Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy

Abstract

Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1. Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

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

“…One illustrative example is milrinone, a non-isoform specific phosphodiesterase 3 (PDE3)-selective inhibitor, which is indicated for the treatment of acute, refractory heart failure, and leads to improvement in symptoms in the short-term but a long-term increase in mortality ( Packer et al, 1991 ). Using an isoform specific PDE3 inhibitor may avoid this problem ( Subramaniam et al, 2023 ) and using the tools outlined in this review during the drug discovery process to determine the effect on specific cAMP nanodomains could minimise similar unanticipated off-target effects in the future.…”

G Protein–Coupled Receptor Signaling: New Insights Define Cellular Nanodomains

“…The resident PDE's identity, activation/inhibition status, and conditions that promote its recruitment or release from a specific location determine the cAMP level within that domain and the cellular response to the extracellular stimulus. Ultimately, the localization of different PDE isoforms in a cell determines the subcellular topography of the cAMP nanodomains (174), which is expected to be dynamic and to undergo remodeling in pathological conditions (14,(175)(176)(177).…”

Compartmentalised cAMP signalling in the primary cilium

“…cAMP can diffuse freely through the TZ, so it is no surprise that its level ultimately equilibrates in the cilium and cytosol. However, the cAMP pathway does rely on the tight spatio-temporal compartmentalisation of its signal, and far less-isolated cAMP subdomains than the primary cilium have been defined ( Surdo et al, 2017 ; Anton et al, 2022 ; Subramaniam et al, 2023 ). For example, at the plasma membrane of cardiac myocytes adrenergic signalling activates PKA to phosphorylate several downstream substrates such as Phospholamban (PLB), Troponin I (TnI), and β2AR, resulting in increased strength of contraction.…”