Developmenal Changes Of Inward Rectifier Potassium Current (IK1)in Murine Fetal Ventricular Cardiomyocytes

Inward rectifier potassium (IK1) currents have strong inward rectifier properties in mammalian hearts. IK1 stabilizes the resting membrane potential and plays a major role during the final phase of action potential (AP) repolarization in the cardiomyocytes. The reduction of IK1 can prolong the action potential duration and lead to long QT syndrome which is also named Anderson's syndrome. On the contrary, the increase of IK1 can shorten the action potential duration(APD), Effective refractory period(ERP) and can also lead to short QT syndrome. The shortening of ERP can lead to Ventricular arrhythmia such as Ventricular tachycardia and ventricular fibrillation, etc.IK1 channels in the heart are believed to be homo- and/or heterotetramers of Kir2.1,Kir2.2 and Kir2.3 subunits from the Kir2 family of inward rectifier potassium channels. Recently, it is believed that the species-dependent and tissue-specific expression of different IK1 channels subunits may contribute to its variability.The embryonic cardiomyocytes which displayed spontaneous occurring APs share some properties of the diseased cardiomyocytes which suffered from hypertrophy or cardiac failure. Thus recently many electrophysiological properties of fetal cardiomyocytes have been discussed intensively, IK1 is one of the interests in some species. During development, the current density of IK1 increases in the rat, rabbit, chick, guinea-pig hearts. However, the expression of IK1 channels in fetal mice is little discussed, especially the detailed subunits basis of IK1 channels during development is unclear. The present study was designed to investigate the electrophysiological properties of IK1 channel with specific inhibitors and the molecular basis of IK1 channels using approaches of whole cell patch clamp, real time PCR, western blot and double-immunofluorescence analysis in developing murine ventricular cardiomyocytes. The findings here may offer a new aspect for better understanding of cardiogenesis and heart disease such as myocardial hypertrophy and heart failure.PARTâ… Functional expression of IK1 in murine ventricular cardiomyocytes during development1. IK1 current densities increased during developmentMethods: The fetal murine ventricles were dissociated enzymatically using collagenase B to get single cardiomyocytes and cultured for 24-36 h. IK1 currents were recorded using whole cell voltage patch clamp technique with specific inhibitors 2 mM Ba²⁺ at EDS and LDS, respectively.Results: IK1 currents recorded with 5.4 mM KCl in external solution was too small to do further evaluation. Larger IK1 currents were obtained with 100 mM K+ in extracellular solution. The inward rectifier potassium current (IK1) conducts inward currents at more negative potentials than the K+ equilibrium potential (EK) but also allows much smaller outward currents at more positive potentials than EK. Peak inward current density measured at -120 mV was -6.72±1.02 pA/pF (n=9) for ventricular cells at EDS and was significantly greater at LDS (-92.29±3.03 pA/pF,n=10, P<0.01). Peak outward current density was 0.68±0.32 pA/pF (n=9) for cells at EDS and was 3.27±1.11 pA/pF at LDS (n=10). The effect of Ba2+ can be reversed by washout in the recording of the currents.2. Inward rectifier properties of IK1 currents increased during developmentMethods: Fetal ventricular cardiomyocytes were prepared and IK1 was recorded as afore-mentioned. The reverse potential was obtained by fitting I-V curve at voltages of -40⁺20 mV with Boltzmann function, the potential activating peak outward current was obtained by fitting I-V curve at voltages of 0⁺40 mV with Gauss function. Inward rectification ratio was calculated with inward and outward current amplitudes (pA/pF) measured at potentials 30 mV negative and 20 mV positive to the respective IK1 reversal potentials. The potentials approximately correspond to -40 mV and 10 mV, respectively. That is, RR (rectification ratio) = IK1 (-40 mV) /IK1 (10 mV).Results: The reversal potential was not significantly different between EDS (-11.55±4.55 mV, n=9) and LDS (-18.41±8.68 mV, n=10). However, with maturation outward current peaked at voltage of 18.32±0.79 mV (n=10) for LDS and at a more positive potential 22.58±2.42 mV (n=9) for EDS. The averaged rectification ratio (RR) (-13.39±1.9) at LDS was as 4-fold large as that at EDS (3.36±0.92), indicating a stronger rectification at LDS..3. IK1 current activation kinetics increased during developmentMethods: Fetal murine ventricular cardiomyocytes were prepared as illustrated above. IK1 currents were recorded using whole cell voltage patch clamp technique with specific inhibitors including 2 mM Ba2+ in response to voltage steps (from -120 mV to -20 mV with 20 mV increments, 100 ms, holding potential -40 mV). The Tau was acquired by fitting IK1 currents with the exponential function to observe the developmental changes of IK1 activation kinetics.Results: With maturation murine embryonic cardiac IK1 displayed different activation kinetics. Although channel activation at hyperpolarized