Cardiomyocyte Dedifferentiation Research Articles

Exploring the interactions between cardiomyocytes and endothelial cells to induce cardiac revascularization and regeneration

Abstract Funding Acknowledgements None. Background The adult heart is unable to regenerate after ischemic injury, primarily due to the incapacity of cardiomyocyte (CM) to re-enter in the cell cycle and endothelial cells (EC) to form new vessels. Interestingly, our recent data have unveiled that the decline of CM proliferation after birth is paralleled by a reduction in angiogenic potential. Nevertheless, the precise mechanisms governing angiogenesis inhibition in the adult heart remain elusive. We hypothesize that the terminal differentiation of CMs after birth leads to the paracrine suppression of EC proliferation. Purpose This study aims to identify membrane or secreted factors produced by adult CMs that inhibit EC proliferation and vessel formation in order to create new therapeutic strategies to induce cardiac revascularization and regeneration. Material and Methods To validate our hypothesis, ECs were co-cultured with CMs at distinct differentiation stages. Leveraging existing RNA sequencing datasets, we generated an interactome between ligands produced by CMs and receptors expressed by cardiac ECs. Through gain and loss of function studies, we identified pivotal anti-angiogenic factors and subsequently knocked-out the corresponding genes in Cas9 mice, with the aim of rescue the angiogenic potential of the adult heart. Concurrently, we assessed the ability of miR199a, a pro-proliferative miRNA known for inducing CM de-differentiation, to elicit EC proliferation and angiogenesis when delivered to the adult heart together with VEGF-A, utilizing adeno-associated viral vectors. Results Our findings revealed that adult CMs impaired EC proliferation. Comprehensive analysis identified 20 membrane and secreted factors, establishing 118 ligand-receptor interactions between CMs and ECs. Notably, KCNJ11 and CD74 emerged as key factors, as validated through gain and loss of function experiments. In vivo knockout of these genes in CMs led to significant increase in EC proliferation and the formation of neo-vessels. Furthermore, CM de-differentiation induced by miR199a demonstrated the capacity to promote EC proliferation and neo-vessel formation upon VEGF-A stimulation. Conclusions Collectively, this work demonstrated that fully mature CMs have a role in limiting angiogenic potential of the adult heart, likely explaining the failure of therapeutic angiogenesis approaches attempted so far. Our results suggest that innovative strategies targeting CM-EC cross-talk or inducing partial CM de-differentiation may open new therapeutic avenues in the field of cardiac regeneration.Adult CMs inhibit EC proliferationCMdedifferentiation rescues angiogenesis

The role of asparagine synthetase in cardiomyocyte dedifferentiation

The role of asparagine synthetase in cardiomyocyte dedifferentiation

Antigen presentation plays positive roles in the regenerative response to cardiac injury in zebrafish

In contrast to adult mammals, adult zebrafish can fully regenerate injured cardiac tissue, and this regeneration process requires an adequate and tightly controlled immune response. However, which components of the immune response are required during regeneration is unclear. Here, we report positive roles for the antigen presentation-adaptive immunity axis during zebrafish cardiac regeneration. We find that following the initial innate immune response, activated endocardial cells (EdCs), as well as immune cells, start expressing antigen presentation genes. We also observe that T helper cells, a.k.a. Cd4+ T cells, lie in close physical proximity to these antigen-presenting EdCs. We targeted Major Histocompatibility Complex (MHC) class II antigen presentation by generating cd74a; cd74b mutants, which display a defective immune response. In these mutants, Cd4+ T cells and activated EdCs fail to efficiently populate the injured tissue and EdC proliferation is significantly decreased. cd74a; cd74b mutants exhibit additional defects in cardiac regeneration including reduced cardiomyocyte dedifferentiation and proliferation. Notably, Cd74 also becomes activated in neonatal mouse EdCs following cardiac injury. Altogether, these findings point to positive roles for antigen presentation during cardiac regeneration, potentially involving interactions between activated EdCs, classical antigen-presenting cells, and Cd4+ T cells.

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.

