Induction Of Cardiomyocyte Proliferation Research Articles

Induction of cardiomyocyte proliferation, a new way forward for true myocardial regeneration?

Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality worldwide and although acute treatment during myocardial infarction has improved significantly, chronic burden of the disease gained much attention recent years (1,2). The major cause of the failing hearts, however, the loss of contractile cardiomyocytes is currently still a problem to overcome.

The role of the tumor necrosis factor (TNF)-related weak inducer of apoptosis (TWEAK) in offspring exposed to prenatal hypoxia.

Exposure to prenatal hypoxia in rats leads to intrauterine growth restriction (IUGR), decreases fetal cardiomyocyte proliferation and increases the risk to develop cardiovascular diseases (CVD) later in life. The tumor necrosis factor-related weak inducer of apoptosis (TWEAK) induces cardiomyocyte proliferation through activation of the fibroblast growth factor-inducible molecule 14 (Fn-14) receptor. The TWEAK/Fn-14 pathway becomes quiescent shortly after birth, however, it becomes upregulated with CVD; suggesting that it could be a link between the increased susceptibility to CVD in pregnancies complicated by hypoxia/IUGR. We hypothesized that offspring exposed to prenatal hypoxia will exhibit reduced cardiomyocyte proliferation due to reduced Fn-14 expression and that the TWEAK/Fn-14 pathway will be expressed in those adult offspring. We exposed pregnant Sprague Dawley rats to control (21% oxygen) or hypoxic (11% oxygen) conditions from gestational days 15 to 21. Ventricular cardiomyocytes were isolated from male and female, control and hypoxic offspring at postnatal day 1. Proliferation was assessed in the presence or absence of r-TWEAK (72 h, 100 ng/ml). Prenatal hypoxia was not associated with differences in Fn-14 protein expression in either male or female offspring. Cardiomyocytes from prenatal hypoxic male, but not female, offspring had decreased proliferation compared with controls. Addition of r-TWEAK increased cardiomyocyte proliferation in all offspring. In adult offspring of all groups, the TWEAK/Fn-14 pathway was not detectable. Cardiomyocyte proliferation was reduced in only male offspring exposed to prenatal hypoxia but this was not due to changes in the Fn-14 pathway. Studies addressing other pathways associated with CVD and prenatal hypoxia are needed.

Cardiac resident macrophages are involved in hypoxia‑induced postnatal cardiomyocyte proliferation.

Induction of cardiomyocyte proliferation, the most promising approach to reverse myocardial attrition, has been gaining importance as a therapy for cardiovascular disease. Hypoxia and macrophages were previously independently reported to promote cardiomyocyte proliferation in mice. However, whether hypoxia promotes cardiomyocyte proliferation in humans, and the association between hypoxia and macrophages in cardiomyocyte proliferation, have not to the best of our knowledge been previously investigated. The present study investigated the cardiomyocyte proliferation in 22 acyanotic and 29 cyanotic patients. Cardiomyocyte proliferation in a hypoxic mouse model (15% O2) was subsequently performed and the macrophage subsets were analyzed. A C-C chemokine receptor type 2 (CCR2) inhibitor was used to increase the number of resident macrophages in order to investigate the effect of macrophages on cardiomyocyte proliferation. The results demonstrated that cardiomyocyte proliferation in the cyanotic infant group was significantly increased compared with the acyanotic infant group and the hypoxia-treated C57BL/6J neonates confirmed the hypoxia-induced cardiomyocyte proliferation. However, hypoxia did not induce the proliferation of isolated cardiomyocytes. Notably, hypoxia treatment increased the number of cardiac resident macrophages in neonate hearts. Furthermore, increasing the number of resident macrophages significantly enhanced cardiomyocyte proliferation. In conclusion, postnatal hypoxia promoted cardiomyocyte proliferation in humans and animals, and cardiac resident macrophages may be involved in this process. Therefore, this novel mechanism may provide a promising strategy for cardiovascular disease treatment.

HASF (C3orf58) is a novel ligand of the insulin-like growth factor 1 receptor

We have recently shown that hypoxia and Akt-induced stem cell factor (HASF) protects the heart from ischemia-induced damage and promotes cardiomyocyte proliferation. While we have identified certain signaling pathways responsible for these protective effects, the receptor mediating these effects was unknown. Here, we undertook studies to identify the HASF receptor. A yeast two-hybrid screen identified a partial fragment of insulin-like growth factor 1 receptor (IGF1R) as a binding partner of HASF. Subsequent co-immunoprecipitation experiments showed that HASF bound to full-length IGF1R. Binding assays revealed a high affinity of HASF for IGF1R. The treatment of neonatal ventricular cardiomyocytes with HASF resulted in the phosphorylation of IGF1R and other proteins known to be involved in IGF1R-mediated signaling pathways. HASF-mediated ERK activation was abrogated by IGF1R pharmacological inhibitors and siRNAs that targeted IGF1R. However, siRNA-mediated knockdown of either IGF2R or the insulin receptor had no effect on HASF-induced cell signaling. Additionally, pharmacologic inhibition of IGF1R impeded HASF's ability to induce cardiomyocyte proliferation. Finally, we documented that in vivo deletion of the IGF1R completely abolished the ability of HASF to promote cardiomyocyte proliferation in an overexpression mouse model providing further evidence in vivo that the IGF1R is the functional receptor for HASF.

