Dapagliflozin Ameliorates Myocardial Ischemia/Reperfusion Injury by Modulating EGFR Signaling and Targeting NCOA4-mediated Ferritinophagy

doi:10.21203/rs.3.rs-5172738/v1

Download PDF

Article

Dapagliflozin Ameliorates Myocardial Ischemia/Reperfusion Injury by Modulating EGFR Signaling and Targeting NCOA4-mediated Ferritinophagy

https://doi.org/10.21203/rs.3.rs-5172738/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

SGLT2 inhibitor dapagliflozin (Dapa) has gained increasing attention in the treatment of myocardial ischemia-reperfusion injury (IRI). However, the mechanism of action of the cardiovascular benefits of Dapa is unclear. The present study aimed to investigate the effects of Dapa on myocardial IRI and the underlying molecular mechanisms. The effects of Dapa on myocardial IRI were investigated using the in vitro perfusion Langendorf model and the in vitro hypoxia/reoxygenation (H/R) cell model. Histological changes, myocardial enzymes, oxidative stress and mitochondrial structure/function were assessed. Mechanistic studies involved various molecular biology methods such as ELISA, immunoprecipitation, western blot, immunofluorescence and Bioinformatics. Our findings demonstrate that Dapa upregulates EGFR phosphorylation, suppresses NHE1 expression in myocardial tissues, modulates NCOA4-mediated ferritinophagy to enhance mitochondrial function, reduces ROS expression, and mitigates myocardial IRI. In the Langendorf model, Dapa effectively attenuates cardiac dysfunction, myocardial injury, mitochondrial damage, and oxidative imbalance induced by ischemia-reperfusion. In vitro experiments revealed that blocking EGFR or autophagy with inhibitors (AG and Baf, respectively) or inducing ferroptosis with Era promotes ROS release, exacerbates mitochondrial injury, and diminishes the protective effects of Dapa. Notably, Era did not affect NCOA4-mediated ferritinophagy. Conversely, the EGFR agonist NSC counteracted these effects, underscoring that Dapa confers cardioprotection by modulating mitochondrial function through EGFR-mediated regulation of NCOA4-mediated ferritinophagy. In summary, Dapa activates EGFR phosphorylation, regulates NCOA4-mediated ferritinophagy, modulates mitochondrial function, and effectively mitigates myocardial IRI. These findings provide a robust theoretical foundation for the clinical application of Dapa in treating cardiovascular conditions.

Biological sciences/Cell biology

Biological sciences/Drug discovery

Biological sciences/Physiology

myocardial ischemia/reperfusion

dapagliflozin

ferritinophagy

EGFR

NCOA4

Myocardial infarction is the result of blocked coronary arteries and is deemed a grave cardiovascular ailment, which can escalate to heart failure or even mortality without timely intervention ¹. Currently, the best and most effective treatment for acute myocardial infarction is rapid and safe restoration of blood supply to the ischemic myocardium ². However, ischemic heart disease can lead to myocardial dysfunction, structural damage, and postischemic myocardial reperfusion injury after rapid restoration of coronary blood flow; this phenomenon is known as myocardial ischemia-reperfusion injury (IRI) ³, which has been identified as a predominant health hazard. Several strategies have been spotlighted in animal models and clinical scenarios to counteract this injury ^3–5. The findings suggested that propionate attenuates angiotensin II-enhanced myocardial ischemia-reperfusion injury via GPR41 ⁶. Dexmedetomidine was beneficial in treating this injury in rats by restricting ferritin deposition, leveraging the SLC7A11/GPX4 axis ⁷. In a previous study, we showed that metformin protected against myocardial ischemia-reperfusion injury post-treatment through the AMPK/NLRP3 inflammatory vesicle pathway ⁸. Due to the unpredictable onset of ischemia, post-ischemia-reperfusion pharmacological treatments are gaining traction. Thus, exploring clinically-established drugs that can mitigate myocardial IRI is a promising avenue.

Dapagliflozin (Dapa), a sodium-glucose cotransporter subtype 2 inhibitor (SGLT2i), is widely used in the treatment of type 2 diabetes in an insulin-independent manner and has a low risk of developing hypoglycemia ⁹. SGLT2 inhibitors are beneficial to major cardiovascular outcomes (cardiovascular death or hospitalization) in patients with and without type 2 diabetes ^10,11. Previous studies have shown that Dapa inhibits IRI in renal and cardiac organs through multiple mechanisms ^12,13. For instance, Dapa exerts protective effects in cardiovascular disease by curtailing inflammatory surges, invigorating mitochondrial metabolism, and stymieing apoptosis ^12,14,15. A recent study indicated that Dapa is effective in ameliorating myocardial IRI by inhibiting ferroptosis via mitogen-activated protein kinase ¹⁶. However, these studies have not yet explored the ferritinophagy pathway leading to ferroptosis in myocardial IRI.

Furthermore, ferroptosis is an iron-centric programmed cell death mode in various cardiovascular disease pathologies. It is intrinsically linked to iron-ion and pivots on iron-ion-induced lipid peroxidation during cell death ¹⁶. Ferritinophagy is a specific type of ferritin-selective autophagic degradation, crucial for the regulation of intracellular iron ion bioavailability ¹⁷. Nuclear receptor coactivator 4 (NCOA4) is one of the specific mediators of selective ferritin degradation. It is highly enriched in autophagosomes and interacts with and targets through ferritin heavy chain 1 (FTH1) ¹⁸. While ferritinophagy is essential for iron homeostasis, overactivation of this process leads to intracellular iron overload and increases cellular susceptibility to ferroptosis ¹⁹. During myocardial IRI, iron accumulation triggers an avalanche of reactive oxygen species (ROS) ²⁰, leading to lipid peroxidation and subsequent ferroptosis ¹⁶. Therefore, a targeted curtailment of ferroptosis can be instrumental in thwarting myocardial IRI. Some studies have explored the use of Dapa to inhibit ferroptosis and protect against myocardial IRI ^16,21,22, however, the exact mechanism is yet to be elucidated. A previous study demonstrated enhanced lipid peroxidation, pronounced iron-ion accumulation, and NCOA4-facilitated ferritinophagy in a rat myocardial IRI model and hypoxia/reoxygenation (H/R) treatment of NRVMs. Interestingly, Baicalein reversed these phenomena ²³. Nevertheless, the interplay between ferritinophagy and ferroptosis activation during myocardial IRI, especially in the context of Dapa, remains uncharted territory.

Therefore, the present study aims to harness Dapa as a post-treatment, probing its regulatory impact on ferritinophagy, potential to minimize ferroptosis, and protective effect against myocardial IRI in animal tissues and cell models. These findings would furnish a robust theoretical foundation for Dapa’s prospective deployment in cardiovascular disease management.

2.1. Chemicals and reagents

Dapagliflozin was obtained from Sigma-Aldrich (SML2804). NSC228155 (NSC) was purchased from Selleck Chemicals (Catalog No.S8312). Tyrphostin AG-1478 (AG) was purchased from Selleck Chemicals (No.S2728). Bafilomycin A1 (BAF) was purchased from Selleck Chemicals (No.S1413). Erastin ²⁴ was purchased from Sigma-Aldrich (571203-78-6). GAPDH was purchased from Biyuntian Company China (1:5000 AF0006). The drugs were individually dissolved in DMSO (0.1%), and they were introduced into the culture medium 12 hours prior to subjecting it to hypoxia/reoxygenation (H/R). Primary antibodies against GAPDH (1:5,000, AF0006) was purchased from Biyuntian Biotechnology (Shanghai, China); Anti-EGFR antibody (ab52894), Anti-EGFR (phospho Y1068) antibody (1:500, ab40815), Anti-STAT3 antibody [EPR787Y] (ab68153), Anti-STAT3 (phospho Y705) antibody [EPR23968-52] (ab267373), Anti-Sodium/Hydrogen Exchanger 1/NHE-1 antibody (ab67314), Anti-Glutathione Peroxidase 4 antibody [1B4] (ab252833), Anti-NCOA4 antibody (ab86707), and Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) were purchased from Abcam (San Francisco, CA, United States). SGLT2 Polyclonal Antibody (Catalog # PA5-101893), SLC7A11 Recombinant Polyclonal Antibody (3HCLC, Catalog # 711589), ACSL4/FACL4 Polyclonal antibody (Catalog: 22401-1-AP), Ferritin Polyclonal Antibody (Catalog # FRTN-101AP) and LC3A/LC3B Polyclonal Antibody (Catalog # PA1-16931) were obtained from Thermo Fisher Scientific (Waltham, MA, United States).

