Clinical information of dataset
From GEO database (NCBI), dataset GSE1145 was screened out and chosen for WGCNA analysis. In this dataset, it contained 90 left ventricle biopsies of patients. These biopsy samples represented 8 subgroups: 1, normal hearts, used as control (Health, n = 11); 2, idiopathic dilated (ID, n = 15); 3, ischemic (IS, n = 11); 4, idiopathic cardiomyopathy (IDCM, n = 12); 5, familial cardiomyopathy (FCM, n = 5); 6, post-partum cardiomyopathy (PCM, n = 4); 7, ischemic cardiomyopathy (ISCM, n = 20); 8, viral cardiomyopathy (VCM, n = 7). Using Affymetrix Human Genome U133 Plus 2.0 Array, the dataset contains expression data of 20,283 target genes for each biopsy sample. Each probe-set was linked with gene symbol through the Affymetrix annotation file GLP570. Besides gene symbols, their related function and clinical traits were also annotated. (Figure.1, Table. S1)
Identified Gene Modules Correlation with Pathological Traits
The cardiomyopathic and health samples were separated in the PCA plot (Figure.S1). Four normal samples (GSM18444/18445/18447/18448) were detected as outliers and ignored in the subsequent analysis. The total 86 samples were used for next step analysis (Figure.1). Co-expression networks with different clinical traits were built using the Pearson correlation analysis (Figure.2A). Calculated with a dynamic tree cutting algorithm, the distinct co-expression modules were identified that significantly related to different pathological features (Figure.2B & Figure.3A). Twenty-three modules were detected through the dataset (Figure.2B, Table.1). The numbers of significant genes containing in modules were varied from 121 to 14938. The correlation significance of module and pathological features was determined by module significance (MS) correlation and statistics p-value, and significant module varied from different subtype cases Through calculation of the linear mixed-effects model, significant modules were identified for specific pathological feature (Figure.3B). The higher value of module eigengene (ME) correlation, the module is closer correlated to cardiomyopathies (Figure.S3A-G). In the idiopathic dilated group, six modules were significantly associated with its status, including turquoise module (t-value = 0.75, p-value = 5e − 17), light-cyan module (t-value = 0.54, p-value = 7e − 08), tan module (t-value = 0.56, p-value = 2e − 08), grey module (t-value = 0.7, p-value = 8e − 14), green module (t-value = 0.45, p-value = 1e − 05) and the blue module (t-value = 0.72, p-value = 8e − 15) (Figure.2B, Figure.3B, Table.1). In ischemic group, six significant modules were identified, including light-yellow module (t-value = 0.43, p-value = 3e − 05), green-yellow module (t-value = 0.41, p-value = 8e − 05), light-green module (t-value = 0.4, p-value = 1e − 04), red module (t-value = 0.35, p-value = 8e − 04), green module (t-value = 0.24, p-value = 0.03) and the blue module (t-value = 0.44, p-value = 2e − 05) (Fig. 2B, Fig. 3B, Table.1). In idiopathic cardiomyopathy (IdCM) group, three modules were significantly linked to pathological trait, listed as module of Magenta (t-value = 0.43, p-value = 4e − 05), Purple (t-value = 0.31, p-value = 0.003) and Brown (t-value = 0.25, p-value = 0.02). In familial cardiomyopathy group, only Magenta module (t-value = 0.22, p-value = 0.04) was significantly correlated to this trait (Figure.2B, Figure.3B). For the Hypertrophic cardiomyopathy (HCM) group, none of module was identified and ignored in next step analysis (Figure.2B). In the Post. Partum cardiomyopathy (PCM) group, Magenta module (t-value = 0.24, p-value = 0.03) was correlated to its trait (Figure.2B, Figure.3B, Table.1). In ischemic cardiomyopathy group, seven modules were significantly correlated to pathological trait, including Black (t-value = 0.56, p-value = 2e − 08), Midnight-blue (t-value = 0.49, p-value = 2e − 06), Dark-red (t-value = 0.43, p-value = 3e − 05), Cyan (t-value = 0.34, p-value = 0.02), Yellow (t-value = 0.57, p-value = 1e − 08), Pink (t-value = 0.29, p-value = 0.007) and Red (t-value = 0.26, p-value = 0.02) (Figure.3B). For Viral cardiomyopathy (VCM), two significant modules, Cyan (t-value = 0.35, p-value = 0.001) and Magenta (t-value = 0.23, p-value = 0.03), were identified (Figure.2B, Figure.3B).
