Impact of chronic intermittent hypoxia on the long non-coding RNA and mRNA expression profiles in myocardial infarction

Abstract

Chronic intermittent hypoxia (CIH) is the primary feature of obstructive sleep apnoea (OSA), a crucial risk factor for cardiovascular diseases. Long non-coding RNAs (lncRNAs) in myocardial infarction (MI) pathogenesis have drawn considerable attention. However, whether CIH participates in the modulation of lncRNA profiles during MI is yet unclear. To investigate the influence of CIH on MI, cardiac damage was assessed by histology and echocardiography, and lncRNA and mRNA integrated microarrays were screened. MI mouse model showed myocardial hypertrophy, aggravated inflammation and fibrosis, and compromised left ventricle function under CIH. Compared with normoxia, 644 lncRNAs and 1084 differentially expressed mRNAs were identified following CIH for 4 weeks, whereas 1482 lncRNAs and 990 mRNAs were altered at 8 weeks. Strikingly, reoxygenation after CIH markedly affected 1759 lncRNAs and 778 mRNAs. Of these, 11 lncRNAs modulated by CIH were restored after reoxygenation and were validated by qPCR. The GO terms and KEGG pathways of genes varied significantly by CIH. lncRNA-mRNA correlation further showed that lncRNAs, NONMMUT032513 and NONMMUT074571 were positively correlated with ZEB1 and negatively correlated with Cmbl. The current results demonstrated a causal correlation between CIH and lncRNA alternations during MI, suggesting that lncRNAs might be responsible for MI aggravation under CIH.

Keywords: chronic intermittent hypoxia; lncRNA; mRNA; myocardial infarction.

Figure 1

CIH modulates cardiac injury post‐infarction. A, Experimental scheme for establishing the mouse model. A representative whole heart image for the quantification of cardiac hypertrophy burden. HW/BW indicates the heart weight/bodyweight. n = 11 for Sham + Air, n = 10 for Sham + CIH, n = 7 for MI + Air and n = 8 for MI + CIH. B and C, Cross sections of heart tissues were stained by HE and Masson, respectively, and cardiomyocyte inflammation and cardiac fibrosis were determined. n = 6 for each group. D, Cardiac function was examined by echocardiography, and representative left ventricular M‐mode echocardiography images are shown. E‐O, Quantification of heart rate, EF, FS, LVAWd (left ventricular diastolic anterior wall), LVAWs (left ventricular systolic anterior wall), LVPWd (left ventricular diastolic posterior wall), LVPWs (left ventricular systolic posterior wall), LVIDd (left ventricular internal diastolic diameter), LVIDs (left ventricular internal systolic diameter), LV Vol‐d (left ventricular diastolic volume) and LV Vol‐s (left ventricular systolic volume). Air indicates normoxia. n = 8 for Sham + Air, n = 7 for Sham + CIH, n = 6 for MI + Air and n = 8 for MI + CIH. *P < .05, **P < .01, ***P < .001. Data are presented as mean ± SEM

Figure 2

Specific lncRNAs and mRNAs regulated by CIH and reoxygenation under MI. A and C, Volcano plots showing the lncRNAs and mRNAs significantly regulated by 4‐week exposure to CIH. The lncRNAs and mRNAs with P ≤ .05 (t test) and fold change in expression ≥ 1.2 were regarded as differentially expressed between the groups. B and D, Heat map depicting the changes in lncRNAs and mRNAs in MI mice after exposure to CIH for 4 wk. E and G, Volcano plots of the significantly different lncRNAs and mRNAs between MI and MI + CIH group at 8 wk. F and H, Heat map of the relative abundance of significantly changed lncRNAs and mRNAs by CIH at 8 wk. I and K, The lncRNAs and mRNAs that varied between MI complicated with CIH (8 wk) and MI with reoxygenation (4 wk) after CIH (4 wk) are shown in the volcano plots. J and L, The relative abundance of lncRNAs and mRNAs in I and K is shown in the heat map. Air indicates normoxia; Reo indicates reoxygenation after CIH; 4W indicates 4 wk; and 8W indicates 8 wk. The red scatters in volcano plots indicate genes up‐regulated by CIH, blue scatters indicate genes down‐regulated, and grey scatters indicate genes that are not different between the groups. The heat map scale indicates the relative abundance of specific genes that were transformed into Z scores. The value of (−log P) was the base 10e negative logarithm of the P‐value. n = 4/group

Figure 3

lncRNAs responsive to CIH post‐infarction. A, Venn diagram showing the number of lncRNAs that are statistically altered by 4 and 8 wk of CIH post‐MI, as well as the lncRNAs regulated by reoxygenation (4 wk) after CIH (4 wk). B, The relative abundance of the 20 overlapping lncRNAs detected by Venn diagram in lncRNA array. n = 4 for each group. C, The expression of 17 out of the 20 overlapping lncRNAs was validated by qPCR analysis. Murine GAPDH gene was used as the housekeeping internal control. The transcript expression was quantified relative to the expression level of GAPDH using the comparative cycle threshold (ΔCt) method. Air indicates normoxia; Reo indicates reoxygenation after CIH; 4W indicates 4 wk; and 8W indicates 8 wk. n = 3/group. *P < .05. Data are presented as mean ± SEM

Figure 4

GO and KEGG pathway analysis for the mRNAs regulated by 4‐wk CIH. A‐C, Data show bioinformatics analysis of the GO terms in biological process, cellular component and molecular function of RNAs enriched in MI with 4 wk of CIH as compared to MI mice with air. D‐F, The GO terms that were deficient in MI with 4 wk CIH. G and H, KEGG pathways that were statistically different between MI with 4 wk CIH and MI with air. n = 4/group

Figure 5

lncRNA‐mRNA co‐expression correlation network and target pathway network of core lncRNAs. A, LncRNA‐mRNA expression correlation network analysis of core lncRNAs (NONMMUT032513 and NONMMUT074571) and their correlated mRNAs regulated by CIH post‐MI. Red nodes represent mRNAs, green blocks represent lncRNAs, and node area represents the value of betweenness centrality. The lines between nodes indicate a correlation, with a solid line representing positive correlation and a blue dotted line representing a negative correlation. B, Network of KEGG pathways associated with the core lncRNA‐linked mRNA. Red nodes represent core pathways, and node area represents the associated gene number, whereas lines represent an interaction between the pathways. C, Network of the core lncRNAs and target pathways. Yellow nodes represent pathways, green blocks represent lncRNAs, and node area represents the value of betweenness centrality. The lines between the nodes indicate a correlation. n = 4/group