2.2. Animals and Langendorff isolated heart perfusion model

All animal experiments followed the ARRIVE guidelines and complied with the UK Animals (Scientific Procedures) Act 1986 and its related regulations. Ethics approval for all animal experiments was obtained from the Institutional Animal Care and Use Committee of Nanchang University, and the experiments were carried out following the guidelines for the Principles of Laboratory Animal Care and Use of Laboratory Animals published by NIH (NIH Publication, 8th Edition, 2011) ²⁵. The licence number of the ethics committee that approved the study is 0064257. Male Sprague – Dawley (SD) rats weighing 180–230 g were acquired from the Medical Laboratory Animal Center at Nanchang University (Nanchang, China). Healthy adult male Sprague-Dawley rats (n = 8 each) were anesthetized with 3% sodium pentobarbital (50 mg/kg) followed by intraperitoneal injection of heparin (1000 IU/kg). After midline thoracotomy, the heart was excised within 1 minute and immediately immersed in 4°C Krebs-Henseleit (K-H) buffer to remove residual blood. Subsequently, the heart underwent retrograde perfusion at a constant pressure of 80 mmHg using a Langendorff apparatus equipped with a modified Krebs-Henseleit (MKH) solution. A mixture of 95% oxygen and 5% carbon dioxide was continuously bubbled through the K-H buffer, which was perfused into the heart at the same pressure to ensure optimal oxygenation. The temperature of the perfusion system was tightly controlled at either 37 ± 0.2°C or 34 ± 0.2°C using a circulating water bath, with specific reference to the study by Koyama et al ²⁶. All hearts were randomly divided into four groups: Sham, no treatment; Dapa, subjected to Dapa post-treatment at a concentration of 5 µM in Supplementary Fig. 1; IRI; IRI + Dapa. According to our previous methods ⁸, hemodynamic indices ± dp/dtmax; left ventricular peak pressure (LVSP) were measured to assess cardiac function. A biosignal acquisition and processing system (U/4C501H Med Lab, Shanghai, China) was used to monitor and record the left ventricular end-diastolic pressure (LVEDP). at equilibrium for 30 min (T0), 30 min (T1), 60 min (T2), 90 min (T3), and 2 h (T4).

2.3. Cell culture

To isolate neonatal rat ventricular myocytes (NRVMs), we followed a procedure involving neonatal SD rats aged 1–3 days (Supplementary Fig. 2). After SD rats were anesthetized with 3% sodium pentobarbital (50 mg/kg) by intraperitoneal injection, cervical dislocation execution followed, the hearts were subjected to a wash with ice-cold Hank's Balanced Salt Solution (HBSS) to eliminate blood. Subsequently, the hearts were finely diced into small fragments and subjected to digestion at 37°C for 30–35 minutes, utilizing the Pierce primary cardiomyocyte isolation kit. This process yielded a single-cell suspension, from which approximately 10–20 × 10⁶ dissociated cardiomyocytes were obtained, originating from 10 neonatal rat hearts. The isolated cardiomyocytes were then seeded into either 6-well plates at a density of 2 × 10⁵ cells/well or 96-well plates at a density of 5 × 10⁴ cells/well. Following a 3 - day cultivation period, the cardiomyocytes were prepared for a variety of experiments. When NRVMs reached 80% confluence, the cells were treated with H/R to mimic the IRI model in vitro. Then, the cells were randomly divided into six groups: HR + Veh, no treatment; HR + Dapa: subjected to Dapa at a concentration of 5 mM; HR + NSC (EGFR agonist); HR + Dapa + AG (EGFR inhibitor); HR + Dapa + Baf (autophagy inhibitor): Baf; HR + Dapa + Era (ferroptosis activator).

2.4. Myocardial infarction size measurement

At the end of reperfusion, the hearts were promptly removed from the Langendorff perfusion device and rapidly frozen at − 80°C for 5 min. Subsequently, 5–6 pieces of heart tissue, each 2-mm-thick, were prepared using a heart cutter and immersed in 1% triphenyltetrazolium chloride (TTC, No. T8877, Sigma, USA) for uniform staining. For accurate results, the staining was carried out in a light-free environment at a constant temperature for 20 min, following which, the tissue pieces were washed with phosphate-buffered saline (PBS) buffer and fixed in 10% formalin for 24 h. Consecutively, the infarcted area appeared off-white, while the non-infarcted area was brick-red in color. The left ventricular infarction volume and the total volume of the left myocardium (myocardial infarction size) were measured using Alpha View gel image software to quantify and compare the area of infarcted tissue and whole myocardial tissue.

2.5. Hematoxylin and eosin (H&E) staining

Myocardial tissue obtained from rats was fixed in 10% paraformaldehyde and subsequently embedded in paraffin. The tissue was then cut into 4 µm sections. These sections underwent dewaxing with xylene twice for 5 minutes each and hydration with 95% ethanol. Following this, hematoxylin was applied for 10 minutes, followed by rinsing with tap water and differentiation in 1% HCl ethanol for 10 seconds. Finally, the sections were stained with 5% eosin for 1–3 minutes. After staining, dehydration was carried out with ethanol for 25 minutes, followed by dewaxing with xylene. The sections were then sealed and dried with neutral resin before observation under a Leica IX71 microscope (×200).

2.6. LDH activity assay, CK-MB and cTnI measurement

Based on a previous study, cardiac lactate dehydrogenase (LDH) activity was measured using an LDH assay kit (Nanjing Jiancheng Biotechnology, China) following the manufacturer’s instructions. LDH release was quantified as units per liter (U/L). After 2 hours of reperfusion following ischemia, left ventricular tissue was harvested. A 10% tissue homogenate in normal saline was centrifuged at 4°C at 3,000 rpm for 15 minutes to obtain the supernatant. Creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) levels were assessed using commercial kits from Nanjing Jiancheng Bioengineering Institute according to the manufacturer’s instructions, and results were expressed as units per liter (U/L).

2.7. Cell viability

The cell viability of NRVMs was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, all steps were performed in strict accordance with the instructions, as described previously ⁸.

2.8. Electron microscopy imaging with parallel Flameng’s score

The cells were fixed in 3% glutaraldehyde and 1% osmium tetroxide, following which they were dehydrated with acetone. Subsequently, the samples were embedded in Epon812 resin and cut into approximately 50-nm-thick slices. To enhance the contrast for imaging, the sliced samples were stained with uranyl acetate and lead citrate. Images were acquired under a JEM-1400PLUS transmission electron microscope. The mitochondria scores were based on the degree of mitochondrial damage. Each mitochondrion was individually scored on a scale of 0–4 (higher scales indicate more severity), reflecting the extent of damage. Finally, the average score of all mitochondria in a given sample was calculated to obtain flameng’s score for that specific sample.

2.9. Evaluation of mitochondrial ROS (mROS)

The mROS were assessed using a MitoSOX™ Red mitochondrial superoxide indicator (YEASEN, Shanghai, China), following the manufacturer’s instructions. 4T1 cells were seeded onto coverslips in a 24-well plate. The cells from all experimental groups were incubated with a 5 µM MitoSOX™ reagent at 37°C for 10 min in the dark. Following the incubation period, the cells were washed three times with warm HBSS. The intensity of ROS ²⁰ was then detected using a fluorescent microscope.

