Figure 2. Depletion of PHD2/3 leads to abnormal activation of CaMKII and increases cardiomyocyte apoptosis induced by ISO.

(A) Mice lacking PHD2 and PHD3 have elevated CaMKII activation compared with WT mice. Phd2/3^fl/fl Cre^+/– and Phd2/3^fl/fl Cre^–/– mice were i.p. injected with tamoxifen once daily for 5 days. Mice were then treated with ISO or PBS for 6 hours on day 7 after the first tamoxifen injection. Heart lysates were immunoblotted with the indicated antibodies. Each lane represents heart lysate from 1 mouse. (B and C) Deletion of PHD2 and PHD3 potentiates ISO-induced apoptosis in cardiomyocytes. Male Phd2/3^fl/fl Cre^+/– and Phd2/3^fl/fl Cre^–/– mice were i.p. injected with tamoxifen once daily for 5 days, followed by ISO infusion with miniosmotic pumps (20 mg/kg/d) for 2 days. Hearts were harvested and fixed for H&E or TUNEL staining (B). Graph in C shows quantitative analyses. n = 5, P < 0.01, 2-tailed Student’s t test. (D and E) Primary isolated cardiomyocytes deficient in PHD2 and PHD3 were also sensitized to ISO-induced apoptosis. Phd2/3^fl/fl cardiomyocytes were isolated and infected with Ad-Cre or LacZ for 2 days and then treated with PBS or ISO for another 24 hours. Myosin and TUNEL staining was then performed. Representative images are shown in D. Quantitative analysis of TUNEL staining from 3 experiments is shown in E. *P < 0.01, 2-way ANOVA. Scale bars: 50 μm (B), 20 μm (D).