Regulation of Mitophagy by RNA Alternative Splicing in the Aging Heart - Aging is associated with structural and functional changes in the heart, a condition commonly referred to as cardiac aging, which is a critical risk factor for cardiovascular diseases. These changes, including fibrosis, hypertrophy, impaired autophagy, and mitochondrial dysfunction, significantly increase the risk of cardiovascular disease. One key process affected by aging is mitophagy, a specialized form of cellular recycling that clears damaged mitochondria, the energy-producing components of cells. A decline in mitophagy leads to mitochondrial dysfunction, making the heart less adaptable to stress and more prone to damage. Emerging evidence suggests that RNA alternative splicing—a cellular process that generates different isoforms of proteins from a single gene—might play a crucial role in regulating mitophagy. Despite its potential significance, little is known about how changes in RNA splicing influence mitophagy, particularly in the aging heart. Preliminary analysis of long-read RNA sequencing has identified significant changes in mitophagy- related gene isoforms in the aging heart. Additionally, using enhanced RNA interactome capture, I identified RNA-binding proteins (RBPs) with significant changes in expression in the aging heart, including components of the spliceosome machinery. This project aims to uncover how alternative splicing regulates mitophagy and identify potential therapeutic targets to combat cardiac aging. Aim 1: Using human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), I will study the role of spliceosome machinery components in mitophagy. Mitophagy indicators, such as mito-Keima and GFP-LC3, CRISPR/Cas9 genome editing technology, along with electron microscopy and long-read RNA sequencing, will be used to investigate how RBPs regulate mitophagy. This aim could position RBPs as promising therapeutic targets to restore mitophagy in aging hearts. Aim 2: I will explore novel splicing isoforms of mitophagy-related genes using iPSC-CMs. This aim will investigate how these novel isoforms impact mitochondrial fission and mitophagy and how their manipulation could restore mitophagy in aging hearts. By uncovering the molecular mechanisms linking RNA splicing to mitophagy, this study could pave the way for innovative therapies to rejuvenate the aging heart and reduce the burden of cardiovascular diseases.