potentials displayed a pseudoinstantaneous components of activation (not fit) a much slower monoexponential current increase was followed at EDS than LDS. The activation Tau at -110 mV at EDS (6.14±0.32 ms, n=9) was much slower than that at LDS (0.85±0.14 ms, n=10, P<0.01). The kinetics of IK1 activation at EDS were similar to those of Kir2.1/Kir2.3 concatemer in tested HEK293 cells while the activation kinetics at LDS were similar to those of Kir2.1-mono in tested HEK293 cells.Partâ…¡The developmental changes of IK1 channel subunits in fetal murine ventricular cardiomyocytes1 Expression of IK1 channel subunits at mRNA level during development Methods: Fetal murine ventricular tissue was isolated from the heart, semi-quantitative RT-PCR, and real time PCR were used to investigate the molecular basis of the IK1 channels at mRNA level.Results: At EDS, Kir2.1 and Kir2.3 were expressed at a comparable level. With maturation, Kir2.1 was upregulated (n=4, P<0.05 EDS vs. LDS) and Kir2.3 was downregulated (n=3, P<0.05 EDS vs. LDS). At LDS, Kir2.1 became the predominant subunit. Kir2.2 was detected at low levels thoughout the fetal development and upregulated (n=5, P<0.01 EDS vs. LDS).2 Expression of IK1 channel subunits at protein level during developmentMethods: Fetal murine ventricular tissue was isolated from the heart, western blotting and double-immunofluorescence techniques were used to investigate the subunits basis of the IK1 at protein levels. Since the Kir2.3 subunits of IK1 channels show a unique slower activation kinetics, a unique"shallow"rectification profile and a smaller single channel conductance than Kir2.1 and Kir2.2 subunits. Moreover Kir2.2 was detected at a low level, and Kir2.1 is the most important subunit to composite IK1 current, we specially investigate the contribution Kir2.1 and Kir2.3 subunits to IK1 channels at protein levels. The functional expression of Kr2.1 and Kir2.3 decided the electrical characterization of IK1 channel, we therefore investigated the expression of Kir2.1 and Kir2.3 at protein level. Results: The developmental changes were consistent with the above observation at mRNA level. Kir2.1 and Kir2.3 were predominant at EDS, while Kir2.1 was upregulated and Kir2.3 was downregulated during development (n=3). The double-label immunofluorescence analysis further revealed the co-localization of Kir2.1 and Kir2.3 in ventricular cardiomyocytes at both EDS and LDS.Partâ…¢Functional contribution of IK1 to action potential in murine ventricular cardiomyocytes during development1 Developmental changes in action potentials of fetal murine ventricular cardiomyoctesMethods: Fetal murine ventricular cardiomyocytes were prepared enzymatically and cultured for 24-36h. Whole cell current clamp technique was used to investigate action potential during development.Results: The embryonic cardiomyocytes displayed spontaneous occurring APs, and the basic parameters of APs changed dramatically during development, especially action potential duration (APD) and velocity of late repolarization phase (Vlrp). APD at 90% of repolarization (APD90), at 50% of repolarization (APD50), at 20% of repolarization (APD20) was 144.5±7.6 ms, 106.3±5.6 ms and 59.9±4.5 ms at EDS, respectively, while APD90, APD50 and APD20 at LDS was 80.3±4.1 ms, 60.8±3.8 ms and 29.6±3.0 ms. Statistics analysis showed the APD at EDS and LDS was significantly diffferent (P<0.05). Velocity of late repolarization phase (Vlrp) at EDS (1.40±0.14 V/s) was slower than that at LDS (2.0±0.06 V/s) (P<0.05). Maximum diastolic potential (MDP) at EDS (61.6±1.7 mV) was almost the same as that at LDS (63.8±1.1 mV). Data at EDS were obtained from 46 independent action potentials and the data at LDS from 22 independent action potentials. 2 Functional contribution of IK1 in ventricular cardiac excitability during development.Methods: Fetal murine ventricular cardiomyocytes were prepared as illustrated above, whole cell current clamp technique was used to investigate the contribution of IK1 currents to action potential during development.Results: Ba2+ application led to a prolongation of APD, depolarization of MDP, and a decrease in velocity of late repolarization phase (Vlrp) at both EDS (n=7, P<0.05) and LDS (n=9, P<0.05). As compared to EDS, the Ba2+ effects on APD90, APD50 and APD20 at LDS was much stronger (P<0.05 EDS vs. LDS). There was no significant difference between the Ba2+ effects on MDP or Vlrp (P>0.05 EDS vs. LDS).Conclusions1 Compared to EDS, IK1 channels at LDS displayed stronger current density, faster activation kinetics and stronger inward rectifier properties.2 The molecular basis of IK1 electrophysiological changes during development: the Kir2.1 upregulated and Kir2.3 downregulated. Kir2.1 and Kir2.3 were predominant at EDS, while Kir2.1 became the predominant subunit at LDS.3 IK1 had different functional contribution to action potential in murine ventricular cardiomyocytes during development. Ba2+ at LDS had much stronger effect on APD90 and APD50 than that at EDS. (P<0.05 EDS vs. LDS). However, there is no difference between EDS and LDS regarding Ba2+ effects on MDP or Vlrp of action potential (P>0.05 EDS vs. LDS).