Strategy For Cardiac Regeneration Research Articles

SMAD3 mediates the specification of human induced pluripotent stem cell-derived epicardium into progenitors for the cardiac pericyte lineage

Understanding the molecular mechanisms of epicardial epithelial-to-mesenchymal transition (EMT), particularly in directing cell fate toward epicardial derivatives, is crucial for regenerative medicine using human induced pluripotent stem cell (iPSC)-derived epicardium. Although transforming growth factor β (TGF-β) plays a pivotal role in epicardial biology, orchestrating EMT during embryonic development via downstream signaling through SMAD proteins, the function of SMAD proteins in the epicardium in maintaining vascular homeostasis or mediating the differentiation of various epicardial-derived cells (EPDCs) is not yet well understood. Our study reveals that TGF-β-independent SMAD3 expression autonomously predicts epicardial cell specification and lineage maintenance, acting as a key mediator in promoting the angiogenic-oriented specification of the epicardium into cardiac pericyte progenitors. This finding uncovers a novel role for SMAD3 in the human epicardium, particularly in generating cardiac pericyte progenitors that enhance cardiac microvasculature angiogenesis. This insight opens new avenues for leveraging epicardial biology in developing more effective cardiac regeneration strategies.

RETRACTED: The novel antibody fusion protein rhNRG1-HER3i promotes heart regeneration by enhancing NRG1-ERBB4 signaling pathway

Stimulating cardiomyocyte proliferation in the adult heart has emerged as a promising strategy for cardiac regeneration following myocardial infarction (MI). The NRG1-ERBB4 signaling pathway has been implicated in the regulation of cardiomyocyte proliferation. However, the therapeutic potential of recombinant human NRG1 (rhNRG1) has been limited due to the low expression of ERBB4 in adult cardiomyocytes. Here, we investigated whether a fusion protein of rhNRG1 and an ERBB3 inhibitor (rhNRG1-HER3i) could enhance the affinity of NRG1 for ERBB4 and promote adult cardiomyocyte proliferation. In vitro and in vivo experiments were conducted using postnatal day 1 (P1), P7, and adult cardiomyocytes. Western blot analysis was performed to assess the expression and activity of ERBB4. Cardiomyocyte proliferation was evaluated using Ki67 and pH 3 immunostaining, while fibrosis was assessed using Masson staining. Our results indicate that rhNRG1-HER3i, but not rhNRG1, promoted P7 and adult cardiomyocyte proliferation. Furthermore, rhNRG1-HER3i improved cardiac function and reduced cardiac fibrosis in post-MI hearts. Administration of rhNRG1-HER3i inhibited ERBB3 phosphorylation while increasing ERBB4 phosphorylation in adult mouse hearts. Additionally, rhNRG1-HER3i enhanced angiogenesis following MI compared to rhNRG1. In conclusion, our findings suggest that rhNRG1-HER3i is a viable therapeutic approach for promoting adult cardiomyocyte proliferation and treating MI by enhancing NRG1-ERBB4 signaling pathway.

Polarizing Macrophage Functional Phenotype to Foster Cardiac Regeneration.

There is an increasing interest in understanding the connection between the immune and cardiovascular systems, which are highly integrated and communicate through finely regulated cross-talking mechanisms. Recent evidence has demonstrated that the immune system does indeed have a key role in the response to cardiac injury and in cardiac regeneration. Among the immune cells, macrophages appear to have a prominent role in this context, with different subtypes described so far that each have a specific influence on cardiac remodeling and repair. Similarly, there are significant differences in how the innate and adaptive immune systems affect the response to cardiac damage. Understanding all these mechanisms may have relevant clinical implications. Several studies have already demonstrated that stem cell-based therapies support myocardial repair. However, the exact role that cardiac macrophages and their modulation may have in this setting is still unclear. The current need to decipher the dual role of immunity in boosting both heart injury and repair is due, at least for a significant part, to unresolved questions related to the complexity of cardiac macrophage phenotypes. The aim of this review is to provide an overview on the role of the immune system, and of macrophages in particular, in the response to cardiac injury and to outline, through the modulation of the immune response, potential novel therapeutic strategies for cardiac regeneration.

