Insulin-like growth factor-binding protein-7 (IGFBP7) links senescence to heart failure

Abstract

Heart failure (HF) is a rising global cardiovascular epidemic driven by aging and chronic inflammation. As elderly populations continue to increase, precision treatments for age-related cardiac decline are urgently needed. Here we report that cardiac and blood expression of IGFBP7 is robustly increased in patients with chronic HF and in an HF mouse model. In a pressure overload mouse HF model, Igfbp7 deficiency attenuated cardiac dysfunction by reducing cardiac inflammatory injury, tissue fibrosis and cellular senescence. IGFBP7 promoted cardiac senescence by stimulating IGF-1R/IRS/AKT-dependent suppression of FOXO3a, preventing DNA repair and reactive oxygen species (ROS) detoxification, thereby accelerating the progression of HF. In vivo, AAV9-shRNA-mediated cardiac myocyte Igfbp7 knockdown indicated that myocardial IGFBP7 directly regulates pathological cardiac remodeling. Moreover, antibody-mediated IGFBP7 neutralization in vivo reversed IGFBP7-induced suppression of FOXO3a, restored DNA repair and ROS detoxification signals and attenuated pressure-overload-induced HF in mice. Consequently, selectively targeting IGFBP7-regulated senescence pathways may have broad therapeutic potential for HF.

Fig. 1. Elevated plasma IGFBP in HF is correlated with chronic inflammation and accelerated cellular senescence.

a, IGFBP7 and NT-proBNP protein concentrations in plasma samples of patients with HFpEF (n = 106), patients with HFrEF (n = 207) and controls (n = 98) are presented as mean ± s.e.m. in scatter plots; one-way ANOVA with Bonferroni correction for multiple comparisons was used to calculate P value. b, ROC curve shows that the addition of IGFBP7 to NT-proBNP II values significantly improved the diagnostic performance in the discrimination of HFpEF from HFrEF. c, Correlation analysis revealed that increased plasma IGFBP7 level in HFpEF correlates with decreased diastolic function of the heart. d, Heat map shows changes of SASP proteins in plasma samples of HFpEF (n = 13), HFrEF (n = 26) and controls (HC) (n = 9); protein expression is shown as fold change against control group. P < 0.05–0.0001 for all panels. e, Relative gene expression of key senescence markers in whole blood samples; CDKN2A (p16), CDKN1A (p21) and TP53 (p53) are shown as fold changes against HC (n = 15 per group). Error bars represent s.e.m. One-way ANOVA with Bonferroni correction for multiple comparisons was used to calculate P values. RA, right atrial; RVSP, right ventricular systolic pressure.

Fig. 2. Cardiac expression of IGFBP7 is increased in patients with HF and mouse models of HF.

a, Representative immunoblotting and quantification show that IGFBP7 protein expression was markedly increased in both heart tissue lysates and plasma samples of patients with HF compared to non-HF controls; expression levels were normalized to total proteinn (n = 4 per group). b,c, Representative confocal microscopy images examined over two independent experiments showing significantly increased IGFBP7 in cardiomyocytes of infarct zone of a patient who suffered from HF after myocardial infarction (MI) compared to normal zone of the same patient (b) and elevated Igfbp7 staining in cardiomyocytes of TAC mouse heart compared to sham control 8 weeks after surgery (c). Heart sections were probed with anti-IGFBP7 antibody and visualized by staining with Alexa Fluor 555 secondary antibody (red), and nuclei were stained with DAPI (blue). In all images, scale bars are 30 μm. d, Representative immunoblotting and quantification show that Igfbp7 protein expression was markedly increased in TAC heart compared to sham heart 8 weeks after surgery; expression levels were normalized to total protein (n = 7 per group). e, Relative Igfbp7 gene expression in TAC and sham mouse hearts is shown as fold changes against sham control (n = 5 per group). f, Elevated serum Igfbp7 was also evidenced in TAC mouse measured by ELISA 8 weeks after TAC (n = 8 per group). g, Representative immunoblots and quantification show that Igfbp7 protein expression is increased in aged C57BL/6 mouse (24-month-old) heart compared to young (3-month-old) heart; relative Igfbp7 protein level is shown as fold changes against young (n = 3 per group). h, Representative immunoblots show that Igfbp7 protein was significantly upregulated in neonatal rat cardiomyocytes (rNCMs) treated with various hypertrophic stimuli in vitro. Gapdh was used as loading control. i,j, Representative confocal microscopy images showing, upon exposure to cardiac stressors, upregulated Igfbp7 (red) relocated toward the plasma membrane in rNCMs (i) and IGFBP7 (red) co-localized with vesicular structures (green) that labeled with FM 1-43FX lipophilic styryl dye in AC16 hCMs in vitro (j). In all images, scale bars are 20 μm. In all panels, error bars represent s.e.m. Unpaired two-tailed t-tests were used to calculate P values. Source data

Fig. 3. Igfbp7 deficiency protects mice from pressure-overload-induced HF.

