ASB1 differential methylation in ischaemic cardiomyopathy. Relationship with left ventricular performance in end stage heart failure patients
Ana Ortega, Estefanía Tarazón, Carolina Gil-Cayuela, Luis Martínez-Dolz, Francisca Lago, José Ramón González-Juanatey, Juan Sandoval, Manuel Portolés, Esther Roselló-Lletí, Miguel Rivera
11 ASB1 differential methylation in ischaemic cardiomyopathy. Relationship with left ventricular performance in end stage heart failure patients
Ana Ortega (1) ¶ , Estefanía Tarazón (1) ¶ , Carolina Gil-Cayuela (1), Luis Martínez-Dolz (2), Francisca Lago (3), José Ramón González-Juanatey (3), Juan Sandoval (4), Manuel Portolés (1), Esther Roselló-Lletí (1) & , Miguel Rivera (1) & . (1) Cardiocirculatory Unit, Health Research Institute La Fe, Valencia, Spain. (2) Heart Failure and Transplantation Unit, Cardiology Department, University and Polytechnic La Fe Hospital, Valencia, Spain. (3) Cellular and Molecular Cardiology Research Unit, Department of Cardiology and Institute of Biomedical Research, University Clinical Hospital, Santiago de Compostela, Spain. (4) Epigenomic Unit, Health Research Institute La Fe, Valencia, Spain. ¶ These authors contributed equally to this work. & These authors also contributed equally to this work. ims:
Ischaemic cardiomyopathy (ICM) leads to impaired contraction and ventricular dysfunction causing high rates of morbidity and mortality. Epigenomics allows the identification of epigenetic signatures in human diseases. We analyse the differential epigenetic patterns of
ASB gene family in ICM patients and relate these alterations to their haemodynamic and functional status.
Methods and Results:
Epigenomic analysis was carried out using 16 left ventricular (LV) tissue samples, 8 from ICM patients undergoing heart transplantation and 8 from control (CNT) subjects without cardiac disease. We increased the sample size up to 13 ICM and 10 CNT for RNA-sequencing and to 14 ICM for pyrosequencing analyses. We found a hypermethylated profile (cg11189868) in the
ASB1 gene that showed a differential methylation of 0.26Δβ, P < 0.05. This result was validated by pyrosequencing technique (0.23Δβ, P < 0.05). Notably, the methylation pattern was strongly related to LV ejection fraction (r = -0.849, P = 0.008) stroke volume (r = -0.929, P = 0.001) and end-systolic and diastolic LV diameters (r = -0.743, P = 0.035 for both). ASB1 showed a down regulation in mRNA levels (-1.2 fold, P < 0.05). Conclusion:
Our findings link a specific
ASB1 methylation pattern to LV structure and performance in end-stage ICM, opening new therapeutic opportunities and providing new insights regarding which is the functionally relevant genome in the ischemic failing myocardium.
Keywords: ischaemic cardiomyopathy; epigenomics; heart failure; left ventricular dysfunction; stroke volume;
ASB1 . INTRODUCTION
There are several large-scale studies deciphering the alterations in human heart failure (HF) proteome and genome, to elucidate the molecular mechanisms involved in the pathophysiology of this syndrome.
1, 2
However, apart from these global changes, there are other omic approaches revealing insights in the regulation of expression patterns in disease. Epigenomics has allowed the identification of epigenetic signatures in human diseases including different types of cancer, neurological disorders
6, 7 or infections. There are also some evidences of their role in cardiovascular diseases.
Moreover, the development of the novel approach MethylationEPIC BeadChip (Infinium) microarray, has improved greatly the coverage, reaching 850,000 CpG methylation sites (850K). Our group has extensively studied different pathophysiological factors in HF, including apoptosis processes and cytoskeletal and cell adhesion molecular alterations
14, 15 that are important to clinical status of patients. Interestingly, the gene family
ASB codifies Ankyrin repeat and SOCS box proteins that mediate, through their specific domains, protein-protein interactions, protein synthesis, and myogenesis and proteasomal degradation processes.
Moreover members of this family have been related to skeletal muscle mass regulation. Taking into account these previous data and the increasing evidence of the influence of epigenetic changes in the pathophysiology of human diseases, we hypothesize that the epigenetic changes in the
ASB gene family may relate to the ischaemic left ventricular (LV) performance. We analysed specific gene methylation changes in patients with end stage ischaemic cardiomyopathy (ICM) compared with those in control (CNT) subjects. Furthermore, we related these alterations with LV function and with invasively calculated stroke volume (SV).
