Introduction: Anthracyclines (ANTC) are potent chemotherapy agents, but may cause chronic irreversible cardiac failure. Recent data suggest cardiac damage due to ANTC begins early and may be a continuum. Cardioprotective modalities effective against acute injury may therefore provide prolonged benefit. Hypoxic Preconditioning (HP), i.e., subjecting cardiomyocytes to sub-lethal hypoxia and reoxygenation before a prolonged lethal ischaemic insult, protects cardiomyocytes by activating the pro-survival Reperfusion Injury Salvage Kinase (RISK) pathway. We examined the hypothesis that HP can protect cardiomyocytes against toxicity of the ANTC compound Doxorubicin (Dox). / Methods: A toxic concentration of Dox was identified in an in vitro model of cardiotoxicity established in isolated adult rat ventricular myocytes (ARVM), using Propidium iodide (PI) permeability and altered morphology including cell-membrane blebbing as markers of celldeath. An in vitro hypoxic preconditioning (HP) protocol was characterised, and the effect of this HP-stimulus on cell-death with subsequent Dox-treatment was assessed. Induction of the RISK pathway by HP was investigated by pre-inhibiting the Phosphoinositide 3-kinase (PI3K)/Akt cascade and the Mitogen Activated Protein Kinase (MAPK) Extracellular signal-regulated Kinase (ERK) 1/2 pathway with 10µM LY294002 (LY) and 30µM PD98059 (PD) respectively. The role of oxidative stress in Dox-toxicity was probed using N-acetyl cysteine, a known Reactive Oxygen Species (ROS)-scavenger. Fluorescence of 30 nM Tetramethylrhodamine methyl ester (TMRM) was used to assess the mitochondrial transmembrane potential (ΔΨm). Laser-induced ROS generation with 3 µM TMRM was used to assess mitochondrial permeability transmembrane pore (mPTP) induction after Dox-treatment, with 1µM Ciclosporine A (CsA) as a positiveprotective control against mPTP induction. Dox-induced cell-death was also reassessed as an agent to protect against mPTP induction. / Results and Discussion: 7.5 μM Dox resulted in significant increase in cell-death (p<0.05) after 18h (44.2 + 4.7% Dox vs 18.7 + 1.5 % Control). HP protected cardiomyocytes from Dox (35.4 ± 1.7% Dox; 14.7 ± 1.5% Dox HP). LY, but not PD, abrogated this HP-induced protection (Dox HP: 16.9 + 1.5%; Dox HP LY: 38.5 + 3.3%; Dox HP PD: 15.9 + 1.3%). ROS-scavenging failed to rescue cardiomyocytes from Dox-toxicity. Dox failed to alter ΔΨm or mPTP-induction. However, although the protective effect of presence of CsA on mPTP-induction was abrogated in cells pre-treated with Dox, concomitant treatment with dox and CsA could not protect ARVMs from dox-toxicity. / Conclusions and future directions: HP protects cardiomyocytes against cell-death due to Dox. Cell-death data suggest this protection is mediated via the PI3K/Akt pathway. The role of MAPK ERK 1/2 against Dox-toxicity in vitro is unclear. Dox-toxicity appears to be independent of oxidative stress, variations in ΔΨm, or vulnerability of the mPTP-induction. Further work to delineate mechanisms of toxicity, and the downstream components of HP-induced protection is necessary