Igfbp7^−/− and WT mice were subjected to TAC or sham surgery and analyzed 2–8 weeks after the operation. HW/TL (a) and LW/TL (b) ratio at 8 weeks (n = 30 WT and KO sham, n = 31 WT TAC and n = 38 KO TAC mice). c, Representative WGA staining of transverse heart (8 weeks) cross-sections showing myocyte cross-sectional area (scale bars, 20 μm) and quantitation (n = 3 WT sham, n = 11 WT TAC, n = 5 KO sham and n = 11 KO TAC images examined over slides from three mice of each group). d, Relative gene expression of Nppb, Nppa and Myh7/Myh6 ratio in TAC and sham heart 8 weeks after surgery are shown as fold changes against WT sham group (n = 9 per group). e, Echocardiographic assessment of cardiac function at 8 weeks after surgery. IVRT and mitral E/E′ ratio measured by transmittal Doppler flow velocity are shown (n = 15 WT sham, n = 16 WT TAC, n = 7 KO sham and n = 13 KO TAC mice). f, Measurement of cardiac function by PV conductance catheterization 8 weeks after sham and TAC surgery. LVEDP and isovolumic relaxation constant (Tau) (Weiss model) are shown (n = 10 WT sham, n = 11 WT TAC, n = 8 KO sham and n = 16 KO TAC mice). g, Representative PSR staining for collagen of transverse heart cross-sections and quantization showing increased collagen deposition in WT TAC heart. This is much attenuated by Igfbp7 deficiency. Scale bars, 500 μm. n = 10 WT sham, n = 8 WT TAC, n = 8 KO sham and n = 7 KO TAC images were examined over slides from three mice of each group. h, Immunoblotting and quantification for Ctgf in heart extracts (n = 6 mice examined over two independent experiments). Gapdh was used as loading control. i, Relative Ctgf/Hprt1 and Tgfb2/Hprt1 expression in TAC and sham heart 2–8 weeks after surgery is shown as fold change against WT sham group (n = 6 mice examined over two independent experiments per group). In all panels, error bars represent s.e.m. One-way ANOVA with Bonferroni correction for multiple comparisons was used to calculate P values. Source data

Fig. 4. IGFBP7 is required for stress-induced cellular senescence.

a–c, Igfbp7^−/− and WT mice were subjected to TAC or sham surgery and analyzed 2–8 weeks after the operation. a, qRT–PCR measurement of relative Il-6 and Il-1β expression shown as fold change against WT sham group in heart samples 8 weeks after surgery (n = 6 WT sham, n = 6 KO sham, n = 6 KO TAC and n = 7 WT TAC mice examined over two independent experiments). b, Measuring of cytokine levels in blood samples of TAC mice 2 weeks or 8 weeks after the operation by ELISA indicated that Igfbp7 deficiency blocked TAC-triggered elevation of Il-6, Tnf-α, Kc/Gro and Il-33 in serum (n = 7 for 8-week samples, n = 3 for 2-week samples). c, Relative telomere length measured by quantitative PCR in 8-week post-surgery mouse heart is shown as fold change against WT sham group (n = 9 WT sham, n = 9 WT TAC, n = 9 KO sham and n = 14 KO TAC mice examined over three independent experiments). d,e, Representative immunoblotting and quantification for cellular senescence markers p16ARC and p21 (CIPI/WAF1) (d) and 53BP1, phosphor (pp53^ser392) and total p53 (e), in either nuclear fraction (p16ARC) or whole heart extracts. Histone H3 or Gapdh were used as loading control. Protein expression is shown as fold change against WT sham group (n = 3 per group). f, qRT–PCR measurement of senescence marker Tp53 and Cdkn1a expression in heart samples 2 weeks or 8 weeks after surgery. Gene expression is shown as fold change against WT sham group (n = 6 mice examined over two independent experiments). g, Representative immunoblotting shows that the innate immune cGAS–STING cytosolic DNA sensing pathway was activated in WT TAC heart. Vinculin was used as loading control. h–i, Igfbp7^−/− and WT mNCMs were subjected to Dox treatment to induce cellular senescence. h, Representative immunoblotting for cellular senescence markers acetylated p53 (acetyl-p53) and total p53 in Dox (1 μM) or Dox (1 μM) + trichostatin A (400 nM) treated mNCMs for 72 hours. Solvent-treated cells were use as control. Gapdh was used as loading control. i, Representative microscopy images showing that Igfbp7 deficiency decreased senescence-associated β-galactosidase-positive cells (blue) in Igfbp7^−/− mNCMs compared to WT mNCMs. Scale bar, 20 μm. In all panels, error bars represent s.e.m. One-way ANOVA with Bonferroni correction for multiple comparisons was used to calculate P values. Source data

