Protective Effects Of Iptakalim And Natakalim On Myocardium And Their Possible Mechanisms

Cardiovascular disease (CVD) remains the principle cause of death in both developed and developing countries, accounting for roughly 20% of all worldwide deaths per year. Some experts claimed that now the outbreak of cardiovascular disease in China is in the "window period". If people do not take effective measures, cardiovascular disease in China will be pandemic in the next 10 years. Now, most of drugs used for treatment of coronary heart disease are anti-ischemic drugs, but sometimes their cardioprotective effects were not very good, and often accompanied by various side effects. Therefore, new anti-ischemic drugs with good effects, targeted strong, and fewer side effects are needed.Early studies have shown that ATP sensitive potassium channel (K_ATP) is an important target against myocardial ischemia and hypoxia. Not only different K_ATP in different organizations play different functions, but their molecular structure, physiology, pathophysiology and pharmacological characters also are different in different organization. Thus, K_ATP opener (KCOs) which activate different subunits will produce different pharmacological effects. Although many researchers believe that K_ATP opener plays cardioprotective effects by reducing necrosis, promote functional recovery after myocardial ischemia, inhibition of myocardial contraction and reduce the ATP hydrolysis, but the mechanism has not yet been fully elucidated. However, the role of mitochondria in cardioprotection attained people's concern and attention in recent years, mitochondrial K_ATP (mitoK_ATP) has become a new target Iptakalim (Ipt) is a new ATP-sensitive potassium channel opener. Previous study in our group showed that: it is a membrane K_ATP opener, and its antihypertensive effect and cardiovascular activity can be against by glibenclamide, a K_ATP antagonist that can be combined with the K_ATP opener specific binding sites; This showed that iptakalim activated membrane K_ATP to protect cells. Another study showed that iptakalim can antagonize H2O2 induced oxidative stress injury of PC12 cells. The protective effect may be related to mitochondrial ATP-sensitive potassium channels. Iptakalim shows selective action on Kir6.1/SUR2B and Kir6.2/SUR2A, but not Kir6.2/SUR1 K_ATP channels. And the selective activity of iptakalim on SUR2B/Kir6.1 subtype of K_ATP channels is 10 times stronger than that on SUR2A/Kir6.2 subtype. Another research demonstrated that iptakalim could antagonize the oxidative stress injury induced by hydrogen peroxide in PC12 cells and that this protective effect of iptakalim might relate to mitochondrial ATP-sensitive potassium channel.However, whether iptakalim can protect cadiocyte from suffering oxidative stress injury and what could be the possible mechanism of this protetive effect have not been reported. Natakalim, a derivative of iptakalim but different from iptakalim, is a SUR2B/Kir6.1 subtype of ATP–sensitive potassium channel selective opener.But the real mechanism by which natakalim protect myocardial cell is still unclear.Therefore, to further study mechanisms of iptakalim and natakalim this study choose diazoxide as a positive control drug and esTab.lished oxidative stress injury model of myocardial cells after myocardial ischemia-reperfusion injury by H2O2; and through the determination of myocardial mitochondrial respiratory function and matrix volume changes, we understood clear pharmacological characteristics of the two drugs. This study will help to find drugs which are tissue selectivity, low side effects, and clear protective effect.Results:1,The protective effects of iptakalim and natakalim on neonate rat cardiac oxidative stress injury The protective effects of iptakalim and natakalim on cells could effectively decrease plasma membrane permeability, decreasing the outleakage of lactate dehydrogenase, with enhanced activity of SOD, effectively enhanced anti-oxidative stress capability and increased survival rate of cells after being injured. Furthermore, the protective effects of these two drugs on cells all could be abolished by 5-HD,a specific blocker of mitochondrial ATP-sensitive potassium channel, and glibenclamide, a blocker of ATP-sensitive potassium channel, indicating