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. 2018 Sep 7;38(5):BSR20180721.
doi: 10.1042/BSR20180721. Print 2018 Oct 31.

Obesity-mediated regulation of cardiac protein acetylation: parallel analysis of total and acetylated proteins via TMT-tagged mass spectrometry

Samantha S Romanick  1   2   3 , Craig Ulrich  3 , Karen Schlauch  4   5 , Andrew Hostler  1 , Jordanna Payne  6 , Rebekah Woolsey  7 , David Quilici  7 , Yumei Feng  2   3 , Bradley S Ferguson  8   2
Affiliations

Obesity-mediated regulation of cardiac protein acetylation: parallel analysis of total and acetylated proteins via TMT-tagged mass spectrometry

Samantha S Romanick et al. Biosci Rep. .

Abstract

Lysine residues undergo diverse and reversible post-translational modifications (PTMs). Lysine acetylation has traditionally been studied in the epigenetic regulation of nucleosomal histones that provides an important mechanism for regulating gene expression. Histone acetylation plays a key role in cardiac remodeling and function. However, recent studies have shown that thousands of proteins can be acetylated at multiple acetylation sites, suggesting the acetylome rivals the kinome as a PTM. Based on this, we examined the impact of obesity on protein lysine acetylation in the left ventricle (LV) of male c57BL/6J mice. We reported that obesity significantly increased heart enlargement and fibrosis. Moreover, immunoblot analysis demonstrated that lysine acetylation was markedly altered with obesity and that this phenomenon was cardiac tissue specific. Mass spectral analysis identified 2515 proteins, of which 65 were significantly impacted by obesity. Ingenuity Pathway Analysis® (IPA) further demonstrated that these proteins were involved in metabolic dysfunction and cardiac remodeling. In addition to total protein, 189 proteins were acetylated, 14 of which were significantly impacted by obesity. IPA identified the Cardiovascular Disease Pathway as significantly regulated by obesity. This network included aconitate hydratase 2 (ACO2), and dihydrolipoyl dehydrogenase (DLD), in which acetylation was significantly increased by obesity. These proteins are known to regulate cardiac function yet, the impact for ACO2 and DLD acetylation remains unclear. Combined, these findings suggest a critical role for cardiac acetylation in obesity-mediated remodeling; this has the potential to elucidate novel targets that regulate cardiac pathology.

Keywords: acetylation/deacetylation; muscle contraction; muscle metabolism; myocardial remodeling; proteomics.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Obesity-mediated cardiac hypertrophy and fibrosis
Hearts were extracted from male C57BL6/J mice on CD or HFD and (A) heart weight and LV weight measured and normalized to tibia length. (B) RNA was isolated from LV tissue and ANP, BNP mRNA expression was analyzed via qPCR from animals on CD and HFD. (C) PicroSirius Red staining of collagen in LV tissue of CD and HFD animals. (D) Quantitation of collagen, fibrosis, present in LV tissue of CD and HFD animals. At last, (E) mRNA expression of Col1 and Ctgf-1 was assessed by qPCR. Student’s t test with Welch’s correction using GraphPad Prism software was used to determine significance. *P<0.05.
Figure 2
Figure 2. Obesity exacerbates cardiac tissue lysine acetylation
Protein was isolated from (A) the LV, (B) gastrocnemius, (C) white epididymal adipose tissue, or (D) liver from mice fed a CD or HFD and immunoblotted for acetyl-lysine (α-rabbit), acetyl-lysine (α-mouse), acetyl-α-tubulin, total α-tubulin, and total H3.
Figure 3
Figure 3. Experimental proteomic workflow to examine the cardiac acetylome
Representation of the experimental workflow to examine the cardiac acetylome. Hearts from obese animal were dissected and protein lysates TMT tagged and assessed via tandem MS prior to data analysis, interpretation, and IPA.
Figure 4
Figure 4. Obesity-mediated regulation of the cardiac proteome by KLF15 and PPARα transcriptional regulators
(A,B) Pathway analysis (IPA) determined potential activation of the transcriptional regulators KLF15 and PPARα nuclear receptors by increased protein expression of downstream targets. Green highlighted proteins indicate a decrease in total protein expression and red indicate an increase in total protein expression. (C) IPA generated interaction map of KLF15 target proteins shown to be acetylated in this report. Green highlighted proteins indicate decreased acetylation and red indicate increased acetylation. Solid lines indicate direct protein–protein interactions, dashed lines indicate potential protein–protein interaction, and arrows imply directionality of protein–protein regulation.
Figure 5
Figure 5. Obesity-mediated PPARα nuclear receptor regulation of the cardiac acetylome
Pathway analysis (IPA) generated an interaction map of PPARα target proteins shown to be acetylated in this report. Green highlighted proteins indicate decreased acetylation and red indicate increased acetylation. Solid lines indicate direct protein–protein interactions, dashed lines indicate potential protein–protein interaction, and arrows imply directionality of protein–protein regulation.
Figure 6
Figure 6. Obesity-mediated regulation of the cardiac acetylome and pathway analysis
IPA generated (A) stacked canonical pathways significantly affected by obesity, (B) comparison of down- and up-regulated acetylated proteins, and (C) the overlapping pathways in which ten or more acetylated proteins are shared.
Figure 7
Figure 7. Protein acetylation impacts proteins involved in the cardiovascular disease pathway
Down-regulation and up-regulation of protein acetylation within the Cardiovascular Disease Pathway impacted by obesity. Green highlighted proteins indicate decreased acetylation, red indicate increased acetylation, and white indicate no significant changes in acetylation. Solid lines indicate direct protein–protein interactions, dashed lines indicate potential protein–protein interaction, and arrows imply directionality of protein–protein regulation.
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