED PELLA INC), dehydrated using an ethanol gradient series, and then soaked and embedded in pure epoxy resin (Fluka Analytical, Sigma). 70 nm-ultrathin sections were cut and placed on the copper grids. The sections on the grids were double-stained with uranyl acetate and lead citrate before being examined under transmission JEM1400 electron microscope (JEOL, Japan).

Mitochondrial stress assay
In this study, a biochemical analyzer was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of H9c2 cells after I/R treatment, and the related indicators were analyzed to determine the effects of IRI on cellular oxidative phosphorylation and glycolysis. A Seahorse XF24 Extracellular Flux Analyzer was used to determine a suitable inoculation density and the optimal carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) concentration for use in normal H9c2 cells, according to the manufacturer's instructions. A mitochondrial stress assay was performed to evaluate the effects of IRI on mitochondrial OCR and ECAR. On the first day, H9c2 cells were plated on 24-well proprietary Seahorse Biosciences plates (2 × 10 5 cells/well), in 250 µL medium, and could adhere and grow overnight before the IRI experiments. Simultaneously, sensor cartridges (Seahorse) were hydrated in XF Calibrant, overnight, at 37°C, without CO 2 . On the second day, the H9c2 growth medium was removed and replaced with assay medium, which was composed of unbuffered DMEM, supplemented with 25 mM D-glucose, 20 mM sodium pyruvate and 4 mM L-glutamine, pH 7.4. Then, H9c2 cells were incubated at 37°C, without CO 2 , for 1 h prior to respiration analysis.
The OCR parameter values were measured after the injection of load compounds, including oligomycin (1 µM), FCCP (2 µM), antimycin (1 µM), and rotenone (1 µM) (Sigma) into the appropriate ports of a hydrated sensor cartridge from the XF Cell Mito Stress Test kit. The mitochondrial function parameters were automatically calculated by the Seahorse XF Mito Stress Test Report Generator. The ECAR measurement method was the same as that for OCR, except the reagents used were glucose (10 mM), oligomycin (1 µM), and 2-deoxyglucose (50 mM) (Seahorse Bioscience, 2009).

Measurement of ROS generation in H9c2 cells
ROS production has been demonstrated to play key roles during energy metabolism disorders and mitochondrial damage under IRI conditions. The effects of IVA on ROS production were investigated by exposing H9c2 cells to IRI conditions with or without IVA pretreatment. According to the ROS kit (Biosharp Co., Ltd., Guangzhou, China), intracellular ROS can oxidize non-fluorescent 2',7'dichlorodihydrofluorescein diacetate (DCF-DA) to generate fluorescent 2',7'-dichlorofluorescein (DCF), which can be measured to determine ROS production. H9c2 cells were washed twice with PBS after receiving the designated treatment and were then incubated with a suitable amount of DCF-DA solution, at a final concentration of 10 µM, in a 37°C incubator, for 30 min. DCF fluorescence was measured in the field of view, using laser scanning confocal microscopy (Carl Zeiss, German), after washing three times with serum-free medium. Four random fields of view were selected by two investigators, and their mean fluorescence intensities were analyzed using Image-J software to indicate the level of ROS.
ATP, ADP, AMP, and NAD + content measurement with HPLC High-performance liquid chromatography (HPLC) was applied to determine the ATP, ADP, AMP, and NAD + contents (Garcia-Tardon and Guigas, 2018). Chromatographic conditions were as follows. HypersilTM ODS-2 (C18) HPLC (5.0 µm, 4.6 × 250 mm). Mobile phase: 0.5% methanol, 99.5% phosphate buffer (10 mmol/L KH 2 PO 4 , pH 7.0). Flow rate: 1.0 mL/min. Column temperature: 30°C. Detection wavelength: 259 nm. Injection volume: 20 µL. The retention time was used to determine the quality, and the peak area was used to determine the quantity. H9c2 cells were collected in 0.2 mL PBS and lysed by repeated freezing and thawing (3 times, 30 min each). Then, 0.2 mL HClO 4 solution (0.5 M) was added, and the pH of the supernatant was adjusted to 7.0. The cells were centrifuged for 25 min, at 4°C, at 12000 rpm. Then, 20 µL of each sample was injected into the HPLC system to identify and analyze the ATP, ADP, AMP, and NAD + contents.

