Cyclin D2-cardiomyocyte transplantation may be a potential therapeutic strategy for the repair of infarcted hearts

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In a large-animal study, researchers have shown that heart attack recovery is aided by injection of heart muscle cells derived from human induced pluripotent stem cell line, or hiPSCs, that overexpress cyclin D2.

This research, published in the journal Circulation, used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has higher clinical relevance to human disease, compared to studies in mice.

An enduring challenge for bioengineering researchers is the failure of the heart to regenerate muscle tissue after a heart attack has killed part of its muscle wall. That dead tissue can strain the surrounding muscle, leading to a lethal heart enlargement.

Heart experts thus have sought to create new tissue – applying a patch of heart muscle cells or injecting heart cells – to replace damaged muscle. Similarly, they have tried to stimulate division of existing heart muscle cells near the damaged area. This current study, led by researchers at the University of Alabama at Birmingham, shows progress in both goals.

After the experimental heart attack, heart tissue around the infarction site was injected with about 30 million bioengineered human cardiomyocytes that were differentiated from hiPSCs. These cells also overexpress cyclin D2, part of a family of proteins involved in cell division.

Compared to control human cardiomyocytes, the cyclin D2-cardiomyocytes showed enhanced potency to repair the heart. They proliferated after injection, and by four weeks, the hearts had less pathogenic enlargement, reduced size of dead muscle tissue and improved heart function.

Intriguingly, the cyclin D2-cardiomyocytes stimulated not only their own proliferation, but also proliferation of existing heart muscle cells around the infarction site of the pig heart, as well as showing angiogenesis, the development of new blood vessels.

“These results suggest that the cyclin D2-cardiomyocyte transplantation may be a potential therapeutic strategy for the repair of infarcted hearts,” said study leader Jianyi “Jay” Zhang, M.D., Ph.D., the chair of Biomedical Engineering, a joint department of the UAB School of Medicine and the UAB School of Engineering.

This ability of the graft cyclin D2-cardiomyocytes to stimulate the proliferation of nearby existing heart cells suggested paracrine signaling, a type of cellular communication where a cell produces a signal that induces changes in nearby cells.

Exosomes – small blebs or tiny vesicles that are released by human or animal cells and contain proteins and RNA from the cells that release them – are one common form of paracrine signaling.

Zhang and colleagues found that exosomes that they purified from the cyclin D2-cardiomyocyte growth media indeed promoted proliferation of cultured cardiomyocytes. In addition, the treated cardiomyocytes were more resistant to programmed cell death, called apoptosis, induced by low oxygen levels.

The exosomes also induced proliferation of various other cell types, including human umbilical vein endothelial cells, human vascular smooth muscle cells and 7-day-old rat cardiomyocytes that have almost undetectable proliferation.

Part of the cargo that exosomes carry are microRNAs, or miRNAs. These short pieces of RNA have the ability to interact with messenger RNA in target cells, and they are robust players of gene regulation in cells. Humans have more than 2,000 miRNAs with different RNA sequences, and these are thought to regulate a third of the genes in the genome.

So, the researchers documented which microRNAs were present in exosomes from the cyclin D2-overexpressing cardiomyocytes and in exosomes from non-overexpressing cardiomyocytes. As expected, they found differences.

Together, the exosomes from both types of cells contained 1,072 different miRNAs, and 651 were common to the two exosome groups. However, 332 miRNAs were found only in the cyclin D2-overexpressing cardiomyocytes, and 89 miRNAs were specific for the non-overexpressing cardiomyocytes.

In preliminary work of characterizing the effects of specific miRNAs, one particular miRNA from the cyclin D2-overexpressing exosomes was shown to stimulate proliferation when delivered into rat cardiomyocytes.

“Thus, as the therapeutic potential of exosomes for improving cardiac function becomes more evident, combining an exosome-mediated delivery of proliferative miRNAs with transplantation of cyclin D2-overexpressing cardiomyocytes, or cell products, could become a new promising strategy for upregulating proliferation of the recipient cardiomyocytes and reducing cardiac fibrosis,” Zhang said.

