In mammals, including humans, the cells that contract the heart muscle and enable it to beat do not regenerate after injury. After a heart attack, there is a dramatic loss of these heart muscle cells and those that survive cannot effectively replicate. With fewer of these contractile cells, known as cardiomyocytes, the heart pumps less blood with each beat, leading to the increased mortality associated with heart disease.
University of Pennsylvania researchers have developed an injectable hydrogel that can deliver microRNAs (miRNAs) directly into heart muscle, triggering proliferation of cardiomyocytes after a heart attack and helping recovery in mouse models. The scientists hope that the technology might represent a new therapeutic approach that exploits miRNAs to promote self-repair of the heart, and potentially other tissues, after injury. “We’re seeing a change in approaches for regenerative medicine, using alternatives to stem cell delivery,” comments co-researcher Jason Burdick, Ph.D., professor in bioengineering in Penn Engineering. “Here, instead of introducing new cells that can have their own delivery challenges, we’re simply turning on repair mechanisms in cells that survive injury in the heart and other tissues.”
The University of Pennsylvania team describes their developments in Nature Biomedical Engineering, in a paper entitled “Sustained miRNA Delivery from an Injectable Hydrogel Promotes Cardiomyocyte Proliferation and Functional Regeneration after Ischaemic Injury.”
Heart disease contributes to 600,000 to 4,000,000 deaths every year in the U.S. and Europe, respectively, and myocardial infarction (MI) is a contributing factor in at least 50% of deaths, the researchers report. A heart attack occurs when blood flow to the heart muscle is compromised, causing death of the contractile cardiomyocytes. These cardiomyocytes can’t regenerate after injury, and are replaced by fibrotic tissue, which means that the heart can’t function and pump blood as effectively. “This limited renewal capacity exacerbates the damage from ischaemic injury during MI, as damaged cells are replaced with fibrotic scar tissue rather than contractile cardiomyocytes,” the authors write.
Approaches to developing cell therapy for replacing lost cardiomyocytes have focused on the potential use of a number of cell types, including mesenchymal stem cells, and embryonic, or induced pluripotent stem cell-derived cardiomyocytes. Alternative techniques have attempted to promote the proliferation of endogenous cardiomycytes using growth factors, small molecules, and gene therapy, and some miRNAs.
The University of Pennsylvania team has previously identified a group of miRNAs (miR-302/367) that induce proliferation in cardiomyocytes by blocking the signaling pathways that inhibit the cells from replicating. One of the challenges of using miRNAs, however, is the ability to deliver the right dose to the right place. And while delivering high doses of miRNAS systemically might increase the amount that reaches the heart tissue, it could also increase the risk of off-target, tumorigenic effects. “Biologic drugs turn over very fast,” comments Edward Morrisey, Ph.D., professor in medicine, member of the cell and molecular biology graduate group, and scientific director of the Penn Institute for Regenerative Medicine in Penn Medicine. “The miRNAs that we used last less than eight hours in the bloodstream, so having a high local concentration has strong advantages.”
The University of Pennsylvania team has now developed a injectable hyaluronic acid hydrogel that can be used to deliver miRNAs directly into heart tissue. “We want to design the right material for a specific drug and application,” Burdick explains. “The most important traits of this gel are that it’s shear-thinning and self-healing. Shear-thinning means it has bonds that can be broken under mechanical stress, making it more fluid and allowing it to flow through a syringe or catheter. Self-healing means that when that stress is removed, the gel’s bonds re-form, allowing it to stay in place within the heart muscle.”
The gel structure also includes attachment sites for miRNAs, which are released into the heart tissue and cardiomyocytes as the gel breaks down. Encapsulation of the miRNAs also protects against degradation, increasing the period of effectiveness, without increasing the likelihood that the molecules will be taken up into other off-target cells.
The researchers first injected a hydrogel carrying a mimic of miRNA-302 (miR-302) into the hearts of normal healthy mice and found that within days the animals’ cardiac tissues showed increased biomarkers of cardiomyocyte proliferation. The team then evaluated the effects of miRNA hydrogel in the Confetti mouse, a rodent model in which individual cardiomyocytes express one of four different fluorescent proteins. The results indicated that individual cardiomyocytes in treated hearts were dividing in response to the miRNA-hydrogel therapy. In subsequent tests, the team injected the miRNA hydrogel directly into the hearts of mice after a heart attack. In these animals, treatment triggered the color-coded cardiomyocytes to proliferate into clusters of the same color cell.
Encouragingly, when the team next looked at the clinical effects of the miRNA-hydrogel therapy, they found that the treated animals showed greater recovery compared with control animals, including a higher ejection fraction and reduced cardiac enlargement, a common consequence of a heart attack, which leads to the development of scar tissue in place of the affected cardiomyocytes.
Importantly, a single injection of the hydrogel led to sustained, local cardiomyocyte proliferation for two weeks. “The present study demonstrates that an engineered hydrogel, designed for injection and sustained delivery of miR-302, promotes both cardiomyocyte proliferation and functional regeneration,” the authors write. “Currently, there are no approved treatments that regenerate myocardium; in this regard, our system may have unique improvements over other existing treatments for MI.”
The team acknowledges that more research will be needed to optimize the gel-miRNA formulation for use in larger animal models of MI. Even so, they suggest, “this study establishes the proof of concept of a technology to permit minimally invasive, sustained miRNA delivery that can be tailored towards other small RNAs for application to cardiac and other tissues.”
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