Effects of the restrictive cardiomyopathy-associated cardiac troponin I K178E mutation on cAMP signalling at the myofilament

Abstract

Abstract Background Catecholaminergic stimulation of cardiac β-adrenergic receptors (β-ARs) and subsequent cAMP signalling control cardiac inotropy and lusitropy. cAMP signalling is known to be compartmentalised to optimise sympathetic control by producing heterogeneous cAMP signals and protein kinase A (PKA) activation at distinct subcellular sites. This signalling may be disrupted in diseases such as restrictive cardiomyopathy (RCM) characterised by diastolic dysfunction, which is associated with certain mutations including the K178E missense mutation in cardiac troponin I (TPNI). The impaired ventricular relaxation associated with this mutation is thought to arise from myofilament calcium hypersensitivity, which can be modulated by PKA-dependent TPNI phosphorylation. However, a detailed understanding of the pathological effects mediated by the K178E-TPNI mutation is lacking. Purpose This study aims to determine whether the K178E-TPNI mutation alters local cAMP/PKA signalling at TPNI, potentially affecting myofilament calcium sensitivity and cardiac myocyte relaxation. Methods Real-time imaging of cAMP was carried out using the Fluorescence Resonance Energy Transfer (FRET)-based sensor CUTie (cAMP Universal Tag for imaging experiments) [1] fused to wild type (WT)- or K178E-TPNI. The sensors were expressed by transfection in neonatal rat ventricular myocytes (NRVMs) and FRET-changes were recorded at the myofilament on application of the β-AR stimulant isoproterenol (ISO). We further assessed the involvement of cAMP hydrolysing phosphodiesterases (PDEs) in the regulation of cAMP levels selectively at TPNI. Results The peak FRET-change and FRET-change after 5mins on ISO application were significantly higher in NRVMs expressing the mutant compared to the WT sensor, as was the rate of cAMP accumulation. A strong trend showed that the rise in FRET following PDE4 inhibition was larger in NRVMs expressing the WT compared to the mutant sensor, suggesting the involvement of this isoform in degrading cAMP locally at TPNI and reduced PDE4 activity in the presence of the K178E mutation. Co-immunoprecipitation analysis in HEK293 cells demonstrated interaction between WT-TPNI and a PDE4D isoform, and a weaker interaction of this isoform with the K178E-TPNI mutant. Conclusions This study shows that the K178E-TPNI mutation attenuates the TPNI/PDE4D interaction and alters the magnitude and kinetics of the local cAMP response at the myofilament. This mechanistic insight may aid the development of therapeutics targeting the TPNI/PDE4D interaction to treat RCM-associated diastolic dysfunction.The TPNI-CUTie SensorFRET Response Trace