Cardiac proteomics reveals the potential mechanism of microtubule associated protein 4 phosphorylation-induced mitochondrial dysfunction
Our previous work suggested that microtubule associated protein 4 (MAP4) phosphorylation led to mitochondrial dysfunction in MAP4 phosphorylation mutant mice with cardiomyopathy, but the detailed mechanism was still unknown. Thus, the aim of this study was to investigate the potential mechanism involved in mitochondrial dysfunction responsible for cardiomyopathy.
The present study was conducted to explore the potential mechanism underlying the mitochondrial dysfunction driven by MAP4 phosphorylation. Strain of mouse that mimicked constant MAP4 phosphorylation (S737 and S760) was generated. The isobaric tag for relative and absolute quantitation (iTRAQ) analysis was applied to the heart tissue. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) were all analyzed on the basis of differential expressed proteins (DEPs).
Among the 72 cardiac DEPs detected between the two genotypes of mice, 12 were upregulated and 60 were downregulated. GO analysis showed the biological process, molecular function, and cellular component of DEPs, and KEGG enrichment analysis linked DEPs to 96 different biochemical pathways. In addition, the PPI network was also extended on the basis of DEPs as the seed proteins. Three proteins, including mitochondrial ubiquitin ligase activator of NF-κB 1, reduced form of nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial and growth arrest, and DNA-damage-inducible proteins-interacting protein 1, which play an important role in the regulation of mitochondrial function, may correlate with MAP4 phosphorylation-induced mitochondrial dysfunction. Western blot was used to validate the expression of the three proteins, which was consistent with iTRAQ experiments.
These findings revealed that the DEPs caused by MAP4 phosphorylation in heart tissue using iTRAQ technique and may provide clues to uncover the potential mechanism of MAP4 phosphorylation-induced mitochondrial dysfunction.
KeywordsMicrotubule associated protein 4 Mitochondria Isobaric tag for relative and absolute quantitation
Differential expressed proteins
Growth arrest and DNA-damage-inducible proteins-interacting protein 1
Isobaric tag for relative and absolute quantitation
Kyoto Encyclopedia of Genes and Genomes
Liquid chromatography-tandem mass spectrometric
Microtubule associated protein 4
Mitogen-activated protein kinase
Mitochondrial ubiquitin ligase activator of NF-κB 1
Reduced form of nicotinamide adenine dinucleotide
NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial
Nucleotide-binding oligomerization domain
Search Tool for the Retrieval of Interacting Genes/Proteins
14-3-3 protein epsilon
Myocardial hypoxia/ischemia is a major risk factor for the development of chronic heart failure, an ultimate sequelae for many forms of heart diseases responsible for the leading cause of death worldwide [1, 2]. Accumulating data appear to support the argument that mitochondrial dysfunction contributes to the onset and progression of cardiac injury followed by heart failure under hypoxic and/or ischemic conditions . Thus, a better understanding of the mechanism underlying cardiomyocyte mitochondrial dysfunction may therefore hopefully yield novel therapeutic targets against these cardiac disorders.
Microtubule associated protein 4 (MAP4) is recognized as a cytosolic protein that binds to tubulin and stimulates their polymerization [4, 5]. MAP4-microtubule binding is regulated through MAP4 phosphorylation, leading to the detachment of MAP4 from microtubules (MTs) and destabilization of the MT as a consequence . We and others have reported that the phosphorylation sites of human MAP4 at S768 and S787 (corresponding to the S737 and S760 in mouse) are critical sites governing its detachment from MTs [7, 8]. Our previous study suggested that the phosphorylated MAP4 (p-MAP4) dissociated from MTs were translocated to mitochondria after hypoxia in cultured neonatal rat cardiomyocytes, which led to mitochondria dysfunction and apoptosis . In addition, the aberrant MAP4 phosphorylation-induced cardiomyocyte mitochondrial dysfunction was still noted in vivo experiments and in heart tissues of patients of tetralogy of Fallot, and the MAP4 phosphorylation mutant mice developed cardiomyopathy at the age of 30–34 and 70–74 weeks, which mitochondrial dysfunction is deemed an important trigger for myocardial impairment and cardiac dysfunction . However, the precise regulation that how MAP4 phosphorylation induced mitochondrial dysfunction is still elusive and it is worthwhile to explore the underlying mechanism.
