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Klf9 is essential for cardiac mitochondrial homeostasis

Nature Cardiovascular Research volume 3, pages 1318–1336 (2024)Cite this article

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Abstract

Mitochondrial dynamics and mitophagy are intimately linked physiological processes that are essential for cardiac homeostasis. Here we show that cardiac Krüppel-like factor 9 (Klf9) is dysregulated in human and rodent cardiomyopathy. Both global and cardiac-specific Klf9-deficient mice displayed hypertrophic cardiomyopathy. Klf9 knockout led to mitochondrial disarray and fragmentation, impairing mitochondrial respiratory function in cardiomyocytes. Furthermore, cardiac Klf9 deficiency inhibited mitophagy, thereby causing accumulation of dysfunctional mitochondria and acceleration of heart failure in response to angiotensin II treatment. In contrast, cardiac-specific Klf9 transgene improved cardiac systolic function. Mechanistically, Klf9 knockout decreased the expression of PGC-1α and its target genes involved in mitochondrial energy metabolism. Moreover, Klf9 controlled the expression of Mfn2, thereby regulating mitochondrial dynamics and mitophagy. Finally, adeno-associated virus–mediated Mfn2 rescue in Klf9-CKO hearts improved cardiac mitochondrial and systolic function. Thus, Klf9 integrates cardiac energy metabolism, mitochondrial dynamics and mitophagy. Modulating Klf9 activity may have therapeutic potential in the treatment of heart failure.

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Fig. 1: Cardiac Klf9 is dysregulated in human and rodent with cardiomyopathy and regulated by nutritional status.
Fig. 2: Cardiac-specific Klf9-deficient mice phenocopy global Klf9-KO mice.
Fig. 3: Klf9 deficiency accelerates heart failure and inhibits mitophagy in response to ANGII infusion.
Fig. 4: Cardiac-specific Klf9 transgene improves cardiac systolic function by promoting mitophagy in ANGII-treated mice.
Fig. 5: Klf9 mediates mitochondrial autophagy in NRCMs.
Fig. 6: Klf9 regulates mitophagy via Mfn2 in MEFs.
Fig. 7: Klf9 regulates mitochondrial dynamics through induction of Mfn1 and Mfn2.
Fig. 8: Mfn2 rescue improves cardiac and mitochondrial function in ANGII-treated Klf9-CKO mice.

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Data availability

All data are available in the main paper, extended data and supplementary data. RNA-seq data are deposited in the Gene Expression Omnibus under accession number GSE278326.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFA0800601 and 2022YFA0806102); the National Natural Science Foundation of China (82230027, 81825004, 81730024 and 82200975); the Scientific and Technological Research Project of Xinjiang Production and Construction Corps (2021AB028); and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-050).

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Author notes

  1. These authors contributed equally: Lei Zhang, Menglin Zhang, Jinlong Huang.

Authors and Affiliations

  1. Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key of Cellular Homeostasis and Disease, Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, China

    Lei Zhang, Menglin Zhang, Jinlong Huang, Jincan Huang, Yujie Zhang, Yinliang Zhang & Yongsheng Chang

  2. National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Lei Zhang & Houzao Chen

  3. Department of Basic Medicine, Shihezi University School of Medicine, Shihezi, China

    Cuizhe Wang & Jun Zhang

  4. Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China

    Xiangwen Xi & Jinwei Tian

  5. The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, China

    Xiangwen Xi & Jinwei Tian

  6. Ningxia Key Laboratory of Stem Cell and Regenerative Medicine, General Hospital of Ningxia Medical University, Yinchuan, China

    Heng Fan

  7. Henan Key Laboratory for Medical Tissue Regeneration, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, China

    Jikui Wang

  8. Division of Cardiovascular Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Dingsheng Jiang

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Contributions

L. Zhang and M. Zhang participated in the animal studies using Klf9-KO, Klf9-CKO and Klf9-TG mice and in vitro studies using NRCMs and MEFs and contributed to data analysis. Jinlong Huang and Jincan Huang prepared adenoviruses encoding shRNA specific to Mfn1 or Mfn2. H.F., Yujie Zhang, Yinliang Zhang, C.W. and X.X. performed in vitro experiments. H.C. contributed to discussion and editing of the manuscript. D.J. provided the clinical heart samples of patients with HCM and healthy donors. Y.C., J.Z. and J.T. designed the research and wrote the manuscript.

