Abstract
The functional role of inositol 1,4,5-trisphosphate (InsP3) 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 InsP3 receptors can translate Ca2+ 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 InsP3-mediated Ca2+ release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca2+ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca2+]i and spontaneous activity was modulated by [InsP3]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 InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 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. Ca2+ released from InsP3Rs is more effective than Ca2+ released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca2+ release efficiently into a depolarization of the membrane potential.
Highlights
In cardiac muscle the expression of inositol-1,4,5-triphosphate receptors (InsP3R) is most abundant during early development [1,2]
To determine whether the changes in [Ca2+]i correlate with changes in membrane voltage (Vm) we recorded action potentials (APs) in Fluo-4/AM loaded embryonic stem cell-derived cardiomyocytes (ESdCs) with the perforated patch technique
Pronounced perinuclear staining was identified, together with extensive endoplasmic reticulum staining throughout the cell that extended to the plasma membrane
Summary
In cardiac muscle the expression of inositol-1,4,5-triphosphate receptors (InsP3R) is most abundant during early development [1,2]. At embryonic and neonatal stages of differentiation, immunostainings indicate that InsP3Rs pre-dominantly locate to the nuclear envelope [4,5,6]. In contrast to the abundance of InsP3Rs in the early developmental stages, their expression decreases towards adulthood; in the adult atrial [13] and ventricular muscle of rat [14], cat [15], and rabbit [16] the expression of InsP3R2 isoforms was demonstrated. During excitation-contraction coupling in the adult cardiac muscle, Ca2+ is released from the sarcoplasmic reticulum mainly through the ryanodine receptor type 2 (RyR2), which is expressed 50 fold higher than InsP3Rs. In contrast InsP3R-mediated signaling has been linked to excitationtranscription coupling. Activation of nuclear InsP3Rs was sufficient for the activation and translocation of the transcription factor
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