Here, in this study, to investigate the differential expressed proteins (DEPs) of aberrant MAP4 phosphorylation in vivo and dig out the underlying mechanism that mediating MAP4 phosphorylation-induced mitochondrial dysfunction which is responsible for cardiomyopathy, we generated mice that mimicked MAP4 hyperphosphorylation at specific sites (S737 and S760). To avoid the complication and establish a potential cause-effect relationship, we studied relative young mice (24 weeks) with only initial injury, using an isobaric tag for relative and absolute quantitation (iTRAQ) followed by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. The significance of the findings with respect to deciphering the potential mechanism that involves MAP4 phosphorylation-induced mitochondrial dysfunction is discussed.
The mutant hyperphosphorylated MAP4 (S737 and S760) knock-in (MAP4 KI) mice were generated as previously described . Animal experiments were performed in line with the United Kingdom Home Office and European Union guidelines and were authorized by the Animal Care Centre of the Army Medical University.
Sample collection, protein extraction, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation
Male wild type (WT) mice and male MAP4 KI littermates were sacrificed at 24 weeks of age (three mice per group), and the heart tissues were stored at − 80 °C. SDT buffer, including 1 mM dithiothreitol (DTT), 4% SDS, and 150 mM Tris-HCl, together with protease inhibitor, was added to the heart tissues, then the tissues were homogenized. After incubation in boiling hot water for 5 min, the mixture was sonicated at 4 °C and incubated for 15 min in 100 °C water. The homogenate was centrifuged at 14,000×g for 15 min at 4 °C, and the supernatant was collected and preserved at − 80 °C. The protein contents were examined with bicinchoninic acid (BCA) protein assay kit . Subsequently, 20 μg samples with loading buffer were boiled for 5 min and separated on SDS-PAGE gel (12%, 250 V for 40 min). Coomassie blue staining was used to detect proteins.
Protein processing and iTRAQ labeling
Protein processing was performed as described previously . Briefly, 100 mM DTT was added to each sample and boiled in hot water for 5 min, then 200 μL UA buffer, including 150 mM Tris-HCl and 8 M urea, was added to the sample at room temperature, and the sample was centrifuged in a 30 kD ultrafiltration filter (Sartorius, Germany) for 15 min at 14,000×g, then 200 μL UA buffer was added and centrifuged again to abandon the filter solution. Subsequently, the filter was vibrated for 1 min at 600×g with 100 μL iodoacetamide (IAA) solution. After incubation for 30 min in the dark, the filter was centrifuged for 15 min at 14,000×g again and rinsed with 100 μL UA buffer for twice. Next, the filter was centrifuged with 100 μL dissolution buffer for twice using the previous condition. Finally, each filter that contained 4 μg trypsin and 40 μL dissolution buffer was vibrated for 1 min at 600×g, kept in 37 °C for 16–18 h, then the resultant peptides were obtained and moved to a new pipe and centrifuged for 15 min at 14,000×g. The peptide content was then analyzed at 280 nm.
Eight-plex iTRAQ reagent was used to label peptide mixture according to the instruction. For the iTRAQ experiments, two technical replicates and biological replicates (n = 3) were exerted per group.
Peptide fractionation with reversed-phase chromatography
Agilent 1260 infinity II high performance liquid chromatography (HPLC) system was applied to remove interfering substances. Buffer A: 10 mM HCOONH4, 5% ACN, pH 10.0; buffer B: 85% ACN, 10 mM HCOONH4, pH 10.0. The peptides were eluted at a flow rate of 1 ml/min with a gradient of buffer B: 0% for 25 min, 0–7% for 5 min, 7–40% for 35 min, 40–100% for 5 min, and a final hold in 100% for 15 min. Elution was supervised by examining the absorbance at 214 nm, and the components were obtained per min (about 36 components). The samples were stored at − 80 °C [12, 13].