Abstract 12975: Metabolic Reprogramming in Adult Cardiomyocytes Promotes Heart Regeneration

Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes. However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. We tested if dedifferentiating or reprogramming adult cardiomyocytes may recover their proliferative capability and thus promote heart regeneration after injury in mice.We bred mice with inducible, cardiac specific expression of the Yamanaka OSKM factors, enabling adult cardiomyocyte reprogramming and dedifferentiation in vivo. Two days after OSKM induction, adult cardiomyocytes presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult cardiomyocytes and recapitulated cardiomyocyte dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to cardiomyocyte injury, thus allowing cell dedifferentiation and proliferation as a regenerative response. (Cheng et al . Circulation 2022).

Hapln1 promotes dedifferentiation and proliferation of iPSC-derived cardiomyocytes by promoting versican-based GDF11 trapping

Hyaluronan and proteoglycan link protein 1 (Hapln1) supports active cardiomyogenesis in zebrafish hearts, but its regulation in mammal cardiomyocytes is unclear. This study aimed to explore the potential regulation of Hapln1 in the dedifferentiation and proliferation of cardiomyocytes and its therapeutic value in myocardial infarction with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) and an adult mouse model of myocardial infarction. HiPSC-CMs and adult mice with myocardial infarction were used as in vitro and in vivo models, respectively. Previous single-cell RNA sequencing data were retrieved for bioinformatic exploration. The results showed that recombinant human Hapln1 (rhHapln1) promotes the proliferation of hiPSC-CMs in a dose-dependent manner. As a physical binding protein of Hapln1, versican interacted with Nodal growth differentiation factor (NODAL) and growth differentiation factor 11 (GDF11). GDF11, but not NODAL, was expressed by hiPSC-CMs. GDF11 expression was unaffected by rhHapln1 treatment. However, this molecule was required for rhHapln1-mediated activation of the transforming growth factor (TGF)-β/Drosophila mothers against decapentaplegic protein (SMAD)2/3 signaling in hiPSC-CMs, which stimulates cell dedifferentiation and proliferation. Recombinant mouse Hapln1 (rmHapln1) could induce cardiac regeneration in the adult mouse model of myocardial infarction. In addition, rmHapln1 induced hiPSC-CM proliferation. In conclusion, Hapln1 can stimulate the dedifferentiation and proliferation of iPSC-derived cardiomyocytes by promoting versican-based GDF11 trapping and subsequent activation of the TGF-β/SMAD2/3 signaling pathway. Hapln1 might be an effective hiPSC-CM dedifferentiation and proliferation agent and a potential reagent for repairing damaged hearts.

Abstract P3073: Interleukin-2 Receptor Gamma Signaling Is Required For The Regenerative Adaptive Immune Response In The Adult Zebrafish Heart

Ischemic heart disease remains a leading cause of death worldwide. Unlike adult mammals, zebrafish have the ability to fully regenerate their heart after injury through processes including epicardium and endocardium activation, coronary revascularization, cardiomyocyte dedifferentiation and proliferation, and scar resolution. Immune cells are critical to remove damaged tissue, sustain repair and initiate regeneration after injury. Our previous data show that blocking antigen presentation impairs cardiac regeneration, pointing at a role for the adaptive immune system during this process. However, the molecular mechanisms underlying a pro-regenerative adaptive immune response remain largely unknown. Based on bulk transcriptomics, single-cell transcriptomics, and qPCR data, we have identified il2rga ( interleukin-2 receptor gamma a ) and jak3 ( janus kinase 3) as potential regulators of the adaptive immune response during cardiac regeneration. We indeed found that il2rga mutants exhibit a reduced expression of T-cell markers, as well as impaired cardiac regeneration, as observed by decreased cardiomyocyte dedifferentiation and proliferation, and defective scar resolution. In addition, il2rga mutants display decreased expression of pro-regenerative factors (e.g., igf1, ngfb, nrg1, pdgfaa, tgfb2, vegfc ). Similarly, loss of Jak3, which functions downstream of Il2rga, also leads to reduced cardiomyocyte dedifferentiation and proliferation, as well as impaired endocardium activation. Bulk transcriptomics further shows the downregulation of key genes involved in the adaptive immune response in il2rga mutants. Specifically, we found that il13 , a recently described cardiomyocyte cell cycle regulator and T helper 2 (Th2) cytokine, is strongly downregulated upon the loss of il2rga . Altogether, our findings reveal the crucial role of Il2rga/Jak3 signaling during cardiac regeneration, thereby opening new avenues to identify and understand the adaptive immune mechanisms capable of sustaining regenerative responses post-cardiac injury.

