Chronic hypoxia is normal occurrence during embryonic development (1). In addition, it is the basic pathophysiological characteristic of individuals with cyanotic congenital heart disease (CHD) or those inhabitants who lived on the plateau for a long period of time (2,3). Circulation overload, chronic hypoxic stress and other complications on infants with cyanotic CHD results in mortality as high as 36.4% (4). Notably, it has been clinically observed that a number of infants with mild to moderate cyanosis have been able to survive for a prolonged period, including to adulthood. They tend to be well adapted to systematic hypoxia and typically demonstrate good cardiac function during the perioperative period of cardiac surgery (5). The observation suggested that the heart may respond to chronic hypoxia, with some protective adaptation. In addition, previous studies have demonstrated that the myocardium may develop a set of endogenous protective mechanisms to attenuate cardiomyocyte apoptosis and to promote potential proliferation during chronic hypoxia (6–8). However, this ability to replenish the lost cardiomyocytes during infarct or in cardiac overload disorders is extremely limited in adults (9). The molecular mechanisms underlying the hypoxic myocardial injury and adaptation remain unclear, which limits clinical treatment of myocardial hypoxia, ischemia and infarction. Therefore, exploring and understanding the molecular mechanisms may aid in providing protection against chronic hypoxic-ischemic heart disease, including coronary heart disease and cyanotic CHD.

Microarray and high-throughput sequencing have been widely used in the diagnosis and treatment of diseases. Among them, next-generation sequencing (NGS) technology is frequently applied to assess cardiovascular diseases. Notably, NGS can be used to identify potential genetic mutations in hereditary heart diseases, analyze transcriptome changes, screen biological targets of disease and identify molecular pathways involved in cardiovascular development and disease (10). The vast amount of data produced by the high-throughput platforms rely on analysis and interpretation using bioinformatics, which is a cutting-edge interdisciplinary approach that links biology, computer science and information technology. Accordingly, the combination of NGS technologies and bioinformatics has made data processing and data mining a reality, and has been extensively applied in the analysis of biological sequences, genome, transcriptome, proteome, metabolome and systems biology (11). Grunert et al (12) performed a comparative study between myocardial specimens of healthy organ donors and patients with tetralogy of Fallot (TOF) congenital cardiac malformations by DNA-targeted resequencing and mRNA-sequencing technology. The study examined the genetic origin and variation of TOF, but did not elucidate the biological significance of these differential expressed genes (DEGs) under chronically hypoxic conditions.

TOF is the most common cyanotic CHD, and chronic hypoxia is an important pathophysiological feature of this disease, in addition to genetic variations and hemodynamic abnormalities (13). Thus, based on the raw NGS data (GSE36761) uploaded by Grunert et al (12) to the Gene Expression Omnibus (GEO) database, the present study identified DEGs in TOF myocardium compared with healthy specimens. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on these DEGs were performed. The present findings may provide indications to molecular mechanisms underlying the injury and adaptation of chronically hypoxic cardiomyocytes.

Chronic hypoxia is the basic pathophysiological state of cyanotic CHD; the heart forms endogenous mechanisms to protect cardiomyocytes against injuries caused by hypoxia (6–8). To identify the effects of chronic hypoxia on the heart, the present study analyzed DEGs in chronically hypoxic myocardium. NGS data was extracted from the GSE36761 data set, and 1,260 DEGs between healthy and chronically hypoxic myocardium were identified using bioinformatics. The DEGs that co-existed or were co-expressed under chronically hypoxic conditions were likely to be involved in similar biological processes (19). Therefore, for better understanding of the interactions between these DEGs, enrichment analysis was performed, which suggested that they were predominantly enriched in GO_BPs such as inflammatory response, cell regeneration and apoptosis. Further analysis of the PPI network also identified six hub genes underlying the myocardial adaptation to chronic hypoxia.

