Catechin relieves hypoxia/reoxygenation‐induced myocardial cell apoptosis via down‐regulating lncRNA MIAT

Abstract Background Catechin protects heart from myocardial ischaemia/reperfusion (MI/R) injury. However, whether catechin inhibits H/R‐induced myocardial cell apoptosis is largely unknown. Objective This study aims to investigate the underlying mechanism of catechin in inhibiting the apoptosis of H/R‐induced myocardial cells. Methods LncRNA MIAT expression was detected by qRT‐PCR. Cell viability of H9C2 cells was detected using CCK‐8 assay. The apoptosis of H9C2 cells was detected by flow cytometry. The interaction between CREB and MIAT promoter regions was confirmed by dual‐luciferase reporter gene assay and ChIP assay. Results In MI/R rats, catechin improved heart function and down‐regulated lncRNA MIAT expression in myocardial tissue. In H/R‐induced H9C2 cells, catechin protected against cell apoptosis, and lncRNA MIAT overexpression attenuated this protective effect of catechin. We confirmed that transcription factor CREB could bind to MIAT promoter region, and catechin suppressed lncRNA MIAT expression through up‐regulating CREB. Catechin improved mitochondrial function and relieved apoptosis through promoting Akt/Gsk‐3β activation. In addition, MIAT inhibited Akt/Gsk‐3β activation and promoted cell apoptosis in H/R‐induced H9C2 cells. Finally, we found catechin promoted Akt/Gsk‐3β activation through inhibiting MIAT expression in H/R‐induced H9C2 cells. Conclusion Catechin relieved H/R‐induced myocardial cell apoptosis through regulating CREB/lncRNA MIAT/Akt/Gsk‐3β pathway.

