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. 2024 May 9;23(1):164.
doi: 10.1186/s12933-024-02254-7.

MAP4K4 exacerbates cardiac microvascular injury in diabetes by facilitating S-nitrosylation modification of Drp1

Yuqiong Chen #  1 , Su Li #  2 , Bo Guan #  3 , Xiaopei Yan  4 , Chao Huang  5 , Yingqiang Du  6 , Fan Yang  7   8 , Nannan Zhang  6 , Yafei Li  6 , Jian Lu  9 , Jiankang Wang  6 , Jun Zhang  6 , Zhangwei Chen  10 , Chao Chen  11 , Xiangqing Kong  12   13
Affiliations

MAP4K4 exacerbates cardiac microvascular injury in diabetes by facilitating S-nitrosylation modification of Drp1

Yuqiong Chen et al. Cardiovasc Diabetol. .

Abstract

Dynamin-related protein 1 (Drp1) is a crucial regulator of mitochondrial dynamics, the overactivation of which can lead to cardiovascular disease. Multiple distinct posttranscriptional modifications of Drp1 have been reported, among which S-nitrosylation was recently introduced. However, the detailed regulatory mechanism of S-nitrosylation of Drp1 (SNO-Drp1) in cardiac microvascular dysfunction in diabetes remains elusive. The present study revealed that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was consistently upregulated in diabetic cardiomyopathy (DCM) and promoted SNO-Drp1 in cardiac microvascular endothelial cells (CMECs), which in turn led to mitochondrial dysfunction and cardiac microvascular disorder. Further studies confirmed that MAP4K4 promoted SNO-Drp1 at human C644 (mouse C650) by inhibiting glutathione peroxidase 4 (GPX4) expression, through which MAP4K4 stimulated endothelial ferroptosis in diabetes. In contrast, inhibition of MAP4K4 via DMX-5804 significantly reduced endothelial ferroptosis, alleviated cardiac microvascular dysfunction and improved cardiac dysfunction in db/db mice by reducing SNO-Drp1. In parallel, the C650A mutation in mice abolished SNO-Drp1 and the role of Drp1 in promoting cardiac microvascular disorder and cardiac dysfunction. In conclusion, our findings demonstrate that MAP4K4 plays an important role in endothelial dysfunction in DCM and reveal that SNO-Drp1 and ferroptosis activation may act as downstream targets, representing potential therapeutic targets for DCM.

Keywords: Cardiac microvascular injury; Diabetic cardiomyopathy; Drp1; MAP4K4; S-nitrosylation.

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Conflict of interest statement

