Nicorandil alleviates apoptosis in diabetic cardiomyopathy through PI3K/Akt pathway

Abstract Nicorandil exerts myocardial protection through its antihypoxia and antioxidant effects. Here, we investigated whether it plays an anti‐apoptotic role in diabetic cardiomyopathy. Sprague‐Dawley rats were fed with high‐fat diet; then single intraperitoneal injection of streptozotocin was performed. Rats with fasting blood glucose (FBG) higher than 11.1 mmol/L were selected as models. Eight weeks after the models were built, rats were treated with nicorandil (7.5 mg/kg day and 15 mg/kg day respectively) for 4 weeks. H9c2 cardiomyocytes were treated with nicorandil and then stimulated with high glucose (33.3 mmol/L). TUNEL assay and level of bcl‐2, bax and caspase‐3 were measured. 5‐HD was used to inhibit nicorandil. Also, PI3K inhibitor (Miltefosine) and mTOR inhibitor (rapamycin) were used to inhibit PI3K/Akt pathway. The results revealed that nicorandil (both 7.5 mg/kg day and 15mg/kg day) treatment can increase the level of NO in the serum and eNOS in the heart of diabetic rats compared with the untreated diabetic group. Nicorandil can also improve relieve cardiac dysfunction and reduce the level of apoptosis. In vitro experiments, nicorandil (100 µmol) can attenuate the level of apoptosis stimulated by high glucose significantly in H9C2 cardiomyocyte compared with the untreated group. The effect of nicorandil on apoptosis was blocked by 5‐HD, and it was accompanied with inhibition of the phosphorylation of PI3K, Akt, eNOS, and mTOR. After inhibition of PI3K/Akt pathway, the protective effect of nicorandil is restrained. These results verified that as a NO donor, nicorandil can also inhibit apoptosis in diabetic cardiomyopathy which is mediated by PI3K/Akt pathway.


| INTRODUC TI ON
The incidence of diabetes is increasing through the years. Its related complications have threatened the survival rate and life quality of diabetic patients. 1 Diabetes is a risk factor for cardiovascular disease and diabetic cardiomyopathy (DCM) is a major complication in diabetic patients. 2,3 Compromised left ventricular contraction or diastolic dysfunction and ventricular hypertrophy are the main features of DCM, independent of hypertension, coronary syndrome and other diseases. 4 Increase of cardiomyocytic apoptosis and deposition of extracellular matrix protein, which are caused by hyperglycaemia and metabolic disturbance are the main pathological changes of heart in diabetes, leading to the development of DCM. 5,6 Myocardial apoptosis plays a vital role in pathogenesis of cardiovascular diseases in the diabetes. 7 It is known that PI3K/ Akt pathway can inhibit apoptosis. 8 When PI3K/Akt pathway is blocked in the heart of the diabetic rats, the protein expression of eNOS and mTOR which can regulate the level of apoptosis is reduced significantly. 9,10 Thus, exploring the underlying mechanism of how PI3K/Akt pathway is mediated and finding effective drugs which can target on cardiomyocyte apoptosis can alleviate the process of DCM.
Nicorandil is an antianginal agent that causes vasodilatation by dual action: one is releasing nitric oxide (NO) and the other is binding to ATP-dependent K channel and opening it. 11,12 Recent studies show that nicorandil can reduce the level of reactive oxygen species(ROS) in endothelial cells and regulate the PI3K/Akt pathway. [13][14][15] However, whether it can alleviate cardiomyocyte apoptosis in diabetic cardiomyopathy has not been reported yet.
In the present study, we investigated the myocardial protective effect of nicorandil on DCM in streptozotocin (STZ)-induced diabetic rats by adjusting the level of cardiac function and remodelling. The possible mechanism underlying the protective effect of nicorandil was also investigated in apoptotic levels of the rats' heart. The potential molecular mechanism was investigated in H9C2 cardiomyocyte.