Statistical analysis
All data are presented as the mean ± standard deviation (SD) and were analyzed using SPSS 20.0 software and Prism 8.0 software. Differences among groups were analyzed with Student's t-test and one-way analysis of variance (ANOVA). p < 0.05 was considered to be significant.

Effects of IRI on energy metabolism in H9c2 cells under physiological conditions
As shown in Figs. 1A and 1C, the optimal H9c2 cell volume was 2.0 × 10 4 , and the optimal concentration of FCCP was 2 µM. When mitochondrial bioenergy metabolism indicators were analyzed, the results showed that the basal respiration (*p < 0.05) and ATP production (**p < 0.01) were reduced, and proton leakage was increased (**p < 0.01) in IRI cells compared with uninjured cells (Fig. 1B). These results indicate that IRI largely inhibits the process of mitochondrial energy metabolism. Simultaneously, to adapt to the hypoxic environment, the glycolytic capacity (*p < 0.05), glycolytic reserves (*p < 0.05), and glycolytic reserves (%) (**p < 0.01), were significantly increased and glycolysis was reduced (**p < 0.01) in IRI cells (Fig. 1D).
IVA treatment increases H9c2 cell survival rate after IRI During the CCK8 assay, H9c2 cells were incubated with different concentrations (0, 1, 10, 20, 100, and 200 µM) of IVA for 2, 12, and 24 h. As shown in Fig. 2A, a significant protective effect was observed for cell proliferation when IVA pretreatment lasted at least 12 h, and the concentration was maintained between 20 and 100 µM (c = 20 µM, *p < 0.05; and c = 100 µM, **p < 0.01). Therefore, pretreatments with either 20 or 100 µM IVA, for 12 h, were used during subsequent experiments. Flow cytometry analysis revealed that the proportion of late apoptotic cells in the IRI group was significantly reduced by IVA pretreatment, suggesting that IVA may play a role in the prevention of apoptosis in the IRI model (Fig. 2B).

Effects of IVA on mitochondrial morphology in myocardial cells after IRI
Electron microscopy results showed that the myocardial cell structure was severely damaged in the IRI group, and many vacuole-like structures were observed. The mitochondria became rounded, swollen, smashed, or even disappeared, and the matrix density deepened (Figs. 3A and 3B). IVA treatment improved the mitochondrial structure damage induced by IRI to a certain degree. In particular, mitochondria treated with IVA at a concentration of 100 µM tended to reduce swollen and vacuole. Moreover, the matrix color was reduced, and the bilayer membrane was relatively intact (Figs. 3C and 3D).
IVA inhibits the IRI-induced effects on ATP, ADP, AMP, NAD + contents and on the AMP/ATP ratio in H9c2 cardiomyocytes As shown in Figs. 4A and 4B, the retention times for ATP, ADP, AMP, and NAD + were 2.6, 3.2, 4.5, and 14.3 min, respectively. The standard curve equation was obtained using the linear regression from the peak area (Y), for ATP, ADP, AMP, and NAD + , to the concentration (X) (Fig. 4C).
The evaluation of the intracellular ATP, ADP, and AMP levels showed that IVA treatment protected the mitochondrial function of H9c2 cells by increasing ATP production and decreasing the ADP and AMP contents. The AMP/ATP ratio decreased significantly in IVA treated cells compared with untreated cells (Figs. 4D and 4E). The exposure of H9c2 cells to IRI significantly reduced ATP generation ( ## p < 0.01), which was reversed by pre-treatment with IVA (20 or 100 µM) for 12 h (**p < 0.01). Additionally, IVA treatments at either 20 or 100 µM abrogated the IRIinduced decrease in NAD + generation (c = 20 µM, *p < 0.05; and c = 100 µM, **p < 0.01). These data suggested that IVA maintained mitochondrial function by upregulating ATP and NAD + expression.

