Remodelling of cAMP dynamics within the SERCA2a microdomain in heart failure with preserved ejection fraction caused by obesity and type 2 diabetes

Abstract

Hearth failure was traditionally considered a disease in which the heart is unable to pump enough blood to support the needs of the body. However, we now understand that there are multiple types of heart failure, traditional heart failure with reduced ejection fraction (HFrEF), heart failure with middle ejection fraction (HFmEF), and heart failure with preserved ejection fraction (HFpEF) which is the focus of this thesis. HFpEF is characterised by the presence of diastolic dysfunction with a normal ejection fraction. Several risk factors may induce HFpEF, with obesity and type 2 diabetes (T2D) being among the main risk factors, independent of hypertension and increasing age. With the incidences of obesity and T2D increasing all over the world and lack of effective therapies to treat HFpEF, we are in urgent need to understand the pathophysiology of HFpEF. The proper regulation of calcium cycling in the heart is essential to maintain both systolic and diastolic function, while many proteins are involved in these processes, sarcoplasmic reticulum (SR) calcium ATPase 2a (SERCA2a) is particularly relevant in context of HFpEF as it regulates diastolic calcium reuptake into the SR. The activity of SERCA2a is inhibited by phospholamban (PLN), with this inhibitory action relieved upon phosphorylation of PLN by protein kinase A (PKA). This process is initiated via beta-adrenergic receptors (β-ARs), which are a class of the seven transmembrane domain G protein-coupled receptor family responsible for stimulating the production of 3',5'-cyclic adenosine monophosphate (cAMP), a vital second messenger required for cell function that activates PKA and exchange protein directly activated by cAMP (Epac). cAMP actions are inhibited in part, by the enzymatic activity of phosphodiesterases (PDEs) which hydrolyse and reduce the levels of cAMP. Altered phosphorylation of PLN and/or SERCA2a activity have been reported in the obese and T2D heart and are associated with the presence of diastolic dysfunction. However, the mechanisms inducing these alterations in PLN/SERCA2a activity remain unclear. We hypothesize that abnormal cAMP dynamics within the SERCA2a microdomain lead to altered control of PLN phosphorylation and subsequent SERCA2a activity, promoting the development of diastolic dysfunction in obesity and T2D induced HFpEF. In order to investigate β-AR/cAMP/SERCA2a/PLN microdomain remodelling in a mouse model of obesity and T2D HFpEF, we generated a novel mouse line on the leprdb background that stably expresses the fluorescence Resonance Energy Transfer (FRET) biosensor Epac1-PLN in the heart. The presence of a HFpEF phenotype was confirmed via echocardiography and cardiac morphology assessment. Ventricular cardiomyocytes (CMs) were isolated from both healthy (db/+) and obese and T2D (db/db) mice in order to assess real time cAMP and calcium handling dynamics in single living cells, via FRET and IonOptix experiments respectively. This work was supported by additional investigation into β-AR/cAMP signalling and mechanisms of SERCA2a microdomain regulation, via western blot, co-immunoprecipitation and qPCR experiments as well as “rescue” experiments with the use of adenoviral gene transfer in isolated CMs. In this work, we uncovered a desensitisation of β1-AR stimulated cAMP amplitudes and substantially increased β2-AR cAMP amplitudes within the SERCA2a microdomain in db/db CMs. Importantly these changes in cAMP levels were accompanied by blunted β1-AR induced acceleration of SR calcium re-uptake and for β2-AR stimulation to result in an acceleration calcium re-uptake and time for sarcomere relaxation. This newly identified role for β2-AR induced lusitropic effects was observed to be mediated by a loss of PDE4 coupling with the β2-AR subtype and direct loss of PDE4B and PDE4D association within the SERCA2a microdomain, with PDE4B over expression abolishing β2-AR induced cAMP amplitudes within the SERCA2a microdomain. These findings provide key insights into the pathophysiology of obesity and T2D induced HFpEF at the microdomain level, providing integral clues for future study and treatment.