Advances in biomaterial-based cardiac organoids

Cardiovascular disease (CVD) is one of the important causes of death worldwide. The incidence and mortality rates are increasing annually with the intensification of social aging. The efficacy of drug therapy is limited in individuals suffering from severe heart failure due to the inability of myocardial cells to undergo regeneration and the challenging nature of cardiac tissue repair following injury. Consequently, surgical transplantation stands as the most efficient approach for treatment. Nevertheless, the shortage of donors and the considerable number of heart failure patients worldwide, estimated at 26 million, results in an alarming treatment deficit, with only around 5000 heart transplants feasible annually. The existing major alternatives, such as mechanical or xenogeneic hearts, have significant flaws, such as high cost and rejection, and are challenging to implement for large-scale, long-term use. An organoid is a three-dimensional (3D) cell tissue that mimics the characteristics of an organ. The critical application has been rated in annual biotechnology by authoritative journals, such as Science and Cell. Related industries have achieved rapid growth in recent years. Based on this technology, cardiac organoids are expected to pave the way for viable heart repair and treatment and play an essential role in pathological research, drug screening, and other areas. This review centers on the examination of biomaterials employed in cardiac repair, strategies employed for the reconstruction of cardiac structure and function, clinical investigations pertaining to cardiac repair, and the prospective applications of cardiac organoids. From basic research to clinical practice, the current status, latest progress, challenges, and prospects of biomaterial-based cardiac repair are summarized and discussed, providing a reference for future exploration and development of cardiac regeneration strategies.

Contractile Force of Transplanted Cardiomyocytes Actively Supports Heart Function After Injury.

Background:Transplantation of pluripotent stem cell–derived cardiomyocytes represents a promising therapeutic strategy for cardiac regeneration, and the first clinical studies in patients with heart failure have commenced. Yet, little is known about the mechanism of action underlying graft-induced benefits. Here, we explored whether transplanted cardiomyocytes actively contribute to heart function.Methods:We injected cardiomyocytes with an optogenetic off-on switch in a guinea pig cardiac injury model.Results:Light-induced inhibition of engrafted cardiomyocyte contractility resulted in a rapid decrease of left ventricular function in ≈50% (7/13) animals that was fully reversible with the offset of photostimulation.Conclusions:Our optogenetic approach demonstrates that transplanted cardiomyocytes can actively participate in heart function, supporting the hypothesis that the delivery of new force-generating myocardium can serve as a regenerative therapeutic strategy.

Can Pediatric Heart Failure Therapy Be Improved? Yes It Can, But….

Given the heterogenous etiology of pediatric heart failure (pHF), evidence-based studies improving pHF are unlikely. A paradigm shift towards updated medicine-based evidence is therefore necessary. In view of the life expectancy of children, cardiac regeneration strategies are required. Therefore, age- and disease-related differences in myocardial (receptor) physiology require individualized precision medicine. First-line diuretic therapy, adopted from the treatment of adults with HF with no chance for recovery, should be questioned in the treatment of pHF with potential for recovery. Inadequate use of diuretics is a common reason for additional stimulation of the neurohumoral axis. Consecutive intravascular volume depletion led to an inadequate treatment with β-blocker and renin–angiotensin–aldosterone antagonists. Given the age-related catecholamine-driven cardiovascular (patho-) physiology, highly selective β1-blockers (bisoprolol) protect against β1-(noradrenaline)-related myocytic apoptosis and necrosis, but allow β2-receptor-mediated myocardial regeneration. Based on its high safety–efficacy profile with rarely seen adverse effects but easily monitorable efficacy by the surrogate of heart rate (reduction), bisoprolol is our first-line drug in infancy. Reduced heart rate economizes the heart and full body oxygen consumption and extends the diastolic filling and coronary perfusion time. Based on our many years of institutional experience, physicians should be encouraged to use β1-selected blockers in infants with dilated cardiomyopathy and hypoplastic left heart syndrome after stage-1 procedure, but also to treat ventricular septal defects with a significant left-to-right shunt. In summary, individualized pHF therapy is the prerequisite for a causal treatment to improve HF symptoms, but above all for the most functional regeneration possible.

