Sirtuin 1 activated by SRT1460 protects against myocardial ischemia/reperfusion injury

Abstract

BACKGROUND:

Ischemia reperfusion usually results in certain degree of damage to the myocardium, which is called myocardial ischemia/reperfusion (I/R) injury.

OBJECTIVE:

Previous studies have found that Sirt1 plays a critical role in I/R injury by protecting cardiac function. SRT1460 is the activator for Sirt1 that participates in the regulation of various diseases. However, whether SRT1460 has any effects on myocardial I/R injury needs further study.

METHODS:

The I/R rat model and H/R H9C2 model were established to simulate myocardial I/R injury. The infarct area of the rat heart was examined through TTC staining. The EF and FS of rats were detected through echocardiography. The levels of CK-MB, LDH, MDA, SOD and CK in cardiac tissues, serum or H9C2 cells were measured using commercial kits. Cell viability was assessed through MTT assay. Apoptosis was determined through flow cytometry analysis. Sirt1 expression was measured through western blot.

RESULTS:

Our work found that SRT1460 reduced the infarct area of the heart induced by myocardial I/R injury. In addition, SRT1460 was confirmed to ameliorate cardiac dysfunction induced by myocardial I/R injury. Further exploration discovered that SRT1460 weakened oxidative stress induced by myocardial I/R injury. Findings from in vitro assays demonstrated that SRT1460 relieved injury of H/R-treated H9C2 cells. Finally, rescue assays proved that Sirt1 knockdown reversed the protective effects of SRT1460 on the injury of H/R-treated H9C2 cells.

CONCLUSION:

Sirt1 activated by SRT1460 protected against myocardial I/R injury. This discovery may offer new sights on the treatment of myocardial I/R injury.

Keywords

Sirt1 SRT1460 myocardial I/R injury

1 Introduction

Myocardial ischemia/reperfusion (I/R) injury results in undesirable cardiovascular outcomes after cardiac arrest or surgery, and is one of the leading causes of mortality in coronary heart disease patients [1, 2]. I/R injury can activate inflammation, contribute to cardiomyocytes apoptosis and then induce injury by accelerating the release of oxygen free radicals and cytokines [3 –5]. Therapeutic strategies to prevent I/R-mediated myocardial injury can improve clinical outcomes in patients with acute myocardial infarction [6, 7]. Therefore, elucidating the key mechanisms for preventing I/R-induced myocardial injury is of great significance for the development of effective therapeutic strategies.

Silent information regulator 2 homolog 1 (Sirtuin-1, Sirt1), is a histone deacetylase dependent on nicotinamide adenine dinucleotide (NAD +), which plays a variety of roles in different biological systems [8, 9]. Past studies have indicated that in liver transplant I/R, Sirt1 weakens inflammation and hepatocyte injury [10]. The Sirt1 signaling pathway relieves renal I/R injury-mediated endoplasmic reticulum stress in type 1 diabetic [11]. MicroRNA-217 targets Sirt1 to aggravate cerebral I/R injury [12]. Especially, some evidence have shown that Sirt1 participates in the progression and development of myocardial injury mediated by I/R. For example, Sirt1 activates eNOS to improve I/R-induced myocardial injury in diabetic rats [13]. After ischemic induction, lumbrokinase-activated Sirt1 suppresses myocardial I/R injury [14]. MiR-155 binds to Sirt1 to stimulate the cardioprotective function of sevoflurane on I/R-mediated myocardial injury [15]. Together these studies suggest up-regulation or activation of Sirt1 is an effective way to improve myocardial I/R injury.

SRT1460 is a highly selective activator for Sirt1, and is more effective than resveratrol and its close sirtuin homologues [16]. It has been demonstrated that SRT1460 participates in the regulation of several diseases. For instance, SRT1460 restrains cell growth and survival in pancreatic cancer [17]. In addition, SRT1460 contributes to type 2 diabetes treatment by serving as an activator of Sirt1 [18]. However, the effects of SRT1460 on myocardial I/R injury needs further exploration.

In this study, we illustrated that Sirt1 activated by SRT1460 protected against myocardial I/R injury, suggesting SRT1460 may be a pivotal regulator in the progression of myocardial I/R injury and a useful biomarker for future treatment.