most effective therapy to reduce the damage of myocardium, which brings oxygen and nutrition back. However, reperfusion can paradoxically cause the exacerbation of ischaemic tissue injury, which is called myocardial ischaemia/reperfusion (MI/R) injury, 3,4 and ultimately increases the overall size of myocardial infarct and leads to poor prognosis for patients with ischaemic heart disease. 5 Myocardial cell apoptosis mainly occurs in the surviving portion of the heart which contributes to the loss of myocardial cells and inhibits left ventricular remodelling after MI/R injury, and the increase in myocardial cell apoptosis is positively correlated with the occurrence of cardiac failure after MI/R injury. 6 So, apoptosis of myocardial cells is a key promoting factor contributing to cardiac failure after MI/R injury. 7,8 Therefore, inhibiting myocardial cell apoptosis is important for the prevention and treatment of MI/R injury.
Catechin is a bioactive polyphenol extracted from green tea that has antioxidant, antiviral and antioncogenic properties and is used to reduce the risk of various diseases. [9][10][11] It has been well studied that EGCG, another bioactive polyphenol extracted from green tea, relieved MI/R injury through mitigating oxidative stress, decreasing apoptotic myocardial cells, reducing myocardial infarct size and ameliorating cardiac dysfunction in MI/R rabbit, mouse and rat models. 12,13 In hypoxia/reoxygenation (H/R)-stimulated cell apoptosis of rat myocardial cells H9C2, EGCG inhibits the H/R-treated cell apoptosis. 12 Although catechin can protect rat heart from MI/R injury, 14 the effect of catechin on H/R-induced myocardial cell apoptosis is largely unknown.
Long non-coding RNAs (lncRNAs) are a class of transcripts with a length longer than 200 nucleotides which can modulate the expressions of genes at epigenetic, translation or transcription level. 15 More and more studies have shown that lncRNAs are involved in regulating myocardial cell apoptosis after MI/R injury. 16,17 Besides, lncRNA is involved in morphine-meditated alleviation of autophage in MI/R injury, 18 indicating that lncRNAs may mediate the protective role of drugs against MI/R injury. LncRNA myocardial infarction-associated transcript (MIAT) is a widely expressed lncRNA that exerts its regulation function in cancers, diabetic retinopathy, atherosclerosis, etc [19][20][21] Recent studies have shown that down-regulation of lncRNA MIAT reduced myocardial cell apoptosis after MI/R injury, indicating targeting MIAT might protect against H/R-induced myocardial cell apoptosis or MI/R injury. 22,23 Several studies have shown that the expression of lncRNA MIAT can be inhibited or promoted by drugs, indicating lncRNA MIAT involves in drug-mediated inhibition or promotion of diseases progression. 21,24 However, whether lncRNA MIAT is involved in catechin-mediated inhibition on MI/R injury is unclear.
In this study, we found that catechin down-regulated lncRNA MIAT expression in myocardial tissue of MI/R rat and H/R-induced myocardial cells. We further found that catechin relieved H/Rinduced myocardial cell apoptosis through regulating CREB/MIAT/ Akt/Gsk-3β pathway, which might clarify the underlying mechanism of catechin in inhibiting H/R-induced myocardial cell apoptosis. were obtained from the laboratory animal centre of Wenzhou Medical University and maintained in a 12-hour light/12-hour dark room with free access to food and water for a week. Rats were divided into four groups, namely sham group, MI/R group, MI/R+Vehicle group and MI/R+Catechin group, with six rats in each group. Rats were intraperitoneally injected with 80 mg/ kg sodium-pentobarbital for anaesthesia. During the whole surgery, rats were placed in a controlled heating pad to keep the body temperature (37 ± 0.2°C) in the supine position, and electrocardiograph (ECG) was monitored in each rat. The establishment of MI/R rat model was conducted according to the previous report. 25 Thoracotomy was performed at the fourth intercostal space, then the pericardium was cut, and a 5-0 Prolene suture was placed around the left anterior descending coronary artery (LAD). The LAD was occluded for 30 minutes. To confirm the ischaemia was successfully developed by occlusion, prompt STsegment changes with progressive ST-segment elevation in at least 3 leads with or without arrhythmia, and decolorization of occluded distal myocardium should be observed. Thirty minutes later, the ligation was released and reperfusion was continued for 24 hours. For rats in sham group, the procedure of surgery was the same as MI/R group, except the suture around the LAD was not tightened. For rats in MI/R+Vehicle group, 3 mL saline was intragastrically administrated into the rats for 10 days before surgery, and the procedure of surgery was the same as MI/R group. For rats in MI/R+Catechin group, 250 mg/kg/d catechin (C 15 H 14 O 6 ; Sigma) was intragastrically administrated into the rats for 10 days before surgery, and the procedure of surgery was the same as MI/R group.

| Electrocardiography
Serial echocardiograms were obtained at baseline (2 days before the occlusion of the LAD) and at 24 hours after surgery.

| Triphenyl-tetrazolium-chloride (TTC) staining and haematoxylin-eosin (HE) staining
Prolene suture around the LAD was retightened, and Evans blue (1%) in PBS was retrogradely perfused into the aorta to visualize the area at risk. After the kill of rats, hearts were collected and frozen at −20°C. Heart tissues were sliced into 2-mm-thick sections, and 1% TTC was added to the sections and incubated at 37°C for 15 minutes. Intracellular dehydrogenases in the normal myocardium could react with TTC, whereas ischaemia area could not react with TTC and remained white. Heart sections stained with TTC were digitally scanned and evaluated using ImageJ software. Infarct size (%) was calculated as the ratio of infarct size/ area at risk.
For HE staining, heart tissues were fixed with 4% paraformaldehyde at 25°C for 24 hours, embedded in paraffin, and sliced into 5-μm-thick sections. Then, Hematoxylin and Eosin Staining Kit (Beyotime Biotechnology) was used for staining according to the manufacturer's instructions.

