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MARK4 controls ischaemic heart failure through microtubule detyrosination

Nature volume 594, pages 560–565 (2021)Cite this article

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Abstract

Myocardial infarction is a major cause of premature death in adults. Compromised cardiac function after myocardial infarction leads to chronic heart failure with systemic health complications and a high mortality rate1. Effective therapeutic strategies are needed to improve the recovery of cardiac function after myocardial infarction. More specifically, there is a major unmet need for a new class of drugs that can improve cardiomyocyte contractility, because inotropic therapies that are currently available have been associated with high morbidity and mortality in patients with systolic heart failure2,3 or have shown a very modest reduction of risk of heart failure4. Microtubule detyrosination is emerging as an important mechanism for the regulation of cardiomyocyte contractility5. Here we show that deficiency of microtubule-affinity regulating kinase 4 (MARK4) substantially limits the reduction in the left ventricular ejection fraction after acute myocardial infarction in mice, without affecting infarct size or cardiac remodelling. Mechanistically, we provide evidence that MARK4 regulates cardiomyocyte contractility by promoting phosphorylation of microtubule-associated protein 4 (MAP4), which facilitates the access of vasohibin 2 (VASH2)—a tubulin carboxypeptidase—to microtubules for the detyrosination of α-tubulin. Our results show how the detyrosination of microtubules in cardiomyocytes is finely tuned by MARK4 to regulate cardiac inotropy, and identify MARK4 as a promising therapeutic target for improving cardiac function after myocardial infarction.

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Fig. 1: MARK4 deficiency preserves cardiac function after myocardial infarction without altering the size of the scar.
Fig. 2: MARK4 expression in cardiomyocytes regulates cardiac contractile function after myocardial infarction.
Fig. 3: MARK4 regulates cardiomyocyte contractility by promoting microtubule detyrosination.
Fig. 4: MARK4 controls microtubule detyrosination through MAP4 phosphorylation to facilitate VASH2 access to microtubules.

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Data availability

All of the associated raw data presented in this paper are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

Custom STED microscope control software used for data collection can be accessed at https://github.com/Gurdon-Super-Res-Lab/STED-Control, and a customized algorithm used for STED image analysis is available at https://github.com/zhaoaite/dynamic_thresholding_algorithm.

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Acknowledgements

The work is supported by a British Heart Foundation (BHF) fellowship grant (FS/14/28/30713) to X.L., an Isaac Newton Trust grant (18.40u) to X.L. and Cambridge BHF Centre of Research Excellence grants (RE/13/6/30180 and RE/18/1/34212) to X.L. X.Y. was supported by a Royal Society Newton Advanced Fellowship grant (NA140277) and is supported by BHF grants (FS/14/28/30713, RE/18/1/34212). Z.M. is supported by BHF chair grant (CH/10/001/27642) and the NIHR Cambridge Biomedical Research Centre. M.A.-N. is supported by German Research Foundation (Deutsche Forschungsgemeinschaft (DFG)) (AM 507/1-1). G.S., E.A. and D.S.J. are supported by a Wellcome Trust collaborative award (203285), a Wellcome Principal Research Fellowship (207496) and core support from the Wellcome Trust (203144) and Cancer Research UK (A24823). The work of H.Z. is supported in part by a Royal Society Newton Advanced Fellowship grant (NA160342).  We thank staff at the phenotyping hub and biochemical assay laboratory of Cambridge University Hospitals; staff at the Phenomics Laboratory and Anne McLaren Building of Cambridge University; B. Prosser for sharing protocols and discussion; M. Zhang for advice on echography data; M. A. Ackers-Johnson from R. Foo’s laboratory for technical advice on isolating and culturing primary mouse cardiomyocytes; S. Ozanne and L. Pantaleao for access and training of ultracentrifuge use; D. Pavlovic for advice on IonOptix; C. Huang for advice on calcium measurements; T. Zhao for his intellectual discussion and reading the manuscript; and X. Luo, who passed away due to COVID-19 during the outbreak in Wuhan, for her support of this project.