2 Materials and methods

2.1 I/R model

All procedures regarding animal handling were approved by the Committee of Panyu Central Hospital (Approval No. 2018-13). The male Sprague-Dawley (SD) rats (N = 36) were randomly separated into 4 groups (N = 6): Sham, I/R + saline, I/R + SRT (25 mg/kg) and I/R + SRT (50 mg/kg). For I/R stimulation, rats were anesthetized by pentobarbital (40 mg/kg, intraperitoneal injection). Cannula was inserted into the left carotid artery to measure the rats’ blood pressure. Echocardiography was utilized to record the EF and FS. The thoracic cage was opened from the 4th intercostal space, next, the pericardium was removed to expose the heart. Next, the left anterior descending coronary arteries (LADs) were occluded with 4-silk sutures for 30 min, followed by reperfusion for 2 h. At last, blood was collected from the abdominal aorta to measure the levels of myocardial injury markers, and the hearts were removed for further experiments. The Sham group received the entire surgery but not LAD ligation. Finally, the infarct size and myocardial marker enzyme leakage were measured. The activities of CK-MB and LDH in cardiac tissue and the levels of LDH, MDA, SOD, CK in the serum of rats were measured using commercial kits.

2.2 2, 3, 5-Triphenyl Tetrazolium Chloride (TTC) staining

TTC solution (Solarbio, Beijing, China) was used to verify the infarct size of the hearts. At first, the heart was removed and sectioned into slices. After rinsing, the slices were put into TTC solution at 37 for 0.5 h in the dark. Finally, images were acquired with a digital camera.

2.3 Detection via echocardiography

The MyLab 30 CV ultrasound system (Esaote S.p.A, Genoa, Italy) and a 10 MHz linear ultrasonic transducer were utilized to estimate the cardiac functions of the rats through the echocardiography. After removing the hair in the thoracic region, the anesthetized rats were placed on a heating plate at 37°C. The ejection fraction (EF) and fractional shortening (FS) were examined.

2.4 Cell culture and H/R H9C2 model

The H9C2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These H9C2 cells were cultured by the use of Dulbecco’s modified Eagle’s medium (DMEM) with fetal bovine serum (FBS), streptomycin and penicillin. The condition was at 37 in a humidified atmosphere with 5% CO2.

For the H/R H9C2 model, cells were put in a hypoxia incubator with 95% N2 and 5% CO2 for 0.5 h. Finally, they were cultured with 5% CO2 for another 2 h according to previous study [19].

2.5 Cell transfection

Short hair RNAs (shRNA) against Sirt1 (Sirt1-sh1 and Sirt1-sh2) and negative control (shNC) were acquired from GenePharm (Shanghai, China). H9C2 cells were transfected with these plasmids by the use of lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). These transfected H9C2 cells were incubated for 48 h for next experiments.

2.6 Western blot

H9C2 cells were lysed through RIPA lysis (Beyotime) buffer. Samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% -skim milk, membranes were incubated with primary antibodies against Sirt1 (ab110304; 1:1000; Abcam) and GAPDH (ab9485; 1:1000; Abcam) overnight at 4. Next, membranes were incubated with HRP-conjugated secondary antibodies (ab205718, 1:20000, Abcam). Bands were observed using the ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester, NY).

2.7 MTT assay

H9C2 cells were seeded into a 96-well plate. Cell viability was assessed through the 3-(4, 5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) assay. After transfection, 10μL MTT solution (Beyotime, Shanghai, China) was supplemented into each well for 4 h incubation. The absorbance at 490 nm was measured after dimethylsulphoxide (DMSO, Sigma) treatment.

2.8 Flow cytometry

The Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK) was used to examine apoptosis. Generally, after rinsing twice with cold PBS solution, H9C2 cells were resuspended. Afterwards, Annexin V-FITC and propidium iodide (PI) were added and incubated in the dark. Finally, apoptosis rate was tested with the use of a flow cytometer (BD Biosciences, San Jose, CA, USA).

2.9 Statistical analysis

The data were presented as the arithmetic mean±SD. Statistical analysis was conducted through SPSS 20.0 (SPSS, Inc., Chicago, IL, USA). Statistical differences between two groups (or among multiple groups) were assessed by the Student’s t-test (or one-way analysis of variance). P < 0.05 was considered as statistically significant.

3 Results

3.1 SRT1460 reduced the infarct area of the heart induced by myocardial I/R injury

To probe the effects of SRT1460 in myocardial I/R injury, the I/R rat model was constructed using LAD ligation and reperfusion for 2 h. Results from TTC staining demonstrated that the infarct area of the hearts was enhanced after I/R stimulation, but this effect could be attenuated by adding SRT1460. In conclusion, I/R stimulation aggravated the infarct area of the hearts, and SRT1460 reduced the infarct area of the heart (Fig. 1).

Fig. 1

SRT1460 reduced the infarct area of the heart induced by myocardial I/R injury. The black column represents the Sham group; the gray column represents the I/R group; the dark gray column represents the I/R + SRT (25 mg/kg) group; the light gray column represents the I/R + SRT (50 mg/kg) group. The infarct area of the rat heart was assessed in Sham, I/R, I/R + SRT (25 mg/kg) and I/R + SRT (50 mg/kg) groups through TTC assay. N = 6. ^**P < 0.01.

