Identification and analysis of circulating long non-coding RNAs with high significance in diabetic cardiomyopathy

Abstract

Diabetic cardiomyopathy (DCM) lacks diagnostic biomarkers. Circulating long non-coding RNAs (lncRNAs) can serve as valuable diagnostic biomarkers in cardiovascular disease. To seek potential lncRNAs as a diagnostic biomarker for DCM, we investigated the genome-wide expression profiling of circulating lncRNAs and mRNAs in type 2 diabetic db/db mice with and without DCM and performed bioinformatic analyses of the deregulated lncRNA-mRNA co-expression network. Db/db mice had obesity and hyperglycemia with normal cardiac function at 6 weeks of age (diabetes without DCM) but with an impaired cardiac function at 20 weeks of age (DCM) on an isolated Langendorff apparatus. Compared with the age-matched controls, 152 circulating lncRNAs, 127 mRNAs and 3355 lncRNAs, 2580 mRNAs were deregulated in db/db mice without and with DCM, respectively. The lncRNA-mRNA co-expression network analysis showed that five deregulated lncRNAs, XLOC015617, AK035192, Gm10435, TCR-α chain, and MouselincRNA0135, have the maximum connections with differentially expressed mRNAs. Bioinformatic analysis revealed that these five lncRNAs were highly associated with the development and motion of myofilaments, regulation of inflammatory and immune responses, and apoptosis. This finding was validated by the ultrastructural examination of myocardial samples from the db/db mice with DCM using electron microscopy and changes in the expression of myocardial tumor necrosis factor-α and phosphorylated p38 mitogen-activated protein kinase in db/db mice with DCM. These results indicate that XLOC015617, AK035192, Gm10435, TCR-α chain, and MouselincRNA0135 are crucial circulating lncRNAs in the pathogenesis of DCM. These five circulating lncRNAs may have high potential as a diagnostic biomarker for DCM.

Figure 1

Histopathological analysis of the hearts of C57BL/6J and db/db mice at 6 and 20 weeks of age. (A) Representative photomicrographs of mouse hearts following a wheat germ agglutinin staining; (B) quantification of cardiomyocyte size; (C) representative photomicrographs of mouse hearts following a Masson’s trichrome staining; (D) quantification of myocardial fibrosis. Wheat germ agglutinin (A,B) and Masson’s trichrome (C,D) were used to stain left ventricular sections of C57BL/6J and db/db mice. In Masson’s trichrome staining, interstitial fibrosis was stained in blue, as indicated by yellow arrows, and ventricular muscle was stained in red. The scale bar represents 50 µm. *P < 0.05 versus C57BL/6J group at 6 weeks old; ^#P < 0.05 versus C57BL/6J group at 20 weeks old (n = 10/group).

Figure 2

The profiling of deregulated lncRNAs in the plasma of db/db mice with and without diabetic cardiomyopathy compared with controls. (A,B) Volcano plots showing differently expressed lncRNAs in 6 and 20-week-old db/db mice, respectively, compared with controls. The db/db mice develoed early diabetic cardiomyopathy at 20 weeks old. The red and green points represent up- and down-regulated lncRNAs, respectively. The horizontal green line depicts *P ≤ 0.05 (n = 12–13 mice/group), whereas the vertical green line shows a twofold change of up and down. (C,D) Hierarchical cluster analysis presenting differentially expressed lncRNAs between 6 and 20-week-old db/db and control mice, respectively. Colors of red and green represent up- and down-regulated lncRNAs with changes larger than twofold, respectively. (E,F) Chromosomal distribution of deregulated lncRNAs in 6 and 20-week-old db/db mice, respectively. Colors of green and orange represent up- and down-regulated lncRNAs.

