Abstract 19597: Cardiac Hypertrophy is Associated With Differential Protein Isoform Expression

Introduction: Alternative splicing contributes significantly to the diversification of the mammalian proteome by enabling one gene to give rise to multiple proteins, e.g., fetal and adult isoforms. Pathological cardiac hypertrophy is commonly associated with the re-expression of fetal genes, whereas mutations in splicing factors can cause cardiomyopathies. Advances in RNA-seq have led to the discovery of many novel isoform transcripts, but the protein-level existence and thus physiological functions of many isoforms are poorly understood. Hypothesis: Cardiac hypertrophy is associated with widespread differential alternative splicing, which can modulate cardiac phenotypes by altering the ratios of protein isoforms expressed in cardiac cells. Methods: We developed an integrated omics approach, which combines re-analysis of RNA-seq data, shotgun proteomics experiments, and computational algorithms to characterize proteome-wide alternative isoform expression. Splice isoforms specific to the C57BL/6J mouse heart were acquired from public ENCODE data (ENCSR000BYQ) and analyzed via a Bowtie-Tophat-rMATS pipeline. We then built a custom software tool to determine transcript translation frames and translate cell-type specific protein sequences. We then searched in-house proteomics datasets on mouse cardiac hypertrophy against the custom sequences to identify protein isoforms during disease progression. Results: We identified 816 distinct splice junction peptides in the mammalian heart proteome, including 41 instances in which unique peptides from both alternative isoforms are simultaneously identified. Our data present the first direct protein-level evidence for the existence of a number of alternative splicing events in the heart. We also observed differential expression of alternative isoforms, and present proteomics evidence of the isoform shift of pyruvate kinase (PKM1/2) and alpha-enolase (ENO1), etc., in cardiac hypertrophy. Conclusions: Integrated proteotranscriptomics approaches have the potential to expand the catalogue of known molecular changes during cardiac remodeling, and may be applied to identify novel disease drivers and drug targets in other heart disease models.