3.2 SRT1460 attenuated cardiac dysfunction

According to the findings of echocardiography, the decreased EF% and FS% in the I/R group were obviously rescued after treating with SRT1460 (Fig. 2A-B). Furthermore, the levels of CK-MB and LDH in cardiac tissues were up-regulated in the I/R group compared with the Sham group, but SRT1460 treatment could offset this effect (Fig. 2C-D). These data illustrated that SRT1460 attenuated cardiac dysfunction.

Fig. 2

SRT1460 relieved cardiac dysfunction induced by myocardial I/R injury. The black column represents the Sham group; the gray column represents the I/R group; the dark gray column represents the I/R + SRT (25 mg/kg) group; the light gray column represents the I/R + SRT (50 mg/kg) group. (A-B): The EF and FS of rats were examined in Sham, I/R, I/R + SRT (25 mg/kg) and I/R + SRT (50 mg/kg) groups through echocardiography. (C-D): The levels of CK-MB and LDH release in cardiac tissues were measured in Sham, I/R, I/R + SRT (25 mg/kg) and I/R + SRT (50 mg/kg) groups using commercial kits. N = 6. ^**P < 0.01.

3.3 SRT1460 alleviated oxidative stress in myocardial I/R injury

Further experiments were done to verify whether SRT1460 affected oxidative stress induced by myocardial I/R injury. As displayed in Fig. 3A, the enhanced level of MDA caused by I/R injury in serum of rats was reversed by adding SRT1460. Additionally, the level of SOD was lower in the I/R group than that in the Sham group, and this effect could be weakened by SRT1460 treatment (Fig. 3B). Levels of LDH and CK were increased in the I/R group, and SRT1460 treatment could suppress this effect (Fig. 3C-D). To sum up, SRT1460 alleviated oxidative stress.

Fig. 3

SRT1460 relieved oxidative stress induced by myocardial I/R injury. The black column represents the Sham group; the gray column represents the I/R group; the dark gray column represents the I/R + SRT (25 mg/kg) group; the light gray column represents the I/R + SRT (50 mg/kg) group. (A–D): The levels of SOD, MDH, CK, LDH in the serum of rats were measured in Sham, I/R, I/R + SRT (25 mg/kg) and I/R + SRT (50 mg/kg) groups using commercial kits. N = 6. ^**P < 0.01.

3.4 SRT1460 relieved the injury of H/R-treated H9C2 cells

Next, we investigated the effects of SRT1460 in H/R H9C2 model. Cell viability was decreased by I/R stimulation, and this effect could be offset by adding SRT1460 (Fig. 4A). Besides, the level of LDH was higher in the I/R group than that in the Control group, and this effect was reversed by SR.

T1460 treatment (Fig. 4B). Moreover, treatment with SRT1460 rescued the increased apoptosis induced by I/R (Fig. 4C). Taken together, SRT1460 relieved the injury of H/R-treated H9C2 cells.

Fig. 4

SRT1460 relieved the injury of H/R-treated H9C2 cells. The black column represents the control group; the gray col-umn represents the H/R group; the dark gray column represents the H/R + SRT (10μM) group; the light gray column represents the H/R + SRT (50μM) group. (A): Cell viability was verified in control, H/R, H/R + SRT (10μM) and H/R + SRT (50μM) gr-oups through MTT assay. (B): The level of LDH was measured in control, H/R, H/R + SRT (10μM) and H/R + SRT (50μM) groups using commercial LDH kit. (C): Apoptosis was assessed in control, H/R, H/R + SRT (10μM) and H/R + SRT (50μM) groups through flow cytometry. ^**P < 0.01.

3.5 Sirt1 knockdown reversed the protective effects of SRT1460 on the injury of H/R-treated H9C2 cells

Rescue assays were performed to test whether Sirt1 knockdown could reverse the protective effects of SRT1460 on the injury of H/R-treated H9C2 cells. At first, the knockdown efficiency of Sirt1 was verified in Fig. 5A through western blot. Cell viability was increased in the H/R + SRT group compared to the H/R group, and this effect was reversed in the H/R + SRT + Sirt1-sh1 group (Fig. 5B). Moreover, knockdown of Sirt1 reversed the decreased LDH level induced by SRT1460 treatment (Fig. 5C). In addition, apoptosis was decreased by SRT1460 treatment, and this effect could be offset by silencing Sirt1 (Fig. 5D).