GO analysis indicated that hypoxia in the myocardium was associated with inflammatory responses activated by neutrophils and monocytes, which involved changes in ECM organization, cell substrate adhesion, monocyte migration and chemotaxis. Consistent with previous studies (20–24), this indicated that chronic hypoxia was associated with inflammation. In accordance with the findings of hypoxia-activated cardiomyocyte mitosis and regeneration (25), GO analysis revealed that the DEGs in hypoxic myocardium were also enriched in cell regeneration.

KEGG pathway analysis indicated that the DEGs identified in the present study were predominantly enriched in cytokine-cytokine receptor interactions, chemokine signaling pathways, ECM-receptor interaction and signaling pathways, such as focal adhesion and apoptosis. Focal adhesions are multi-protein structures that contain integrin that can mechanically connect the cytoplasmic actin skeleton of different cells to the ECM (26,27). Thus, ECM signals can be transmitted by focal adhesion kinase (FAK) into the cell, thereby mediating cell adhesion, migration, survival, cell cycle regulation, proliferation and apoptosis (28). Notably, hypoxia induces the activation of the FAK signaling pathway in cardiomyocytes, promotes the adhesive interaction between cardiomyocytes and ECMs (29) and may result in cell proliferation and survival. However, the DEGs in the present study were also enriched in the apoptosis pathway, which also supports the findings that hypoxia may induce cardiomyocyte apoptosis (30). Therefore, it is notable that these DEGs were associated with regeneration, focal adhesion and apoptosis, which was indicative of the homeostasis between loss and renewal of cardiomyocytes exposed to hypoxia.

To further understand the intrinsic significance of the 1,260 DEGs, that is, the interactions between them, a PPI network was constructed. The data identified six hub genes with correlated degree scores of >25 through the network correlation analysis, including GNB4, CCR2, CCR1, PF4, CTNNB1 and JUN. Notably, JUN and CTNNB1 were identified to be upregulated. GNB4 is the β-4 subunit of guanine nucleotide-binding protein (G protein), an important regulator of the α subunit of G protein and some signal transduction receptors and effectors (31). It is typically co-expressed with the γ subunit to form dimers that regulate important effector systems, including the N-type Ca²⁺ channel, the G protein-gated inwardly-rectifying K⁺ channel and phospholipase (32,33). Downregulation of GNB4 in chronically hypoxic myocardium is associated with ion channel-associated heart rate and contractility (34), reduction of G-protein-coupled receptors such as chemokine receptors (20), and apoptosis (35). CCR2 and CCR1 are predominantly expressed on the surface of inflammatory cells, including lymphocytes, monocytes and macrophages; they are types of G protein-coupled chemokine receptors that mediate the infiltration of neutrophils following ischemia (36). The downregulation of CCR2 and CCR1 in response to hypoxia was in accord with a previous study (20). The reduction of chemokine receptor expression seemed to imply that less inflammatory cells (such as mononuclear macrophages and neutrophils) migrated into the hypoxic myocardium, thereby alleviating inflammation, improving adverse cardiac remodeling and reducing apoptosis (37,38). This may possibly be considered a protective adaptation of the myocardium to chronic hypoxia (39,40). PF4 is released from α-granules of activated platelets; not only are the activated platelets associated with cell apoptosis and survival (41), but also PF4 itself has been linked to apoptosis. Notably, PF4 inhibits tumor growth and tumor angiogenesis (42) and induces tumor cell apoptosis (43). Furthermore, as the small CXC chemokine and agonist of CCR1, PF4 promotes chemotaxis in inflammatory cells, including neutrophils and monocytes (44,45). Downregulation of PF4 under chronic hypoxia may be connected with apoptosis and the reduction of chemokine receptors in myocardium. Cytoplasmic β-catenin, encoded by the CTNNB1 gene, is a key protein in the Wnt signaling pathway; this is a conserved pathway during animal evolution and development, yet the specific genes it regulates depend on the cell type and background (46). In adult hearts, the Wnt signaling pathway is quiescent under normal conditions but is activated following myocardial injury and participates in cardiac remodeling (47). Once exposed to hypoxia, hypoxia inducible factor-1α (HIF-1α) competitively inhibits the formation of β-catenin/T-cell factor-4 complex and the downstream transcriptional activities in colorectal tumor cells, resulting in G₁ arrest in the cell cycle. However, β-catenin also enhances HIF-1α-mediated transcription and helps the tumor cells with survival and adaptation to hypoxia (48). A previous study has reported that the Wnt/β-catenin signaling pathway may promote proliferation and protect cardiomyocytes from apoptosis, though little direct evidence exists to date (49). Therefore, the present study hypothesized that activation of the Wnt/β-catenin pathway may have an inhibitory effect on apoptosis under chronic hypoxia.

