Labdane diterpenes protect against anoxia/reperfusion injury in cardiomyocytes: involvement of AKT activation

Abstract

Several labdane diterpenes exert anti-inflammatory and cytoprotective actions; therefore, we have investigated whether these molecules protect cardiomyocytes in an anoxia/reperfusion (A/R) model, establishing the molecular mechanisms involved in the process. The cardioprotective activity of three diterpenes (T1, T2 and T3) was studied in the H9c2 cell line and in isolated rat cardiomyocyte subjected to A/R injury. In both cases, treatment with diterpenes T1 and T2 protected from A/R-induced apoptosis, as deduced by a decrease in the percentage of apoptotic and caspase-3 active positive cells, a decrease in the Bcl-2/Bax ratio and an increase in the expression of antiapoptotic proteins. Analysis of cell survival signaling pathways showed that diterpenes T1 and T2 added after A/R increased phospho-AKT and phospho-ERK 1/2 levels. These cardioprotective effects were lost when AKT activity was pharmacologically inhibited. Moreover, the labdane-induced cardioprotection involves activation of AMPK, suggesting a role for energy homeostasis in their mechanism of action. Labdane diterpenes (T1 and T2) also exerted cardioprotective effects against A/R-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals (PI3K/AKT pathways, ERK1/2 and AMPK) and inhibition of apoptosis.

A labdane diterpene exerts ex vivo and in vivo cardioprotection against post-ischemic injury: Involvement of AKT-dependent mechanisms

“…We have recently reported that several diterpenes exert anti-inflammatory and cytoprotective actions [15][16][17]. In particular, labdane diterpenes exerted cardioprotective effects against ischemia/reperfusion (I/R)-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals and inhibition of death pathways [16]. In the present study we show a remarkable cardioprotective effect of diterpene 1 (DT1) in rat models of I/R when administered at reperfusion.…”

“…The hearts were removed and mounted on a Langendorff-perfusion apparatus as previously described [16]. Briefly, the ascending aorta was cannulated and a retrograde perfusion was maintained at a constant flow with different Ca 2+ -free Tyrode solutions.…”

“…Rat cardiomyocytes were isolated using an enzymatic technique, as previously reported [16]. Briefly, rats were heparinized (4 U/g IP) and injected with buprenorphine (0.01 mg/kg).…”

“…LDH was determined spectrophotometrically in the flow obtained after perfusion of the Langendorff heart [16]. Caspase-3 activity was determined in protein extracts using a specific substrate (Ac-DEVD-AMC), according to the supplier's instructions (BD Biosciences).…”

“…Diterpenes are compounds with a broad spectrum of biological activities [12][13][14]. We have recently reported that several diterpenes exert anti-inflammatory and cytoprotective actions [15][16][17]. In particular, labdane diterpenes exerted cardioprotective effects against ischemia/reperfusion (I/R)-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals and inhibition of death pathways [16].…”