CaMKIIγ advances chronic intermittent hypoxia-induced cardiomyocyte apoptosis via HIF-1 signaling pathway

Fig. 1. (A) The changes of rats BW in the Normoxia group and CIH group. (B) The average BW of rats in different time point. (C) The HW of rats in the Norxiama and CIH group. (D) The HW/BW ratio of two groups

Fig. 2. (A) HE staining in the CIH group and Normoxia group. (B) The echocardiography of rats in two groups. (C) Masson staining showed the muscle fibers were red, and the collagenous fibers were blue. (D) Sirius-red staining showed the muscle fibers were yellow, and the collagenous fibers were red. Representative photographs of rat heart slice (original magnification ×20)

Fig. 3. (A) The expression of CaMKIIγ protein in the Normoxia group and CIH group were detected by western blots. CaMKIIγ protein expression was significantly increased in the CIH group. (B) Quantitative CaMKIIγ protein analysis in two groups. (C) Representative immunohistochemical staining of CaMKIIγ in rats. Black arrows indicate CaMKIIγ expression in cardiomyocytes (Original magnification ×20). (D) Quantification of the CaMKIIγ positive cells in the Normoxia group and CIH group. (E) Representative immunofluorescence staining photographs among α-actinin (green), CaMKIIγ (red), and Dapi (blue), (original magnification×20). (F) Number of CaMKIIγ positive cells

Fig. 4. The expression of CaMKIIγ in H9c2 cells were detected by western blots and immunofluorescence staining. (A) Compared with the Control group, the CaMKIIγ expression in the CIH + 8 h group was markedly increased. (B) Tendency of CaMKIIγ protein expression in the Control group and CIH + 8 h group. (C) Representative immunofluorescence staining of CaMKIIγ (green) and mean fluorescence intensity analysis (D) in the Control group and CIH + 8 h group (original magnification×10)

Fig. 5. The apoptosis of cardiomyocytes in vivo after CIH. The immunohistochemical staining of Bcl-2 (A) and Bax (C) in rats (Original magnification×20). Compared with the Normoxia group, the Bcl-2 (B) expression in the CIH group was markedly decreased, however, the Bax (D) expression was notably increased. (E) TUNEL assay showed the cardiomyocytes apoptosis were mainly occurred in the nucleus. (F) Statistical results of positive cells. Black arrow indicate TUNEL-positive cells (Original magnification×20)

Fig. 6. CaMKIIγ advanced cardiomyocytes apoptosis. (A) Representative examples of TUNEL staining in H9c2 cells (Original magnification×40). White arrow indicate TUNEL-positive cells in H9c2 cells. (B) TUNEL positive H9c2 cells in the CIH + 8 h group are significant higher, compared with the Control group. (C) Representative examples of CaMKIIγ (green) with Caspase 3 (red) and Dapi (blue) staining. The co-localizations (yellow) of CaMKIIγ with Caspase 3 in H9c2 cells were observed (Original magnification ×40)

Fig. 7. CaMKIIγ regulated HIF-1 signaling pathway. HIF-1 expression in H9c2 cells were detected by western blots and immunofluorescence staining. (A) Western blot showed the HIF-1 expression was notably increased in the CIH + 8 h group. (B) Tendency in two groups. (C) Representative immunofluorescence staining of HIF-1 (green) and mean fluorescence intensity analysis (D) in the Control group and CIH + 8 h group (Original magnification×10). (E) Representative examples of CaMKIIγ (green) with HIF-1 (red) and Dapi (blue) staining. The co-localizations of CaMKIIγ with HIF-1 (yellow) were observed in H9c2 cells (Original magnification ×20)