Background
Hypertrophic cardiomyopathy (HCM) represents the most common inherited cardiac disease with an estimated prevalence of 0.2 % in the general population [
1,
2]. Classically, HCM is morphologically characterized by varying degrees of myocardial hypertrophy, myocyte disarray and interstitial fibrosis [
3,
4]. It is also a leading cause of sudden cardiac death in young people, including athletes [
5]. Approximately 25 % of individuals with HCM demonstrate left ventricular outflow tract (LVOT) obstruction [
6]. It has been accepted that HCM is caused predominately by genetic variants. To date, around 1400 mutations have been identified as being responsible for HCM pathology and more than 60 % of genetic variants occurred in 9 sarcomeric genes, including
MYH7,
MYL2 and
MYBPC3 [
7‐
10]. However, the molecular mechanisms that underlie the pathogenesis of HCM remain largely unknown.
Long non-coding RNAs (lncRNAs) are defined as the transcripts of more than 200 nucleotides that structurally resemble mRNAs but do not encode proteins [
11]. The Encyclopedia of DNA Elements (ENCODE) project has reported that >90 % of the genome can be transcribed and the non-coding transcripts account for approximately 98 % [
12]. Normally, lncRNAs are involved in a variety of biological processes, such as cell-cycle control, differentiation, apoptosis, chromatin remodeling as well as epigenetic regulation [
13,
14]. Dysregulation of lncRNAs has been reported in numerous human diseases, including cancer [
15] and neurological diseases [
16]. However, dysregulation of lncRNAs in patients with HCM has never been reported. In the present study, we hypothesized that lncRNAs were dysregulated in hypertrophied myocardial tissues.
In order to reveal the potential roles of lncRNAs in the pathogenesis of HCM, we performed microarray analysis to identify dysregulated lncRNAs and mRNAs in HCM patients, compared to control subjects.
Methods
Ethical statement
All subjects provided written informed consent prior to participation and all procedures in the present study were approved by the Ethics Committee of the second people’s hospital of Sichuan.
Tissues from patients with HCM and healthy individuals
Hypertrophied myocardial tissues used in this study were obtained from 7 HCM patients (4 males and 3 females) with severe symptoms (New York Heart Association functional classes III or IV) who underwent septal myectomy at outpatient department of Chengdu first people’s hospital (from May 2013 to December 2013). All patients were diagnosed by two experienced clinicians, based on two-dimensional echocardiography demonstrating an unexplained left ventricular hypertrophy (diastolic interventricular septal thickness ≥ 15 mm and the ratio of septal to posterior wall thickness ≥1.3) in the absence of another cardiac or systemic disease capable of producing a similar magnitude of hypertrophy. Besides, 5 normal myocardial tissues, excised from 5 healthy individuals (3 males and 2 females) at autopsy who voluntarily donated their body for research to the Center of Forensic Medicine in West China, were used as a control group. It is worth noting that we didn’t perform genetic scanning of 1400 known candidate genes in patients.
The following data was collected for all subjects: gender, age at diagnosis or death, heart rate, family history of hypertrophic cardiomyopathy, previous angina pectoris, systolic blood pressure (SBP) and diastolic blood pressure (DBP) (Table
1). Diseases that would affect cardiac structures and functions were absent in any individual. These diseases included, but not limited to, primary hypertension with duration of more than 10 years, secondary hypertension, ischemic heart disease, congenital heart disease, rheumatic heart disease, aortic stenosis and other similar diseases.
