Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

Despite the high prevalence of ischemic heart diseases worldwide, no antibody-based treatment currently exists. Starting from the evidence that a specific isoform of the Bone Morphogenetic Protein 1 (BMP1.3) is particularly elevated in both patients and animal models of myocardial infarction, here we assess whether its inhibition by a specific monoclonal antibody reduces cardiac fibrosis. We find that this treatment reduces collagen deposition and cross-linking, paralleled by enhanced cardiomyocyte survival, both in vivo and in primary cultures of cardiac cells. Mechanistically, we show that the anti-BMP1.3 monoclonal antibody inhibits Transforming Growth Factor β pathway, thus reducing myofibroblast activation and inducing cardioprotection through BMP5. Collectively, these data support the therapeutic use of anti-BMP1.3 antibodies to prevent cardiomyocyte apoptosis, reduce collagen deposition and preserve cardiac function after ischemia.


SUPPLEMENTARY FIGURE 1. Characterization of anti BMP1.3 antibody a.
Coomassie staining of DMP-1 full-length and its cleaved fragments incubated for the indicated time points with BMP1.3 recombinant protein, either alone or in combination with anti-BMP1.3 antibody. The first two lanes show DMP-1 stability between time 0 (immediate loading on gel) and after 20 hour-incubation in the same buffer. b. Coomassie staining of DMP-1 full-length and its cleaved fragments incubated for the indicated time points with BMP1.1 recombinant protein, either alone or in combination with anti-BMP1.3 antibody. In a and b experiments were repeated independently three times with similar results. c, d. Pharmacokinetic curve of the monoclonal mouse anti-BMP1.3 antibody, showing its concentration in the serum after a single dose intravenous (c) or intraperitoneal (d) injection in three rats over 384 hours. For details see Methods.
SUPPLEMENTARY FIGURE 2. Additional parameters of cardiac function in isoproterenol-treated rats and infarcted mice. a. Schematic representation of anti-BMP1.3 antibody administration (arrowheads) and echocardiography in mice subjected to MI by left anterior descendent (LAD) coronary artery ligation (black timeline) and rats treated with isoproterenol (Ip, grey timeline). b. Quantification of Troponin-T levels in plasma of control rats (sham), rats treated with isoproterenol alone (control) or in combination with anti-BMP1.3 antibody (150 µg/kg) at the indicated time points. c-h. Quantification of Fractional shortening (c) end-systolic Left Ventricular Internal Diameter (d, LVIDs), end-diastolic Left Ventricular Internal Diameter (e, LVIDd), end-systolic Left Ventricular Anterior Wall thickness (f., LVAWs), end-diastolic Left Ventricular Anterior Wall thickness (g, LVAWd) and Left Ventricular Anterior Wall thickening (h) in infarcted mice in the absence of treatment (control) or treated with the indicated dose of anti-BMP1.3 antibody. Data in b-h are shown as mean±s.e.m. Sample number is indicated inside or above each bar. Statistical significance was determined using two-way ANOVA followed by Tukey's multiple comparison test in b-h. Source data are provided as a Source Data file.

SUPPLEMENTARY FIGURE 3. Comparative analysis of cardiac function in mice treated with different anti-fibrotic drugs after MI.
Evaluation of the ejection fraction in mice subjected to MI and randomly assigned to the following experimental groups: i) IgG1 (isotype control); ii) anti-BMP1.3 antibody; iii) anti-TGFβ1 antibody, iv) SB-431542 (small molecule blocking multiple TGFβ receptors). Data are shown as mean±s.e.m. Sample number is indicated inside or above each bar. Statistical significance was determined using two-way ANOVA followed by Bonferroni's multiple comparison test. Source data are provided as a Source Data file.

Validation of BMP1.3 ELISA kit
To validate our home-made ELISA, we followed the procedures indicated by Andreasson et al. 2015Andreasson et al. (doi.org/10.3389/fneur.2015). First, we generated a standard curve using human recombinant BMP1.3 (8 biological replicates). As shown in Supplementary Figure 8, the assay provided reliable results in the 1,56 -100 ng/mL range. Shown are mean ± SD.

SUPPLEMENTARY FIGURE 8.
We then estimated the precision of the assay. First, we determined the following precision profile, based on back-calculated standard concentrations from 8 independent runs.

SUPPLEMENTARY FIGURE 9.
As evident from the profile curve in Supplementary Figure 9, the assay was precise in the 20-80 ng/mL range. Additional precision experiments were performed on three different pooled samples divided in 15 aliquots. Five aliquots for each sample were analyzed in three independent runs in three different days. Results are shown in Supplementary Table 1.  To investigate if the concentration-response relationship was similar in the calibration curve and in samples, we performed a recovery test, by adding three different concentrations (5, 10 and 15 ng/sample) of recombinant BMP1.3 to human plasma samples and calculating the recovery using the following formula: % recovery =