Abstract 275: Quantification of the Mitochondrial Protein Interactome in Failing Hearts

Abstract

Mitochondrial dysfunction is a maladaptive mechanism in the progression of heart failure. However, the cause of mitochondrial dysfunction during cardiac stress remains elusive. To understand the pathogenic role of mitochondria in heart failure, a systems biology approach was used to quantify the protein interaction network by comparing mitochondria isolated from healthy and failing mouse heart. This is achieved by Protein Interaction Reporter (PIR) technology consisting of a cleavable, MS-identifiable, isotope labelled cross-linker, which allows identification and quantification of interacting peptide pairs. Heart failure was induced in three month old, wild-type C57Bl6/NCrl mice using chronic pressure overload by transverse aortic constriction (TAC, n=8) surgery. Age-matched Sham-operated (Sham, n=8) mice were used as controls. Four weeks post-surgery, cardiac function was measured by echocardiography. TAC hearts showed significantly lower contractile function than Sham (fractional shortening 49±2.1% vs 13±4.0%), and were dilated (LVID 2.7±0.34mm vs 3.4±0.46mm). Cardiac hypertrophy of TAC hearts (HW/TL 5.0±0.35 vs 9.1±0.58) was associated with greater lung edema (LW wet/dry 3.9±0.49 vs 4.9±0.24). Freshly isolated mitochondria from TAC and Sham hearts were cross-linked with heavy- or light-isotope labelled crosslinkers, digested, enriched, and analyzed by LC/MS 3 . Released peptide spectra were identified and categorized by known molecular functions using a MitoCarta 2.0 constructed database. We identified 3502 non-redundant cross-linked peptide pairs from 297 proteins, involving 1758 lysine residues linked in 2571 lysine-lysine interactions. From 3502 total links, we detected 2304 intra- and 1016 inter-protein links. Inter-protein changes between TAC/Sham were most prevalent in the electron transport chain and ATP synthasome networks. Intra-protein changes were evident in TCA cycle, fatty acid oxidation, and nucleotide transport, suggesting conformational remodeling. Interactome changes in amino acid metabolism, mitochondrial transcription and translation pathways were undetected. A complete picture of mitochondrial protein landscape in the failing heart will lead us to novel mechanisms and therapy.