Experiments were exerted on a Q Exactive mass spectrometer (Thermo Fisher, USA) that was coupled to Easy Nano Liquid Chromatography apparatus (nLC, Thermo Fisher, USA). The peptide mixture was transferred to a C18 reversed-phase column (nanoViper, Thermo Fisher, USA). The sample was subjected to the 60 min gradients of buffer B: 0–6% for 5 min, 6–28% for 40 min, 28–38% for 5 min, 38–100% for 5 min, and a final hold in 100% for 5 min. The data were collected using a data-dependent top 10 most abundant precursor ions from the survey scan (350–1800 m/z) for higher-energy collisional dissociation fragmentation. Survey scans were set to 70,000 resolution at 200 m/z, the target was 3e6 and the maximum fill time was 50 ms. The spectra resolution for higher-energy collisional dissociation was set to 17,500 at 200 m/z and the isolation window was 2 m/z. The normalized collision energy was 30 eV [12, 13].
Data and bioinformatics analysis
MASCOT engine (version 2.5, Matrix Science, UK) in the Proteome Discoverer 2.1 (Thermo Electron, USA) was used for LC-MS/MS spectra analysis, screening against a mouse sequence database (Uniprot_MusMusculus_83374_20170809.fasta). The following parameters were applied; peptide mass tolerance was 20 ppm, fragment mass tolerance was 0.1 Da, trypsin with missed cleavages was 2, iTRAQ 8plex (N-term) with fixed modification, iTRAQ 8plex (K), variable modification of oxidation (M), and iTRAQ 8plex (Y). Ninety-nine percent confidence was applied for data analysis as determined by false discovery rate ≤ 1%.
The DEPs were defined as the following criteria: p < 0.05 and fold change ≤ 0.833 or ≥ 1.2. The DEPs were analyzed through the Gene Ontology (GO) platform (http://www.geneontology.org/), and protein grouping was analyzed based on functional notes using the GO terms for cellular component (CC), biological process (BP), and molecular function (MF) [14, 15]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) platform (http://www.genome.jp/kegg/pathway.html) was used for pathway analysis [16, 17]. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software (http://string.embl.de/) was applied to protein-protein interaction (PPI) analysis. Cluster 3.0 software was applied to hierarchical cluster analysis.
Validation by western blot (WB)
WB analysis was used to ensure the reality of the iTRAQ results. A tissue protein extraction reagent (T-PER, Thermo Scientific, USA) was applied to tissue homogenate. To discard insoluble protein, the mixture was centrifuged at 4 °C for 15 min by 16,000×g. SDS-PAGE gels (8–12%) were used for protein separation. After separation, proteins were moved to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany) and followed by 5% nonfat-dried milk blocking, then incubated at 4 °C for 12–18 h with required antibodies. Target proteins were examined by chemiluminescence detection kit (GE Healthcare, USA). The antibodies were used as follows: mitochondrial ubiquitin ligase activator of NF-κB 1 (Mul1) (ab84067, Abcam), NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial (Ndufs1) (ab169540, Abcam), growth arrest and DNA-damage-inducible proteins-interacting protein 1 (Gadd45gip1) (16260-1-AP, Proteintech), MAP4 (Cat# A301-489A, Bethyl), and α-tubulin (11224-1-AP, Proteintech).
Primary cell culture
Neonatal mouse cardiomyocytes and fibroblasts were cultured as our previous study described .
The experimental data were showed as mean ± standard deviation (SD). The differential analysis of DEPs was tested by two-tailed Student’s t test, and p < 0.05 represented statistically significant.
