Endothelial MICU1 alleviates diabetic cardiomyopathy by attenuating nitrative stress-mediated cardiac microvascular injury

Abstract

Background: Myocardial microvascular injury is the key event in early diabetic heart disease. The injury of myocardial microvascular endothelial cells (CMECs) is the main cause and trigger of myocardial microvascular disease. Mitochondrial calcium homeostasis plays an important role in maintaining the normal function, survival and death of endothelial cells. Considering that mitochondrial calcium uptake 1 (MICU1) is a key molecule in mitochondrial calcium regulation, this study aimed to investigate the role of MICU1 in CMECs and explore its underlying mechanisms.

Methods: To examine the role of endothelial MICU1 in diabetic cardiomyopathy (DCM), we used endothelial-specific MICU1^ecKO mice to establish a diabetic mouse model and evaluate the cardiac function. In addition, MICU1 overexpression was conducted by injecting adeno-associated virus 9 carrying MICU1 (AAV9-MICU1). Transcriptome sequencing technology was used to explore underlying molecular mechanisms.

Results: Here, we found that MICU1 expression is decreased in CMECs of diabetic mice. Moreover, we demonstrated that endothelial cell MICU1 knockout exacerbated the levels of cardiac hypertrophy and interstitial myocardial fibrosis and led to a further reduction in left ventricular function in diabetic mice. Notably, we found that AAV9-MICU1 specifically upregulated the expression of MICU1 in CMECs of diabetic mice, which inhibited nitrification stress, inflammatory reaction, and apoptosis of the CMECs, ameliorated myocardial hypertrophy and fibrosis, and promoted cardiac function. Further mechanistic analysis suggested that MICU1 deficiency result in excessive mitochondrial calcium uptake and homeostasis imbalance which caused nitrification stress-induced endothelial damage and inflammation that disrupted myocardial microvascular endothelial barrier function and ultimately promoted DCM progression.

Conclusions: Our findings demonstrate that MICU1 expression was downregulated in the CMECs of diabetic mice. Overexpression of endothelial MICU1 reduced nitrification stress induced apoptosis and inflammation by inhibiting mitochondrial calcium uptake, which improved myocardial microvascular function and inhibited DCM progression. Our findings suggest that endothelial MICU1 is a molecular intervention target for the potential treatment of DCM.

Keywords: Cardiac microvascular endothelial cells (CMECs); Diabetic cardiomyopathy; Endothelial permeability; Inflammatory response; MICU1; Nitrative stress.

Fig. 1

MICU1 expression is decreased in CMECs of diabetic mice. (A) Cell type-specific MICU1 mRNA expression in different organ tissues and hearts of adults. The MICU1 mRNA levels in different organ tissues and hearts of adults were determined by mining single-cell RNAseq data deposited in the Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000107745-MICU1/single+cell+type/heart+muscle). Different cardiac cell types are colored according to clusters and expressed by uniform manifold approximation and projection. (B) qRT-PCR analysis of MICU1 mRNA levels in the CMECs of diabetic db/db mice and wild-type littermate mice (n = 6, 6-, 12- and 18-week-old mice per group). (C) Western blotting analysis of the protein expression levels of MICU1 and β-actin in the CMECs of diabetic db/db mice and wild-type littermate mice (n = 6, 6-, 12- and 18-week-old per group). (D) qRT-PCR analysis of MICU1 mRNA levels in the CMECs treated with HGHF (n = 3 per group). (E) Western blotting analysis of MICU1 expression in CMECs treated with HGHF (n = 3 per group), **p < 0.01

Fig. 2

Endothelial-specific knockout of MICU1 aggravates diabetes-induced cardiac dysfunction in mice. (A) Western blotting for protein levels of MICU1 in CMECs of WT and MICU1^ecKO mice (aged 18 weeks). (B) The heart weight of different groups. (C) The ratios of heart weight to tibia length (HW/TL) for different groups. (D) Representative echocardiographic images of the LV in different 18-week-old mice. (E, F, G and H) Echocardiographic assessment of heart rate (E), Echocardiographic assessment of LV fractional shortening (LVFS; F), LV end diastolic diameter (LVIDd; G) and LV diastolic posterior wall thickness (LVPWd; H) in different mice (n = 6, 18 weeks old per group). (I) Cardiomyocyte cross-sectional area was analyzed using hematoxylin and eosin staining and wheat germ agglutinin staining in hearts from different mice (n = 6, 18 weeks old per group); scale bar = 20 μm. (J) Comparative quantitative analysis of cardiomyocyte CSA. (K) Representative images and comparative quantitative analysis of Masson trichrome staining of hearts from different mice (n = 6, 18 weeks old per group); scale bar = 20 μm. *p < 0.05, **p < 0.01

Fig. 3

Endothelial-specific knockout of MICU1 exacerbates cardiac microvascular injury in diabetic mice. (A) A TUNEL assay was performed to assess CMECs apoptosis, scale bar = 20 μm. (B) Percentages of TUNEL-positive nuclei over total number of nuclei (n = 6 per group). (C) Percentages of TUNEL positive CMECs over total number of CD31-positive nuclei (n = 6 per group). (D) Cardiac microvascular perfusion is indicated by the ratio of lectin-positive microvessels (green) to CD31-positive microvessels (red) (n = 6 per group); scale bar = 20 μm. (E) Quantification of Lectin positive microvessels is shown (n = 6 per group). (F) Immunofluorescence staining of VE-cadherin was performed to observe the changes in cardiac microvessel endothelial integrity and barrier function. scale bar = 50 μm. (G) Comparative quantitative analysis of fluorescence intensity of VE-cadherin (n = 6 per group). (H) Representative fluorescence images for intercellular adhesion molecule (ICAM)-1. scale bar = 50 μm. (I) Comparative quantitative analysis of fluorescence intensity of ICAM-1 (n = 6 per group). **p < 0.01