Fig. 5. IGFBP7 promotes cardiac senescence by simulating IGF-1R/IR-dependent suppression of FOXO3a.

Igfbp7^−/− and WT mice were subjected to TAC or sham surgery and analyzed 8 weeks after the operation. Representative immunoblotting (a–d) and quantification (g) for activation of IGF-1 receptor (a), shown by the ratio of phosphor-IGF-1R to total IGF-1R; IRS-1 (b), shown by the ratio of phosphor-IRS-1 to total IRS-1; its downstream activation of Akt (c), shown by the ratio of phospho-Akt to total Akt; and inactivation of FoxO3a transcription factor (d), shown by the ratio of phospho-FoxO3a and total FoxO3a to Gapdh. Gapdh was used as loading control. Representative immunoblotting (e–f) and quantification (g) show that downregulation of FoxO3a targets Ddb1 in WT mouse hearts (e), and acetylation of Sod2 (Sod2^K68A), as shown by Sod2^K68Ac to total Sod2, was upregulated in WT TAC hearts (f). Total protein was used as loading control. Protein expression is shown as fold change against WT sham group (n = 3 for pIgf-1R^b, pIrs1³¹⁸, pAkt³⁰⁸, Ddb1 and Sod2^k68Ac over one experiment; n = 6 for pIrs1^S612 over two independent experiments; and n = 9 for pAkt^S473, pFoxO3a^S253 and total FoxO3a over three independent experiments). h, Increased catalase activity in Igfbp7^−/− TAC hearts as measured by catalase activity assay (n = 5 per group). i, Relative expression of key FoxO3a-targeted genes Gadd45a, Ddb1, Sod2, Cad and cdkn1b in heart samples 8 weeks after surgery. Gene expression is shown as fold change against WT sham group (n = 9 per group over three independent experiments). In all panels, error bars represent s.e.m. One-way ANOVA with Bonferroni correction for multiple comparisons was used to calculate P values. j, Diagram shows proposed IGFBP7 action in the stress myocardium. Source data

Fig. 6. Inhibition of IGFBP7 suppressed IGF1R/AKT-induced FOXO3a inactivation.

a, Knockdown of IGFBP7 reduced Ang II-induced CDKN1a and TP53 upregulation in hCMs. Gene expression is shown as fold change against control siRNA-treated hCMs (n = 3 per group). Error bars represent s.e.m. One-way ANOVA with Bonferroni correction for multiple comparisons was used to calculate P values. b, Representative microscopy images showing that IGFBP7 knockdown decreased senescence-associated β-galactosidase-positive cells in hCMs. Scale bar, 20 μm. c, Representative confocal microscopy images showing that IGFBP7 (red) co-localized with IGF-1Rβ (green) in hCMs. Nuclei were stained with DAPI (blue). Scale bars are 10 μm in all images. d, Knockdown of IGFBP7 by siRNA in hCMs blocked IGF-1, and insulin induced AKT phosphorylation; representative immunoblotting of phosphor-AKT and total AKT is shown. Gapdh was used as loading control. e, Representative immunoblotting shows that knockdown of IGF-1R by siRNA in AC16 human cardiomyocytes has the same effect as IGFBP7 knockdown in regulating AKT/FOXO3a signaling, which indicates that IGFBP7 regulates AKT/FOXO3a signaling through IGF-1R. f–h, AC16 hCMs were infected with ad-IGFBP7-6xHis and subjected to immunoprecipitation with anti-6xHis Dynabeads; the pulldown was analyzed by western blot with indicated antibodies; and the co-immunoprecipitation pulldown bands are indicated by red arrows. Representative immunoblotting shows that both IGF-1Rβ (f) and IR (g) were co-immunoprecipitated by anti-6xHis pulldown, whereas deletion of the N-terminal IGFBP motif, as in Ad-ΔIGFBP-6xHis-infected cells, reduced IGFBP7 co-immunoprecipitation with IGF-1Rβ (h). Immunoblotting with anti-IGFBP7 or anti-6xHis tag was used as control. c, Control. Source data

Fig. 7. AAV9-mediated cardiac-myocyte-specific knockdown of Igfbp7 rescued TAC-induced HF in mice.

