Mechanism Of α_1B-adrenergic Receptor Activation In Cardiac Hypertrophy And Heart Failure

Cardiovascular disease and cerebrovascular disease are collectively known as Cardiovascular disease.They include hypertension,coronary atherosclerotic heart disease（coronary heart disease）,hyperlipidemia,cerebral hemorrhage,cerebral infarction,angina pectoris and myocardial infarction.With the improvement of living standards,the incidence of cardiovascular and cerebrovascular diseases is increasing year by year.Myocardial hypertrophy is an independent risk factor of cardiovascular disease and the main reason for the increasing morbidity and mortality of cardiovascular disease.α_1B-adrenergic receptor(adrenergic receptors,α_1B-AR)excessive activation can lead to the occurrence and development of myocardial hypertrophy.However,the pathogenesis of myocardial hypertrophy remains unclear.It is speculated to be closely related toα_1B-AR activation and its mediated signaling pathway.Therefore,it is of great theoretical and practical significance to clarify the key regulatory factors ofα_1B-AR activation in the clinical study of the occurrence of cardiac hypertrophy and the development of drugs to reverse cardiac hypertrophy.α_1B-AR is a classic of G protein coupled receptor（G protein-coupled receptors,GPCRs）,the structure is one of a single polypeptide chain,contains a extracellular amino terminal（N terminal）,7 from 22 to 28 hydrophobic amino acid composition of alpha helix formation of transmembrane domain structure,and one of intracellular carboxyl terminal（C terminal）.However,the C terminal ofα_1B-AR is a unique domain,and important phosphorylation sites are located in the C terminal region ofα_1B-AR.The mechanism by which C-terminal truncation affects biological activity and physiological function ofα_1B-AR is still unclear.This study intends to construct 7 C-terminal truncatedα_1B-AR active probes to elucidate the mechanism of C-terminal truncation affecting biological activity ofα_1B-AR from the aspects of fluorescence resonance energy transfer（FRET）changes,receptor dimerization and downstream ERK activity changes.Meanwhile,the effect of C-terminal truncation ofα_1B-AR on the physiological function of myocardial cells was studied in an in vitro model of myocardial mast cells.Results showed that FRET molecular probes with different lengths at the C-terminal played different roles in FRET signal change and activation of the downstream ERK1/2 pathway.When the length of C-terminal is less than 30aa,there is almost no FRET signal change or ERK activation.When the length of C-terminal was between 50aa and 90aa,significant FRET signal changes and ERK activation could be detected.When the C-terminal length was 90aa,FRET signal change was significantly reduced.Asα_1B-AR-C-tail-90aa retains all important phosphorylation sites at the C-terminal,agonists are able to induce time-dependent changes in signal and increased ERK activation inα_1B-AR-C-tail-90aa FRET.In addition,we also found that agonist-induced ERK activation ofα_1B-AR-C-tail-90aa is PKC-orβ-arrestin 2-dependent and EGFR-,SRC-,or endocytosis-independent.Thus,changes in the C-terminal length of theα_1B-adrenergic receptor play an important role in receptor activation and receptor function.Further,we investigated the effect ofα_1B-adrenergic receptor dimerization on receptor activation.First,we built the molecules probe with a mutation can’t combine agonistα_1B-AR(α_1B-D125A),then study the influence of the homologous dimer on receptor function.We found thatα_1B-AR-WT/α_1B-D125A homologous dimers formation can make up for theα_1B-D125A defects cannot be effectively combined with agonist,apparent recovery ofα_1B-D125A molecules probe biological activity.At the same time,the formation ofα_1B-L151D/α_1B-D125A homologous dimer can also make up for the defect thatα_1B-D125A cannot effectively bind with agonist and partially restore the biological activity ofα_1B-adrenergic receptor molecular probe.Moreover,the formation of CXCR4-WT/α_1B-D125A heterodimer significantly restored the biological activity ofα_1B-adrenergic receptor molecular probe.The above results indicate that homologous or heterodimer can affect the activation of the receptor.In addition,the effect ofα_1B-AR dimerization on the physiological function of cardiac myocytes was preliminarily studied in an in vitro model of cardiac mast cells.Results showed thatα_1B-AR dimerization can significantly promote the expression ofβ-MHC protein and mRNA,indicating thatα_1B-AR dimerization can promote the occurrence of cardiac hypertrophy.Here,we systematically studied the factors affecting activation ofα_1B-AR,including C-terminal truncation and receptor dimerization.This study provides a theoretical basis for elucidation of the molecular mechanism ofα_1B-AR activation in myocardial hypertrophy and a new strategy for the development of cardiovascular drugs with lower side effects.