| Myocardial infarction (MI) is one of the most life-threatening cardiovasvular diseases. Today, it remains a suspending problem to improve survival of patients after acute MI, although various therapies have been applied to control mortality soon after MI attacks. The classic appreciation of MI risk stratification was limited to depressed ventricular function, long-standing complex ventricular arrhythmias, and the extent of coronary atherosclerosis. Arterial baroreflex (ABR) is one of the most important self-regulatory mechanisms of the cardiovascular system. Its function may be expressed as baroreflex sensitivity (BRS), and can be abolished by sinoaortic denervation (SAD). Recently, many studies focused on the relationship between the ABR dysfunction and the pathogenesis and poor prognosis of many cardiovascular diseases, such as chronic heart failure, stroke, endotoxic shock, ventricular arrhythmia, and atherosclerosis. In 1988, the research teams of Schwartz and La Rovere reported that BRS was closely correlated with mortality after acute MI in patients and dogs. The mortality was increased in MI patients or dogs with a low ABR function. Thus, we speculate, improving ABR function might be a new target to improve the outcome of MI. However, the mechanism underlying the relationship between impaired ABR and increased post-MI mortality remains unclear.It was reported that nicotine may stimulate proliferation of tumor cells and angiogenesis of endothelial cells viaα7 subunit of nicotinic acetylcholine receptors (α7-nAChR). However, the link between ABR andα7-nAChR and the contribution of endogenous ACh to angiogenesis in ischemic heart remain unknown. Ketanserin is an antihypertensive drug with affinity for both 5-HT2A andα1 receptors, and a small dose of ketanserin could improve BRS in spontaneously hypertensive rats (SHR) without blood pressure reduction. It will be interesting to know whether restoration of impaired baroreflex with ketanserin could improve the angiogenesis and cardiac function in ischemic heart. The present study was designed first to test whether ABR dysfunction might attenuate the angiogenesis in ischemic heart in rats, and then investigate the underlying mechanisms. We proposed a novel pathway,"vagus-ACh-α7-nAChR", to explain the angiogenesis involved in the influence of ABR function on prognosis of MI. Ketanserin was used to improve ABR function of rats and further to ameliorate prognosis of MI through enhancement of angiogenesis, a new possible drug target for improving prognosis of MI.Experiment 1: Influence of BRS on survival time of MI rats. Five weeks after ligature of the coronary artery of SD rats, BRS was determined. Totally 52 rats were divided into two groups according to the mean BRS value (0.60 ms/mmHg). The survival time within 500 days after BRS measurement was recorded and analyzed. The mortality in rats with BRS<0.60 ms/mmHg was significantly higher than that in rats with BRS >0.60 ms/mmHg. There existed an obvious difference in survival time between two groups, which was expressed by Kaplan–Meier survival curves; Time needed for half of the rats to die was longer in group BRS–high than that in group BRS–low; BRS in the <250-day group was significantly lower than that in the >250-day group when compared according to the survival time; Result of univariate regression analysis showed that the survival time was positively correlated with BRS.Experiment 2: Hemodynamics and cardiac vagal activity in SAD rats. Two weeks after SAD operation, blood pressure (BP) and heart rate (HR) were recorded. Cardiac vagal tone was evaluated by detection of the increase in HR caused by atropine sulfate (0.03 mg/kg, iv). Then coronary artery ligature was performed one week later. Expression of vesicular acetylcholine transporter (VAChT) in tissue membrane proteins in involved area of myocardium was detected by western blotting in rats one week after MI operation or those without MI operation. There was no difference in BP and HR between two groups; BRS and increase in HR caused by atropine were significantly lower in SAD rats than those in sham-operated rats; SAD rats, either received MI operation or not, expressed less VAChT in myocardium.Experiment 3: Influence of ABR dysfunction on angiogenesis in MI rats. Two weeks after SAD operation, coronary artery ligature was performed. Angiogenesis was determined through detection of capillary density in involved area of myocardium two weeks after MI operation by immunohistochemistry. Expression of VEGF in tissue whole proteins in involved area of myocardium was detected by ELISA. Capillary density in SAD rats was 64.4% of that in sham-operated ones. Results of ELISA showed that VEGF levels in SAD rats were significantly lower than that in sham-operated rats (1st, 3rd, 7th day after MI). Since the most predominant difference appeared on the 3rd day after MI, VEGF content on the 3rd day after MI was validated by western blotting, and similar result was obtained.Experiment 4: Involvementα7-nAChR in angiogenesis in vivo. Expression ofα7-nAChR in tissue membrane proteins in involved area of myocardium, as mentioned above, was detected one week after MI operation by western blotting, and was significantly lower in SAD rats compared with that in sham-operated ones. Capillary density in involved area of myocardium was detected two weeks afterα7-nAChR–/– mice and wild type mice underwent MI operation. Results of immunohistochemistry presented attenuated angiogenesis in involved area of myocardium inα7-nAChR–/– mice.Experiment 5: Effects of acetylcholine (ACh) chloride on functions of cardiac microvascular endothelial cells (CMECs). (1) Effect on secretion of VEGF in CMECs: Results of ELISA showed that ACh chloride promoted secretion of VEGF dose-dependently (1-1000 nM). (2) Effect on phosphorylation of VEGFR2 in CMECs: Results of western blotting showed that ACh chloride stimulated phosphorylation of VEGFR2 time-dependently (0-60 min). (3) Effect on migration of CMECs: ACh chloride induced tube formation of CMECs in vitro dose-dependently (1-1000 nM). When pretreated with antagonists, compared with control group, mecamylamine and methyllycaconitine inhibited the above-mentioned effect of ACh chloride significantly, but atropine had no effect.Experiment 6: Signaling pathways involved in ACh chloride-induced angiogenesis of CMECs. Results of western blotting showed that ACh chloride stimulated phosphorylation of Src, Akt and ERK1/2 time-dependently (0-60 min), and promoted expression of HIF-1αdose-dependently (1-1000 nM). Results of ELISA showed that inhibitors for Src, PKC, MEK, PI3K or mTOR can all inhibit ACh chloride-induced secretion of VEGF.Experiment 7: Effects of ketanserin on angiogenesis and heart function in SHR. Ketanserin was daily administrated to SHR in a dose of 0.3 mg/kg in food to improve ABR function. Similar experiments to those on SAD rats were done. In addition, ejection fraction (EF) of heart was evaluated through echocardiography 3 months after MI operation. EF≤45% was set as standard for successful MI operation.(1) Effect on hemodynamics and cardiac vagal activity: BRS and increase in HR caused by atropine were significantly higher in SHR treated with ketanserin than those in control group. And there was no difference in BP and HR between two groups. SHR treated with ketanserin, either received MI operation or not, expressed more VAChT in myocardium.(2) Effect on post-MI angiogenesis: Results of immunohistochemistry presented enhanced angiogenesis in involved area of myocardium in SHR treated with ketanserin. Results of ELISA and western blotting showed that expression of VEGF in SHR treated with ketanserin on the 3rd day after MI was significantly higher than that in control group.(3) Effect on expression ofα7-nAChR and heart function: Compared with control group, SHR treated with ketanserin presented much moreα7-nAChR expression in ischemic myocardium. Improved EF of infracted hearts was also observed in SHR treated with ketanserin 3 months after MI operation.In conclusion, our results demonstrate, for the first time, that ABR dysfunction attenuates angiogenesis in rat ischemic heart via the vagus-ACh-α7-nAChR pathway. These findings may provide a mechanistic basis for restoring baroreflex function that leads to increased angiogenesis in ischemic heart as a novel strategy in the treatment of MI.
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