Fig. 5

Sirt1 knockdown reversed the protective effects of SRT1460 on the injury of H/R-treated H9C2 cells. The black column represents the shNC group; the light gray column represents the Sirt-sh1 group; the dark gray column represents the Sirt-sh2 group. (A): The knockdown efficiency of Sirt1 was measured through western blot. The black column represents the H/R group; the light gray column represents the H/R + SRT group; the dark gray column represents the H/R + SRT + Sirt-sh1 group. (B): Cell viability was measured in H/R, H/R + SRT and H/R + SRT + Sirt-sh1 groups through MTT assay. (C): The level of LDH was measured in H/R, H/R + SRT and H/R + SRT + Sirt-sh1 groups using commercial LDH kit. (D): Apoptosis was assessed in H/R, H/R + SRT and H/R + SRT + Sirt-sh1 groups through flow cytometry. ^**P < 0.01.

4 Discussion

Myocardial I/R injury is a common pathophysiological change after many types of cardiovascular surgery [20]. Myocardial I/R injury could lead to the changes of cellular environment, including inflammatory response, oxidative stress, and cellular Ca2+ overload [21 –23]. Current clinical treatment of I/R-mediated myocardial injury is still unsatisfactory due to incomplete understanding of its mechanisms [24]. Researches have indicated that Sirt1 plays a critical role in many diseases, including I/R-induced myocardial injury [10–15 , 18]. Several different compounds, such as SRT2183, SRT1720, SRT1460, and resveratrol serve as SIRT activator to influence multiple diseases. For example, Sirt1 activator SRT2183 weakens cell growth in glioma [25]. In sepsis, SRT1720 improves inflammation and organ injury by acting as a Sirt1 activator [26]. Sirt1 activator SRT1720 regulates NF-κB signaling to weaken severe acute pancreatitis mediated by sodium taurocholate [27]. Resveratrol stimulates Sirt1/FoxO1 pathway to facilitate osteogenesis [28]. These SIRT activators also have been reported to affect I/R injury. For instance, Sirt1 activator Srt1720 relieves intestinal I/R-induced organ injury [29]. Sirt1 activator resveratrol relieves acute kidney injury caused by I/R [30]. Sirt1 activator SRT2104 attenuates diabetes-mediated cardiac I/R injury [31]. Similar to these studies, SRT1460 also has been discovered to play a regulatory role in some diseases by serving as a Sirt1 activator [16, 17]. However, the function of SRT1460 on myocardial I/R injury needs further exploration. Similar to the results of other Sirt1 activators in myocardial I/R injury, our work found that SRT1460 reduced the infarct area of the heart induced by myocardial I/R injury. In addition, SRT1460 was confirmed to relieve cardiac dysfunction induced by myocardial I/R injury.

Oxidative stress refers to a state of imbalance between oxidation and anti-oxidation in the body, which tends to oxidize and results in irreversible damage to cells or tissues [32, 33]. The aggravation of oxidative stress, manifested as oxidative imbalance, leads to the increase of inflammatory damage [34]. It has been widely accepted that oxidative damage caused by excess free radicals may be critical participators in the progression of myocardial I/R injury. For instance, lncRNA Gpr19 targets miR-324-5p/Mtfr1 axis in acute myocardial infarction to aggravate oxidative stress and apoptosis [35]. NFκB inactivates Nrf2-ARE pathway to increase necrosis and I/R injury mediated by oxidative stress [36]. IL-23 aggravates oxidative stress reactions and inflammatory responses to exacerbate myocardial I/R injury [37]. Oridonin reduces oxidative stress to improve myocardial injury mediated by I/R [38]. Further exploration was done in this work to verify the influences of SRT1460 on oxidative stress in I/R-mediated myocardial injury. Similar to these previous studies, our study discovered that the oxidative stress induced by myocardial I/R injury was affected by SRT1460.

Apoptosis is an autonomic and orderly mechanism of cell death, which is indispensable for various biological processes such as tissue homeostasis, developmental shaping, and elimination of unwanted cells [39, 40]. Ischemia and reperfusion can lead to necrosis and apoptosis of cardiomyocytes [41, 42]. Our findings from in vitro assays demonstrated that SRT1460 improved the injury of H/R-treated H9C2 cells by increasing cell viability and decreasing LDH level as well as apoptosis. Finally, rescue assays proved that Sirt1 knockdown reversed the protective role of SRT1460 on the injury of H/R-treated H9C2 cells.

To sum up, our study for the first time confirmed that Sirt1 activated by SRT1460 protected against myocardial I/R injury. This novel discovery may offer a useful marker for the treatment of myocardial I/R injury. In the future, more experiments will be done to further probe the role of SRT1460 in myocardial I/R injury progression.

Funding

Not applicable.

Competing interests

The authors state that there are no conflicts of interest to disclose.

Ethics approval

Ethical approval was obtained from the Ethics Committee of Panyu Central Hospital (Approval No.2018-13).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

Shanjun Zhao and Lei Yu designed the study, supervised data collection. Shanjun Zhao analyzed the data, interpreted the data. Lei Yu prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Footnotes

Acknowledgments

Not applicable.

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