Β-adrenergic signalling in the neonatal ventricular cardiomyocytes of the popdc2 w188X mutant and characterization of the popdc2 w188X mutant mouse thesis submitted for the degree of doctor of philosophy by
File(s)
Author(s)
Gruscheski, Lena
Type
Thesis or dissertation
Abstract
The β-adrenergic receptor - cyclic adenosine 3’,5’-monophosphate (βAR-cAMP) signalling transduces the extracellular sympathetic stimulus to intracellular effector proteins to adapt cardiac output during stress or exercise. The transmembrane Popeye domain containing (POPDC) protein family represent a novel class of cAMP effectors of which POPDC1 and POPDC2 are abundantly expressed in the working myocardium and in the cardiac conduction system. Dysregulation of POPDC expression has been associated with a defective heart rate response to stress. A nonsense variant of POPDC2 (POPDC2W188X) discovered in patients was selected to generate a knock-in mouse model to study the role of POPDC proteins in the βAR-cAMP-mediated stress response of cardiac pacemaking and conduction.
POPDC2W188X mutant tissue and cultured neonatal mouse cardiomyocytes (NMCM) displayed a loss of Popdc2 transcripts and a loss and mislocalisation of POPDC1 proteins. To understand how this loss affected sinoatrial pacemaking, isolated POPDC2W188X mutant hearts were studied by Langendorff perfusion. The POPDC2W188X mutant demonstrated an increase in heart rate at baseline, and a tachy-bradycardia in response to βAR activation. A dysregulation of β2AR signalling was identified in cultured POPDC2W188X mutant NMCM, resulting in low cAMP nanodomain signalling as revealed by different Förster resonance energy transfer sensors following sympathetic activation but not at baseline. POPDC proteins appear to be specifically involved in βAR signalling as cAMP nanodomain signalling was not impaired in mutant NMCM when the prostaglandin receptor was stimulated. Likewise, direct stimulation of adenylyl cyclases by forskolin was not impacted by the POPDC2W188X mutation. The phenotype was rescued by pertussis toxin suggestive of abnormal Gi (inhibitory G-protein)-coupling of the β2AR in mutant NMCM. Interfering with the interaction of phosphodiesterase 4A and POPDC1 using a disruptor peptide in wild type NMCM was shown to mimic the POPDC2W188X phenotype. These results start unravelling the intricate role of POPDC proteins within the βAR signalling complexes.
POPDC2W188X mutant tissue and cultured neonatal mouse cardiomyocytes (NMCM) displayed a loss of Popdc2 transcripts and a loss and mislocalisation of POPDC1 proteins. To understand how this loss affected sinoatrial pacemaking, isolated POPDC2W188X mutant hearts were studied by Langendorff perfusion. The POPDC2W188X mutant demonstrated an increase in heart rate at baseline, and a tachy-bradycardia in response to βAR activation. A dysregulation of β2AR signalling was identified in cultured POPDC2W188X mutant NMCM, resulting in low cAMP nanodomain signalling as revealed by different Förster resonance energy transfer sensors following sympathetic activation but not at baseline. POPDC proteins appear to be specifically involved in βAR signalling as cAMP nanodomain signalling was not impaired in mutant NMCM when the prostaglandin receptor was stimulated. Likewise, direct stimulation of adenylyl cyclases by forskolin was not impacted by the POPDC2W188X mutation. The phenotype was rescued by pertussis toxin suggestive of abnormal Gi (inhibitory G-protein)-coupling of the β2AR in mutant NMCM. Interfering with the interaction of phosphodiesterase 4A and POPDC1 using a disruptor peptide in wild type NMCM was shown to mimic the POPDC2W188X phenotype. These results start unravelling the intricate role of POPDC proteins within the βAR signalling complexes.
Version
Open Access
Date Issued
2024-02-29
Date Awarded
2025-07-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Brand, Thomas
Gorelik, Julia
Sponsor
British Heart Foundation
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)