Establishment Of An Engineered Cardiac Tissue Model Of Diabetic Cardiomyopathy And Study On The Pathogenesis Of Ferroptosis

Aims:Rodent diabetic models,used to understand the pathophysiology of diabetic cardiomyopathy(DCM),remain several limitations.Engineered cardiac tissues(3D-ECTs)have emerged as robust 3D in vitro models to investigate cardiac injury and repair,structure-function relationships.Advanced glycation end-products(AGEs),produced through glycation of proteins or lipids in response to hyperglycemia,are important pathogenic factor for the development of DCM.Ferroptosis is a newly identified form of regulated cell death characterized by iron mediated phospholipid peroxidation and excessive oxidative stress,both of which are implicated in the pathogenesis of DCM.Therefore,we aimed to develop a murine based 3D-ECT model with AGEs as inducer to mimic DCM-featured structure-function abnormalities in animal model and define the role of ferroptosis in the pathogenesis of DCM.Methods:We used AGEs to treat 3D-ECT composed of neonatal murine cardiac cells and observed the functional,cellular and molecular alterations in different groups of3D-ECT.Furtherly,we define the role of ferroptosis in the pathogenesis of DCM by examining the expression of key characteristics and regulators of ferroptosis in mice with DCM and the newly-established ex vivo DCM model.Results:(1)AGEs at 150 μg/ml did not cause significant cytotoxicity(necrosis,detected by medium Lactate dehydrogenase or apoptosis,detected by cleaved caspase 3 and TUNEL staining),but negatively impacted 3D-ECT function by 9-day treatment;(2)AGEs triggered markers of fibrosis(transforming growth factor β,α-Smooth Muscle Actin,CTGF,Collagen I-α1,Collagen III-α1,and Fn1)and hypertrophy(Nppa and Myh7);(3)AGEs increased oxidative stress(3-nitrotyrosine,4-hydroxynonenal,heme oxygenase-1,catalase,and superoxide dismutase 2)and inflammation response(plasminogen activator inhibitor-1 and tumor necrosis factor,nuclear factor-κB,intercellular adhesion molecule-1)in 3D-ECT;(4)AGEs-induced pathogenic responses were all attenuated by the AGE receptor antagonist FPS-ZM1 or the antioxidant glutathione precursor N-acetylcysteine.(5)AGEs induced ferroptosis in 3D-ECTs,as reflected by increased Ptgs2 and MDA levels and decreased Ferritin and SLC7A11 levels.Typical morphological changes of ferroptosis in cardiomyocytes were evidenced by transmission electron microscopy.Inhibition of ferroptosis prevented remodelling and dysfunction in AGEs-treated 3D-ECT and type 2 diabetic mice.(6)Activation of NRF2 by sulforaphane(SFN)inhibited cardiac cell ferroptosis in both AGE-treated 3D-ECTs and heart of DCM mice by upregulating ferritin and SLC7A11 levels.Protective effect of SFN on ferroptosis in the ex vivo and mouse models of DCM was AMPK-dependent since the protective effect of SFN on ferroptosis was abolished in AMPK inhibitor-treated 3D-ECTs or AMPKα2-knock out mice.Conclusions:(1)AGEs-treated murine 3D-ECTs recapitulate key features of DCM’s functional,cellular and molecular pathogenesis,may serve as an in vitro model to investigate the pathogenesis and protective strategies of DCM.(2)Ferroptosis plays an essential role in the pathogenesis of DCM.(3)SFN prevents ferroptosis and associated pathogenesis via AMPK-mediated NRF2 activation.