2.10. Measurement of the mitochondrial membrane potential (MMP)

Cellular MMP was assessed using tetramethylrhodamine, methyl ester (TMRM) staining. Pre-diluted TMRM dye was added to the cells following established protocols, adhering to the manufacturer's recommended concentration and incubation times. Subsequently, stained cells or tissues were examined under a fluorescence microscope to visualize TMRM fluorescence distribution and intensity. Quantitative analysis of fluorescence images was conducted using image analysis software to quantify alterations in MMP. The experimental methods for these MMP assessments were based on the research conducted by Salie et al ²⁷.

2.11. Mitochondrial respiratory oxygen consumption rate (OCR) detection

The OCR was measured following the instructions of the Seahorse XF Cell Mito Stress Test Kit (catalog number 103015-100, Agilent, Delaware, USA). Briefly, one day before the experiment, 1.5 × 10⁴ cells were seeded into each well of Seahorse Xfe96-well plates and incubated at 37°C under 5% CO₂ for 24 h. The sensor cartridge was hydrated overnight in a CO₂-free incubator at 37°C. Seahorse XF DMEM was prepared as follows: 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate. Metabolic regulation drugs were configured at the specified concentrations: 1.5 µM oligomycin for the A dosing hole, 2 µM fluorocarbonyl cyanide phenylhydrazone (FCCP) for the B dosing hole, and 5 µM rotenone/antimycin A for the C dosing hole. Finally, the mitochondrial breathing program of Seahorse Xfe96 Analyzer (Agilent, Delaware, USA) was selected to detect and analyze cellular mitochondrial respiration and function.

2.12. Bioinformatics analysis

The public dataset GSE4105 from the gene expression synthesis (GEO, https://www.ncbi.nlm.nih.gov/geo/) was used to obtain the genes of IRI [23193258]. Ferroptosis-related genes (FRGs) were acquired from the FerrDB website [36305834], and the targets of Dapa were predicted via Swiss target prediction [24792161]. To investigate the expression difference of genes in IRI and determine the expression at the mRNA level, we conducted differential expression analysis using the “limma” package in R software to identify the differentially expressed genes (DEGs) in GSE4150 between IRI and normal groups [25608792]. The absolute values of log2 fold-change (|log2FC|) > 1 and P-value < 0.05 were used to define the threshold of DEGs. To visualize the results of differential expression analysis, the “ggplot2” package in R software was used to create a volcano plot. Finally, the intersection candidates of DEGs, FRGs, and targets of Dapa were regarded as hub indicators. The R software package “clusterProfiler” was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and to obtain the Gene Ontology (GO) enrichment results of biological processes, cellular components, and molecular function [23740750].

2.13. Western blot

To extract total protein from each group of cells, the following protocol was followed: Cells were lysed on ice using RIPA lysis buffer for a duration of 10 minutes. The lysate was then centrifuged at 13,000 × g for 10 minutes to facilitate protein extraction. The protein concentration was determined using a BCA assay kit. To denature the protein, it was mixed with loading buffer and boiled at 100°C for 5 minutes. Protein samples, each containing 30 µg of protein, were separated using 10% SDS‒PAGE gel electrophoresis. The separated proteins were subsequently transferred to polyvinylidene fluoride (PVDF) membranes at a constant current of 250 mA. The PVDF membrane was blocked with 5% skimmed milk at room temperature for 1 hour. The membrane was then incubated overnight at 4°C in a shaker with the primary antibody. For the subsequent step, goat anti-rabbit or mouse IgG was utilized as the secondary antibody and incubated with the membrane at room temperature for 2 hours. Finally, the membranes were exposed to the ECL color solution, and the chemiluminescent signals were captured using a chemiluminescence imager.

2.14. Iron measurements

Cells (2×10⁶) were rapidly homogenized in iron assay buffer using an Iron Assay kit (Colorimetric, ab83366, Sigma Aldrich). Following the procedure, the solution was subjected to centrifugation at 13,000 × g for 10 minutes at 4°C in order to eliminate insoluble material. Subsequently, the absorbance of the solution was measured at a wavelength of 593 nm using a microplate fluorometer from Bio-Rad Laboratories, Inc.

2.15. Myocardial histopathological staining

For immunohistochemical (IHC) staining, the adjacent slice was used, and a heat-induced antigen retrieval step was employed. All tissue sections were deparaffinized and hydrated, and endogenous peroxidase activity was blocked using hydrogen peroxide. The negative control was treated with phosphate-buffered saline (PBS) instead of the primary antibody. After washing with PBS, the slides were incubated with 3,3′-diaminobenzidine tetrahydrochloride (DAB, ZSGB-BIO, Beijing, China) to visualize the antigen-antibody complexes. Finally, the tissue sections were observed using a light microscope (LM, BX51, Olympus Co., Japan).

2.16. Immunoprecipitation (IP) assay

Briefly, an equivalent of 5 µg of anti-NCOA4 or anti-FTH1 antibody was incubated with cell lysates at 4°C overnight. The samples were incubated with protein A agarose beads with rotation at 4°C for 3 h. Subsequently, the immune complexes were isolated by centrifugation at 3,000 rpm for 5 minutes, washed three times with lysis buffer, and incubated at 95°C for 5 min. The IP complexes were subjected to Western blot analysis using specific primary antibodies.

2.17. Cell apoptosis detection

Apoptosis in the myocardium and NRVMs was assessed using TdT- mediated biotinylated nick end Labeling (TUNEL) staining. Rat myocardial tissue was fixed with 4% paraformaldehyde at 4°C, dehydrated with ethanol, embedded in paraffin for sectioning, and washed twice with PBS (pH 7.35). Following the kit’s instructions, the TUNEL reagent was added to the tissue sections, and the mixture was incubated at 37°C for 1 h in the dark. 4,6-diamidine-2-phenylindole (DAPI) was used to stain the nuclei. Then, the sections were incubated with DAPI at room temperature for 10 min and rinsed twice with PBS. The apoptotic cells were detected under a fluorescent microscope. In TUNEL staining, apoptotic cells showed green fluorescence, while the nuclei exhibited blue fluorescence due to DAPI staining.

2.18. Statistical analysis

Data were expressed as means ± SEM. All experimental data were analyzed by using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. P values < 0.05 were considered statistically signifcant. Statistical analysis was conducted using GraphPad Prism software (version 9.5).

3.1 Dapa reduces myocardial infarct size and improves haemodynamic performance after ischaemia-reperfusion injury in rats

To confirm the cardioprotective impact of Dapa post-treatment against myocardial IRI, we assessed myocardial infarction size using 1% TTC. In the IRI group, the ratio of myocardial infarction volume was approximately 50%, whereas in the IRI + Dapa group, this ratio was significantly reduced to 29%. (P < 0.05; Fig. 1A and 1B). In addition, histological examination using H&E was performed, as depicted in Fig. 1C. The results demonstrated that in the Sham group, cardiomyocytes were regularly arranged with distinct nuclei and no signs of inflammatory cell infiltration. Conversely, in the IRI group, mouse hearts exhibited disorganized cardiomyocytes, extensive necrosis, widened intercellular spaces, significant myocardial damage, and limited survival of cardiomyocytes. However, in the IRI + Dapa group, there was a notable improvement with relatively orderly cardiomyocyte arrangement and a significant reduction in both the severity and extent of necrosis and damage. Furthermore, analysis of myocardial enzyme profiles revealed that serum levels of serum LDH, CK-MB, and cTnI were significantly lower in the IRI + Dapa group compared to the IRI group (P < 0.05, Fig. 1D).

In addition, we evaluated the impact of Dapa on hemodynamic parameters during myocardial ischemia-reperfusion injury in rats. Initially, there were no significant differences in ± dp/dtmax, left ventricular systolic pressure (LVSP), and left ventricular end-diastolic pressure (LVEDP) between the two groups at baseline. However, during the 30 minutes (T1), 60 minutes (T2), 90 minutes (T3), and 120 minutes (T4) of reperfusion, ±dp/dtmax, LVSP, and LVEDP showed significant differences (P < 0.05). Specifically, ±dp/dtmax and LVSP were significantly higher, while LVEDP was significantly lower (P < 0.05) in the IRI + Dapa group compared to the IRI group across all reperfusion stages. Gross observation at T4 indicated that LVEDP was 7 times higher in the IRI group than in Sham (P < 0.05), and 5.5 times higher in the IRI + Dapa group than in Sham (P < 0.05) (Table 1). These findings underscore that Dapa exerts a protective effect against myocardial IRI in rats.

