Myocardial tissue cell damage induced by myocardial ischemia/reperfusion (MI/R) notably elevates the mortality rate, increases the complications of patients with myocardial infarction and decreases reperfusion benefit in patients suffering from acute myocardial infarction. Roflumilast protect against cardiotoxicity. Therefore, the present study aimed to investigate the effect of roflumilast on MI/R injury and the underlying mechanisms. To simulate MI/R
Acute myocardial infarction (AMI) is acute ischemic necrosis of myocardium occurring due to coronary artery disease, which can lead to fatal complications, and even mortality in severe cases (
Roflumilast is a phosphodiesterase-4 (PDE-4) inhibitor. The US Food and Drug Administration has approved roflumilast for the treatment of severe chronic obstructive pulmonary disease because of its strong anti-inflammatory and immunomodulatory properties (
Mitochondrial dysfunction is a key cause of MIRI and the main mechanisms include decreased mitochondrial ATP production (
Therefore, the aim of the present study was to investigate the effect of roflumilast on MIRI as well as to discuss the underlying mechanisms. It was hypothesized that roflumilast could alleviate MIRI by improving mitochondrial dysfunction by activating the AMPK signaling pathway.
The chemical structure of roflumilast was determined by PubChem (
The experimental protocol for animal studies was reviewed and approved by the Committee for the Ethics of Animal Experiments, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center (approval no. 2021-807; Shenzhen, China). The MI/R rat model was established as previously described (
H9C2 cells were obtained from the American Tissue Culture Collection (ATCC; cat. no. CRL-1446). H9C2 cells were cultured in ATCC-formulated Dulbecco's Modified Eagle's Medium (cat. no. ATCC 30-2002) supplemented with 10% fetal bovine serum (cat. no. ATCC 30-2020) in 95% air and 5% CO2 at 37˚C.
For H/R stimulation, H9C2 cells were grown in an anoxic chamber with 5% CO2 and 95% N2 for 6 h and then in a normal chamber with 95% air and 5% CO2 for 12 h at 37˚C.
For roflumilast treatment, cultured cells were pre-incubated with roflumilast (1.0, 2.5 and 5.0 µM; Adooq Bioscience) or compound C (10 µM; an inhibitor of the AMPK signaling pathway; MedChemExpress, United States) for 30 min at 37˚C before H/R treatment.
The MI area in each group was observed by TTC staining. Following storage at -18˚C for 15 min, the heart tissue perpendicular to the coronary sulcus was cut into five equal pieces. Following incubation with 1% TTC solution (Sigma-Aldrich; Merck KGaA) for 10 min at 37˚C and 10% neutral buffered formalin (Thermo Fisher Scientific, Waltham, MA) for 90 min at 37˚C in the dark, all slices (1 mm) were imaged using a digital camera. The MI areas were determined by Image-Pro Plus image analysis software (version 4.1; Media Cybernetics, Inc.). Finally, calculation of MI area was conducted according to the following formula: Myocardial infarct size (%)=(infarct area/whole heart area) x100%.
The cardiac tissue samples collected from the left ventricle were fixed in 4% paraformaldehyde overnight at 4˚C, dehydrated in ascending ethanol gradient and embedded in paraffin for 50-60 min at room temperature. Subsequently, the embedded cardiac tissues were cut into 4 µm slices, followed by staining with 0.5% hematoxylin for 5 min and eosin for 2 min at room temperature. Then, the sections were mounted and observed under a light microscope (magnification, x100/400; Olympus Corporation BX53).
The mitochondrial membrane potential in cardiac tissue and H9C2 cells was detected using the JC-1 staining kit (cat. no. C2006; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. In brief, the isolated cardiomyocyte suspension and H9C2 cells were stained with 2.5 mg/ml JC-1 solution for 20 min at 37˚C. Subsequently, the cells were washed with JC-1 staining buffer twice and observed using a fluorescence microscope (magnification, x200; Olympus Corporation BX63).
