Fig 2: ATF4 expression in H 2 O 2 and thapsigargin treated cardiomyocytes and in electrically stimulated cardiomyocytes. (a) Real-time PCR of ATF4 mRNA expression in cultured HL-1 cardiomyocytes in response to treatment with 0, 0.05, or 0.1 µl/ml H2O2 (n = 3), real-time PCR of ATF4 mRNA expression in cultured cardiomyocytes in response to treatment with 1 µM thapsigargin (n = 3), and real-time PCR of cardiomyocytes stimulated electrically with 4 Hz for 20 h (n = 3). Representative western blots showing protein expression of ATF4 in response to H2O2 treatment (b) and in response to thapsigargin treatment (c). (d) Cardiomyocytes were electrically stimulated with 4 Hz for 20 h and ATF4 protein expression was detected. (e) MTT assay displaying viability of cardiomyocytes treated with H2O2 (n = 3), 1 µM thapsigargin (n = 3), and paced HL-1 cardiomyocytes (n = 5). * indicates p < 0.05.

Fig 3: Gene expression microarray analysis. (a) The number of genes compared to control (GFP) that are up- (green) and downregulated (red) in cardiomyocytes overexpressing ATF4 wild type (wt) or ATF4?RK with and without electrical stimulation (fold change = 1.2 or =0.83, and p value = 0.05). (b) Pathways enriched in electrically stimulated ATF4wt overexpressing HL-1 cardiomyocytes versus unstimulated ATF4wt overexpressing HL-1 cardiomyocytes (upper panel), electrically stimulated ATF4wt overexpressing cardiomyocytes versus stimulated GFP overexpressing cardiomyocytes (middle panel), and ATF4wt versus GFP overexpressing cardiomyocytes (lower panel).

Fig 4: Immunohistological analysis of ATF4 expression in atrial tissue obtained from patients with sinus rhythm and with atrial fibrillation. (a) Representative images of HE staining and staining with antibodies directed against ATF4 (dark brown). Bar, 50 µm. (b) Percentage of ATF4 positive nuclei in histological specimen obtained from patients in sinus rhythm compared to patients with atrial fibrillation. * indicates p < 0.05.

Fig 5: ATF4 overexpression in fibroblasts. (a) Representative western blots showing ATF4 expression in murine fibroblasts overexpressing GFP, ATF4wt, or ATF4?RK. (b) Cell proliferation was assessed by BrdU assay (n = 4). * indicates p < 0.05.