Table 1

**Hemodynamics in vitro experiments**
	Group	T₀	T₁	T₂	T₃	T₄
+dp/dtmax (mmHg/s)	Sham Dapa IRI IRI + Dapa	2593 ± 135 2609 ± 196 2424 ± 206 2631 ± 176	2584 ± 172 2596 ± 113 1419 ± 136^# 1671 ± 209^#	2579 ± 193 2601 ± 234 1187 ± 152^# 1360 ± 100^#	2592 ± 209 2671 ± 122 1044 ± 117^# 1289 ± 157^#	2493 ± 65 2578 ± 138 844 ± 87^# 1217 ± 150^#&
-dp/dtmax (mmHg/s)	Sham Dapa IRI IRI + Dapa	2547 ± 320 2568 ± 242 2725 ± 361 2706 ± 208	2643 ± 213 2646 ± 196 1513 ± 106^# 1565 ± 72^#	2639 ± 165 2622 ± 232 1235 ± 165^# 1341 ± 108^#	2647 ± 155 2465 ± 196 1168 ± 89^# 1251 ± 159^#	2736 ± 239 2674 ± 257 902 ± 139^# 1074 ± 229^#
LVSP (mmHg)	Sham Dapa IRI IRI + Dapa	96 ± 9 88 ± 7 90 ± 9 90 ± 15	94 ± 7 88 ± 9 48 ± 4^# 51 ± 5^#	87 ± 7 93 ± 10 41 ± 4^# 50 ± 5^#	92 ± 9 90 ± 5 35 ± 2^# 40 ± 4^#	94 ± 11 89 ± 8 31 ± 5^# 37 ± 5^#
LVEDP (mmHg)	Sham Dapa IRI IRI + Dapa	6.3 ± 0.5 5.9 ± 0.5 6.0 ± 0.9 6.4 ± 0.7	6.1 ± 0.6 6.3 ± 0.6 37.8 ± 1.8^# 30.0 ± 5.0^#&	6.3 ± 0.4 6.3 ± 0.6 37.8 ± 1.8^# 30.0 ± 5.0^#&	6.3 ± 0.4 6.6 ± 0.6 40.1 ± 4.3^# 33.3 ± 3.5^#&	6.0 ± 0.5 6.3 ± 0.8 45.6 ± 3.7^# 34.0 ± 5.0^#&
*P < 0.05 vs T₀; #P < 0.05 vs Sham group; &P < 0.05 vs IRI group. Values are means ± standard deviation. n = 6 / group. LVSP, left ventricular peak pressure; LVEDP, left ventricular end diastolic pressure. T₀, equilibration; T₁, 30 min after reperfusion; T₂, 60 min after reperfusion; T₃, 90 min after reperfusion; T₄, 2h after reperfusion. SHAM, sham control; IRI, ischemia reperfusion injury; Dapa, dapagliflozin.

3.2 Dapa improves mitochondrial function and attenuates myocardial ischaemia-reperfusion injury-mediated mitochondrial damage and oxidative stress

Numerous studies have demonstrated that oxidative stress and mitochondrial injury play pivotal roles in myocardial ischemia-reperfusion injury. To explore further the cardioprotective effects of Dapa and their relationship with oxidative stress, we assessed ROS levels, mitochondrial injury, and mitochondrial function in rat myocardial tissues across all experimental groups. Electron microscopy revealed that rat cardiomyocytes in the IRI group exhibited significant mitochondrial structural damage, flameng’s scoring approximately 4–5 times higher than the Sham group, whereas the IRI + Dapa group showed preserved mitochondrial structure comparable to the Sham group (P < 0.05; Fig. 2A, 2B). Additionally, measurements of mROS using MitoSOX™ Red indicated elevated ROS levels in the IRI group compared to both Sham and IRI + Dapa groups (P < 0.05; Fig. 2C, 2D). Mitochondrial injury often leads to reduced mitochondrial membrane potential (MMP). Analysis of MMP in NRVMs using tetramethylrhodamine, methyl ester, and perchlorate (TMRM) staining showed MMP levels were 4-fold higher in the Sham group than the IRI group, and 2-fold higher in the IRI + Dapa group compared to the IRI group (P < 0.05; Fig. 2E, 2F). Moreover, mitochondrial function assessments, including basal respiration, maximum respiration, ATP production, and spare respiratory capacity using a hippocampometer assay, demonstrated significant improvements in the IRI + Dapa group compared to the IRI group (P < 0.05; Fig. 2G-L). These findings collectively indicate that Dapa enhances myocardial ischemia-reperfusion injury prognosis by mitigating mitochondrial damage, improving mitochondrial function, and reducing oxidative stress injury.

3.3 Dapa is involved in improving myocardial IRI by regulating the EGFR signaling pathway

Myocardial mitochondrial injury profoundly impacts mitochondrial function and oxidative stress levels, with ferroptosis contributing to a marked reduction in mitochondrial membrane potential. Previous research has highlighted the role of dapa in modulating ferroptosis, which is crucial in the context of myocardial IRI. Building on this understanding, our study aims to elucidate the precise mechanisms through which Dapa modulates iron overload to mitigate myocardial IRI. Herein, we used bioinformatics analysis to identify the target of Dapa and explore the mechanism underlying the treatment of myocardial IRI-mediated ferroptosis. The differential expression analysis of hub indicators in the myocardial IRI and normal groups with respect to myocardial IRI and Dapa retrieved 316 DEGs (100 down- and 216 up-regulated) (Fig. 3A, 3B, Supplementary Table). Intriguingly, only one gene (EGFR) was identified in the intersection of the genes related to myocardial IRI, ferroptosis, and Dapa, regarded as hub indicators (Fig. 3B). Furthermore, base on the median expression value of EGFR in the IRI samples, all the 6 IRI samples were divided into the High-Expression EGFR and Low-Expression EGFR group. The DEGs between the above two group were regarded as the potential downstream genes of EGFR in IRI samples. GO enrichment analysis for the downstream genes of EGFR indicated that EGFR plays a significant role in maintaining the stability of the extracellular matrix (Fig. 3C). Subsequently, we assessed EGFR levels using western blot, revealing that phosphorylation of EGFR expression in the IRI + Dapa group was four times higher than that in the IRI group (P < 0.05; Fig. 3D, 3E). Furthermore, signal transducer and activator of transcription 3 (STAT3), identified as a downstream signaling molecule of EGFR ²⁸, exhibited a five-fold increase in expression levels in the IRI + Dapa group compared to the IRI group (P < 0.05; Fig. 3D, 3E). In addition, cardiomyocytes and cardiac tissues lack SGLT2 receptor proteins, suggesting that SGLT2 inhibitors may exert their protective effects in the heart through alternative pathways, this aligns with our findings (Fig. 3F, 3G). Study shows that SGLT2 inhibitors indirectly modulate cardiomyocytes and may ameliorate arrhythmias by inhibiting the activity of myocardial sodium-hydrogen exchange proteins 1 (sodium-hydrogen exchange protein 1, Na+/H + exchange protein 1, NHE1) ^29–31. The results indicated that the expression level of NHE1 in the IRI group was three times higher compared to the IRI + Dapa group. Taken together, these data suggest that EGFR could be a target through which Dapa exerts a protective effect in myocardial IRI.

