Development of a Novel Proteomic Approach for Mitochondrial Proteomics from Cardiac Tissue from Patients with Atrial Fibrillation

Abstract

Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting approximately 2.2 million Americans. Because several studies have suggested that changes in mitochondrial function and morphology may contribute to AF, we developed a novel proteomic workflow focused on the identification of differentially expressed mitochondrial proteins in AF patients. Right human atrial tissue was collected from 20 patients, 10 with and 10 without AF, and the tissue was subjected to hydrostatic pressure cycling-based lysis followed by label-free mass spectrometric (MS) analysis of mitochondrial enriched isolates. Approximately 5% of the 700 proteins identified by MS analysis were differentially expressed between the AF and non-AF samples. We chose four differentially abundant proteins for further verification using reverse phase protein microarray analysis based on their known importance in energy production and regulatory association with atrial ion channels: four and a half LIM, destrin, heat shock protein 2, and chaperonin-containing TCP1. These initial study results provide evidence that a workflow to identify AF-related proteins that combines a powerful upfront tissue cell lysis with high resolution MS for discovery and protein array technology for verification may be an effective strategy for discovering candidate markers in highly fibrous tissue samples.

Proteomics of Atrial Fibrillation

“…Cytoskeletal proteins, desmin and actin, were found to be increased, and this corresponded to findings in other studies. 15,20,39 In addition, the metabolic enzyme, 3-oxoacid transferase, was upregulated in AF (left atrial appendage only), consistent with other groups. 15,39 This enzyme plays an important role in ketone body metabolism, suggesting a potential increased role of ketone bodies as a substrate for ATP production in AF.…”

“…They grouped these into 3 classifications: cytoskeletal and myofilament-associated protiens, proteins involved with energy metabolism, and those linked to oxidative stress. Although proteins associated with energy metabolism have been demonstrated by other proteomic studies, only 1 protein, pyruvate kinase, overlapped between this study and that of Goudarzi et al 39 Furthermore, this 40 and previous work by Mayr et al 15 highlighted differential expression of the antioxidant peroxiredoxin 1, being up-and downregulated, respectively. These differing findings may be because of several reasons, which will be discussed later.…”

“…Recent work by Goudarzi et al 39 zoomed in on the mitochondrial proteome in preference to whole tissue homogenates in human RAA. The authors focused on mitochondrial proteomics, predominantly as a result of an earlier study by Mayr et al 15 This is of particular importance because a role for metabolic remodeling has been suggested in the pathophysiology of AF, along with mitochondrial dysfunction.…”

The Use of High-Throughput Technologies to Investigate Vascular Inflammation and Atherosclerosis

“…93 In addition, the importance of ERK1 and ERK2 phosphorylation was shown in vitro in bovine aortic ECs after stimulation with oxidized phospholipids using liquid chromatography-mass spectrometry analysis. 94 Supplementally, proteomics has also been proven to be of emerging interest in atrial fibrillation-associated stroke, 3 cerebral infarction, 95 and focal cerebral ischemic injury, 96 identifying potential marker proteins such as HSP2 or HSP60. Furthermore, proteomic screening of prediagnostic plasma samples from postmenopausal women receiving a hormone therapy described B2M and insulin-like growth factor binding protein 4 as risk markers for CAD and stroke, respectively.…”

“…About 80% of strokes are ischemic, and atherosclerosis, but cardioembolism mostly triggered by atrial fibrillation-and small-vessel disease are also very common causes. 2,3 The multifaceted pathomechanisms underlying all these diseases necessitate a holistic study approach. Therefore, in vitro experiments, which lack the complex organization of an organism, need to be combined with clinical and animal studies.…”