| Dual-luciferase reporter assay
Dual-luciferase reporter assay was used to detect the interaction between CREB and MIAT promoter region. The wild-type MIAT promoter luciferase (Luc) and mutant MIAT promoter luciferase were constructed by Genomeditech Co. Ltd. HEK293T cells were seeded in 24-well plates, and then, luciferase reporter constructs (300 ng/well) were cotransfected into HEK293T cells with 600 ng/ well pcDNA-NC and pcDNA-CREB using Lipofectamine 2000 (Invitrogen). Forty-eight hours later, Dual-Luciferase Reporter Assay kit (Promega) was used to measure the relative luciferase activity.

| Chromatin Immunoprecipitation (ChIP) assay
ChIP assay was conducted using Pierce™ Agarose ChIP Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

| Western blot
Total proteins were isolated from H9C2 cells using RIPA buffer Abcam) at 25°C for 1 hour. The intensity of bands was analysed using ChemiDoc MP imaging system (BIO-RAD).

| Statistical analysis
Statistics were calculated using SPSS 17.0 (Chicago, IL, USA), and all data were presented as mean ± standard deviation (SD).
Statistical analysis between groups or among groups was analysed using Student's t test or one-way ANOVA followed by Bonferroni post hoc test. P value < .05 was considered statistically significant.

| Catechin improved heart function of myocardial ischaemia/reperfusion (MI/R) rat and down-regulated lncRNA MIAT expression in myocardial tissue
According to the analysis of data from echocardiography, we   Figure 2C).
Next, we further investigated whether MIAT was involved in protection of myocardial cell injury mediated by catechin. As described above, compared with the control group, MIAT was obviously increased ( Figure 2D), cell activity was obviously decreased ( Figure 2E) and apoptosis was obviously increased in the H/R group ( Figure 2F  increased after mutation of binding sites 1 + 2 than mutation of binding site 1, and the luciferase activity was significantly increased after mutation of binding sites 1 + 2 + 3 + 4 than mutation of binding sites 1 + 2 + 3, indicating binding sites 2 and 4 play important roles in the interaction of CREB with MIAT promoter region ( Figure 3C). CREB occupancy on the MIAT promoter was detected by ChIP ( Figure 3D), indicating that CREB could bind to binding sites 2 and 4. We divided H9C2 cells into four groups: H/R group, H/R+Catechin group, H/R+Catechin+si-NC and H/R+Catechin+si-CREB group. We found that the CREB protein was significantly increased and the MIAT was significantly decreased in the H/R+Catechin group compared with the H/R group ( Figure 3D,E).
However, after transfection of si-CREB, CREB protein was significantly reduced while MIAT was significantly elevated (Figure 3D,E).

| Catechin increased myocardial mitochondrial membrane potential (MMP), decreased intracellular ROS level and cytochrome c level under H/R condition, and lncRNA MIAT overexpression abolished the effects of catechin
As shown in Figure 4A Figure 4B). Protein levels of Bax, Bcl-2, cleaved F I G U R E 3 Catechin suppressed lncRNA MIAT expression through up-regulating CREB. A, H9C2 cells were divided into control group, H/R group and H/R+Catechin group. CREB protein level was detected by Western blot. **P < .01 vs control; #P < .05 vs H/R. B, H9C2 cells were divided into si-NC, si-CREB, pcDNA-NC and pcDNA-CREB groups. qRT-PCR was used to detect MIAT expression. **P < .01 vs si-NC or pcDNA-NC. C, Wild-type MIAT promoter luciferase (Luc) and mutant MIAT promoter luciferase were constructed, and they cotransfected into HEK293T cells with pcDNA-NC and pcDNA-CREB. The luciferase activity was detected by dual-luciferase reporter gene assay. *P < .05; **P < .01 vs pcDNA-NC. D, CREB occupancy on the MIAT promoter was detected by ChIP assay. IgG was used as negative control, H3 was used as positive control. H9C2 cells were divided into H/R group, H/R+Catechin group, H/R+Catechin+si-NC group and H/R+Catechin+si-CREB group. E, CREB protein level was detected by Western blot. F, MIAT expression was detected by qRT-PCR. **P < .01, *P < .05 vs H/R; ##P < .01 vs H/R+Catechin+si-NC. N = 3 caspase-3, cleaved caspase-9, cytosolic and mitochondrial cytochrome c were also detected, and results showed that catechin