that both plasmalemma ATP-sensitive potassium channel and mitochondrial ATP-sensitive potassium channel were involved in the protective effects of iptakalim and natakalim on cells.2,Effects of iptakalim and natakalim on respiratory functions of mitochondria isolated from rat cardiac muscle(1)Effect of iptakalim on the respiratory function of rat cardiac mitochondria①Iptakalim had no influence on R4,R3 and RCR of NADH respiratory chain. But it could increase P/O of rat cardiac mitochondria;②As for FAD respiratory chain, iptakalim could accelerate its R4 respiratory rate, leading to decreased RCR which indicated a mild uncoupling effect. Diazoxide had the similar uncoupling effect as iptakalim did. But the uncoupling effect of iptakalim was more obvious than that of diazoxide (P<0.05). The effect of iptakalim on FAD respiratory chain could be abolished by 5-HD, a specific blocker of mitochondrial ATP-sensitive potassium channel, indicating the involvement of mitoK_ATP opening in the effect of iptakalim on FAD respiratory chain;③Iptakalim could decrease the light absorbance of cardiac mitochondria at 540nm, and its effect could be abolished by 5-HD, indicating that this effect of iptakalim was induced by opening mitoK_ATP channel. Diazoxide could also decrease the light absorbance of cardiac mitochondria at 540nm, and its effect could be abolished by 5-HD, verifying that diazoxide is a mitoK_ATP channel opener.(2)Effect of natakalim on the respiratory function of cardiac mitochondria①The effect of natakalim on the respiratory function of cardiac mitochondria was different from that of iptakalim. Natakalim had no significant influence on R3 of NADH respiratory chain. But it could enhance R4, leading to decreased RCR. Furthermore, this mild uncoupling effect induced by natakalim could be abolished by 5-HD. Natakalim had no significant influence on P/O of cardiac mitochondria;②Natakalim could accelerate R4 respiratory rate of FAD respiratory chain, leading to decreased RCR which indicated a mild uncoupling effect. Furthermore, the effect of natakalim on FAD respiratory chain could be abolished by 5-HD;③Natakalim could decrease the light absorbance of cardiac mitochondria at 540nm, and its effect could be abolished by 5-HD, indicating that this effect of natakalim was induced by opening mitoK_ATP channel.From these observations, we came to conclusions as follow:Iptakalim and natakalim all could prevent cadiocyte from being injured by oxidative stress induced by hydrogen peroxide. And these protective effects of the two drugs could abolished by 5-HD and glibenclamide, a blocker of ATP-sensitive potassium channel. These results demonstrated that mitoK_ATP channel might involved in the anti-myocardial damage effects of iptakalim and natakalim.Iptakalim and natakalim, with different characteristics, all could influence the respiratory functions of mitochondria isolated from cadiocyte. Iptakalim could only enhance P/O of NADH respiratory chain. Natakalim could decrease the RCR of NADH respiratory chain. And this mild uncoupling effect could abolished by 5-HD, which demonstrated that mitoK_ATP channel might involved in it. As for FAD respiratory chain, iptakalim and natakalim all could induce mild uncoupling effects which could be abolished by 5-HD, indicating the role played by mitoK_ATP channel opening in these effects.Iptakalim and natakalim all could decrease the light absorbance of cardiac mitochondria at 540nm, and their effects could be abolished by 5-HD, indicating that the effects of iptakalim and natakalim were induced by opening mitoK_ATP channel.In summary, we demonstrated for the first time the protective effects of iptakalim and natakalim on cadiocyte oxidative stress injury induced by hydrogen peroxide in neonate rats and that mitoK_ATP channel might involved in these protective effects. Besides, we demonstrated for the first time that iptakalim and natakalim could regulate respiratory function of mitochondria isolated from rat cadiocyte and that 5-HD could block their effects,indicating the involvement of mitoK_ATP channel opening in these regulative effects.These findings provided experimental evidences to develop iptakalim and natakalim into new myocardial ischemic antagonists with good curative effect, high selectivity and less side effects.