AAV9-mediated shRNA knockdown of Igfbp7 in cardiac myocytes by echo-guided LV injection of AAV9-Igfbp7-shRNA in post-TAC mice hearts. a,b, At day 3 after surgery and injection, mCherry staining of heart sections tracked AAV9-mCherry-U6-mIgfbp7-shRNA to cardiomyocytes (a) (scale bars, 50 μm); meanwhile, qRT–PCR analysis showed that there is a significantly decreased Igfbp7 mRNA expression in the hearts of AAV9-mCherry-U6-mIgfbp7-shRNA-injected mice (n = 4) compared to AAV9-scrmb-shRNA-injected mouse hearts (n = 4) (b). c–g, Four weeks after TAC and injection, Igfbp7 knockdown attenuated TAC-induced HW/BW and LW/BW increase in AAV9-mCherry-U6-mIgfbp7-shRNA-injected mice (n = 11) compared to control shRNA-injected mice (n = 9) (c). d, Representative WGA staining of transverse heart cross-sections showing myocyte cross-sectional area, which demonstrates that Igfbp7 knockdown attenuated TAC-induced cardiac myocyte enlargement. Scale bars, 50 μm. e, Representative PSR staining of heart sections shows increased collagen deposition in control AAV9-scrmb-shRNA-injected TAC hearts, and this is much attenuated by Igfbp7 knockdown with AAV9-mCherry-U6-mIgfbp7-shRNA. Scale bars are 4 mm for top panels and 100 μm for bottom panels. f, Echocardiographic analysis shows improved cardiac function in AAV9-mCherry-U6-mIgfbp7-shRNA-injected mice (n = 11) compared to control shRNA-injected mice (n = 9). g, qRT–PCR revealed that there is a trend of decreased key senescence gene Trp53 and Cdkn1a expression in AAV9-mCherry-U6-mIgfbp7-shRNA-injected mice (n = 7) compared to control shRNA-injected mice (n = 9). In all panels, error bars represent s.e.m. Unpaired two-tailed t-tests were used to calculate P values.

Fig. 8. Antibody-mediated IGFBP7 depletion in vivo in mice rescued TAC-induced HF.

a, Representative immunoblotting showing that blocking IGFBP7 by neutralizing anti-IGFBP7 antibody in human AC16 cardiomyocytes attenuates IGF-1-induced AKT activation and the phosphorylation of FOXO3a. Total protein was used as loading control. b–j, C57BL/6 mice were subjected to TAC operation and immediately received subcutaneous injection of either 20 μg of rabbit monoclonal IGFBP7 antibody or 20 μg of anti-rabbit IgG isotopy control, followed by analysis at a variety of timepoints after the operation. b, ELISA results show that anti-IGFBP7 antibody injection diminished TAC-induced Igfbp7 protein induction in both heart and serum 5 days after treatment (n = 4 per group). c, HW/TL and LW/TL ratio at 4 weeks (n = 14 for each group). d, Representative WGA staining of 4-week post-treatment transverse heart cross-sections showing myocyte cross-sectional area. e, Representative PSR staining of 4-week post-treatment transverse heart cross-sections shows increased collagen deposition in control IgG-injected TAC heart, and this is much attenuated by Igfbp7 depletion with antibody. In both d and e, scale bars are 2 mm for top panels and 50 μm for bottom panels. f, Transmittal Doppler flow velocity assessment of cardiac function at 4 weeks after TAC and antibody injection; IVRT and mitral E/A ratio are shown (n = 14–15 per group). g, Measurement of cardiac function by PV conductance catheterization at 4 weeks after surgery further indicated that antibody treatment preserved LV function; isovolumic relaxation constant (Tau) (Weiss model) and LVEDP are shown (n = 9–10 per group). h, Representative immunoblotting and quantification for activation of Akt, shown by the ratio of phospho-Akt to total Akt, and inactivation of FoxO3a, shown by the ratio of phospho-FoxO3 and total FoxO3. Total protein was used as loading control. Protein expression was shown as fold change against control IgG-treated group (n = 3 per group). i, Relative expression of key FoxO3a target genes Gadd45a, Ddb1, Sod2 and cdkn1b in heart samples 4 weeks after TAC and antibody injection. Gene expression is shown as fold change against control IgG-treated group (n = 12–15 per group). j, Representative immunoblotting and quantification showing that cellular senescence markers acetylated p53 (Ap53^L379) and total p53 and p16ARC, in whole heart extracts; total protein was used as loading control. Protein expression is shown as fold change against control IgG group (n = 3 per group). In all panels, error bars represent s.e.m. Unpaired two-tailed t-tests were used to calculate P values. Ab, antibody; Ctl, Control. Source data