Abstract P1014: Cardiomyocyte Energetic Capacity Governs Responses To Cell Cycle Stimulation

Background: Induction of cardiomyocyte (CM) proliferation is a promising strategy for cardiac regeneration. We previously showed that the overexpression of four cell cycle factors (4F), i.e. cyclin B1, CDK1, cyclin D1 and CDK4, induces CM proliferation. Temporal profiling of the single cell transcriptomes of 4F- and LacZ-overexpressing CMs showed a specific population with higher levels of CD36 that was more responsive to the 4F cell cycle induction. In addition, P7 primary CMs isolated from CD36 KO mice demonstrated 50% less response to the 4F compared with the WT controls (Abouleisa et al., Circulation, 2022). These data indicate that CD36 may be required to prime CMs to proliferate. CD36 is known to internalize fatty acids to be utilized for energy production. Here, we aim to investigate the direct effect of CM energetic capacity on their response to cell cycle stimulation. Methods and Results: Carnitine palmitoyl transferase -1 (CPT-1) is a rate limiting enzyme for the β-oxidation of fatty acids. Knockdown of CPT-1 using siRNA significantly reduced mitochondrial ATP production by 10% and led to a significant reduction in cell cycle markers (PHH3, EDU) in response to the 4F (PHH3; control siRNA+4F:24±1% vs CPT1 siRNA+4F:15±1%, EDU; control siRNA+4F:22±2% vs CPT1siRNA+4F:16±1%, P<0.01, n=4). Small molecule inhibition of CPT1 using etomoxir significantly decreased mitochondrial ATP production and suppressed CM responses to the 4F. Furthermore, overexpression of CPT-1 using viral vectors resulted in significant increase in ATP production and increased response to cell cycle stimulation using the 4F. Supplementing the culture medium with (1μmol) L-carnitine significantly increased mitochondrial ATP production and enhanced the CM response to the 4F. Finally, to increase ATP availability in CMs directly, we overexpressed a light-activated mitochondrial proton pump (MT-ON). CMs expressing MT-ON showed a significant increase in mitochondrial ATP levels and enhanced cell cycle induction in CMs transduced with 4F (PHH3; 4F:18±2% vs 4F+MT-ON:27±3%, EDU; 4F:18±1% vs 4F+MT-ON:30±4 %, P<0.01, n=4). Conclusions: These findings suggest that an energy threshold is required for CMs to progress into the cell cycle.

Metabolic Determinants in Cardiomyocyte Function and Heart Regenerative Strategies.

Heart disease is the leading cause of mortality in developed countries. The associated pathology is characterized by a loss of cardiomyocytes that leads, eventually, to heart failure. In this context, several cardiac regenerative strategies have been developed, but they still lack clinical effectiveness. The mammalian neonatal heart is capable of substantial regeneration following injury, but this capacity is lost at postnatal stages when cardiomyocytes become terminally differentiated and transit to the fetal metabolic switch. Cardiomyocytes are metabolically versatile cells capable of using an array of fuel sources, and the metabolism of cardiomyocytes suffers extended reprogramming after injury. Apart from energetic sources, metabolites are emerging regulators of epigenetic programs driving cell pluripotency and differentiation. Thus, understanding the metabolic determinants that regulate cardiomyocyte maturation and function is key for unlocking future metabolic interventions for cardiac regeneration. In this review, we will discuss the emerging role of metabolism and nutrient signaling in cardiomyocyte function and repair, as well as whether exploiting this axis could potentiate current cellular regenerative strategies for the mammalian heart.

