Isolation Of Rat Cardiomyocytes And Effects Of Astragalus Saponins On Ion Channels

Objective:1. To develop and optimize a calcium tolerant adult rat ventricular myocytes isolation method for patch-clamp experiments and record sodium channel current (Ina)、 transient outward potassium current (Ito) and L-type calcium channel current (ICa,L).2. To investigate the electrophysiological effects of total saponins of astragalus(TSA) on INa、Ito and ICa,L in rat ventricular myocytes, including I-V curves、activation kinetics、inactivation kinetics and recovery kinetics, to investigate the possible effects and mechanism of TSA on heart.Methods:Langendroff perfusion apparatus and collagenase I are used to isolate ventricular myocytes from male Sprague-Dawley rats. TSA was added into the suspension of the myocytes. Whole cell patch-clamp technique was used to record the INa、Ito and ICa,L in the ventricular myocytes.Results:1. A large amount of rod-shaped rat cardiomyocytes with clear cross striations and high activity were acquired with a ratio of sixty percent to eighty percent. About thirty percent to forty percent of the cardiomyocytes were dead after calcium reintroduction, the remainders are quiescent with clear transverse striation. We successfully record the typical INa、Ito and ICa,L in the ventricular myocytes. INa is an inward current, which can be blocked by TTX. Its activation and deactivation are fast. The I-V curves show that its activation potential、peak potential and reversal potential are -60mV、-30mV、+40mV, respectively. Ito is an outward current, which can be blocked by 4-AP. Its activation and deactivation are fast, too. The I-V curves show that its activation potential and peak potential are -30mV and +70mV. As the voltage increases, the current becomes larger and larger. ICa,L is an inward current, which can be blocked by CdCl2 It activates quickly and deactivates slowly. The I-V curves show that its activation potential、 peak potential and reversal potential are -30mV、 OmV、+50mV, respectively.2. TSA(10μg/mL 30μg/mL 100μg/ml) inhibited INa in a concentration-dependent manner, with the inhibition rate of (26.15±6.74)%、 (37.40±6.91)%. (54.35±5.90)%, respectively. The INa I-V curve was shifted upwards by TSA without changes of their active, peak, reverse potentials. 100μg/ml TSA had no significant effect on INa steady-state activation curve. 100μg/ml TSA turned INa steady-state inactivation curve to left, which made inactivate more quickly. 100μg/ml TSA turned INa recovery curve to right, which took longer to reactivate.3. TSA(10μg/ml、30μg/ml、100μg/ml) inhibited Ito in a concentration-dependent manner, with the inhibition rate of (36.60±10.07)%、(51.71±8.81)%、(62.66±6.38)%, respectively. The Ito I-V curve was shifted downwards by TSA without changes of their active, peak, reverse potentials. 100μg/ml TSA had no significant effect on Ito steady-state activation curve. 100μg/ml TSA turned Ito steady-state inactivation curve to left, which made inactivate more quickly. 100μg/ml TSA turned Ito recovery curve to right, which took longer to reactivate.4. TSA(10μg/mL 30μg/ml) had no significant effects on ICa,L, while 100μg/ml TSA inhibited Ica,L with the inhibition rate of (33.16±3.84)%. The Ica,L I-V curve was shifted upwards by TSA without changes of their active, peak, reverse potentials. 100μg/ml TSA had no significant effect on Ica,L steady-state activation curve. 100μg/ml TSA turned ICa,L steady-state inactivation curve to left, which made inactivate more quickly.100μg/ml TSA turned Ica,L recovery curve to right, which took longer to reactivate.Conclusion:1.It is an convenient and reliable method to isolate adult rat cardiomyocytes with normal electrophysiological characteristics. The cardiomyocytes can be used to study ion channels with patch clamp.2. TSA can block INa、Ito、ICa，L with different degrees.