Extended Data Fig. 1. IGFBP7 is independently informative of processes contributing to HF from pro-BNP.

(a) Correlation analysis revealed there is poor correlation between the level of IGFBP7 and NT-proBNP, whether in the HFpEF, HFrEF or the overall HF population (r value between 0.30 and 0.40). (b) Correlation analysis revealed there is reasonable correlation between myocardial levels of IGFBP7 and circulating levels of IGFBP7, with r value of 0.7. Correlation analyses were performed with GraphPad Prism 9 and the Pearson registration analysis were summarized in the right panel next to each plot.

Extended Data Fig. 2. Hypertrophic stress induced IGFBP7 elevation were more evidenced in cardiac myocytes.

(a) Igfbp7 protein expression was markedly increased in TAC heart compared with sham heart 8 weeks post-surgery measured by ELISA, shown as ng/ml of normalized whole heart lysate; n = 4 per group, unpaired two-tailed Student t-test was used to calculate p value. (b–c) Selected confocal microscope images of multiplex IHC with either (b) Igfbp7 and isolectin GS-IB4 (endothelial marker), or (c) Igfbp7 with Vimentin (myofibroblast marker) shows TAC induced increase of Igfbp7 protein was more evidenced in cardiac myocytes; less in cardiac microvascular endothelial cells and myofibroblasts. 8 weeks post sham or TAC mouse heart sections were stained with rabbit polyclonal anti-Igfbp7 antibody (abcam ab74169), visualized with Alexa Flour 647 chicken anti-rabbit IgG (ThermoFisher A21443), counter stained with Alexa Flour 568 conjugated Isolectin GS-IB4 (ThermoFisher, I21412) to visualize microvasculature or anti-vimentin antibody to visualize myofibroblast. Scale bar = 30 μm in panel b, and 20 μm in panel c. (d) Representative immunoblots showing increased IGFBP7 protein expression was more evidenced in primary human cardiac myocytes (hCM) than cardiac fibroblasts (hCF) and cardiac microvascular endothelial cells (hCMEC) (all from PromoCell). Total proteins were used as loading controls. (e) qRT–PCR analysis shows there is a significantly increased Igfbp7 mRNA expression in hCM compared with hCF and hCMEC. IGFBP7 expression was shown as fold change again non-treated hCM. The data was from two independent treated cell culture experiment. Error bars represent s.e.m., one-way ANOVA with Bonferroni correction for multiple comparisons were used to calculate p values. Source data

Extended Data Fig. 3. Igfbp7 deficiency protects mice from pressure overload induced cardiac dysfunction.

(a) Heart weight/body weight (HW/BW) and lung weight/body weight (LW/BW) ratio at 8 weeks, n = 30 WT and KO sham, 31 WT TAC and 38 KO TAC mice, one-way ANOVA with Bonferroni multiple comparisons was used to calculate p values. (b) Representative images of pressure–volume loop recordings of WT and Igfbp7^-/- TAC and sham mice, respectively. (c) Echocardiographic assessment of cardiac function at 8 weeks post-surgery, ejection fraction (EF) % and corrected LV mass were shown. n = 15 WT sham, 16 WT TAC, 7 Ko sham and 13 KO TAC mice, one-way ANOVA with Bonferroni multiple comparisons was used to calculate p values. In all panels error bars represent s.e.m.

Extended Data Fig. 4. Inhibition of IGFBP7 suppressed IGF1R/AKT induced FOXO3a inactivation.