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Author notes

  1. These authors contributed equally: Xian Yu, Xiao Chen

Authors and Affiliations

  1. Department of Medicine, Cardiovascular Division, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK

    Xian Yu, Hannah Chenoweth, Marc Clement, James Harrison, Christian Doreth, Ziad Mallat & Xuan Li

  2. Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Xiao Chen

  3. Department of Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany

    Mamta Amrute-Nayak

  4. The Gurdon Institute, University of Cambridge, Cambridge, UK

    Edward Allgeyer, George Sirinakis & Daniel St Johnston

  5. Department of Genetics, University of Cambridge, Cambridge, UK

    Edward Allgeyer, George Sirinakis & Daniel St Johnston

  6. College of Computer Science and Technology, Qingdao University, Shandong, China

    Aite Zhao

  7. Department of Medicine, Experimental Medicine and Immunotherapeutics Division, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK

    Thomas Krieg

  8. School of Informatics, University of Leicester, Leicester, UK

    Huiyu Zhou

  9. Department of Biology, Southern University of Science and Technology, Shenzhen, China

    Hongda Huang

  10. Muroran Institute of Technology, Muroran, Japan

    Kiyotaka Tokuraku

  11. Université de Paris, Institut National de la Santé et de la Recherche Médicale, U970, PARCC, Paris, France

    Ziad Mallat

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Contributions

X.Y., X.C., M.A.-N., A.Z., H.Z. and X.L. designed the experiments. X.Y., X.C., M.A.-N., E.A., A.Z., H.C., M.C., J.H. and X.L. performed the experiments. X.Y., X.C., M.A.-N., H.C., A.Z., H.Z. and X.L. analysed the data. X.Y. performed all of the in vivo experiments using mouse models and contractility assays, and wrote the relevant Methods section. X.C. performed the primary cardiomyocyte isolation, fractionation, real-time PCR, western blotting and imaging experiments. M.A.-N. performed in vitro microtubule co-sedimentation assay, analysed the data and wrote the relevant Methods section. E.A. configured the custom STED system, performed STED imaging and wrote the relevant Methods section. G.S. provided assistance for STED imaging. A.Z. and H.Z. wrote the STED image analytic code, analysed STED data and wrote the relevant Methods section. H.C. assisted with tissue collection, performed tissue sectioning and staining assays, and optimized some of the experimental conditions. M.C. assisted with part of the tissue collection, staining and analysis. J.H. provided some technical supports on mouse experiments. C.D., H.C. and X.L. performed the initial test. H.H. and K.T. provided recombinant proteins. T.K. provided initial training for the LAD model. D.S.J. provided the super-resolution imaging platform. Z.M. independently had the idea and supported the initiation of the project (the mouse in vivo work was performed under the Procedure Project Licence of Z.M.). X.Y., X.C., Z.M. and X.L. interpreted the data for important intellectual contents. X.L. conceived idea, designed and initiated the project, established the collaboration, supervised the project, and wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Xuan Li.

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The authors declare no competing interests.

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Peer review information Nature thanks Leslie Leinwand and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Timeline of experimental design.

a, Timeline of the experimental design for Fig. 1d, e. Investigation of the effect of total MARK4 deficiency on cardiac function using the model of LAD coronary artery ligation to induce myocardial infarction. Echocardiography (Echo) and histological analyses were performed at the indicated time points. b, Timeline of the experimental design for Fig. 2a–c. Investigation of the effect of total MARK4 deficiency on cardiac function at 24 h after myocardial infarction. Echocardiography, circulating cardiac troponin and histological analyses were performed at the indicated time point. c, Timeline of the experimental design for Fig. 2d and Extended Data Fig. 3a, b. Investigation of the effect of MARK4 expression in haematopoietic cells on cardiac function using the LAD ligation model. BM, bone marrow; BMT, bone marrow transplantation. Echocardiography analysis was performed at the indicated time points. d, Timeline of the experimental design for Fig. 2e and Extended Data Fig. 3c–e. Investigation of the effect of MARK4 expression in cardiomyocytes on cardiac function using the LAD ligation model. Tm, tamoxifen. Mark4 cKO, Mark4 conditional knockout mice. Echocardiography analysis was performed at the indicated time points.

