Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two‐dimensional polyacrylamide gel electrophoresis and mass spectrometry

Abstract Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta‐oxidation, electron transport chain and Krebs cycle to be down‐regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D‐PAGE and nanoLC‐MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down‐regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D‐PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181.


| INTRODUCTION
Obstructive sleep apnea (OSA) is characterized by repeated cessation in respiration (apnea) in the upper airway during sleep lasting at least 10 seconds. Apneas occur as a result of temporary pharyngeal collapse or narrowing, resulting in decreased blood oxygen saturation. 1 OSA has been associated with headache, daytime sleepiness, obesity, depression, arthritis, type 2 diabetes mellitus, arteriosclerosis, atherosclerosis, hypertension, atrial arrhythmia and sudden cardiac death. 1,2 Although OSA has been closely associated with atrial arrhythmia, atrial enlargement, increased P wave duration and decreased atrial voltage; little is known about the molecular pathways causing these pathologies. 3,4 Many animal models do not address both the obstructive and hypoxemic components of OSA in conscious, free roaming animals. In order to accurately reproduce the effects of OSA as observed in the clinic, we have employed a surgical model involving a silicone obstructive device implanted in the trachea of conscious, free roaming rats. 3 Here, we used 2D PAGE coupled with nanoLC-MS/MS as a complementary approach to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. While the 1D-PAGE approach compared the whole atrial proteomes from the severe OSA, moderate OSA and controls, 3 the 2D-PAGE approach allowed us to identify only the dysregulated proteins from these conditions. Furthermore, 2D-PAGE also allowed us to identify different protein isoforms, already demonstrated in others' work. 5 In this study, we not only found that the entire glycolytic pathway and Krebs cycle are down-regulated, but also found evidence that additional enzymes involved in the beta-oxidation, electron transport chain and Krebs cycle anaplerotic reactions were also down-regulated. Other protein dysregulations identified are involved in metabolic, structural or inflammatory pathways, suggesting that these proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia.

| MATERIALS AND METHODS
The rats and the rat model setup ( Figure S1) and the sample preparation method used in this study was described previously. 3,6 Atrial homogenates of control (n = 2), moderate (n = 2) and severe (n = 2) apnea were analysed by 2D PAGE and nanoLC-MS/MS analysis. A total of 2293 protein spots were compared for statistical differences, of which 208 spots having a fold increase or decrease of ≥1.7 and a P ≤ 0.05, or a fold increase or decrease of ≥3.0 were selected for nanoLC-MS/MS analysis, as previously described. 6 Raw data were processed as in, 6 using NCBInr rat (rattus) database. The mass spectrometry proteomics data have been deposited to the Pro-teomeXchange Consortium via PRIDE partner repository with dataset identifier PXD011181 and 10.6019/PXD011181. 7

| RESULTS
Comparison of the protein pattern between severe OSA versus control and moderate OSA versus control was done using 2 biological replicates for each condition. Two Coomassie and 1 silver stained gels were run for each of the 6 samples (2 controls, 2 moderates and 2 severe samples). Figure S2 shows an example of one Coomassie stained gel for each sample and Figure 1 shows the silver stained 2D PAGE gel images of the 3 conditions in duplicates. Figure S3 depicts the 2D gel difference image of averaged severe apnea vs averaged control where the spots increased in severe are shown in blue and the spots decreased in severe are shown in red. A summary and comparison of all the proteins identified from the spots of severe, moderate and control gels are shown in Tables S1-S3. A few important proteins dysregulated between severe apnea, moderate apnea and control conditions, their function and their possible association with OSA disease are discussed below.
Down-regulated proteins in severe apnea samples when compared to control samples are shown in Figure S4B

| Dysregulated proteins in moderate apnea samples, compared to controls
Up-regulated proteins in moderate apnea samples when compared to controls are shown in Figure S5A. Some of these proteins include Additionally, many enzymes associated with aerobic metabolic pathways like glycolysis, Krebs cycle and anaerobic respiration were dysregulated in severe and moderate apnea when compared to control which was also identified to be dysregulated in our 1-D analysis.

| Protein charge heterogeneity on 2D PAGE
Proteins expressed from a single gene can undergo different posttranslational modifications (PTMs), 8 such as acetylation, methylation, phosphorylation or nitrosylation. These modifications alter the pI of the proteins with a slight change in the molecular weight which will induce a spot shift in the 2D gels across the pH gradient. 9 Hence, the protein products can migrate to multiple spots on the gel. This can be observed in our study where we have found the same protein to be both up-regulated and down-regulated in severe and/or moderate when compared to control. For example, protein AR was found to be up-regulated in severe when compared to control in spot no. 1516 (Table S1) as well as down-regulated in spot no. 1485 and 1489 (Table S1). The different spots are observed for the same protein because of its charge heterogeneity caused by the PTMs, suggesting that OSA is not only a dysregulation of the levels of proteins, but also the proteins' PTMs.

| DISCUSSION
Apnea has also been found to cause myocardial dysfunction. 10 12,16 While in our previous study that employed 1D-PAGE followed by nanoLC-MS/MS analysis, we focused on a comparison of the full proteomes of the atrial tissues from control, moderate apnea and severe apnea, in current study that employed 2D-PAGE followed by nanoLC-MS/MS analysis, we focused only on the proteins that are dysregulated in the atrial tissues from control, moderate apnea and severe apnea. Therefore, the two studies could not only confirm, but also complement each other and produced also a more accurate representation of the proteomic changes as they occur in the atria. This is also evidenced by the large number of dysregulated identified proteins in 2D-PAGE, which greatly complements our previous study. 3

Control
Moderate Severe   Specifically, in the 1D-PAGE study we found that some of the enzymes involved in the aerobic and anaerobic glycolysis, as well as in the Krebs cycle are down-regulated.
In the current 2D-PAGE study, additional enzymes involved in the aerobic glycolysis (Krebs cycle, electron transport chain), anaplerotic reactions (enzymes involved in the replenishment of the metabolites, ie oxaloacetate, alpha-keto-glutarate, succinylCoA) involved in the Krebs cycle and beta-oxidation were also found, suggesting that the OSA-induced oxygen deprivation of the heart induced down-regulation of the entire glycolytic pathway, Krebs cycle, anaplerotic reactions, electron transport chain and beta-oxidation of the fatty acids.
For example, citrate synthase, which plays an important role as the first enzyme in the citric acid cycle by converting oxaloacetate and acetyl CoA to citrate and CoA-SH 17 is down-regulated in various types of heart failure. 12 In addition, mitochondrial dysfunction and citrate synthase down-regulation have been shown to be exacerbated by atrial fibrillation. 11 In our study, not only citrate synthase, but also the entire glycolytic pathway were down-regulated. A comparative summary of the dysregulated enzymes in these pathways in our 1D-PAGE 3 and our current 2D-PAGE experiments is shown in Figure 2.
Overall, we believe our model accurately recreates the cardiac effects of OSA as it occurs in humans. This model also led to identification of protein dysregulations that may indicate a potentiation for atrial arrhythmia, decreased atrial compliance, increased inflammation and risk of cardiomyopathy. This pilot study and methodology will be used as a baseline criterion to compare the protein dysregulations in severe apnea and moderate apnea with control using many biological and technical replicates.

S U P P O R T I N G I N F O R M A T I O N
Additional supporting information may be found online in the Supporting Information section at the end of the article.