Abstract P1105: Dissecting The Roles Of Vegfba And Pgfb In Zebrafish Cardiac Regeneration

Vascular endothelial growth factor (VEGF) family members promote angiogenesis, regulate inflammation, antagonize oxidative stress, and regulate lipid metabolism, which present potential therapeutic value for coronary heart disease. However, all of their roles during cardiac regeneration are still largely unknown. Unlike mammals, zebrafish are able to fully regenerate their heart after injury. In zebrafish, Vegf factors are regulated in different manners after cardiac cryoinjury, indicating functional diversity during cardiac regeneration. Here, we focus on vegfba and placental growth factor b ( pgfb ), which have been much less investigated in regenerative contexts, and were found to be persistently down-regulated in injured hearts before 5 days post cryoinjury (dpci) according to our previous dataset. We generated HOTcre transgenic lines to control the overexpression of vegf genes in a spatially and temporally restricted fashion during cardiac regeneration. Global overexpression of vegfba had no significant effect on coronary vessels, whereas it promoted the proliferation of cardiomyocytes at 7 dpci. In addition, global overexpression of pgfb significantly increased the vessel coverage in the wound at both 96 hpci and 7 dpci. These results suggest their possible roles in promoting myocardial regeneration and revascularization. However, myocardium-specific overexpression of vegfba and pgfb significantly reduced coronary vessel coverage within the injured area and decreased the proliferation and dedifferentiation of cardiomyocytes, indicating possible disruption of revascularization and regeneration caused by their misexpression. To further study the mechanism underlying these phenotypic differences, we are performing transcriptomic analysis to identify the differentially regulated genes and related signaling pathways. This project will provide new candidate cues that may help understand the mechanisms of coronary revascularization and cardiac regeneration in zebrafish, with potential therapeutic implications of achieving better directed and enhanced regeneration upon coronary heart disease.

Abstract P3174: Loss Of Hmgcs2-mediated Ketogenesis Accelerates Cardiac Aging

Introduction: We have previously reported that the ketogenic enzyme HMGCS2 regulates metabolic changes to ameliorate cardiac damage by promoting adult cardiomyocyte dedifferentiation and proliferation after myocardial infarction (Cheng et al. Circulation , 2022). Intriguingly, ketogenic diet has been shown to prolong the lifespan of aged mice. However, whether the loss of HMGCS2 links to cardiac aging remains unclear. Hypothesis: We tested if loss of HMGCS2 results in metabolic change and heart dysfunction and leads to premature cardiac aging. Methods and Results: Conventional HMGCS2 knock-out (KO) mice were generated by inserting loxP sites flanking exon 2 of HMGCS2 to eliminate HMGCS2 protein production. HMGCS2 expression and β-hydroxybutyrate production were eliminated in the livers and hearts in HMGCS2 KO mice. Echocardiographic analysis and intraventricular catheterization did not display heart functional differences between wild-type and HMGCS2 KO mice at 3-month-old. However, growing with age, HMGCS2 KO mice revealed a declining survival rate, accompanied by worse diastolic and systolic functions including reduced left ventricle ejection fraction, myocardial performance index, fractional shortening, stroke volume, end-diastolic dimension, end-systolic dimension, isovolumic relaxation time, and left ventricular diastolic time constant (Tau) in HMGCS2 KO mice at 12-month-old. Histological analyses also exhibited aging-related phenotypes in the HMGCS2 KO heart at 12-month-old, including cardiac hypertrophy and fibrosis. Furthermore, mitochondrial analyses indicated mitochondrial impairments of function and structure. Conclusion: These results suggest that loss of HMGCS2 accelerates cardiac aging with declining cardiac and mitochondrial function.

Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

Cardiomyocyte maturation and its reversal during cardiac regeneration.

Cardiovascular disease is a leading cause of death worldwide. Due to the limited proliferative and regenerative capacity of adult cardiomyocytes, the lost myocardium is not replenished efficiently and is replaced by a fibrotic scar, which eventually leads to heart failure. Current therapies to cure or delay the progression of heart failure are limited; hence, there is a pressing need for regenerative approaches to support the failing heart. Cardiomyocytes undergo a series of transcriptional, structural, and metabolic changes after birth (collectively termed maturation), which is critical for their contractile function but limits the regenerative capacity of the heart. In regenerative organisms, cardiomyocytes revert from their terminally differentiated state into a less mature state (ie, dedifferentiation) to allow for proliferation and regeneration to occur. Importantly, stimulating adult cardiomyocyte dedifferentiation has been shown to promote morphological and functional improvement after myocardial infarction, further highlighting the importance of cardiomyocyte dedifferentiation in heart regeneration. Here, we review several hallmarks of cardiomyocyte maturation, and summarize how their reversal facilitates cardiomyocyte proliferation and heart regeneration. A detailed understanding of how cardiomyocyte dedifferentiation is regulated will provide insights into therapeutic options to promote cardiomyocyte de-maturation and proliferation, and ultimately heart regeneration in mammals.

Metabolic Changes Associated With Cardiomyocyte Dedifferentiation Enable Adult Mammalian Cardiac Regeneration.

Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo. Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

Abstract 10234: HMGCS2-Mediated Ketogenesis Induces Adult Cardiomyocyte Dedifferentiation and Enables Cardiac Regeneration

Introduction: Human cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, some animals readily regenerate lost myocardium via a process involving CM dedifferentiation, which unlocks their proliferative capacity. Methods: Inspired by this concept, we generated mice with inducible, CM-specific expression of the Yamanaka factors (OSKM), enabling adult CM dedifferentiation in vivo . We profiled changes in CM gene expression by microarray, measured metabolite production by LC-MS and 13 C NMR, and used adeno-associated virus (AAV) to over-express target genes. Cardiac function was measured by echocardiography and intraventricular catheterization. Results: Two days following OSKM induction, adult CMs exhibited a dedifferentiated phenotype and increased proliferation in vivo . Microarray analysis revealed alterations of metabolism-related pathways, particularly an increase of the ketogenic enzyme HMGCS2. Analysis of metabolites confirmed that HMGCS2-induced ketogenesis in adult CMs allowed a metabolic switch from fatty acid oxidation towards ketone utilization. AAV-driven overexpression of HMGCS2 in OSKM mice induced more adult CM dedifferentiation and increased the proliferation rate by two-fold compared to controls. The same phenomenon was observed after myocardial infarction, specifically in the border zone. On the contrary, CM-specific HMGCS2 knockout mice showed poorer baseline cardiac function and impaired recovery following injury. Forced expression of HMGCS2 rescued cardiac function in these mice, thus demonstrating a role of ketogenesis in CM dedifferentiation and proliferation. Conclusions: These data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to mammalian cardiac injury, allowing cell dedifferentiation and proliferation as a regenerative response.

Abstract 12829: Biomimetic Cardiac Tissue Culture Model to Emulate Cardiac Physiology and Pathophysiology Ex Vivo

