This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Cardiovascular diseases (CVDs) are a group of conditions that affect the heart and blood vessels, and they are the leading cause of death worldwide. According to the World Health Organization, CVDs account for over 17 million deaths each year, which is approximately 31% of all deaths worldwide (1). Hypertrophic cardiomyopathy (HCM) is a common genetic cardiac disease with a distribution of 1:200-1:500 in the general population (2). HCM is also associated with various severe symptoms including heart failure, valvular heart disease, arrhythmias, heart attack, and left ventricular hypertrophy. Diagnosing heart disorders is often difficult, and HCM is typically confirmed by cardiovascular imaging combined with echocardiography. However, diagnosis remains challenging for clinicians because of the diversity in disease manifestations along with differences in ventricular mass or morphologic expression across patients (3). The limited self-renewal capacity of myocardial cells complicates survival evaluation for patients with heart failure (4). Therefore, understanding the biological processes leading to cardiomyocyte hypertrophy and apoptosis is crucial for developing effective therapies (5). Proteomic studies have provided insights into the mechanisms underlying CVDs, especially in the development of biomarkers.
Current protein biomarkers for heart diseases include troponin I and T, myoglobin, creatine kinase, and creatine kinase‐ myoglobin (6). Natriuretic peptides are the most extensively studied but have not been used as biomarkers in clinical applications (7). Abnormal troponin expression is widely used as a diagnostic and prognostic marker for heart diseases. Troponin I and troponin T are highly sensitive in clinical samples, but their expression levels differ in patients with various heart syndromes.
Acute myocardial infarction (AMI), also known as a heart attack, is an obstruction condition that occurs when blood flow is abruptly disrupted, resulting in heart tissue or coronary artery injury (8). We have discovered obscurin and titin as biomarkers from human plasma using liquid chromatography with tandem mass spectrometry for AMI, which showed higher sensitivity than troponin I and T (9). Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which have equivalent secretory profiles in cardiomyocyte under tension response, have been used as biomarkers to diagnose AMI and acute coronary syndrome in humans. However, the understanding of their biochemistry is limited, and post-translational modifications in their metabolism result in unsuccessful detection (10). In this study, we established an
ET-1 and Ang II-treated immortalized human cardiomyocytes (T0445 cell line) were evaluated to determine their hypertrophic responses. The expression of sarcomeric α-actinin resulted in cytoskeletal actin rearrangement and increased cell surface area. Both hypertrophic reagents (11) showed a very similar increase in cell surface area after 4 h and 24 h of treatment, followed by the visible enlargement of actin fibrous tissue compared to untreated control cells (Fig. 1A). Moreover, the average cell surface areas of ET-1- and Ang II-treated cells were 3.5-fold higher than those of normal cells (Fig. 1B). Thus, cardiomyocyte hypertrophy was successfully induced by 200 nM ET-1 and 1 μM Ang II in T0445 cells. Immunocytochemistry revealed increased cell size and thickened cell membrane in the treated group compared to the control group. The phenotypical changes may include increased cell size, primary changes in the structures of the heart muscle cells, and alterations in the contractile properties of the heart. The significant change in cell size after treatment confirmed the induction of the disease model (Fig. 1A, B).
Western blots were performed to validate the successful induction of hypertrophy in the cardiomyocyte model; we confirmed the expression of secretome proteins at different time points over the treatment courses. The secretome of samples treated with 200 nM ET-1 showed significantly increased BNP expression after 15 min, 1 h, and 2 h, whereas ANP expression increased after 1, 2, 6, and 24 h of treatment (Fig. 1C, D). In contrast, the 1 μM Ang II-treated secretome displayed a notable enhancement of BNP levels after 1, 2, 12, and 24 h, along with increased ANP expression after 15 min, 1 h, and 2 h of treatment (Fig. 1E, F). BNP and ANP levels fluctuated according to time variations, and reproducibility increased 24 h after treatment.
Proteomic analysis of hypertrophied and non-hypertrophied cardiomyocytes revealed distinct protein profiles. All total ion chromatogram data are shown in (Supplementary Fig. 1), and the spectral count for all samples was 10 × E9.
A total of 200-300 proteins were identified from secretome proteins using Proteome Discoverer. Of these, 32 proteins with elevated levels were selected (Supplementary Table 1), showing the highest value of cross-correlation score (Xcorr) consistency for reproducibility in both ET-1 and Ang II treatment (fold change >1.4). We identified several proteins that have already been proposed as cardiac hypertrophy biomarkers, such as talin-1 (12), filamin-A (13), follistatin-related protein 1 (14), profilin-1 (15, 16), and vimentin (17, 18). Our results showed the upregulation of six 14-3-3 protein isoforms (beta, epsilon, gamma, theta, zeta/delta, and eta) in both ET-1- and Ang II-treated cardiomyocytes.
