Gata4 regulates cardiac fibrosis: a new mechanism to block fibroblast migration and expansion

Background: Cardiac fibrosis is an adaptive response to cardiac injury. Cardiac fibrogenesis is executed by the conversion from quiescent cardiac fibroblasts (CFs) to extracellular matrix producing myofibroblasts (MFs). GATA binding protein 4 ( Gata4 ), one of the earliest genes expressed during heart development, plays critical roles in cardiomyocyte proliferation, differentiation, survival, and hypertrophy. In the current study, we found a significant number of Gata4 positive (Gata4 + ) non-myocytes in hearts after myocardial infarction (MI). We used transgenic mouse model and artificial intelligence (AI) powered live cell imaging approaches to test the hypothesis that Gata4 expression in heart acts as a negative regulator of cardiac fibrosis following MI. METHOD: MFs-specific lineage tracing mouse models, including Postn -MerCreMer(iCre): R26 tdTomato (tdT) control mice and Postn iCre : Gata4 fl/fl : R26 tdT mice were utilized. MI was induced via left anterior descending (LAD) coronary artery ligation. Trichrome staining assessed scar size, wall thickness, and chamber dilation (5 sections per heart). MFs derived from Gata4 fl/fl mice were transduced with adenovirus expressing Cre recombinase to delete Gata4. To investigate the role of Gata4 in cardiac fibrosis, Collagen I and αSMA immunohistochemistry staining were performed to evaluate fibrogenesis. CRISPR/Cas9 was used to knockout Gata4 in fibroblasts. Live-cell imaging and Nikon NIS-Elements AI-based analysis quantified fibroblast migration and morphology before and after Gata4 ablation. (>100 cells). Results: We observed that the number of Gata4 + non-myocytes markedly increased following MI, whereas relatively few non-myocytes expressed Gata4 in the uninjured heart. These Gata4 + non-myocytes mainly co-expressed the CFs marker Pdgfr-α but did not express the endothelial cell marker CD31, indicating they are CFs rather than endothelial cells. Interestingly, when challenged with MI, cardiac fibroblast specific Gata4 knockout hearts showed greater production of fibrotic proteins (e.g. Collagen I, α-SMA), higher Trichrome staining signals, larger infarct sizes (over 20% larger than controls, p < 0.005), and reduced contractile function (over 15% reduction compared to controls, p < 0.05). In vitro, Gata4 knockout CFs spontaneously exhibited increased production of Collagen I and other fibrogenic gene expressions (like Col1a1, Col1a2, Col3a1, Tgfb1, etc.). Additionally, AI-based path analysis revealed that Gata4 knockout cells exhibited significantly increased migration distance and speed. Specifically, their average path lengths reached 633 µm, which is 32% higher than that of control cells (p < 0.0001). Their path speeds increased to 0.013 µm/s, representing a 27.5% increase over the control group (p < 0.0001). Similarly, line lengths and line speeds were 38.7% (p < 0.0001) and 22.6% (p < 0.0002) higher, respectively, compared to controls, indicating enhanced migratory capacity due to Gata4 deficiency. Furthermore, knockout of Gata4 led to fibroblast enlargement, with cell sizes being 35.7% larger than controls (p < 0.0001). This enlargement suggests enhanced cellular activation and fibrogenic potential, contributing to increased fibrosis. Conclusions: Taken together, our study suggests that Gata4 expression in CF-derived cells in the heart is essential to regulate post-injury cardiac fibrosis. Our results also revealed that Gata4 plays an important role in regulating fibrotic gene expression, fibroblast migration and morphology. These findings highlight the Gata4 signaling pathway as a potential therapeutic target to mitigate cardiac fibrotic remodeling and preserve cardiac function post-MI. This research is supported by American Heart Association grant 23CDA1045959 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.