Characterization of early age-associated remodelling of cAMP-phosphodiesterases and β-adrenergic regulation of excitation–contraction coupling

Abstract

The most common form of heart failure (HF) in the elderly is HF with preserved ejection fraction (HFpEF), characterized by impaired filling and relaxation of the heart. Ageing is accompanied by an activation of the sympathetic nervous system and β-adrenergic (β-AR) desensitization, followed by reduced cardiomyocyte contractility, yet little is known about the early involved mechanisms. We aimed to study cAMP-phosphodiesterase (PDE) signaling changes upon β-AR stimulation in an early ageing model and its contribution to excitation–contraction coupling. Wistar rats at 3 and 15 months of age were used as young vs. early aged rat model. Ventricular myocytes were isolated and expression of senescent marker genes (p15/16/p21) and phosphodiesterases (PDEs) was assessed by qPCR. Total cAMP-PDE activity as well as PDE3 and PDE4 specific activities were measured by radioimmunoassay. Sarcomere shortening and Ca2+ transients were assessed in field-stimulated (1 Hz) cardiomyocytes loaded with the Ca2+ dye, Fura 2 (1 μM). In parallel, cells were infected with adenoviruses encoding the cytosolic cAMP FRET sensor, EpacSH187. Concentration-response curves to the β-AR agonist isoproterenol (Iso) were obtained. The early ageing phenotype of our model was demonstrated through a physiological increase in heart weight that was back to normal when divided by tibia length and through the absence of detectable p15/16/p21 levels. The expression of PDE2A, PDE3A, PDE4A, PDE4B and PDE4D at the transcript level was not modified in young vs. aged cardiomyocytes ( n = 7/group). Total PDE activity, as well as specific PDE3 and PDE4 activities tended to be increased in cardiomyocytes from aged vs. young rats, although it did not reach statistical significance ( n = 8/group). This was accompanied by a significantly diminished efficacy of Iso to increase cytosolic cAMP levels and Ca2+ transient amplitude in cardiomyocytes from old vs. young rats ( n = 5–9/group, P < 0.05, extra sum-of-squares F test). However, basal and Iso-stimulated sarcomere shortening were similar in cardiomyocytes from aged vs. young rats. Our data show that cardiomyocyte contractility is preserved in 15 months old rats despite decreased Ca2+ transients and suggest a precocious alteration of cAMP-PDEs at this early ageing stage. Further experiments are being pursued to depict the mechanisms underlying depressed Ca2+ transients and increased myofilament Ca2 + sensitivity in early aging.