Microtubule-mediated regulation of β2AR translation and unction in failing hearts

Background: Beta-1 adrenergic receptor (β 1 AR)- and Beta-2 adrenergic receptor (β 2 AR)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac β-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that β-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. Methods: The localization pattern of β-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on β-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible β-AR translation sites in cardiomyocytes. The mechanism by which β-AR mRNA is redistributed post–heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post–myocardial infarction and detubulated cardiomyocytes. Results: β 1 AR and β 2 AR mRNAs show differential localization in cardiomyocytes, with β 1 AR found in the perinuclear region and β 2 AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β 2 AR transcripts toward the perinuclear region. The close proximity between β 2 AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of β 2 AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both β 1 AR and β 2 AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to β-AR mRNA redistribution and impaired β 2 AR function in failing hearts. Conclusions: Asymmetrical microtubule-dependent trafficking dictates differential β 1 AR and β 2 AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 β-ARs on the plasma membrane. The localization pattern is altered post–myocardial infarction, resulting from t-tubule remodeling, leading to distorted β 2 AR-mediated cyclic adenosine monophosphate signaling

Ticagrelor inverse agonist activity at the P2Y 12 receptor is non-reversible versus its endogenous agonist adenosine 5´-diphosphate.

BACKGROUND AND PURPOSE: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y 12 receptor (P2Y 12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y 12 R. EXPERIMENTAL APPROACH: Studies were performed in human platelets, with P2Y 12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y 12 R activation. KEY RESULTS: Initial studies revealed that a range of P2Y 12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y 12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y 12 R function. The P2Y 12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y 12 R than other P2Y 12 R ligands. CONCLUSION AND IMPLICATIONS: Ticagrelor binding to P2Y 12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y 12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures. </p