“Altogether, our data suggest that cardiac cell therapy, involving cardiomyocytes with enhanced proliferation capacity, may become an efficacious future strategy for myocardial repair and prevention of congestive heart failure in patients with acute myocardial infarctions.”


Molecular Signals that Stimulate Cardiomyocyte Proliferation

Given the sharp decline in cell cycle activity and loss of regenerative potential in adult mammalian hearts, considerable effort has been made to understand the cellular mechanism(s) that underlie cardiomyocyte cell division. A number of groups have determined that within the cardiomyocyte, signaling from growth factors, intrinsic pathways, microRNAs and cell cycle regulators stimulate cardiogenesis following injury (Fig. 2).

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Figure 2:
Molecular signals that stimulate cardiac repair.Exogenous signaling by growth factors, such as NRG/ERBB and IGF family members, activates intracellular pathways that promote cardiomyocyte (CM) proliferation and regeneration mechanisms. The Hippo kinase pathway is an intrinsic signaling pathway that triggers inhibitory phosphorylation of the CM proliferation protein Yap. This leads to either Yap sequestration at the plasma membrane with the dystrophin glycoprotein complex (DGC) or in the cytosol with 14–3-3 (dashed arrows). Treatment of CMs with agrin or anti-Hippo (shSalv) molecules promotes localization of dephosphorylated Yap to the nucleus to drive expression of CM renewal genes. MicroRNAs (miRs) can positively or negatively regulate CM proliferation, and development of anti-miR antagomiR molecules have innovated cardiac repair strategies. The CM cell cycle can be reactivated via modulation of regulators such as Cyclin A2 and member of the Cyclin B and Cyclin D families. Delivery of Wee1 and TGFb inihibitor molecules enhances cyclin/CDK-induced cardiac repair. CM proliferation: positive regulators (green), negative regulators (red). . (Illustration Credit: Ben Smith).

Cell Cycle Regulators
As neonatal life ends, cardiac expression of cell cycle regulators such as cyclins and cyclin-dependent kinases is largely silenced32. During the cell cycle, Cyclin A2 positively regulates G1/S and G2/M transitions, whereas Cyclin D2 drives G1/S progression. Overexpression studies in mice suggest that these genes promote proliferation of mature cardiomyocytes in adult hearts. Constitutive cardiac expression of Cyclin A2 in transgenic mice produces cardiomegaly due to the upregulation of cardiomyocyte proliferation69.

Following ischemic injury, Cyclin A2 transgenic mice show less ventricular dilation and enhanced cardiac function, concomitant with cell cycle reentry in infarct and border zone myocardium70. Studies in rats and pigs revealed that viral delivery of Cyclin A2 to infarcted hearts was protective to ischemic injury71, 72.

Likewise, transgenic overexpression of Cyclin D1 and D2 promotes cardiomyocyte cell cycle activity in infarcted hearts as evidenced by increased multinucleation, DNA synthesis, and proliferation73, 74. Recent investigation of the cardiomyocyte cell cycle reveals that combinatorial overexpression of cell-cycle regulators CDK1/CCNB/CDK4/CCND (referred to as 4F) activates proliferation of post-mitotic cardiomyocytes75.

In this study, the authors used rigorous lineage tracing to determine that viral delivery of 4F induced cardiomyocyte proliferation, cell survival and functional recovery to infarcted hearts75. The replacement of CDK1/CCNB with Wee1 and TGFβ inhibitor molecules further enhanced cell-cycle re-entry of cardiomyocytes75, supporting the notion that cell cycle reactivation, if used in a safe manner, may be useful as a therapeutic strategy for cardiac repair.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534162/


More information: Meng Zhao et al, Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction, Circulation (2021). DOI: 10.1161/CIRCULATIONAHA.120.049497

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