Figure 3

Differential expression Profile of circulating mRNA in db/db mice with and without early diabetic cardiomyopathy compared with controls. (A,B) Volcano plots showing deregulated mRNAs in db/db mice at 6 and 20 weeks of age, respectively compared with age-matched controls. The red and green points represented up- and down-regulated mRNAs, respectively. The horizontal green line depicts *P ≤ 0.05, whereas the vertical green line shows a twofold change of up and down. (C,D) Hierarchical clustering analysis demonstrating differently expressed mRNAs between 6 and 20-week-old db/db mice and controls, respectively. Colors of red and green represented up- and down-regulated mRNAs with changes larger than twofold, respectively. (E,F) Chromosomal distribution of deregulated circulating mRNAs in 6 and 20-week-old db/db mice, respectively. Colors of green and orange represented up- and down-regulated mRNAs.

Figure 4

Connections of five deregulated circulating lncRNAs with mRNA transcripts in db/db mice with and without diabetic cardiomyopathy. (A) The network of lncRNA-mRNA co-expression in 6-week-old db/db mice; (B) the network of lncRNA-mRNA co-expression in 20-week-old db/db mice. The network represents the co-expression correlations between the significantly differentially expressed lncRNAs and mRNA transcripts. Five lncRNAs having maximum connections with differentially expressed genes were taken to construct the co-expression network (Pearson correlation > 0.995 or <  − 0.995 and P < 0.05). Circles, squares, and V shapes indicate lncRNA transcripts, transcription factors, and mRNA transcripts, respectively. Solid arrows and dashed lines indicate positive and negative correlation, respectively, whereas red and green colors represent up- and down-regulated transcripts. The width of the line is based on Person’s value (stronger correlation corresponds to more width) and color of the line depicts the significance. Nodes indicate lncRNAs or mRNAs.

Figure 5

Gene ontology (GO) analysis of top 5 deregulated circulating lncRNAs in db/db mice with diabetic cardiomyopathy. The network represents the GO pathway terms specific for mRNA genes having co-expression relationship with the significantly differentially expressed top 5 lncRNAs having maximum connections in the co-expression network (Pearson correlation > 0.995 or <  − 0.995 and P < 0.05) at 20 weeks of ag. Functionally grouped networks with GO terms as node are grouped based on kappa score level (> 0.03), the most significant groups are shown. The node size represents the term enrichment significance. Different GO clusters are shown in different color whereas functionally related groups partially overlap. The color gradient represents the gene proportion of each cluster associated with that term. Nodes indicate lncRNAs or mRNAs.

Figure 6

Transmission electron microscopy micrographs of myocardial myofilaments and mitochondria in db/db mice with diabetic cardiomyopathy and controls. (A) Non-diabetic control ventricle showed well-defined sarcomeres (yellow line) with distinguishable Z-lines (Z) and M-lines (M) throughout the tissue; (B) non-diabetic control ventricle showed well-organized sarcomeres and mitochondria (Mito); (C) the sarcomeric structure of db/db mice with DCM was less organized, with blurred Z-lines and absent M lines; (D) in db/db mice with DCM, Z-lines were disarrayed, the sarcomeres were damaged, and mitochondrial volumes were decreased. Scar bar: 500 nm.

Figure 7

Up-regulated expression of tumor necrosis protein-α (TNF-α) and phosphorylated p-38 mitogen-activated protein kinase (p-p38 MAPK) in the myocardium of db/db mice with diabetic cardiomyopathy. (A) The expression of myocardial TNF-α. Top: representative Western blot bands of TNF-α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bottom: the ratio of TNF-α/GAPDH; (B) the expression of myocardial p-p38 MAPK and p38 MAPK. Top: representative western blot bands of p-p38 MAPK, p38 MAPK, and GAPDH as control. Bottom: the ratio of p-p38-MAPK/p38 MAPK. *P < 0.05 versus C57BL/6J groups (n = 5 mice/group). The Western blot bands were cropped from Figure S6, which were pointed by red arrows. In Figure S6, the original images of Western blot bands included C57BL/6J and db/db mice at 6 weeks of age in addition to 20-week-old mice, and several Western blot experiments were put on a big membrane.