The lncRNA Malat1 regulates microvascular function after myocardial infarction in mice via miR-26b-5p/Mfn1 axis-mediated mitochondrial dynamics

Abstract

Rationale: Myocardial infarction (MI) is a leading cause of cardiovascular mortality globally. The improvement of microvascular function is critical for cardiac repair after MI. Evidence now points to long non-coding RNAs (lncRNAs) as key regulators of cardiac remodelling processes. The lncRNA Malat1 is involved in the development and progression of multiple cardiac diseases. Studies have shown that Malat1 is closely related to the regulation of endothelial cell regeneration. However, the potential molecular mechanisms of Malat1 in repairing cardiac microvascular dysfunction after MI remain unreported.

Methods and results: The present study found that Malat1 is upregulated in the border zone of infarction in mouse hearts, as well as in isolated cardiac microvascular endothelial cells (CMECs). Targeted knockdown of Malat1 in endothelial cells exacerbated oxidative stress, attenuated angiogenesis and microvascular perfusion, and as a result decreased cardiac function in MI mice. Further studies showed that silencing Malat1 obviously inhibited CMEC proliferation, migration and tube formation, which was at least in part attributed to disturbed mitochondrial dynamics and activation of the mitochondrial apoptosis pathway. Moreover, bioinformatic analyses, luciferase assays and pull-down assays indicated that Malat1 acted as a competing endogenous RNA (ceRNA) for miR-26b-5p and formed a signalling axis with Mfn1 to regulate mitochondrial dynamics and endothelial functions. Overexpression of Mfn1 markedly reversed the microvascular dysfunction and CMEC injuries that were aggravated by silencing Malat1 via inhibition of excessive mitochondrial fragments and mitochondria-dependent apoptosis.

Conclusions: The present study elucidated the functions and mechanisms of Malat1 in cardiac microcirculation repair after MI. The underlying mechanisms of the effects of Malat1 could be attributed to its blocking effects on miR-26b-5p/Mfn1 pathway-mediated mitochondrial dynamics and apoptosis.

Keywords: Cardiac microvascular dysfunction; Malat1; Mfn1; Myocardial infarction; miR-26b-5p.

Fig. 1

Silencing Malat1 in CMECs aggravated myocardial infarction (MI) and microvascular dysfunction. Time course of the relative expression of Malat1 in cardiac tissues and CMECs in the infarct border zone and remote areas after MI (A, B). One-week overall survival curve (C). Analysis of brain natriuretic peptide (BNP) levels in serum (D). The M-mode of echocardiography images and the data on left ventricular end-diastolic diameter (LVEDD), ejection fraction (EF) and fractional shortening (FS) for each group (E). Heart weight to body weight ratio (HW/BW) for each group (F). HE staining, Masson staining and quantification of cardiac fibrosis (G, H). Analyses of cardiac troponin T (cTnT) concentration and lactate dehydrogenase (LDH) activity in serum (I, J). Microvascular perfusion of the border zone was indicated by the ratio of lectin-perfused vessels (green) to CD31-positive ECs (red) (K). Immunofluorescence staining for eNOS (L). Nitric oxide (NO) content in the border zone of each group (M). Immunofluorescence staining for VEGFR2 (N). eNOS expression and phosphorylation at Ser¹¹⁷⁷ in the border zone were analysed by Western blotting (O). VEGFR2 expression and phosphorylation at Tyr¹¹⁷⁵ in the border zone were analysed by Western blotting (P). *p < 0.05 compared with the sham group, #p < 0.05 compared with the MI + Scr-shRNA group. n = 6 in each group.