Extended Data Fig. 2 MARK4 expression, α-tubulin posttranslational modifications and changes in the inflammatory response after myocardial infarction.

a, Representative confocal images of primary cardiomyocytes (CMs) isolated from Mark4−/− or control mice at baseline or at day 3 after myocardial infarction. Scale bar, 20 μm. b, Levels of pro-inflammatory cytokines at day 3 after myocardial infarction (n = 6 per group). c, LVEF at day 3 after myocardial infarction (n = 4 per group). d, e, Western blots of detyrosinated α-tubulin in cell lysates of cardiomyocytes isolated from wild-type mice at day 3 after myocardial infarction or post-sham surgery (S), with the lysates of the remaining cells from the same hearts used as control. d, Representative western blots. e, Ratio of detyrosinated α-tubulin over total α-tubulin quantified using western blot data from biologically independent samples. Sham group, n = 4 mice; myocardial infarction group, n = 5 mice. f, g, Western blots of cell lysates from the isolated cardiomyocytes of Mark4−/− or control mice at day 3 after myocardial infarction to detect detyrosinated α-tubulin, polyglutamylated α-tubulin (Polyglu-tub), acetylated α-tubulin (Ace-tub) and α-tubulin. f, Representative western blots. g, Ratio of detyrosinated α-tubulin, polyglutamylated α-tubulin or acetylated α-tubulin over total α-tubulin quantified using western blot data from biologically independent samples (n = 3 mice per group). b, The box bounds represent the 25th and 75th percentiles, the middle line shows the median, the whiskers show the minimum and maximum, and individual replicates are shown as circles. c, e, g, Data are mean ± s.e.m. b, c, e, g, Two-tailed unpaired t-tests were used; P values are indicated.

Source data

Extended Data Fig. 3 Validation of the mouse models for MARK4 selective expression in either haematopoietic cells or cardiomyocytes.

a, b, Confirmation of MARK4 deficiency in CD45+ cells of chimeric wild-type mice reconstituted with bone marrow (BM) cells from Mark4−/− mice (strategy is shown in Extended Data Fig. 1c). a, Representative image with arrows pointing to CD45+ cells in the infarct area. Scale bars, 20 μm (a). b, Quantification of the percentage of MARK4+ cells (green) within CD45+ cells (red). n = 3 mice per group. c, Confirmation of Mark4 deletion in cardiomyocytes (strategy is shown in Extended Data Fig. 1d). Real-time PCR of Mark4 level in primary cardiomyocytes isolated from Myh6-mcm+/−;Mark4fl/fl (also known as αMHC-mcm+/−;Mark4fl/fl) (n = 4) and control mice (n = 3) at day 7 after the last tamoxifen injection. d, Assessment of LVEF of a different group (compared with Fig. 2e) of Mark4 cKO and control mice (n = 6 per group) at day 1 after myocardial infarction. e, Infarct size at 24 h after myocardial infarction (n = 6 per group). Scale bar, 2 mm. b–e, Data are mean ± s.e.m.; two-tailed unpaired t-test; P values are indicated.

Source data

Extended Data Fig. 4 The effect of MARK4 deficiency on sarcomere length, peak shortening, velocity and calcium transients in cardiomyocytes before and after myocardial infarction.

a–f, Contractility assay of single primary cardiomyocytes isolated at baseline or at day 3 after myocardial infarction from the following groups: Mark4+/+ baseline (n = 4 mice, n = 45 cardiomyocytes examined over 4 independent experiments), Mark4−/− baseline (n = 3 mice, n = 45 cardiomyocytes examined over 3 independent experiments), Mark4+/+ myocardial infarction (n = 5 mice, n = 54 cardiomyocytes examined over 5 independent experiments) and Mark4−/− myocardial infarction (n = 6 mice, n = 57 cardiomyocytes examined over 6 independent experiments). a, Colour denotation of samples. b, Resting sarcomere length. c, d, Average sarcomere shortening traces were compared. e, f, Average velocity traces (dSL/dT). g–m, Calcium influx assay on single cardiomyocytes isolated from Mark4−/− or control mice at baseline or at day 3 after myocardial infarction in the following groups: Mark4+/+ baseline group (n = 2 mice, n = 34 cardiomyocytes examined over 2 independent experiments), Mark4−/− baseline group (n = 2 mice, n = 33 cardiomyocytes examined over 2 independent experiments), Mark4+/+ myocardial infarction group (n = 4 mice, n = 65 cardiomyocytes examined over 4 independent experiments) and Mark4−/− myocardial infarction group (n = 3 mice, n = 58 cardiomyocytes examined over 3 independent experiments). g, Basal Ca2+ level. h, Amplitude level of Ca2+ transients. i, Ca2+ release speed during contraction. j, Ca2+ reuptake speed during contraction. k, Ca2+ elevation time. l, Ca2+ reuptake time. m, Traces of Ca2+ kinetic curves. b, g–l, The box bounds represent the 25th and 75th percentiles, the middle line shows the median, the whiskers show the minimum and maximum, and individual cardiomyocytes are shown as circles. b, g–l, Two-way ANOVA with Bonferroni post hoc correction for multiple comparisons; P values are indicated.

