Developmental Changes Of ATP-sensitive K⁺ Channels (K_ATP) In Mouse Embryonic Cardiomyocytes

Objective: ATP-sensitive K+ channels (KATP) might be involved in the hypoxia resistance of fetal cardiomyocytes, therefore we aimed to investigate the developmental change of KATP in mouse embryonic myocardial cells, including the electrophysiological characteristics and the functional contributions.Methods: Mouse embryos of 10.5 days and 17.5 days were taken as early (EDS) and late (LDS) developmental stage, respectively. The single mouse embryonic cardiomyocytes were dissociated enzymatically using collagenase B and cultured for 24-36h before electrophysiological recordings. The specific openers of KATP channel Pinacidil and Cromakalim, as well as the specific inhibitor Glibenclamide, were used to indentify IKATP. The current-clamp was used to record the action potentials and observe the functional contributions of IKATP during development, the whole-cell voltage-clamp (Extracellular K+ 140 mM, holding potential 0mV, test pulse from -100 to 40mV, step 20mV, duration 500ms) was applied to study the electrophysiological characteristics of IKATP. Finally, RT-PCR was performed investigate the expression levels of KATP subunits Kir6.1 , Kir6.2 and the regulatory subunits SUR1, SUR2A .Results: Data acquired from action potential recordings showed that the specific opener Pinacidil shortened the action potential duration (APD) of most working cardiomyocytes while no effects on maximum diastolic potential (MDP). However, it strongly hyperpolarized the MDP of LDS working cardiomyocytes. Whole cell recordings revealed that there was no significant difference of the IKATP current density between EDS and LDS fetal cardiomyocytes, although the expression of Kir6.1, Kir6.2, SUR1 and SUR2 at mRNA level were all increased with the development. Moreover, the IKATP inward rectifier characteristics of the EDS cardiomyocytes was stronger than LDS cells.Conclusions: With maturation, the expression of KATP channel subunits upregulated whereas the current density did not change significantly but the inward rectifier characteristics of the KATP channels reduced. The conflictions of IKATP electrophysiological characteristics and the levels of molecular expression may rise from the different combination of each subunits, or else the different levels of protein translation.