Fig. 2

Silencing Malat1 compromised CMEC functions by accelerating mitochondrial fragmentation and related apoptosis under hypoxia. Time course of the relative expression of Malat1 under hypoxia (A). Relative cell viability was measured by the CCK-8 assay (B). Nitric oxide (NO) content in CMECs (C). Cellular ROS (green) and mtROS (red) in CMECs were measured and quantified (D). Mitochondria were stained with MitoTracker (red), and mitochondrial morphology was quantified (E). The expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins in CMECs (F). Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay (G). TUNEL staining of CMECs and the percentage of TUNEL-positive CMECs (red) were quantified (H). The expression of apoptosis-associated proteins in CMECs (I, J). *p < 0.05 compared with the control group, #p < 0.05 compared with the hy + siNC group. n = 5 in each group.

Fig. 3

The lncRNA Malat1 targeted miR-26b-5p to act as a molecular sponge in CMECs. The predicted binding sites of Malat1 and miR-26b-5p and the mutated binding sites are indicated (A). Time course of the expression of miR-26b-5p in tissues and CMECs isolated from the border zone and remote areas of MI (B–C). Time course of the expression of miR-26b-5p in CMECs under hypoxia (D). Relative expression of miR-26b-5p in tissues and CMECs isolated from the border zone after silencing Malat1 (E, F). Relative expression of miR-26b-5p in CMECs under hypoxia after silencing Malat1 (G). Luciferase activities of reporter vectors containing luciferase genes and a fragment of Malat1 RNA containing wild-type or mutated miR-26b-5p binding sites (H). Malat1 was associated with miR-26b-5p. CMECs were transfected with biotinylated wild-type miR-26b-5p (Bio-miR-26b-5p-WT) or biotinylated mutant miR-26b-5p (Bio-miR-26b-5p-Mut) (I). The RIP assay for Malat1 was performed with an anti-AGO2 antibody in CMECs transfected with NC or mimics, and the expression of Malat1 and miR-26b-5p was detected (J). *p < 0.05 compared with the sham or NC group, #p < 0.05 compared with the adjacent group. n = 5 in each group.

Fig. 4

miR-26b-5p negatively regulated CMEC functions under hypoxic injury. Relative cell viability was measured by the CCK-8 assay (A). Nitric oxide (NO) content in CMECs (B). Cellular ROS (green) and mtROS (red) in CMECs were measured and quantified (C). Mitochondria were stained with MitoTracker (red), and mitochondrial morphology was quantified (D). The expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins in CMECs (E). Mitochondrial membrane potential (ΔΨm) was measured by the JC-1 assay (F). TUNEL staining of CMECs and the percentage of TUNEL-positive CMECs (red) were quantified (G). The expression of apoptosis-associated proteins in CMECs (H, I). *p < 0.05 compared with the hy + mimic-NC group, #p < 0.05 compared with the hy + inhibitor-NC group. n = 5 in each group.Relative cell viability was measured by the CCK-8 assay (A). Nitric oxide (NO) content in CMECs (B). Cellular ROS (green) and mtROS (red) in CMECs were measured and quantified (C). Mitochondria were stained with MitoTracker (red), and mitochondrial morphology was quantified (D). The expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins in CMECs (E). Mitochondrial membrane potential (ΔΨm) was measured by the JC-1 assay (F). TUNEL staining of CMECs and the percentage of TUNEL-positive CMECs (red) were quantified (G). The expression of apoptosis-associated proteins in CMECs (H, I). *p < 0.05 compared with the hy + mimic-NC group, #p < 0.05 compared with the hy + inhibitor-NC group. n = 5 in each group.

Fig. 5

Inhibiting miR-26b-5p alleviated Malat1 deletion-aggravated EC injuries under hypoxia. Relative cell viability was measured by the CCK-8 assay (A). Nitric oxide (NO) content in CMECs (B). Cellular ROS (green) and mtROS (red) in CMECs were measured and quantified (C). Mitochondria were stained with MitoTracker (red), and mitochondrial morphology was quantified (D). The expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins in CMECs (E). Mitochondrial membrane potential (ΔΨm) was measured by the JC-1 assay (F). TUNEL staining of CMECs and the percentage of TUNEL-positive CMECs (red) were quantified (G). The expression of apoptosis-associated proteins in CMECs (H, I). *p < 0.05 compared with the siNC + inhibitor-NC group, #p < 0.05 compared with the hy + siNC + inhibitor-NC group, & p < 0.05 compared with the hy + siMalat1+inhibitor-NC group. n = 5 in each group.

