Cardioprotective effect of genetic deletion of Atg5 gene or pharmacological autophagy inhibition in mouse model of experimental autoimmune myocarditis

Abstract Background Myocarditis is a severe inflammatory heart disease that can lead to heart failure and post-inflammatory dilated cardiomyopathy (DCM). The experimental autoimmune myocarditis (EAM) mouse model is efficient tool for studying this disease. Autophagy is an intracellular degradation process essential for maintaining cardiac homeostasis. However, its role in myocarditis and its progression to post-inflammatory DCM remains unclear. Purpose This study investigates whether autophagic dysfunction affects development of myocarditis and its progression to DCM. Using cell type-specific genetic deletion of the Atg5 gene or pharmacological inhibition of autophagy with bafilomycin A1 (BafA1), we assessed the impact of autophagy in EAM. Methods Myocarditis was induced in wild-type Balb/c and haploinsufficient Cdh5-Cre x Atg5-flox/null, and LysM-Cre x Atg5-flox/null mice using alpha myosin heavy chain (α-MyHC) emulsified in complete Freund’s adjuvant. In a complementary approach, Balb/c mice were treated with pharmacological autophagy inhibitor BafA1 (2.5µg/injection/mice/every other day) in preventive (days 0-21) or therapeutic (days 21-40) approach. Histological and immunohistochemical staining was used to evaluate the severity of inflammation at day 21. Cardiac function was analysed on day 40 of EAM using echocardiography. Furthermore, the effect of BafA1 was studied in vitro using TCR-M transgenic mice, which overexpress a T cell receptor specific for α-MyHC, and cardiac microvascular endothelial cells (CMVECs) isolated from Balb/c mice. Cellular and molecular changes were analyzed through flow cytometry, ELISA, and proliferation assays. Results In the EAM model, we characterized the acute phase of inflammation by an infiltration of CD3 and CD45 positive cells into the myocardium, as well as increased autophagy markers, such as Beclin-1 and Lc3b. Genetic deletion of Atg5 in LysM-Cre × Atg5-flox/null mice resulted in reduced levels of pro-inflammatory cytokines (Il-6, Il-1β, Tnfα) and inflammatory CD3+ T cells in the myocardium at day 21. Similarly, in Cdh5-Cre x Atg5-flox/null mice, at day 21 we observed decreased inflammatory score, cardiac CD45+ cells, and autophagy marker Lc3b. Moreover, preventive pharmacological inhibition of autophagy with BafA1 protected mice from the left ventricular dysfunction in the post-inflammatory DCM phase (d40 of EAM). Our in vitro studies pointed out that BafA1 has inhibited T cell proliferation, and suppressed expression of the adhesion proteins ICAM and VCAM in CMVECs. Conclusions The results obtained from Atg5-deletion and BafA1 treatment models indicates that autophagy promotes myocarditis development and its progression to DCM.The role of autophagy in myocarditis