Enriched Genes Significance related to different cardiomyopathies.
Compared the module memberships (MM) correlation and Genes Significance (GS) among the all significant modules, the module with most significant value was defined as the best candidate for pathological traits correlation analysis (Table 1, Fig. 3B), respectively. These candidates were listed as turquoise module (cor = 0.77, p < 1.0e − 200, GS = 0.4244) (Idiopathic dilated group, Figure.S3A), light-yellow module (cor = 0.13, p = 3.0e − 05, GS = 0.2733) (Ischemic group, Figure.S3B), magenta module (cor = 0.65, p = 1.5e − 114, GS = 0.2783) (Idiopathic cardiomyopathy group, Figure.S3C), magenta module (cor = 0.31, p = 1.7e − 22, GS = 0.1502) (Familial cardiomyopathy group, Figure.S3D), magenta module (cor = 0.30, p = 4.2e − 21, GS = 0.1583) (Post. partum cardiomyopathy group, Figure.S3E), yellow module (cor = 0.63, p < 1.0e − 200, GS = 0.3259) (Ischemic cardiomyopathy group, Figure.S3F) and cyan module (cor = 0.39, p = 8.2e − 24, GS = 0.2245) (Viral cardiomyopathy group, Figure.S3G), respectively. In addition, the scatter plot of multiple module memberships (MM) was plotted against the Genes Significance (GS) in each significant module, and the point was represented each gene contained in a module.
Hierarchical Clustering of Eigengene Profiles with cardiomyopathies Traits
Based on the ME’s values, the hierarchical clustering was performed between all modules and different cardiomyopathies traits to identify their relationships. Furthermore, the eigengene dendrogram analysis was performed to build the correlation of candidate module with different subtypes of cardiomyopathy feature, respectively (Figure.S4A-G). In the idiopathic dilated group, turquoise module was tightly clustered with idiopathic dilated (Figure. S4A). In the ischemic group, green yellow and light yellow modules were the closest branch clustered with ischemic (Figure.S4B). In previous step analysis, green-yellow module (t-value = 0.41, p-value = 8e − 05, GS = 0.2418) was the secondary higher correlation with ischemic status (Figure.2B, Figure.3B). It suggests that genes containing in green-yellow module would be involve the progress of ischemic. In idiopathic cardiomyopathy group, modules of brown, magenta and purple were clustered with idiopathic cardiomyopathy in a separate branch, and magenta and purple module allocated in same cluster (Figure.S4C). It suggests that module of purple and magenta would be the top two significant associated with cardiomyopathy status (Figure.2B, Table.1). In the familial cardiomyopathy and post. partum cardiomyopathy groups, modules of brown, magenta and purple were tightly clustered with familiar cardiomyopathy, while the magenta module was the most significant associated with disease status (Figure.S4D-4E). In the hypertrophic cardiomyopathy group, no module was associated with its pathological feature. In the ischemic cardiomyopathy group, although module of black and midnightblue were clustered in closer branch, the yellow module was allocated in the adjacent branch (Figure.S4F). Combined with the module-trait relationship correlation and gene significance results, it suggested that module of black, midnightblue and yellow were significant associated with ischemic cardiomyopathy (Figure.2B, Figure. 3B, Table. 1). The module of brown and blue were ignored for next analysis as with higher negative GS values (Table.1, Figure. 2B). The yellow module was the most significant correlation to ischemic cardiomyopathy. In the viral cardiomyopathy group, the modules of magenta, purple and brown were clustered with viral cardiomyopathy in a separated branch, and cyan module had the highest GS value associated with pathological feature (t-value = 0.35, p-value = 1e − 03, GS = 0.2245) (Figure.2B, Figure. 3B, Supporting Figure.4G). It suggested that magenta module containing genes involve the progress of viral cardiomyopathy.