Corresponding authors

Correspondence to Jinwei Tian, Jun Zhang or Yongsheng Chang.

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All authors declare no competing interests. There are no conflicts of interest in the submission of this manuscript, which was approved by all authors for publication. We also confirm that this work is original. It has not been published elsewhere and is not currently under consideration for publication elsewhere.

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Nature Cardiovascular Research thanks Roberta Gottlieb and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Loss of Klf9 function predisposes mice to hypertrophic cardiomyopathy.

a, Left, Western blotting analysis of Klf9 in WT and Klf9 KO hearts. Right, Quantification of Klf9 protein levels (n = 3). b, Left, B- and M-mode echocardiographic analysis of WT and Klf9 KO mice. Right, Quantitative group data showing the changes of FS, EF and LV mass (n = 6). c, Ratios of hearts or lungs weight to tibia length of WT and Klf9 KO mice (n = 6). d, H&E staining of WT and Klf9 KO hearts. e, Left, WGA staining of WT and Klf9 KO hearts. Right, Quantitative data of cardiomyocyte cross-sectional area (CSA) (n = 6). f, qRT-PCR analysis of mRNA levels of ANF, BNP, and β-MHC in WT and Klf9 KO hearts (n = 6). g, Left, Masson’s trichrome staining of WT and Klf9 KO hearts. Right, Quantification of blue-staining replacement fibrosis (n = 6). h, Left, TUNEL staining of WT and Klf9 KO hearts. Right, Quantitative data for apoptosis positive cells (n = 6). i, Heart rates, systolic and diastolic blood pressures of WT and Klf9 KO the mice (n = 6). Data are shown as mean ± SEM; 2-tailed Student’s t test (a, b, c, e, f, g, h, i).

Source data

Extended Data Fig. 2 Loss of Klf9 function impairs the cardiac mitochondrial function.

a, Volcano plot indicating the genes that were differentially expressed in WT versus Klf9 KO hearts. In red are the genes significantly up-regulated and in blue are the genes significantly down-regulated. b, KEGG pathway enrichment analysis of downregulated genes in Klf9 KO hearts. Orange dots represent the number of genes of each annotated pathway and the length of each column is based on the p values. c, Volcano plots showing the GSEA of GO cellular component categories of differentially expressed genes (DEGs). Gene sets related to mitochondria are marked. Sizes of the dots represent the number of genes in the gene sets and the transparency represent the FDR of the enrichment. d, Top hits of GSEA analysis from c showing the Klf9 positive correlation with the mitochondria. NES, normalized enrichment score; FDR, false discovery rate. e, GSEA analysis of disease category of DEGs. f-i, Heat-map, qRT-PCR (n = 6) and Western blotting analysis (n = 3) of the expression of genes involved in FA oxidation, oxidative phosphorylation, mitochondrial biogenesis, mitochondrial dynamics, autophagy and mitophagy. j, ChIP assay of heart extracts showing the enrichment of Klf9 proteins at the promoter region of PGC-1α (n = 4). k, TEM analysis of hearts from the indicated mice. l, Quantitative data of mitochondrial size distribution and content based on TEM images (n = 6). m, n, Left, Flow cytometric analyses of size (forward scatter, FSC) (m) and transmembrane electrical potential (DiOC6 intensity) (n) of isolated cardiac mitochondria from the indicated hearts. Right, Quantitative data of FSC and DiOC6 intensity (n = 6). o, Left, Oxygen consumption rates (OCR) showing the respiration of isolated mitochondria from the indicated hearts. Right, Quantification of basal and FCCP induced maximal respiration (n = 9). p, Myocardial ATP levels in the indicated hearts (n = 12). q, Left, Mitochondrial ROS production of isolated mitochondria from the indicated hearts measured with MitoSOX. Right, Quantitative data of ROS intensity (n = 6). Data are shown as mean ± SEM; 2-tailed Student’s t test (g, h, i, l, m, n, p, q), 2-way ANOVA (j, o).

