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Correcting mitochondrial loss mitigates NOTCH1-related aortopathy in mice

Nature Cardiovascular Research volume 4, pages 235–247 (2025)Cite this article

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Abstract

Loss-of-function mutations in NOTCH1 were previously linked to thoracic aortopathy, a condition for which non-surgical treatment options are limited. Based on clinical proteome analysis, we hypothesized that mitochondrial fusion and biogenesis in aortic smooth muscle cells (SMCs) are crucial for regulating the progression of NOTCH1-related aortopathy. Here we demonstrate that SMC-specific Notch1 knockout mice develop aortic pathology, including stiffening, dilation and focal dissection. These changes are accompanied by decreased expression of MFN1/2 and TFAM, mirroring findings in human patients. SMC-specific deletion of Mfn1 and/or Mfn2 genes recapitulates the aortopathy seen in Notch1-deficient mice. Prophylactic or therapeutic approaches aimed at increasing mitochondrial DNA copy number, either through AAV-mediated overexpression of Mfn1/2 or oral treatment with mitofusion activators teriflunomide or leflunomide, help mitigate or slow the progression of aortopathy in SMC-Notch1−/− mice. Our findings provide a molecular framework for exploring pharmacological interventions to restore mitochondrial function in NOTCH1-related aortopathy.

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Fig. 1: Proteomic analysis of human ascending aorta.
Fig. 2: Ablation of Notch1 or Mfn1 or Mfn2 or Mfn1/2 in SMCs induces aortic pathology in mice.
Fig. 3: Early intervention with SMC-specific AAV9-mediated Mfn1/2 delivery mitigates the progression of aortic dilation and stiffening in SMC-Notch1−/− mice.
Fig. 4: Early interventions with teriflunomide or leflunomide mitigate the progression of aortic dilation and stiffening in SMC-Notch1−/− mice.
Fig. 5: Alterations in aortic protein profile resulting from mitofusin elevation and the mechanistic explanation of pharmacotherapy management.

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Data availability

The proteome dataset can be obtained from the Proteomics Identification Database (PRIDE) (PXD044019). Source data are provided with this paper.

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Acknowledgements

We thank the staff of the Department of Laboratory Animal Science of Fudan University, Shanghai Medical College, for their assistance in animal experiments. We thank the staff of the mass spectrometry technology facility of Fudan University, Shanghai Medical College, for their assistance in proteomics. We thank the Core Facility of Shanghai Medical College, Fudan University. This work was supported by National Natural Science Foundation of China 82370472, 51927805, 82070482 and 82200525; the Innovation Program of Shanghai Municipal Education Commission (2023ZKZD07); the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01); and the Science and Technology Commission of Shanghai Municipality (17JC1400200). ChatGPT 3.5 was used to correct grammatical and spelling errors.

Author information

Author notes

  1. These authors contributed equally: Yuyi Tang, Jingjing Zhang, Yixuan Fang, Kai Zhu.

Authors and Affiliations

  1. Shanghai Fifth People’s Hospital and Institutes of Biomedical Sciences Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China

    Yuyi Tang, Jingjing Zhang, Yixuan Fang, Jingqiao Zhu, Zhuxin Xie, Shan Zhang, Guoquan Yan, Shaowen Liu, Xin Liu, Wenjing Han, Yue Xin, Chenxi Yang, Peiyun Zhou, Chenxi He, Fei Lan, Dan Ye, Fa-Xing Yu, Yanhui Xu & Weijia Zhang

  2. State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai, China

    Jingjing Zhang & Weijia Zhang

  3. Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai, China

    Jingjing Zhang, Ce Huang & Weijia Zhang

  4. Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, China

    Kai Zhu, Wenrui Ma, Mieradilijiang Abudupataer, Hao Lai & Chunsheng Wang

  5. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China

    Yang Liu

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Contributions

Conceptualization: W.Z. Methodology: Y.L., G.Y., F.-X.Y., D.Y. and Y.H.X. Investigation: Y.T., J.J.Z., Y.F., C.H., S.Z., S.L., Z.X., W.M., M.A., H.L., K.Z., C.W., P.Z., C.H., J.Q.Z., X.L., W.H., Y.X. and C.Y. Clinical demographics: K.Z. and W.M. Clinical specimen collection: K.Z. and M.A. Histology and ultrasound experiments: Y.T. Proteomics experiments: J.J.Z. Western blot experiments: J.J.Z. and S.Z. Visualization: Y.T. and J.J.Z. Funding acquisition: W.Z. and K.Z. Project administration: Y.T. and W.Z. Supervision: W.Z. Writing—original draft: W.Z., Y.T., J.J.Z., Y.F. and C.H. Writing—review and editing: F.L., D.Y., Y.X., F.-X.Y. and W.Z.

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Correspondence to Weijia Zhang.

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Extended data

Extended Data Fig. 1 SMC-specific NOTCH1 or MFN1/2 KO validation and ultrasound of aorta in SMC-Notch1−/− and Myh11-CreERT2 mice.