Functions and pathways enrichment analysis
Blast through GenClip2, the enrichments analysis of significance genes contained in the correlated module were summarized as bar chart, including Biological Process, Molecular Function and Cellular Component (Ischemic, Figure.S5A-A; Idiopathic Cardiomyopathy, Figure.S5B-A; Familial Cardiomyopathy, Figure.S5C-A; Post-Partum Cardiomyopathy, Figure.S5D-A; Ischemic Cardiomyopathy, Figure.S5E-A; Viral Cardiomyopathy, Figure.S5F-A).
The Biological Processes were mainly concentrated in subgroups of cellular macromolecular metabolic process, protein metabolic process, organic substance metabolic process and macromolecule modification. The genes significant enriched in molecular functions were summarized and listed (Table.S4). The molecular functions were linked to endoplasmic reticulum functions, cell functions (migration, death, growth, division), DNA binding, protein-protein interaction, kinases activity and signal transduction, iron binding and nucleotide binding, etc. The cellular components were mainly enriched in membrane-bounded organelle, intracellular organelle part, etc. The pathways concentrated on mitogen-activated protein kinase (MAPK) signaling pathway, protein processing in endoplasmic reticulum, regulation of actin cytoskeleton, etc. These results suggested that dysregulation of cardiac functions would be associated with metabolism abnormal and accelerated progress of cardiomyopathies. Furthermore, through gene network literature mining and clustering analysis, these significance genes were clustered and labelled according to cellular functions and pathological feature keywords literature corresponding (Ischemic, Fig.S5A; Idiopathic Cardiomyopathy, Fig.S5B; Familial Cardiomyopathy, Fig.S5C; Post-Partum Cardiomyopathy, Fig.S5D; Ischemic Cardiomyopathy, Fig.S5E; Viral Cardiomyopathy, Fig.S5F).
Moreover, the network and connectivity of significance genes were identified as node-connection map, which was correlated to different traits (Ischemic, Figure.S5A-B; Idiopathic Cardiomyopathy, Figure.S5B-B; Familial Cardiomyopathy, Figure.S5C-B; Post-Partum Cardiomyopathy, Figure.S5D-B; Ischemic Cardiomyopathy, Figure.S5E-B; Viral Cardiomyopathy, Figure.S5F-B). The key regulatory genes were labelled with purple border, which was reported in pathological cases, and majority of genes involved the pathway were labelled as un-reported. More interesting, no related key regulatory gene was identified in Post-Partum cardiomyopathy group.
Identification of hub genes for cardiomyopathies
Through co-expression network (MM-GS) filtered, the candidates of hub gene were identified in different groups (5 genes in Ischemic group, Figure.4A; 113 genes in Idiopathic Cardiomyopathy group, Figure.4B; 41 genes in Familiar Cardiomyopathy group, Figure.4C; 65 genes in Post-Partum Cardiomyopathy group, Figure.4D; 83 genes in Ischemic Cardiomyopathy group, Figure.4E; 60 genes in Viral Cardiomyopathy group, Figure.4F).
Calculated by the PPI network method, the candidates of hub gene were summarized (12 genes in Ischemic group, Figure.4A; 120 genes in idiopathic cardiomyopathy group, Figure.4B; 277 genes in Familiar Cardiomyopathy group, Figure.4C; 119 genes in Post-Partum Cardiomyopathy group, Figure.4D; 348 genes in Ischemic Cardiomyopathy group, Figure.4E; 49 genes in Viral Cardiomyopathy group, Figure.4F). These real hub genes were determined as described in method section (Table.3), and the numbers of real hub genes were listed (Figure. 4A-F). The Idiopathic Dilated group was dismissed for further analysis as no identified real hub gene. Through Venn diagrams analysis, three common axes of hub genes were discovered among these cardiomyopathic groups (Figure.4G).
The first axis was PICALM, which shared by Ischemic Cardiomyopathy, Idiopathic Cardiomyopathy and Post. Partum Cardiomyopathy groups (Figure.4G), and significantly up-regulated in Idiopathic Cardiomyopathy and Ischemic Cardiomyopathy groups (Table.3). PICALM is key regulator in iron homeostasis, clathrin-mediated endocytosis 15,16. Overexpression of PICALM impaired endocytosis of Transferrin (Tf) Receptor (TfR) and Epidermal Growth Factor Receptor (EGFR) and disturbed the iron homeostasis 16,17. Up to now, it is still illusive that the exactly role and deregulatory mechanism of PICALM in cardiomyopathies. It is strongly suggesting that PICALM work as potential novel biomarker and therapy target for these subcases of cardiomyopathies.
