Experimental Study On The Effects Of Exogenous Phosphocreatine On Ion Channels Of Ischemic Myocyte And The Mechanism For Treatment Of Heart Failure

Objective: This study sought to investigate the whole process of patch clamp which comprise acute ventricular myocyte isolation, establishment of whole-cell configuration and recording of ion channels.Method: The single ventricular myocyte was obtained from heart of guinea pig, which was retrogradely perfused with tyrode and enzyme solution in turn on a Langendorff apparatus. Cells were stored in normal Tyrode solution for at least 1 hour, rod-shaped ventricular myocyte with clear striations were selected for experiment. Myocytes were placed in a chamber on a stage of an inverted microscope(TE300, Nikon, Japan) and continuously perfused with test solution at a speed of 2ml/min at 37℃. Micropipettes were pulled by a two-step puller (p-97, Sutter, USA), which had a tip resistance of 2~5M@ when filled with pipette solution. After the whole-cell configuration was achieved, membrane current was recorded in voltage clamp mode. The protocol of experiment and data acquisition were performed with Pclamp8.2 software (Clampex8.2 and Clampfit8.2, Axon Instrument Co, USA) running on a personal computer. The protocol for Ica,L recording involved a holding potential of -40mV with 250ms depolarizing voltage-clamp pulse going from -40mV to +60mV in 10 mV steps, the protocol for INa involved a holding potential of -90mV with 60ms depolarizing voltage-clamp pulse going from -80mV to +60mV in 10 mV steps, the protocol for IK involved a holding potential of -90mV with 200ms depolarizing voltage-clamp pulse going from -90mV to +60mV in 10 mV steps.Results: 70% acute isolation myocytes presented rod-shape, clear striations and calcium-resistance character, which could be used for current recording. Threshold for Ica,L activation was around -25-30mV, maximum activation was attained at about -10~+10mV, the course of activation and inactivation was slow. Threshold for iNaWas around -60-50mV, peak current was attained at about -30~-40mV, the time course of activation and inactivation was rapid. IK was activated around -60~-50mV, current was elevated in correlation with the increasing voltage. IK comprised many components which presented different electrophysiological property. Conclusion: The establishment of whole-cell patch clamp configuration and single ventricular isolation played a important role for accurate recording of ion currents. Ica,L, iNa and IK were voltage dependent channels, accurate recording of these currents was of importance for the further experiment.