The collected peripheral blood (15 ml) was centrifuged at 2,072 x g at 4˚C for 10 min to separate the serum. Heart muscle damage indicators, including aspartate transaminase (AST), creatine kinase-myocardial band (CK-MB) and lactate dehydrogenase (LDH) in serum were detected by AST (cat. no. C010-2-1), CK-MB (cat. no. A032-1-1) and LDH (cat. no. A020-2-2) assay kits (all Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions, respectively.
The concentrations of IL-1β and IFN-γ in myocardial tissue and in cell supernatant were measured using ELISA kits for IL-1β (cat. no. E-EL-R0012c; Elabscience Biotechnology, Inc.) and IFN-γ (cat. no. E-EL-R0009c; Elabscience Biotechnology, Inc.) according to the manufacturer's instructions.
The activity of malondialdehyde (MDA) and superoxide dismutase (SOD) in myocardial tissue and H9C2 cells were measured using MDA (cat. no. S0131S; Beyotime Institute of Biotechnology) and SOD assay kits (cat. no. S0109; Beyotime Institute of Biotechnology) according to the manufacturer's instructions.
The detection of ATP concentration in H9C2 cells was conducted using ATP assay kit (cat. no. S0026; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, H9C2 cells were collected and mixed with cell lysis buffer for 10 min at 4˚C, followed by centrifugation at 12,000 x g at 4˚C for 5 min. Subsequently, cell supernatant was incubated with 100 µl kit solution at room temperature for 5 min and the ATP levels in the cell supernatant were detected using a LuminMax-C luminometer (Maxwell Sensors Inc.).
Protein from cardiac tissues and H9C2 cells was obtained using RIPA lysis buffer (Beyotime Institute of Biotechnology) and qualified with a BCA detection kit (Beyotime Institute of Biotechnology). A total of 25 µg/lane protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane. Following blocking with 5% BSA (Beyotime Institute of Biotechnology) at room temperature for 2 h, the membrane was incubated with primary antibodies targeting AMP-activated protein kinase alpha (AMPKα; cat. no. 5831; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p-)AMPKα (cat. no. 50081; 1:1,000; Cell Signaling Technology, Inc.), SIRT1 (cat. no. ab189494; 1:1,000; Abcam), Bcl-2 (cat. no. ab196495; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), PINK1 (cat. no. ab186303; 1:1,000; Abcam), DRP1 (cat. no. 8570; 1:1,000; Cell Signaling Technology, Inc.), p-DRP1 (cat. no. 4867; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. 93473; 1:1,000; Cell Signaling Technology, Inc.) overnight at 4˚C. Then, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. ab6721; 1:2,000; Abcam) or goat anti-mouse IgG (cat. no. ab6789; 1:2,000; Abcam) for 1 h at room temperature. Finally, the bands were examined with ECL reagent (Beyotime Institute of Biotechnology) and band density was quantified using ImageJ Software (version 1.46; National Institutes of Health).
H9C2 cells were inoculated into a 96-well plate at a density of 1x103 cells/well. H9C2 cells were treated with roflumilast (1.0, 2.5 and 5.0 µM) for 24 h at 37˚C and induced by H/R. H9C2 cells in each well were mixed with 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) and incubated at 37˚C for 1 h. The optical density at 450 nm was detected by a spectrophotometer (Bio-Rad Laboratories, Inc.).
The apoptosis of H9C2 cells was determined by
The opening of the mPTP was detected using a calcein-loading/cobalt chloride (CoCl2)-quenching system. Briefly, 2x105 H9C2 cells seeded in a 6-well plate were treated with 1 µM calcein and 2 mM CoCl2 for 20 min at 37˚C in the dark. After washing with PBS, H9C2 cells were observed and imaged using a confocal laser scanning microscope (model no. LSM 880; Carl Zeiss AG; magnification, x100). The mean green fluorescence intensities in the mitochondria were quantified using ImageJ Software (version 1.46; National Institutes of Health). The changes of green fluorescence intensity in the mitochondria were the index of mPTP opening.
