Developmental Changes In Delayed Rectifier Potassium Current And Action Potential In Guinea-pig Ventricular Myocytes

Cardiac ion channels across the surface membrane underlie the proper contraction and thereby pumping of the heart. Disorders of ion channels, called'cardiac channelopathies'can be inherited due to mutations in the genes encoding these channels or acquired upon exposure to drugs that block cardiac K+ channels or myocardial disease (such as cardiac hypertrophy and heart failure induced by a variety of initiating factors). Changes in the properties or the functional expression of myocardial ion channels can lead to changes in action potential waveforms, synchronization, and/or propagation, thereby predisposing the heart to potentially life-threatening arrhythmias, resulting in the disturbances of heart pump and, in the extreme case, sudden cardiac death that makes up a major cause of human death. There is, therefore, considerable interest in delineating the molecular, cellular, and systemic mechanisms contributing to the generation and maintenance of normal cardiac rhythms, as well as in understanding how these mechanisms are altered in the diseased myocardium.Previous studies have shown that the types of cardiac ion channels vary considerably among species and different cardiac regions of the heart. The postnatal development of cardiac ion channels results in a dynamic changes of action potential morphology and duration. In addition, as age increased, changes take place in the electrophysiological properties of some important ionic currents including current amplitude and density, channel kinetics or gating characteristics, resulting in the alterations in cardiac action potential shape and duration. Therefore, the changes of cardiac ion channels during the developmental period attract much attention.A variety of repolarizing potassium currents are determinant for ventricular action potential duration, mainly including the transient outward potassium current (Ito), the delayed rectifier potassium current (IK) and the inward rectifier potassium current (IK1). Studies have shown that the density of Ito in rodent ventricular myocytes is significantly increased from childhood to adulthood, accompanied by an enhancement in the expression of channel protein Kv4.2/Kv4.3. The inward rectifier potassium current (IK1), a current mianly responsible for resting potential also shows upregulation from neonatal towards adult due to the regulation of multi-amines, such as spermine and spermidine. Thus, the action potential undergoes a developmental change, manifested by a shortening duration and increased resting membrane potential.The delayed rectifier K+ currents (IK) are the major repolarizing outward currents of ventricular action potentials in mammalian species, including humans and consist of rapidly and slowly activating components (IKr and IKs, respectively). Human ether-a-go-go-related gene (hERG or KCNH2) and KCNQ1 respectively encodes the pore formingα-subunit of IKr, IKs channel, and it is generally considered that KCNE2 and KCNE1 respectively encodes theβ-subunits of IKr, IKs channel.The type and/or expression patterns of the K+ channels that participate in the genesis of the cardiac action potential vary considerably among species. The delayed rectifier K+ currents are the primary determinants of action potential repolarization in the large mammalian myocardium. Despite the guinea pig heart are widespread used to study the function and regualtion of IKr, IKs channel, no comprehensive study has been reported regarding the functional properties of these channels throughout postnatal development in guinea pig ventricular myocytes.By using patch-clamp technique, the present study was to investigate the electrophysiological characterization of the action potential configuration and the delayed rectifier K+ currents inlcuding IKr, IKs present in guinea pig ventricular myocytes throughout postnatal development. Understanding these developmental changes and the factors regulating them might give significant insight into the mechanisms controlling specific cardiac functions such as cardiac K+ channel physiology. 1. Developmental changes in delayed rectifier potassium currents of guinea pig ventricular myocytesObjective: To observe the developmental changes in delayed rectifier potassium current IK and its two main components IKr, IKs in guinea pig ventricular myocytesMethods: (1) Isolation of single ventricular myocytes: Single ventricular myocytes isolated from Day-2, Day-14 and adult guinea pig heart were dissociated by enzymatic dispersion. The hearts were rapidly removed, and retrogradely perfused through the aorta on a modified Langendorff apparatus with the following solutions containing (in mM): NaCl 140, KCl 5.4, MgCl2 1, Glucose 10, HEPES 10 (pH adjusted to 7.4 with NaOH) for 5 min. The hearts were then digested with the enzymatic solution containing 0.4g/L collagenase typeⅡin calcium-free Tyrode solution by circulating perfusion for 5~15 min, At the end of the perfusion, the ventricles were removed from the heart, and rod shape single myocytes from mid-myocarfium were obtained by gentle agitation and stored in KB solution (in mM): KOH 80, KCl 40, KH2PO4 25, MgSO4 3, L-glutamic acid 50, taurine 20, EGTA1, Glucose 10, HEPES 10 (pH adjusted to 7.2 with KOH) at 4℃until being used within 6–8 h.