Source data

Extended Data Fig. 5 The effect of TTL overexpression, or PTL treatment, on contractility of Mark4−/− cardiomyocytes after myocardial infarction.

a–i, Adenovirus (Adv)-mediated overexpression (o.e.) of TTL in cardiomyocytes isolated from Mark4−/− or control Mark4+/+ mice at day 3 after myocardial infarction, with overexpression of a null vector as control (Ctrl). a, Representative western blot. b–i, Contractility assay of single cardiomyocytes with overexpression in the following groups: Mark4+/+ myocardial infarction Adv-Null (n = 3 mice, n = 75 cardiomyocytes examined over 3 independent experiments), Mark4+/+ myocardial infarction Adv-TTL (n = 3 mice, n = 69 cardiomyocytes examined over 3 independent experiments), Mark4−/− myocardial infarction Adv-Null (n = 3 mice, n = 74 cardiomyocytes examined over 3 independent experiments) and Mark4−/− myocardial infarction Adv-TTL (n = 3 mice, n = 73 cardiomyocytes examined over 3 independent experiments). b, Colour denotation of samples. c, Resting sarcomere length. d–f, Average sarcomere shortening traces. g–i, Average velocity traces (dSL/dT). j–s, Contractility assay of single cardiomyocytes isolated at day 3 after myocardial infarction with the following treatments: Mark4+/+ myocardial infarction DMSO (n = 3 mice, n = 46 cardiomyocytes examined over 3 independent experiments), Mark4+/+ myocardial infarction PTL (n = 3 mice, n = 67 cardiomyocytes examined over 3 independent experiments), Mark4−/− myocardial infarction DMSO (n = 3 mice, n = 55 cardiomyocytes examined over 3 independent experiments) and Mark4−/− myocardial infarction PTL (n = 3 mice, n = 64 cardiomyocytes examined over 3 independent experiments). j, Colour denotation of samples. k, Resting sarcomere length. l, Sarcomere peak shortening. m–o, Average sarcomere shortening traces. p, Pooled data of contraction velocity and relaxation velocity. q–s, Average velocity traces (dSL/dT). c, k, l, p, The box bounds represent the 25th and 75th percentiles, the middle line shows the median, the whiskers show the minimum and maximum and individual cardiomyocytes are shown as circles. c, k, l, p, Two-way ANOVA test with Bonferroni post hoc correction for multiple comparisons; P values are indicated.

Source data

Extended Data Fig. 6 The association of MAP4 or VASH2 with polymerized microtubules.

a, Protein sequence alignment between human MAP4 (NP002366) and mouse MAP4 (NP001192259). KXGS motifs (highlighted with red frames) within the tubulin-binding repeats (highlighted with yellow, brown, dark brown and purple frames) of MAP4 are MARK4 substrate sites. S941 of human MAP4 (S914 of mouse MAP4) and S1073 of human MAP4 (S1046 of mouse MAP4) are conserved phosphorylation sites within the KXGS motifs. b, Schematic illustration of the possible association between MAP4 and microtubules before or after MARK4-dependent phosphorylation. Unphosphorylated MAP4 binds to microtubules. After MARK4-dependent phosphorylation of S914 of mouse MAP4 at the microtubule weak binding site, MAP4 makes allosteric changes. After MARK4-dependent phosphorylation of S1046 of mouse MAP4 at the microtubule anchor site, MAP4 detaches from microtubules. c, Representative gel image of 4R-MAP4 (1–4 μM) binding to the polymerized microtubules (5 μM) in a microtubule co-sedimentation assay. d, Quantification of the binding shown in c. n = 7 samples examined over 3 independent experiments (1 μM); n = 4 samples examined over 3 independent experiments (2 μM); n = 6 samples examined over 3 independent experiments (3 μM); n = 3 samples examined over 3 independent experiments (4 μM). e, Representative gel image of VASH2–SVBP (0.5–2 μM) binding to the polymerized microtubules (2.5 μM) in a microtubule co-sedimentation assay. |f, Quantification of the binding shown in e. n = 7 samples examined over 5 independent experiments (0.5 μM); n = 7 samples examined over 5 independent experiments (1 μM); n = 4 samples examined over 3 independent experiments (1.5 μM); n = 4 samples examined over 3 independent experiments (2 μM). d, f, Data are mean ± s.e.m.; one-way ANOVA; P values are indicated.

