P3488Mechanistic insight on the cardioprotective effect of levosimendan against doxorubicin induced cardiomyopathy: Pivotal role of PKA signaling

Abstract

Abstract Background Levosimendan (LEVO) an inodilator indicated for the treatment of heart failure exerts multifaceted cardioprotective effects. Case-studies indicate protection against doxorubicin (DXR)-induced cardiotoxicity, but this effect remains elusive. We have previously shown that LEVO exerts cardioprotection against DXR-induced cardiomyopathy in a rat in vivo model, in a PKA/PKG-dependent manner. Purpose We sought to elucidate the mechanism of LEVO's induced cardioprotection and clarify the contribution of PKG and PKA pathways converging onto phospholamban (PLN). Methods As previously observed, LEVO at a dose of 24μg/kg protects against DXR cardiotoxicity, with protein kinase B (Akt)/ endothelial nitric oxide synthase (eNOS) and protein kinase A and G (PKA/PKG) pathways emerging as the main contributors to cardioprotection. Moreover, phospholamban seems to be the end-target of the signaling cascade. To verify the contribution of phospholamban, phospholamban deficient mice (PLN−/−) were assigned to PLN−/−/DXR (18mg/kg) and PLN−/−/DXR+LEVO (acute) (LEVO bolus, 24 μg/kg) groups for 14 days. Echocardiographic analysis was conducted in all groups and protocols. Furthermore, in order to solidify the mechanism of LEVO-mediated cardioprotection, primary adult ventricular murine cardiomyocytes (AVMCs) were isolated and treated with doxorubicin or/and LEVO as well with L-NAME, DT-2 and H-89 (eNOS, PKG and PKA inhibitors, respectively) and cardiomyocyte-toxicity was assessed. Results In the transgenic PLN−/− mice, LEVO did not exert cardioprotection, whilst the co-administration of doxorubicin and levosimendan led to an impaired Left ventricular function [FS (%): PLN−/−/Control: 39.01±0.42 vs PLN−/−/DXR: 38.12±0.51 in (NS); PLN−/−/DXR+LEVO: 35.38±0.86 (**p<0.01 vs PLN−/−/Control, *p<0.05 vs PLN−/−/DXR]. The latter data suggest that phospholamban is crucial for LEVO's cardioprotective effect. Finally, by investigating the contribution of different molecular pathways -shown to be induced by LEVO in vivo- on the AVMCs, we found that only PKA inhibition by H-89, abrogated LEVO-mediated cytoprotection, indicating that the effect is cAMP-PKA dependent. Conclusions Single-dose LEVO prevented DXR cardiotoxicity through a cAMP-PKA-phospholamban pathway, highlighting the role of inotropy in DXR cardiotoxicity. These preclinical data can stand as promising grounds for further clinical investigations.