Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart

Abstract

The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion-channel functional expression. The present study addressed regionally defined differential ion-channel expression in the non-diseased human heart with a genomic approach. High-throughput real-time RT-PCR was used to quantify the expression patterns of 79 ion-channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non-diseased human donor hearts. Two-way non-directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left- versus right-sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cav alpha2 delta2, Nav beta1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression-portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na(+),K(+)-ATPase, NCX1, SERCA2, and the Ca(2+)-handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin-alpha. Nav1.5 and Nav beta1 were more strongly expressed in the endocardium. For selected genes, RT-PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion-channel subunit-gene expression in the non-diseased human heart. Our data point to significant regionally determined ion-channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion-channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion-current expression may be primarily transcriptional.

Figure 1. Two way hierarchical agglomerative clustering applied to 79 genes (vertically) and to 5 left and right atria (LA and RA), 7 left and right ventricles (LV and RV) and 8 Purkinje fibre samples (horizontally)

The input consisted of the ratio for each patient and gene versus reference gene. Each gene is represented by a single row of coloured boxes and each patient by a single column. The entire gene clustering is shown on the left. Three selected gene clusters are shown on the right (A–C) containing relevant genes for Purkinje fibres and working myocardium discrimination (A, C), and for atrial–ventricular discrimination (B). Each colour patch in the map represents the gene expression level in one sample from one patient, with expression levels ranging from bright green (lowest) to bright red (highest). Missing values are coded as silver.

Figure 2. Two-way hierarchical agglomerative clustering applied to 79 genes (vertically) and to 7 left epicardial (Lepi) and left endocardial (Lendo), and 8 right epicardial (Repi) and right endocardial (Rendo; horizontally) samples

Same overall format as in Fig. 1.

Figure 3. Expression profile of Ca²⁺ channels, connexins and Na⁺ channels in the human heart

Differentially expressed genes in atria versus ventricles (A), in Purkinje fibres versus RV (B) and in epicardium versus endocardium (C). Data are expressed as percentage difference. a, P < 0.05; b, P < 0.01; and c, P < 0.001.

Figure 6. Expression profile of pumps, exchangers and Ca²⁺ handling proteins in the human heart

Same presentation and symbols as in Fig. 3.

Figure 7. Western-blot analysis of key channel proteins

Left, representative Western blots probed with anti-Cav1.3, anti-Kv1.5, anti-Kv4.3, anti-KChIP2, anti-TWIK1 and anti-Cx40 antibodies. GAPDH is shown as loading control. Expected molecular masses are indicated. Right, mean ±s.e.m. ion-channel subunit protein expression values normalized to that of GAPDH. a, P < 0.05; b, P < 0.01; and c, P < 0.001 for LA versus LV and for Lepi versus Lendo, n= 4 tissue samples per group.

Figure 4. Expression profile of K⁺ channel α- and β-subunits in the human heart

Same presentation and symbols as in Fig. 3.

Figure 5. Expression profile of Kir, HCN and Cl⁻ channels in the human heart

Same presentation and symbols as in Fig. 3.

Figure 8. Schematic diagram of the heart illustrating statistically significant gene-expression differences that create an ion-channel ‘signature’ for various cardiac regions

The absolute expression levels of genes that are significantly more strongly expressed in a region of interest relative to the reference region indicated are provided by colour coding: genes expressed > 20-fold the reference gene (HPRT) in the tissue/region of interest are indicated in bold and genes expressed at > 185 times the reference gene level are shown in bold red. Values shown are differences that were statistically significant for both right and left-sided comparisons for atrium versus ventricle and epicardium versus endocardium.