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Global Translational Medicine Proteomic analysis of heart in metabolic cardiomyopathy

will be affected by type 2 diabetes by the year 2040 . An In the present study, we aimed to identify the key
[1]
increasing number of evidence indicates that patients differentially expressed proteins (DEPs) and pathways
who suffer from glucometabolic perturbations are more involved in high-fat diet (HFD)-induced MC by analyzing
susceptible to metabolic cardiomyopathy (MC), which is the proteome of hearts from the indicated mice. We
independent to the presence of hypertension, coronary compared the DEPs between samples from control and
artery disease, and other comorbidities [2,3] . Over 80% of HFD groups. Interestingly, we identified 90 proteins
patients who suffered from heart failure are either obese which were only detected in the control group and 18
or diabetic [4,5] . Notably, the prevalence of MC is expected proteins which were only detected in the HFD group. In
to rise in the next decades. In patients with glucometabolic these DEPs, most of them belong to the metabolic related
perturbations, the accumulation of lipotoxic by-products process. Our results revealed the differentially expressed
in cardiac myocyte could lead to myocyte apoptosis and proteome related to the progression of MC, which could
contractile dysfunction, eventually resulting in MC. be a potential therapeutic target for MC.
Detrimental effects on cardiomyocytes in patients with
obesity and type 2 diabetes include changes in tissue 2. Materials and methods
metabolism, substrate utilization, inflammation, and 2.1. Mice
oxidative stress. These effects are thought to induce heart
failure [6-9] . However, the underlying mechanisms remain Eight-week-old male wild-type mice (Chongqing Tengxin
unclear and therapies of MC are yet to be developed. [2] Biotechnology Co., Ltd) were fed a standard diet and HFD
(60% kcal fat; Research Diets, New Brunswick, NJ, USA),
The molecular mechanisms underlying MC are respectively, for 5 months in a specific pathogen-free
mainly based on metabolic dysregulation, inflammation, environment provided by the Experimental Animal Center
fibrosis, oxidative stress, and apoptosis. Although free of Southwest Medical University. The mice were separated
fatty acids are the preferred energy substrate for cardiac into two groups, the control group and the HFD group.
cells, alternative fuel sources, such as glucose, lactate, or The mice were sacrificed to collect the hearts, and all the
ketone bodies, are also ultilized by the heart to balance heart tissue were immediately labeled and stored at −80°C
the energy supply and by-products overproduction. until protein extraction. Animal protocols were approved
In obese or diabetic people, mitochondrial fatty acid by Institutional Animals Ethics Committees of Southwest
β-oxidation is increased due to the hyperglycemia and Medical University (Approval No. 20220225-014).
insulin resistance, which is regulated by the PPAR family-
mediated transcriptional machinery. For example, the 2.2. Preparation of samples for liquid chromatography-
down-regulation of GLUT4 reduces the uptake of glucose tandem mass spectrometry (LC-MS/MS)
in the cardic cells . Peroxisome proliferator-activated To prepare samples for LC-MS/MS, we performed an
[10]
receptor-gamma coactivator-1 alpha, estrogen-related in-solution digestion by trypsin; the protocol is as follows:
receptor alpha, NFE2 like BZIP transcription factor 2 (i) 8 M urea was added to 300 µg lysates; (ii) proteins
(NFE2L2), or nuclear respiratory factor 1 (NRF1) and were reduced with 5 mM dithiothreitol and incubated for
nuclear factor erythroid 2-related factor 2 (NRF2), which 45 min at 56°C to reduce disulfide bonds; (iii) mixture
are up-regulated in obesity and diabetes, could promote was cooled to room temperature and alkylated with
fatty acid oxidation and shut down glucose oxidation [11,12] . iodoacetamide to a final concentration of 20 mM; (iv)
It is well known that the increased fatty acid oxidation the mixture was incubated for 30 min in the dark at room
will lead to lipotoxicity, which subsequently activates the temperature; (v) the mixture was diluted 8-fold with 1 M
proinflammatory transcription factor such as nuclear urea using 10 mM triethylammonium bicarbonate and
factor kappa B (NF-κB). NF-κB could increase the subsequently digested using 1:20 (w/w) trypsin at 37°C
downstream targets, such as activator protein 1, nuclear overnight; and (vi) Oasis HLB Cartridge 30 mg (Waters
factor of activated T-cells, or NF-κB itself, which carry Corporation, Milford, MA, USA) was used to desalt the
out numerous autocrine activities, including the secretion tryptic digests, and it was lyophilized for the subsequent
of cytokines and chemokines . Increased inflammation MS/MS analysis.
[13]
finally impairs myocardial tissues and causes cardiac
remodeling by interstitial fibrosis. In addition, increase 2.3. Data processing and parameters
fatty acid β-oxidation-induced oxidative stress stimulates LC-MS/MS was used for analysis for the six sets of digested
the proinflammatory transcription factors and the peptides. The dried peptides were re-dissolved in 30 µL 0.1%
activation of mitogen-activated protein kinase, involving formic acid in UHQ water. The nano-LC-MS experiments
the proapoptotic c-Jun N-terminal kinase and p38, which were performed using AB Sciex 5600+ mass spectrometer.
promotes cell death in MC . The sample was applied onto a high-performance liquid
[14]

Volume 1 Issue 2 (2022) 2 https://doi.org/10.36922/gtm.v1i2.137