ARVC and DCM are two types of cardiomyopathy, both manifests as arrhythmia, heart failure and sudden death. ARVC usually involving the right ventricle, but there are also reports of left ventricular involvement , while DCM is involved in left ventricle as well as right ventricle. According to the similar clinical features of these disease and they both damage the ventricular muscle, we aim to detect their shared and specific molecular mechanisms. With the development of sequences of micro array technology, bioinformatics analysis helps us detect potential mechanism of disease. Here, the shared and specific potential mechanisms of ARVC and DCM were identified by using bioinformatics analysis based on GSE29819 micro-array dataset.
The shared pathways of ARVC and DCM compared to NF
The shared DEGs were enriched on the 25 pathways, as shown in Figure 3. Cytokines play important role in regulation of immune function and are associated with the occurrence and development of a large number of human diseases, and many cardiovascular diseases have been shown to be associated with uncontrolled cytokines . The activity of intracellular Janus kinases (JAKs) is associated with the assembly of the cytokine-receptor complex in the classic cytokine signaling pathway . JAKs phosphorylate and activate the signal transducer and activator of transcription (STAT), which subsequently modulates gene expression [26, 27]. IL-2, IL-4, and IL-6 are the most common types of cytokine; among them, IL-6 first forms a dimer with IL-6R prior to binding its cognate receptor gp130, which is constitutively associated with JAK family tyrosine (Tyr) kinases and can be phosphorylated by JAKs. In addition, IL-6 also activates phosphatidylinositol 3-kinase (PI3K) pathways and extracellular signal-regulated kinase (ERK)1/2, following recruitment of SH2-containing protein Tyr phosphatase 2 to JAK-phosphorylated gp130 . PI3Kα/Akt signaling leads to phosphorylation of Nav1.5 on a site that regulates its gating properties, thus suppressing persistent INa. At some point, the decrease of Nav1.5 on the cell surface may result in a decrease in peak INa after the inhibition of PI3Kα, which could slow action potential conduction and further induce arrhythmia if large enough . These factors interact with pathways, some of which, if unregulated or unbalanced, may induce arrhythmias.
Many previous reports have proposed theories related to ARVC and DCM, including immunity, apoptosis, and gene mutation-related theories [30, 31]. FoxO activity is involved in immune response, apoptosis, oxidative stress, aging, and other biological processes, mainly regulated by the PI3K/Art signaling pathway . Similarly, the p53 signal pathway and TNF signal pathway are important components of apoptosis pathways. Consistent with previous reports, our study found the FoxO signal pathway, the p53 signal pathway, and TNF signaling pathways among the shared genes. The results suggest that immunity and apoptosis may be the common pathogenesis of ARVC and DCM, and intervention based on cell immunity and apoptosis may have significance for the prevention and treatment of ARVC and DCM.
Electrical remodeling is an important mechanism of arrhythmia and heart failure. After the integrity of myocardial fibers is destroyed, the anisotropy of electrical activity increases, which promotes conduction disorders, inducing arrhythmias and heart failure. Shimizu H. et al  revealed that overload of Ca2+ can destroy the integrity of myocardial fibers by activating a calcineurin-FoxO-MuRF1-proteasome signaling pathway. Mota R. et al  reported a novel link between atrogin-1-mediated regulation of FoxO1/3 activity, reduced collagen deposition, and fibrosis in the aged heart. Bagchi AK. et al  showed that under stress, IL-10-mediated toll-like receptor 4 (TLR4) signaling suppresses apoptosis as well as fibrosis, while TLR2 has the opposite effect. Similarly, the hypoxia-inducible factor-1 (HIF-1) signal pathway also takes part in the development of fibrosis . In the present study, the FoxO signaling pathway, HIF-1 signaling pathway, and toll-like receptor signaling pathway were all discovered to be shared DEGs. Thus, pathway-based interventions may help preserve the integrity of primary myocardial fibers, improve electrical remodeling, and reduce the occurrence of heart failure in patients with ARVC or DCM.
The specific pathways of ARVC compared to NF and DCM compared to NF
Specific pathways of ARVC compared to NF were identified in our study. Among them, the ECM-receptor interaction had already been proven to be involved in the fibrosis of myocardium . The rest of the pathways tended toward neuro-regulation (neuroactive ligand-receptor interaction, serotonergic synapse, dopaminergic synapse, Fc gamma R-mediated phagocytosis) and infection (Leishmaniasis and Staphylococcus aureus infection). By contrast, the specific pathways of DCM compared to NF were related to ATP-binding cassette (ABC) transporters and glycerolipid metabolism. ABC transporters like ABCA1, ABCA4, and ABCA5 are all expressed in human platelets, and they regulate platelet function . The ATP-binding cassette transporter P-glycoprotein (ABCB1) may affect the bioavailability and elimination of digoxin, while ABCA8 and ABCA9 are indispensable components of the ATP-sensitive potassium (KATP) channel . Glucose and lipid metabolism is important for myocardial cells, and some research shows that metabolic disorders are a cause of chronic heart failure, and that several parameters are even biological indicators of prognosis . According to our results, development of ARVC and DCM occur via their unique pathways, and these pathways may provide some evidence to support targeted intervention for the two diseases, respectively.