The expression of 17 proteins was significantly downregulated in the secretome of hypertrophied cardiomyocytes in both treatments (ET-1 and Ang II) with reproducibility (Supplementary Table 2). Isoform 8 of filamin-B, alpha-2 HS-glycoprotein, glutathione S-transferase P, and T-complex protein 1 subunit beta and epsilon were reduced by 100-fold in hypertrophied cardiomyocytes compared to normal cells.
Canonical pathway analysis categorized the dramatically altered protein groups from ET-1- (Fig. 2A) and Ang II-treated hypertrophied cardiomyocytes (Fig. 2B). Among the most abundant signaling categories, 14-3-3-mediated signaling had the highest significance in both hypertrophied cardiomyocyte groups, whereas Hippo signaling was decreased. Protein-protein interaction networks analyzed by ingenuity pathway analysis (IPA) explored a tight and robust interaction network of the identified 14-3-3 family (Supplementary Fig. 2A). Furthermore, our data revealed that 14-3-3 binding inactivates the Hippo signaling pathway by sequestering the YAP/TAZ function in the cytoplasm (Supplementary Fig. 2B).
To verify the utility of 14-3-3 proteins as organ-specific biomarkers for cardiac hypertrophy, we analyzed the expression of six isoforms (Supplementary Table 3) from the secretome of different cell lines, including MKN-1 and HFE-145, Hep-3B and Hepa-RG, A549 and L132, and T0445, in control and hypertrophy treatment samples. In this context, we evaluated the relative intensity of the six 14-3-3 protein isoforms by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) coupled with multiple reaction monitoring (MRM). Peptide ions were selected for fragmentation using MS/MS, and spectrum MS/MS sequencing data are shown in Supplementary Fig. 3.
The expression of 14-3-3 protein-gamma was extremely low, and was inconsistently detected in the samples. Therefore, we compared the intensity of five 14-3-3 isoforms between different cell lines including cardiomyocytes without hypertrophy induction by measuring the peak area ratios of one fragment ion from the internal standard (beta-actin) and the endogenous peptide. All five 14-3-3 protein isoforms were elevated in hypertrophy-induced cardiomyocytes, as revealed by MRM. The abundances of 14-3-3 protein-beta and protein-zeta/delta (Fig. 3A, C) showed the highest significant difference (>10-fold) intensity among all isoforms while 14-3-3 protein theta (Fig. 3E) exhibited a lower expression (<2 fold) than other isoforms across cell lines. The expression of 14-3-3 protein-eta and protein-epsilon (Fig. 3B, D) were similar to those of different organ cells. The levels of 14-3-3 proteins were also measured in human plasma using ELISA (8 samples each from healthy controls and patients with AMI) and were elevated in all samples of patients with AMI compared to those of healthy controls (Supplementary Fig. 4).
Dual quantitative and qualitative analyses were used to confirm the biomarker candidates 14-3-3 protein-zeta/delta using synthesized peptide standards. 14-3-3 protein-zeta/delta was selected because its concentration in the secretome was significantly higher in induced cardiac hypertrophy cell lines than in other organ cell lines. The gradient and MS operating conditions and quantitative mass spectrometry with an MRM assay employed one unique peptide among the three transition ions monitored are presented in Supplementary Table 4. Representative MRM chromatograms obtained from 14-3-3 protein-zeta standard (Fig. 4A), T0445 control cell line (Fig. 4B), T0445 hypertrophied cell lines (Fig. 4C, D), and human plasma samples (Fig. 4E, F).
MRM assays were performed in 30 AMI and 23 healthy control plasma sample pools. The mass chromatogram peak areas from each run were calculated for 14-3-3 protein-zeta expression. The scatterplot distribution of the logarithm of average MRM peak areas (a.u.) according to the AMI/control status of the pool (Fig. 4G). Levels of 14-3-3 protein-zeta were found to be higher in the AMI group relative to those in the control group. Importantly, ROC curve analysis revealed AUC values of 0.854 (95% confidence interval (CI) 0.696-0.949) with a Youden index J of 0.6923, indicating the potential to use 14-3-3 protein-zeta expression as a biomarker for AMI diagnosis (Fig. 4H). These results demonstrated a strong correlation between spectral count-derived abundance and MRM peak area, indicating sensitivity and specificity of 69.2 and 100 respectively. Considering these findings, we identified a label-free quantitative analysis-based peptide that can differentiate between control and AMI groups with high specificity from logistic regression-based prediction modeling in clinical samples.