3.4 Dapa protects against myocardial IRI by regulating ferroptosis

The results showed that IRI surgery significantly increased the generation of Fe²⁺, while dapa reduced intracellular ferrous iron overload. (P < 0.05, Fig. 4A). SLC7A11/GPX4 axis is recognized as the primary defense mechanism against ferroptosis. Immunohistochemical staining of myocardial sections from all groups showed the expected results. The positive staining was decreased in the myocardium sections of the IRI group compared to the sham group (P < 0.05; Fig. 4B, 4C). However, compared to the IRI group, GPX4 staining in the Dapa + IRI myocardium sections was markedly increased (P < 0.05, Fig. 4B, 4C). Moreover, Western blot analysis indicated that the expression levels of GPX4 and SLC7A11 were lower in the IRI group than in the sham and Dapa + IRI groups (P < 0.05; Fig. 4D, 4E).ACSL4, a lipid metabolism enzyme required for ferroptosis, showed higher expression levels in the IRI groups compared to the sham and Dapa + IRI groups (P < 0.05; Fig. 4D, 4E). These findings suggested that Dapa regulates iron metabolism and inhibits ferroptosis.

3.5 Dapa promotes NCOA4-mediated ferritinophagy and reduces increased myocardial IRI-induced ferroptosis

Subsequently, we verified the levels of ferroptosis and ferritinophagy in the cardiac tissue. As shown in Fig. 5A, 5B, compared to the sham group, the protein level of NCOA4 was increased, and the FTH1 level was decreased in the IRI group (P < 0.05). However, in the Dapa + IRI group, the protein level of NCOA4 was decreased and the level of FTH1 was increased compared to the IRI group (P < 0.05). Among autophagy proteins, the level of LC3-II/I was in the order of IRI group > Dapa + IRI group > sham group (P < 0.05). Moreover, the trend of P62 protein levels was the same as that of NCOA4 protein among the groups. Furthermore, to further investigate the interaction between NCOA4 and FTH1, the binding intensity of NCOA4 and FTH1 was assessed by IP. Figure 5C, 5D shows that the IRI group showed upregulated NCOA4 protein and downregulated FTH1 protein levels (P < 0.05). Conversely, the Dapa + IRI group showed an opposite trend in protein levels compared to the IRI group, suggesting NCOA4 and FTH1 complex formation.

3.6 Activation of Dapa-mediated EGFR phosphorylation improves mitochondrial function and ferritinophagy against H/R-induced injury

Our preliminary experimental results suggested that SGLT2 inhibitors promote EGFR phosphorylation activation, ameliorate ferritinophagy-mediated ferroptosis, and improve myocardial IRI. To confirm these findings, we conducted additional experiments using H/R-induced NRVMs treated with various agents to validate the mechanism of the protective effect of Dapa on H/R cardiomyocytes. CCK-8 assay results showed that the cell activity of H/R cardiomyocytes was significantly higher after Dapa administration and EGFR agonist NSC 228155 (NSC) treatment ³². Compared to the H/R + NSC group, the cellular activity of H/R cardiomyocytes treated with Dapa and EGFR inhibitor AG-1478 ³³ was reduced ³⁴, suggesting that Dapa improves H/R cardiomyocytes by activating EGFR (P < 0.05; Fig. 6A). Next, we added autophagy inhibitor Bafilomycin A1 (BAF) ³⁵ and ferroptosis agonist Erastin ^{24 36} respectively. The cellular activity of cardiomyocytes was significantly lower in the H/R + Dapa + AG, H/R + Dapa + BAF (autophagy inhibitor), and H/R + Dapa + ERA (ferroptosis agonist) groups compared to that in the H/R + Dapa group. Next, we found that the application of AG, BAF, and ERA reversed the protective effects of Dapa. Consistent with our animal data, we observed that Dapa significantly reduces LDH release and Fe²⁺ expression (P < 0.05; Fig. 6B, 6E) and reduced cardiomyocyte apoptosis and ROS generation (P < 0.05; Fig. 6C, 6D, 6F, 6G). However, Dapa did not exert any cardioprotective effect against H/R injury when EGFR and autophagy were inhibited and ferroptosis was activated (P < 0.05; Fig. 6B–6G).

Next, to detect mitochondrial function, we conducted fluorescence staining to detect the expression level of the translocase of the outer mitochondrial membrane 20 (TOM20) protein and found that the addition of H/R + Dapa and H/R + NSC significantly increased the TOM20 protein expression compared to the H/R + Veh group (P < 0.05; Fig. 7A, 7B). However, when AG, BAF, and ERA were added, the TOM20 protein level was significantly reduced (P < 0.05; Fig. 7A, 7B). Furthermore, we examined the mitochondrial OCR function in vitro. As shown in Fig. 7C–7H, basal respiration, maximum respiration, proton leak, ATP production, and spare respiratory capacity were significantly improved (P < 0.05; Fig. 7D, 7F, 7G, 7H) in the presence of Dapa and NSC compared to the H/R + Veh group. Conversely, basal respiration, maximum respiration, ATP production, and spare respiratory capacity were suppressed when AG, BAF, and ERA were added (P < 0.05; Fig. 7D, 7F, 7G, 7H). These results suggested that Dapa improves mitochondrial function by mediating the EGFR pathway, thereby protecting the cardiomyocytes from H/R-induced injury.

As shown in Fig. 8A, 8B, we examined the phosphorylation level of EGFR protein to further understand the correlation between EGFR and NHE1 and found that compared to the control group, the phosphorylation level of EGFR protein was elevated in the HR + Dapa and HR + NSC groups; however, this phenomenon was reversed by the EGFR inhibitor AG (P < 0.05; Fig. 8A, 8B). Also, GPX4 showed a similar trend. Additionally, NHE1 protein expression was decreased in the HR + Dapa and HR + NSC groups. On the other hand, HR + Dapa + AG upregulated NHE1 expression, which was not reversed in the HR + Dapa + BAF and HR + Dapa + ERA groups (P < 0.05; Fig. 8A, 8B). Next, we detected ferritinophagy-associated proteins by immunoblotting. The results showed decreased levels of NCOA4 and LC3-II/LC3-I (P < 0.05, Fig. 8C, 8D), whereas those of FTH1 were elevated (P < 0.05) in cardiomyocytes under the H/R + Dapagliflozin treatment. Moreover, in the H/R + Dapa + AG and H/R + Dapa + BAF groups, the expression of NCOA4 (P < 0.05), LC3-II/LC3-I (P < 0.05; Fig. 8C, 8D), and P62 (P < 0.05; Fig. 8C, 8D) was upregulated compared to the H/R + Dapa group,, while that of FTH1 (P < 0.05, Fig. 8C, 8D) was downregulated in the H/R + Dapa + AG group. These findings indicated that ferritinophagy is involved in Dapa-regulated H/R cardiomyocyte protection.

To the best of our knowledge, this is the first mechanistic study investigating the modulatory effects of Dapa post-treatment on the EGFR pathway-mediated mitochondrial injury and ferritinophagy in myocardial IRI. Herein, we combined bioinformatics analysis with ex vivo experiments to systematically investigate the role of Dapa in the EGFR signaling pathway in ferritinophagy during myocardial IRI. Dapa is a novel oral hypoglycemic agent applied in clinical practice. Previous studies have demonstrated that Dapa exerts cardioprotective effects through multiple mechanisms ^12,21,22. Notably, Dapa pre- and post-treatments reduce myocardial IRI by counteracting inflammation, bolstering mitochondrial integrity, adjusting autophagy, and influencing ferroptosis ²³. Therefore, the current study expands on the previous work by elucidating the pathogenesis of myocardial IRI and the therapeutic pathway of Dapa.

Langendorff's heart, an in vitro paradigm, facilitates the assessment of heart contraction strength and rate, thereby circumventing the intricacies of an entire organism. In this study, we determined the extent of myocardial IRI by examining myocardial infarction size, extent of myocardial injury, and pertinent myocardial injury enzyme markers. The results showed that Dapa post-treatment inhibits myocardial infarct size, improves myocardial injury extent, and decreases myocardial injury-related enzymes LDH, CK-MB, and cTnl levels. Some studies have shown the protective effects of Dapa on cardiomyocytes by improving mitochondrial function, while our study proved that post-treatment with Dapa improves mitochondrial structure and function in cardiac muscle tissues via decreased ROS generation, optimized mitochondrial membrane potential, and elevated mitochondrial oxidative phosphorylation, thus protecting the cardiomyocytes.

