An in‐Depth Analysis of the PAK1 Autophagy Signaling Pathway in H9C2 Cardiomyoblasts

Abstract

P21‐activated serine/threonine Kinase 1 (PAK1) plays a critical role in cardiomyocyte survival under numerous stressful conditions. Autophagy is a cellular process that promotes homeostasis by removing and replacing antiquated or damaged cellular components. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria to maintain a pool of healthy mitochondria. We have previously demonstrated that PAK1 is essential for maintaining autophagy and mitophagy activities. However, the downstream mediators have not yet been discovered. In this study, we explored the mechanisms of PAK1‐dependent autophagy and mitophagy by determining the protein expression levels of the major regulators of autophagy and mitophagy. H9c2 cardiac myoblasts were treated with siRNA to knockdown the expression of PAK1. Western blot analysis showed that PAK1 knockdown substantially reduced the protein expression levels of ATG5‐12 complex, an essential promotor of autophagosome formation, TFEB, a master regulator of lysosome biogenesis and autophagy, and p62, an autophagy receptor for ubiquitinated cargos. All these changes are expected to reduce autophagy activity. In addition, PAK1 knockdown also reduced the expression of mitophagy receptor FUNDC1 and diminished the association of p62 with mitochondria, which coincided with reduced mitophagy activity. Collectively, these results suggest that the ability of PAK1 knockdown to inhibit autophagy and mitophagy is mediated by reduced expression levels of several important regulators of autophagy and/or mitophagy pathways. Future research is warranted to determine whether restoring the expression levels of these target genes can overcome the inhibition of autophagy and mitophagy by PAK1 deficiency.