A Tissue Repair Protein to Improve Cardiomyocyte Survival after Acute Myocardial Infarction

Introduction: Myocardial infarction (MI) is the leading cause of death in the United States. MI results in extensive cardiac cell death, and timely intervention could reduce the severity of cardiac damage. However, enhancing cardiomyocyte survival under the ischemic conditions of MI remains a significant challenge. In response, we have developed an innovative drug delivery system designed for use during the acute phase of MI, which shows promise in efficiently rescuing cardiac cells under ischemic conditions. This system incorporates a cardiomyocyte-targeted, tissue repair protein Mitsugumin 53 (MG53) within an injectable hydrogel. MG53 is known for enhancing cell survival by repairing the cell membrane and activating prosurvival pathways. The targeting feature allows for specific delivery to cardiomyocytes, enhancing their survival.

Materials and

Methods: The cardiomyocyte-targeting MG53 (CT-MG53) was engineered by genetically attaching a cardiomyocyte-binding sequence to the C-terminus of MG53. The protein was expressed in E. coli BL21(DE3) cells harboring the requisite plasmid and was labeled using a Dylight 633 fluorescent kit. Neonatal cardiomyocytes were isolated from 3-5 day old pups and cultured on collagen-coated plates. To assess CT-MG53 binding, cardiomyocytes were treated with 1 μM of the protein for 3 hours and subsequently stained with α-actinin. For in vivo studies, 0.1 mL of a 200 μg/mL CT-MG53 solution was injected into the myocardium of mice post-MI induction. Hearts were harvested and imaged using IVIS 24 hours post-injection. To evaluate cell survival under ischemic conditions, treated neonatal cardiomyocytes were cultured in DMEM at 1% oxygen for 5 days, with survival quantified using a double-stranded DNA (dsDNA) assay kit. The injectable, fast gelation, and thermosensitive hydrogel was synthesized by free radical polymerization of N-isopropylacrylamide, acrylate-oligolactide, 2-hydroxyethyl methacrylate and 4-(acryloxymethyl)-phenylboronic acid pinacol ester.

Results, Conclusions, and Discussions: The synthesis of CT-MG53 was verified by SDS-PAGE. In vitro experiments demonstrated effective binding of CT-MG53 to cardiomyocytes. Post-delivery, CT-MG53 showed greater retention in the infarcted myocardium compared to non-targeted MG53, as confirmed by IVIS imaging. Under ischemia-mimicking conditions, CT-MG53 significantly enhanced cardiomyocyte survival, with efficacy comparable to that of non-targeted MG53. To administer CT-MG53 following acute MI, we designed a release system utilizing an injectable, rapidly gelling, and biodegradable hydrogel as the carrier. The rapid gelation property ensures that the hydrogel, along with the encapsulated CT-MG53, retains effectively within the infarct area upon injection. The CT-MG53 was able to gradually release from the hydrogel. 65% of the encapsulated protein was released over a period of four weeks (Figure 1). Given these characteristics, we anticipate that the CT-MG53 delivery system will effectively aid in the rescue of cardiac cells when deployed in infarcted hearts post-MI.

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