Direct Reprogramming of Adult Human Cardiac Fibroblasts into Induced Cardiomyocytes Using miRcombo.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through microRNAs (miRNAs) is a new emerging strategy for myocardial regeneration after ischemic heart disease. Previous studies have reported that murine fibroblasts can be directly reprogrammed into iCMs by transient transfection with four miRNAs (miRs-1, 133, 208 and 499 - termed "miRcombo"). While advancement in the knowledge of direct cell reprogramming molecular mechanism is in progress, it is important to investigate if this strategy may be translated to humans. Recently, we demonstrated that miRcombo transfection is able to induce direct reprogramming of adult human cardiac fibroblasts (AHCFs) into iCMs. Although additional studies are needed to achieve iCM maturation, our early findings pave the way toward new therapeutic strategies for cardiac regeneration in humans. This chapter describes methods for inducing direct reprogramming of AHCFs into iCMs through miRcombo transient transfection, showing experiments to perform for assessing iCM generation.

Natural Melanin/Alginate Hydrogels Achieve Cardiac Repair through ROS Scavenging and Macrophage Polarization.

The efficacy of cardiac regenerative strategies for myocardial infarction (MI) treatment is greatly limited by the cardiac microenvironment. The combination of reactive oxygen species (ROS) scavenging to suppress the oxidative stress damage and macrophage polarization to regenerative M2 phenotype in the MI microenvironment can be desirable for MI treatment. Herein, melanin nanoparticles (MNPs)/alginate (Alg) hydrogels composed of two marine‐derived natural biomaterials, MNPs obtained from cuttlefish ink and alginate extracted from ocean algae, are proposed. Taking advantage of the antioxidant property of MNPs and mechanical support from injectable alginate hydrogels, the MNPs/Alg hydrogel is explored for cardiac repair by regulating the MI microenvironment. The MNPs/Alg hydrogel is found to eliminate ROS against oxidative stress injury of cardiomyocytes. More interestingly, the macrophage polarization to regenerative M2 macrophages can be greatly promoted in the presence of MNPs/Alg hydrogel. An MI rat model is utilized to evaluate the feasibility of the as‐prepared MNPs/Alg hydrogel for cardiac repair in vivo. The antioxidant, anti‐inflammatory, and proangiogenesis effects of the hydrogel are investigated in detail. The present study opens up a new way to utilize natural biomaterials for MI treatment and allows to rerecognize the great value of natural biomaterials in cardiac repair.

Developing novel cardiac regenerative strategies.

Developing novel cardiac regenerative strategies.

Direct reprogramming induces vascular regeneration post muscle ischemic injury

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes—Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)—together with three reprogramming-helper genes—dominant-negative (DN)-TGFβ, DN-Wnt8a, and acid ceramidase (AC)—to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

Applications of Omics Technologies in Regenerative Therapies for Cardiac Repair

Lately, cardio regenerative therapeutic approaches in clinic have received strong boost from promising research advances in stem cells. Researchers have shown applications of the first generation of clinical trials using cell-based therapies in the heart that have been performed with bone marrow and adipose tissue derived mesenchymal stem cells shortcomings in the first generation cells. Second generation cell therapies are considered superior and being considered towards the use of cardiac-committed cell populations. These include cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes. However, translating the research laboratory results along with pre-clinical data into effective clinical treatments is still thought to be challenging. This is due to the existing lack of knowledge on the regenerative mechanisms of action of these therapeutic products in addition to the stringent regulatory and safety concerns. This has prompted researchers to consider advanced analytical methods for characterization of such complex products and a deep understanding of their therapeutic effects at the cell and molecular level. This characterization is considered very critical to overcome the challenges and make these cells compatible with the advanced therapies. To this end, omics technologies including proteomics and glyco(proteo)mics that are based on state-of-the-art mass-spectrometry have shown tremendous promise to generate novel data on cell biology along with the required assessment of cell based-products that are applied in cardiac regeneration strategies. In this perspective, we have described proteomics and glyco(proteo)mics and discuss the impact of omics technologies on cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes biology cardiac regenerative therapies.

