CUGBP1, a crucial factor for heart regeneration in mice

The mammalian heart is capable of achieving perfect regeneration following cardiac injury through sustained cardiomyocyte proliferation during the early period after birth. However, this regenerative capacity is lost by postnatal day 7 and throughout adulthood. CUGBP1 is critical for normal cardiac development but its role in heart regeneration remains unclear. Cardiac CUGBP1 levels are high in the early postnatal period and soon downregulate to adult levels within 1 week following birth in mice. The simultaneously diminished regenerative capacity and CUGBP1 levels by postnatal day lead us to hypothesize that CUGBP1 may be beneficial in heart regeneration. In this study, the function of CUGBP1 in heart regeneration was tested by a heart apex resection mouse model. We demonstrate that cardiac inactivation of CUGBP1 impairs neonatal heart regeneration at P1, in turn, replenishment of CUGBP1 levels prolong regenerative potential at P8 and P14. Furthermore, our results imply that the Wnt/β-catenin signaling and GATA4 involve in the CUGBP1 modulated neonatal heart regeneration. Altogether, our findings support CUGBP1 as a key factor promoting post-injury heart regeneration and provide a potential therapeutic method for heart disease.Subject terms: Cell proliferation, Self-renewal     

Regenerative potential of mouse embryonic stem cell-derived PDGFRα+ cardiac lineage committed cells in infarcted myocardium

BACKGROUND Pluripotent stem cell-derived cardiomyocytes(CMs) have become one of the most attractive cellular resources for cell-based therapy to rescue damaged cardiac tissue.AIM We investigated the regenerative potential of mouse embryonic stem cell(ESC)-derived platelet-derived growth factor receptor-α(PDGFRα)+ cardiac lineagecommitted cells(CLCs), which have a proliferative capacity but are in a morphologically and functionally immature state compared with differentiated CMs.METHODSWe induced mouse ESCs into PDGFRα+ CLCs and αMHC+ CMs using a combination of the small molecule cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197.We implanted PDGFRα+ CLCs and differentiated αMHC+ CMs into a myocardial infarction(MI) murine model and performed functional analysis using transthoracic echocardiography(TTE) and histologic analysis.RESULTS Compared with the untreated MI hearts, the anterior and septal regional wall motion and systolic functional parameters were notably and similarly improved in the MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs based on TTE.In histologic analysis, the untreated MI hearts contained a thinner ventricular wall than did the controls, while the ventricular walls of MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs were similarly thicker compared with that of the untreated MI hearts. Furthermore, implanted PDGFRα+ CLCs aligned and integrated with host CMs and were mostly differentiated into α-actinin+ CMs,and they did not convert into CD31+ endothelial cells or αSMA+ mural cells.CONCLUSION PDGFRα+ CLCs from mouse ESCs exhibiting proliferative capacity showed a regenerative effect in infarcted myocardium. Therefore, mouse ESC-derived PDGFRα+ CLCs may represent a potential cellular resource for cardiac regeneration.     

Cardiomyocyte proliferation,a target for cardiac regeneration

Cardiac diseases, characterized by cardiomyocyte loss, lead to dramatic impairment of cardiac function and ultimately to congestive heart failure. Despite significant advances, conventional treatments do not correct the defects in cardiac muscle cell numbers and the prognosis of congestive heart failure remains poor. The existence, in adult mammalian heart, of low but detectable cardiomyocyte proliferative capacities has shifted the target of regenerative therapy toward new therapeutical strategy. Indeed, the stimulation of terminally differentiated cardiomyocyte proliferation represents the main therapeutic approach for heart regeneration. Increasing evidence demonstrating that the loss of mammalian cardiomyocyte renewal potential shortly after birth causes the loss of regenerative capacities, strongly support the hypothesis that a detailed understanding of the molecular mechanisms controlling fetal and postnatal cardiomyocyte proliferation is essential to identify targets for cardiac regeneration. Here, we will review major developmental mechanisms regulating fetal cardiomyocyte proliferation and will describe the impact of the developmental switch, operating at birth and driving postnatal heart maturation, on the regulation of adult cardiomyocyte proliferation, all these mechanisms representing potential targets for cardiac repair and regeneration.     