Protein identification and quantification
GO analysis of DEPs
KEGG pathway analysis of DEPs
WB validation of iTRAQ data
In our previous studies, we firstly demonstrated that p-MAP4 was a trigger factor that contributed to cardiomyocyte mitochondrial dysfunction, which led to the cardiac dysfunction and pathological remodeling [7, 9]. However, the detailed mechanism of how MAP4 phosphorylation triggered the mitochondrial dysfunction was still unclear. In the present study, for the first time, we investigated the DEPs between WT and MAP4 KI mice using iTRAQ-based global proteomics and LC-MS/MS analysis. We identified 72 DEPs in MAP4 KI mice.
In particular, several DEPs involved in mitochondrial dysfunction, which have not been reported correlated with MAP4, were identified and analyzed on the basis of PPI and their characteristics, thereby providing the underlying candidate molecular mechanisms that involved in MAP4 phosphorylation-induced mitochondrial dysfunction.
Further bioinformatics analysis, including GO analysis, KEGG assessment, and PPI were evaluated to reveal the characteristics of DEPs. The GO analysis revealed that cellular process, binding, and cell part were the most abundant categories in BP, MF, and CC, respectively. GTP binding, guanyl ribonucleotide binding, and guanyl nucleotide binding were the three most enriched GO terms. In KEGG analysis, several cardiomyopathy-related pathways, such as dilated cardiomyopathy, metabolic pathways, calcium signaling pathway, adrenergic signaling in cardiomyocytes, arrhythmogenic right ventricular cardiomyopathy, and hypertrophic cardiomyopathy were detected, indicating that MAP4 phosphorylation may correlate with these diseases. However, the detailed role of MAP4 phosphorylation in such diseases is still unknown.
Mitochondrial dysfunction [9, 18, 19, 20] has been recognized as one of the most important reasons for the development of cardiomyocyte injury. Our previous work revealed that MAP4 phosphorylation led to cardiomyocyte mitochondrial dysfunction under different stimuli (e.g., hypoxia, ischemia, and transverse aortic constriction) [7, 9, 21]. Here, in the results of KEGG analysis, both metabolic pathway and calcium signaling pathway were tightly correlated with the mitochondrial dysfunction, which may provide fundamental opening and evidence for future studies to reveal the mechanism of MAP4 phosphorylation-induced mitochondrial dysfunction. We also acquired some DEPs with thyroid hormone signaling pathway, adrenergic signaling in cardiomyocytes, arrhythmogenic right ventricular cardiomyopathy, nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, and rap1 signaling pathway. We cannot exclude the possibility that these proteins may be triggers and have a certain capacity to switch on or exaggerate the role of key effector molecules, and further studies should be carried out.
In the current study, three mitochondrial proteins, including Mul1, Gadd45gip1, and Ndufs1, which were involved in the regulation of mitochondrial dysfunction, were chosen from DEPs to validate by WB. Mul1 has been known to be involved in the regulation of multiple proteins, including anti-apoptotic proteins, and is predominantly expressed in mitochondria of heart tissue . The previous study reported that Mul1 interacted with multiple mitochondrial proteins, and overexpression of Mul1 promoted NF-κB activation, which increased the transcription of anti-apoptotic proteins and exhibited the inhibitory effects on mitochondrial apoptosis of cardiomyocyte, in contrast, the Mul1 inhibition led to susceptibility to mitochondrial apoptosis and dysfunction . Ndufs1, the largest subunit of complex 1, which located at the mitochondrial inner membrane. The protein exhibited reduced form of nicotinamide adenine dinucleotide (NADH) dehydrogenase activity and oxidoreductase activity, which transferred electrons from NADH to the respiratory chain to exert oxidative phosphorylation . The mutation or knockdown of Ndufs1 impaired mitochondrial oxygen consumption, increased reactive oxide species production, and decreased oxidative phosphorylation, contributing to progressive mitochondrial dysfunction followed by various diseases [25, 26]. Gadd45gip1 was newly identified as de novo components in large subunit of mitochondrial ribosome and played an important role in genomic stability, DNA repair, cell cycle regulation, and apoptosis. It was widely expressed in the heart, thyroid, and lymph node . Previous data demonstrated that elevated Gadd45gip1 expression promoted activation of tumor protein p53 (p53), a pro-apoptotical protein, and its target genes in the suppression of cell growth