P605Unraveling tight spatiotemporal regulation of 5-HT4 receptor-mediated inotropic effects using targeted FRET-based cAMP sensors

Abstract

Background: We have previously reported that an inotropic response (increase in contractility) to serotonin through 5-HT4 receptors appears in the ventricle of failing rat and human hearts. The inotropic response is ~80% of that to β-adrenergic receptor (β-AR) stimulation in failing rat hearts, yet the increase in cAMP levels is only ~20% compared to β-AR stimulation in cardiac myocytes. As another extreme, EP prostanoid receptor activation has long been considered to yield large increases in myocyte cAMP levels without a positive inotropic response, although some recent data have made this less clear, at least in mouse heart. It thus appears that cAMP produced through stimulation of 5-HT4 receptors and a subset of β-ARs is more tightly coupled to a cardiac inotropic response, e.g. through localization of the produced cAMP closer to the contractile apparatus, compared to EP prostanoid receptors. We have previously found that cAMP produced following 5-HT4 receptor stimulation is degraded by both phosphodiesterase 3 (PDE3) and PDE4, whereas the 5-HT4-mediated inotropic response is primarily limited by PDE3, indicating that localized degradation of cAMP is a primary factor that confines compartmentation of cAMP signaling. Methods: Experiments were performed on cardiac myocytes from left ventricle from Wistar rats with heart failure 6 weeks after myocardial infarction or sham-operated animals. We measured local concentrations of cAMP in both normal and failing cardiac myocytes using FRET (Fluorescence Resonance Energy Transfer)-based cAMP sensors specifically targeted to subcellular compartments. Results and conclusion: We find that in failing cardiac myocytes, 5-HT4 receptor stimulation results in a larger increase in cAMP close to the contractile compartment compared to compartments not associated with contraction, whereas β-AR stimulation increases both local and global levels of cAMP. Inhibiting PDE3 enhances both 5-HT4 receptor- and β-AR-mediated increases in cAMP, supporting the notion that PDEs define the contractile compartment. Thus, both production and degradation of cAMP regulating inotropic effects appear to be tightly controlled.