| PrefaceEstrogen (E) is one kind of steroid hormone whose main component is estradiol (E2). Estradiol has a andβtwo types, andβtype shows strong biological activity. Numerous studies show that estrogen has high research value in the treatment of menopausal syndrome, anti-osteoporosis, prevention of cardiovascular disease and protective effect on the central nervous system in addition to regulation of reproductive system. Cardiovascular disease is the main reason for death in postmenopausal women. There are about 53% women who are more than 50 years old died of cardiovascular disease. Application of estrogen replacement therapy (ERT) for postmenopausal women can decrease the incidence of cardiovascular disease by 35%-50%, it suggest that estrogen has a protective effect against cardiovascular disease. Its protective mechanism is unclear. In recent years, it was discovered that estrogen may act directly on cardiac and vascular smooth muscle, resulting in negative inotropic effects, dilate blood vessels, and there is evidence that estrogen can inhibit calcium channel current.ERT can lead to the risk of tumor in breast, ovary and endometrium. Therefore, in-depth study of the role of estrogen on cardiovascular protection, to find ideal ERT drugs, and to minimize the side effects, it will play an important role for prevention and treatment of cardiovascular diseases. Therefore, we research the impact of 17β-estradiol(17P-E2) on L-type calcium channel current (ICa-L) of ventricular myocytes with patch-clamp technique. To analyze the protective effect and the mechanism of 17β-estradiol on cardiovascular from the electrophysiological point of view, and to provide experimental basis for the development of safe and effective female hormone replacement drugs and clinical treatment.Materials and Methods1. Animals and Groups Select healthy adult guinea pigs (300-400) g, female. Experiments were divided into five groups, control group:Records ICa-L of guinea pig ventricular myocytes when use normal extracellular fluid perfuse; Three groups apply 17(3-E2:Records ICa-L of guinea pig ventricular myocytes when apply 17β-E2 (10-9,10-8,10-7mol/L); 17β-E2+Receptor antagonist groups:17β-E2 (10-7mol/L) pretreatment with the estrogen receptor antagonist tamoxifen (TAM) (10-7mol/L), then records ICa-L of guinea pig ventricular myocytes.2. Experimental InstrumentsPatch-clamp amplifier, DAC, Micromanipulator, Inverted microscope, Microelectrode drawn apparatus, Micro-electrode polishing apparatus, Vibration platform, Langendorff perfusion apparatus, Small animal respirator, NC super thermostat bath, Low-speed centrifuge, Ultrasonic cleaning, Constant temperature water bath, pH meter, Electronic balance, Magnetic stirrer, Surgical instruments.3. Solution and DrugCalcium-free Tyrode's solution(mmol/L), Calcium Tyrode's solution(mmol/L), KB solution(mmol/L), Electrode internal solution, Collagenase,17(3-Estradiol, Protease, Tamoxifen.4. The separation of guinea pig ventricular myocytesAnesthetize the guinea pig with 1% pentobarbital sodium according to 100mg/kg by intraperitoneal injection, tracheal intubation, thoracic aorta isolated and intubated, and connected isolated heart to the Langendorff perfusion apparatus, preparation of ventricular myocytes using enzymatic adopt the method of mechanical dissociation.5. The recording method of ICa-LDrop the cell suspension in box that on the top of the inverted microscope, select a good single ventricular myocytes that static, rod-shaped, stripes and clear, refractive, recording ICa-L with whole-cell recording mode.6. The measurement method of capacitance valueOpen a recorded sample data of ICa-L Select the voltage lines below clamping voltage. Determine the n-Sweep, and select the n-Sweep in the Select Sweep dialog box which in the View dialog box, press OK button. Adjustment 1,2,3,4 cursor, then select the appropriate item in the statistics dialog box, press the OK button, the corresponding area value under the curve generate in the Result dialog box. The area value divided 10 should be capacitor value.7. Data processing and statistical analysisAll data analyse and measure use pCLAMP 10.0. Experimental Results show with Mean±SD, make t test for two groups of the administration group and blank control group. P<0.05 means the difference is significant, P<0.01 means the difference is highly significant.Results1. The effects of 17β-E2 on ICa-LThe results show that the peak current density of ICa-L values from (-3.5±1.4) pA/pF decreased to (-2.5±0.2) pA/pF, (-2.1±0.7) pA/pF and (-1.3±0.5) pA/pF after treated by 17β-E2 (10-9,10-8,10-7mol/L) (Compare with control group, P＜0.01, n=5). The peak current density of ICa-L decreased to 26.8%,40% and 62.9%.2. The effects of 17(3-E2 on ICa-L after pretreatment by TAMAfter pretreated by TAM(10-7mol/L),17β-E2 (10-7mol/L) make peak current density of ICa-L decrease from (-3.5±1.4) pA/pF to (-1.2±0.5) pA/pF(Compare with control group, P＜0.01, n=5). The peak current density of ICa-L decreased to 65.7%. Compared with the 17β-E2 (10-7mol/L) with no TAM, the peak current density of ICa-L was no significant change (P>0.05, n=5).DiscussionCalcium channel is one of the most important membrane proteins on myocardial cell membrane. It can be divided into long-activated (L-type) and transient-activated (T type) according to the molecular biology of calcium channels and calcium current kinetics. The slow calcium inward current caused by L-type calcium channel opening and calcium influx play an important role in cardiac action potential repolarization and the process of coupling excitation-contraction. It is important factor to determine the myocardial cell action potential duration.In this study, we use whole-cell patch clamp recording technique to analyze the effects of estrogen on ICa-L of guinea pig ventricular myocytes. It was found that 17β-E2 can inhibit this current and the inhibition was concentration dependent, suggesting that estrogen may shortened the action potential duration by inhibiting the ICa-L.Inhibition of inward calcium current or the promotion of outward potassium currents lead to shorten the action potential plateau, estrogen's role in promoting the outflow of potassium is not clear.17β-E2 inhibition of Ica-L, to shorten the action potential duration, leading to reduced Ca2+influx, decreased myocardial contractility, reduced cardiac work and myocardial oxygen consumption. In addition, the lower intracellular Ca2+is conducive to mitochondrium discharge the accumulation Ca2+, to reduce Ca2+overload, play a protective effect on the heart.It was found that 17β-E2 pretreated by TAM, the inhibition of 17β-E2 on ICa-L did not changed, suggesting that the inhibition of 17β-E2 on ICa-L is not mediated by receptors. Suggest that the effect may be direct through a certain point which in calcium ion channels or in Calmodulin binding to play biological effects.It was found that 17β-E2 of physiological concentrations can affect, without being blocked by specific estrogen receptor TAM, indicating the effects of 17β-E2 on ICa-L is not through the process of gene transcription, it play biological effects through acting on the calcium channel directly or through a particular way.Conclusion1.17β-E2 can inhibit ICa-L in isolated guinea-pig ventricular myocytes in a concentration-dependent manner.2.17β-E2 receptor antagonist did not abolish the effects of E2 on ICa-L.3.17β-E2 at physiological concentrations can play the above effects.
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