Isolated heart and preparation
Ths study was approved by the Animal Care and Use Committee of Wenzhou Medical College. Adult Male Sprague-Dawley rats, weighing 250 to 300 g, were anesthetized using 5% chloral hydrate (7 ml/kg intraperitioneal injection). The carotid artery is cut to allow rapid and thorough exsanguination. The hearts were rapidly removed intact with a short aortic remnant. While in this Tyrode solution the aorta is cannulated, one should make sure that there are no air bubbles trapped in the cannula. The cannulated heart is then mounted on a Langendorff perfusion apparatus with constant flow. After rapid excision, the heart was mounted in a modified Langendorff system and then perfused with the nominally Ca2 + -free Tytode solution (NaCl 137 mM、KCl 5.4 mM、MgCl2 1.0 mM、NaH2PO4 0.33 mM、HEPES 10 mM、Glucose 10 mM, and PH7.35 with NaOH) for 5 min at room temperature (20-25)°C. The perfusate reservoirs and column are glass jacketed, allowing the temperature to be maintained at 37°C by means of a recirculating water bath. All of the perfusion solutions are equilibrated with 100% oxygen. The heart is then retrogradely perfused with the nominally Ca2 + -free Tyrode solution which causes cessation of the heartbeat until the blood is washed out. This was followed by perfusion with enzyme solution, containing 1 mg/ml collagenase (Sigma, typeI) in nominally Ca2 + -free Tytode solution, followed for 10 ~ 15 min. The softened heart was removed from the column, and the left ventricle was dissected in modified KB (KCl 40 mM, KH2PO4 20 mM, MgSO4 3.0 mM, KOH 80 mM, Glutamate 50 mM, Taurine 20 mM, HEPES 10 mM, glucose 10 mM, EGTA 0.5 mM, and pH 7.35 with KOH). The cells were maintained in modified KB solution and stabilized at room temperature for 1 hour.
Equipments and methods
An EPC-10 patch clamp amplifier (HEKA, Germany) was used for application of the whole-cell patch clamp in the ventricular myocytes. Pulse stimulation and data acquisition were recorded by Pulse 8.0 software (HEKA, Germany). Patch pipettes were pulled from glass tubing with a 1.5 mm outer diameter (SUTTER, USA) by the use of micro-electrodes (P-97, SUTTER, USA), and the tip was heated to give a resistance of 1.5-2.5 MΩ when filled with the specific, appropriate solution (see below). Using an inverted microscope, microelectrodes were directed to the ventricular myocytes by a three-dimensional micromanipulator (MP-285, SUTTER, USA). A Giga-seal was formed after vacuum suction. The patch membrane was broken after fast capacitance compensation by the provision of additional suction with subsequent construction of a whole-cell recording. To reduce the instantaneous current charging/discharging, and to minimize clamping errors, the slow capacitance compensation and series resistance compensation were settled at 70% - 80%. Leakage currents were subtracted by the P/4 method.
Electrophysiology
1. For recording INa, the external solution was composed of the following, in mM: Choline-Cl 120, NaCl 20, MgCl2 1.0, HEPES 5, Glucose 10, CsCl 4.6, pH 7.35 with CsOH. the internal solution was composed of the following, in mM: CsCl 120、NaCl 10、MgCl2 1.0、Na2ATP 5.0、EGTA 10、HEPES 10, pH 7.3 with CsOH.
The potential was held at -90 mV, INa was evoked by 25 ms, and accompanied by -30 mV square-wave depolarizing pluses. The stimulation program of current density-voltage curve was as follows: under a holding potential (Vh) of -90 mV, the step clamp voltage (Vs) was stimulated from -90 mV to +50 mV by a step of 10 mV with a 50 ms duration, and a 0.2 Hz stimulation frequency.
2. For recording ICa-L, the external solution was composed of the following, in mM: Choline-Cl 140, MgCl2 1.0, CaCl2 2.0, HEPES 5, Glucose 10, CsCl 4.6, TEA-Cl 10, pH 7.35 with CsOH. the internal solution was composed of the following, in mM: CsCl 120、MgCl2 1.0、MgATP 5.0、EGTA 10、HEPES 10 TEA-Cl 10, pH 7.3 with CsOH.
The potential was held at -40 mV, and ICa-L was evoked by 150 ms, accompanied by 0 mV square-wave depolarizing pluses. The stimulation program of current density-voltage curve was as follows: under a Vh of -40 mV, the Vs was stimulated from -40 mV to +50 mV by a step of 10 mV with a 250 ms duration, and a 0.2 Hz stimulation frequency.
3. For recording Ito, the external solution was composed of the following, in mM: NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 1.0, NaH2PO4 0.33, HEPES 10, Glucose 10, CdCl2 0.3, pH t7.35 with NaOH.the internal solution was composed of the following, in mM: KCl 140、MgCl2 1.0、K2ATP 5.0、EGTA 10、HEPES 5,pH 7.3 with KOH.
The potential was held at -90 mV, Ito was evoked by 20 ms, 50 mV square-wave depolarizing pluses. The stimulation program of current density-voltage curve was: under a Vh of -90 mV, and INa was eliminated by 20 ms, with -40 mV depolarizing pluses; the Vs was stimulated from -40 mV to +50 mV by a step of 10 mV with a 400 ms duration and a 0.2 Hz stimulation frequency.
A control curve of I-V was collected before drug perfusion (T0). Then 100 umol/L bupivacaine and the mixture of 100 umol/L bupivacaine and 0.15 μg/ml epinephrine were added into the reservoirs respectively from superfusion systerm (DADVC-8PP,ALA SCIENTIFIC, USA). The DAD-VC systems go out with a Micromanifold consisting of 8 tubes of polyamide coated quartz glass of 100 um ID. The Micromanifold enables up to 8 solutions from the reservoirs to flow into a small common space of less than 1ul. The Micromanifold with a micromanipulator can easily be moved around the cell preparation and pointed at the target cell. The user must be careful to aim the output so that it completely bathes the cell under study. So during the study there is no motion of the output tip to be dealt with and there is no need to have all the solutions flowing out and contaminating the preparation solution during an experiment. After the cell surface perfusion with 100 μmol/L bupivacaine for 10 seconds. The peak current and I-V curve were recorded (T1). The microperfusion tube was then swithed by another reservoir prefilled with 100 μmol/L bupivacaine and 0.15 ug/ml epinephrine after the model was successfully made by 100 μmol/L bupivacaine. The peak current and I-V curve were then recorded at the time (T2) when the cells’ surface were perfusion with 100 μmol/L bupivacaine and 0.15 ug/ml epinephrine for 10 seconds.
To eliminate the error among cells, the size of the ion currents was represented by the current density, which was the ratio of current intensity and cell membrane capacitance (pA/pF). Data were stored in the hard disk for the measurement and analysis. Raw current data were analyzed and measured by pulse 8.0.