Injectable human recombinant collagen matrices limit adverse remodeling and improve cardiac function after myocardial infarction

Abstract

Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium's mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.

Fig. 1

Preparation and physical characterization of injectable rHC matrices. a Schematic depicting the components, delivery, and reparative properties of the injectable material presented in this study. b Denaturation temperatures (T_d) (°C; n = 3), (c) in vitro degradation in collagenase (mg/min; n = 3), and (d) viscosity measurements (Pa·s; n = 7) for rHCI and rHCIII matrices. e Pore size distribution for the rHCI (gray bars) and rHCIII (black bars) matrices calculated from 250 individual pores per sample. Inset: Representative Cryo-SEM images of the rHC matrices. Scale bar = 200 µm. f Representative images of ex vivo imaging of MI mouse hearts at 2 h, 2 days, or 7 days, after treatment with the rHC matrices, labeled using Alexa-Fluor®594-NHS dye. Bottom histograms display the average total fluorescence emission for the different time points (n = 4). Also shown are representative histological sections of the myocardium at 2 days post injection. Arrows indicate representative regions containing the collagen matrices labeled with the fluorescent dye. P-values were determined by a two-tailed t test. For b–d , ± data are presented as the mean ± SD and in f ± corresponds to SEM. Source data are provided as a Source Data file. For b–e, n indicates number of hydrogel batches. For f, n is the number of mice per group

Fig. 2

rHC matrices improve cardiac function post-MI and restore mechanical properties. a Left ventricular ejection fraction (LVEF) at baseline (time of treatment delivery) and at 28 days post treatment (n = 11 for PBS, n = 15 for rHCI, and n = 13 for rHCIII). Significance determined by a two-way ANOVA followed by Holm’s correction for multiple comparisons. b–f The change in (b) fractional area change (FAC), (c) end-systolic volume (ESV), (d) end-diastolic volume (EDV), (e) stroke volume (SV), and (f) cardiac output (CO) between baseline and 28 days post-treatment (n = 11 for PBS, n = 15 for rHCI, and n = 13 for rHCIII). P-values were determined by an ANOVA followed by Holm-corrected multiple comparisons. g Young’s modulus (YM) for scar/border zone tissue measured at 2 and 28 days (n = 3–4). h Left: Strain force reached at the aortic valve closure within the mid anterior LV wall at 2 days post injection for the different experimental groups (n = 5–12). Right: Representative longitudinal strain curves for the PBS and rHCI groups. The yellow traces correspond to the anterior mid region. Significance determined by a one-way ANOVA followed by Dunnett’s comparison as well as a two-tailed Student’s t test within a treatment group over time. The data are presented as the mean ± SEM. Source data are provided as a Source Data file. For a–h, n indicates the number of mice per group

Fig. 3

Morphology of MI hearts is improved by rHCI treatment. a Representative images of hearts harvested 28 days after treatment (bar = 2 mm). b Scatter plot for the heart mass/tibia length ratio measured 28 days post injection (n = 8–9). P-values were determined by a one-way ANOVA followed by Holm’s multiple comparisons to PBS control. c Representative Masson’s trichrome-stained sections of hearts harvested at 28 days (bar = 1 mm). d Wall thickness of the remote myocardium measured in Masson’s trichrome-stained tissue sections at 28 days post treatment (n = 7–8). P-values were determined by one-way ANOVA using Holm’s multiple comparison. e Scar size (% of LV) measured at 28 days post treatment in Masson’s trichrome-stained tissue sections (n = 7–8). P-values were determined by multiple regression analysis, including treatment group (rHCI, rHCIII and PBS) and baseline LVEF. rHCI and rHCIII as compared with PBS were significant predictors of infarct size, in addition to baseline LVEF. The correlation coefficient for the model is 0.6. The data are presented as the mean ± SEM. Source data are provided as a Source Data file. For a–e, n indicates the number of mice per group

Fig. 4

rHC matrix increases vascularity and preserves cardiomyocytes post-MI. a Immunohistochemistry analysis and quantification (number/mm²) of capillary and arteriole number in the myocardial scar and border zone after treatment with PBS, rHCI, or rHCIII. CD31, red; α-SMA, green; DAPI, blue (n = 6–7). b Immunohistochemistry analysis and quantification of total area (in mm²) positive for cardiac troponin I (cTnI) staining in the border zone after treatment with PBS, rHCI, or rHCIII. cTnI, red; wheat germ agglutinin, green; DAPI, blue. c Immunohistochemistry analysis and quantification of connexin 43 (Cx43) expression (percent area) in the remote area after treatment with PBS, rHCI, or rHCIII. Cx43, green; cTnI, red; DAPI, blue (n = 6–7). P-values were determined by one-way ANOVA using Holm’s multiple comparison. The data are presented as the mean ± SEM. Source data are provided as a Source Data file. For a–c, n indicates the number of mice per group

Fig. 5

rHC matrix treatment alters bone marrow cell and macrophage response. a Immunohistochemistry analysis and quantification of the number of CD206⁺ macrophages (per mm²) in the scar, border zone and remote myocardium of MI hearts treated with PBS, rHCI, or rHCIII. CD206, green; DAPI, blue (n = 6–7). b Flow-cytometry analysis for the number of GFP⁺, GFP⁺F4/80⁺, GFP⁺CD38⁺, and GFP⁺CD11b⁺ cells recruited from the bone marrow to the left ventricle of Cxcr3-EGFP MI mice 2 days after PBS, rHCI, or rHCIII treatment (n = 9–10). P-values were determined by one-way ANOVA using Holm’s multiple comparison. The data are presented as the mean ± SEM. Source data are provided as a Source Data file. For a, b, n indicates the number of mice per group

Fig. 6

rHCIII matrix increases Ly6C^hi monocyte mobilization and accumulation in the heart. Flow-cytometry plots show F480 and Ly6C expression of mononuclear cells (CD45⁺CD11b⁺Ly6G^-) in the blood (a) and heart (b) of mice at 2 days post treatment. Macrophages were considered F480⁺, while monocytes were F480⁻ and classified based on the expression of the Ly6C marker. The data are presented as the mean ± SEM (n = 5–7). P-values were determined by a one-way ANOVA for effect of treatment followed by Holm’s correction for multiple comparisons. Source data are provided as a Source Data file. For a, b, n indicates the number of mice per group

Fig. 7

rHC matrices support wound-healing macrophages. a The number of adherent macrophages after culture on rHC matrices (n = 4). b Number of macrophages that migrated through 300 µm thickness rHC matrices (n = 4). c M1 and M2 macrophage polarization determined by CD86 and CD206 expression, respectively, after culture on rHC matrices (n = 4). d MMP1 and Arg1 mRNA expression in macrophages cultured on rHC matrices for 72 h relative to TCPS (n = 4). e Percentage of dead macrophages (7-AAD⁺ cells) after 3 h incubation with H₂O₂ (n = 4). f Percentage of dead neonatal rat ventricular myocytes (NRVMs) after 3 h incubation with H₂O₂ (n = 4). P-values were determined by a one-way ANOVA using Holm’s multiple comparison (c, d, e, and f), or by a two-tailed t test (a and b). All data are presented as the mean ± SD. Source data are provided as a Source Data file. For a–e, n indicates the number of mice (mouse bone marrow) per group. For f, n indicates the number of hydrogel batches