In this study, we identified phosphorylation and inflammatory responses as the two biological pathways associated with HF, using RNA-Seq and whole exome sequencing. We also showed that mutations in patients with HF are located in TTN, OBSCN, NOD2, CDH2, MAP3K5, and SLC17A4, and the genes that showed upregulated expression in patients with HF were S100A12, S100A8, S100A9, PFDN5, and TMCC2.
Technological advances have enabled the use of whole exome sequencing for the study of human diseases. However, the cost per base of next-generation sequencing platforms still precludes the generation of large sample sizes of completely sequenced genomes with high coverage [14]. In comparison, the gene chip technology is much cheaper. Therefore, there has been considerable focus on whole exome sequencing as the first step, with gene chip as the validation step [15]. RNA-Seq is used for transcriptome profiling using deep-sequencing technologies, and it generates millions of short sequences in a single run. These fragments, or ‘reads’, can be used to measure gene expression levels and to identify novel splicing events [16]. In this study, we comprehensively used whole exome sequencing, gene chip, and RNA-Seq technology to find the gene expression changes in patients with HF. Both whole exome sequencing and RNA-Seq identified phosphorylation and inflammatory response as the two key pathways associated with HF. These results were further verified using gene chip analysis.
Many eukaryotic cell functions, including signal transduction, cell adhesion, gene transcription, RNA splicing, apoptosis, and cell proliferation, are regulated via protein phosphorylation [17]. The expression of cardiac phosphatases is increased in patients with end-stage HF [18]. Muscle contraction and its molecular motor myosin are regulated through the phosphorylation of cardiomyocyte cytoskeletal proteins, such as the regulatory myosin light chain (MLC2). Decreased levels of phosphorylated MLC2 (MLC2-P) have been observed in HF [19, 20]. HF top-down quantitative proteomics has identified the phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for chronic HF [21]. cMyBP-C phosphorylation clearly has a direct effect on the contractile properties of the heart, sarcomere organization, and its ability to attenuate the development of HF [22]. Chronic protein kinase A (PKA) hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction [23]. The p38 MAPK pathway is a potential target in the therapeutic regimens for infarction, hypertrophy, and HF [24]. Consistent with results of these previous reports, the current study elucidated the role of altered phosphorylation in HF, using RNA sequencing and whole exome sequencing.
In addition, we demonstrated alteration of inflammation processes in HF. The mechanisms that drive the development of HF can be divided into four broad categories [25]. The first is based on traditional risk factors, such as ischemic injury, hypertension, and metabolic syndrome; the second includes genetic cardiomyopathies; the third is based on valve dysfunction. In these three categories, the initial insult is not immune-based, rather, activation of immune system is a secondary response; and the fourth category is immune-based, which includes autoimmune and infectious (viral and bacterial) triggers. Therefore, HF cannot occur without inflammation. Upregulation of pro-inflammatory cytokines has been implicated in the progression of HF, and elevated inflammatory markers constitute important risk factors for HF [26]. The roles of interleukin, endothelin, and other cytokines are still under investigation, and their overexpression has been indicated to be harmful under certain conditions [27]. In this study, we not only showed an inflammatory response in HF, but also highlighted the altered expression of a series of chemokines, such as CD163, sCD30 TNFRSF8, gp130 sIL-6Ra, sTNF-R1, sTNF-R2, BCA-1, CTACK, eotaxin-2, fractalkine, IL-8, IP-10, I-TAC, MCP-1, MIG, MIP-1a, MIP-3a, MPIF-1, and TECK, as well as cytokines in the plasma of patients with HF.
Gene chip verification using a bigger sample size identified that the mutations in patients with HF were mainly in TTN, OBSCN, NOD2, CDH2, MAP3K5, and SLC17A4. The two most significantly mutated genes were TTN and OBSCN. Mutation in the TTN gene locus 2q31 has been implicated in human skeletal muscle and heart diseases for over 15 years [28]. However, only recently, TTN mutation was found to be associated with HF [29]. Mutations in OBSCN variants are also relatively common in inherited cardiomyopathies. Unique OBSCN variants have been found in a group of 30 end-stage failing hearts [30], wherein the frequency was similar to that for TTN-truncating mutations, which were proposed to be the major alterations associated with patients with HF. Interestingly, obscurin (encoded by OBSCN) interacts with titin (encoded by TTN) through the N-terminal domain, which in turn interacts with M-line complexes of titin and myomesin, hence, enhancing the binding and contributing to stability [31]. In addition, titin and obscurin contain signaling domains close to their C-terminal (a protein serine/threonine kinase domain in titin, and a Rho-GTPase GTP-GDP exchange factor domain in obscurin) that can be coupled to two serine/threonine protein kinase domains [32]. Phosphorylation of a specific sequence in the cardiac isoform (N2B region) by cAMP-dependent or cGMP-dependent protein kinases results in acute reduction in stiffness [33]. Based on the gene chip analysis, we identified that nearly 70% of mutations were related to TTN and nearly 40% of mutations were related to OBSCN. Therefore, targeting TTN and OBSCN as therapeutic strategies will be highly significant. The pathological increase in passive stiffness caused by TTN and OBSCN mutations may be reversed by PKA and cGMP-dependent protein kinase (PKG) [34]. In addition, We also found NOD2, CDH2, MAP3K5, and SLC17A4 mutations in HF here. NOD2 plays a key role in the immune response to intracellular bacterial lipopolysaccharides [35]. CDH2 encodes a classical cadherin and is a member of the cadherin superfamily, generating a calcium-dependent cell adhesion molecule. The glycoprotein MAP3K5 is a member of the MAPK signaling cascade, abundantly expressed in the human heart [36]. SLC17A4 is a sodium/phosphate cotransporter in the intestinal mucosa and plays an important role in the absorption of phosphate from the intestine [37]. The significance of mutations in the genes encoding these four proteins as therapeutic targets will require further validation, as strong mutations in these genes were not identified in this study.
S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and are majorly secreted from neutrophils, monocytes, and macrophages [4]. S100 proteins are recognized for their potential in inflammation [38]. S100A8/A9 is homologous to S100A12, which provides a significant predictive value for one-year mortality in elderly patients with severe HF [39]. Our group was the first to prove, in a previous study, that S100A12 is a potential biomarker for the prediction of HF. Upregulation of S100A12, S100A8, and S100A9 in patients with HF was consistent across our studies. Further, S100A8, S100A9, and S100A12 could be potential indicators in evaluating HF [40, 41]. In addition, PFDN5 and TMCC2 were upregulated in RNA-Seq, which was further verified using real-time PCR. PFDN5 encodes a member of the prefoldin alpha subunit family, and TMCC is a novel protein of the endoplasmic reticulum. Misfolded proteins are upregulated in patients with chronic HF, and misfolded PFDN5 and TMCC2 are promising biomarkers for the prediction of HF.
Our study revealed the key pathways and molecules implicated in HF; however, it has several limitations. First, the initial population used for RNA-Seq and whole exome sequencing was modest, which decreased the sensitivity and accuracy of detecting the changes in expression levels and gene variants that do not make large contributions to HF. Second, the SNP coverage was relatively low when we used a large sample to verify the variants.
In conclusion, we verified that phosphorylation and inflammation are associated with HF, and confirmed that compounds partly inhibiting protein phosphatase and inflammation can have cardiac protective effects.