ALKBH5 regulates cardiomyocyte proliferation and heart regeneration by demethylating the mRNA of YTHDF1.

N6-methyladenosine (m6A) RNA modification, a dynamic and reversible process, is essential for tissue development and pathogenesis. However, the potential involvement of m6A in the regulation of cardiomyocyte (CM) proliferation and cardiac regeneration remains unclear. In this study, we aimed to investigate the essential role of m6A modification in heart regeneration during postnatal and adult injury.Methods and results: In this study, we identified the downregulation of m6A demethylase ALKBH5, an m6A “eraser” that is responsible for increased m6A methylation, in the heart after birth. Notably, ALKBH5 knockout mice exhibited decreased cardiac regenerative ability and heart function after neonatal apex resection. Conversely, forced expression of ALKBH5 via adeno-associated virus-9 (AAV9) delivery markedly reduced the infarct size, restored cardiac function and promoted CM proliferation after myocardial infarction in juvenile (7 days old) and adult (8-weeks old) mice. Mechanistically, ALKBH5-mediated m6A demethylation improved the mRNA stability of YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1), thereby increasing its expression, which consequently promoted the translation of Yes-associated protein (YAP). The modulation of ALKBH5 and YTHDF1 expression in human induced pluripotent stem cell-derived cardiomyocytes consistently yielded similar results.Conclusion: Taken together, our findings highlight the vital role of the ALKBH5-m6A-YTHDF1-YAP axis in the regulation of CMs to re-enter the cell cycle. This finding suggests a novel potential therapeutic strategy for cardiac regeneration.

WNT-targeted compound and phytoestrogen promoted cardiogenic differentiation of human induced pluripotent stem cells (hiPSCs) in vitro

Background and objectives: Despite the advances made in the prevention and treatment of cardiovascular diseases (CVD) in the last decade, they are still the leading cause of death in males at the rate of 50% worldwide. Considering the protective role of estrogen to decrease CVD rates in young females, it was suggested that using hormone therapy can be considered to improve heart regeneration. Using in vitro induced pluripotent stem cells (iPSCs) has become one of the most significant tools in CVD treatment in both genders. We design a novel optimal protocol for the differentiation of iPSCs to cardiomyocytes which may be valuable for CVD treatment in men. Methods: Human iPSCs were initially cultivated on mouse embryonic fibroblasts and then, transferred to a specific culture medium for differentiation process. In vitro differentiation of iPSCs into cardiomyocytes was induced at three phases on RPMI-1640 medium including CHIR99021 (5 µM) on days 0–3, BMP4 (20 ng/mL), and bFGF (100 ng/mL) on days 3–5, 10 µM of XAV939 on 6–8, and phytoestrogen + ascorbic acid on days 8–13. Scanning electron microscopy and Real-time PCR using specific primers were applied to confirm produced cardiomyocytes. Results: We found that the simultaneous use of small chemical molecules such as CHIR99021 and XAV 939, growth factors, such as BMP4, bFGF, and herbal-derived phytoestrogen from red clover could efficiently differentiate hiPSCs from the mesoderm and cardiomyocytes after 13 days. Using phytoestrogen increased the induction of cardiac markers including cTnT and GATA-4 in a shorter time; consequently, the proposed formulation has the potential to be used in developing a novel approach for cardiac repair or regeneration. Conclusion: Presented data indicated that the serial use of XAV939 and phytoestrogen at different times and stages can successfully induce cardiogenesis from hiPSCs. Thus, the proposed approach can be used for improved translational strategies for cardiac regeneration with fewer side effects.

