Biology subjects

Nikolaev, V.

Publications and source records attributed to Nikolaev, V..

4 recordsLinked to original sources

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↗

bioRxiv2026

Neprilysin inhibition reduces microtubule detyrosination in cardiomyocytes through a cGMP-PRKG1-VASH1 axis

Microtubule detyrosination and re-tyrosination on the C-terminus of -tubulin are mediated by the vasohibin (VASH)-small vasohibin-binding protein (SVBP) complex and tubulin tyrosine ligase (TTL), respectively. Elevated levels of detyrosinated -tubulin (dTyr-tub) are observed in heart failure, and reducing this modification improves cardiac function, suggesting that clinically used heart failure therapies may modulate microtubule detyrosination. We investigated whether sacubitrilat and valsartan, the active components of the angiotensin receptor-neprilysin inhibitor LCZ696, influence dTyr-tub levels in endothelin-1 (ET1)-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While both sacubitrilat and valsartan prevented hypertrophy, only sacubitrilat prevented ET1-induced dTyr-tub accumulation. RNA sequencing revealed that sacubitrilat normalized several ET1-induced dysregulated pathways. Sacubitrilat slightly increased cyclic guanosine 3,5-monophosphate (cGMP) levels and lowered dTyr-tub, whereas inhibition or knockdown of the cGMP-dependent protein kinase 1 (PRKG1) increased dTyr-tub level. Mechanistically, PRKG1 alpha phosphorylated native VASH1. Incubation of microtubules with the VASH1-SVBP complex containing wild-type VASH1 increased detyrosination, while incubation of the complex containing a VASH1 phosphomimic, in which seven C-terminal serine residues were mutated to glutamate (VASH1-7E) did not. Consistently, overexpression of VASH1-7E gave rise to lower dTyr-tub level than overexpression of a non-phosphorylatable form of VASH1 (VASH1-7A) in hiPSC-CMs deficient in VASH1. In conclusion, these findings identify a cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes. Our work provides mechanistic insight into how neprilysin inhibition may contribute to therapeutic benefit in heart failure. One Sentence SummaryWe establish a neprilysin-cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes.

cell biology↗

bioRxiv2023

The phosphodiesterase 2A regulates lymphatic endothelial development via cGMP-mediated control of Notch signaling

During vascular development endothelial junctions mature and vessel integrity is established to form the endothelial barrier. The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we uncover the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic, but not blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, while large blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, while cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to downregulation of NOTCH signaling. Vice versa, DLL4-induced NOTCH activation restores junctional maturation in PDE2A-deficient lymphatic endothelial cells. Our data demonstrate that PDE2A selectively modulates a crosstalk between cGMP and NOTCH signaling to finetune lymphatic development and suggest that PDE2A may be a druggable target to control lymphatic leakage and regeneration.

developmental biology↗

bioRxiv2019

Direct monitoring of cAMP at the cardiac ryanodine receptor using a novel targeted fluorescence biosensor mouse

RationaleCyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon {beta}-adrenergic receptor ({beta}-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating the cAMP-dependent protein kinase (PKA) phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac ryanodine receptor type 2 (RyR2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum (SR) often occurring upon RyR2 hyperphosphorylation by PKA and calcium/calmodulin-dependent kinase.\n\nObjectiveWe sought to establish a real time approach capable of directly visualizing cAMP and its pathological changes in the vicinity of RyR2 by generating a proper targeted biosensor and transgenic mouse model to express it in adult cardiomyocytes.\n\nMethods and ResultsWe generated transgenic mice expressing a novel targeted fluorescent biosensor for RyR2-associated cAMP in adult mouse cardiomyocytes. In healthy cardiomyocytes, {beta}1-AR but not {beta}2-AR stimulation strongly increased local RyR2-associated cAMP levels. However, in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of phosphodiesterases (PDEs) 2, 3 and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable {beta}2-AR-induced cAMP signals, increased SR calcium leak and arrhythmia susceptibility.\n\nConclusionsOur new targeted biosensor expressed in transgenic mice can visualize cAMP levels in the vicinity of cardiac RyR2 in healthy and diseased cardiomyocytes. In the future, this novel biosensor can be used to better understand alterations of RyR2-associated cAMP in cardiovascular diseases and local actions of new therapies.

cell biology↗