Background: Fibroblast/ myofibroblast differentiation following acute myocardial infarction (MI) is important for myocardial healing but also causes adverse remodeling and stiffening of the myocardium leading to cardiac dysfunction and heart failure. Myofibroblasts are characterized by periostin, alpha-smooth muscle actin (a-SMA), and extracellular matrix protein expression. Moreover, infiltration and differentiation of specific monocyte/ macrophage (Ly6C^hi and Ly6C^lo) subsets regulate TGF-ß/SMAD3 mediated trans-differentiation of myofibroblasts. The transcription factor forkhead box O 3 (FOXO3a) has been recently shown to inhibit hypertrophic cardiac remodeling and immune cell differentiation.

Purpose: We hypothesized that FOXO3a, a key regulator of cell size, immunity, and cell differentiation, might inhibit transdifferentiation of fibroblasts into myofibroblasts as well as attenuate the differentiation of reparative macrophages following MI.

Methods: Acute myocardial infarction was induced in Foxo3a^-/- mice and wild-type littermates (FVB/N background) by permanent LAD ligation. Cardiac function was determined by transthoracic echocardiography at baseline as well as 4 and 14 days post-MI. Cardiac injury markers were analyzed by ELISA and CBA assay in plasma and myocardium. Monocyte/ macrophage subsets were analyzed by flow cytometry on day 4 after MI. Myofibroblast and monocyte differentiation were determined by single nucleus RNA-sequencing (snRNA-seq) on day 14 after MI. IP/IF was used to test the direct interaction between FOXO3a and SMAD3 in vitro.

Results: Foxo3a^-/- mice showed significant improved survival despite similar infarct size and injury post- MI. The survival advantage was in part due to reduced cardiac ruptures and sudden cardiac death. Foxo3a^-/- mice exhibited an attenuated immune response characterized by diminished IL-6 and TNFa expression as well as reduced cardiac pro-inflammatory Ly6C^hi monocyte but enhanced reparative Ly6C^lo macrophage accumulation. In line with these findings on day four post-MI, sn-RNAseq revealed differentially expressed, i.e. upregulated pro-fibrotic genes in myofibroblasts of Foxo3a^-/- mice as well as increased numbers of reparative macrophages and myofibroblasts important for stabilizing matrix interactions at 14days post-MI. These changes were accompanied by decreased left ventricular function as well as increased wall stiffness in Foxo3a^-/- mice at this time. Mechanistically, in vitro studies showed enhanced transdifferentiation of myofibroblasts in Foxo3a^-/- mice following stretch or TGFb incubation that was due to enhanced interaction with the remodeling transcription factor SMAD3.

Conclusion: Our data identify Foxo3a as a master regulator of cardiac remodeling coordinately affecting macrophage responses and myofibroblast transdifferentiation following acute MI. These important findings suggest that deficiency of Foxo3a provides early remodeling preventing cardiac rupture but also contributes to adverse remodeling in the later stages resulting in impaired cardiac function and enhanced fibrosis.