IVA mitigates IRI-induced ROS production
The antioxidant potential of IVA against intracellular ROS generation was investigated using the DCF-DA assay. The production of ROS following the IRI of H9c2 cells was measured in the absence or presence of IVA (20 and 100 µM). As shown in Figs. 5A and 5B, the amount of intracellular ROS generation in the IRI model group increased significantly relative to that in the control group, indicating that the model successfully induced oxidative stress ( ## p < 0.01). As shown in Figs. 5C and 5D, IVA treatment reduced IRI-induced ROS production (c = 20 µM, *p < 0.05; and c = 100 µM, **p < 0.01).
IVA pretreatment effect on AMPK, SIRT1, and PGC-1α mRNA expression after IRI induced in H9C2c cells The RT-PCR analysis (Fig. 6) indicated that IRI reduced the expression levels of SIRT1 and PGC-1α mRNA at the mRNA level, which was reversed by IVA (c = 20 µM, *p < 0.05; and c = 100 µM, **p < 0.01). These results were consistent with previous findings, which confirm the downregulation of NAD + improved by IVA pretreatment in IRI induced in H9c2 cardiomyocytes. Additionally, no significant change in the expression levels of AMPK mRNA was observed (p > 0.05).
IVA activates the AMPK/SIRT1/PGC-1α signaling pathway As shown in Fig. 7, in comparison with the IRI group, the IVA-treated group showed increased AMPK activation (c = 20 µM, *p < 0.05; and c = 100 µM, *p < 0.01). The expression levels of SIRT1 and PGC-1α decreased in H9c2 cardiomyocytes induced by IRI. However, a significant recovery of their expression levels was observed by IVA pretreatment.

Discussion
Our study indicated that the bioenergy metabolism characteristics of myocardial cells are altered after IRI. In addition to relieving mitochondrial damage, the administration of IVA improved cell energy metabolism, reduced oxidative stress, increased cell viability, and inhibited cardiomyocyte apoptosis. The experimental results showed that this protection was closely related to the AMPK/SIRT1/PGC-1α signaling pathway. IVA reduces oxidative stress and improves myocardial mitochondrial energy metabolism by regulating the AMPK/SIRT1/PGC-1α signaling pathway in the H9c2 cardiomyocyte IRI model.
During myocardial IRI, energy metabolism disorder causes hypoxia-reperfusion injury (Gary and Edmonton, 2000). Reperfusion is characterized by an early and a late phase, during which, due to reduced adenylate cyclase activity and intracellular cAMP levels, free radicals, such as ROS, are generated. The mechanisms of cell death are triggered; ROS can further aggravate existing tissue damage (Lei et al., 2015). The beta-blocker esmolol reduces myocardial damage after resuscitation by regulating apoptosis, acting as an antioxidant, and improving energy metabolism . Preclinical studies have suggested that reductions in ROS formation, caused by the inhibition of NADPH oxidase, may be the underlying mechanism through which IVA confers cardioprotection. IVA reduced myocardial cell injury by decreasing ROS bursts during the period of reperfusion (Bolli et al., 2013;Heusch, 2009). This evidence suggests that IVA may also have a cardioprotective mechanism that mitigates IRI by protecting cardiomyocyte mitochondrial function, especially mitochondria-mediated cellular energy metabolism and oxidative stress.
Mitochondria are considered to act as the energy pool for cardiomyocytes and are directly related to energy metabolism. Mitochondrial changes are closely related to myocardial damage, such as changes in mitochondrial structure, biosynthesis abnormalities, the excessive accumulation of ROS, and the excessive consumption of NAD + (Ong et al., 2013; Pei et al., 2016;Di Lisa et al., 2001;Fujio et al., 2011). The activation of the AMPK signaling pathway is a MIRI protection mechanism, with important pathophysiological significance, and represents one of the signaling pathways that has been increasingly explored in a recent study focused on MIRI (Qi and Young, 2015).
During I/R, the available oxygen and ATP production in the myocardium is insufficient, leading to the accumulation of ADP and AMP and an increase in the AMP/ATP ratio. AMPK becomes activated when AMP binds with the regulatory nucleotide-binding domain of the AMPK gamma subunit (Salt and Hardie, 2017). Activated AMPK regulates many downstream molecules and reduces MIRI by optimizing myocardial energy metabolism, inhibiting oxidative stress, apoptosis, and regulating autophagy and anti-inflammatory activities (Daskalopoulos et al., 2016;Qi and Young, 2015). During ischemia, SIRT1 mediates AMPK activation, and in turn, AMPK regulates SIRT1 activity by regulating NAD + levels (Ruderman et al., 2010;Wang et al., 2018). In addition, AMPK and SIRT1 cooperate to activate the expression of PGC-1α which can effectively resist cellular oxidative stress. The upregulation of PGC-1α can reduce oxidative stress damage, however the downregulation of PGC-1α directly affects mitochondrial dysfunction (Ham and Raju, 2017). This study showed the increased activation of AMPK (p-AMPK) in cells after IRI, and IVA further increased p-AMPK levels, suggesting that IVA can upregulate p-AMPK expression. Meanwhile, the expression levels of SIRT1 and PGC-1α mRNA and protein with the intracellular ATP levels, were also significantly upregulated by IVA treatment. Some studies have confirmed that the AMPK/SIRT1/PGC-1α signaling pathway plays a crucial role during energy metabolism and oxidative stress which are closely related to the protection of cardiac function The effects of IVA on the AMP/ATP ratio. All data are expressed as the mean ± SD, N ≥ 3. Compared with the control group, # p < 0.05, ## p < 0.01. Compared with the IRI group, *p < 0.05, **p < 0.01. (Chau et al., 2010;Tian et al., 2019). Therefore, the protective effects of IVA may be related to the AMPK/SIRT1/PGC-1α signaling pathway.
Currently, many pharmacological functions for IVA have been reported (Xiang et al., 2019;Yu et al., 2018). For example, IVA was shown to prevent cartilage matrix degradation caused by inflammation and oxidative stress. IVA also alleviated cardiac fibrosis and hypertrophy via the PI3K/AKT/mTOR/p70S6K pathway. In clinical studies, intravenous IVA after percutaneous coronary interventions (PCIs), performed after ST-elevation myocardial infarction (STEMI), was associated with notable reductions in left All data are expressed as the mean ± SD, N ≥ 3. Compared with the control group, # p < 0.05, ## p < 0.01. Compared with the IRI group, *p < 0.05, **p < 0.01. FIGURE 6. Effect of IVA pretreatment on the expression levels of AMPK, SIRT1, and PGC-1α mRNA after IRI induced in H9c2 cells. All data are expressed as the mean ± SD, N ≥ 3. Compared with the control group, # p < 0.05, ## p < 0.01. Compared with the IRI group, *p < 0.05, **p < 0.01. ventricular ESV and EDV when compared with placebo treatments (Steg et al., 2013). In addition, IVA was associated with an improved left ventricular remodeling in reperfused STEMI patients, based on cardiac magnetic resonance assessments (Gerbaud et al., 2014). These findings showed that the mechanism of action underlying IVA cardioprotection may be more complex because IVA may have a wide range of clinical applications. Myocardial disorders after cardiac arrest and resuscitation are associated with high mortality after resuscitation (Herlitz et al., 1995;Laver et al., 2004). Our study examined the beneficial mechanisms underlying IVA treatment, thus identifying novel mechanisms and potential targets associated with the protection of the mitochondrial bioenergetic process for the development of future PRMD treatments.