Table 1
Clinical characteristics of HCM patients (n = 7) and controls (n = 5)
Gender | F | M | M | F | F | M | M | F | F | M | M | M | 0.92 |
Age (years) | 51 | 39 | 49 | 60 | 42 | 54 | 43 | 48 | 54 | 31 | 41 | 40 | 0.29 |
Family history of hypertrophic cardiomyopathy (Y or N) | Y | N | N | N | Y | Y | Y | N | N | N | N | N | 0.04 |
Heart rate (bpm) | 80 | 98 | 74 | 73 | 70 | 62 | 67 | 74 | 84 | 59 | 61 | 60 | 0.29 |
Previous angina pectoris | N | N | Y | Y | Y | N | N | N | N | N | Y | N | 0.41 |
SBP (mmHg) | 114 | 122 | 112 | 117 | 121 | 123 | 115 | 127 | 110 | 126 | 129 | 131 | 0.11 |
DBP (mmHg) | 82 | 62 | 65 | 70 | 75 | 74 | 71 | 75 | 80 | 71 | 82 | 91 | 0.09 |
NYHA Classes | 3 | 4 | 3 | 4 | 3 | 3 | 3 | N.A. | N.A. | N.A. | N.A. | N.A. | |
Myocardial specimens were quickly excised and snap-frozen in liquid nitrogen. Tissues were homogenized in TRIZOL reagent (Invitrogen, USA) using a Qiagen Tissuelyser. Total RNA was extracted in accordance with the manufacturer’s protocol and then quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). RNA integrity of each sample was assessed by denaturing agarose gel electrophoresis.
RNA labeling and array hybridization
The expression of lncRNAs and mRNA was determined using Arraystar Human LncRNA Microarray v2.0 (CapitalBio, China). Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, USA) with minor modifications for all 12 samples. First, rRNA was removed from total RNA using mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre Biotechnologies, USA). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias using the random priming method. The labeled cRNAs were purified using an RNeasy Mini Kit (Qiagen, Germany) and then hybridized with the specific probes on the Human LncRNA Array v2.0. Positive probes for housekeeping genes and negative probes are printed onto the array for quality control. The hybridized arrays were washed, fixed, and scanned with an Agilent DNA Microarray Scanner G2505C.
Microarray data analysis
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX software package (version 11.5.1, Agilent Technologies). Differentially expressed lncRNAs and protein-coding mRNAs between patient group and control group were identified through Volcano Plot filtering (fold change > 2 and P < 0.05).
Real-time PCR
Single-strand cDNA was synthesized by AMV Reverse Transcriptase Kit (Promega, USA) in accordance with the manufacturer’s instructions. Real-time PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). The PCR conditions included an initial step at 95 °C for 30 s, followed by 40 cycles of amplification and quantification (95 °C for 5 s, 60 °C for 5 s). Each DNA sample was performed in triplicates in a final volume of 25 μl containing 1 μl of cDNA and 400 nM of forward and reverse gene-specific primers. Relative gene expression levels were quantified based on the cycle threshold (Ct) values and ACTIN was used as an internal control. For quantitative results, expression of each gene was represented as a fold change using the following mathematical model: Fold change = (Etarget)ΔCttarget (Control - Sample)/(Eref)ΔCtref (Control - Sample). Etarget and Eref are the PCR efficiency of target gene transcript and reference gene transcript, respectively; ΔCttarget is the Ct deviation of control – sample of the target gene transcript; ΔCtref is the Ct deviation of control – sample of the reference gene transcript. All primer pairs are available upon request.
Functional group analysis
Gene Ontology (GO) analysis was performed to explore the functions of differentially expressed coding genes identified in this study by using the Database for Annotation, Visualization and Integrated Discovery (DAVID;
http://david.abcc.ncifcrf.gov/) [
17,
18]. Pathway analysis was used to place differentially expressed coding genes according to Kyoto Encyclopedia of Genes and Genomes (KEGG), Biocarta and Reactome (
http://www.genome.jp/kegg/). Generally, Fisher’s exact test and v2 test were used to classify the GO category and select the significant pathway. Besides, the threshold of significance was defined by the P-value and false discovery rate (FDR).
Statistical analysis
The statistical significance of microarray data was analyzed in terms of fold change using the Student’s t-test and FDR was calculated to correct the P-value. FC ≥ 2 and P < 0.05 were used to screen the differentially expressed lncRNAs and coding mRNAs. For other statistical analysis, GraphPad Prism 5 software and Microsoft Office software were applied. Stusent’s t-test was applied for comparison of two groups and differences with P < 0.05 were considered statistically significant.
