Fig. 7

Arginine methylation of CaMKII at R9 and R275 is important for PRMT1-mediated suppression. a A schematic diagram of CaMKIId domain structure. b Identification of CaMKII methylation sites by mass spectrometry. His-tagged CaMKIId was coexpressed with PRMT1 in 293T cells and purified, followed by mass spectrometry analysis. The result indicated that arginine residues 9 and 275 of CaMKIId are methylated. c Asymmetric methylation levels of wild-type (WT) CaMKIId or arginine to alanine mutants at amino acid 9 or/and 275 (R9A, R275A, R9/275A). d Relative levels of asymmetric methylation of WT and mutants from experiments shown in c. N = 3. Data represent means ± SD. ^***P < 0.001, Student’s t-test. e Protein analysis of p-CaMKII levels of WT CaMKII or mutants transfected NRVM in the presence of PRMT1 overexpression. f Relative levels of p-CaMKII proteins from experiments shown in e. n = 3. Data represent means ± SD.^***P < 0.001, one-way ANOVA. g Representative images for immunostaining of NRVM cells transfected with MYC-tagged WT CaMKII, mutant (Red) and HA-tagged PRMT1 (magenta). Cardiomyocytes were labeled with α-Actinin (green). White dash line indicates cell border. Size bar: 50 μm. h Quantification of the cell surface area from three independent experiments as shown in g. The cell number plotted in the graph per each lane is as following: n = 133 (control); 142 (WT + control); 140 (WT + PRMT1); 137 (R9A + PRMT1); 142 (R275A + PRMT1); 143 (R9/275A + PRMT1). Data represent means ± SD. ^***P < 0.001, one-way ANOVA. i Immunoblotting for ANP expression in NRVM cells expressing WT or CaMKII T287D with PRMT1. j Quantification of ANP protein levels from experiments shown in i. n = 3. Data represent means ± SD. ^*P < 0.05, Student’s t-test. k A working model summarizing the regulatory mechanism of CaMKII activity and cardiac function by PRMT1-mediated methylation of CaMKII at R9 and 275