Figure 3.

hCT1 engages a restricted activation of the intrinsic caspase-mediated cell death pathway. (A, B) Primary cardiomyocytes were treated for 1 h with control serum-free medium (Ctrl), hCT1 (0.5 nM), or PE (100 μM) in the presence or absence of the caspase 9 inhibitor (z-LEHD-fmk, 20 μM). Caspase 9 was significantly activated with hCT1 and PE versus control (n = 3; ^*P < 0.05 and ^**P < 0.01) and this was decreased in the presence of z-LEHD-fmk (n = 3; ^*P < 0.05). (C) Cardiomyocytes were treated with hCT1 (0.5 nM), PE (100 μM), or hCT1+PE for 15 min and 24 h and analyzed by western immunoblotting. hCT1 caused a moderate increase in caspase 3 activity compared to PE at 24 h. Caspase 3 activity also decreased with combined PE and hCT1 stimulation (arrows). GAPDH was the loading control. (D) Cardiomyocytes were transfected with reporter plasmids under the control of NF-κB or Mef2 promoters and luciferase activity was measured after treatment with: control serum-free medium (Ctrl), hCT1 (0.5 nM), PE (100 μM), or procaspase 3-activating compound 1 (PAC-1; 25 μM). hCT1 treatment resulted in NF-κB and Mef2 activation at 1 and 3 h versus control (n = 4; ^* P < 0.05 and ^**P < 0.01); and at 24 h, only NF-κB activity was sustained (n = 4; ^*P < 0.05). However, PE and PAC-1 treatment caused significant activation of Mef2 after 24 h (n = 4; ^**P < 0.01 and ^****P < 0.0001). Co-stimulation of hCT1/PE and hCT1/PAC-1 significantly reduced Mef2 activity compared to PE and PAC-1 alone (n = 4; ^****P < 0.0001 and ^*P < 0.05, respectively). (E, F) Similar procedure as in (A) above; however, with 24 h treatment. hCT1 and PE significantly increased cell area and the pro-hypertrophic marker ANP versus control (n = 3; ^***P < 0.001 and ^****P < 0.0001) and this was significantly attenuated in the presence of z-LEHD-fmk (n = 3;*P < 0.05 and ^**P < 0.01). (G, H) Cardiomyocytes were infected for 24 h with an adenovirus (AdV) encoding the caspase inhibitor, p35-AdV, prior to inducing hypertrophy with hCT1 or PE for 24 h. GFP-AdV was used as a control. p35-AdV significantly inhibited hCT1 and PE induced hypertrophy (n = 3; ^**P < 0.01 and ^****P < 0.0001, respectively) and inhibited ANP expression (n = 3; ^**P < 0.01 and ^*P < 0.05, respectively). Both p35-AdV and GFP-AdV were used at a mean of infectivity (MOI) of 20. (I-K) Similar procedure as in (E, F) above; however, casein kinase 2 (CK2) activity was blocked using TBBt (50 μM). CK2 inhibition significantly increased cell size and ANP expression (n = 3; ^*P < 0.05) while reducing length:width ratio (n = 3; ^**P < 0.01) when compared to hCT1 treatment alone.