Abstract 17357: Direct Cardiac Reprogramming Using Combinatorial Modified mRNA

Introduction: Despite various clinical modalities, ischemic heart disease remains among the leading causes of mortality and morbidity worldwide. The elemental problem is the immense loss of cardiomyocytes (CMs) post-myocardial infarction (MI). Reprogramming non- cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells in vivo is a promising strategy for cardiac regeneration: however, the traditional viral delivery method hampered its application into clinical settings due to low and erratic transduction efficiency. Methods: We used a modified mRNA (modRNA) gene delivery platform to deliver different stoichiometry of cardiac-reprogramming genes (Gata4, Mef2c, Tbx5 and Hand2) together with reprogramming helper genes (Dominant Negative (DN)-TGFβ, DN- Wnt8a and Acid ceramidase (AC)), named 7G, to induce direct cardiac reprogramming post myocardial infarction (MI). Results: Here, we identified 7G modRNA cocktail as an important regulator ofthe cardiac reprogramming. Cardiac transfection with 7G modRNA doubled cardiac reprogramming efficiency (57%) in comparison to Gata4, Mef2C and Tbx5 (GMT) alone (28%) in vitro . By inducing MI in our lineage tracing model, we showed that one-time delivery of the 7G-modRNA cocktail reprogrammed ~25% of the non-CMs in the scar area to CM- like cells. Furthermore, 7G modRNA treated mice showed significantly improved cardiac function, longer survival, reduced scar size and greater capillary density than control mice 28 days post-MI. We attributed the improvement in heart function post modRNA delivery of 7G or 7G with increased Hand2 ratio (7G-GMT Hx2) to significant upregulation of 15 key angiogenic factors without any signs of angioma or edema. Conclusions: 7G or 7G GMT HX2 modRNA cocktails boosts de novo CM-like cells and promotes cardiovascular regeneration post-MI. Thus, we highlight that this gene delivery approach not only has high efficiency but also high margin of safety for translation to clinics.

Melatonin promotes cardiomyocyte proliferation and heart repair in mice with myocardial infarction via miR-143-3p/Yap/Ctnnd1 signaling pathway.

The neonatal heart possesses the ability to proliferate and the capacity to regenerate after injury; however, the mechanisms underlying these processes are not fully understood. Melatonin has been shown to protect the heart against myocardial injury through mitigating oxidative stress, reducing apoptosis, inhibiting mitochondrial fission, etc. In this study, we investigated whether melatonin regulated cardiomyocyte proliferation and promoted cardiac repair in mice with myocardial infarction (MI), which was induced by ligation of the left anterior descending coronary artery. We showed that melatonin administration significantly improved the cardiac functions accompanied by markedly enhanced cardiomyocyte proliferation in MI mice. In neonatal mouse cardiomyocytes, treatment with melatonin (1 μM) greatly suppressed miR-143-3p levels. Silencing of miR-143-3p stimulated cardiomyocytes to re-enter the cell cycle. On the contrary, overexpression of miR-143-3p inhibited the mitosis of cardiomyocytes and abrogated cardiomyocyte mitosis induced by exposure to melatonin. Moreover, Yap and Ctnnd1 were identified as the target genes of miR-143-3p. In cardiomyocytes, inhibition of miR-143-3p increased the protein expression of Yap and Ctnnd1. Melatonin treatment also enhanced Yap and Ctnnd1 protein levels. Furthermore, Yap siRNA and Ctnnd1 siRNA attenuated melatonin-induced cell cycle re-entry of cardiomyocytes. We showed that the effect of melatonin on cardiomyocyte proliferation and cardiac regeneration was impeded by the melatonin receptor inhibitor luzindole. Silencing miR-143-3p abrogated the inhibition of luzindole on cardiomyocyte proliferation. In addition, both MT1 and MT2 siRNA could cancel the beneficial effects of melatonin on cardiomyocyte proliferation. Collectively, the results suggest that melatonin induces cardiomyocyte proliferation and heart regeneration after MI by regulating the miR-143-3p/Yap/Ctnnd1 signaling pathway, providing a new therapeutic strategy for cardiac regeneration.

