Journal Article

The SGLT-2 inhibitor empagliflozin improves myocardial strain, reduces cardiac fibrosis and pro-inflammatory cytokines in non-diabetic mice treated with doxorubicin

Conte, Mariarosaria, Quagliariello, Vincenzo, De Laurentiis, Michelino, Rea, Domenica, Paccone, Andrea, Canale, Maria Laura, Altucci, Lucia, Barbieri, Antonio, Carbone, Andreina, Maurea, Nicola, Botti, Gerardo, Monti, Maria Gaia

2021

Overview

Background Empagliflozin (EMPA), 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 EMPA in non-diabetic patients through the involvement of biochemical pathways that are still to be deeply analysed. We aimed to evaluate the effects of EMPA 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 EMPA. 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 Ca 2+ homeostasis. Moreover, pro-inflammatory studies 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), EMPA (EMPA, n = 6) or doxorubicin combined to EMPA (DOXO-EMPA, n = 6). DOXO was injected intraperitoneally. Ferroptosis and xanthine oxidase were studied before and after treatments. Cardiac function studies, including EF, FS and radial/longitudinal strain were analysed through transthoracic echocardiography (Vevo 2100). Cardiac fibrosis and apoptosis were histologically studied through Picrosirius red and TUNEL assay, respectively and quantified through pro-collagen-1α1, MMP-9 and Caspase-3 expression. Tissue NLRP3, MyD88 and cytokines were also quantified before and after treatments through ELISA methods. Results Cardiomyocytes exposed to doxorubicin increased the intracellular Ca 2+ content and expression of several pro-inflammatory markers associated to cell death; co-incubation with EMPA reduced significantly the magnitude of the effects. In preclinical study, EMPA increased EF and FS compared to DOXO groups (p < 0.05), prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin (RS) 30.3% in EMPA-DOXO vs 15.7% in DOXO mice; LS − 17% in EMPA-DOXO vs – 11.7% in DOXO mice (p < 0.001 for both). Significant reductions in ferroptosis, xanthine oxidase expression, cardiac fibrosis and apoptosis in EMPA associated to DOXO 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 (p < 0.001). Conclusion EMPA reduced ferroptosis, fibrosis, apoptosis and inflammation in doxorubicin-treated mice through the involvement of NLRP3 and MyD88-related pathways, resulting in significant improvements in cardiac functions. These findings provides the proof of concept for translational studies designed to reduce adverse cardiovascular outcomes in non-diabetic cancer patients treated with doxorubicin.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: 4-Hydroxynonenal / Acids / Angiology / Animals / Anti-Inflammatory Agents - pharmacology / Antidiabetics / Antifibrotic Agents - pharmacology / Apoptosis / Apoptosis - drug effects / Benzhydryl Compounds - pharmacology / Breast cancer / Calcium (intracellular) / Calcium homeostasis / Cardio-Oncology / Cardiology / Cardiomyocytes / Cardiotoxicity / Caspase-3 / Cell culture / Cell death / Cell Line / Clinical trials / Collagen / Congestive heart failure / Cytokines / Cytokines - metabolism / Diabetes / Diabetes mellitus / Disease Models, Animal / Doxorubicin / Echocardiography / EMPA / Enzyme-linked immunosorbent assay / Female / Ferroptosis / Ferroptosis - drug effects / Fibrosis / Gelatinase B / Glucose transporter / Glucosides - pharmacology / Heart Diseases - chemically induced / Heart Diseases - drug therapy / Heart Diseases - metabolism / Heart Diseases - pathology / Homeostasis / Inflammasomes / Inflammasomes - metabolism / Inflammation / Inflammation Mediators - metabolism / Interleukins / Intracellular / Kidneys / Lipid peroxidation / Malondialdehyde / Medicine / Medicine & Public Health / Mice / Mice, Inbred C57BL / Myeloid Differentiation Factor 88 - metabolism / Myocytes, Cardiac - drug effects / Myocytes, Cardiac - metabolism / Myocytes, Cardiac - pathology / NLR Family, Pyrin Domain-Containing 3 Protein - metabolism / Original Investigation / Patients / Signal Transduction / Sodium-Glucose Transporter 2 Inhibitors - pharmacology / Ventricular Function, Left - drug effects