GraphPad (version 8.0.1; GraphPad Software, Inc.; Dotmatics) was used to analyze the experimental data. Data are shown as the mean ± the standard deviation from three independent experiments. The comparisons between multiple groups were conducted by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
The chemical structure of roflumilast is shown in
The H&E staining results revealed that the myocardial fiber structure was damaged, vascular walls were broken and hemocyte infiltration was apparent in the MI/R group. However, MI/R rats pre-injected with roflumilast exhibited mild tissue damage (
The ratio of green and red fluorescence intensity was significantly increased in MI/R rats compared with control rats; this was reversed by roflumilast treatment, indicating that roflumilast decreased depolarization of the mitochondrial membrane (
The expression of p-AMPK and SIRT1 in the cardiac tissue of rats subjected to MI/R were significantly decreased compared with the control group, while roflumilast treatment promoted the expression of p-AMPK and SIRT1 in a dose-dependent manner (
After H9C2 cells were treated with roflumilast, their viability was unchanged, indicating that roflumilast has no significant effect on H9C2 cells at these concentrations (
Roflumilast significantly suppressed MDA and significantly upregulated SOD in H/R-induced H9C2 cells, while compound C significantly impaired the function of roflumilast (
Roflumilast treatment significantly decreased the ratio of green and red fluorescence intensity in H/R-induced H9C2 cells compared with that in the H/R group; this was increased following the administration of compound C (
The present study demonstrated that roflumilast treatment decreased MIRI
The rhythmic contraction of cardiomyocytes consumes a lot of energy, and 90% of ATP is produced by mitochondria. Therefore, maintaining good mitochondrial morphology and function is crucial for the survival and normal function of cardiomyocytes (
When the energy crisis of the body is caused by stress conditions (ischemia, hypoxia, oxidative stress and other factors), the ATP levels in the body decrease or the newly generated ATP cannot rapidly replace its consumption by tissues and organs, resulting in the insufficient energy supply in cells and the activation of AMPK (
A previous study indicated that roflumilast could mitigate inflammation, oxidative stress and apoptosis in the acute lung injury of rabbits (
However, there were certain limitations to the current study. Firstly, cardiac functional studies, such as imaging and cardiac echo, were not performed. Secondly, female rats were not included in the MIRI model. Finally, the target of roflumilast in the MIRI model was not determined. These factors should be considered in further studies.
In conclusion, the present study demonstrated that roflumilast could alleviate MI in rats subjected to MI/R and attenuate H/R-induced oxidative stress, inflammatory response and mitochondrial damage in H9C2 cells by activating the AMPK signaling pathway.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ZH designed and conceived the study. BL conducted the experiments, analyzed the data and drafted the manuscript. ZH and BL confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The experimental protocol for the animal studies was reviewed and approved by the Committee for the Ethics of Animal Experiments, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center (approval no. 2021-807; Shenzhen, China).
Not applicable.
The authors declare that they have no competing interests.
Roflumilast decreases MI/R-induced myocardial infarction. (A) Chemical structure of roflumilast was obtained from PubChem. (B) Representative images of myocardial infarct size demonstrated by TTC staining. The non-stained areas (white or pale) indicate infarct areas; TTC-stained areas (red) indicate non-infarct areas. The activities of (C) CK-MB, (D) LDH and (E) AST in rat serum were detected by corresponding assay kits. ***P<0.001 vs. Control. ##P<0.01 and ###P<0.001 vs. MI/R. ++P<0.01 and +++P<0.001 vs. MI/R + roflumilast (1 mg/kg). MI/R, myocardial ischemia/reperfusion; TTC, 2,3,5-triphenyltetrazolium chloride; CK-MB, creatine kinase-myocardial band; LDH, lactate dehydrogenase; AST, aspartate transaminase; MI, myocardial infarction.
