Hypoadiponectinemia-induced upregulation of microRNA449b downregulating Nrf-1 aggravates cardiac ischemia-reperfusion injury in diabetic mice

Abstract

Diabetes enhances myocardial ischemic/reperfusion (MI/R) injury via an incompletely understood mechanism. Adiponectin (APN) is a cardioprotective adipokine suppressed by diabetes. However, how hypoadiponectinemia exacerbates cardiac injury remains incompletely understood. Dysregulation of miRNAs plays a significant role in disease development. However, whether hypoadiponectinemia alters cardiac miRNA profile, contributing to diabetic heart injury, remains unclear. Methods and Results: Wild-type (WT) and APN knockout (APN-KO) mice were subjected to MI/R. A cardiac microRNA profile was determined. Among 23 miRNAs increased in APN-KO mice following MI/R, miR-449b was most significantly upregulated (3.98-fold over WT mice). Administrating miR-449b mimic increased apoptosis, enlarged infarct size, and impaired cardiac function in WT mice. In contrast, anti-miR-449b decreased apoptosis, reduced infarct size, and improved cardiac function in APN-KO mice. Bioinformatic analysis predicted 73 miR-449b targeting genes, and GO analysis revealed oxidative stress as the top pathway regulated by these genes. Venn analysis followed by luciferase assay identified Nrf-1 and Ucp3 as the two most important miR-449b targets. In vivo administration of anti-miR-449b in APN-KO mice attenuated MI/R-stimulated superoxide overproduction. In vitro experiments demonstrated that high glucose/high lipid and simulated ischemia/reperfusion upregulated miR-449b and inhibited Nrf-1 and Ucp3 expression. These pathological effects were attenuated by anti-miR-449b or Nrf-1 overexpression. In a final attempt to validate our finding in a clinically relevant model, high-fat diet (HFD)-induced diabetic mice were subjected to MI/R and treated with anti-miR-449b or APN. Diabetes significantly increased miR-449b expression and downregulated Nrf-1 and Ucp3 expression. Administration of anti-miR-449b or APN preserved cardiac Nrf-1 expression, reduced cardiac oxidative stress, decreased apoptosis and infarct size, and improved cardiac function. Conclusion: We demonstrated for the first time that hypoadiponectinemia upregulates miR-449b and suppresses Nrf-1/Ucp3 expression, promoting oxidative stress and exacerbating MI/R injury in this population. Dysregulated APN/miR-449b/oxidative stress pathway is a potential therapeutic target against diabetic MI/R injury.

Keywords: Adiponectin; Antioxidant; Diabetes; Ischemia/reperfusion; microRNA.

Figure 1.. Myocardial microRNA (miRNAs) expression profiles in animals subjected to myocardial ischemia-reperfusion.

A. Hierarchal clustering heat map showing all differentially expressed microRNAs in WT and APN-KO mice hearts. B. Venn diagram showing 23 miRNAs was the same over-crossed from WT and APN-KO comparation (Up panel). miRNA expression fold change was calculated between WT and APN-KO Sham over MI/R (Down panel). C. Digital PCR validation using miRNA-specific primer assays for miR-449b in cardiac tissues. **p<0.01 versus MI/R, respectively; ##p < 0.01 versus WT-MI/R group. Statistical significance was evaluated by a two-way ANOVA. Post hoc pairwise tests for indicated group pairs were performed after Tukey correction. MI/R, myocardial ischemia/reperfusion.

Figure 2.. Myocardial infarct size and apoptosis detection after myocardial ischemia-reperfusion in WT, APN-KO, and HFD mice.