Fig. 6

Mfn1 is a direct downstream mRNA of miR-26b-5p in CMECs. The predicted binding sites of Malat1 and miR-26b-5p and the mutated binding sites are indicated (A). Luciferase activities of reporter vectors containing luciferase genes and a fragment of Mfn1 RNA containing wild-type or mutated miR-26b-5p binding sites (B). Mfn1 was associated with miR-26b-5p. CMECs were transfected with biotinylated wild-type miR-26b-5p (Bio-miR-26b-5p-WT) or biotinylated mutant miR-26b-5p (Bio-miR-26b-5p-Mut) (C). Concentration gradients of the miR-26b-5p mimic and inhibitor were transfected into CMECs, and quantitative analysis of the alteration of miR-26b-5p and the gene and protein expression of Mfn1 was performed (D-E). *p < 0.05 compared with the NC group, #p < 0.05 compared with the adjacent group. n = 5 in each group.

Fig. 7

Mfn1 positively regulated CMEC functions after hypoxic injury. Relative cell viability was measured by the CCK-8 assay (A). Nitric oxide (NO) content in CMECs (B). Cellular ROS (green) and mtROS (red) in CMECs were measured and quantified (C). Mitochondria were stained with MitoTracker (red), and mitochondrial morphology was quantified (D). The expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins in CMECs (E). Mitochondrial membrane potential (ΔΨm) was measured by the JC-1 assay (F). TUNEL staining of CMECs and the percentage of TUNEL-positive CMECs (red) were quantified (G). The expression of apoptosis-associated proteins in CMECs (H, I). *p < 0.05 compared with the control group, #p < 0.05 compared with the hy + siNC group, & p < 0.05 compared with the hy + pcDNA3.1 group. n = 5 in each group.

Fig. 8

Mfn1 reversed mitochondrial damage and apoptosis induced by silencing Malat1 in CMECs under hypoxia. Relative protein expression of Mfn1 in CMECs (A). Relative cell viability was measured by the CCK-8 assay (B). Nitric oxide (NO) content in CMECs (C). Cellular ROS (green) and mtROS (red) in CMECs were measured and quantified (D). Mitochondria were stained with MitoTracker (red), and mitochondrial morphology was quantified (E). The expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins in CMECs (F). Mitochondrial membrane potential (ΔΨm) was measured by the JC-1 assay (G). TUNEL staining of CMECs and the percentage of TUNEL-positive CMECs (red) were quantified (H). The expression of apoptosis-associated proteins in CMECs (I, J). *p < 0.05 compared with the siNC + pcDNA3.1 group, #p < 0.05 compared with the hy + siNC + pcDNA3.1 group, & p < 0.05 compared with the hy + siMalat1+pcDNA3.1 group. n = 5 in each group.

Fig. 9

Overexpressing Mfn1 reversed the aggravation of myocardial infarction and microvascular dysfunction induced by silencing Malat1 in MI hearts. One-week overall survival curve (A). The M-mode of echocardiography images and the data on left ventricular end-diastolic diameter (LVEDD), ejection fraction (EF) and fractional shortening (FS) for each group (B, C). Heart weight to body weight ratio (HW/BW) of each group (D). Analysis of serum brain natriuretic peptide (BNP) levels (E). HE staining, Masson staining and quantification of cardiac fibrosis (F, G). Analysis of serum cardiac troponin T (cTnT) concentration and lactate dehydrogenase (LDH) activity (H, I). Microvascular perfusion of the border zone was indicated by the ratio of lectin-perfused vessels (green) to CD31-positive ECs (red) (J). Immunofluorescence staining for eNOS (K). Nitric oxide (NO) content in the border zone of each group (L). Immunofluorescence staining for VEGFR2 (M). eNOS expression and phosphorylation at Ser¹¹⁷⁷ in the border zone were analysed by Western blotting (N). VEGFR2 expression and phosphorylation at Tyr¹¹⁷⁵ in the border zone were analysed by Western blotting (O).*p < 0.05 compared with the sham + Scr-shRNA + vector group, #p < 0.05 compared with the MI + Scr-shRNA + vector group, & p < 0.05 compared with the MI + Malat1-shRNA + vector group. n = 6 in each group.