Cardiomyocytes and cardiac tissues lack SGLT2 receptor proteins, suggesting that SGLT2 inhibitors may exert their protective effects in the heart through alternative pathways. One such proposed pathway involves targeting NHE1 ³⁷. Specifically, Dapagliflozin (Dapa) may exert its protective effects by reducing cytoplasmic calcium ([Ca2+]) and sodium ([Na+]) levels mediated by NHE1 in primary neonatal rat cardiomyocytes, while also increasing mitochondrial calcium ([Ca2+]). Other studies have underscored the role of NHE1 receptors in the cardiovascular benefits of SGLT2 inhibitors, including in conditions such as heart failure, diabetic cardiomyopathy, and myocardial infarction ^38–40. Our study similarly found no SGLT2 protein expression in myocardial tissue. Importantly, we observed that NHE1 expression levels were 3–4 times higher in the IRI group compared to the IRI + Dapa group, consistent with findings in cell culture and previous research. Thus, our investigation focused on elucidating how SGLT2 inhibitors specifically act via NHE1 levels in experimental animals and cardiomyocytes.

Ferroptosis has been identified as a distinct iron-dependent, non-apoptotic regulated cell death caused by the collapse of GSH-dependent antioxidant defense and the accumulation of lipid peroxides that elevate ROS ⁴¹. Accumulation of iron within cells can result in heightened oxidative stress, a significant characteristic of ferroptosis. Oxidative stress triggers an elevation in intracellular ROS, which subsequently damages both the mitochondrial outer and inner membranes. This decline in mitochondrial membrane potential not only disrupts ATP synthesis and cellular energy metabolism but also exacerbates ROS production, initiating a detrimental cycle that further compromises mitochondrial function. Intriguingly, a recent study demonstrated that Dapa ameliorates myocardial IRI via ferroptosis ¹⁶. These studies put forth the mechanism underlying Dapa-mediated cardiomyocyte ferroptosis in the myocardial IRI model by regulating iron overload. Subsequently, we employed bioinformatics to unravel Dapa targets and the putative mechanisms against myocardial IRI-induced ferroptosis. After consolidating insights from multiple databases, we identified targets affiliated with Dapa. To better understand the mechanism of action of Dapa in the treatment of myocardial IRI, we enriched and analyzed these 60 targets. Their biological processes and modular functions were mainly involved in the regulation of EGFR activity. GSE4150, a public dataset from the gene expression compendium, unveiled core targets intrinsic to the EGFR pathway. Cross-referencing with the ferroptosis database highlighted the association of these targets with ferroptosis. Consequently, our examination revealed that less severe myocardial IRI post-Dapa administration is achieved by inhibiting ferroptosis and modulating protein expression and phosphorylation of the EGFR pathway.

Several studies have delved into the inhibition of ferroptosis as a strategy to mitigate cardiac IRI ^42–47. Some findings indicated that ferroptosis exacerbates tissue necrosis and cellular apoptosis in myocardial IRI, and ferritinophagy plays a significant role in this process ^23,48. In 1970s, this degradation process was distinguished as “autophagy” utilizing high-resolution transmission electron microscopy ⁴⁹. ferritinophagy, a novel type of autophagy, has similar physiological roles ⁵⁰. NCOA4 selectively recognizes the FTH subunit of ferritin, and FTH1 R23 is an essential structure for ferritin binding to NCOA4. The degradation of ferritin is induced by NCOA4-mediated phagocytosis, which then releases free iron, a key factor contributing to ferroptosis ^19,23,51. In the present study, we demonstrated that Dapa post-treatment inhibits ferritinophagy. Concurrently, our immunoprecipitation data revealed the role of Dapa in amplifying the NCOA4-FTH1 nexus. These results confirmed the protective role of Dapa via ferritinophagy-mediated ferroptosis in myocardial IRI.

According to a recent study, microRNA-122a exacerbates intestinal IRI by amplifying pyroptosis via the EGFR-NLRP3 signaling axis ⁵². In addition, Xu et al. demonstrated that morphine prevents myocardial IRI by improving myocardial mitochondrial function by activating the EGFR pathway ⁵³. EGFR is a membrane receptor tyrosine kinase that plays a crucial role in cell proliferation, survival, differentiation, and metabolism. EGFR’s internal tyrosine kinase machinery is activated, ushering in a cascade of intracellular signaling dynamics and modulating cellular functions ⁵⁴. Subsequently, we validated this phenomenon in isolated cells using the EGFR agonist NSC, the antagonist AG, the autophagy inhibitor BAF, and the ferroptosis agonist ERA, respectively. The activation of EGFR using NSC resulted in reduced myocardial apoptosis and necrosis, decreased LDH levels, and decreased Fe²⁺ and ROS levels in cardiomyocytes, which is consistent with the myocardial protective effects of Dapa. Importantly, the results indicated that the degree of myocardial apoptosis and necrosis in cardiomyocytes increased, which affected mitochondrial function and the occurrence of ferritinophagy, leading to a significant increase in Fe²⁺ and ROS levels, elevated NCOA4, P62, and LC3-II/I ratios, and decreased FTH1 content. The autophagy inhibitor BAF had a similar effect to the EGFR inhibitor AG. Interestingly, the ferroptosis inducer ERA nullified Dapa protection, but did not affect autophagy-related protein expression, indicating that the EGFR signaling conduit, positioned upstream of ferritinophagy-mediated iron-induced cell death, is at the heart of the defensive role of Dapa in myocardial IRI. Collectively, our results suggested that the EGFR signaling pathway plays a critical role in Dapa-mediated protection against ferritinophagy-driven ferroptosis induced by myocardial IRI. The molecular mechanism of Dapa against myocardial IRI is complex and involves multiple molecular pathways. Together, these findings highlighted the action of Dapa prowess in combat against myocardial IRI and stifling ferroptosis via activation of EGFR and its affiliated ferritinophagy. Hence, targeting EGFR and its orchestrated ferritinophagy-driven ferroptosis emerge as a promising therapeutic avenue for treating clinical ischemic heart disease.

The present study presents robust evidence underscoring the pronounced cardioprotective role of Dapa in non-diabetic myocardial IRI. The safeguarding mechanism appears intricately linked to diminished ferritinophagy-driven ferroptosis, orchestrated through adept modulation of the EGFR pathway. Such insights pave the way for SGLT2 inhibitors as potential therapeutic candidates in warding off myocardial IRI injuries (Fig. 9). Taken together, EGFR, upstream of ferritinophagy, may be a potential target for the treatment of myocardial IRI.

DAPA dapagliflozin

IRI ischemia-reperfusion injury

EGFR epidermal growth factor receptor

NRVMs Neonatal rat ventricular myocytes

HR hypoxia/reoxygenation

LVSP left ventricular peak pressure

LVEDP left ventricular end-diastolic pressure

TMRM tetramethylrhodamine, methyl ester

NHE1 Na+/H+ exchange protein 1

NSC NSC228155

AG Tyrphostin AG-1478

BAF Bafilomycin A1

ERA Erastin

NCOA4 Nuclear receptor coactivator 4

FTH1 ferritin heavy chain 1

ROS Reactive oxygen species

IP Immunoprecipitation

LDH Lactate dehydrogenase

TTC triphenyltetrazolium chloride

OCR oxygen consumption rate

Author contributions

Peng Yu & Jitao Ling: Conceptualization, Methodology, Funding acquisition. Zhou Xu & Jing Zhang: Writing-Original draft preparation. Kaibo Hu & Fuwei Liu: Visualization. Feng Chen & Yuting Wu: Investigation. Panpan Xia & Yixuan Chen: Supervision. Deju Zhang & Xiao Liu: Software, Validation. Tieqiu Huang & Yuliang Zhan: Reviewing and Funding.

Acknowledgments

The graphical abstracts were created with BioRender software (BioRender.com).