Roflumilast attenuates MI/R-induced myocardial injury. (A) Changes of pathological injury of myocardial tissue were detected by H&E staining. The arrows indicated hemocyte infiltration. (B) Inflammatory factors (IL-1β and IFN-γ) in myocardial tissue were detected by corresponding assay kits. ***P<0.001 vs. Control. #P<0.05 and ###P<0.001 vs. MI/R. ++P<0.01 vs. MI/R + roflumilast (1 mg/kg). MI/R, myocardial ischemia/reperfusion.
Roflumilast attenuates myocardial mitochondrial damage induced by MI/R. (A) Changes in mitochondrial membrane potential were detected by JC-1 staining. (B) Levels of oxidative stress markers in myocardial tissue were detected by assay kits. ***P<0.001 vs. Control. ##P<0.01 and ###P<0.001 vs. MI/R. +++P<0.001 vs. MI/R + roflumilast (1 mg/kg). MI/R, myocardial ischemia/reperfusion; MDA, malondialdehyde; SOD, superoxide dismutase.
Roflumilast activates the AMPK signaling pathway in MI/R injury. The expression of AMPK signaling pathway-associated proteins in myocardial tissue was detected by western blotting. ***P<0.001 vs. Control. #P<0.05 and ###P<0.001 vs. MI/R. ++P<0.01 vs. MI/R + roflumilast (1 mg/kg). AMPK, AMP-activated protein kinase; MI/R, myocardial ischemia/reperfusion; p, phosphorylated; SIRT1, sirtuin 1.
Roflumilast mitigates damage of H/R to H9C2 cell viability by activating the AMPK signaling pathway. (A) Effect roflumilast on H9C2 cell viability was analyzed using CCK-8 assay. (B) Viability of H/R-induced H9C2 cells treated with roflumilast was analyzed by CCK-8 assay. (C) Expression AMPK signaling pathway-related proteins in H/R-induced H9C2 cells with roflumilast treatment was detected by western blotting. (D) Apoptosis of H/R-induced H9C2 cells with roflumilast and compound C treatment was analyzed by TUNEL assay. (E) Expression of apoptosis-related proteins in H/R-induced H9C2 cells with roflumilast and compound C treatment was detected by western blotting. ***P<0.001 vs. Control. #P<0.05, ##P<0.01 and ###P<0.001 vs. H/R. ++P<0.01 and +++P<0.001 vs. H/R + roflumilast (5 µM). H/R, hypoxia/reoxygenation; AMPK, AMP-activated protein kinase; CCK-8, Cell Counting Kit-8; p, phosphorylated; SIRT1, sirtuin 1.
Roflumilast activates the AMP-activated protein kinase signaling pathway to alleviate oxidative stress and the inflammatory response of H9C2 cells induced by H/R. (A) Oxidative stress in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by assay kits. (B) Inflammatory factors in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by assay kits. ***P<0.001 vs. Control. ###P<0.001 vs. H/R. ++P<0.01 and +++P<0.001 vs. H/R + roflumilast (5 µM). H/R, hypoxia/reoxygenation; MDA, malondialdehyde; SOD, superoxide dismutase.
Roflumilast activates the AMP-activated protein kinase signaling pathway to decrease mitochondrial damage of H9C2 induced by H/R. (A) Changes of mitochondrial membrane potential in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by JC-1 staining. (B) ATP levels in H/R-induced H9C2 cells with roflumilast and compound C treatment were detected by ATP assay kit. (C) mPTP in H/R-induced H9C2 cells with roflumilast and compound C treatment was examined via a calcein-loading/CoCl2-quenching system (magnification, x100). (D) Quantification of mPTP opening level. (E) Expression of mitochondrial regulatory proteins in H/R-induced H9C2 cells with roflumilast and compound C treatment was determined by western blotting. ***P<0.001 vs. Control. ###P<0.001 vs. H/R. +P<0.05 and +++P<0.001 vs. H/R + roflumilast (5 µM) group. H/R, hypoxia/reoxygenation; mPTP, mitochondrial permeability transition pore; PINK1, PTEN-induced kinase 1; p, phosphorylated; DRP1, dynamin-related protein 1.