A recent study using a large animal model has shown that recovering from a heart attack can be improved by injecting spheroids of heart muscle cells. These cells are derived from human induced pluripotent stem cells (hiPSCs) that have been modified to overexpress cyclin D2 and lack human leukocyte antigen classes I and II. This research, which appeared in the journal Circulation Research, utilized a pig model for heart attacks, as pig hearts are anatomically and physiologically more similar to human hearts compared to mouse hearts, making the findings more relevant for human conditions.
Researchers from the University of Alabama at Birmingham (UAB), led by Jianyi “Jay” Zhang, M.D., Ph.D., and Lei Ye, M.D., Ph.D., developed specialized hiPSCs, known as KO/OEhiPSCs. When these cells were transformed into cardiomyocyte spheroids and implanted into pig hearts after ischemia/reperfusion injury, the results showed markedly enhanced heart function and a reduced size of infarcts after four weeks.
“It’s well understood that the size of the infarct correlates directly with how the left ventricle remodels post-infarction and the risk of developing heart failure,” said Zhang. “In our study, at four weeks following ischemia/reperfusion, we noted a significant 35.8 percent reduction in the infarct area in pigs treated with KO/OEhiPSC-cardiomyocyte spheroids compared to those treated with a basic medium, as well as compared to those treated with wildtype-hiPSC-cardiomyocyte spheroids.”
The improvements observed resulted from an unexpected finding: the stimulation of endogenous heart muscle cells within the pig hearts. This discovery is significant since mammalian heart muscle cells typically lose their ability to divide shortly after birth. Consequently, following a heart attack, the heart cannot regenerate muscle cells in the scarred areas left behind. Many previous trials involving the injection of muscle cells into damaged hearts have faced challenges due to these cells not successfully integrating and proliferating.
In this study, the implanted spheroids did not remain in the pig hearts, yet marked improvements in heart function and decreased infarct size were still evident. Although initial engraftment was observed at one week, by week four it had almost completely vanished. Instead, researchers noted a considerable increase in the proliferation of existing pig cardiomyocytes—heart muscle cells that normally do not divide in healthy adult hearts. These cells expressed higher levels of markers for cellular division and genes involved in DNA replication. Additionally, they showed increased activity in three signaling pathways: the Mitogen-Activated Protein Kinase pathway, the HIPPO/YAP pathway, and the Transforming Growth Factor Β pathway.
The research team examined the cell-surface receptors connected to these pathways and evaluated the extracellular proteins that interact with these receptors. They found no significant differences in the expression of extracellular proteins in the endogenous cardiomyocytes. This indicated that the observed increased proliferation might stem from extracellular proteins produced by injected KO/OEhiPSC-cardiomyocytes.
Cytokine profiling of the KO/OEhiPSC-cardiomyocytes identified follistatin, a type of autocrine glycoprotein, as a likely factor stimulating the proliferation of heart muscle cells. Follistatin was found to be secreted in high amounts by the KO/OEhiPSC-cardiomyocytes. In laboratory tests, human cardiomyocytes significantly increased in number, with a 30 percent rise when treated with follistatin compared to control groups. In an in vivo mouse heart attack model, UAB researchers observed that injecting follistatin prompted the proliferation of adult mice cardiomyocytes after a myocardial infarction. Further studies confirmed that follistatin influences the HIPPO/YAP signaling pathway, which promotes cardiomyocyte growth.
“To our knowledge, this is the first research showing that follistatin triggers the proliferation of hiPSC-cardiomyocytes as well as cardiomyocytes from adult mammalian hearts,” stated Zhang. “The specific mechanisms by which follistatin encourages cardiomyocyte proliferation still need to be understood.”
The need for new treatments for heart attack patients is critical—heart failure accounts for 13 percent of worldwide deaths, with half of all heart failure patients passing away within five years. When coronary arteries are blocked during a heart attack, the heart muscle cells die. As the muscle tissue is replaced with dense scar tissue that receives little blood flow, the affected heart loses its ability to contract, leading to heart enlargement, a gradual decline in pumping capacity, an increased risk of dangerous heart rhythms, and ultimately end-stage heart failure.
This study builds upon earlier research from 2021 by Zhang and colleagues, which indicated that heart attack recovery could be facilitated through the injection of muscle cells that overexpress cyclin D2, although those experiments were conducted in immunocompromised mice. The current study utilized hypoimmunogenic and cyclin D2-overexpressing hiPSC-cardiomyocytes in a large animal model, promoting the potential for clinical application and improved effectiveness of this innovative treatment strategy.
“This emphasizes the considerable potential of KO/OEhiPSC-cardiomyocytes in encouraging the proliferation of endogenous cardiomyocytes in the hearts of adult patients,” Zhang noted.
Co-authors of the study “Follistatin from hiPSC-cardiomyocytes promotes myocyte proliferation in pigs with postinfarction LV remodeling,” along with Ye and Zhang, include Yuhua Wei, Gregory Walcott, Thanh Nguyen, Xiaoxiao Geng, Bijay Guragain, Hanyu Zhang, Akazha Green, Manuel Rosa-Garrido, and Jack M. Rogers from UAB’s Department of Biomedical Engineering; as well as Daniel J. Garry from UAB’s Department of Medicine, Division of Cardiovascular Disease.
This research was supported by grants from the National Institutes of Health: HL114120, HL131017, HL134764, HL160476, and HL49137.
At UAB, Zhang is the holder of the T. Michael and Gillian Goodrich Endowed Chair of Engineering Leadership. The Biomedical Engineering department collaborates closely with the Marnix E. Heersink School of Medicine and the UAB School of Engineering. Medicine is a department under the Heersink School of Medicine.