Fig. S1

Transfection efficiency and expression of target genes. Immunofluorescence staining of His-tagged AAV-shMalat1 (green) and CD31-labelled CMECs (red) and quantitative analysis of the percentage of His-positive CMECs (A). Transfection efficiency of AAV-shMalat1 in mouse CMECs (B). Relative expression of Malat1 in cardiac tissues and CMECs in the border zone 7 days after AAV-shMalat1 injection (C, D). The silencing efficiency of siMalat1 in CMECs under normoxia in vitro (E). Relative expression of Malat1 in CMECs under hypoxia after siMalat1 transfection (F). Transfection efficiency of the miR-26b-5p mimic and miR-26b-5p inhibitor in CMECs under normoxia in vitro (G). Relative expression of miR-26b-5p in CMECs under hypoxia after transfection of the miR-26b-5p mimic and miR-26b-5p inhibitor (H). PCR and Western blot results of the silencing efficiency of Mfn1 in CMECs under normoxia (I, J). PCR and Western blot results of the overexpression efficiency of Mfn1 in CMECs under normoxia (K, L). PCR and Western blot results of Mfn1 expression after knocking out or overexpressing Mfn1 under hypoxia (M, N). Immunofluorescence staining of Flag-tagged AAV-Mfn1 (green) and CD31-labelled CMECs (red) and quantitative analysis of the percentage of Flag-positive CMECs (O). Transfection efficiency of AAV-Mfn1 in mouse CMECs (P, Q). Gene and protein expression of Mfn1 in mouse CMECs (R–S). *p < 0.05 compared with sham, control or NC, #p < 0.05 compared with the adjacent group. n = 5 in each group

Fig. S2

Malat1-mediated mitochondrial homeostasis enhanced cardiac microcirculation resistance after MI via the miR-26b-5p/Mfn1 axis. The Malat1/miR-26b-5p/Mfn1 axis is involved in mitochondrial dynamics in CMECs, which may contribute to restoring mitochondrial dynamics and inhibiting mitochondria-dependent apoptosis in CMECs under hypoxia

Fig. S3

Silencing Malat1 in CMECs aggravated oxidative stress, apoptosis and mitochondrial dysfunction after myocardial infarction (MI). MDA content, total SOD activities, GSH and GSSG levels, and ROS and H₂O₂ generation in the border zone (A). Relative expression of mitochondrial apoptosis-related genes and mitochondrial fusion- and fission-related genes (B). *p < 0.05 compared with the sham group, #p < 0.05 compared with the MI + Scr-shRNA group. n = 6 in each group

Fig. S4

Silencing Malat1 compromised CMEC functions by inhibiting cellular proliferation, migration and promoting oxidative stress Cell proliferation was measured by EdU staining, and the percentage of EdU-positive CMECs (red) was quantified (A). CMEC migration ability was measured by the Transwell assay (B). Images and quantitative analysis of the tube formation assay (C). MDA content, GSH and GSSG levels, and total SOD and Mn-SOD activities of CMECs (D). Relative expression of mitochondrial fusion/fission-related genes (E). Quantification of the expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins (F). Expression of mitochondrial apoptosis-related genes (G). Quantification of the expression of apoptosis-associated proteins (H). *p < 0.05 compared with the control group, #p < 0.05 compared with the hy + siNC group. n = 5 in each group

