Simultaneous Activation of PKA and Epac is Cardioprotective in Neonatal and Adult hearts

Abstract

Cardiac vulnerability to ischaemia/reperfusion (I/R) changes during development; 14-day old rat heart being least vulnerable compared to adult or younger postnatal hearts. The underlying mechanisms are not known, thus design of age-specific optimal cardioprotective interventions is difficult. Recent work has shown activation of cAMP/PKA/Epac signalling pathway is cardioprotective in adult hearts. Whether this also mediate protection in postnatal hearts is unknown. This will be addressed in this thesis. Wistar rats of 7, 14, and 28 days postnatal & adults were used. Cardiac proteome was examined using TMT-MS/MS and bioinformatic analysis. Langendorff perfused hearts were exposed to I/R and injury assessed by measuring cardiac enzyme release and infarction by staining with tetrazolium chloride. Isolated cardiomyocytes were used in suspension to study simulated reperfusion injury (H2O2 & 3mM Ca2+) and viability measured by Trypan blue staining. Superfused field stimulated isolated cardiomyocytes were exposed to metabolic inhibition and contraction measured using edge tracking whilst Ca2+ transients measured using Fura-2-AM. Isolated mitochondria were used to study mitochondrial permeability transition pore (MPTP) sensitivity to Ca2+. Selective cAMP analogues were used to directly activate PKA, Epac or both. Isoproterenol a β-Adrenergic receptor (β-AR) agonist, was used to stimulate cAMP/PKA/Epac signalling pathways. Proteomics showed age-related variation in networks concerned with β-adrenergic signalling, Ca2+ homeostasis, mitochondrial regulation, and antioxidant activity. Simultaneous activation of PKA & Epac conferred cardioprotection in whole heart and in cardiomyocytes across the age ranges. In mitochondria from both adult and 14-day hearts, perfusion with isoprenaline or activation of both PKA and Epac reduced MPTP sensitivity to Ca2+. Despite significant molecular and cellular developmental changes and vulnerability to I/R, direct or indirect β-AR signalling involving Epac and PKA activation protects against acute injury in both adult and immature hearts and cardiomyocytes and this is linked to reduced MPTP sensitivity to Ca2+.

Date of Award	1 Oct 2019
Original language	English
Awarding Institution	University of Bristol
Supervisor	Clive H Orchard (Supervisor) & Saadeh Suleiman (Supervisor)