Background: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2 ^WT CMs or CCND2 ^OE CMs, respectively) and injected into infarcted pig hearts. Methods: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2 ^WT CMs or CCND2 ^OE CMs (3×10 ⁷ cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. Results: The number of the engrafted CCND2 ^OE CMs exceeded that of the engrafted CCND2 ^WT CMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2 ^WT CMs or the delivery vehicle, the administration of CCND2 ^OE CM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2 ^OE CM-treated than from the CCND2 ^WT CM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2 ^OE CMs (CCND2 ^OE Exos) than from the CCND2 ^WT CMs (CCND2 ^WT Exos). As demonstrated by our study, CCND2 ^OE Exos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2 ^OE Exos versus CCND2 ^WT Exos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. Conclusions: We demonstrated that enhanced potency of CCND2 ^OE CMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2 ^OE CMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

The failure of adult cardiomyocytes to reproduce themselves to repair an injury results in the development of severe cardiac disability leading to death in many cases. The quest for an understanding of the inability of cardiac myocytes to repair an injury has been ongoing for decades with the identification of various factors which have a temporary effect on cell‐cycle activity. Fetal cardiac myocytes are continuously replicating until the time that the developing fetus reaches a stage of maturity sufficient for postnatal life around the time of birth. Recent reports of the ability for early neonatal mice and pigs to completely repair after the severe injury has stimulated further study of the regulators of the cardiomyocyte cell cycle to promote replication for the remuscularization of injured heart. In all mammals just before or after birth, single‐nucleated hyperplastically growing cardiomyocytes, 1X2N, undergo ≥1 additional DNA replications not followed by cytokinesis, resulting in cells with ≥2 nuclei or as in primates, multiple DNA replications (polyploidy) of 1 nucleus, 2X2(+)N or 1X4(+)N. All further growth of the heart is attributable to hypertrophy of cardiomyocytes. Animal studies ranging from zebrafish with 100% 1X2N cells in the adult to some strains of mice with up to 98% 2X2N cells in the adult and other species with variable ratios of 1X2N and 2X2N cells are reviewed relative to the time of conversion. Various structural, physiologic, metabolic, genetic, hormonal, oxygenation, and other factors that play a key role in the inability of post‐neonatal and adult myocytes to undergo additional cytokinesis are also reviewed.

The meager regenerative capacity of adult mammalian hearts appears to be driven by the proliferation of endogenous cardiomyocytes; thus, strategies targeting mechanisms of cardiomyocyte cell cycle regulation, such as the Hippo/Yes-associated protein (Hippo/Yap) pathway, could lead to the development of promising therapies for heart disease. The pharmacological product TT-10 increases cardiomyocyte proliferation by upregulating nuclear Yap levels. When intraperitoneal injections of TT-10 were administered to infarcted mouse hearts, the treatment promoted cardiomyocyte proliferation and was associated with declines in infarct size 1 week after administration, but cardiac function worsened at later time points. Here, we investigated whether encapsulating TT-10 into poly-lactic-co-glycolic acid nanoparticles (NPs) before administration could extend the duration of TT-10 delivery and improve the potency of TT-10 for myocardial repair. TT-10 was released from the TT-10–loaded NPs for up to 4 weeks in vitro, and intramyocardial injections of TT-10–delivered NPs stably improved cardiac function from week 1 to week 4 after administration to infarcted mouse hearts. TT-10–delivered NP treatment was also associated with significantly smaller infarcts at week 4, with increases in cardiomyocyte proliferation and nuclear Yap abundance and with declines in cardiomyocyte apoptosis. Thus, NP-mediated delivery appears to enhance both the potency and durability of TT-10 treatment for myocardial repair.

Background: The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes‐associated protein, 5SA [active] mutant), but persistent overexpression of proliferation‐inducing genes could lead to hypertrophy and arrhythmia, whereas off‐target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Methods and Results: Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte‐specific modified mRNA translation system ( ^YAP5SA CM‐SMRTs or ^GFP CM‐SMRTs, respectively). YAP5SA‐cardiomyocyte specificity was confirmed via in vitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced‐ pluripotent stem cells and in human umbilical‐vein endothelial cells, and the regenerative potency of ^YAP5SA CM‐SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced‐pluripotent stem cells‐cardiomyocytes, YAP was abundantly expressed for 3 days after ^YAP5SA CM‐SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near‐background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after ^GFP CM‐SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced‐pluripotent stem cells‐cardiac fibroblasts and human umbilical‐vein endothelial cells on day 1 after ^GFP CM‐SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left‐ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in ^YAP5SA CM‐SMRTs–treated mice than in vehicle‐treated control animals, and ^YAP5SA CM‐SMRTs appeared to promote cardiomyocyte proliferation. Conclusions: The CM‐SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

In mammals, because cardiomyocytes withdraw from cell-cycle activities shortly after birth, the heart cannot repair the damage caused by a myocardial injury; thus, understanding how cardiomyocytes proliferate is among the most important topics in cardiovascular sciences. In newborn neonatal mammals, when a left ventricular injury is applied in hearts earlier than postnatal day 7, the cardiomyocytes actively proliferate and regenerate lost myocardium in the following weeks. The regulators promoting cardiomyocyte proliferation were discovered by analyzing transcriptomic data generated from models. Most of these regulators support the mRNA production of cell-cycle machinery, yet the mRNA requires translation into functional proteins under the regulation of RNA-binding proteins (RBPs). In this work, we performed a meta-analysis to study the relationship between RBP expression and cardiomyocyte proliferation. To identify RBPs associated with mouse and pig cardiomyocyte proliferation, the single-nuclei RNA sequencing (snRNA-seq) data from regenerating mouse and pig hearts were reanalyzed via an Autoencoder focusing on RBP expression. We also generated and analyzed new bulk RNA-seq from two human-induced pluripotent stem cell-derived (hiPSC) cardiomyocyte (hiPSC-CM) cell lines; the first cell line was harvested sixteen days after differentiation, when the cells still actively proliferated, and the second cell line was harvested one hundred and forty days after differentiation, when the cells ceased cell cycle activity. Then, the RBP associated with mouse, pig, and hiPSC-CM were compared across species. Twenty-one RBPs were found to be consistently upregulated, and six RBPs were downregulated in proliferating mouse, pig, and hiPSC-derived cardiomyocytes. Among upregulated RBPs across species, an immunofluorescence-based imaging analysis validated the significant increase in the proteins of DHX9, PTBP3, HNRNPUL1, and DDX6 in pig hearts with proliferating CMs. This meta-analysis in all species demonstrated a strong relationship between RBP expression and cardiomyocyte proliferation.