Fig. S5

miR-26b-5p negatively regulated CMEC proliferation, migration and promoted oxidative stress Cell proliferation was measured by EdU staining, and the percentage of EdU-positive CMECs (red) was quantified (A). CMEC migration ability was measured by the Transwell assay (B). Images and quantitative analysis of the tube formation assay (C). MDA content, GSH and GSSG levels, and total SOD and Mn-SOD activities of CMECs (D). Relative expression of mitochondrial fusion/fission-related genes (E). Quantification of the expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins (F). Expression of mitochondrial apoptosis-related genes (G). Quantification of the expression of apoptosis-associated proteins (H). *p < 0.05 compared with the hy + mimic-NC group, #p < 0.05 compared with the hy + inhibitor-NC group. n = 5 in each group

Fig. S6

Inhibiting miR-26b-5p alleviated Malat1 deletion-aggravated EC dysfunction and oxidative stress under hypoxia. Cell proliferation was measured by EdU staining, and the percentage of EdU-positive CMECs (red) was quantified (A). CMEC migration ability was measured by the Transwell assay (B). Images and quantitative analysis of the tube formation assay (C). MDA content, GSH and GSSG levels, and total SOD and Mn-SOD activities of CMECs (D). Relative expression of mitochondrial fusion/fission-related genes (E). Quantification of the expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins (F). Expression of mitochondrial apoptosis-related genes (G). Quantification of the expression of apoptosis-associated proteins (H). *p < 0.05 compared with the siNC + inhibitor-NC group, #p < 0.05 compared with the hy + siNC + inhibitor-NC group, & p < 0.05 compared with the hy + siMalat1+inhibitor-NC group. n = 5 in each group

Fig. S7

Mfn1 positively regulated CMEC proliferation, migration and inhibited oxidative stress after hypoxic injury Cell proliferation was measured by EdU staining, and the percentage of EdU-positive CMECs (red) was quantified (A). CMEC migration ability was measured by the Transwell assay (B). Images and quantitative analysis of the tube formation assay (C). MDA content, GSH and GSSG levels, and total SOD and Mn-SOD activities of CMECs (D). Relative expression of mitochondrial fusion/fission-related genes (E). Quantification of the expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins (F). Expression of mitochondrial apoptosis-related genes (G). Quantification of the expression of apoptosis-associated proteins (H). *p < 0.05 compared with the control group, #p < 0.05 compared with the hy + siNC group, & p < 0.05 compared with the hy + pcDNA3.1 group. n = 5 in each group

Fig. S8

Mfn1 reversed EC dysfunction and oxidative stress induced by silencing Malat1 in CMECs under hypoxia. Relative gene expression of Mfn1 in CMECs (A). Cell proliferation was measured by EdU staining, and the percentage of EdU-positive CMECs (red) was quantified (B). CMEC migration ability was measured by the Transwell assay (C). Images and quantitative analysis of the tube formation assay (D). MDA content, GSH and GSSG levels, and total SOD and Mn-SOD activities of CMECs (E). Relative expression of mitochondrial fusion/fission-related genes (F). Quantification of the expression of mitochondrial fusion/fission-related proteins and mitophagy-related proteins (G). Expression of mitochondrial apoptosis-related genes (H). Quantification of the expression of apoptosis-associated proteins (I). *p < 0.05 compared with the siNC + pcDNA3.1 group, #p < 0.05 compared with the hy + siNC + pcDNA3.1 group, & p < 0.05 compared with the hy + siMalat1+pcDNA3.1 group. n = 5 in each group

Fig. S9

Overexpressing Mfn1 reversed the aggravation of oxidative stress, apoptosis and mitochondrial dysfunction induced by silencing Malat1 in MI hearts. MDA content, total SOD activities, GSH and GSSG levels, and ROS and H₂O₂ generation in the border zone (A). Relative expression of mitochondrial apoptosis-related genes and mitochondrial fusion/fission-related genes (B). *p < 0.05 compared with the sham + Scr-shRNA + vector group, #p < 0.05 compared with the MI + Scr-shRNA + vector group, & p < 0.05 compared with the MI + Malat1-shRNA + vector group. n = 6 in each group