Fig. 1: cAMP generation at the Golgi distinctly regulates cardiomyocyte relaxation in neonatal mouse cardiomyocytes.
From: Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools
a, Illustration of the roles of subcellular cAMP/PKA signaling hubs in regulating cardiomyocytes contraction/relaxation. Blue light stimulation of Golgi-bPAC (TGNP-bPAC) generates cAMP to increase PLB phosphorylation and promote cardiac muscle relaxation b, Representative images of Golgi-bPAC (red) and Golgi marker (green), visualized by SNAP and GM130 antibodies and DAPI staining. Scale bar = 10 μm. n = 14, 2 biological replicates c, cAMP generation mediated by Golgi-bPAC in neonatal cardiomyocytes. Cells were stimulated with blue light for 3 min and 5 min or FSK (20 μM) for 5 min and then 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. n = 8 and 6 biological replicates form Golgi-bPAC and FSK-treated cardiomyocytes, respectively. d, Representative phosphorylation profiles of RyR2, TnI and PLB induced by Golgi-bPAC in mouse neonatal cardiomyocytes. The protein levels of pRyR2 Ser2808, pTnI Ser23/Ser24 and pPLB Ser16/Thr17 were analyzed in the Golgi-bPAC-expressing mouse neonatal cardiomyocytes kept in the dark or exposed to 0.34 μW cm−2 or 3.2 μW cm−2 blue light for 3 min. The protein level of Golgi-bPAC was analyzed using the SNAP antibody. The protein level of CSQ2 was used as a loading control. e, The band intensities of pRyR2, pTnI and pPLB were normalized to CSQ2 intensity and then reported as a percentage of the highest value in the groups. The quantified data from different experiments are presented as mean ± s.e.m. The P values were calculated by one-way ANOVA. n = 7 biological replicates. f, The subcellular localization of pRyR2, pTnI, PLB and pPLB upon stimulation of Golgi-bPAC with 0.34 μW cm−2 blue light in mouse neonatal cardiomyocytes. The protein localizations of pRyR2 Ser2808, pTnI Ser23/Ser24, total PLB and pPLB Ser16/Thr17 antibodies were visualized (green) with Golgi marker stained by GM130 (red). n = 25, 3 biological replicates. Scale bar = 10 μm.