This study elucidated the role of the 14-3-3 protein family in HCM using nano-LC-MS/MS. Proteomic analysis revealed significant upregulation of 14-3-3 proteins in the secretome of the hypertrophy-induced heart cell line compared to the control. As shown in Supplementary Tables 1 and 2, filamin B is a cytoplasmic actin-binding protein that has been found in human skeletal disorders and impaired microvascular environments (19). T-complex protein 1 is a chaperonin that plays an important role in the folding of cytoskeletal proteins (20). Histone 1.5 is a linker protein that binds and affects chromatin (21). Histone 1.3 elevation has been reported as a prognostic biomarker for pancreatic ductal adenocarcinoma (22). Histone proteins appear to be downregulated by anti-apoptotic cell reactions. As shown in Supplementary Table 2, gelsolin, alpha-2-macroglobulin, and elongation factor 1-alpha 1 were downregulated. Gelsolin is an actin-binding protein known for its role in cell motility and apoptosis (23).
Considering the complex underlying pathogenic factors associated with cardiomyocyte hypertrophy, we investigated the secretome to identify proteins related to this condition. The study of secretome, which consists of extracellular proteins, has gained significant attention in the research community owing to their potential to reveal biomarkers and new therapeutic targets (24). The 14-3-3 isoform proteins are expressed in high levels in the brain, skeletal muscles, heart, and embryonic stem cells with a wide range of roles in cell signaling. After platelet activation, 14-3-3 protein-zeta was localized in and secreted from dense granules (25). Transport vesicles budding from the endoplasmic reticulum carry these secreted proteins from the inside of the cell to the cell surface. From there, the vesicles fuse with the plasma membrane, releasing 14-3-3 protein into the extracellular space (26).
14-3-3 protein-zeta constrains the insulin secretion in patients with type 2 diabetes by regulating mitochondrial function. Mice that expressed dominan t-negative 14-3-3 mutant protein in the myocardium showed increased occurrence of cardiac hypertrophy, fibrosis, and inflammation after diabetes induction (27). Thus, modulating 14-3-3 protein expression may serve as a novel therapeutic approach against diabetes-associated cardiovascular complications. However, the exact underlying mechanism is not fully elucidated due to its diverse structure and protein interaction. Further studies are required to assess the functional role of 14-3-3 protein-zeta in cardiac hypertrophy.
This study showed that several signaling pathways were activated or inhibited in response to HCM. The IPA results revealed that the most activated upstream regulators were glycolysis, actin cytoskeletal signaling, and 14-3-3 mediated signaling. In contrast, the most inhibited regulator was the Hippo signaling pathway, with a z-score <−2. The Hippo signaling pathway controls organ size by suppressing cell proliferation, limiting cell size, and inducing apoptosis. The inactivation of this pathway or activation of its downstream effectors has been reported to improve cardiac regeneration (28, 29). Studies have also established the crucial role of the actin cytoskeleton in regulating the Hippo-YAP/TAZ signaling pathway (30).
Consistent with the proteomic analysis data obtained from the cardiomyocyte secretome, 14-3-3 protein-zeta was significantly upregulated in the hypertrophy model. Therefore, we further performed an
The immortalized human cardiomyocyte cell line T0445 was obtained from Applied Biological Materials Inc. (Abm, Richmond, BC, Canada). The human normal lung epithelial cell line (L132), human lung cancer cell line (A549), human gastric cancer cell line (MKN-1), human normal gastric cell line (HFE-145), and human liver cancer cell line (Hep3B) were obtained from the Korea Cell Line Bank (KCLB, Seoul, South Korea). The human normal liver cell line (Hepa RG) was obtained from Thermo Fisher Scientific (Massachusetts, USA). All cell lines were regularly cultured in RPMI, DMEM, Williams medium E media (Gibco, USA), and Prigrow I media (ABM, Richmond, BC, Canada) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). Culture plates were maintained at 37°C with 5% CO2 in a humidified cell incubator.
Hypertrophic agonists ET-1 and Ang II were purchased from Sigma-Aldrich (Darmstadt, Germany). Cardiomyocytes were treated with 1 μM Ang II and 200 nM ET-1 (Sigma-Aldrich, Darmstadt, Germany) diluted in an appropriate medium.