Introduction: There is an urgent need for a reliable in vitro system to accurately replicate the cardiac physiological and pathological environment for drug testing. The limited availability of human heart tissue culture systems has led to inaccurate interpretations of cardiac-related drug effects. Our group developed a culture system for pig/human heart slices that enables functional and structural viability for 6 days using continuous electrical stimulation and enriched media. This culture system can more reliably detect cardiotoxic effects compared to hiPSC-CMs. However, by day 10, the slices undergo cardiomyocyte dedifferentiation and fibrotic remodeling making them inadequate for long-term drug testing. Hypothesis: To determine whether incorporating physiological mechanical, and humoral cues within the culture system can fully emulate cardiac physiology and pathophysiology in culture. Methods and results: We have developed a novel c ardiac t issue c ulture m odel (CTCM) that can electro-mechanically stimulate heart slices with physiological (0 to 80 mmHg) preload and afterload during each cardiac cycle. After 12 days in culture, this approach alone partially improved the viability of heart slices but did not maintain the structural integrity. Therefore, following a small molecule screening, we found that incorporating 100 nM tri-iodothyronine (T3) and 1 μM dexamethasone (Dex) into our culture media preserved the structural integrity of the slices for 12 days. When combined with T3/Dex, the CTCM system fully maintained the transcriptional profile, viability, metabolic activity, and structural integrity for 12 days at the same levels as fresh heart tissue. Furthermore, to test the ability of the CTCM to emulate cardiac hypertrophy, increasing the peak pressure to 140 mmHg resulted in induced cardiac hypertrophy within 6 days in culture as indicated by a 3-fold increase in NT-ProBNP release, increased NFATC4 and P-ERK expression, and increased cardiomyocyte cross-sectional area. Conclusions: This new CTCM heart slice culture system incorporating physiological electromechanical and humoral cues can emulate cardiac physiology and pathophysiology in culture, which could be used for drug screening for new therapeutics and cardiotoxins.

A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration.

Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

A Vegfc-Emilin2a-Cxcl8a Signaling Axis Required for Zebrafish Cardiac Regeneration.

Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential. We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration. We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration. These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.

The Role of Oncostatin M and Its Receptor Complexes in Cardiomyocyte Protection, Regeneration, and Failure.

Oncostatin M (OSM), a member of the interleukin-6 family, functions as a major mediator of cardiomyocyte remodeling under pathological conditions. Its involvement in a variety of human cardiac diseases such as aortic stenosis, myocardial infarction, myocarditis, cardiac sarcoidosis, and various cardiomyopathies make the OSM receptor (OSMR) signaling cascades a promising therapeutic target. However, the development of pharmacological treatment strategies is highly challenging for many reasons. In mouse models of heart disease, OSM elicits opposing effects via activation of the type II receptor complex (OSMR/gp130). Short-term activation of OSMR/gp130 protects the heart after acute injury, whereas chronic activation promotes the development of heart failure. Furthermore, OSM has the ability to integrate signals from unrelated receptors that enhance fetal remodeling (dedifferentiation) of adult cardiomyocytes. Because OSM strongly stimulates the production and secretion of extracellular proteins, it is likely to exert systemic effects, which in turn, could influence cardiac remodeling. Compared with the mouse, the complexity of OSM signaling is even greater in humans because this cytokine also activates the type I leukemia inhibitory factor receptor complex (LIFR/gp130). In this article, we provide an overview of OSM-induced cardiomyocyte remodeling and discuss the consequences of OSMR/gp130 and LIFR/gp130 activation under acute and chronic conditions.

OSKM-mediated reversible reprogramming of cardiomyocytes regenerates injured myocardium

Cellular reprogramming has rapidly become a promising methodology to generate new cardiomyocytes from non-cardiomyocyte cell types. Using the transient expression of OSKM factors, Chen et al. demonstrate a unique reprogramming strategy involving the modulation of the resident adult cardiomyocyte identity to an immature proliferative state (Science 373:1537–40, 2021). This OSKM-mediated reversion results in the adoption by adult murine cardiomyocytes of a transcriptional profile similar to cardiomyocytes found in developing hearts, as well as increased proliferative capacity of these reprogrammed cardiomyocytes compared to mature cardiomyocytes. Furthermore, this novel approach enhances the regeneration of adult murine hearts post-myocardial injury. Although concerns and questions remain, the encouraging results of this study advance the field of cardiac regeneration by providing a new technique to generate cardiomyocytes as well as insights into cardiomyocyte dedifferentiation and its relation to proliferation.