and tumor development in human cancer cells . The p53 was a downstream target of p38/mitogen-activated protein kinase (MAPK) and was known to induce mitochondrial dysfunction of cardiomyocyte upon the activation of p38/MAPK . In our previous study, we have demonstrated that p38/MAPK activation induced MAP4 phosphorylation in cardiomyocyte . Thus, we inferred that the MAP4 KI mice-induced increased Gadd45gip1 was also contaminated with p53 activation, which may be a pathway involved in cardiomyocyte mitochondrial dysfunction. Besides, in the DEPs, secretory protein including thrombospondin 1, and transcription factor, such as signal transducer and activator of transcription 1, have been reported to be associated with mitochondrial function [30, 31]. Whether they are associated with the observed changes of the above-mentioned three mitochondrial proteins are still unclear and deserve further research. The other secretory proteins, including immunoglobulin heavy variable 1–82, immunoglobulin heavy variable 1–26, transforming growth factor beta-1-induced transcript 1 protein, serum amyloid A-2 protein and serum amyloid A-1 protein, and transcription factors, such as NFX1-type zinc finger-containing protein 1 and endothelial differentiation-related factor 1, showed little correlation with cardiac mitochondrial function; while for other transcription factor, such as signal transducer and activator of transcription 1, has been reported to be associated with cardiac mitochondrial function ; however, the potential effect had little interaction with Mul1, Ndufs1, and Gadd45gip1, the detailed mechanism need to be investigated in future study.
In addition, the PPI analysis also revealed that 14-3-3 protein epsilon (Ywhae), which mediated signal transduction by binding to a multitude of functionally diverse signaling proteins, including phosphoserine-containing proteins, kinases, and transmembrane receptors [32, 33], was the center of the network in mice. In the network, Ywhae directly interacted with MAP4, Gadd45gip1, and Ndufs1, respectively. And Mul1 was interacted with Znfx1, which was also revealed a direct correlation with Ywhae. Furthermore, the protein expression of Mul1, Ndufs1, and Gadd45gip1 were validated by WB, which was consistent with the iTRAQ experiments. Thus, these findings provided a further link between MAP4 and Gadd45gip1, Ndufs1, and Mul1, suggesting possible molecular targets involved in MAP4 phosphorylation-induced cardiomyocyte mitochondrial dysfunction, and these screened DEPs may provide us new directions in mechanism exploration in future studies. Even though there are still some limitations in the present study, the screened proteins involved in mitochondrial function and other cardiac-specific function are limited to only a couple and the extent of change is less than twofold for most of these proteins, so the cause and effect relationship between these proteins and cardiac mitochondrial dysfunction still needs to be demonstrated in future studies. In addition, a relatively small number of DEPs were detected, which may be related to the young mice that we used, if we used elder mice, the phenotypes would be more obvious and the more DEPs may be detected.
In conclusion, iTRAQ proteomic analysis was used to assess the cardiac protein expression profile, and 72 DEPs were detected in MAP4 KI mice, including 12 upregulated and 60 downregulated proteins as compared to WT littermates. GO analysis, KEGG assessment, and PPI analysis were all evaluated to reveal the characteristics of DEPs. And three proteins were chosen to validate, which were possibly correlated with MAP4 phosphorylation-induced cardiomyocyte mitochondrial dysfunction. Further study is warranted to clarify the precise mechanism for MAP4 phosphorylation-induced cardiomyocyte mitochondrial dysfunction.
This work was supported by the Key Program of National Natural Science Foundation of China (No.81430042).
Availability of data and materials
The data during the present study are available from the corresponding authors.
JH and YH supervised the work. LL and JH designed the experiments. LL and JZ analyzed the data and performed the experiments with help from QZ. LL, JH, and YH co-wrote the manuscript. All authors have discussed the results and commented on the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal experiments were performed in accordance with United Kingdom Home Office and European Union guidelines and were approved by the Animal Care Centre of the Army Medical University.
Consent for publication
The authors declare that they have no competing interests.
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