The secondary axis, contained genes of PRKACB, MOB1A, CDC40, were shared in Post. Partum Cardiomyopathy and Idiopathic Cardiomyopathy groups. In addition, these genes (PRKACB, MOB1A, CDC40) were significantly overexpressed in Idiopathic cardiomyopathy group, and MOB1A was up-regulated in Post. Partum cardiomyopathy group (Table.3).These genes were linked to the cAMP (cyclic AMP)-dependent protein kinase A (PKA) mediated the exciting-contraction coupling in cardiomyocytes18, and regulated microtubule stability, cell cycle and cell proliferation & migration, and restrained cardiomyocyte proliferation and size via Hippo pathway19,20. PRKACB (protein kinase cAMP-activated catalytic subunit β gene) was linked to congenital heart defect with abnormal over-expression21. MOB1A (MOB kinase activator 1A) was required for cytokinesis through regulating microtubule stability. It worked as binding partners as well as co-activators of Ndr family protein kinases and mediated phosphor-recognition in core Hippo pathway that restrains cardiomyocyte proliferation during development to control cardiomyocyte size19,20. Overexpression of MOB1A induces centrosomes fail to split and cell size dysregulation22. CDC40 (Cell Division Cycle 40), a splicing factor of cell division cycle 40 homolog, regulates cell cycle and cell proliferation and migration23. Overexpression of CDC40 causes abnormally cell proliferation and migration, and linked with carcinogenesis24.
The third axis consisted of five genes (CREB1, DBT, NCOA2, NUDT21, PIK3C2A) and were overlapped among three groups of Familial / Idiopathic / Post. Partum Cardiomyopathy (Figure.4G). The CREB1 (cAMP-responsive element-binding protein) had been identified as the transcription factor and mediated cAMP stimulation by multiple extracellular signals, such as growth factors and hormones. The CREB1 was the key regulator in heart and linked with heart disease via cAMP-PKA pathway dysregulation 25,26. The DBT (dihydrolipoamide branched chain transacylase E2) is an inner-mitochondrial enzyme complex regulated to degrade the branched-chain amino acids isoleucine, leucine, and valine27. The DBT was reported as clinical diagnostics biomarker for patients with dilated cardiomyopathy via caused mitochondria dysfunction 28. NCOA2 (nuclear receptor coactivator 2) is a transcriptional coactivator that functional aid for nuclear hormone receptors, including steroid, thyroid, retinoid, and vitamin D receptors. NCOA2 promotes muscle cells maintenance and growth, eventually regulates in cardiac cTnT levels29,30. Overexpression of NCOA2 regulated cell proliferation in cardiomyopathy30,31. NUDT21 (nudix hydrolase 21) is a novel of cell fate regulator by alternative polyadenylation chromatin signaling, and suppression of NUDT21 will enhance the cell pluripotent, facilitated trans-differentiation into stem cell32. NUDT21 regulates cell proliferation through ERK pathway33. Up to now, little knows about the function of NUDT21 in cardiomyocytes. PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha) is an enzyme belong to phosphorylate the 3'-OH of inositol ring of phosphatidylinositol (PI) superfamily and regulates multiple signaling pathways. PIK3C2A is mainly expressed in endothelial cells, vascular endothelium, and smooth muscle34. Lower expression of PIK3C2A in peripheral blood was used as significant biomarker for acute myocardial infarction patients 35. More interesting, these hub genes indicated different expression pattern. The expression level of DBT, NCOA2, NUDT21 and PIK3C2A were significantly upregulated in Idiopathic cardiomyopathy group, and PIK3C2A was up-regulated in Familiar cardiomyopathy group (Table.3). It hints that these hub genes play different regulatory pattern in the progress of these subtype’s cardiomyopathies.
