Protection against reperfusion injury by 3′,4′-dihydroxyflavonol in rat isolated hearts involves inhibition of phospholamban and JNK2

Background: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts. Methods: We assessed the effect of global ischemia (20 min) and reperfusion (5-30 min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10 mu M) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection. Results: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30 min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca2+/calmodulin. Conclusions: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca2+ from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis. (C) 2017 Elsevier B.V. All rights reserved.