The transmural gradient in the transient outward current (Ito) is an important determinant of the normal sequence of cardiac action potential repolarization. Ito is a fast activating and inactivating K+ current present in the cardiac myocytes of most mammalian species and its density is much higher in the epicardium than in the endocardium of the free ventricular wall.
Recently, we have proposed that the gradient in Ito is determined by the uneven expression of the K+ channel β subunit gene KChIP2 (K+ channel interacting protein 2) across the ventricular wall. In both human and canine heart, KChIP2 mRNA expression parallels the gradient in Ito, while the α subunit Kv4.3 mRNA is expressed evenly across the ventricle (Rosati et al. 2001).
A recent study (Deschenes et al. 2002) has cast doubt on our hypothesis, reporting that the KChIP2 protein expression is uniform across the canine and human ventricles. We have re-examined this issue and have found that KChIP2 protein levels tightly match (R 2 = 0.91) those of KChIP2 mRNA and parallel the density of Ito in nine different regions of the canine ventricle.
These conflicting results can be reconciled only by assuming that one set of data is artifactual, i.e. due to the cross-reaction of the anti-KChIP2 antibody with a different protein. Using microarrays, we show that only 0.1 % of the genes expressed in human heart are upregulated in the epicardium of the left ventricle relative to endocardium. Therefore, the probability of our results being due to a non-specific cross-reaction is extremely low. This possibility becomes even lower when it is taken into account that the KChIP2 protein distribution matches the KChIP2 mRNA distribution throughout the ventricles.
RNA isolation, RNase protection assays, myocyte preparation and electrophysiological recordings were performed as previously reported (Rosati et al. 2001). Western blot experiments were conducted according to Guo et al. (2002). The microarray experiment was performed using an Affymetrix Human genome GeneChip Set (U133) and data analysis was conducted according to Irizarry et al. (2002). Donor animals were humanely killed. All animal procedures were approved by the Institutional Animal Care and Use Committeee of SUNY at Stony Brook. Human RNA had been previously isolated after approval of the University’s ethical committee (Rosati et al. 2001).