Nanodomain cAMP signalling in cardiac pathophysiology: potential for developing targeted therapeutic interventions.

3', 5'-cyclic adenosine monophosphate (cAMP) mediates the effects of sympathetic stimulation on the rate and strength of cardiac contraction. Beyond this pivotal role, in cardiac myocytes cAMP also orchestrates a diverse array of reactions to various stimuli. To ensure specificity of response, the cAMP signaling pathway is intricately organized into multiple, spatially confined, subcellular domains, each governing a distinct cellular function. In this review, we describe the molecular components of the cAMP signalling pathway, how they organized are inside the intracellular space and how they achieve exquisite regulation of signalling within nanometer-size domains. We delineate the key experimental findings that lead to the current model of compartmentalised cAMP signaling and we offer an overview of our present understanding of how cAMP nanodomains are structured and regulated within cardiac myocytes. Furthermore, we discuss how compartmentalized cAMP signaling is affected in cardiac disease and consider the potential therapeutic opportunities arising from understanding such organization. By exploiting the nuances of compartmentalized cAMP signaling, novel and more effective therapeutic strategies for managing cardiac conditions may emerge. Finally, we highlight the unresolved questions and hurdles that must be addressed to translate these insights into interventions that may benefit patients.

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signalling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the α-1 agonist phenylephrine (PE, 3 µM) resulted in a FRET change 21.20 ± 7.43 % and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 µM) resulted in a peak of 20.31 ± 6.74 %. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5µM) or Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. These data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets.

Investigating G-protein coupled receptor signalling with light-emitting biosensors.

G protein-coupled receptors (GPCRs) are the most frequent target of currently approved drugs and play a central role in both physiological and pathophysiological processes. Beyond the canonical understanding of GPCR signal transduction, the importance of receptor conformation, beta-arrestin (ß-arr) biased signalling, and signalling from intracellular locations other than the plasma membrane is becoming more apparent, along with the tight spatiotemporal compartmentalisation of downstream signals. Fluorescent and bioluminescent biosensors have played a pivotal role in elucidating GPCR signalling events in live cells. To understand the mechanisms of action of the GPCR-targeted drugs currently available, and to develop new and better GPCR-targeted therapeutics, understanding these novel aspects of GPCR signalling is critical. In this review, we present some of the tools available to interrogate each of these features of GPCR signalling, we illustrate some of the key findings which have been made possible by these tools and we discuss their limitations and possible developments.

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.