Regulatory Effects Of Salvianic Acid A On The Ion Channels And Contractility In Myocytes

Objectives: L-type Ca2+ channels(LTCCs) are mediators and regulators of Ca2+ influx and are pivotal in the function and dysfunction of cardiac myocytes. LTCCs are located in sarcolemma and are activated by membrane depolarization, but intracellular Ca2+-dependent inactivation limits Ca2+ influx during action potential. L-type calcium current(ICa-L) is important in heart function because it triggers excitation–contraction coupling, modulates action potential shape and is involved in cardiac arrhythmia. The inhibitory effects of calcium channel blockers on LTCCs can result in decrease of Ca2+ influx and the intracellular calcium, reduction of cardiac contractility and reduction of the myocardial oxygen consumption.The slowly activating delayed rectifier potassium current(IKs) and the rapidly activating delayed rectifier potassium current(IKr) play an important roles in the repolarization of human cardiac action potential. Loss or reduction of IKs and IKr channels function, due to genetic mutations in the genes or electrical remodeling in diseased heart, breaks down the balance of the channels, thus induces a decrease in the K+ outflow and net repolarizing current. The reduced repolarization reserve results in delayed repolarization, lengthened action potential duration and prolongation of QT interval in the electrocardiogram accompany with the malignant arrhythmia syncope and the sudden death. Inhibitors of IKs and IKr may lengthen QT interval and induce the arrhythmia.Salvia miltiorrhiza has been used in clinical therapy in China for thousands of years, which is widely used in the treatment of hypertension, coronary heart disease, myocardial infarction, blood circulation diseases, and other cardiovascular diseases. More and more studies showed that salvianolic acid A(SAA), the main water-soluble component in S. miltiorrhiza, has a variety of pharmacological effects, including inflammation and tumor inhibition, neuro and myocardial protection, immunity improvement, etc. In the early stage of our work, we found that salvia miltiorrhiza injection could block LTCCs. SAA, as one of the main components in salvia miltiorrhiza injection, may be the material basis for salvia miltiorrhiza injection exerting cardioprotective effects via inhibition of LTCCs.The aim of the study is to discuss the cardioprotection mechanism of SAA. The patch clamp technique is used to observe the regulatory effects of SAA on LTCCs in rat ventricular myocytes and IKs, IKr expressed in HEK293. Ion Optix is used to observe contractility in rat ventricular myocytes.Methods:(1) Isolation of adult rat ventricular myocytes The adult male Sprague-Dawley rats were anesthetized, enzyme digestion was performed on the rapidly excised rat heart on a Langendorff apparatus with with Ca2+-free Tyrode’s solution containing 0.02% collagenase II at 37°C. Finally, the left ventricles were removed and placed in a beaker filled with Kreb’s(KB) solution and minced. Single myocyte was harvested after filtration through a nylonmesh(pore size 200 μm) and stored in Kreb’s solution(at room temperature) for at least 1 h before gradually increasing the concentration of Ca2+ in the KB solution to 1.8 m M before the experiment. After inducing cardiac ischemia through injecting pituitrin intravenously by tail vein within 10 min, experiments with rat ischemic ventricular myocytes were performed, the heart was removed and used for experiments as normal rat ventricular myocytes above. All experiments were implemented within 6 h after isolation. The whole-cell patch clamp technique is used to record the effects of SAA on ICa-L and its kinetic process.