In the present study, the role of JUN in hypoxic myocardium became the primary focus. The transcription factor c-Jun, which is encoded by the oncogene JUN and belongs to the basic-region leucine zippers (bZIP) family, constitutes the activator protein-1 (AP-1) in the form of homodimers or heterodimers with other bZIPs, thereby regulating transcription and cellular activities, including proliferation, apoptosis, tumorigenesis and tissue morphogenesis (50). Through the GO and KEGG pathway enrichment analyses, it was identified that c-Jun was enriched in the biological function of ‘regeneration’ and the signaling pathways of ‘apoptosis’ and ‘focal adhesion’. It seemed paradoxical that c-Jun functioned in promoting survival as well as apoptosis in hypoxic myocardium. As an important endpoint of mitogen-activated protein kinase (MAPK) cascade activation, the DNA binding and transcriptional activity of c-Jun are enhanced following activation by JNK phosphorylation at Ser63/73. Following this, it forms AP-1, transcribes the downstream proteins (51) and mediates the expression or activation of pro-apoptotic genes, including Fas ligand, insulin-like growth factor binding protein 4, tumor necrosis factor (TNF)-α, B cell lymphoma-2 homologous antagonist/killer (52), p53 upregulated modulator of apoptosis (53) and Bim (54). Conversely, c-Jun has also been demonstrated to have anti-apoptotic properties. Endogenous c-Jun could upregulate survivin, counteract apoptosis, maintain mammary epithelial cell survival (55) and prevent TNF-α-induced hepatocyte apoptosis by antagonizing the activity of the pro-apoptotic gene p53 (56). Additionally, it works with signal transducer and activator of transcription 3 to inhibit Fas transcription, thereby reducing the sensitivity of tumor cells to apoptosis (57). Another study identified that c-Jun transcriptionally inhibits the tumor suppressor phosphatase and tensin homolog, which in turn activates the Akt signaling pathway and promotes fibroblast survival (58). In addition to its functions in apoptosis, c-Jun also promotes cell cycle progression through direct transcriptional activation of cyclin D1 (59) and repression of p53 (60). Previous studies have demonstrated that the MAPK subfamily JNK and its target c-Jun are activated by phosphorylation, which induces cardiomyocyte apoptosis under hypoxia stress (6,7). However, the downstream mechanisms underlying apoptosis caused by c-Jun remains to be elucidated. Therefore, whether c-Jun takes part in anti-apoptotic effects during hypoxic myocardial apoptosis is worthy of further investigation. One possible explanation for the opposing nature of pro- and anti-apoptosis (or pro-survival) is that c-Jun may activate anti-apoptotic programs under hypoxia to inhibit the excessive cardiomyocyte apoptosis caused by c-Jun itself and other factors. In addition, c-Jun may even promote cell cycle progression and proliferation to replenish the lost cardiomyocytes subjected to hypoxia. In this way, a new cellular homeostasis under hypoxia could be maintained. Therefore, the role of c-Jun in promoting survival and apoptosis may be the adaptation of myocardial tissue to chronic hypoxia, that is, some mechanism of self-protection and self-repair.

In conclusion, data mining was used in the present study to reveal the biological significance of DEGs in hypoxic myocardium, and genes associated with the life or death of myocardium were screened. The results provided a series of potential targets for studying mechanisms underlying the cellular adaptation to chronic hypoxia. However, the specific molecular mechanisms require further biological validation in in vitro or in in vivo experiments.