Many cardiac genes undergo alternative splicing to produce cardiac vs. skeletal, and adult vs. fetal isoforms. Differential regulation of alternative splicing patterns is a central component of the altered genetic program during cardiac remodeling after ischemic injuries. Alternative splicing has been shown to causally modulate the severity of cardiac remodeling, and thus represents a potential therapeutic target to halt disease progression. Nevertheless, although a large number of alternative isoform transcripts have now been discovered in the cardiac genome, current knowledge on the protein coding potential and hence molecular functions of these alternative isoform is poor. The majority of discovered isoforms were yet to be detected at the protein level -- therefore the effects of alternative isoforms on protein abundance, motif features, and protein-protein interactions are virtually unknown. This knowledge gap obstructs a fuller understanding on the impact of alternative splicing on the myocardium in health and disease. Proteomics technologies have advanced in recent years and are now well positioned to transform cardiovascular sciences, but the detection of isoform proteins remains a substantial challenge. We and others have recently shown that transcriptomics (RNA-seq) and proteomics (mass spectrometry) data may be combined to probe more deeply into the cardiac proteome. In this project, we will combine the respective strengths of RNA-seq (modeling transcript isoforms) and proteomics (providing protein-level evidence) to examine cardiac alternative splicing. Specifically, we will apply this RNA-seq-guided- proteomics approach to: (i) define the landscape of alternative protein isoforms in fetal and adult hearts; (ii) identify disease signature isoforms in remodeling hearts using mouse models of myocardial infarction and isoproterenol stimulation; (iii) examine the functional consequence of protein isoforms by identifying isoform- specific protein-protein interactions and modulators of isoform expression in disease; and (iv) elucidate the impact of isoform-specific overexpression/knockdown on myocyte functional phenotypes. We anticipate the completion of the proposed studies will open new avenues into understanding the role of alternative splicing in heart diseases.
Alternative splicing is a biological phenomenon in which one gene can encode for multiple protein products to perform diverse functions. Following ischemic injury, the failing heart is associated with differential alternative splicing of many genes involved in metabolism and cell death processes, but the functional significance of many isoforms remains unknown. Our goal here is to examine the dynamic changes and functional consequences of alternative isoforms at the protein level, and to leverage the resulting information to better understand the progression of heart diseases.