Fig. 4

MICU1 deficiency aggravates the apoptosis, inflammatory response and nitrification stress of CMECs treated with HGHF. (A) KEGG pathway enrichment analysis based on these 496 overlapping DEGs. The top 10 enriched pathways ranked by the number of DEGs in the pathways are shown. (B) Heatmap showing the expression levels of DEGs belonging to apoptosis and the NF-κB signaling pathway in RNA-Seq. The color scale depicts the range of log2-fold changes in gene expression. (C) Six selected DEGs in this pathway were validated using qRT-PCR (n = 6 per group). (D) Flow cytometry of apoptosis using annexin V and propidium iodide (PI) staining in CMECs treated as indicated (n = 6 per group). (E) Changes in the expression of NLRP3, NF-κB, Caspase 1, IL-1β,TNF-α and Cleaved caspase 1as assessed by Western blotting (n = 4 per group). (F) Western blotting of eNOS, p-eNOS, iNOS and β-actin in CMECs (n = 4 per group). (G) Analysis of 3-NT Elisa results of CMECs (n = 6 per group). (H) FITC-dextran clearance was measured to assess changes in endothelial permeability (n = 6 per group). *p < 0.05, **p < 0.01

Fig. 5

MICU1 knockout aggravates endothelial dysfunction by promoting nitrification stress mediated by mitochondrial calcium uptake in endothelial cells. (A) Quantitative determination of Ca²⁺ content in mitochondria of CMECs from the indicated groups (n = 6 per group). (B) Mitochondrial membrane potentials were analyzed using JC-1 staining in CMECs treated as indicated. Flow cytometry results are presented (n = 6 per group). (C). Representative Western blotting of eNOS, p-eNOS, iNOS and β-actin in CMECs (n = 4 per group). (D) Analysis of 3-NT Elisa results of CMECs (n = 6 per group). (E) Flow cytometry of apoptosis using annexin V and PI staining in CMECs treated as indicated (n = 6 per group). (F) Changes in the expression of NLRP3, NF-κB, Caspase 1, IL-1β, TNF-α and Cleaved caspase 1 assessed using Western blotting (n = 4 per group). (G) FITC-dextran clearance was measured to assess changes in endothelial permeability (n = 6 per group). **p < 0.01

Fig. 6

Upregulation of MICU1 expression alleviates HGHF-induced endothelial cell injury by inhibiting nitrification stress. (A) Representative Western blotting of eNOS, p-eNOS, iNOS and β-actin in CMECs (n = 4 per group). (B) Mitochondrial membrane potentials were analyzed using JC-1 staining in CMECs treated as indicated. Flow cytometry results are presented (n = 6 per group). (C) Analysis of 3-NT Elisa results of CMECs (n = 6 per group). (D) Flow cytometry of apoptosis using annexin V and PI staining in CMECs treated as indicated (n = 6 per group). (E) Changes in the expression of NLRP3, NF-κB, Caspase 1, IL-1β, TNF-α and Cleaved caspase 1 as assessed by Western blotting (n = 4 per group). (F) FITC-dextran clearance was measured to assess changes in endothelial permeability (n = 6 per group). *p < 0.05, **p < 0.01

Fig. 7

Cardiac endothelial-specific overexpression of MICU1 alleviates diabetes-induced cardiac microvascular injury in diabetic mice. (A) A TUNEL assay was performed to assess CMECs apoptosis, scale bar = 20 μm. (B) Percentages of TUNEL-positive nuclei over total number of nuclei (n = 6 per group). (C) Percentages of TUNEL positive CMECs over total number of CD31-positive nuclei (n = 6 per group). (D) Cardiac microvascular perfusion is indicated by lectin-positive microvessels (green) to CD31-positive microvessels (red), scale bar = 20 μm. (E) Quantification of Lectin positive microvessels is shown (n = 6 per group). (F) Immunofluorescence staining of VE-cadherin was performed to observe changes in cardiac microvessel endothelial integrity and barrier function, scale bar = 50 μm. (G) Comparative quantitative analysis of fluorescence intensity of VE-cadherin (n = 6 per group). (H) Representative fluorescence images of intercellular adhesion molecule ICAM-1, scale bar = 50 μm. (I) Comparative quantitative analysis of fluorescence intensity of ICAM-1 (n = 6 per group). **p < 0.01

Fig. 8

Cardiac endothelial-specific overexpression of MICU1 alleviates diabetes-induced cardiac dysfunction in diabetic mice. (A) The heart weight of different groups. (B) The ratios of heart weight to tibia length (HW/TL) for different groups. (C) Representative echocardiographic images of the LV in different mice (aged 18 weeks). (D, E, F and G) Echocardiographic assessment of heart rate (D), Echocardiographic assessment of LV fractional shortening (LVFS; E), LV end diastolic diameter (LVIDd; F) and LV diastolic posterior wall thickness (LVPWd; G) in different mice (n = 6, 18-week-old per group). (H) Cardiomyocyte cross-sectional area was analyzed using hematoxylin and eosin staining and wheat germ agglutinin staining in hearts from different mice (n = 6, 18-week-old per group); scale bar = 20 μm. (I) Comparative quantitative analysis of cardiomyocyte CSA. (J) Representative images and comparative quantitative analysis of Masson trichrome staining of hearts from different mice (n = 6 per group); scale bar = 20 μm. *p < 0.05, **p < 0.01