Extended Data Fig. 7 Association of VASH2 with microtubules in cardiomyocytes before and myocardial infarction, and the effect of MAP4 knockdown.

a, Subcellular fractionation of primary cardiomyocytes isolated from mice at baseline or after myocardial infarction. Western blotting of the fractions from CEB or PEB. b, Representative western blots of the free-tubulin fraction (F1) and extraction from the stable pellet fraction (F2) obtained using a conventional fractionation method. c–e, Western blots of CEB or PEB fractions of wild-type (WT) cardiomyocytes at baseline or after myocardial infarction. c, Representative western blots (derived from the same experiment). d, Quantification of pMAP4(S1046) in CEB, pMAP4(S914) in PEB and VASH2 levels in PEB (n = 5 mice at baseline, n = 6 mice after myocardial infarction, blots were processed in parallel). e, Correlation between VASH2 level in the PEB fraction and pMAP4 levels. f–i, Wild-type cardiomyocytes after myocardial infarction transduced with adenovirus-mediated Map4 or control shRNA. f, Representative western blots of CEB or PEB fraction, and Coomassie-stained gels loaded with the same amounts of proteins. g, Quantification of VASH2 levels in PEB (n = 3 mice examined over 3 experiments per group). h, i, STED images of VASH2 and α-tubulin in the cardiomyocytes after knocking down MAP4. h, Representative images. Scale bar, 2 μm. i, Pearson correlation coefficient of VASH2 and α-tubulin signals, percentage of VASH2 signals on the polymerized microtubules and percentage of VASH2 signals off the polymerized microtubules, in the following groups: control shRNA (n = 2 mice, n = 35 cardiomyocytes examined over 2 independent experiments) and Map4 shRNA (n = 2 mice, n = 27 cardiomyocytes examined over 2 independent experiments). d, g, i, Data are mean ± s.e.m.; two-tailed unpaired t-test. e, Two-tailed correlation test. P values are indicated.

Source data

Extended Data Fig. 8 The status of VASH2 and MAP4 in cardiomyocytes before and after myocardial infarction.

a, Subcellular fractionation of wild-type cardiomyocytes, isolated from mice after myocardial infarction and transduced with adenovirus-mediated Vash2 or control shRNA. Representative western blots of fraction in PEB, with the same membrane stained with Ponceau S. b, Representative western blot of PEB extractions denatured in the presence or absence of urea from cardiomyocytes after myocardial infarction. c, d, STED images of MAP4 and α-tubulin in cardiomyocytes of Mark4−/− or control mice at baseline or after myocardial infarction. c, Representative images. Oligomerized puncta are indicated within the square frames. Scale bar, 2 μm. d, Quantification of the presence of the MAP4 oligomerized puncta in the following groups: Mark4+/+ baseline (n = 2 mice, n = 22 cardiomyocytes examined over 2 independent experiments), Mark4+/+ myocardial infarction (n = 2 mice, n = 26 cardiomyocytes examined over 2 independent experiments) and Mark4−/− myocardial infarction (n = 2 mice, n = 21 cardiomyocytes examined over 2 independent experiments). e, f, Western blot of native gels loaded with samples in CEB of cardiomyocytes isolated at baseline or after myocardial infarction. e, The presence of pMAP4(S1046) and total MAP4 is indicated. f, Coomassie-stained native gel loaded with the same amounts of proteins as used in e. g, Western blot of the CEB fraction denatured in the presence of urea, with a Coomassie-stained denaturing gel loaded with the same amounts of protein. h, Western blot of fractions in PEB of cardiomyocytes isolated from Mark4−/− or control mice after myocardial infarction, with a Coomassie-stained gel loaded with the same amounts of proteins. i, Quantification of VASH2 and desmin levels in PEB fraction (n = 4 mice per group). j, Correlation between desmin and VASH2 levels in PEB. d, i, Data are mean ± s.e.m.; two-tailed unpaired t-test. j, Two-tailed correlation test. P values are indicated.