METHODS
Cardiac tissue samples
Epigenomic experiments were conducted with 8 LV tissue samples from ICM patients undergoing cardiac transplantation and 8 non-diseased donor hearts (CNT), increasing the sample size for RNA-sequencing (13 ICM) and pyrosequencing (14 ICM). Clinical history, hemodynamic study, ECG, and Doppler echocardiography data were available. These data were collected by physicians blind to the subsequent analysis of LV function that was carried out. Patients were functionally classified according to the NYHA criteria and were receiving medical treatment following the guidelines of the European Society of Cardiology. All CNTs had normal LV function (LVEF>50%) and none had any history of cardiac disease. Samples were obtained from non-diseased donor hearts that had been rejected for cardiac transplantation owing to size or blood type incompatibility. Donors died of either cerebrovascular or motor vehicle accidents. Tissue samples were collected from near the apex of left ventricle, maintained in 0.9% NaCl, and preserved at 4 °C for a maximum of 4.4±3 h after the coronary circulation loss. Then, were stored at −80°C until used. Appropriate handling and rapid sample collection and storage, by our on call (24 h) team over the last 10 years, led to the obtaining of high quality samples (RIN≥9 and DNA ratios 260/280 ~1.8 and 260/230 ~2.0). This study was approved by the Ethics Committee (Biomedical Investigation Ethics Committee of La Fe University Hospital of Valencia, Spain), and was conducted in accordance with the guidelines of the Declaration of Helsinki. Signed informed consent was obtained from each patient.
DNA extraction, quality assessment and Infinium MethylationEPIC BeadChip
DNA was extracted using a modified phenol-chloroform protocol.
The epigenomic study (Infinium MethylationEPIC BeadChip platform (Illumina) used the HumanMethylation450 BeadChip protocol. Methylation level was displayed as beta values ranging from 0–1. Beta-values with detection P >0.01 were removed from the analysis. The raw data were normalized and background corrected. The resulting raw data (IDATs) were normalized and background corrected using the methylation module (1.9.0) in GenomeStudio (v2011.1) software. Validation by pyrosequencing
RNA-sequencing and computational analysis
These protocols were performed as described in. The data presented in this manuscript have been deposited in NCBI’s Gene Expression Omnibus (GEO) Statistical methods
Data were analysed as previously described. P <0.05 was considered statistically significant. CpGs with Δβ≥±0.1 were considered differentially methylated. RESULTS Clinical characteristics of patients
For epigenomic studies we analyzed 8 ICM samples, patients were all men with a mean age of 53±5 years, and 8 CNT samples, 63% men with a mean age of 59±20 years. Sample size was increased for RNA-sequencing (13 ICM), all men with a mean age of 54±7 years and 10 CNT, 80% men with a mean age of 47±16 years. And also for pyrosequencing (14 ICM), all men with a mean age of 53±6 years. Patients had a NYHA classification of III-IV and were diagnosed with comorbidities including hypertension and diabetes mellitus. Comorbidities and other echocardiographic data were not available for the CNT group, in accordance with the Spanish Organic Law on Data Protection 15/1999. Clinical characteristics of patients are shown in Table 1.