Novel insights into cardiac regeneration based on differential fetal and adult ovine heart transcriptomic analysis.

The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies.NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration.

Abstract 423: Cardiomyocyte Maturation and Multinucleation in Postnatal Swine

Objectives: Cardiomyocyte (CM) cell cycle arrest and decline of mononucleated diploid CMs have been implicated in loss of regenerative potential in postnatal mouse hearts. Sarcomeric and extracellular matrix (ECM) maturation also occur concurrently in mice, influencing CM proliferative arrest. Recent studies show a 3-day neonatal period of cardiac regeneration in pigs similar to mice, but the dynamics of postnatal pig CM growth are unknown. Our objective is to explore cardiac cell cycling, growth and maturation in postnatal pigs to understand the events guiding loss of cardiac regenerative capacity in large mammals. Methods & Results: Left-ventricular tissue from farm pigs (White Yorkshire-Landrace) at Postnatal day (P)0, P7, P15, P30, 2 months (2mo) and 6mo were utilized. CM dissociations revealed predominant CM mononucleation at birth in swine, with persistence of ~50% (1186/2537 cells, n=5) mononucleated CMs at P15, and ~10% (227/1785 cells, n=4) at 2mo. By 6mo, pig CMs are entirely multinucleated, exhibiting 4-16 nuclei per cell. Assessing hypertrophic growth in dissociated pig CMs revealed longitudinal CM growth relative to increased nucleation at all ages. However, onset of diametric hypertrophy only occurs beyond 2mo. When nuclear pHH3 and mRNA expression of cell cycle genes was assessed, pig hearts show robust cell-cycling up to 2mo. Also, fetal TNNI1 and MYH6 are active up to 2mo in pig hearts. Ongoing studies on collagen remodeling indicate ECM remodeling in swine occurs beyond P7. Future studies are designed to identify nuclear ploidy in postnatal pig CMs and measure cytokinesis. Conclusions: Cardiac maturational events are staggered over a 2-6 month postnatal window in pigs, with older pig hearts exhibiting extensive CM multinucleation and differences in longitudinal versus diametric CM growth. These fundamental variations in CM growth characteristics are important to consider when designing preclinical trials for cardiac regenerative strategies in pigs. Also, despite a similar period of regenerative capacity as mice, pig hearts do not undergo loss of CM cell cycling and mononucleation until 2mo after birth. Utilizing pigs may thus offer unique opportunities to study aspects of heart regeneration unavailable in other animal models.

Cardiac regeneration with pluripotent stem cell-derived cardiomyocytes and direct cardiac reprogramming.

Cardiovascular disease is the leading cause of death globally. Cardiomyocytes (CMs) have poor regenerative capacity, and pharmacological therapies have limited efficacy in severe heart failure. Currently, there are several promising strategies for cardiac regeneration. The most promising approach to remuscularize failing hearts is cell transplantation therapy using newly generated CMs from exogenous sources, such as pluripotent stem cells. Alternatively, approaches to generate new CMs from endogenous cell sources in situ may also repair the injured heart and improve cardiac function. Direct cardiac reprogramming has emerged as a novel therapeutic approach to regenerate injured hearts by directly converting endogenous cardiac fibroblasts into CM-like cells. Through cell transplantation and direct cardiac reprogramming, new CMs can be generated and scar tissue reduced to improve cardiac function; therefore, cardiac regeneration may serve as a powerful strategy for treatment of severe heart failure. While substantial progress has been made in these two strategies for cardiac regeneration over the past several years, challenges remain for clinical translation. This review provide an overview of previous reports and current challenges in this field.