LTQ-Orbitrap Velos Pro combined with the EASY-nLC1000 system (ThermoFisher Scientific, USA) was used for proteomic profiling. Peptide separation was achieved using an Acclaim PepMap EASY-Spray analytical column (75 μm × 50 cm, nanoViper, 100 Å, C18, 2 μm) (Thermo Scientific, USA). The CM and human plasma samples were tested using a UHPLC-MS/MS system connected to an LTQ Orbitrap Velos Pro and separated using a 4 μm Proteo Phenomenex (90 Å pore, LC Column 250 × 4.6 mm) (Jupiter, USA). The spray voltage was +3.9 kV, and the eluted peptides were examined. MRM was used to quantitatively analyze six 14-3-3 protein isotypes and beta-actin simultaneously.
The MS/MS data obtained from sample analysis were searched using the Proteome Discoverer v2.2 (ThermoFisher Scientific, USA) search engine against the SEQUEST algorithm with amino acid sequences in the SwissProt database (2017). The results were searched using the Percolator for scoring. The followed database parameters were applied: trypsin digestion of the enzyme, fragment ion mass tolerance of 0.6 Da, precursor ion mass tolerance of 10 ppm, allowance of a maximum of 2 missed cleavages, and carbamidomethyl and oxidation as variable modifications with respective masses of +57.021 Da and +15.995 Da. The peptide matching criteria were set as a SEQUEST HT score of >1, and Xcorr criteria of >1.2 for (+1) peptides, >1.9 for (+2) peptides, and >2.3 for (+3) peptides. A unique peptide number of at least 2 per protein was required to ensure reliability. Differences between the two groups were considered statistically significant at P < 0.05.
All identified differentially expressed proteins in hypertrophied and non-hypertrophied cardiomyocytes were subjected to protein pathway analysis using IPA tools (http://www.ingenuity.com) (Qiagen, Germany). The z-score value (>2.0) was predicted as the activation state of canonical pathways, whilst the inhibition state was defined by z-score <−2.0.
This study was supported by the National Research Foundation of Korea (NRF) and funded by the Ministry of Science and ICT, Republic of Korea (no. NRF-2017R1A2B2004398, NRF-2021R1A2C209370611), and the Korea Institute of Science and Technology (KIST) Institutional Program (2E31623).
Western blot, ELISA, immunocytochemistry analysis, sample preparation, in-solution trypsin digestion, data availability, and data processing are described in the supplementary information.
The Institutional Review Board of the Korea University Medical Center approved the sample collection and analysis (KUGH 12118-005).
The authors have no conflicting interests.
Fig. 1. Immunocytochemistry and western blot analysis of BNP and ANP expressions in T0445 cells treated with 200 nM ET-1 and 1 μM Ang II. (A) Immunostaining results show α-actinin (green) and nucleus (blue). (B) Bar chart showing relative signal intensity on average cell surface area. (C) Western blotting of ET-1-treated secretome. (D) Relative expression changes of ET-1-treated secretome. (E) Western blotting of Ang II-treated secretome. (F) Relative expression changes of Ang II-treated secretome.
Fig. 2. Identification of differentially expressed proteins in canonical pathways identified using Ingenuity Pathway Analysis of treated secretome. (A) ET-1-treated secretome. (B) Ang II-treated secretome. Bar charts representing fold-change in protein intensity between the treated group and untreated T0445 cells. Columns represent canonical pathways.
Fig. 3. Relative intensity of 14-3-3 protein isoforms in the secretome of hypertrophied cardio myocytes together with different organ cells. (A) 14-3-3 protein-beta. (B) 14-3-3 protein-theta. (C) 14-3-3 protein-zeta/delta. (D) 14-3-3 protein-epsilon. (E) 14-3-3 protein-etha.
Fig. 4. Representative selected reaction monitoring chromatograms and performance in human plasma samples of 14-3-3 protein-zeta. (A) 14-3-3 protein-zeta standard. (B) T0445 secretome. (C) ET-1-treated T0445 secretome. (D) Ang-II-treated T0445 secretome. (E) Healthy control human plasma. (F) AMI human plasma. (G) Box-and-whiskers plot showing the levels of 14-3-3 protein-zeta in healthy controls (n = 23) and patients with AMI (n = 30). (H) ROC analysis distinguishing the diagnostic accuracy of 14-3-3 protein-zeta expression between patients with AMI and healthy controls.
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Author ORCID Information Min-Jung Kang
Funding Information National Research Foundation of Korea Ministry of Science and ICT, South Korea Korea Institute of Science and Technology |
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