(a) Decreased Igf-1 level in Igfbp7^-/- mice hearts and serum as measured by Igf-1 Elisa assay 2 weeks post-surgery. n = 6 for heart lysates, and n = 3 for serum. Error bars represent s.e.m., one-way ANOVA with Bonferroni multiple comparisons was used to calculate p values. (b) Representative immunoblot shown Igfbp7 deficiency diminished Igf-1 induced Akt and FoxO3a phosphorylation in mouse neonatal cardiomyocytes (mNCM). Gapdh was used as loading control. Source data

Extended Data Fig. 5. Requirement of IGFBP motif in modulating IGF-1R/IRS signaling.

(a) Diagrams representing 6xHis-tagged WT (ad-IGFBP7) and mutant IGFBP7 (ad-ΔSP, ad-ΔIGFBP, ad-ΔKazal and ad-ΔIgLD) adenovirus (Ad) constructs. (b,c) AC16 cells were infected with Ad-hIGFBP-6xhis for 48 hours. (b) Representative immunoblot shows IGF-1R and IR binding adaptor protein IRS1 and IRS2 were pulldown by IP with anti-6xHis Dynabeads. (c) Representative immunoblot shows IGFBP7 were pull down by reverse IP with anti-IR antibody; The co-IP poll-down bands were indicated by red arrows. (d,e) AC16 cells were infected with Ad-hIGFBP7 (WT), its mutant forms and control (ad-GFP) for 48 hours. (d) Representative immunoblot shows deletion of IGFBP motif, reduced IGFBP7 co-IP with IGF-1Rβ. (e) Representative immunoblot shown ad infected 6xHis-tagged hIGFBP7 and its mutant forms were readily detected by immunoblot with anti-6His antibody as indicated by red cycle; and deletion of IGFBP motif, diminished AKT phosphorylation. Red cycle indicated down regulation of phosphor-Akt. GAPDH was used as loading control.

Extended Data Fig. 6. In silicon analysis of publicly available scRNA-seq dataset.

In silicon analysis of publicly available scRNA-seq dataset of normal adult mouse heart reveals Igfbp7 mRNA is expressed in multiple types of cells of the heart. (a) Unbiased clustering of scRNA-seq data from normal adult mouse hearts. (b) Featureplot showing expression of Igfbp7 in multiple cardiac cell populations.

Extended Data Fig. 7. Selection and testing the specificity of IGFBP7 antibody for in vivo neutralization of IGFBP7.

(a) Representative immunoblot shown seven IGFBP7 antibodies were tested for its binding efficiency to native recombinant human IGFBP7 protein (hIGFBP7) by IP in cell free system, and IGFBP7 recombinant rabbit monoclonal antibody (clone 65, Invitrogen), as indicated by red cycle, has been selected. The IP were pull down with dynabeads protein G immunoprecipitation kit, and blot with anti-6xHis antibody. The first wash through were used as Input. (b–c) Representative immunoblot shown blocking IGFBP7 by neutralizing anti-IGFBP7 antibody attenuate IGF-1 induced AKT activation in both (b) primary human cardiac myocytes (hCM) and (c) mouse neonatal cardiac myocyte (mNCM). Total proteins labeled by no stain protein labeling reagent was used as loading control. (d) Representative immunoblotting shows Rm anti- IGFBP7 antibody (clone 68) is only binding to recombinant human IGFBP7 protein (rhIGFBP7) but not recombinant human IGFBP3 protein (rhIGFBP3); and the Rm anti-IGFBP3 antibody (ERP18680-153) is only binding to rhIGFBP3, but not hrIGFBP7. Source data

Extended Data Fig. 8. Antibody-mediated IGFBP7 inhibition in vivo rescued TAC-induced HF.

(a) Mortality rate of IGFBP7 antibody and control IgG treated TAC mice is shown by Kaplan–Meier plots as the survival proportion of each cohort of mice. Survival incidence in mice over the indicated follow-up period was shown as the ratio of the number of mice survived/total mice analyzed for each group. This represents 70% (7/10) for IGFBP7 antibody+TAC mice, 50% (5/10) for control IgG+TAC mice on day 40. (b) Heart weight/body weight and lung weight/body weight ratio at 4 weeks, n = 14 for each group, unpaired two-tailed t-tests was used to calculate p values. (c–e) Representative images of (c) Transmittal doppler flow velocity, (d) PW tissue doppler and (e) pressure–volume loop recordings of TAC + antibody and TAC + IgG mice, respectively.