A. Left ventricular ejection fraction (LVEF%), and fractional shorting (FS%) were determined by echocardiography in WT and APN-KO mice subjected to MI/R with anti-miR-449b or miR-449b mimic (miR-449b OE) administration. **p < 0.01 vs. vehicle+MI/R group in WT mice, .##p < 0.01 vs. vehicle+MI/R group in APN-KO mice. n=10/group. B. CK-MB measurement of WT and APN-KO mice subjected to MI/R with miR-449b OE or anti-miR-449b administration. *p < 0.05 versus WT-MI/R group; **p < 0.01 versus APN-KO-MI/R group. For A and B, statistical significance was evaluated by one-way ANOVA with Tukey test for post hoc pairwise tests. C. Percentage of infarct size expressed as a percentage of the area risk (AAR). **p < 0.01 versus WT-MI/R group; ##p < 0.01 versus APN-KO-MI/R group. Statistical significance was evaluated by two-way ANOVA with Tukey test for post hoc pairwise tests. D. DNA ladder induced by MI/R on WT and APN-KO mice with miR-449b mimic or anti-miR-449b administration. E. Percentage of apoptosis expressed as a percentage of cell death. **p < 0.01 versus WT-MI/R group; ##p < 0.01 versus APN-KO-MI/R group. n=6–10/group, 3 independent experiments. Statistical significance was evaluated by two-way ANOVA with Tukey for post hoc pairwise tests. F. Cleaved Caspase 3 evaluated by Western blot in WT and APN-KO mice with miR-449b mimic or anti-miR-449b administration. **p < 0.01 versus WT-MI/R group; ##p < 0.01 versus APN-KO-MI/R group. n=6/group, 3 independent experiments. Statistical significance was evaluated by two-way ANOVA with Tukey for post hoc pairwise tests. OE, overexpression; MI/R, myocardial ischemia/reperfusion. APN-KO, Adiponectin Knockout.

Figure 3.. Oxidative stress evaluation in animal model.

A. Venn diagram shows that 72 targets were the same over-crossed from predicted targets for miR-449b. B. Go analysis showing signaling pathways regulated by miR-449b targeting genes. C. DHE assay (up panel) and Lucigenin assay detection (down panel) for superoxide generation in WT, APN-KO, and HFD mice subjected to MI/R with anti-miR-449b administration. **p < 0.01 versus MI/R group, respectively; #p < 0.05 & ## p < 0.01 versus WT-MI/R group. D. Caspase 3 activity evaluations in generation in WT, APN-KO, and HFD mice subjected to MI/R with anti-miR-449b administration. **p < 0.01 versus MI/R group, respectively; #p < 0.05 & ## p < 0.01 versus WT-MI/R group. For C and D, statistical significance was evaluated by two-way ANOVA. Post hoc pairwise tests for indicated group pairs were performed after Tukey correction. E. Superoxide generation in cultured NRCMs subjected to SI/R with anti-miR-449b administration. *p < 0.05 & **p < 0.01 versus control group; ## p < 0.01 versus SI/R group. F. Apoptosis evaluation by TUNEL stain assay (green) in HGHL-induced diabetic NRCMs with anti-miR-449b administration. DAPI (blue). **p < 0.01 versus control group; ## p < 0.01 versus SI/R group. G. Cleaved Caspase 3 evaluated by Western blot in HGHL-induced diabetic NRCMs with anti-miR-449b administration. **p < 0.01 versus control group; ## p < 0.01 versus SI/R group. n=6–11/group, 3 independent experiments. For E-G, statistical significance was evaluated by one-way ANOVA. Tukey tests were used to correct for multiple comparisons. I/R, myocardial ischemia/reperfusion; SI/R, Simulated ischemia/reperfusion; HGHL, High glucose, and high lipids; KD, Knockdown.

Figure 4.. Luciferase activity assay for 3-UTR in NFE2L1 (nuclear factor erythroid 2-related factor 1, Nrf-1).

A. Luciferase assay showing co-transfected with luciferase constructs harboring 3’-UTR sequences from either wild-type (WT) or mutated (Mut) target Nrf-1 with anti-miR-449b or miR-449b mimic in NRCMs. **p < 0.01 versus Nrf1-3’UTR¹-WT; ##p < 0.01 versus Nrf1-3’UTR²-WT. B. Luciferase assay showing co-transfected with luciferase constructs harboring 3’-UTR sequences from either wild-type (WT) or mutated (Mut) target Ucp3 with anti-miR-449b or miR-449b mimic in NRCMs. **p < 0.01 versus Ucp3-3’UTR¹-WT; ##p < 0.01 versus Ucp3-3’UTR²-WT. n=6–9 per group, 3 independent experiments. Statistical significance was evaluated by two-way ANOVA. Post hoc pairwise tests for indicated group pairs were performed after Tukey correction.