Methylation profile of
ASB family and gene expression analysis of
ASB1 gene
We analyzed the methylation status (β-values) of CpGs belonging to the
ASB gene family between 8 ICM patients and 8 CNT donors using the 850K methylation array. Analysis of CpG differential methylation revealed the presence of only one hypermethylated CpG site of all
ASB family, located in the
ASB1 gene (chr2:239344401-239344627) with a Δβ>0.1. The hypermethylated CpG site (cg11189868), displayed a differential methylation profile of 0.26Δβ, P <0.05 (Figure 1A). We also validated these results through pyrosequencing, observing a 0.23Δβ, P <0.05 (Figure 1B). Further, we performed an analysis of ASB1 mRNA levels through RNA-sequencing and we found a downregulation of
ASB1 gene expression of -1.2 fold, P <0.05 (Figure 1C). Relationships between
ASB1 differential methylation and LV function and performance
We sought to investigate the potential relationships between
ASB1 differential methylations and expression and hemodynamic and echocardiographic parameters of ICM patients. Interestingly, the differential methylation pattern of
ASB1 cg11189868 was strongly linked to SV ( r = -0.929, P = 0.001) and LVEF ( r = -0.849, P = 0.008) (Figure 2). This ASB1 methylation profile also related to end-systolic and end-diastolic LV diameters ( r = -0.743, P = 0.035 for both). DISCUSSION
In this study, we analyse the methylation profile of
ASB gene family in ICM patients, showing the presence of a differentially methylated CpG site located at the
ASB1 gene. None of the other
ASB family genes showed methylation changes. This analysis demonstrates the presence, not previously reported, of a strong association between a differentially methylated pattern, validated by pyrosequencing, of
ASB1 gene in ICM subjects with the hemodynamic status, LV performance and cardiac function of these patients. In previous studies, we have analysed the transcriptomic changes in cytoskeletal components of HF patients, showing important alterations and links with LV dysfunction. Importantly, the gene coding of ankyrin repeat domain 1, ANKRD1 , showed relationships with functional status of these patients, indicating a relevant role of this ankyrin gene in HF.
ASB gene family codify Ankyrin repeat and SOCS box proteins, being involved in protein-protein interactions acting as adaptors that target proteins for proteasomal degradation. Scant data are available about the specific function of
ASB1 , relating it with alterations in spermatogenesis, moreover, no studies have been conducted in cardiac tissues, but its protein superfamily has relevant implications in controlling the skeletal muscle contractile apparatus structural fixation and adequate regulation of differentiation steps. ASB2 has been implicated as a negative regulator of skeletal muscle mass through the TGF-β pathway, indicating that increased levels prevents hypertrophy. Our results and the structural domain similarity in this gene family suggest a similar function for
ASB1 in the heart muscle. As demonstrated, gain of methylation of
ASB1
CpG island closely relates to LV function, dimensions, and output, and none of the other 18
ASB family genes show such change, indicating that an increased degree of methylation may be an indicator of deteriorating hemodynamic and cardiac function. In contrast, the
ASB1 gene expression calculated by means of the RNA-sequencing technique does not showed any LV significant relationships, suggesting a prominent role for this DNA methylation, maybe related to an unknown specific function in coding.
Conclusions
Our findings strongly link a specific
ASB1 methylation pattern to LV morphology and performance in end stage ICM, and provide new insight and raising questions regarding which is the functionally relevant genome for the ischaemic failing myocardium. SOURCE OF FUNDING
This work was supported by the National Institute of Health “Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III” [PI13/00100; PI14/01506], CIBERCV [CB16/11/00261], the European Regional Development Fund (FEDER), and RETICS [12/0042/0003].
ACKNOWLEDGMENTS
The authors thank the Transplant Coordination Unit (University and Polytechnic Hospital La Fe) for their help in obtaining the samples.
CONFLICTS OF INTEREST
The authors declare no conflict of interests. REFERENCES
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ICM, ischaemic cardiomyopathy; NYHA, New York Heart Association; BMI, body mass index; LVEF, ejection fraction; LVESD, left ventricular end-systolic diameter; LVEDD, left ventricular end-diastolic diameter.
ICM (n=8) ICM (n=13) ICM (n=14)
Epigenomics RNA sequencing Pyrosequencing
Age (years) 53±5 54±7 53±6 Gender male (%) 100 100 100 NYHA class III-IV III-IV III-IV BMI (kg/m ) 28±3 26±4 28±4 Haemoglobin (mg/dL) 14±2 14±3 14±2 Haematocrit (%) 44±4 41±6 42±5 Total cholesterol (mg/dL) 152±43 162±41 171±46 Prior hypertension (%) 25 30 39 Prior smoking (%) 88 84 92 Diabetes mellitus (%) 63 38 54 LVEF (%) 24±6 24±4 23±5 LVESD (mm) 57±8 55±7 56±7 LVEDD (mm) 65±7 64±7 64±7 LEGENDS
Figure 1. Differentially methylated profile of
ASB1 and gene expression in ICM patients. A.
Methylation pattern of the
ASB1 gene in ICM patients showing the expansion of the differentially methylated CpG sites between ICM and CNT. B . Validation of DNA methylation CpG island by pyrosequencing. C. Gene expression analysis of
ASB1 gene through RNA-sequencing. CNT, control; ICM, ischaemic cardiomyopathy; TSS, transcription start site. *P<0.05.