The pathways of ARVC compared to DCM
Compared to the pathways based on the shared DEGs, some of the pathways that are enriched in ARVC vs. DCM are unique, including ABC transporters, signaling pathways that regulate the pluripotency of stem cells, type II diabetes mellitus, fat digestion and absorption, bile secretion, complement and coagulation cascades, and inflammatory bowel disease (IBD). Type II diabetes mellitus, fat digestion and absorption, and bile secretion are correlated with glucose and lipid metabolism. As we mentioned above, metabolic syndrome is a risk factor for heart failure. Metabolic disorders such as those related to glucose, fat, or protein metabolism may contribute to heart failure. The complement system is a major element of immune response, and also plays an important role in the development of IBD . Whether the complement system activated by inflammatory bowel disease has any effect on the development of ARVC or DCM needs further study.
Hub genes of the PPI network
As the Figure 5 and Figure 6 show, we identified the top 10 hub genes of the ARVC vs. NF and DCM vs. NF, and ARVC vs. DCM, respectively. These genes are involved with metabolism, inflammation, immune, cell apoptosis, or other critical biological processes. Proenkephalin (PENK), which is related to renal function, is a stable endogenous opioid biomarker and has been reported to be a prognostic indicator of heart failure . What’s more, it is also a modulator of IL-10 , a cytokine involved in inflammation and immune response, which also have important roles in cardiovascular disease. BDKRB2 is related to hypertension as a target of angiotensin II type 1 receptor signaling, and its polymorphism is related to the glucose metabolism [44, 45]. Endothelial APLNR is critical for apelin signaling and its glucose-lowering effects . CCR3 and CCR5 are chemokine receptors, and the former plays a pivotal role in leukocyte chemotaxis . CXC chemokines regulate the recruitment of neutrophils via CXCR1 and CXCR2 in humans, and CXCR2 recognizing CXCL1 and CXCL2 to promote the bioactive IL-1β production, regulating inflammasome activation [48, 49]. In addition, CXCL10 has demonstrated a novel function in mediating monocyte production of pro-inflammatory cytokines . Another gene, CXCR4, was postulated to mediate atherosclerosis and inflammation in a recent study . FPR1 encodes a G protein-coupled receptor of mammalian phagocytic cells, neutrophil activation and functional responses . Finally, mutation of GRM8 gene has been reported associated with schizophrenia and depressive disorder, but there is little study about the relationship between this gene and cardiovascular disease . These genes played its role in immunity and/or inflammation, which have been demonstrated to be related to the development of cardiovascular disease.
In the context of the hub genes of the PPI network based on ARVC vs. DCM, RPS3A was a key factor in modulating the brown fat-specific gene UCP-1 and carbon metabolic enzymes in EAT for preventing CAD . CDK11p46 and RPS8 are associated with each other, and both are involved in cell apoptosis; similarly, over-expression of RPS14 can inhibit Rb phosphorylation and result in cell cycle arrest and senescence [55, 56]. It is well known that diabetes and renal dysfunction can both affect cardiac function, but interestingly, RPS12 has been identified as a pathogenic gene of diabetic kidney disease by a genome-wide association study (GWAS) . Finally, the mutations of RPL18A and RPL31 have been proven to be associated with Diamond-Blackfan anemia (DBA) , and RPL15 was demonstrated as a new gene involved in DBA , which may decrease the blood supply to myocardial cells to some degree. Results from Arthurs C et al suggested that expression of RPS21 increased in in malignant tissue and may serve as biomarkers for cancer . Similarly, RSL21 has been reported may be a biomarker of cervical intra-epithelial neoplasia 1 , and loss of the heterozygosity and decreased expression of RPL14 might be an earlier event in the tumorigenesis of the esophagus . Thus, these genes may be involved in the development of tumors, but their role in cardiovascular disease is poorly known.
Although the function and the pathway of the above hub genes have been reported previously, they have not been verified before, as a large enough sample of myocardium tissues with ARVC and DCM is difficult to collect. Meanwhile, it can also be inferred that since the mechanisms of ARVC and DCM are regulated by multiple genes and multiple pathways, they require more comprehensive and targeted intervention.
Clinical implications of this study
ARVC and DCM are two special cardiomyopathies with complex clinical manifestations and difficulty in treatment. Micro-array dataset helps us identify the shared pathways of ARVC and DCM, such as Chemokine signal pathways, PI3K-Akt signal pathways, FoxO signal pathway, TNF signaling pathways, Toll-like receptor signal pathways, and HIF-1 signaling pathway. Besides, the specific pathways that may be involved in the development of ARVC and DCM, such as ECM-receptor interaction, Jak-STAT signal pathway, and ABC transports were also identified. These pathways may affect the development of ARVC and DCM on immunity, apoptosis, voltage-gated channel, electrical remodeling, and fibrosis of ventricular muscle. Further, the top hub genes were also identified, and a part of them are new genes that may be associated in ARVC and DCM, like GRM8, RPS21, RPL21 and RPL14. Our findings may help reveal the potential mechanism of ARVC and DCM, and these genes and pathways may be potential targets of interference on development of ARVC and DCM.
There are some limitations to our study. Firstly, the samples from non-failing donors may differ from the normal population, which may limit the applicability of our results between patients with ARVC or DCM and normal population. Secondly, since genes interact with each other, we artificially screened the intersection of different groups of DEGs for further analysis and may have inadvertently excluded some genes with potential links, which may make the analysis of diseases one-sided to some degree. Thirdly, our study was only based on the GSE29819 dataset, and the results need to be validated with a further, more rigorous investigation and a large sample size.