Source data

Extended Data Fig. 3 PGC-1α rescue increases mitochondrial energy metabolism and improves the cardiac function in Klf9-CKO mice at baseline.

a, Schematic illustration of the experimental design to study the effects of PGC-1α rescue on cardiac and mitochondrial function of Klf9-CKO hearts. b, Left, Western blotting analysis of Klf9 and PGC-1α protein levels in the hearts from Ctrl and AAV-GFP or AAV-PGC-1α injected Klf9-CKO mice. Right, Quantification of Klf9 and PGC-1α protein levels (n = 3). c, B- and M-mode echocardiographic analysis of Ctrl and AAV-GFP or AAV-PGC-1α injected Klf9-CKO mice. d, Quantitative group data showing the changes of FS, EF and LV mass (n = 6) of Ctrl and AAV-GFP or AAV-PGC-1α injected Klf9-CKO mice. e, Ratios of hearts or lungs weight to tibia length of Ctrl and AAV-GFP or AAV-PGC-1α injected Klf9-CKO mice (n = 6). f, Left, WGA staining of hearts from above mice. Right, Quantitative data for cardiomyocyte CSA (n = 6). g, Left, Masson’s trichrome staining of hearts from above mice. Right, Quantification of blue-staining replacement fibrosis (n = 6). h, H&E staining of hearts from the hearts from above mice. i, qRT-PCR analysis of genes involved in mitochondrial metabolism in hearts from above mice (n = 6). j, TEM analysis of hearts from above mice (The red arrows indicate the fragment of mitochondria). k, Quantitative data of mitochondrial size distribution (Left) and content (Right) based on TEM images (n = 6). l, Left, Flow cytometric analyses of transmembrane electrical potential (DiOC6) of isolated cardiac mitochondria from above hearts. Right, Quantitative data of DiOC6 intensity (n = 6). m, Left, OCR showing the respiration of isolated mitochondria treated with oligomycin (Oligo), FCCP and rotenone/antimycin A (Rot-AA). Right, Quantification of basal and FCCP induced maximal respiration (n = 9). n, Quantification of myocardial ATP levels of above mice hearts (n = 6). o, Left, Mitochondrial ROS production of isolated mitochondria from above hearts measured with MitoSOX. Right, Quantitative data of ROS levels (n = 6). Data are shown as mean ± SEM; 2-way ANOVA (b, d, e, f, g, i, k, l, m, n, o).

Source data

Extended Data Fig. 4 Klf9 deficiency accelerates heart failure and mitochondrial senescence with aging.

a, Representative gross morphology of 24-month-old Ctrl and Klf9-CKO mice. b, Left, B- and M-mode echocardiographic analysis of 12-month-old Ctrl and Klf9-CKO mice. Right, Quantitative data showing the changes of FS, EF and LV mass (n = 6). c, Ratios of hearts or lungs weight to tibia length of 12-month-old Ctrl and Klf9-CKO mice (n = 6). d, Left, WGA staining of 12-month-old Ctrl and Klf9-CKO hearts. Right, Quantitative data for cardiomyocyte CSA (n = 6). e, Left, Masson’s trichrome staining of 12-month-old Ctrl and Klf9-CKO hearts. Right, Quantification of blue-staining replacement fibrosis (n = 6). f, Left, TUNEL staining of 12-month-old Ctrl and Klf9-CKO hearts. Right, Quantitative data for apoptosis positive cells (n = 6). g, TEM of 12-month-old Ctrl and Klf9-CKO hearts. h, i, Quantitative data of mitochondrial size distribution (h) and content (i, n = 6) based on TEM images. j, Quantification of mitochondrial protein content of 12-month-old Ctrl and Klf9-CKO hearts (n = 20). k-m, Left, Flow cytometric analysis of size (FSC) (k), transmembrane electrical potential (DiOC6) (l) and ROS production (MitoSOX) (m) of isolated cardiac mitochondria from 12-month-old Ctrl and Klf9-CKO hearts. Right, Quantitative data of FSC, DiOC6 and MitoSOX intensity (n = 6). n, qRT-PCR analysis of expression levels of genes involved in mitochondrial biogenesis in 12-month-old Ctrl and Klf9-CKO hearts (n = 6). o, Left, Western blotting analysis of proteins involved in the mitochondrial unfolded protein response (mtUPR) in 12-month-old Ctrl and Klf9-CKO hearts. Right, Quantification of AFG3L2, LONP1 and HSP60 protein levels (n = 3). Data are shown as mean ± SEM; 2-tailed Student’s t test (b, c, d, e, f, i, j, k, l, m, n, o).

