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. 2013 Jul 2;8(7):e67513.
doi: 10.1371/journal.pone.0067513. Print 2013.

The Cardiac Acetyl-Lysine Proteome

Free PMC article

The Cardiac Acetyl-Lysine Proteome

D Brian Foster et al. PLoS One. .
Free PMC article


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 Ca(2+)-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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Proteomic Work-flow.
Asterisks denote that since the data from the microsomal and plasma membrane fraction yielded few new sites or proteins, it was combined with data from the cytosolic fraction for the purposes of discussion.
Figure 2
Figure 2. The Dataset.
Panel A. Distribution of acetylated proteins by experimental subfraction. Panel B. Distribution by biological replicate. Panel C. Comparison with published global-scale mammalian lysine acetylomes including: (*) mouse liver , (&) human acute myeloid leukemia cell line, MV4-11 , and (%) human liver . Panel D. Comparison with the (@) mouse liver mitochondrial lysine acetylome of Schwer et al .
Figure 3
Figure 3. The Cardiac Lysine Acetylome.
Acetyl-proteins are grouped first by cellular component and then by molecular function. Asterisks (*) designate acetyl-proteins found in all three biological replicates. Acetylation sites are numbered according to guinea pig sequence. The numbering of orthologous human lysine residues generally tracks very closely with guinea pig sequences. Orthologous human lysines are found in Table S2.
Figure 4
Figure 4. Functional Annotation and Enrichment analysis.
Panel A. Distribution of acetylation sites by cellular component. Panel B. Distribution of acetylation sites by biological process. Data for panels A and B were taken from Figure 3 and expressed as percentages of the total number of sites. Panel C. Network of gene ontology annotations for molecular function. Node color represents the magnitude of corrected p-value, a continuous variable that provides a measure of enrichment (see legend). Node size is proportional to the number of genes associated with each GO-term. The layout is broadly grouped by similar molecular functions, including catalytic activity, ligand binding and transport activity.
Figure 5
Figure 5. Immunodetection of Cardiac Lysine Acetylation.
Panel A. Guinea pig cardiac myofilaments were prepared essentially as described by Solaro et al. . 30 µg of protein was subjected to electrophoresis and immunoblotting with both monoclonal and polyclonal antibodies to acetylated lysine as described in the Methods section. Lanes with immunoreactive bands are juxtaposed with the corresponding exposure of a parallel control experiment conducted with >2% w/v acetylated BSA in the primary antibody incubation step. Panels B and C. Macromolecular respiratory complexes were resolved as described in the methods. Up to 20 µg of protein from the 30% w/v sucrose fraction (enriched Complex I, Panel B) and the 22.5% w/v sucrose fraction (enriched complex V, Panel C) were probed for immunoreactivity to anti-acetylated lysine as described for Panel A.
Figure 6
Figure 6. Lysine Acetylation of Two Key Proteins in EC Coupling.
Panel A depicts a structural ribbon model of guinea pig SERCA2a with helices in cyan and beta strands in magenta; the membrane domain, the phosphorylation domain (P-domain) and the nucleotide binding domain (N-domain) are shown. Residues that are important for nucleotide binding are shown in salmon hue. The positions of lysine residues acetylated in our study (K464, K510, K533) are confined to the distal end of the cytoplasmic nucleotide-binding domain (N-domain), and are highlighted in red. Panel B depicts a model of guinea pig myosin heavy chain beta S1 head. Helices and beta strands are shown in pale cyan for simplicity. The positions of lysines acetylated in the dataset are highlighted. Magenta residues represent acetylation sites identified by the most spectra. Red residues were identified by fewer spectra. Solid blue lysine residues are sites of acetylation that are mutated in HCM. Spotted blue residues indicate positions of HCM mutation immediately adjacent to an acetylation site. Brown residues denote the positions of lysine residues found to be acetylated in previous studies by Samant et al . Lysines highlighted in the discussion are numbered for clarity in each panel.

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