Statin and resveratrol in combination induces cardioprotection against myocardial infarction in hypercholesterolemic rat

Abstract

Hypercholesterolemia (HC) is a common health problem that significantly increases risk of cardiovascular disease. Both statin (S) and resveratrol (R) demonstrated cardioprotection through nitric oxide-dependent mechanism. Therefore, the present study was undertaken to determine whether combination therapy with statin and resveratrol is more cardioprotective than individual treatment groups in ischemic rat heart model. The rats were fed with 2% high cholesterol diet and after 8 weeks of high cholesterol diet the animals were treated with statin (1 mg/kg bw/day) and resveratrol (20 mg/kg bw/day) for 2 weeks. The rats were assigned to: (1) Control (C), (2) HC, (3) HCR, (4) HCS and (5) HCRS. The hearts, subjected to 30-min global ischemia followed by 120-min reperfusion were used as experimental model. The left ventricular functional recovery (+dp/dt(max)) was found to be significantly better in the HCRS (1926+/-43), HCR (1556+/-65) and HCS (1635+/-40) compared to HC group (1127+/-16). The infarct sizes in the HCRS, HCS and HCR groups were 37+/-3.6, 43+/-3.3 and 44+/-4.2 respectively compared to 53+/-4.6 in HC. The lipid level was found to be decreased in all the treatment groups when compared to HC more significantly in HCS and HCRS groups when compared to HCR. Increased phosphorylation of Akt and eNOS was also observed in all the treatment groups resulting in decreased extent of cardiomyocyte apoptosis but the extent of reduction in apoptosis was more significant in HCRS group compared to all other groups. In vivo rat myocardial infarction (MI) model subjected to 1 week of permanent left descending coronary artery (LAD) occlusion documented increased capillary density in HCR and HCRS treated group when compared to HCS treatment group. We also documented increased beta-catenin translocation and increased VEGF mRNA expression in all treatment groups. Thus, we conclude that the acute as well as chronic protection afforded by combination treatment with statin and resveratrol may be due to pro-angiogenic, anti-hyperlipidemic and anti-apoptotic effects and long-term effects may be caused by increased neo-vascularization of the MI zone leading to less ventricular remodeling.

Figure 1

A–1E. Post ischemic ventricular recovery of resveratrol and statin treatment in hypercholesterol rats compared to control. The results (LVDP-1A, heart rate-1B, +dp/dt-1C, aortic flow-1D, coronary flow-1E) are shown in six animals per group. *P<0.05 compared to control, †P<0.05 compared to hypercholesterol group, #P<0.05 compared to HCRS group. Blank bar represents control group. Dark upward diagonal bars represent Hypercholesterol group. Dashed horizontal bars represent resveratrol treated group. Black bar represents statin treated group. Dark horizontal lines represent combination group.

Figure 2

Infarct size of the hearts, expressed as a percentage of the area at risk in rat myocardium subjected to 30 min of ischemia followed by 2 hours of reperfusion. Results are expressed as mean ± Std. error of six hearts per group. *P<0.05 compared with control, †P<0.05 compared with HC group, #P<0.05 compared with HCRS group.

Figure 3

Cardiomyocyte apoptosis by TUNEL assay. Representative pictures show immunohistochemical staining of extended DNA. A: shows the extent of cardiomyocyte apoptosis between the comparative groups. B: shows the graphical representation of cardiomyocyte apoptosis. Data were expressed as a percentage of the total particular cell nuclei on counts per 100 high power fields. Values are mean ± Std. error (n=6). *P<0.05 compared to control, †P<0.05 compared to HC group, #P<0.05 compared to HCRS group. 100 hpf - represents 100X high power field.

Figure 4

Representative Western blot showing the protein expression (A) Phosphorylated AKT and non-phosphorylated AKT. *P<0.05 compared to control, †P<0.05 compared to HCRS group (B) Phosphorylated eNOS and non-phosphorylated eNOS. *P<0.05 compared to control, †P<0.05 compared to HCRS group.

Figure 5

Representative immunohistochemical staining for β-catenin. Nuclei were counterstained with hematoxylin. Arrows indicate nuclear translocation of β-catenin between the comparative groups.

Figure 6

Graph represents Real Time RT-PCR analysis for VEGF mRNA between the comparative groups. *P<0.05 compared to control, †P<0.05 compared to hypercholesterol group, #P<0.05 compared to HCRS group.

Figure 7

Effect of resveratrol and statin on capillary density. Quantitative analysis of capillary density in the peri-infarct area 7 days after Myocaridal Infarction. Results are expressed as mean ± Std. error of six hearts per group. *P<0.05 compared to control, †P<0.05 compared to hypercholesterol group, # P<0.05 compared to HCRS group.