Type II diabetes mellitus (DM) remains one of the major causes of morbidity and mortality in the industrialized world. Several animal and human studies have found reactive oxygen species (ROS) as one of the key mediators of signal transduction during DM and DM-mediated cardiovascular disease (CVD). In addition to redox-specific post-translational modifications (PTM), accumulating evidence suggests the involvement of ROS in accelerating non-specific ubiquitination of cardiac proteins as a label for protein degradation. Our group has defined changes in several protein PTMs in DM and DM-mediated CVD using Langendorff perfused rat hearts and begun to understand how these various PTMs cross-talk with each other. Here, we examined the ubiquitin-modified cardiac proteome in DM.
Rats were fed a standard chow citrate (CC) (12% fat) or high fat (HF) (42% fat) diet for 8 weeks, with DM induced in 50% of the animals after 4 weeks utilising a low dose of streptozotocin (STZ; 55mg/kg); a pancreatic β-cell toxin. At the cessation of the feeding protocol, animals were euthanised and hearts excised. Cardiac hemodynamic performance was measured in hearts subjected to non-ischemic perfusion. Myocardial protein lysates were subjected to Western blotting using anti-ubiquitin antibodies and peptides enriched via immunoprecipitation using anti-diGly remnant antibodies for analysis by LC-MS/MS. Western blotting revealed gross changes in ubiquitination across the different groups. We identified 2,043 and 1,575 unique ubiquitination sites, originating from 669 and 579 unique proteins in CC and HFDM, respectively. These modified proteins were mapped to functional Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, identifying enriched regulatory pathways such as metabolic, proteasome, glycolysis/gluconeogenesis, cardiac muscle contraction and dilated/hypertrophic cardiomyopathy pathways. Our data suggest that the rat cardiac ubiquitinome is sensitive to diet and DM, indicating a possible role for ubiquitination in this disease.