The fourth axis of hub genes (HNRNPC, UEVLD) were shared by Familiar Cardiomyopathy and Idiopathic Cardiomyopathy groups, and significantly overexpressed in Idiopathic cardiomyopathy group (Table.3). HNRNPC (heterogeneous nuclear ribonucleoprotein C) is RNA binding protein that belong to ubiquitously expressed heterogeneous nuclear ribonucleoproteins subfamily, and mediates pre-mRNAs transport and metabolism between cytoplasm and nucleus 36,37 and overexpression caused cells multi-nucleation 38. UEVLD (EV and lactate/malate dehydrogenase domain-containing protein) involves the protein degradation and dysregulated linked with metabolic disease 39. In this study, the expression level of HNRNPC and UEVLD were significantly up-regulated in Idiopathic cardiomyopathy group (Table.3). Furthermore, through different significant expression analysis, the significant changed hub genes were summarized (Table.3, p < 0.05). Combined these results together, it hints that these significantly expressed Hub genes play dominant role and work as common key regulatory nodes in progress of cardiomyopathies.
Disease Signature Genes Identification and expression analysis
The filtered disease signature genes were summarized with the functional annotation of genetic dysregulation correlated to heart diseases phenotypes, including ten signature genes in the ischemic group and viral cardiomyopathy group, forty signature genes among the group of familiar cardiomyopathy, Post-partum cardiomyopathy and Idiopathic cardiomyopathy, and 69 signature genes in the ischemic cardiomyopathy group (Table.4). Through Venn Diagram analysis, the common signature genes were determined among different groups (Figure.5A). Four signature genes (MDM4, CFLAR, RPS6KB1, PKD1L2) were shared by Ischemic and Ischemic Cardiomyopathy group (Table.4, Figure.5A) 40–43. Ischemic cardiomyopathy group did share eight disease signature genes (MAPK1, MAPK11, MAPK14, LMNA, RAC1, PECAM1, XIAP, CREB1) with Post. Partum/Familiar/Idiopathic Cardiomyopathy groups, which dysregulated in cardiomyopathies 26,44, and genes expression level of MAPK1, MAPK11, LMNA, RAC1 were significantly up-regulated in these cardiomyopathies groups (Table.5, Figure.5A). Two signature genes (TFAM, RHEB) were shared between Viral Cardiomyopathy and Post. Partum / Familiar /Idiopathic Cardiomyopathy groups, which involved in development of cardiac hypertrophy 45 and Mitochondrial Cardiomyopathy 46.
Furthermore, through different significant expression analysis, the significantly changed signature genes were summarized (Fig. 5D-F, Table.5). In ischemic group, MDM4 gene was significantly upregulated (FC = 1.0495, p = 0.0037) (Table 5, Figure.5B), which genetic deletion associated with cardiomyopathy 43. In viral cardiomyopathy group, COA5 was overexpressed (FC = 1.087485, p < 0.0001) (Table 5, Figure.5C), which was upregulated in Ischemia/Reperfusion Injury caused cardiomyopathy 47. In idiopathic cardiomyopathy group, seven genes (ADAM10, RAB1A, TFAM, FGF2, ELMOD2, GUF1, ABCC9) were significantly up-regulated, while 8 genes (FHL1, CTNNA3, PDLIM5, LMNA, SIRT4, YME1L1, RHEB, GNB1L) were down-regulated (Figure.5D, Table.5). In idiopathic cardiomyopathy group, four down-regulated genes (NEBL, FHL1, FHL2, SIRT4) and 5 up-regulated genes (GUF1, ELMOD2, ABCC9, FGF2, YME1L1) were significantly changed (Figure.5E, Table. 5). In post-partum cardiomyopathy group, 11 genes (ATL3, ADAM10, ELMOD2, FGF2, GUF1, YMEIL1, up-regulated; FHL1, CTNNA3, PTPN11, GNB1L, SIRT4, down-regulated) were significantly changed (Figure.5F, Table.5). In Ischemic cardiomyopathy group, 31 genes are significantly changed expression, including 12 genes (RAC1, FKTN, EDNRB, ZBTB33, TXN, RALGAPA1, PSEN1, LAMP2, UBR5, SCN4B, SMAD1, MYO6) down-regulated and 19 genes up-regulated expression (POLRMT, AVP, GATA4, CACNB2, MAPK1, NOS3, LAMA3, SOD2, LMNA, MAP1LC3A, MAPK14, TCAP, LRP4, BAD, DES, AKAP8, CASP9, HSPB1, SNTA1) (Figure.5G, Table.5). These results suggest that these common disease signature genes work as novel biomarker and be potential key regulators of the cardiomyopathy progress.