Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2: A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection

Abstract

ATP-sensitive K(+) (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100nM) for 24h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K(+) current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10mM), which was accompanied by increased in K(+) current in response to DNP (10mM). Cytoprotection afforded by phenylephrine (100nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K(+) conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes.

Keywords: Cardiomyocytes; Cardioprotection; H9c2 cells; Preconditioning; SUR2B.

Fig. 1

Pretreatment with phenylephrine up-regulate SUR2B/Kir6.2 leading to increase in number of functional K_ATP channels. (A) Bar graphs representing cycling threshold of the real time RT-PCR progress curves of K_ATP channel-forming subunits. Each bar represents mean ± SEM (n = 6). *P < 0.05. (A1) Relative expression ratio (control/phenylephrine ratio of mRNA levels calculated from data shown in A) of K_ATP channel subunits (as depicted on the graph). Each bar represents mean ± SEM (n = 6). (B) Original whole cell membrane currents for cells kept under control conditions (control) and pretreated with phenylephrine (100 nM). (B1) Bar graphs of K⁺ current density at +80 mV under conditions corresponding to (B). Each bar represents mean ± SEM (n = 5). *P < 0.05. (C) Original Western blots with anti-SUR2B antibody of anti-Kir6.2 immunoprecipitate pellets from membrane fractions obtained from H9c2 cells under control conditions and when pretreated with phenylephrine (100 nM) and corresponding graph. Each bar represents mean ± SEM (n = 3–4). *P < 0.05.

Fig. 2

Pretreatment with phenylephrine increases survival of cells and whole cell K⁺ current in response to DNP. (A) A bar graph showing a percentage of survival in control cells and cells pretreated with phenylephrine (100 nM) exposed to DNP (10 mM). Each bar represent mean ± SEM (n = 6). *P < 0.05. (B) Original perforated patch clamp whole cell membrane currents in response to DNP (10 mM) for cells kept under control conditions (control) and pretreated with phenylephrine (100 nM). (B1) Bar graphs of K⁺ current density at +80 mV under conditions corresponding to (B). Each point represents mean ± SEM (n = 6). *P < 0.05.

Fig. 3

Kir6.2AFA inhibits cytoprotection afforded by phenylephrine. A bar graph showing a percentage of survival in cells infected with Kir6.2AFA kept under control conditions and pretreated with phenylephrine (100 nM) exposed to DNP (10 mM). Each bar represent mean ± SEM (n = 6).