Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle

Abstract

Expression of four members of the KChIP family of potassium channel beta subunits was examined in canine heart. Only one member of the gene family, KChIP2, was expressed in heart. There was a steep gradient of KChIP2 mRNA expression across the canine ventricular free wall. KChIP2 mRNA was 25-fold more abundant in the epicardium than in the endocardium, and this gradient paralleled the gradient in transient outward current (Ito) expression. In contrast, Kv4.3 potassium channel alpha subunit mRNA was expressed at equal levels across the ventricular wall. There was no difference in the pharmacological sensitivity of epicardial and endocardial Ito channels to flecainide, suggesting that the current is produced by the same channel in the two tissues. A similar gradient of KChIP2 expression was found across the ventricular wall of human heart, but not rat heart. It is concluded that transcriptional regulation of the KChIP2 beta subunit gene, rather than the Kv4.3 [alpha] subunit gene, is the primary determinant regulating the transmural gradient of Ito expression in the ventricular free wall of canine and human heart.

Figure 1

A, Kv4.3 mRNA expression across the left ventricular free wall of canine heart determined by RNase protection analysis. En, endocardium; M, midmyocardium; Ep, epicardium; P, probe; t, negative control tRNA; cyc, cyclophilin. There was 5 μg of total RNA in each sample and the x-ray film was exposed overnight. B, histogram comparing the relative abundance of Kv4.3 mRNA transcripts in endocardium, midmyocardium and epicardium. Error bars indicate s.e.m. (n = 4). C, recordings of I_to obtained from endocardial, midmyocardial and epicardial myocytes isolated from canine left ventricular free wall. The currents were elicited in response to 500 ms depolarizing voltage steps (traces are truncated in the figure) ranging from -20 to +50 mV in 10 mV increments, from a holding potential of -70 mV at a frequency of 0.1 Hz. D, histogram comparing the magnitude of I_to in endocardial, midmyocardial and epicardial myocytes. I_to was measured at +50 mV and was determined as the difference between the peak current and the plateau current at 500 ms. Data were normalized to cell capacitance. Error bars indicate s.e.m. (n = 11-14).

Figure 2

A, KChIP1, KChIP2, KChIP3 and KChIP4 mRNA expression in the left ventricular free wall of canine heart determined by RNase protection analysis. Only KChIP2 is expressed at detectable levels in canine left ventricle. The weak, lower protected band in the brain sample for KChIP3 is due either to a polymorphism or to a single-base error in the probe. V, left ventricle; C, positive control brain cortex; P, probe; t, negative control tRNA; cyc, cyclophilin. There was 5 μg of total RNA in the ventricular samples and 2.5 μg of total RNA in the cortical samples. B, KChIP2 mRNA expression across the left ventricular free wall of canine heart. En, endocardium; M, midmyocardium; Ep, epicardium. There was 5 μg of total RNA in each of the samples. C, histogram comparing the relative abundance of KChIP2 mRNA transcripts in endocardium, midmyocardium and epicardium. Data points for each independent experiment were normalized relative to expression in epicardium. Errors bars are s.e.m. (n = 4).

Figure 3

A, effect of flecainide on I_to in epicardial and endocardial myocytes isolated from canine left ventricular free wall. I_to elicited in a representative epicardial (top) and endocardial (bottom) myocyte by a 300 ms voltage step to +50 mV, in control conditions or in the presence of 100 μm flecainide, as indicated. B, comparison of the dose-response curve for flecainide block of I_to in endocardial and epicardial myocytes. Plotted data points represent the mean values (±s.e.m.) for the percentage blockade of I_to (measured as the current-time integral) for different flecainide concentrations in epicardial (•, n = 4) and endocardial (○, n = 3) myocytes. Data points were fitted with the Hill equation. There was no significant difference in the IC₅₀ for flecainide block for the two cell types (IC₅₀= 12.6 ± 1.2 and 10.8 ± 2.7 μm for endocardial and epicardial myocytes, respectively).

Figure 4

A, KChIP2 and Kv4.3 mRNA expression across the left ventricular free wall of human heart determined by RNase protection analysis. B, KChIP2 and Kv4.2 mRNA expression across the left ventricular free wall of rat heart. Pa, papillary muscle; En, endocardium; M, midmyocardium; Ep, epicardium; P, probe; t, negative control tRNA; cyc, cyclophilin.