Revuelta, D. | bioRxiv Science

bioRxiv2026

Submembrane geography dictates GPCR signalling compartmentation and physiological function

G-protein-coupled receptors (GPCRs) are key mediators of intercellular signalling and targets for the vast majority of current therapeutics. Numerous biochemical and biophysical studies have uncovered that individual GPCRs activated at different subcellular locations can mediate distinct functional effects due to compartmentalization of the downstream second messengers in subcellular nanodomains. However, the reasons why a single receptor subtype activated at the same subcellular compartment such as plasma membrane leads to various functional responses have remained poorly understood. Here, we used beta1-adrenergic receptor and cardiomyocytes as classical examples of a therapeutically relevant GPCR and highly compartmentalized cell, to study communication between membrane receptors and subcellular cAMP nanodomains based on a combination of nanoscale scanning ion conductance microscopy assisted local receptor activation with Foerster resonance energy transfer based live cell imaging of the cAMP pathway. Unexpectedly, this approach revealed that beta1-adrenoceptors located in cardiomyocyte T-tubular membrane are strongly coupled to the regulation of cardiac contractility, whereas receptors residing in the outer non-tubular membrane preferentially engage nuclear nanodomains linked to hypertrophy. This unique mechanism of spatially segregated signalling was maintained in failing rodent and human hearts, indicating that it is conserved across species and preserved in disease. Finally, we explored potential therapeutic targeting of this mechanism by generating submembrane-specific beta-blockers acting exclusively at the outer non-tubular membrane. These improved compounds could ameliorate hypertrophy and cardiac remodeling without reducing contractility in vitro and in vivo. Together, these findings uncover an unexpected level of spatial organization of GPCR signaling within the same subcellular compartment and show that not only its identity but also its submembrane location determines cellular signaling and functional response engaged by the same GPCR.

pharmacology and toxicology↗

bioRxiv2025

Targeting Troponin I Phosphorylation for Precision Treatment of Diastolic Dysfunction

BackgroundDefective cardiac relaxation (diastolic dysfunction) is prevalent in heart failure, particularly in the context of diabetes, obesity, hypertension, and ageing, and is associated with increased mortality. Yet there is no available treatment that directly targets this abnormal relaxation. Phosphorylation of troponin I by cAMP-dependent protein kinase A (PKA) promotes relaxation, but global PKA activation has widespread, undesirable effects. Here we investigated whether selectively enhancing troponin I phosphorylation could improve relaxation without engaging the broader PKA signalling network. MethodsTo resolve cAMP signalling at subcellular scales, we combined real-time cAMP measurements with FRET-based genetically encoded reporters targeted to defined nanodomains, alongside biochemical and genetic approaches. Protein-protein interaction surfaces were mapped using peptide-array technology, which guided the design of a disruptor peptide to displace specific interactions. The peptides effects on cardiomyocyte function were assessed in vitro using rodent and human cardiomyocytes with biochemical assays, real-time imaging, and work-loop analysis. In vivo effects were evaluated through echocardiography and haemodynamic measurements. ResultsWe identified a previously unrecognized regulatory mechanism in which the cAMP-hydrolyzing enzyme PDE4D9 interacts with troponin I and restricts local cAMP levels within a nanometre-scale domain, thereby selectively controlling troponin I phosphorylation. We found that PDE4D9-troponin I association is markedly increased in cardiac disease in both rodents and humans. A peptide that displaces PDE4D9 from troponin I in a mouse model of heart failure selectively enhances troponin I phosphorylation, accelerates cardiomyocyte relaxation, and prevents diastolic dysfunction, without compromising systolic contractility. ConclusionsThese findings reveal a highly specific regulatory nanodomain governing troponin I phosphorylation and points to a first-in-class, mechanism-based therapeutic strategy with the potential to address a major unmet contributor to heart failure. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe identify a previously unrecognized, nanometre-scale regulatory mechanism in which PDE4D9 binds troponin I to locally suppress cAMP, limit troponin I phosphorylation and attenuate relaxation. C_LIO_LIWe demonstrate that this mechanism becomes maladaptive in disease: PDE4D9-troponin I interaction is markedly upregulated in failing hearts in both rodents and humans, which can mechanistically explain the impaired cardiac relaxation. C_LIO_LIWe show that this interaction is druggable: a rationally designed peptide can selectively displace PDE4D9, restore localized cAMP-PKA signalling, and normalize myocardial relaxation without global cAMP perturbation. C_LIO_LIDisplacement of PDE4D9 from TPNI improves relaxation with no impact on the calcium transient amplitude and without adversely affecting systolic output and cardiac reserve. C_LI What Are the Clinical Implications?O_LIDiastolic dysfunction, a major cause of heart failure with no targeted treatments, may be amenable to therapies that modulate cAMP signalling with subcellular precision rather than global pathway modulation. C_LIO_LISelective disruption of PDE4D9-troponin I binding offers a mechanism-based approach with potential to restore diastolic function while minimizing off-target effects typical of GPCR-directed therapies. C_LIO_LIBy enhancing relaxation while preserving systolic function and cardiac reserve, this approach may overcome limitations of myofilament-targeted therapies currently under evaluation for diastolic dysfunction that exhibit negative inotropic effects. C_LIO_LIMore broadly, this work establishes a platform for targeting localized signalling domains, a concept that may extend beyond cardiovascular disease to other pathological conditions in which dysregulated cAMP contributes to dysfunction. C_LI

cell biology↗