Associate Professor, Physical Therapy and Rehabilitation Sciences
University of Kansas Medical Center
Proteomic Approach to Study Diabetic Heart Protein Posttranslational Modification (2005-07)
Six percent of the U.S. population suffers from diabetes mellitus, with the greatest mortalities caused by cardiovascular complications. Diabetic cardiomyopathy, the weakening of the heart muscle or a change in heart muscle structure, is a chronic condition and is characterized by impaired function and alterations in the morphological structure of the heart muscle. The molecular mechanisms underlying this pathology are not well understood, however hyperglycemia is thought to be the main etiological factor in its development.
Many proteins in the diabetic heart show profoundly altered function, due to changes in their expression level and/or structure. Changes in posttranslational modifications of proteins have been linked to a variety of disease states; however, this area is poorly explored in the malfunctioning diabetic heart. Nonetheless we found a dramatic increase (5-29 fold) in protein acetylation in cardiac muscle from diabetic rats using immunoblots of heart homogenates probed with an antibody that specifically recognizes lysine residues acetylated on the epsilon-amino group. This effect was observed in two different rat models for type 1 diabetes. However, the identities of the acetylated proteins were unknown. Since lysine acetylation is an important regulatory mechanism in eukaryotes, we speculate that excessive cardiac protein acetylation contributes to the development of diabetic cardiomyopathy.
Our hypothesis is that diabetes causes increased acetylation of cardiac proteins, and that this posttranslational modification may be responsible for altered protein function. We propose to investigate this hypothesis through two Specific Aims:
- We will identify the acetylated proteins and localize the acetylation sites. The experiments to accomplish this aim will include protein fractionation, separation using 2D-PAGE, in-gel digestion followed by protein identification and localization of acetylated sites using MALDI-TOF mass spectrometry at the COBRE Protein Structure Core Laboratory.
- We will explore the mechanism for increased protein acetylation in diabetic heart. This aim will be addressed by determining relative activities of enzymes participating in both acetylation and deacetylation of proteins, identifying whether de novo protein synthesis is involved, and quantifying differences in protein expression in control and diabetic hearts using differential 2D-PAGE. Identification of proteins acetylated in the diabetic heart will lead to a better understanding of the molecular pathways involved in the development of diabetic cardiomyopathy.