The SGLT-2 inhibitor empagliflozin improves myocardial strain, reduces cardiac fibrosis and pro-inflammatory cytokines in non-diabetic mice treated with doxorubicin
Authors: Quagliariello Vincenzo , De Laurentiis Michelino , Rea Domenica , Barbieri Antonio , Monti Maria Gaia , carbone andreina , Paccone Andrea , Canale Maria Laura , Botti Gerardo , Maurea Nicola 
Published: February 2021

Abstract
BackgroundEmpagliflozin, a selective inhibitor of the sodium glucose co-transporter 2, reduced the risk of hospitalization for heart failure and cardiovascular death in type 2 diabetic patients in the EMPA‐REG OUTCOME trial. Recent trials evidenced several cardio-renal benefits of empagliflozin in non-diabetic patients through the involvement of biochemical pathways that are still to be deeply analyzed . We aimed to evaluate the effects of empagliflozin on myocardial strain of non-diabetic mice treated with doxorubicin (DOXO) through the analysis of NLRP3 inflammasome and Myd88-related pathways resulting in anti-apoptotic and anti-fibrotic effects.Methods Preliminary cellular studies were performed on mouse cardiomyocytes (HL-1 cell line) exposed to doxorubicin alone or combined to empagliflozin. The following analysis were performed: determination of cell viability (through a modified MTT assay), study of intracellular ROS production, lipid peroxidation (quantifying intracellular malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed: expression of NLRP3 inflammasome, MyD88 myddosome and p65/NF-κB associated to secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin (DOXO, n=6), empagliflozin (EMPA, n=6) or doxorubicin combined to empagliflozin (DOXO-EMPA, n=6). DOXO was injected intraperitoneally. Radial and longitudinal strain were analysed through transthoracic echocardiography (Vevo 2100). Cardiac fibrosis and apoptosis were histologically studied through Picrosirius red and TdT-mediated dUTP nick-end labelling (TUNEL) assay, respectively. Tissue NLRP3, Myd88 and cytokines were quantified after treatments.ResultsCardiomyocytes exposed to doxorubicin increased the intracellular Ca2+ content and expression of several pro-inflammatory markers associated to cell death; co-incubation with empagliflozin reduced significantly the magnitude of the effects. In preclinical study, empagliflozin prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin (radial strain (RS) 30.3 % in EMPA-DOXO vs 15.7% in DOXO mice ; longitudinal strain (LS) -17% in EMPA-DOXO vs -11,7% in DOXO mice (p<0.001 for both). A significant reduction of cardiac fibrosis and apoptosis were also seen. A reduced expression of pro-inflammatory cytokines, NLRP3, MyD88 and NF-kB in heart, liver and kidneys was also seen in DOXO-EMPA group compared to DOXO mice (p<0.001)ConclusionEmpagliflozin reduced fibrosis, apoptosis and inflammation in doxorubicin-treated mice through the involvement of NLRP3 and MyD88-related pathways, resulting in a significant improvement of myocardial strain. These findings provides the proof of concept for translational studies designed to reduce adverse cardiovascular outcomes in non-diabetic cancer patients treated with doxorubicin.