Leonurine Research On The Role Of Ion Channels Of Rat Cardiac Myocytes

Leonurine (Leo) is the main plant alkaloid present in Chinese motherwort (also called Yi-Mu-Cao in the traditional Chinese medicine) for the treatment of dysmenorrhea, menoxenia and some other gynecological disorders in women for hundreds of years. Leo was found to possess a variety of pharmacological actions. Recently, pharmceutical preparations based on motherwort have also been used for the treatment of myocardial ischemia in China. However, the underlying mechanism remains elusive. In the present study, we investigate the effects of Leo on the ionic transport across cell membrane in single ventricular myocytes of adult Sprague-Dawley(SD) rats with the advanced patch-clamp technique. We found that Leo reversibly inhibited L-type Ca²⁺ current (I_(Ca),L). The inhibition was concentration- dependent (the IC₅₀ value of Leo was 18μM, at the holding potential of-40 mV). Leo up to 200μM had no significant effect on the fast-inactivating Na⁺ current (I_Na) and K⁺ current (I_K). Action potential duration (both APD 50 and APD 90) was decreased by 20 and 100μM Leo. Among these findings, the inhibitory effect on the calcium channel is of our great interest since those blockers have well-known cardioprotective effect. We further explore the mechanisms underlying this calcium blocking phenomenon. Two reasons may be attributed to the reduced calcium current. One is the effect of Leo on the channel kinetics of calcium channel, the other is the effect of Leo on the amount of calcium channel protein.In the presence of 20 and 100μM Leo, both the activation and steady-state inactivation curves of I_(Ca),L were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I_(Ca),LL from inactivation state was delayed. The results suggest that the channel kinetics play a role to reduce the calcium current by Leo. We then study whether the mRNA and protein levels forα_1C subunits of the L-type calcium channel are controlled by Leo. Reverse transcription- polymerase chain reaction (RT-PCR) and Western blot assays were used to determine the transcription and protein expression levels of these genes in cultured neonatal rat cardiac myocytes. In Leo (5,10, 20, 50, 100 and 200μM)-treated myocytes, transcription of genes encoding theα_1C subunits was reduced. The decreases in transcription level forα_1C subunits genes were 11.9% , 28.4% , 47.8% , 50.8% , 47.8% and 46.3% of control, respectively. Pretreatment with Leo (5, 10, 20, 50, 100 and 200μM) also blocked the protein expression ofα_1C subunit. The decreases in protein level were 8.4% , 28.2% , 47.9% , 60.1% , 66.2% and 59.2% of control, respectively. These results suggest that Leo significantly affect transcriptional and translational regulation of the L-type calcium channel in myocyte. The expression ofα_1C subunits is under coordinate transcriptional control.Our conclusion is Leo exerts the cardioprotective effect by inhibiting the L-type calcium current in myocyte, at least in part, through the mechanism of affecting calcium channel kinetics and protein expression.