(a) Representative images of immunohistochemistry staining with NOTCH1 in the aorta of control and SMC-Notch1−/− mice. (b) Quantitative PCR results of control and SMC-Notch1−/− mice aortas after removing aortic adventitia (n=3). (c) Schematic detailing the locations measured during echocardiographic analysis, and schematic illustrating the calculation of aortic strain and aortic pulse wave velocity. (d) Representative aortic ultrasound images of control mice and SMC-Notch1−/− mice without or with tamoxifen injection (n=5 per group). (e) Representative images of immunohistochemistry staining with MFN1 or MFN2 in the aorta of control and SMC-Mfn1/2−/− mice (n=3 per group). Data are represented as mean ± standard deviation, p values were determined by two-tailed Studentʼs t-tests. * represents P<0.05, ** represents P<0.01. All mice were male.

Source data

Extended Data Fig. 2 SMC-specificity of the AAV9 vector.

(a) GFP of AAV9 vector expression in tissue sections from aortas and hearts of mice without or with AAV9-Mfn1/2 treatments (n=5). (b) Representative fluorescence images of mitotracker staining of aorta tissue sections without or with AAV9-mediated Mfn1/2 delivery (n=5). Elastic fiber in aortic sections showed strong autofluorescence in all images.

Extended Data Fig. 3 Ultrasound measurement methodology and representative ultrasound images.

Representative aortic ultrasound images of Myh11-CreERT2 mice and SMC-Notch1−/− mice with or without treatments at the age of 40 weeks.

Extended Data Fig. 4 Early intervention with SMC-AAV9-mediated Mfn1 or Mfn2 delivery mitigates the progression of aortic dilation and stiffening in SMC-Notch1−/− mice, but less effectively than the delivery of both Mfn1 and Mfn2.

(a) Experimental design of mice with AAV9-mediated Mfn1 or Mfn2 delivery. (b) Representative macroscopic images of the heart and aorta of euthanized at 40 weeks old mice. (c) Statistical analysis of diameters of mice thoracic aorta. (d) Statistical analysis of diameters of mice abdominal aorta. (e) Statistical analysis of pulse wave velocity and circumferential cyclic strain of the mice aortas (Myh11-CreERT2, n=8; SMC-Notch1−/−+AAV9-vector, n=9; SMC-Notch1−/−+AAV9-Mfn1, n=6; SMC-Notch1−/−+AAV9-Mfn2, n=6; SMC-Notch1−/−+AAV9-Mfn1/2, n=7). (f) Representative histologic staining with HE, EVG and Alcian blue in aorta of 40 weeks old mice (n=6). The ultrasound data of control mice and SMC-Notch1−/− mice without or with AAV-Mfn1/2 in this Extended Data Fig. 4c–e and main text Fig. 3c-e were identical (that is, representing the same data) for comparison. Thoracic aortic diameter is presented in box and whisker plots showing maximal and minimal values and 75th and 25th percentiles. Histograms show mean± standard deviation. Statistical significance was assessed by Multiple t tests-one per row (with Holm-Sidak correction) (c) and two-tailed Studentʼs t-tests (d,e). * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001. All mice were male and subjected to tamoxifen injection.

Source data

Extended Data Fig. 5 Representative histologic staining with HE and EVG showing focal dissection in the aortas of three SMC-Notch1−/− mice at the age of 40 weeks, and lethal dissection in the aortas of SMC-Mfn1/2−/− mice.

The focal dissections were histologically characterized by a loss of continuity in at least three adjacent inner elastic laminae of the tunica media. Black arrows depict focal dissections. The dissections in SMC-Notch1−/− mice were primarily located in regions close to the heart (root to the ascending aorta). The dissections in SMC-Mfn1/2−/− mice could be found in different regions of aortas in different mice. All mice were male and subjected to tamoxifen injection.

Extended Data Fig. 6 Blood pressure and cardiac function measurements.

(a) Blood pressure measurements at ages of 20 and 30 weeks (n=6). (b) Blood pressure measurements at ages of 40 weeks (n=6). (c) Color flow Doppler echocardiography at the outflow tract and M-mode image depicting aortic valve function. Data are represented as mean ± standard deviation, p values were determined by two-tailed Studentʼs t-tests.

Source data

Extended Data Fig. 7 Intervention effects of SMC-specific AAV9-mediated Mfn1/2 delivery, teriflunomide or leflunomide in SMC-Notch1−/− mice, comparing treatments starting at 20 weeks of age versus those starting at 11 weeks of age.