(2) Potassium current recording: The delayed rectifier potassium currents were recorded by using whole-cell patch-clamp technique at room temperature (23℃~25℃) in isolated single ventricular from Day-2, Week-2 and adult guinea pig heart. The myocytes were superfused with the solution containing (mM): NaCl 132, KCl 4, MgCl2 1.2, CaCl2 1.8, Glocose 5, HEPES 10 (pH 7.4 with NaOH). Pipettes were filled with the following solution (mM): KCl 140, Mg-ATP 4, MgCl2 1, EGTA 5, HEPES 10 (pH 7.2 with KOH). Nimodipine (Nim, 1μM) was added in external solution to block L-type Ca2+ channels. Both Na+ and T-type Ca2+ channels were inactivated by a holding potential of -40 mV. The delayed rectifier potassium currents IK was elicited by a 3-second depolarizing step to 50 mV in 10 mV increment, followed by a 2-second repolarizing step to -40 mV. IKs was then recorded as above pulse protocol after adding a specific IKr channel blocker E-4031(2μM) to the external solution. IKr was obtained by subtracting IKs tail from IK tail current. Cell capacitance was calculated from uncompensated capacity current transient elicited by a 10-mV hyperpolarizing voltage step from a holding potential of -40 mV. All averaged values presented were mean±SEM. Statistical comparisons were made using Student's paired t-tests. A value of P<0.05 was considered statistically significant.Results: (1) The density of IK tail current in guinea pig ventricular myocytes was significantly increased from the day-2 neonate to the adult. At +50 mV, the density of IK tail current was 1.72±0.12 pA/pF in the day-2 neonate, 2.86±0.16 pA/pF in the week-2 neonate, and 3.95±0.18 pA/pF in the adult (P < .05).(2) The density of IKs tail current in ventricular myocytes also exhibited a significantly enhancement from the day-2 neonate to the adult. At +50 mV, the density of IKs tail current was 1.18±0.12 pA/pF in the day-2 neonate, 1.95±0.19 pA/pF in the week-2 neonate, and 3.08±0.19 pA/pF in the adult (P<0.05) and tooks up 67.7%, 67.7%, and 77.8% of corresponding IK, respectively.(3) The density of IKr tail current in ventricular myocytes was upregulated from the day-2 to week-2 neonate. At +50 mV, the density of IKr tail current was 0.55±0.14 pA/pF in the day-2 neonate and 0.91±0.16 pA/pF in the week-2 neonate (P<0.05). However, the density of IKr tail current, 0.88±0.15 pA/pF in the adult was not different from that in the week-2 neonate (P>0.05).Conclusion: (1) The density of IK underwent significant up-regulation during postnatal life in guinea pig ventricular myocytes.(2) The idensity of IKr and IKs was also increased during the developmental period. The development of IKr was completed within the first 2 weeks of life, whereas the density of IKs gradually increased from the day-2 neonate to the adult. The different pattern of developmental changes of K+ currents suggests that regulatory factors taking place during development are major determinants of the functional role of K+ channels in cardiac repolarization.2. Developmental changes in action potentials of guinea pig ventricular myocytes Objective: To observe the developmental changes in ventricular action potential of guinea pigMethods: (1) Isolation of single ventricular myocytes: The method was the same as the first part.(2) Action potential recording: Action potentials were recorded in ventricular myocytes using the perforated patch technique. Action potentials were evoked at a rate of 1 Hz with suprathreshold current pulse of 4?6 ms duration applied via patch electrodes in the current-clamp mode. The resting potentials, action potential amplitude and APD90 (time of 90% repolarization) were measured.Results: (1) The resting potential in guinea pig ventricular myocytes was respectively -80.1±1.5, -81.6±1.8 and -78.9±1.3 mV for 2 days, 14 days and adult animals. No statistical difference among the three groups (P> 0.05);(2) The action potential amplitude in guinea pig ventricular myocytes was respectively 127.9±2.1, 129.3±3.0 and 131.7±2.5 mV for 2 days, 14 days and adult animals. No statistical difference among the three groups (P> 0.05);(3) The APD90 in guinea pig ventricular myocytes was significantly prolonged from the day-2 neonate to the adult, with 159.7±9.8 ms in the day-2 neonate, 188.4±14.3 in the week-2 neonate, and 203.8±12.8 ms in the adult (P<0.05). The prolongation of the action potential hints an up-regulation of depolarizing current in ventricular myocytes during the development from the neonate to the adult.Conclusion: (1) The action potential duration underwent a significant prolongation during postnatal life although there was a developmental increase in repolarizing K+, suggesting of an up-regulation of depolarizing current in guinea pig ventricular myocytes during the development from the neonate to the adult.(2) There was no developmental changs in the resting potential and action potential amplitude in guinea pig ventricular myocytes.