Limitations
This study had the following limitations. First, this study only illustrated the effect of IVA on aerobic phosphorylation. Whether IVA can improve myocardial I/R effects associated with the anaerobic oxidation of glucose and increased anaerobic glycolysis remains unknown. Further investigation of these aspects is worthy of an in-depth study in the future. Second, this study did not use signaling pathway inhibitors to confirm the link between IVA and the AMPK/SIRT1/ PGC-1α signaling pathway. Third, this study only examined the role of the AMPK/SIRT1/PGC-1α signaling pathway, which may not be the only pathway through which IVA affects PRMD. Therefore, further research is necessary in the future to elucidate these questions.

Conclusions
In conclusion, IVA can protect against myocardial energy metabolism disorders and reduce oxidative stress, ultimately improving myocardial function. The effects of IVA may be regulated by the AMPK/SIRT1/PGC-1α signaling pathway. Compared with traditional heart-rate-lowering betablockers, IVA represents a viable and innovative treatment option for reducing mortality after CPR, and it may have a wider range of clinical applications beyond the treatment of cardiac disorders.
Acknowledgement: This study was performed at the Central Laboratory of the Second Affiliated Hospital of Anhui Medical University. We thank Tianfeng Hua for her outstanding revision of this article and Mingfei Wu and Peizhong Kong for their outstanding technical support. We thank Lisa Giles, Ph.D., from Liwen Bianji, Edanz Editing China (www. liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. Availability of Data and Materials: The datasets used or analysed during the current study are available from the corresponding author on reasonable request.
Funding Statement: This study was supported by a research grant from the National Natural Science Foundation Youth Graph showing the p-AMPK/AMPK ratios. (C) Graph showing the relative protein expression levels of SIRT1 and PGC-1α. All data are expressed as the mean ± SD, N ≥ 3. Compared with the control group, # p < 0.05, ## p < 0.01. Compared with the IRI group, *p < 0.05, **p < 0.01.