Source data

Extended Data Fig. 5 Klf9 is required for cardiac functional adaptation to hemodynamic pressure overload.

a, Ratios of hearts or lungs weight to tibia length (n = 6). b, qRT-PCR analysis of mRNA levels of ANF, BNP and β-MHC in hearts from Ctrl and Klf9-CKO mice treated with saline or ANGII (n = 6). c, Left, WGA staining of hearts from Ctrl and Klf9-CKO mice treated with saline or ANGII. Right, Quantitative data for cardiomyocyte CSA (n = 6). d, Left, Masson’s trichrome staining of hearts from Ctrl and Klf9-CKO mice treated with saline or ANGII. Right, Quantification of blue-staining replacement fibrosis (n = 6). e, Left, TUNEL staining of hearts from Ctrl and Klf9-CKO mice treated with saline or ANGII. Right, Quantitative data for apoptosis positive cells (n = 6). f, qRT-PCR analysis of mRNA levels of genes involved in mitochondrial energy metabolism in hearts from above mice (n = 6). Data are shown as mean ± SEM; 2-way ANOVA (a, b, c, d, e).

Source data

Extended Data Fig. 6 Cardiac-specific Klf9 transgene prevents cardiac hypertrophy, fibrosis and cardiomyocytes apoptosis in ANGII-treated mice.

a, Ratios of hearts or lungs weight to tibia length (n = 6). b, qRT-PCR analysis of mRNA levels of ANF, BNP and β-MHC in hearts from Ctrl and Klf9-TG mice treated with saline or ANGII (n = 6). c, Left, WGA staining of hearts from Ctrl and Klf9-TG mice treated with saline or ANGII. Right, Quantitative data for cardiomyocyte CSA (n = 6). d, Left, Masson’s trichrome staining of hearts from Ctrl and Klf9-TG mice treated with saline or ANGII. Right, Quantification of blue-staining replacement fibrosis (n = 6). e, Left, TUNEL staining of hearts from Ctrl and Klf9-TG mice treated with saline or ANGII. Right, Quantitative data for apoptosis positive cells (n = 6). Data are shown as mean ± SEM; 2-way ANOVA (a, b, c, d, e).

Source data

Extended Data Fig. 7 Klf9 regulates ANGII induced hypertrophy of NRCMs.

a, Confocal imaging of NRCMs infected with Ad-shCtrl or Ad-shKlf9 for 24 hours, followed by treatment with ANGII (1 μM) or PBS for 48 hours. α-Actinin staining was performed to identify cells. b, Quantification of cell size of 30 NRCMs in each group (n = 22). c, Quantification of myocardial ATP levels in NRCMs as described in B (n = 12, n value refers to independent experiments). d, qRT-PCR analysis of mRNA levels of hypertrophic genes in NRCMs in A (n = 6, n value refers to independent experiments). e, Confocal imaging of NRCMs infected with Ad-GFP or Ad-Klf9 for 24 hours, followed by treatment with ANGII (1 μM) or PBS for 48 hours. α-Actinin staining was performed to identify cells. f, Quantification of cell size of 30 NRCMs in each group (n = 22). g, Quantification of myocardial ATP levels of NRCMs infected with Ad-GFP or Ad-Klf9, followed by treatment with ANGII or PBS (n = 12, n value refers to independent experiments). h, qRT-PCR analysis of mRNA levels of hypertrophic genes in NRCMs in E (n = 6, n value refers to independent experiments). Data are shown as mean ± SEM; 2-way ANOVA (b, c, d, f, g, h).