Nerve Growth Factor Stimulates Cardiac Regeneration via Cardiomyocyte Proliferation in Experimental Heart Failure

Although the adult heart likely retains some regenerative capacity, heart failure (HF) typically remains a progressive disorder. We hypothesise that alterations in the local environment contribute to the failure of regeneration in HF. Previously we showed that nerve growth factor (NGF) is deficient in the failing heart and here we hypothesise that diminished NGF limits the cardiac regenerative response in HF. The capacity of NGF to augment cardiac regeneration was tested in a zebrafish model of HF. Cardiac injury with a HF phenotype was induced in zebrafish larvae at 72 hours post fertilization (hpf) by exposure to aristolochic acid (AA, 2.5 µM, 72–75 hpf). By 168 hpf, AA induced HF and death in 37.5% and 20.8% of larvae respectively (p<0.001). NGF mRNA expression was reduced by 42% (p<0.05). The addition of NGF (50 ng/ml) after exposure to AA reduced the incidence of HF by 50% (p<0.01) and death by 65% (p<0.01). Mechanistically, AA mediated HF was characterised by reduced cardiomyocyte proliferation as reflected by a 6.4 fold decrease in BrdU+ cardiomyocytes (p<0.01) together with features of apoptosis and loss of cardiomyocytes. Following AA exposure, NGF increased the abundance of BrdU+ cardiomyocytes in the heart by 4.8 fold (p<0.05), and this was accompanied by a concomitant significant increase in cardiomyocyte numbers. The proliferative effect of NGF on cardiomyocytes was not associated with an anti-apoptotic effect. Taken together the study suggests that NGF stimulates a regenerative response in the failing zebrafish heart, mediated by stimulation of cardiomyocyte proliferation.     

Molecular basis of functional myogenic specification of Bona Fide multipotent adult cardiac stem cells

Ischemic Heart Disease (IHD) remains the developed world’s number one killer. The improved survival from Acute Myocardial Infarction (AMI) and the progressive aging of western population brought to an increased incidence of chronic Heart Failure (HF), which assumed epidemic proportions nowadays. Except for heart transplantation, all treatments for HF should be considered palliative because none of the current therapies can reverse myocardial degeneration responsible for HF syndrome. To stop the HF epidemic will ultimately require protocols to reduce the progressive cardiomyocyte (CM) loss and to foster their regeneration. It is now generally accepted that mammalian CMs renew throughout life. However, this endogenous regenerative reservoir is insufficient to repair the extensive damage produced by AMI/IHD while the source and degree of CM turnover remains strongly disputed. Independent groups have convincingly shown that the adult myocardium harbors bona-fide tissue specific cardiac stem cells (CSCs). Unfortunately, recent reports have challenged the identity and the endogenous myogenic capacity of the c-kit expressing CSCs. This has hampered progress and unless this conflict is settled, clinical tests of repair/regenerative protocols are unlikely to provide convincing answers about their clinical potential. Here we review recent data that have eventually clarified the specific phenotypic identity of true multipotent CSCs. These cells when coaxed by embryonic cardiac morphogens undergo a precisely orchestrated myogenic commitment process robustly generating bona-fide functional cardiomyocytes. These data should set the path for the revival of further investigation untangling the regenerative biology of adult CSCs to harness their potential for HF prevention and treatment.     

Treatment of Glucocorticoids Inhibited Early Immune Responses and Impaired Cardiac Repair in Adult Zebrafish