Figure 5.. Nrf-1 was regulated by anti-miR-449b and miRNA mimic administration.

A. Real-time PCR analysis of miR-449b in NRCMs **p < 0.01 versus HG/HL group; &&p < 0.01 versus HG/HL+SI/R group; ##p < 0.01 versus HG/HL+SI/R+miR-449b KD group. B. Nrf-1 and Ucp3 expression levels were detected in NRCMs after simulated ischemia-reperfusion (SI/R) with anti-miR-449b or miR-449b mimic administration. **p < 0.01 versus HG/HL group; &&p < 0.01 versus HG/HL+SI/R group; ##p < 0.01 versus HG/HL+SI/R+miR-449b KD group. For A and B, statistical significance was evaluated by one-way ANOVA. Tukey tests were used to correct for multiple comparisons. C. DHE assay (up panel) and Lucigenin assay detection (down panel) for superoxide generation in NRCMs transfected with harboring Nrf-1 plasmid followed by SI/R challenges. **p < 0.01 versus vehicle group, respectively, ##p < 0.01 versus control+SI/R group. n=6/group, 3 independent experiments. Statistical significance was evaluated by two-way ANOVA. Post hoc pairwise tests for indicated group pairs were performed after Tukey correction. Nrf-1, Nuclear factor erythroid 2-related factor 1. HGHL, high glucose and high lipid.

Figure 6.. APN administration upregulated Nrf1 and Ucp3’s level preventing HFD-induced cardiac MI/R injury.

A. Nrf1 level in APN administrated WT and HFD mice subjected to MI/R. Representative Western blot, upper panel; Bar graph quantification, down panel. **p < 0.01 versus WT+MI/R group, ##p < 0.01 versus HFD+MI/R group, n = 6/group. B. Ucp3 level in APN administrated HFD mice subjected to MI/R. Representative Western blot, upper panel; Bar graph quantification, down panel. **p < 0.01 versus WT+MI/R group, ##p < 0.01 versus HFD+MI/R group, n = 6/group. C. Determination of Cleaved Caspase 3 level in HFD-induced diabetic animals with APN administration followed by MI/R. **p < 0.01 versus WT+MI/R group, ##p < 0.01 versus HFD+MI/R group, n = 6/group. D. DHE assay (up panel) and Lucigenin assay detection (down panel) for superoxide generation in HFD-induced diabetic animals with APN administration followed by MI/R challenges. **p < 0.01 versus WT+MI/R group, ##p < 0.01 versus HFD+MI/R group, n = 11/group. For A-D, statistical significance was evaluated by one-way ANOVA. Tukey tests were used to correct for multiple comparisons. HFD, high-fat diet; MI/R, myocardial ischemia/reperfusion; APN, Adiponectin.

Figure 7.. miR-449b regulation altered cardiomyocyte apoptosis in HFD-induced diabetic animals.

A. Anti-miR-449b administration reduced infarct size in HFD animals followed by MI/R. B. Quantification of infarct size of AAR shown in A. **p < 0.01 versus scramble+MI/R group, n = 10/group. C. CK-MB determination in anti-miR-449b administrated HFD animals followed by MI/R. **p < 0.01 versus scramble+MI/R group, n = 6/group. D. Cardiac function evaluations were determined by echocardiography in anti-miR-449b administrated HFD animals followed with MI/R. **p < 0.01 versus scramble+MI/R group, n = 10/group. For B-D, statistical significance was evaluated by one-way ANOVA. Tukey tests were used to correct for multiple comparisons. E. Correlation between IHD and miR-449b analyzed via multivariate logistics regression analysis. F. ROC curve illustrating miR-449b diagnostic ability for IHD. IHD, ischemic heart disease. ROC, Receiver operating characteristic.