Notch1 Signaling Negatively Modulates Repolarizing Kv Currents in Cardiomyocytes

Notch1 is a critical signaling pathway during embryonic and early postnatal development and activity of this transduction system gradually declines after birth. Conversely, outward K + Kv currents are progressively increased in myocytes during postnatal life leading to shortening of the action potential (AP) duration and acquisition of the mature electrical phenotype. Thus, we tested the possibility that Notch1 signaling modulates the electrophysiological properties of developing cells by interfering with Kv current densities. For this purpose, molecular and physiological studies were conducted in vitro using mouse neonatal myocytes (NMs). These assays were complemented with tests in adult cells obtained from a conditional mouse model of Notch1 intracellular domain (NICD) overexpression. NMs were treated with a γ-secretase inhibitor (DAPT) to prevent the cleavage of Notch1 receptor and subsequent translocation of the active NICD to the nucleus. By RT-PCR, Notch1 targets Hes1 and Hey1 were reduced with DAPT-treatment, confirming the effective perturbation of Notch1 signaling. Importantly, inhibition of Notch signaling led to upregulation of the three splice variants of the Kv channel-interacting proteins 2 (KChIP2) gene, which controls the appearance of outward Kv currents in myocytes. By patch-clamp, Kv currents were identified in only 15% of NMs (n=20) cultured for 1-2 days in in control condition, whereas this fraction increased to 50% (n=18) in cells treated with DAPT. Thus, Notch inhibition promotes the expression of KChIP2 and appearance of Kv currents. To clarify these in vitro findings, the consequences of ectopic activation of Notch1 signaling on the electrical behavior of adult myocytes were established. By patch-clamp, NICD-overexpressing myocytes presented a >2-fold longer duration of the early repolarization phase of the AP, with respect to cells from wild type hearts. This alteration was coupled with a >50% reduction of Kv currents I to and I Kslow1 in NICD-overexpressing cells, with respect to wild-type myocytes. Thus, Notch signaling represses KChIP2 and Kv currents in cardiomyocytes representing an important modulator of the electrical phenotype of the developing heart.