| Catechin improved mitochondrial function and relieved apoptosis of myocardial cells through promoting Akt/Gsk-3β activation under H/R condition
Under H/R condition, catechin significantly increased pAKT/AKT ratio and pGsk-3β/Gsk-3β ratio ( Figure 5A), indicating catechin promoted Akt/Gsk-3β activation. To observe whether catechin exerted its protective role in myocardial cells through promoting Akt/Gsk-3β activation, LY294002 (a potent PI3K inhibitor) was used to treat H9C2 cells. We found that under H/R and catechin treatment, LY294002 significantly inhibited cell viability of H9C2 cells ( Figure 5B), promoted the apoptosis of H9C2 cells ( Figure 5C), inhibited MMP in H9C2 cells ( Figure 5D), increased intracellular ROS level ( Figure 5E) and up-regulated cytosolic and mitochondrial cytochrome c protein levels ( Figure 5F). These findings indicated that under H/R condition, catechin improved mitochondrial function and reduced apoptosis of H9C2 cells through promoting Akt/Gsk-3β activation.

| MIAT inhibited Akt/Gsk-3β activation and promoted the apoptosis of myocardial cells under H/R condition
As shown in Figure 6A

| D ISCUSS I ON
LncRNAs are the cellular responding molecules for bioactive polyphenol treatment in various diseases. For example, studies showed that lncRNAs are dysregulated after EGCG treatment in lung cancer, nasopharyngeal carcinoma, prostatic hyperplasia, osteoarthritis, etc [33][34][35] However, whether lncRNAs mediated the protective role of catechin, a kind of bioactive polyphenol that has a variety of pharmacological and biological properties and plays a protective role in MI/R injury, 14 against H/R-induced myocardial cell apoptosis or MI/R injury is largely unknown. In this study, we found that lncRNA MIAT expression was down-regulated after catechin treatment in myocardial Recently, studies have shown that abnormally expressed lncRNAs were closely related with mitochondrial dysfunction. 39 For example, lncRNA HOTAIR knockdown increased ROS production and caused mitochondrial swelling in HeLa cells, indicating lncRNA HOTAIR knockdown led to mitochondrial dysfunction. 40  Akt/Gsk-3β pathway has been shown to be involved in modulating oxidative stress and apoptosis, and the activation of Akt/Gsk-3β pathway preserved heart function and alleviated MI/R injury. 45,46 Activation of Akt/Gsk-3β pathway also inhibited the apoptosis and oxidative stress of H/R-induced H9C2 cells. 47,48 Importantly, the activation of Akt/Gsk-3β pathway is involved in the cardioprotection function of EGCG. 49 In this study, we clarified that the Akt/Gsk-3β pathway was activated by catechin and this activation was mediated by MIAT down-regulation in H/R-induced H9C2 cells. To the best of our knowledge, no other studies figured out catechin improved mitochondrial function and reduced the apoptosis of H/R-induced H9C2 cells through regulating lncRNA MIAT/Akt/Gsk-3β pathway, which revealed the underlying mechanism of catechin in protecting against H/R-induced myocardial cell apoptosis.
In conclusion, this study showed that catechin relieved H/Rinduced myocardial cell apoptosis through regulating CREB/lncRNA MIAT/Akt/Gsk-3β pathway, which clarified the underlying mechanism of catechin in inhibiting H/R-induced myocardial cell apoptosis, and might provide potential targets for relieving MI/R injury.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
For some reasons, the findings of this study are available do not support availability.