| Animal
Sixty Sprague-Dawley rats (100-120 g) were randomly allocated into four groups (n = 15). All rats were kept with a light-dark cycles at 23°C. The control group were fed with the basal diet and the other three groups were fed with high-fat diet (HF diet; 16% fat and 0.30% cholesterol). Four weeks after the HF diet, we performed intraperitoneal insulin tolerance test (IPITT) and intraperitoneal glucose tolerance test (IPGTT) to indentify insulin-resistant rats. Single intraperitoneal injection of streptozotocin (STZ;40 mg/kg, solarbio, China) to rats with insulin resistance was performed to induce the diabetic models. Then we measured the fasting blood glucose (FBG) 7 days after the injection. Only rats with FBG ≥11.1 mmol/L were considered as a successful diabetic model. 16,17 After 8 weeks of high blood glucose, the rats were given nicorandil through drinking water at the concentration of 7.5 mg/kg day and 15 mg/kg day respectively.
To constantly administer the amount of nicorandil, the concentration of nicorandil in the drinking water was adjusted every 4 days along with as per the water intake volume. We performed IPITT and IPGTT and killed all rats 4 weeks later after nicorandil treatment. All experimental protocols were approved by the law of Shandong University Animal Care Committee.

| Cardiac function
Cardiac function of rats was measured by the Vevo 770 imaging system with the RMB710 transducer (VisualSonics, Toronto, Canada). The echocardiography parameters involved the left ventricular end-diastolic dimension (LVEDd), left ventricular ejection fraction(LVEF), peak E to peak A ratio(E/A), early (e') to late (a') diastolic velocity ratio(e/a), peak E to early (e') ratio(E/e') and the fractional shortening(FS).

| Histology staining
Hearts were fixed with the 4% paraformaldehyde and embedded with paraffin, which were sliced to 4 µm for haematoxylin and eosin (HE) staining. We performed masson's trichrome and sinus red to measure the level of fibrosis. We used the antibody of collagen III (novusbio, NB600-594SS), collagen I (novusbio, NBP1-30054) for immunohistochemistry. Briefly, primary antibody of collagen I and collagen III were used to incubate the sections overnight at 4°C overnight and then sections were washed with phosphate buffered saline (PBS) and incubated in secondary antibody for 30  After incubation, digoxigenin antibody buffer was added before DAB incubation. PBS was used to wash residual buffer one last time.

| Western blot
Protein from rat heart or cells was separated through 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, USA). Five percent non-fat milk was used to block the membrane for 1 hr at room temperature and then the protein on the membrane was incubated overnight with primary antibody at 4°C. Secondary antibodies were used for 1 hr at room temperature and enhanced chemiluminescence (Millipore, USA) was used for exposure via Amersham Imager 600 (General Electric Company, USA).We used the antibody against bcl-2(abcam, ab180665), bax(novusbio, NBP1-78977SS), cleaved caspase-3(abcam, ab216995), caspase-3(abcam, ab216995), collagenIII

| Statistical analysis
All analyses were carried out in Prism 6.0 (Graphpad) and SPSS 20.0. One-way ANOVA was used to compare the difference among groups and unpaired t-test was used for the difference between two groups. Each experiment was repeated at least three times and data were shown as means ± standard deviation (SD). Two-tailed P < 0.05 was treated as statistically significant.

| Basic characteristic of type 2 diabetic rats
We performed intraperitoneal insulin tolerance test (IPITT) and intraperitoneal glucose tolerance test (IPGTT) to rats after high-fat diet for 4 weeks. Insulin resistance occurred in the high-fat diet F I G U R E 1 Characteristic of type 2 diabetic rats. IPGTT(A) and IPITT(B) were performed after 4 weeks of high-fat diet. AUC were calculated in control and HF group. After nicorandil treatment for 4 weeks, IPGTT(C) and IPITT(D) were performed in four groups. AUC were calculated in 4 groups. *P < 0.05 compared with the control group (HF) group，the mean area under the receiver operating characteristic curve (AUC) increased significantly in HF group compared with the control group ( Figure 1). Then insulin-tolerant rats were injected intraperitoneally with STZ. IPITT and IPGTT experiments were performed among groups after treatment of nicorandil for 4 weeks. There was no significant difference in blood glucose between the nicorandil group and untreated DM group. The blood pressure of the untreated DM group appeared to be higher than that of the nicorandil group, but this difference did not reach statistical significance (Table 1).

| Nicorandil alleviated left ventricular hypertrophy and myocardial structural changes in rats with diabetes
Compared with the control group, the heart of rats with diabetes presented the characteristic of pathological hypertrophy, the ratio of heart weight to bodyweight increased, and the diameter of cardiomyocytes increased significantly. Compared with the untreated group, both treatments by nicorandil with the concentration of 7.5 mg/kg day and 15 mg/kg day can reduce the ratio of heart weight to bodyweight and alleviate ventricular hypertrophy (Figure 2A and 2). Also, the diameter of cardiomyocytes in rats decreased significantly after nicorandil treatment ( Figure 2C).