(a) Experimental design of AAV9-mediated Mfn1/2 treatments. (b) Statistical analysis of diameters of mice aortic root, ascending aorta, and aortic arch at the age of 40 weeks. (c) Statistical analysis of diameters of mice abdominal aorta. (d) Statistical analysis of pulse wave velocity and circumferential cyclic strain of the mice aortas (SMC-Notch1−/−+AAV-vector, n=9; SMC-Notch1−/−+AAV- Mfn1/2, 11&25w twice AAV, n=7 and SMC-Notch1−/−+AAV- Mfn1/2, 20w one-time AAV, n=6, b-d). (e) Quantification of aortic medial thickness and elastin breaks (SMC-Notch1−/−+AAV-vector, n=5; SMC-Notch1−/−+AAV- Mfn1/2, 11&25w twice AAV, n=4 and SMC-Notch1−/−+AAV- Mfn1/2, 20w one-time AAV, n=6). (f) Experimental design of teriflunomide and leflunomide treatments. (g) Statistical analysis of diameters of mice aortic root, ascending aorta, and aortic arch at the age of 40 weeks. (h) Statistical analysis of diameters of mice abdominal aorta. (i) Statistical analysis of pulse wave velocity and circumferential cyclic strain of the mice aortas (SMC-Notch1−/−+vehicle or + Teriflunomide/Leflunomide (11-40w), n=8 and SMC-Notch1−/− + Teriflunomide/Leflunomide (20-40w), n=6, g-i). (j) Quantification of aortic medial thickness and elastin breaks (SMC-Notch1−/−+vehicle or + Teriflunomide/Leflunomide (11-40w), n=5 and SMC-Notch1−/− + Teriflunomide/Leflunomide (20-40w), n=6). (k) Representative histologic staining with HE, EVG and Alcian blue in the mice aorta (SMC-Notch1−/−-no treatment, n=5; SMC-Notch1−/− + Teriflunomide/Leflunomide (20-40w), n=6; SMC-Notch1−/−+AAV- Mfn1/2, 20w one-time AAV, n=6). Elastic breaks are marked by red arrows. The ultrasound and histological data of SMC-Notch1−/− mice and those treated from 11 to 40 weeks in this Extended Data Fig. 7a-j and main text Fig. 3c–g and Fig. 4c–g were identical (that is, representing the same data) for comparison. Thoracic aortic diameter is presented in box and whisker plots showing maximal and minimal values and 75th and 25th percentiles. Histograms show mean± standard deviation. Statistical significance was assessed by Multiple t tests-one per row (with Holm-Sidak correction) (b,g) and two-tailed Studentʼs t-tests (c-e, h-j). ns represents no significance, * represents P<0.05, ** represents P<0.01. All mice were male and subjected to tamoxifen injection.

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Extended Data Fig. 8 Western blots of the aortas of mice subjected to boosting mitofusin treatments or not (n=3).

a–b, Representative immunoblot analysis (a) and quantification (b) of MFN1, MFN2, TFAM, TOMM20 and NDUFB8. Data are represented as mean ± standard deviation, p values were determined by two-tailed Studentʼs t-tests. Uncropped western blots and source data are provided as a Source Data file.

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Extended Data Fig. 9 Early intervention with SMC-specific AAV9-mediated Tfam delivery mitigates the progression of aortic dilation and stiffening in SMC-Notch1−/− mice.

(a) Experimental design of AAV9-Tfam treatment (n=7). (b) Statistical analysis of diameters of mice aortic root, ascending aorta, and aortic arch. (c) Statistical analysis of diameters of mice abdominal aorta. (d) Statistical analysis of pulse wave velocity and circumferential cyclic strain of the mice aortas. For comparison, data from groups of control (n=8) and SMC-Notch1−/− mice without (n=9) and with AAV9-Mfn1/2 treatments (n=7) were included. (e) Representative histologic staining with HE, EVG and Alcian blue in the mice aorta. (f) Quantification of aortic medial thickness and elastin breaks (SMC-Notch1−/−, n=5 and SMC-Notch1−/−+AAV-Tfam, n=7). (g) Western blots of the aortas of mice subjected to boosting TFAM treatments by SMC-specific AAV9-Tfam or not (n=3). (h) Flow cytometry analyses of TOMM20. Five mice thoracic aortas were combined for one test of TOMM20 staining and flow cytometry. The ultrasound and histological data of control and SMC-Notch1−/− mice without and with AAV9-Mfn1/2 treatments in this Extended Data Fig. 9b–f and main text Fig. 3c–g were identical (that is, representing the same data) for comparison. Thoracic aortic diameter is presented in box and whisker plots showing maximal and minimal values and 75th and 25th percentiles. Histograms show mean± standard deviation. Statistical significance was assessed by Multiple t tests-one per row (with Holm-Sidak correction) (b) and two-tailed Studentʼs t-tests (c,d,f,g). * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001. All mice were male and subjected to tamoxifen injection. Uncropped western blots and source data are provided as a Source Data file.

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Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables 1–5

Supplementary Table 1. Clinical demographics of the patients included in this study. Supplementary Table 2. Human ascending aortic proteome with lists of differentially expressed proteins (DEPs), pathways and functions. Supplementary Table 3. Pearsonʼs correlation coefficient between NOTCH1 and other proteins (P < 0.05). Supplementary Table 4. Ultrasonographic measurements of aortic valvular and cardiac function. Supplementary Table 5. Mice aortic proteome with a list of DEPs.

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Tang, Y., Zhang, J., Fang, Y. et al. Correcting mitochondrial loss mitigates NOTCH1-related aortopathy in mice. Nat Cardiovasc Res 4, 235–247 (2025). https://doi.org/10.1038/s44161-024-00603-z

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