Myocardial injury, such as myocardial infarction (MI), can lead to drastic heart damage. Zebrafish have the extraordinary ability to regenerate their heart after a severe injury. Upon ventricle resection, fibrin clots seal the wound and serve as a matrix for recruiting myeloid-derived phagocytes. Accumulated neutrophils and macrophages not only reduce the risk of infection but also secrete cytokines and growth factors to promote tissue repair. However, the underlying cellular and molecular mechanisms for how immune responses are regulated during the early stages of cardiac repair are still unclear. We investigated the role and programming of early immune responses during zebrafish heart regeneration. We found that zebrafish treated with an anti-inflammatory glucocorticoid had significantly reduced heart regenerative capacities, consistent with findings in other higher vertebrates. Moreover, inhibiting the inflammatory response led to excessive collagen deposition. A microarray approach was used to assess the differential expression profiles between zebrafish hearts with normal or impaired healing. Combining cytokine profiling and immune-staining, our data revealed that impaired heart regeneration could be due to reduced phagocyte recruitment, leading to diminished angiogenesis and cell proliferation post-cardiac injury. Despite their robust regenerative ability, our study revealed that glucocorticoid treatment could effectively hinder cardiac repair in adult zebrafish by interfering with the inflammatory response. Our findings may help to clarify the initiation of cardiac repair, which could be used to develop a therapeutic intervention that may enhance cardiac repair in humans to compensate for the loss of cardiomyocytes after an MI.     

LRP6 downregulation promotes cardiomyocyte proliferation and heart regeneration

The adult mammalian heart is thought to be a terminally differentiated organ given the postmitotic nature of cardiomyocytes. Consequently, the potential for cardiac repair through cardiomyocyte proliferation is extremely limited. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor that is required for embryonic heart development. In this study we investigated the role of LRP6 in heart repair through regulation of cardiomyocyte proliferation. Lrp6 deficiency increased cardiomyocyte cell cycle activity in neonatal, juvenile and adult mice. Cardiomyocyte-specific deletion of Lrp6 in the mouse heart induced a robust regenerative response after myocardial infarction (MI), led to reduced MI area and improvement in left ventricular systolic function. In vivo genetic lineage tracing revealed that the newly formed cardiomyocytes in Lrp6-deficient mouse hearts after MI were mainly derived from resident cardiomyocytes. Furthermore, we found that the pro-proliferative effect of Lrp6 deficiency was mediated by the ING5/P21 signaling pathway. Gene therapy using the adeno-associated virus (AAV)9 miRNAi-Lrp6 construct promoted the repair of heart injury in mice. Lrp6 deficiency also induced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our study identifies LRP6 as a critical regulator of cardiomyocyte proliferation, which may lead to the development of a novel molecular strategy to promote myocardial regeneration and repair.Subject terms: Cell-cycle exit, Cytokinesis     

Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than its damage. Here, we characterize the cellular response and regenerative capacity of the zebrafish heart after cryoinjury, an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). Localized damage was induced in 25% of the ventricle by cryocauterization (CC). During the first 24 hours post-injury, CC leads to cardiomyocyte death within the injured area and the near coronary vasculature. Cell death is followed by a rapid proliferative response in endocardium, epicardium and myocardium. During the first 3 weeks post-injury cell debris was cleared and the injured area replaced by a massive scar. The fibrotic tissue was subsequently degraded and replaced by cardiac tissue. Although animals survived CC, their hearts showed nonhomogeneous ventricular contraction and had a thickened ventricular wall, suggesting that regeneration is associated with processes resembling mammalian ventricular remodeling after acute MI. Our results provide the first evidence that, like mammalian hearts, teleost hearts undergo massive fibrosis after cardiac damage. Unlike mammals, however, the fish heart can progressively eliminate the scar and regenerate the lost myocardium, indicating that scar formation is compatible with myocardial regeneration and the existence of endogenous mechanisms of scar regression. This finding suggests that CC-induced damage in zebrafish could provide a valuable model for the study of the mechanisms of scar removal post-MI.     

Getting to the heart of regeneration in zebrafish

A scientific and clinical prerogative of the 21st century is to stimulate the regenerative ability of the human heart. While the mammalian heart shows little or no natural regeneration in response to injury, certain non-mammalian vertebrates possess an elevated capacity for cardiac regeneration. Adult zebrafish restore ventricular muscle removed by surgical resection, events that involve little or no scarring. Recent studies have begun to reveal cellular and molecular mechanisms of this regenerative process that have exciting implications for human cardiac biology and disease.     

