Cardioprotective Properties of Humoral Factors Released From Rat Hearts Subject to Ischemic Preconditioning

Abstract

Myocardial protection can be achieved by transfer of coronary effluent from ischemically preconditioned to non-preconditioned hearts. This study was designed to test the hypothesis that preconditioned effluent from rat hearts purified by Sep-Pak C-18 cartridges could induce remote cardioprotection against ischemia/reperfusion (I/R) injury through the activation of protein kinase C signaling pathway. Buffer-perfused rat hearts were subject to 30 min ischemia and 60 min reperfusion. The myocardial I/R injury was assessed by postischemic contractile function recovery and infarct size. The protective effect of coronary effluent collected during ischemic preconditioning (IPC) was tested in non-preconditioned hearts in presence or absence of a PKC inhibitor, chelerythrine. Infarct size was 17 +/- 2% in preconditioned versus 37 +/- 1% in control hearts (P < 0.001). Hearts perfused with fresh preconditioned effluent had infarct sizes of 16 +/- 3% versus 36 +/- 1% in hearts treated with non-preconditioned effluent. The cardioprotective effect was lost when the effluent was left at room temperature during 24 h (infarct size, 40 +/- 3%) or heated to 70 degrees C (26 +/- 4%, P < 0.05) or 100 degrees C (39 +/- 1%, P < 0.001). The lyophilized effluent was stable for 30 days, and its purification in a Sep-Pak C-18 column resulted in a hydrophobic fraction that reduced the infarct size to 17 +/- 2% versus 38 +/- 2% for the hydrophilic fraction. Chelerythrine (100 microM) inhibited the reduction of infarct size induced by IPC (35 +/- 4%) or hydrophobic fraction (37 +/- 3%). Recovery of the contractile function at reperfusion was higher in preconditioned group (74 +/- 6% versus 17 +/- 7% in control, P < 0.001) and hydrophobic fraction (66 +/- 7% versus 8 +/- 4% in hydrophilic fraction, P < 0.001). Similarly, chelerythrine was able to abrogate the contractile function recovery (12 +/- 6%, P < 0.001 versus preconditioned group and 19 +/- 7%, P < 0.001 versus hydrophobic fraction). In conclusion, the cardioprotective factors released in the coronary effluent by IPC are thermolabile hydrophobic substances with molecular weights higher than 3.5 kDa and acting through PKC activation.