Conflicts of interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethical staement:

All animal experiments followed the ARRIVE guidelines and complied with the UK Animals (Scientific Procedures) Act 1986 and its related regulations. This experiment was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. The permit number is 0064257.

Funding

This work was supported by the Natural Science Foundation of China (No. 82160371 to J.Z., No.82100869 and No.82360162 to P.Y., 202201011395 to X. L); Natural Science Foundation of Guangdong (202201011395 to X. L); Basic and Applied Basic Research Project of Guangzhou (202201011395 to X.L); Natural Science Foundation in Jiangxi Province grant [20224ACB216009 to J.Z];the Jiangxi Province Thousands of Plans (No. jxsq2023201105 to P.Y); the Science and Technology Project of Jiangxi Province (No.20203BBGL73188); and the Hengrui Diabetes Metabolism Research Fund (No. Z-2017-26-2202-4 to P.Y).

Thygesen, K. et al. Fourth Universal Definition of Myocardial Infarction (2018). J Am Coll Cardiol 72, 2231-2264, doi:10.1016/j.jacc.2018.08.1038 (2018).
Bhatt, D. L., Lopes, R. D. & Harrington, R. A. Diagnosis and Treatment of Acute Coronary Syndromes: A Review. Jama 327, 662-675, doi:10.1001/jama.2022.0358 (2022).
Davidson, S. M. et al. Multitarget Strategies to Reduce Myocardial Ischemia/Reperfusion Injury: JACC Review Topic of the Week. J Am Coll Cardiol 73, 89-99, doi:10.1016/j.jacc.2018.09.086 (2019).
Fernandez Rico, C. et al. Therapeutic Peptides to Treat Myocardial Ischemia-Reperfusion Injury. Front Cardiovasc Med 9, 792885, doi:10.3389/fcvm.2022.792885 (2022).
Neri, M., Riezzo, I., Pascale, N., Pomara, C. & Turillazzi, E. Ischemia/Reperfusion Injury following Acute Myocardial Infarction: A Critical Issue for Clinicians and Forensic Pathologists. Mediators Inflamm 2017, 7018393, doi:10.1155/2017/7018393 (2017).
Deng, F. et al. Propionate alleviates myocardial ischemia-reperfusion injury aggravated by Angiotensin II dependent on caveolin-1/ACE2 axis through GPR41. Int J Biol Sci 18, 858-872, doi:10.7150/ijbs.67724 (2022).
Yu, P. et al. Dexmedetomidine post-conditioning alleviates myocardial ischemia-reperfusion injury in rats by ferroptosis inhibition via SLC7A11/GPX4 axis activation. Hum Cell 35, 836-848, doi:10.1007/s13577-022-00682-9 (2022).
Zhang, J. et al. Metformin protects against myocardial ischemia-reperfusion injury and cell pyroptosis via AMPK/NLRP3 inflammasome pathway. Aging (Albany NY) 12, 24270-24287, doi:10.18632/aging.202143 (2020).
Dhillon, S. Dapagliflozin: A Review in Type 2 Diabetes. Drugs 79, 1135-1146, doi:10.1007/s40265-019-01148-3 (2019).
Cowie, M. R. & Fisher, M. SGLT2 inhibitors: mechanisms of cardiovascular benefit beyond glycaemic control. Nat Rev Cardiol 17, 761-772, doi:10.1038/s41569-020-0406-8 (2020).
Fathi, A., Vickneson, K. & Singh, J. S. SGLT2-inhibitors; more than just glycosuria and diuresis. Heart Fail Rev 26, 623-642, doi:10.1007/s10741-020-10038-w (2021).
He, S. et al. Dapagliflozin Protects Methamphetamine-Induced Cardiomyopathy by Alleviating Mitochondrial Damage and Reducing Cardiac Function Decline in a Mouse Model. Front Pharmacol 13, 925276, doi:10.3389/fphar.2022.925276 (2022).
Heerspink, H. J., Perkins, B. A., Fitchett, D. H., Husain, M. & Cherney, D. Z. Sodium Glucose Cotransporter 2 Inhibitors in the Treatment of Diabetes Mellitus: Cardiovascular and Kidney Effects, Potential Mechanisms, and Clinical Applications. Circulation 134, 752-772, doi:10.1161/circulationaha.116.021887 (2016).
Hou, Y. C., Zheng, C. M., Yen, T. H. & Lu, K. C. Molecular Mechanisms of SGLT2 Inhibitor on Cardiorenal Protection. Int J Mol Sci 21, doi:10.3390/ijms21217833 (2020).
Xie, Y. et al. Mechanisms of SGLT2 Inhibitors in Heart Failure and Their Clinical Value. J Cardiovasc Pharmacol 81, 4-14, doi:10.1097/fjc.0000000000001380 (2023).
Chen, W. et al. Dapagliflozin alleviates myocardial ischemia/reperfusion injury by reducing ferroptosis via MAPK signaling inhibition. Front Pharmacol 14, 1078205, doi:10.3389/fphar.2023.1078205 (2023).
Li, N. et al. Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury. Free Radic Biol Med 160, 303-318, doi:10.1016/j.freeradbiomed.2020.08.009 (2020).
Fang, Y. et al. Inhibiting Ferroptosis through Disrupting the NCOA4-FTH1 Interaction: A New Mechanism of Action. ACS Cent Sci 7, 980-989, doi:10.1021/acscentsci.0c01592 (2021).
Santana-Codina, N., Gikandi, A. & Mancias, J. D. The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis. Adv Exp Med Biol 1301, 41-57, doi:10.1007/978-3-030-62026-4_4 (2021).
Li, W. et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation. J Clin Invest 129, 2293-2304, doi:10.1172/jci126428 (2019).
Lahnwong, S. et al. Acute dapagliflozin administration exerts cardioprotective effects in rats with cardiac ischemia/reperfusion injury. Cardiovasc Diabetol 19, 91, doi:10.1186/s12933-020-01066-9 (2020).
Tanajak, P. et al. Cardioprotection of dapagliflozin and vildagliptin in rats with cardiac ischemia-reperfusion injury. J Endocrinol 236, 69-84, doi:10.1530/joe-17-0457 (2018).
Fan, Z., Cai, L., Wang, S., Wang, J. & Chen, B. Baicalin Prevents Myocardial Ischemia/Reperfusion Injury Through Inhibiting ACSL4 Mediated Ferroptosis. Front Pharmacol 12, 628988, doi:10.3389/fphar.2021.628988 (2021).
Dhaibar, H. A. et al. Exposure to Stress Alters Cardiac Gene Expression and Exacerbates Myocardial Ischemic Injury in the Female Murine Heart. Int J Mol Sci 24, doi:10.3390/ijms241310994 (2023).
Deng, W. et al. Mild therapeutic hypothermia upregulates the O-GlcNAcylation level of COX10 to alleviate mitochondrial damage induced by myocardial ischemia-reperfusion injury. J Transl Med 22, 489, doi:10.1186/s12967-024-05264-x (2024).
Koyama, T., Temma, K. & Akera, T. Reperfusion-induced contracture develops with a decreasing [Ca2+]i in single heart cells. Am J Physiol 261, H1115-1122, doi:10.1152/ajpheart.1991.261.4.H1115 (1991).
Salie, R. et al. Melatonin protects against ischaemic-reperfusion myocardial damage. J Mol Cell Cardiol 33, 343-357, doi:10.1006/jmcc.2000.1306 (2001).
Andreadou, I., Bell, R. M., Bøtker, H. E. & Zuurbier, C. J. SGLT2 inhibitors reduce infarct size in reperfused ischemic heart and improve cardiac function during ischemic episodes in preclinical models. Biochim Biophys Acta Mol Basis Dis 1866, 165770, doi:10.1016/j.bbadis.2020.165770 (2020).
Bayes-Genis, A. et al. Decoding empagliflozin's molecular mechanism of action in heart failure with preserved ejection fraction using artificial intelligence. Sci Rep 11, 12025, doi:10.1038/s41598-021-91546-z (2021).
Uthman, L. et al. Class effects of SGLT2 inhibitors in mouse cardiomyocytes and hearts: inhibition of Na(+)/H(+) exchanger, lowering of cytosolic Na(+) and vasodilation. Diabetologia 61, 722-726, doi:10.1007/s00125-017-4509-7 (2018).
Yu, Y. W. et al. Sodium-Glucose Co-transporter-2 Inhibitor of Dapagliflozin Attenuates Myocardial Ischemia/Reperfusion Injury by Limiting NLRP3 Inflammasome Activation and Modulating Autophagy. Front Cardiovasc Med 8, 768214, doi:10.3389/fcvm.2021.768214 (2021).
Sakanyan, V. et al. Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells. Sci Rep 4, 3977, doi:10.1038/srep03977 (2014).
Itoh, H. et al. Metabolically Healthy Obesity and the Risk of Cardiovascular Disease in the General Population　- Analysis of a Nationwide Epidemiological Database. Circ J 85, 914-920, doi:10.1253/circj.CJ-20-1040 (2021).
Weglicki, W. B., Kramer, J. H., Spurney, C. F., Chmielinska, J. J. & Mak, I. T. The EGFR tyrosine kinase inhibitor tyrphostin AG-1478 causes hypomagnesemia and cardiac dysfunction. Can J Physiol Pharmacol 90, 1145-1149, doi:10.1139/y2012-023 (2012).
Yuan, N. et al. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica 100, 345-356, doi:10.3324/haematol.2014.113324 (2015).
Zhao, Y. et al. The Role of Erastin in Ferroptosis and Its Prospects in Cancer Therapy. Onco Targets Ther 13, 5429-5441, doi:10.2147/ott.S254995 (2020).
Uthman, L. et al. Direct Cardiac Actions of Sodium Glucose Cotransporter 2 Inhibitors Target Pathogenic Mechanisms Underlying Heart Failure in Diabetic Patients. Front Physiol 9, 1575, doi:10.3389/fphys.2018.01575 (2018).
Jiang, K. et al. Cardioprotective mechanism of SGLT2 inhibitor against myocardial infarction is through reduction of autosis. Protein Cell 13, 336-359, doi:10.1007/s13238-020-00809-4 (2022).
Lin, K. et al. Direct cardio-protection of Dapagliflozin against obesity-related cardiomyopathy via NHE1/MAPK signaling. Acta Pharmacol Sin 43, 2624-2635, doi:10.1038/s41401-022-00885-8 (2022).
Zhang, H. et al. Empagliflozin Decreases Lactate Generation in an NHE-1 Dependent Fashion and Increases α-Ketoglutarate Synthesis From Palmitate in Type II Diabetic Mouse Hearts. Front Cardiovasc Med 7, 592233, doi:10.3389/fcvm.2020.592233 (2020).
Gan, B. Mitochondrial regulation of ferroptosis. J Cell Biol 220, doi:10.1083/jcb.202105043 (2021).
Chen, H. Y. et al. ELAVL1 is transcriptionally activated by FOXC1 and promotes ferroptosis in myocardial ischemia/reperfusion injury by regulating autophagy. Mol Med 27, 14, doi:10.1186/s10020-021-00271-w (2021).
Chen, H. Y. et al. Correction: ELAVL1 is transcriptionally activated by FOXC1 and promotes ferroptosis in myocardial ischemia/reperfusion injury by regulating autophagy. Mol Med 28, 96, doi:10.1186/s10020-022-00529-x (2022).
Fu, C. et al. PFKFB2 inhibits ferroptosis in myocardial ischemia/reperfusion injury through AMPK activation. J Cardiovasc Pharmacol, doi:10.1097/fjc.0000000000001437 (2023).
Ju, J. et al. Circular RNA FEACR inhibits ferroptosis and alleviates myocardial ischemia/reperfusion injury by interacting with NAMPT. J Biomed Sci 30, 45, doi:10.1186/s12929-023-00927-1 (2023).
Li, W., Li, W., Wang, Y., Leng, Y. & Xia, Z. Inhibition of DNMT-1 alleviates ferroptosis through NCOA4 mediated ferritinophagy during diabetes myocardial ischemia/reperfusion injury. Cell Death Discov 7, 267, doi:10.1038/s41420-021-00656-0 (2021).
Xu, S. et al. Naringenin alleviates myocardial ischemia/reperfusion injury by regulating the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) /System xc-/ glutathione peroxidase 4 (GPX4) axis to inhibit ferroptosis. Bioengineered 12, 10924-10934, doi:10.1080/21655979.2021.1995994 (2021).
Shan, X. et al. The Protective Effect of Cyanidin-3-Glucoside on Myocardial Ischemia-Reperfusion Injury through Ferroptosis. Oxid Med Cell Longev 2021, 8880141, doi:10.1155/2021/8880141 (2021).
De Duve, C. & Wattiaux, R. Functions of lysosomes. Annu Rev Physiol 28, 435-492, doi:10.1146/annurev.ph.28.030166.002251 (1966).
Tang, M., Chen, Z., Wu, D. & Chen, L. Ferritinophagy/ferroptosis: Iron-related newcomers in human diseases. J Cell Physiol 233, 9179-9190, doi:10.1002/jcp.26954 (2018).
Mancias, J. D., Wang, X., Gygi, S. P., Harper, J. W. & Kimmelman, A. C. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 509, 105-109, doi:10.1038/nature13148 (2014).
Wang, F. et al. MicroRNA-122a aggravates intestinal ischemia/reperfusion injury by promoting pyroptosis via targeting EGFR-NLRP3 signaling pathway. Life Sci 307, 120863, doi:10.1016/j.lfs.2022.120863 (2022).
Xu, J. et al. Morphine Prevents Ischemia/Reperfusion-Induced Myocardial Mitochondrial Damage by Activating δ-opioid Receptor/EGFR/ROS Pathway. Cardiovasc Drugs Ther 36, 841-857, doi:10.1007/s10557-021-07215-w (2022).
Liu, X., Wang, P., Zhang, C. & Ma, Z. Epidermal growth factor receptor (EGFR): A rising star in the era of precision medicine of lung cancer. Oncotarget 8, 50209-50220, doi:10.18632/oncotarget.16854 (2017).