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Extended Data Fig. 9 MARK4 overexpression regulates MAP4 phosphorylation, and the presence of MAP4 oligomers in the cytosolic fraction.

a–c, Subcellular fractionation of wild-type cardiomyocytes transduced with adenovirus to overexpress Mark4 or a null control. a, Representative western blots of fractions in CEB or PEB (derived from the same experiment). b, Quantification of pMAP4(S1046) in CEB and VASH2 level in PEB (n = 5 mice per group, blots were processed in parallel). c, Correlation between VASH2 level in the PEB fraction and pMAP4 levels. d, e, STED images of MAP4 and α-tubulin in wild-type cardiomyocytes at baseline transduced with adenovirus to overexpress Mark4 or a null control. d, Representative images. Scale bar, 2 μm. e, Quantification of MAP4 oligomerized puncta in the following groups: overexpression of control (n = 2 mice, n = 20 cardiomyocytes examined over 2 independent experiments) and overexpression of Mark4 (n = 2 mice, n = 24 cardiomyocytes examined over 2 independent experiments). d, e, Data are mean ± s.e.m.; two-tailed unpaired t-test. c, Two-tailed correlation test. P values are indicated.

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Extended Data Fig. 10 VASH2 status in cardiomyocytes before and after myocardial infarction, and the schematic summary of the results.

a, b, STED images of VASH2 and α-tubulin in wild-type cardiomyocytes at baseline or after myocardial infarction. a, Representative images. Scale bar, 2 μm. b, Pearson correlation coefficient of VASH2 and α-tubulin signals, percentage of VASH2 signals on the polymerized microtubules and percentage of VASH2 signals off the microtubules in the following groups: wild-type baseline (n = 4 mice, n = 38 cardiomyocytes examined over 2 independent experiments) and wild-type myocardial infarction (n = 38 cardiomyocytes of n = 6 mice, n = 38 cardiomyocytes examined over 3 independent experiments). c, Real-time PCR of cardiomyocytes after myocardial infarction from the following groups: Mark4+/+ myocardial infarction (n = 5 mice) and Mark4−/− myocardial infarction (n = 6 mice). d, Quantification of VASH2 mean fluorescence intensity (MFI) within the cell area (region of interest (ROI)) using the STED images from the following groups: Mark4+/+ myocardial infarction (n = 6 mice, n = 38 cardiomyocytes examined over 3 independent experiments) and Mark4−/− myocardial infarction (n = 6 mice, n = 47 cardiomyocytes examined over 3 independent experiments). b–d, Data are mean ± s.e.m.; two-tailed unpaired t-test. P values are indicated. e, A working model for the MARK4-dependent regulation of microtubule detyrosination after myocardial infarction. After ischaemic injury, increased MARK4 phosphorylates MAP4 at its KXGS motifs. Phosphorylated MAP4 either changes its conformation on the polymerized microtubules or detaches itself from the polymerized microtubules to form oligomerized MAP4 structures in the cytosol. The phosphorylation of MAP4 by MARK4 allows for space access of VASH2 to the polymerized microtubules, thereby promoting α-tubulin detyrosination. As a consequence, the increased level of detyrosinated microtubules causes a reduction in the contractile function of the cardiomyocyte.

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Supplementary information

Supplementary Figures

This file contains Supplementary Figs 1-2. Supplementary Fig. 1 contains immunoblots, and loading controls (Coomassie stained gels and Ponceau S stained membranes) associated with the data presented in the Figures and Extended Data Figures. Supplementary Fig. 2 shows the gating strategy for infiltrating immune cells post-myocardial infarction.

Reporting Summary

Supplementary Table 1

The effect of MARK4 deficiency on cardiac parameters after myocardial infarction (MI). M-mode echocardiography was acquired at baseline, and week 1 (W1), week 2 (W2), week 4 (W4) post-myocardial infarction (MI). All statistical tests were Two-way ANOVA with Bonferroni post-hoc correction for multiple comparisons. All data are mean±s.e.m. P>0.05 is considered statistically non-significant.

Supplementary Table 2

The effect of MARK4 deficiency on immune cells infiltrating the hearts after myocardial infarction (MI). Immune cells were collected from hearts at day 3 post-MI or post-sham surgery. Data were analyzed using unpaired t-test (two-tailed), and presented as mean±s.e.m. P>0.05 is considered statistically non-significant.

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Yu, X., Chen, X., Amrute-Nayak, M. et al. MARK4 controls ischaemic heart failure through microtubule detyrosination. Nature 594, 560–565 (2021). https://doi.org/10.1038/s41586-021-03573-5

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