Abstract 21208: Subcompartments of the Nucleus are Differentially Modulated During the Development of Cardiac Hypertrophy and Failure in the Mouse
The protein constituents of biological subcompartments of the cardiac nucleus are unknown. Because of the importance of altered gene regulation during cardiac hypertrophy, failure and other diseases, understanding the totality of molecules regulating chromatin is a fundamental step towards systems-level therapies to remodel the genome. Nuclei were isolated and distinct populations of proteins enriched using a mild salt and detergent buffer (nucleoplasm), followed by extraction in 1% SDS (chromatin). DNA-associated proteins were enriched by acid extraction (0.4N H2SO4). Proteins were detected and quantified by high resolution mass spectrometry. We identified 464 proteins from intact nuclei, 784 in nucleoplasm, 415 in chromatin and 466 in acid-extract, with only 164 proteins in common between these four fractions (biological replicates=2, technical rep=2/group). Upon evaluation of the genomic hotspots contributing proteins to the nuclear proteome, the data indicate proteins from genes on all 21 (1–19 plus × and Y) mouse chromosomes with a marked enrichment from chromosomes 11 and 13, the latter of which contain the majority of the histone genes. We identified 22 histone protein isoforms, representing each phylogenetic branch. Multiple isoforms of core (H2A/H2B/H3/H4) and linker (H1) histones were identified including four novel cardiac variants, two of which have never been described at the protein level. Gene ontology analysis indicated a significant enrichment of functionalities related to transcription, translation, membrane transport, cell differentiation and nucleosome assembly. In the setting of heart failure induced by transverse aortic constriction, we observed a 57% decrease in methylation of lysine 9 on histone H3 (a repressive mark) concomitant with a 150% increase in lysine 4 methylation (an activating mark), both indicative of a transition from hetero- to euchromatin. Mass spec data revealed 13 total modifications on histone H3, 8 of which are known and 5 of which have never before been reported in any cell type. These investigations have fundamental implications for how the proteome regulates the genome in the normal heart and reveal the quantitative changes in nuclear proteins during the progression from hypertrophy to failure.
- © 2010 by American Heart Association, Inc.