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Dapagliflozin Ameliorates Myocardial Ischemia/Reperfusion Injury by Modulating EGFR Signaling and Targeting NCOA4-mediated Ferritinophagy

Status:

Version 1

Abstract

Figures

1. Introduction

2. MATERIALS AND METHODS

2.1. Chemicals and reagents

2.2. Animals and Langendorff isolated heart perfusion model

2.3. Cell culture

2.4. Myocardial infarction size measurement

2.5. Hematoxylin and eosin (H&E) staining

2.6. LDH activity assay, CK-MB and cTnI measurement

2.7. Cell viability

2.8. Electron microscopy imaging with parallel Flameng’s score

2.9. Evaluation of mitochondrial ROS (mROS)

2.10. Measurement of the mitochondrial membrane potential (MMP)

2.11. Mitochondrial respiratory oxygen consumption rate (OCR) detection

2.12. Bioinformatics analysis

2.13. Western blot

2.14. Iron measurements

2.15. Myocardial histopathological staining

2.16. Immunoprecipitation (IP) assay

2.17. Cell apoptosis detection

2.18. Statistical analysis

3. Results

3.1 Dapa reduces myocardial infarct size and improves haemodynamic performance after ischaemia-reperfusion injury in rats

3.2 Dapa improves mitochondrial function and attenuates myocardial ischaemia-reperfusion injury-mediated mitochondrial damage and oxidative stress

3.3 Dapa is involved in improving myocardial IRI by regulating the EGFR signaling pathway

3.4 Dapa protects against myocardial IRI by regulating ferroptosis

3.5 Dapa promotes NCOA4-mediated ferritinophagy and reduces increased myocardial IRI-induced ferroptosis

3.6 Activation of Dapa-mediated EGFR phosphorylation improves mitochondrial function and ferritinophagy against H/R-induced injury

4. Discussion

5.Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1