Heart attack recovery enhanced by human stem cell spheroids, study shows

In a large-animal model study, researchers have found that heart attack recovery is aided by injection of heart muscle cell spheroids derived from human induced pluripotent stem cells, or hiPSCs, that overexpress cyclin D2 and are knocked out for human leukocyte antigen classes I and II. This research, published in the journal Circulation Research, used a pig model of heart attacks. Pig hearts more closely resemble the human heart in size and physiology, and thus have a higher clinical relevance to human disease, compared to studies in mice.

University of Alabama at Birmingham researchers, led by Jianyi "Jay" Zhang, M.D., Ph.D., and Lei Ye, M.D., Ph.D., generated the human leukocyte antigen-knockout and cyclin D2-overexpressing hiPSCs, called ^KO/OEhiPSCs. When ^KO/OEhiPSCs were differentiated into cardiomyocyte spheroids and implanted into the pig hearts that had undergone ischemia/reperfusion injury, the ^KO/OEhiPSC-cardiomyocyte implantation resulted in significantly improved cardiac function and reduced infarct size after four weeks.

It is widely acknowledged that the infarct size is linearly related to the severity of post-infarction left ventricle remodeling and the occurrence of heart failure. In our current study, at week 4 after ischemia/reperfusion, we observed a significant 35.8 percent decrease in the infarct area in the hearts treated with the ^KO/OEhiPSC-cardiomyocyte spheroids compared with those treated with basal medium, and a significant reduction compared with wildtype-hiPSC-cardiomyocyte spheroid-treated pigs."

Jianyi "Jay" Zhang, M.D., Ph.D.

These improvements were due to a surprising finding - proliferation of endogenous heart muscle cells in the pig hearts. This is particularly noteworthy because shortly after birth mammalian heart muscle cells lose their ability to divide. Thus, a damaged heart after a heart attack cannot repair itself by growing new muscle cells in the scar area left by the heart attack. Many previous pig model preclinical trials to inject new heart muscle cells into the damaged heart have been blunted by general failure of the cells to engraft and grow.

In the current study, the spheroids that the UAB researchers injected failed to persist, despite finding significant improvement in heart function and infarct size in the damaged pig hearts. Engraftment was seen at week 1, but was nearly undetectable at week 4. Instead, the researchers found significant increases in the proliferation of endogenous pig cardiomyocytes, those preexisting heart muscle cells that are unable to divide in normal adult hearts. These proliferating cells showed elevated expression levels of cellular proliferation markers, and they expressed genes for DNA replication. The pig cardiomyocyte cells also showed upregulation of three signaling pathways - the Mitogen-Activated Protein Kinase pathway, the HIPPO/YAP pathway and the Transforming Growth Factor Β pathway.

Researchers analyzed the heart cells for cell-surface receptors that are associated with the three pathways, and they looked for differential extracellular protein expression of proteins that interact with those cell-surface markers receptors. They did not see differential expression of the extracellular proteins in the endogenous cardiomyocytes. This suggested that the enhanced proliferation of the endogenous heart muscle cells might instead be due to extracellular proteins produced by the injected ^KO/OEhiPSC-cardiomyocytes.

Cytokine arrays of the ^KO/OEhiPSC-cardiomyocytes identified follistatin, an autocrine glycoprotein, as the potential inducer of the heart muscle cell proliferation. Follistatin was found to be highly secreted by ^KO/OEhiPSC-cardiomyocytes. In cell culture, human cardiomyocytes significantly proliferate, and the total number cardiomyocytes increased by 30 percent when treated with follistatin as compared to the control groups. In an in vivo mouse model of heart attacks, the UAB researchers found that injected follistatin induced proliferation of adult mouse cardiomyocytes after myocardial infarction. Other experiments confirmed that follistatin targets the HIPPO/YAP signaling pathway to promote the growth of cardiomyocytes.

"To our knowledge, this is the first report demonstrating that follistatin promotes the proliferation of hiPSC-cardiomyocytes and cardiomyocytes from adult mammalian hearts," Zhang said. "The mechanisms by which follistatin activates cardiomyocyte proliferation have yet to be deciphered."

The need for a new therapy for heart attack patients is great. Heart failure is responsible for 13 percent of deaths worldwide, and half of patients with heart failure die within five years. Blockage of coronary arteries in a heart attack leads to death of the cardiomyocyte heart muscle cells. When that muscle tissue is replaced by dense scar tissue with little blood circulation, the infarcted heart loses contractile power, leading to heart enlargement, progressive loss of pumping ability, increased chance of ventricular arrhythmias and clinical end-stage heart failure.

The current study advances a 2021 study by Zhang and colleagues that showed heart attack recovery could be aided by injection of heart muscle cells that overexpress cyclin D2. However, these experiments were done in immunocompromised mice. The current study developed and tested hypoimmunogenic and cyclin D2-overexpressing hiPSC-cardiomyocytes in a large-animal model for possible clinical translation and enhanced therapeutic efficacy of this promising treatment approach.

"This highlights the significant potential of ^KO/OEhiPSC-cardiomyocytes to stimulate endogenous cardiomyocyte proliferation in the hearts of adult patients," Zhang said.

Co-authors with Ye and Zhang in the study, "Follistatin from hiPSC-cardiomyocytes promotes myocyte proliferation in pigs with postinfarction LV remodeling," are Yuhua Wei, Gregory Walcott, Thanh Nguyen, Xiaoxiao Geng, Bijay Guragain, Hanyu Zhang, Akazha Green, Manuel Rosa-Garrido and Jack M. Rogers, UAB Department of Biomedical Engineering; and Daniel J. Garry, UAB Department of Medicine, Division of Cardiovascular Disease.

Support came from National Institutes of Health grants HL114120, HL131017, HL134764, HL160476 and HL49137.

At UAB, Zhang holds the T. Michael and Gillian Goodrich Endowed Chair of Engineering Leadership. Biomedical Engineering is a joint department in the Marnix E. Heersink School of Medicine and the UAB School of Engineering. Medicine is a department in the Heersink School of Medicine.