Discussion
In the present study, we performed a comprehensive analysis of dysregulated lncRNAs by comparing the transcriptome profiles of hypertrophied myocardial tissues from HCM patients and normal myocardial tissues. A total of 8435 lncRNAs and 8404 mRNAs were detected. We identified 965 up-regulated and 461 down-regulated lncRNAs, and summarized their general characteristics and functional annotations. Thus, our study could provide a comprehensive understanding of lncRNAs in HCM patients and help to elucidate the molecular mechanisms of HCM.
To our knowledge, several proteomic and transcriptome profiling of HCM have been performed and a number of dysregulated genes have been identified before [
20‐
23] (Additional file
3). For example, Lim et al. identified 36 dysregulated genes involved in cytoskeletal proteins, protein synthesis, redox system, ion channels and those with unknown function in HCM [
20]. We compared the list of HCM-related genes between our study and previous studies and found that some genes appeared both in our study and previous studies, including
AFG3L2, AGPAT9, CDC14B, CMKLR1, COX4I1, GFM1, GLUD1, LDHB, LYRM4, MRPL22, NCAM1, SLC25A46, SS18L2, TACO1, TMEM135, TMEM41B and ZFAND1.
However, there were also many genes which occurred only in previous studies but not in our study. We believed that discrepancy of expression profiling of mRNA and lncRNA between our study and previous studies was mainly derived from the following several reasons. First, as we know, the RNA and protein profiling fluctuated dramatically during different stages of diseases’ progress [
24,
25]. In different progess stage of HCM, the RNA profiling might be variable and this would be the major reason. Second, time-dependent degradation of nucleic acids confers great difficulty to expression analysis when postmortem tissues were used. In our study, the normal myocardial tissues were excised from healthy formalin-fixed postmortem individuals 3–7 days after death, which might lead to significant RNA degradation [
26,
27]. Inhomogenous RNA degradation would result in the change of relative expression of RNAs, Third, different detection platforms may have huge impact on the profiling result and this is why subsequent experimental verification is essential. Most of the previous studies used Affymetrix platform for profiling analysis, while, in our study, Arraystar Human LncRNA Microarray v2.0 was used. The molecular mechanisms responsible for HCM have been extensively explored. However the pathogenesis of this disease is still largely unknown. Most of the genes discovered in HCM were protein-coding while non-coding genes were scarcely reported. Technological improvements have contributed to reveal the importance of lncRNA underling various diseases [
28]. Up till now, both transcriptome sequencing [
29] and microarrays [
30] have been applied to identify differential lncRNA expression. To the best of our knowledge, only one lncRNA,
Myheart, was reported to be involved in cardiac hypertrophy in previous studies.
Myheart could protect heart from pathological hypertrophy by its interaction with chromatin [
31,
32]. Our study could greatly enrich the lncRNAs spectrums involved in the pathogenesis of HCM.
The Gene Ontology project provides a controlled vocabulary to describe gene attributes unbiasedly [
18]. Interestingly, in our study, these dysregulated genes were mainly involved in translational regulation, including translational elongation, ribonucleoprotein complex, ribosome and structural constituent of ribosome. As we know, once the translational process was disrupted, the levels of numerous proteins would be influenced, which might have profound effects on normal physiological processes [
33,
34]. However, no previous study of HCM was concentrated on translational regulation and we hold the opinion that it would be quite meaningful to investigate translational dysregulation in HCM. Pathway analysis identified 7 distinct pathways, including ribosome and oxidative phosphorylation. `It has been reported that CREB contributes to physiological hypertrophy by enhancing expression of genes in complex I of oxidative phosphorylation [
35]. Many more studies are needed to fully understand the molecular mechanisms of HCM in the future.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WY and HXW conceived of this study and participated in the design of the study. HXW oversaw all aspects of the research. WY, YL and FWH performed RNA extraction and real-time PCR analysis. WY analyzed the microarray data and drew figures and tables. WY performed statistic analysis and wrote the manuscript; HXW revised it. All authors contributed to and have approved the final manuscript.