(2) Cell culture HEK 293 cells were cultured at 37 °C, 5% CO2 and 95% air in Dulbecco’s Modified Eagle’s Medium(DMEM, Invitrogen) supplemented with 10% fetal bovine serum and subcultured every 1–2 days. HEK 293 cells require lipfectin transfection to express human hearts KCNQ1/KCNE1(IKs) potassium channels. HEK 293 cells expressing IKr can be directly used in the patch clamp experiment. The perforated patch clamp technique is used to record the effects of SAA on IKs and IKr and their kinetic process.(3) Measurements of cell contractions The heart of the adult male Sprague-Dawley rat was rapidly excised, single myocyte was harvested through enzyme digestion. All experiments were implemented within 6 h after isolation. In addition, myocyte calcium and contractility systems were used to measured contractility in isolated adult rat myocytes.Results:1 Confirmation of ICa-LICa-L was elicited by different levels of test pulses between-60 m V and +60 m V in 10 m V step from a holding potential of-80 m V. As a specific LTCC blocker, Ver almost completely blocked the currents(P < 0.05, compared with control).2 Reversible effects of SAA on ICa-LSAA decreased the Ca2+ current effectively with the inhibition rate of 35.5 ± 0.9%(P < 0.05, compared with control). However, the Ca2+ current recovered nearly to the level of the control by washout(19.8 ± 2.7%)(n = 5).3 Dose-dependent effects of SAA on ICa-LICa-L was progressively suppressed by increasing concentrations of SAA. The inhibition rates of 3×10-6, 10-5, 3×10-5, 10-4 and 3×10-4 M SAA were 9.5 ± 0.9%, 21.0 ± 2.1%, 29.6 ± 1.8%, 34.6 ± 1.9% and 39.5 ± 1.9%, respectively(n = 5).4 Effects of SAA on current-voltage relationship of ICa-LThe current-voltage curves shifted upward with a significant increase in the application of SAA, indicating SAA inhibited ICa-L in a dose-dependent manner. However, activated potential, peak potential and reversal potential of ICa-L were not significantly changed.5 Effects of SAA on steady-state activation and inactivation of ICa-LSAA did not alter the activation and inactivation gating properties of the cardiac Ca2+ channel. Values at V1/2 and the slope factor(k) of the normalized activation conductance curves after application of 0, 3×10-6, 3×10-5, 3×10-4 M SAA were-10.40 ± 0.64 m V/7.03 ± 0.58,-9.53 ± 0.69 m V/6.99 ± 0.62,-8.16 ± 0.69 m V/6.99 ± 0.61,-8.61 ± 0.69 m V/6.88 ± 0.61, respectively(P > 0.05, n = 5). Values at V1/2 and the slope factor(k) of the normalized inactivation conductance curves after application of 0, 3×10-6, 3×10-5, 3×10-4 M SAA were-32.03 ± 0.15 m V/4.62 ± 0.13,-31.34 ± 0.08 m V/ 4.84 ± 0.08,-30.75 ± 0.01 m V/4.98 ± 0.01,-30.94 ± 0.02 m V/5.08 ± 0.02, respectively(P > 0.05, n = 5).6 Effects of SAA on ICa-L of ischemic ventricular myocytesThe peak amplitude of ICa-L was decreased by 35.2 ± 0.5% by SAA derivatives at 3×10-4 M(P < 0.05,n = 5). The results were consistent with the normal cells.7 Effects of SAA on IKsSAA at 3×10-4 M had no significant effects on the expressed IKs at all test potentials 2.68 ± 0.9%(P = NS, n = 6).8 Effects of SAA on IKrSAA at 3×10-4 M had no significant effects on the expressed IKr at all test potentials 3.80 ± 0.5%(P = NS, n = 6).9 The inhibition effects of SAA on cell shorteningThe results indicate that SAA at the concentration of 3×10-7 M and 10-6 M could significantly inhibit cell shortening by 33.48 ± 0.75% and 92.98 ± 0.48%, respectively(P < 0.05, compared with control).Conclusions:In this study, we focus on the mechanism of SAA on cardiovascular protection and provide a theoretical basis for the further study and clinical application of SAA. This study systematically demonstrated the significant concentration-dependent effects of SAA on adult rat cardiac myocytes. We provide evidence to support that SAA protects the heart by inhibiting ICa-L and decreasing myocardial contractility, thereby reduces the myocardial oxygen consumption. Meanwhile, SAA at 3×10-4 M had little effects on the expressed IKs and IKr at all test potentials, thus means SAA may exert cardioprotective effects without causing drug-induced LQTS.