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

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.


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!5 integrin 1:250, Cat#ab150361, Abcam Antibodies used in this study were optimized for their use. The endpoints for optimal dilutions depended the assay (histology vs. flow cytometry). Dilutions used in our study are described in our manuscript, and further experimental protocols can be obtained directly from our team.
Neonatal rat ventricular myocytes (NRVMs) Sprague-Dawley rats and bone marrow-derived macrophages C57BL/6J mice. Those cells were isolated freshly in our laboratories.
NVRMs were isolated following a protocol described in J Physiol 593, 1147-1157 (2015), Dr. Liang is an expert on this protocol. As per the macrophages, those were isolated following a protocol described in CSH protocols 2008CSH protocols , pdb.prot5080 (2008.
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Monocyte subset experiment: Two days post-treatment injection mice were sacrificed by CO2 inhalation followed by cervical dislocation. Blood was collected from mice through cardiac puncture in a 50 mM EDTA solution and RBCs were lysed with red blood cell (RBC) lysis buffer according to the manufacturer's protocol (Biolegend 420301). Hearts were perfused with PBS and cells were isolated from harvested mouse hearts using a digestion buffer containing: DNAse I (50 U/µL; Sigma D5025), collagenase type II (400 U/mL; ThermoFisher 17101015), collagenase D (0.15 U/mL; Sigma 11088866001) and hyaluronidase (10 U/mL; Sigma H3506). Following an hour digestion at 37ºC, isolated heart cells were passed through a 70 µm filter. Finally, harvested mouse spleens were mashed and triturated through a 70 µm filter followed by incubation with red blood cell ( with Zombie Aqua fixable viability dye (1:500, Biolegend 423101) for 20 min at room temperature. Next, Fc receptors were blocked with TruStain X reagent (1:100, Biolegend 103319) for 10 min at room temperatue. The cells were then incubated with an antibody cocktail for 45 min at room temperature containing CD45-APC/Fire 750 , CD11b-PE/Cy7, Ly6G-PerCP/Cy5.5, CD3-PE, B220-AF488, F480-AF647 and Ly6C-BV421. Cx3cr1 Experiment: To evaluate the recruitment of circulating mononuclear cells to the myocardium following rHC treatment, B6.129P-Cx3cr1 tm1Litt/J mice (Cx3cr1-EGFP) were purchased from the Jackson Laboratory and used as previously described 61. Briefly, at 1 week post-MI, mice received treatment of 50"l of PBS, rHCI or rHCIII, as described above. Animals were sacrificed 2 days after treatment and hearts were perfused with PBS and the right ventricle and the apical region of the left ventricle were collected. The tissues were rinsed with HBSS and digested in 2.4U/ml dispase I (Roche) and 1mg/ml Collagenase B (Roche) for 40 min at 37°C . Samples were washed 3 times with PBS, centrifuged for 5 min at 400g, and the isolated cells were prepared for flow cytometry . Cells were labeled with APC anti-mouse/human CD11b, PE anti-mouse Ly-6G/6C, PE/Cy5 anti-mouse F4/80, PE/Cy7 anti-mouse CD38 and Alexa Fluor® 700 anti-mouse CD206, following the manufacturer-recommended dilutions.