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Abstract

Background: Despite recent advancements, heart failure remains the leading cause of death in the Western world. Since fibrosis is a central and deleterious feature of cardiac remodelling leading to heart failure, there is a great interest in identifying new targets that can ameliorate or even reverse cardiac fibrosis. The canonical role of the eukaryotic elongation factor 1 alpha (eEF1A) is to deliver aa-tRNAs to the ribosome; in addition, it holds non-canonical functions in regulating autophagy, aggresome formation, and proteasome activity.

Methods and Results: This study aimed to investigate the potential role of eEF1A in promoting pro-fibrotic activity in fibroblasts. Fibroblasts express mainly eEF1A1 and only minimal amounts of the eEF1A2 isoform. Mouse embryonic fibroblasts (MEF) were isolated from eEF1A1/A2 flox/flox mice and incubated with either a control (Adβgal) or a Cre-recombinase expressing adenovirus (Ad. Cre). A strong downregulation (>70%) of total eEF1A both at the mRNA and protein levels, which was followed by a significant downregulation of fibrosis- associated genes, such as Col1a1, αSMA, Col3a1, FN, and Sm22 at the mRNA as well as at the protein level (Col1a1, αSMA) was observed. As expected, global protein synthesis activity (assessed with the SUnSET assay) was downregulated by around 50% in MEFs with downregulation of eEF1A (eEF1A-KD). A harringtonine run-off assay indicated a reduced translational elongation speed in the KD cells. These findings of a considerable downregulation of extracellular matrix proteins (αSMA, SM22, FN, Col1a1, and Col3a1) were confirmed upon approximately 50% downregulation of eEF1A in adult cardiac fibroblasts (AMcFB) by quantitative analysis using Western blot and RT-qPCR. Next, various cellular-based assays were performed to assess the effect of eEF1A on the functional properties of fibroblasts (MEF), and observed a significant decrease in proliferation and migration capacity after eEF1A knock-down (KD) was observed. Investigations into autophagy in eEF1A-KD cells (MEF) revealed an upregulation in p62 (by +50%) and LC3b (by +500%) compared to control cells. Given the crucial role of ER stress in maintaining proteostasis and this study's finding of an upregulation of the unfolded protein response (UPR) following eEF1A knockdown, the UPR could rescue proteostasis under these circumstances. Therefore, this study suggests that further in-depth analysis is essential to elucidate the underlying mechanism and the adaptive responses triggered by the downregulation of eEF1A in fibroblasts.

Conclusions: Counteracting eEF1A expression reduces general protein synthesis and triggers autophagy in fibroblasts, while predominantly reducing pro-fibrotic activity in these cells in vitro. Therefore, targeting eEF1A by specific inhibitors could be an anti-fibrotic therapeutic strategy in the future.