Fig. 4: β1AR autoantibody specifically activates plasma membrane-localized β1AR and regulates RyR2 phosphorylation in neonatal mouse cardiomyocytes.
From: Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools
a, The amino acid similarity between the second extracellular loop of β1AR and an autoantibody (AAb) epitope region (blue color labeled) found in patients with dilated cardiomyopathy (DCM; top), representative images of HEK293 cells transfected with SNAP-tagged β1AR and nanobody-based biosensor for βARs (Nb80-GFP) before and after 100 nM AAb stimulation (bottom). Nb80-GFP is recruited to the plasma membrane-localized β1AR after 2.5 min (arrowheads). n = 35, 3 biological replicates, Scale bar = 10 μm. b, cAMP generation mediated upon 33 nM and 100 nM AAb treatment in neonatal cardiomyocytes. Cells were stimulated for 15 min and lysed for direct cAMP determination by ELISA. cAMP concentrations were normalized to the relative protein concentrations in the cell lysate of each sample. The quantified data are represented as mean ± s.e.m. The P values were calculated by one-way ANOVA, C, control group. n = 6 biological replicates. c, Representative western blots of RyR2, TnI and PLB phosphorylation profiles regulated by β1AR AAb in mouse neonatal cardiomyocytes. Mouse neonatal cardiomyocytes were treated with 10 nM and 33 nM β1AR AAb or 20 μM FSK for 15 min. d, The protein levels of pRyR2 Ser2808, pTnI Ser23/Ser24 and pPLB Ser16/Thr17 were analyzed. The protein level of pRyR2 was normalized with the protein level of CSQ2 and then reported as a percentage of the highest value in the FSK-treated group (20 μM). The quantified data from different experiments were presented as mean ± s.e.m. The P values were calculated by one-way ANOVA. n = 5 biological replicates.
