Ischaemic Preconditioning Protects Cardiomyocytes from Anthracycline-Induced Toxicity via the PI3K Pathway

Figures

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  Fig. 1. a The protective effect of simulated ischaemic preconditioning (sIPC) against a range of doxorubicin (Dox) concentrations. Primary adult rat cardiomyocytes were subject to 18 h Dox treatment at the indicated concentrations, with or without preceding simulated ischaemic preconditioning (sIPC). sIPC prevented the increase in cell death from 7.5 or 10 μM Dox (** p < 0.01, *** p < 0.001 n = 5). b Inhibiting PI3K/Akt but not MAPK/ERK1/2 eliminated sIPC-mediated protection against cell death caused by 18 h exposure to 7.5 μM doxorubicin. Cells were treated with LY294002 to inhibit PI3K/Akt or PD98059 to inhibit MAPK/ERK1/2, throughout exposure to sIPC and Dox as in panel a. (*** p < 0.001 n = 5)

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  Fig. 2. a Western blot analysis demonstrated that sIPC activated PI3K/Akt in cardiomyocytes, and that LY294002 (LY) inhibited this activation. Insulin, used as a positive control, strongly activated PI3K/Akt, and this was decreased by LY294002 treatment (n = 3). b Western blot analyses for MAPK/ERK1/2 similarly revealed increased phosphorylation in response to sIPC and to insulin respectively, which was abrogated by treatment with PD98059 (n = 3)

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  Fig. 3. a A ROS scavenger (NAC) did not affect Dox-induced cardiomyocyte-death. Cardiomyocytes were exposed to 18 h 7.5 μM Dox or 1 μM H2O2 in the presence or absence of 7.5 μM NAC, prior to analysis of cell death. NAC rescued cardiomyocytes from H2O2 toxicity, but had no effect on Dox-induced death (n = 3, ** p < 0.01, *** p < 0.001)

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  Fig. 4. a Exposure to doxorubicin did not alter the sensitivity of the mPTP to oxidative stress. Cardiomyocytes were exposed to 1 μM CsA or 7.5 μM Dox for 18 h, before loading with a photo-sensitising concentration of 3 μM TMRM and subjecting cells to laser-induced oxidative stress. The time to mPTP opening was measured and was significantly increased by CsA in control cells (CsA) but not in those cells that had been exposed to Doxorubin (Dox + CsA) (p < 0.05, n = 7). b 1 μM CsA did not protect cardiomyocytes against exposure to 18 h 7.5 μM Dox. Cell death was measured at the end of 18 h with vehicle (Con) or Dox (p < 0.05, n = 5)

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  Fig. 5. a 2 μM doxorubicin (Dox) increased cell death in HeLa cancer cells significantly, but preconditioning did not protect them (n = 5). b Akt was not phosphorylated in response to sIPC in HeLa cells