All the authors have approved the final article and declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Endothelial-specific knockdown of MAP4K4 ameliorated myocardial microcirculation injury and prevented ventricular remodeling after long-term diabetes. Four-week-old male db/db mice and age-matched db/m mice were transfected with AAV9-shMAP4K4 or AAV9-shNC for 24 weeks. A–B Protein expression and quantitative analysis of MAP4K4 in myocardial tissue and primary CMECs from db/m and db/db mice. C The transfection efficiency of AAV9-shMAP4K4 was measured by western blotting in primary CMECs. D Cardiac microvascular density was assessed by immunofluorescence staining of CD31, and microvascular blood flow was assessed by a lectin-FITC perfusion assay. Scale bar = 25 μm. E Quantitative analysis of NO content in myocardial tissues. F Total and phosphorylated eNOS protein levels. G Protein expression of VCAM-1 and ICAM-1. H Quantitative analysis of EB leakage. I–J Statistical analysis of the LVEF, LVFS, and LVEDD. K Quantitative analysis of serum BNP levels. L Cardiac fibrosis and cardiomyocyte cross-sectional area were detected by Masson trichrome staining and WGA staining, respectively. Scale bar = 70 μm. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four to six biological replicates were performed, and the results are indicated in scatter plots
Fig. 2
Fig. 2
MAP4K4 knockdown ameliorated cardiac microvascular endothelial dysfunction under HG/FFA conditions. Cultured CMECs were transfected with LV-shMAP4K4 for 48 h and exposed to HG/FFA conditions. A–B Representative western blot and related statistical analysis of MAP4K4 protein expression in CMECs under HG/FFA conditions. C–F The transfection efficiency of LV-shMAP4K4 under control and HG/FFA conditions was measured via western blotting. G Relative cell viability was determined by a CCK-8 assay. H Statistical analysis of LDH release. I Representative images of the transwell assay and statistical analysis of migrated cells. Scale bars: 100 μm. J Total and phosphorylated VEGFR2 protein expression and quantitative analysis of VEGFR2. K Statistical analysis of NO release. L Total and phosphorylated protein expression and quantitative analysis of eNOS. M Statistical analysis of FITC-dextran permeability in the indicated groups. N TEER was analyzed to assess the barrier function of endothelial cells. O Protein expression and quantitative analysis of VCAM-1 and ICAM-1. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four to six biological replicates were performed, and the results are indicated in scatter plots
Fig. 3
Fig. 3
MAP4K4 knockdown improved mitochondrial morphology and function. HCMECs were transfected with LV-shMAP4K4 for 48 h and exposed to HG/FFA conditions for 72 h. A Mitochondrial morphology (red) was visualized by MitoTracker staining, and mitochondrial length was quantified. Scale bar = 10 μm. B Mitochondrial membrane potential was detected by TMRM staining. C Mitochondrial respiratory activity was assessed using a Seahorse analyzer. The baseline OCR, ATP-linked OCR, maximal OCR and spare respiratory OCR were quantitatively analyzed. D Intracellular ROS (green) and mitoROS (red) were photographed and quantitatively analyzed. Scale bar = 25 μm. E Quantitative analysis of the CoQ10 concentration in CMECs. F Quantification of the mtDNA copy number in each group. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four to six biological replicates were performed, and the results are indicated in scatter plots
Fig. 4
Fig. 4
MAP4K4 silencing inhibited the mitochondrial translocation of Drp1 and downregulated SNO-Drp1. HCMECs were exposed to HG/FFA conditions for 72 h, with or without transfection with LV-shMAP4K4. A Relative mRNA expression of mitochondrial dynamics-related genes. B Representative immunoblotting images showing the protein expression, phosphorylation, and S-nitrosylation of Drp1. C Representative immunoblotting images of Bnip3, Mief1, Senp5, and Mfn1. D Mitochondrial and cytoplasmic expression of MAP4K4 and Drp1, Drp1 phosphorylation at Ser616, Drp1 phosphorylation at Ser637, and SNO-Drp1 were assessed by western blotting and statistically analyzed under HG/HFFA conditions. E Mitochondrial and cytoplasmic expression of Drp1, Drp1 phosphorylation at Ser616, Drp1 phosphorylation at Ser637, and SNO-Drp1 were assessed by western blotting and statistically analyzed after silencing MAP4K4. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four biological replicates were performed, and the results are indicated in scatter plots
Fig. 5
Fig. 5