Cardiac regeneration: epicardial mediated repair

The hearts of lower vertebrates such as fish and salamanders display scarless regeneration following injury, although this feature is lost in adult mammals. The remarkable capacity of the neonatal mammalian heart to regenerate suggests that the underlying machinery required for the regenerative process is evolutionarily retained. Recent studies highlight the epicardial covering of the heart as an important source of the signalling factors required for the repair process. The developing epicardium is also a major source of cardiac fibroblasts, smooth muscle, endothelial cells and stem cells. Here, we examine animal models that are capable of scarless regeneration, the role of the epicardium as a source of cells, signalling mechanisms implicated in the regenerative process and how these mechanisms influence cardiomyocyte proliferation. We also discuss recent advances in cardiac stem cell research and potential therapeutic targets arising from these studies.     

Non-coding RNAs in cardiomyocyte proliferation and cardiac regeneration: Dissecting their therapeutic values

Cardiovascular diseases are associated with high incidence and mortality, contribute to disability and place a heavy economic burden on countries worldwide. Stimulating endogenous cardiomyocyte proliferation and regeneration has been considering as a key to repair the injured heart caused by ischaemia. Emerging evidence has proved that non-coding RNAs participate in cardiac proliferation and regeneration. In this review, we focus on the observation and mechanism that microRNAs (or miRNAs), long non-coding RNAs (or lncRNAs) and circular RNA (or circRNAs) regulate cardiomyocyte proliferation and regeneration to repair a damaged heart. Furthermore, we highlight the potential therapeutic role of some non-coding RNAs used in stimulating CMs proliferation. Finally, perspective on the development of non-coding RNAs therapy in cardiac regeneration is presented.     

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.     

BMP and Notch Signaling Pathways differentially regulate Cardiomyocyte Proliferation during Ventricle Regeneration

Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration.     

LRP5 regulates cardiomyocyte proliferation and neonatal heart regeneration by the AKT/P21 pathway

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low‐density lipoprotein receptor‐related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac‐specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin‐dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.     

Improving cardiac regeneration after injury: Are we a step closer?

During regeneration, lost functional tissue can, in general, be replaced by different mechanisms, including proliferation of terminally differentiated cells or through differentiation of resident stem cells. It is a well-accepted dogma that the mammalian heart cannot efficiently regenerate upon injury as a consequence of insufficient oxygen supply. This is in sharp contrast to the hearts of adult zebrafish or newts that are able to replace lost ventricular tissue. Novel data indicate that the young murine heart also has the ability to regenerate within the first week after birth using mechanisms apparently quite similar to those observed in fish. This now provides us with a good starting point to identify the molecular mechanisms that led to the loss of the regenerative capacity of the adult mammalian heart. These future studies will also indicate whether it will be possible to reawaken the regenerative capability of cardiomyocytes in the human heart by treatment with selected pharmaceuticals.     

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction¹. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life^2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues^4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury^6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex^2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish^8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.     

Cardiac regeneration as an environmental adaptation

Heart failure is a devastating disease that affects more than 26 million individuals worldwide and has a 5-year survival rate of less than 50%, with its development in part reflecting the inability of the adult mammalian heart to regenerate damaged myocardium. In contrast, certain vertebrate species including fish and amphibians, as well as neonatal mammals, are capable of complete cardiac regeneration after various types of myocardial injury such as resection of the ventricular apex or myocardial infarction, with this regeneration being mediated by the proliferation of cardiomyocytes, dissolution of temporary fibrosis, and revascularization of damaged tissue. In an effort to identify regulators of cardiac regeneration and to develop novel therapeutic strategies for induction of myocardial regeneration in the adult human heart, recent studies have adopted an approach based on comparative biology. These studies have pointed to cellular or tissue responses to environmental cues—including activation of the immune system, the reaction to mechanical stress, and the adoption of oxidative metabolism—as key determinants of whether the heart undergoes regeneration or nonregenerative scar formation after injury. We here summarize recent insight into the molecular mechanisms as well as environmental and systemic factors underlying cardiac regeneration based on the findings of inter- or intraspecific comparisons between regenerative and nonregenerative responses to heart injury. We also discuss how recent progress in understanding the molecular, systemic, and environmental basis of cardiac regeneration in a variety of organisms may relate to multiple scientific fields including ecology, evolutionary as well as developmental biology.