Evaluating effects of the protein components of an infusible decellularized extracellular matrix material for targeting and treating leaky vasculature following myocardial infarction

Introduction: Current treatments for myocardial infarction (MI) and ischemia reperfusion (I/R) injury fail to address the overreactive inflammatory response, resulting in negative left ventricular (LV) remodeling and heart failure. Previous decellularized extracellular matrix materials have demonstrated pro-regenerative effects but require direct administration for delivery. We recently developed an infusible ECM material (iECM) derived from porcine LV myocardium, which can be delivered through intracoronary infusion, targets the leaky vasculature of the infarct, and exerts immunomodulatory and pro-regenerative effects. To better understand the mechanism of action of iECM, components were isolated and their biochemical properties, in vitro biological effects, and in vivo targeting ability were assessed.

Materials and

Methods: iECM was manufactured by first digesting decellularized porcine LV myocardium, separating large particulates through centrifugation, then filtering the supernatant through a 0.2 µm filter. Size exclusion chromatography (SEC) was used to quantify the molecular weight of iECM components and separate components into fractions. Protein and peptide composition of iECM fractions were analyzed through SDS-PAGE and morphology was characterized by transmission electron microscopy (TEM). Fractions were analyzed for their biochemical properties, including free amines and thiols, which can mediate reactive oxygen species scavenging, and evaluated for their ability to promote survival of cardiomyocytes undergoing oxidative stress in vitro. For in vivo biodistribution, iECM and iECM fractions (10 mg/mL) were delivered through simulated intracoronary infusion immediately after reperfusion in a rat I/R model of MI. One and 3 days post infusion, rats were euthanized, and the heart and satellite organs were collected for analysis through near infrared imaging and immunohistochemical staining.

Results, Conclusions, and Discussions:

Results: SEC analysis of iECM revealed a bimodal distribution with either high molecular weight (HMW) components (>80 kDa) or peptide sized components ( < 5 kDa) (fig. 1A). Following fractionation at a molecular weight cutoff of 30 kDa, biochemical analysis demonstrated differences in free amines and free thiols between peptide and HMW fractions, with the peptide components of iECM containing more free amines and thiols. Protein composition analysis of iECM fractions also showed differences, with HMW iECM components appearing similar to fibrillar collagens based on findings from SDS-PAGE and TEM. Despite differences in structure and biochemical properties, the iECM fractions’ ability to promote cell survival was the same when tested in vitro. However, significant differences in the iECM fractions’ ability to localize to the site of infarct were observed, with HMW components localizing primarily to the infarct, similar to complete iECM (fig. 1B, 1D, 1E), while sub-10kDa components were spread throughout the heart, with no preference to the infarcted LV (fig 1B, 1C, 1F).

Conclusions: Based on studying the components of iECM, a possible mechanism of targeting to the LV following MI has been elucidated. High molecular weight proteins with characteristics akin to fibrillar collagen appear to more readily target the infarct compared to the peptide sized components of iECM, suggesting their importance in the reduction in vascular permeability, and immunomodulatory and pro-regenerative effects previously observed. Ongoing studies will evaluate the in vivo biological effects of iECM fractions following administration.

Acknowledgements (Optional): This work was supported in part by the NHLBI through training grant T32HL0074444.