Source data

Extended Data Fig. 8 Mfn2 rescue improves the cardiac and mitochondrial function in Klf9-CKO mice at baseline.

a, Schematic illustration of the experimental design to study the effects of Mfn2 rescue on cardiac and mitochondrial function of Klf9-CKO hearts. b, Left, Western blotting analysis of Klf9 and Mfn2 protein levels in hearts from Ctrl and Klf9-CKO mice infected with AAV-GFP or AAV-Mfn2. Right, Quantification of protein levels of Klf9 and Mfn2 (n = 3). c, B- and M-mode echocardiographic analysis of Ctrl and Klf9-CKO mice infected with AAV-GFP or AAV-Mfn2. d, Quantitative group data showing the changes of FS, EF and LV mass of Ctrl and Klf9-CKO mice infected with AAV-GFP or AAV-Mfn2 (n = 6). e, Ratios of hearts or lungs weight to tibia length of Ctrl and Klf9-CKO mice infected with AAV-GFP or AAV-Mfn2 (n = 6). f, H&E staining of hearts from above mice. g, Left, WGA staining of hearts from above mice. Right, Quantitative data for cardiomyocyte CSA (n = 6). h, Left, Masson’s trichrome staining of hearts from above mice. Right, Quantification of blue-staining replacement fibrosis (n = 6). i, Respective TEM of hearts above mice. j, Quantitative data for mitochondrial size distribution and content (n = 6) based on TEM images. k, Left, Flow cytometric analyses of transmembrane electrical potential (DiOC6). Right, Quantitative data of DiOC6 intensity (n = 6, n value refers to independent experiments). l, Left, OCR showing the respiration of isolated cardiac mitochondria from above mice. Right, Quantification of basal and FCCP induced maximal respiration (n = 9). m, Quantification of myocardial ATP levels of hearts from above mice (n = 6, n value refers to independent experiments). n, Left, ROS production (MitoSOX) of cardiac mitochondria isolated from above mice. Right, Quantitative data of MitoSOX intensity (n = 6, n value refers to independent experiments). Data are shown as mean ± SEM; 2-way ANOVA (b, d, e, g, h, j, k, l, m, n).

Source data

Extended Data Fig. 9 Mfn2 rescue prevents the cardiac hypertrophy, fibrosis and cardiomyocytes apoptosis in ANGII-treated Klf9-CKO mice.

a, Left, Western blotting analysis of Klf9 and Mfn2 in hearts from Ctrl and Klf9-CKO mice infected with AAV-GFP or AAV-Mfn2 and treated with ANGII. Right, Quantification of protein levels of Klf9 and Mfn2 (n = 3). b, Left, WGA staining of hearts from Ctrl and Klf9-CKO mice infected with AAV-GFP or AAV-Mfn2 and treated with ANGII. Right, Quantitative data for cardiomyocyte CSA (n = 6). c, Ratios of hearts or lungs weight to tibia length (n = 6). d, qRT-PCR analysis of mRNA levels of ANF, BNP and β-MHC in hearts from above mice (n = 6). e, Left, Masson’s trichrome staining of hearts from above mice. Right, Quantification of blue-staining replacement fibrosis (n = 6). f, Left, TUNEL staining of hearts from above mice. Right, Quantitative data for apoptosis positive cells (n = 6). Data are shown as mean ± SEM; 2-way ANOVA (a, b, c, d, e, f).

Source data

Extended Data Fig. 10 Proposed model of Klf9 regulating mitochondrial energy metabolism, mitochondrial dynamics and mitophagy in cardiomyocytes.

Cardiac Klf9 is dysregulated in hypertrophic hearts, and Klf9 serves as a critical transcriptional regulator of mitochondrial homeostasis in the heart. Klf9 controls mitochondrial energy metabolism via induction of PGC-1α, while it regulates mitochondrial dynamics and mitophagy through Mfns and Mfn2/Parkin, respectively.

Source data

Supplementary information

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Supplementary Tables 1–4, Supplementary Figs. 1–7 and full unedited gel of western blotting.

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Statistical Source Data for supplementary figures.

Supplementary Tables 1–4.

Supplementary Table 1. Clinical characteristics of patients with HCM in Excel format. Supplementary Table 2. qPCR primer sequences in Excel format. Supplementary Table 3. ChIP–qPCR primer sequences in Excel format. Supplementary Table 4. Plasmid construction primer sequences in Excel format.

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Source data

Statistical Source Data for Figs. 1–8 and Extended Data Figs. 1–9.

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Zhang, L., Zhang, M., Huang, J. et al. Klf9 is essential for cardiac mitochondrial homeostasis. Nat Cardiovasc Res 3, 1318–1336 (2024). https://doi.org/10.1038/s44161-024-00561-6

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  • DOI: https://doi.org/10.1038/s44161-024-00561-6

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