Spatially Defined InsP(3)-Mediated Signaling in Embryonic Stem Cell-Derived Cardiomyocytes

The functional role of inositol 1,4,5-trisphosphate (InsP(3)) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP(3) receptors can translate Ca(2+) release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP(3)-mediated Ca(2+) release. [InsP(3)](i) was monitored with the FRET-based InsP(3)-biosensor FIRE-1 (Fluorescent InsP(3) Responsive Element) and heterogeneity in sub-cellular [InsP(3)](i) was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP(3) 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+) transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+)](i) and spontaneous activity was modulated by [InsP(3)](i). A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP(3) by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP(3) signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+) released from InsP(3)Rs is more effective than Ca(2+) released from RyRs to enhance I(NCX). The results support the hypothesis that in ESdCs InsP(3)Rs form a functional signaling domain with NCX that translates Ca(2+) release efficiently into a depolarization of the membrane potential.