| Protective effects of nicorandil on heart contractile and diastolic dysfunction
Cardiac function in DM rats was examined by ultrasound.   Figure 5D).

| Nicorandil relieves apoptosis through PI3K/ AKT pathway
The phosphorylation level of the PI3K/AKT pathway was inhibited in the high-glucose group compared with the normal glucose group. Compared with the untreated HG group, the phosphorylation level of PI3K/AKT pathway protein was increased in the nicorandil treatment group, and the ratio of Bax/Bcl-2 and cleaved caspase-3/ caspase-3 was decreased. After blocking this pathway with PI3K inhibitor and mTOR inhibitor, nicorandil treatment was given, but the ratio of Bax/Bcl-2 and cleaved caspase-3/caspase-3 did not decrease significantly compared with the untreated group, indicating that this pathway plays an important role in nicorandil-induced reduction of apoptosis. The eNOS expression was also measured, the results showed that the ratio of p-eNOS/eNOS can increase after the nicorandil treatment and the PI3K inhibitor and mTOR inhibitor blocked this function to some degree. The TUNEL-positive cells is accordance with the expression of apoptosis related proteins (Figure 8). Nicorandil is a K+-ATP channel opener with a function of nitrate. 12

| D ISCUSS I ON
It has been proven that nicorandil has numerous protective effects on the cardiovascular system, including reducing the ventricular preload and afterload, improvement of myocardial perfusion, prevention of Ca2+ overload by opening K+-ATP channels, anti-inflammatory and anti-apoptosis effects. 24,25 Our study found that nicorandil can increase the level of nitric oxide in the serum of diabetic coronary heart disease (CHD) patients significantly (Appendix 1). However, whether nicorandil can directly play a role in the pathological process of diabetic cardiomyopathy has not been reported. In our study, type 2 diabetic models were established successfully and nicorandil was used on rats 8 weeks after high blood glucose. Dosage of 7.5 mg/kg day and 15 mg/kg day were used in our nicorandil-treated groups. We found that although nicorandil had no significant effect on blood glucose, it significantly improved myocardial remodelling by reducing the ratio of heart weight/ bodyweight and decreasing the transverse diameter of myocardial So we wondered the potential mechanism in the function of nicorandil on DCM. We measured the fibrosis and apoptosis level in the heart of four groups and found that after the treatment of nicorandil, the level of fibrosis and apoptosis in the heart decreased significantly compared with untreated diabetic group, indicating that nicorandil can alleviate the process of fibrosis and apoptosis in the heart of DM. We also measured the NO and ADMA levels in serum and found that nicorandil can promote the level of NO and reduce the ADMA in serum compared with the untreated diabetic rats. Also, the ratio of p-eNOS/eNOS increased in the nicorandil-treated group.
All results in vivo suggest that the nicorandil treatment and the Akt pathway was reduced after high-glucose stimulation, which is consistent with previous studies. Interestingly, when H9C2 cardiomyocyte was stimulated with high glucose, the phosphorylation level of this pathway was raised after nicorandil intervention. When we used PI3K/Akt pathway inhibitors on H9C2 cardiomyocyte, we examined the levels of apoptosis-related proteins and found that the anti-apoptotic effect of nicorandil disappeared after inhibition of the PI3K/Akt pathway.
In conclusion, it is more clear about the mechanism of how nicorandil protects high-glucose exposed cardiomyocytes from apoptosis. Nicorandil can produce NO which takes part in various actions to protect myocardial damage in diabetes. Moreover, we have also found that nicorandil can promote eNOS activity in the process of protecting cardiomyocytes. In the environment of high glucose, nicorandil can promote the phosphorylation levels of eNOS, mTOR which can be blocked in HG by activating the PI3K/Akt pathway. These proteins play an important role in inhibiting apoptosis and alleviating DCM.
However, it still requires further investigation to explore the specific mechanism of nicorandil in regulating the PI3K/Akt pathway.

| CON CLUS ION
In summary, nicorandil plays an important role in the progression of diabetic cardiomyopathy; it can directly relieve cardiac remodelling and improve cardiac function through inhibiting fibrosis and apoptosis in the heart of type 2 diabetic rats. Nicorandil can not only release NO directly but also promote the activity of eNOS and mTOR by increasing the phosphorylation of PI3K/Akt pathway. These mechanisms play an important role in the process of nicorandil alleviating apoptosis in cardiomyocyte caused by hyperglycaemia.

ACK N OWLED G EM ENTS
The authors thank Yun Zhang for his help in editing the manuscript.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.