MAP4K4 modulated the SNO-Drp1 through GPX4. A–B HCMECs were transfected with LV-shMAP4K4 for 48 h and exposed to HG/FFA conditions for 72 h. Representative western blot and statistical analysis of S-nitrosylation modification-related proteins. C HCMECs were cotransfected with LV-shMAP4K4 and LV-shGPX4 and subjected to HG/FFA injury. SNO-Drp1 was assessed by western blotting. D HCMECs were transfected with LV-GPX4 or treated with L-NAME and then subjected to HG/FFA injury. SNO-Drp1 was assessed by western blotting. E HCMECs were treated with LV-shMAP4K4 or L-NAME and subjected to HG/FFA injury. SNO-Drp1 expression was then assessed by western blotting. F The mitochondrial and cytoplasmic expression of Drp1 and the S-nitrosylation and phosphorylation of Drp1 were assessed by western blotting. G Quantitative analysis of GSH, GSSG, H2O2 and ROS levels in the indicated groups. H Quantitative analysis of the MDA, LPO, and iron content in the indicated groups. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four biological replicates were performed, and the results are indicated in scatter plots
Fig. 6
Fig. 6
The C644A mutation inhibited SNO-Drp1 and reversed the effects of MAP4K4 on CMEC dysfunction under HG/FFA conditions. A Relative SNO-Drp1 levels were assessed in the Drp1-WT cell line, the Drp1-C505A knock-in cell line and the Drp1-C644A knock-in cell line, with or without HG/FFA injury. B MAP4K4 was overexpressed in Drp1-WT and Drp1-C644A knock-in cells treated with HG/FFA. The protein expression, S-nitrosylation and phosphorylation of Drp1 were assessed by western blotting. C Quantitative analysis of GSH, GSSG, and ROS levels in the indicated groups. D Quantitative analysis of the MDA, LPO, and ferrous iron content in the indicated groups. E–F Relative cell viability was determined by the CCK-8 assay, and cytotoxicity was measured by the LDH release assay. G Representative images of the transwell assay. Scale bars: 100 μm. H The expression and phosphorylation of VEGFR2 were assessed by western blotting. I Statistical analysis of FITC-dextran permeability. J TEER was used to assess the barrier function of endothelial cells. K Protein expression and quantitative analysis of p-eNOS, eNOS, VCAM-1 and ICAM-1. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four to six biological replicates were performed, and the results are indicated in scatter plots
Fig. 7
Fig. 7
C650 mutation inhibited Drp1 SNO, attenuated cardiac microcirculatory injury and ameliorated ventricular remodeling in db/db mice. Four-week-old male db/db mice and age-matched db/m mice were transfected with AAV9-Drp1-WT or AAV9-Drp1-C650A for 24 weeks. A Cardiac microvascular density was detected by immunofluorescence staining of CD31, and microvascular blood flow was assessed by a lectin-FITC perfusion assay. Scale bar = 25 μm. B Quantitative analysis of NO content in myocardial tissues. C Total and phosphorylated protein expression of eNOS and VEGFR2. D Total and phosphorylated VE-cadherin protein levels. E Quantitative analysis of EB leakage in each group. F Statistical analysis of the LVEF, LVFS, LVEDD and E/A ratio in the indicated groups. G Quantitative analysis of serum BNP levels. H Cardiac fibrosis and the cardiomyocyte cross-sectional area were detected by Masson trichrome staining and WGA staining, respectively. Scale bar = 70 μm. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four to six biological replicates were performed, and the results are indicated in scatter plots
Fig. 8
Fig. 8
MAP4K4 contributed to myocardial ferroptosis by facilitating Drp1 S-nitrosylation and phosphorylation-induced activation in vivo. Four-week-old male db/db mice received 3 mg/kg DMX-5804 orally three times per week for 24 weeks. A-C Total, mitochondrial, and cytoplasmic levels of MAP4K4, Drp1, Drp1 phosphorylated at Ser616, Drp1 phosphorylated at Ser637, and S-nitrosylated Drp1 were assessed by western blotting. D Cardiac microvascular density was detected by immunofluorescence staining of CD31, and microvascular blood flow was assessed by a lectin-FITC perfusion assay. Scale bar = 25 mm. E Total and phosphorylated protein expression of eNOS and VEGFR2. F Total and phosphorylated VE-cadherin protein expression and the protein expression of VCAM-1 and ICAM-1. G Cardiac fibrosis and cardiomyocyte cross-sectional area were detected by Masson trichrome staining and WGA staining, respectively. Scale bar = 70 mm. H Statistical analysis of the LVEF. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Four to six biological replicates were performed, and the results are indicated in scatter plots
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