Abstract
In the heart, lysine acetylation has been implicated in processes ranging from transcriptional control of pathological remodeling, to cardioprotection arising from caloric restriction. Given the emerging importance of this post-translational modification, we used a proteomic approach to investigate the broader role of lysine acetylation in the heart using a guinea pig model. Briefly, hearts were fractionated into myofilament-, mitochondrial- and cytosol-enriched fractions prior to proteolysis and affinity-enrichment of acetylated peptides. LC-MS/MS analysis identified 1075 acetylated peptides, harboring 994 acetylation sites that map to 240 proteins with a global protein false discovery rate <0.8%. Mitochondrial targets account for 59% of identified proteins and 64% of sites. The majority of the acetyl-proteins are enzymes involved in fatty acid metabolism, oxidative phosphorylation or the TCA cycle. Within the cytosolic fraction, the enzymes of glycolysis, fatty acid synthesis and lipid binding are prominent. Nuclear targets included histones and the transcriptional regulators E1A(p300) and CREB binding protein. Comparison of our dataset with three previous global acetylomic studies uniquely revealed 53 lysine-acetylated proteins. Specifically, newly-identified acetyl-proteins include Ca2+-handling proteins, RyR2 and SERCA2, and the myofilament proteins, myosin heavy chain, myosin light chains and subunits of the Troponin complex, among others. These observations were confirmed by anti-acetyl-lysine immunoblotting. In summary, cardiac lysine acetylation may play a role in cardiac substrate selection, bioenergetic performance, and maintenance of redox balance. New sites suggest a host of potential mechanisms by which excitation-contraction coupling may also be modulated.
Highlights
Acetylation of lysine residues on histones was first recognized as a post-translational modification nearly 50 years ago [1]
Other rodents have strikingly different electrophysiological and Ca2+ handling properties that make it difficult to extrapolate findings to the human disease process. Other large animals, such as the rabbit, dog or cat could provide relevant information; at much greater expense. Another major advantage is that the basic properties of guinea pig cardiac physiology have been extensively characterized previously, and the aortic banding model of hypertrophy and failure has been validated in prior studies, including changes occurring in ion channels and Ca2+ handling that are similar to findings in human failing hearts
The dataset may not necessarily be comprehensive, as it is subject to any biases that may arise from the specificity of the acetyl-lysine antibody chosen for affinity-peptide enrichment, though this strategy was recently used to identify as many as 15,474 sites from 16 rat tissues [67]
Summary
Acetylation of lysine residues on histones was first recognized as a post-translational modification nearly 50 years ago [1]. Families of histone acetyltransferases and deacetylases have been discovered, and nuclear protein acetylation has emerged as paramount in chromatin remodeling and transcriptional regulation [2]. The advent of new proteomic tools has permitted global scale assessments of lysine acetyl-proteomes [3,4,5,6]. From these studies, it has become apparent that lysine acetylation is a widespread, evolutionarily conserved post-translational modification whose scope rivals phosphorylation. We identified acetylproteins unique to the cardiac proteome by mass spectrometry and validated them by immunoblotting
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