Reprogramming‐derived gene cocktail increases cardiomyocyte proliferation for heart regeneration

Although remnant cardiomyocytes (CMs) possess a certain degree of proliferative ability, efficiency is too low for cardiac regeneration after injury. In this study, we identified a distinct stage within the initiation phase of CM reprogramming before the MET process, and microarray analysis revealed the strong up‐regulation of several mitosis‐related genes at this stage of reprogramming. Several candidate genes were selected and tested for their ability to induce CM proliferation. Delivering a cocktail of three genes, FoxM1, Id1, and Jnk3‐shRNA (FIJs), induced CMs to re‐enter the cell cycle and complete mitosis and cytokinesis in vitro. More importantly, this gene cocktail increased CM proliferation in vivo and significantly improved cardiac function and reduced fibrosis after myocardial infarction. Collectively, our findings present a cocktail FIJs that may be useful in cardiac regeneration and also provide a practical strategy for probing reprogramming assays for regeneration of other tissues.

Hypoxia induces heart regeneration in adult mice.

The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.

Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment.

The cardiac microenvironment includes cardiomyocytes, fibroblasts and macrophages, which regulate remodeling after myocardial infarction (MI). Targeting this microenvironment is a novel therapeutic approach for MI. We found that the natural compound derivative, BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime) modulated the cardiac microenvironment to exert a therapeutic effect on MI. Using a series of co-culture studies, BIO induced proliferation in cardiomyocytes and inhibited proliferation in cardiac fibroblasts. BIO produced multiple anti-fibrotic effects in cardiac fibroblasts. In macrophages, BIO inhibited the expression of pro-inflammatory factors. Significantly, BIO modulated the molecular crosstalk between cardiac fibroblasts and differentiating macrophages to induce polarization to the anti-inflammatory M2 phenotype. In the optically transparent zebrafish-based heart failure model, BIO induced cardiomyocyte proliferation and completely recovered survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel therapeutic effects of BIO. We identified the mechanism of BIO as differential modulation of p27 protein expression and potent induction of anti-inflammatory interleukin-10. In a rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed modulation of the cardiac microenvironment by BIO, with increased presence of anti-inflammatory M2 macrophages. Our results demonstrate that BIO produces unique effects in the cardiac microenvironment to promote recovery post-MI.

Abstract 299: Natural Product Derivative Bio Promotes Recovery After Myocardial Infarction via Unique Modulation of the Cardiac Microenvironment

Rationale: Myocardial infarction (MI) induces cardiac remodeling, which is regulated by the cardiac microenvironment and results in scarring and loss of cardiac function. Targeting the microenvironment represents a novel therapeutic approach. Objective: To investigate the therapeutic effect of a natural compound derivative, BIO [(2’Z,3’E)-6-Bromoindirubin-3′-oxime], on cardiac microenvironment cells and remodeling post-MI. Methods and Results: Using a series of co-culture studies, BIO was shown to induce proliferation in cardiomyocytes and, conversely, inhibit proliferation in cardiac fibroblasts. In addition, BIO produced anti-fibrotic effects in the fibroblasts, such as reduced motility and expression changes in fibrosis-related factors. In macrophages, BIO inhibited the expression of pro-inflammatory inducible nitric oxide synthase. Interestingly, BIO modulated molecular crosstalk between cardiac fibroblasts and differentiating macrophages to increase expression of anti-inflammatory M2 macrophage markers. In the optically transparent zebrafish-based model of heart failure, BIO induced cardiomyocyte proliferation to recover survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel, potential therapeutic effects of BIO on remodeling. We characterized these novel effects of BIO as differential modulation of p27 expression and potent induction of interleukin-10 in microenvironment cells. In rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed a greater presence of anti-inflammatory M2 macrophages in the infarction zone. BIO treatment also reduced serum levels of pro-fibrotic interleukin-6. Conclusions: BIO differentially activates signaling pathways in cardiomyocytes, cardiac fibroblasts and cardiac macrophages to reduce fibrosis, increase cardiomyocyte proliferation and promote recovery post-MI.

Towards regenerating the mammalian heart: challenges in evaluating experimentally induced adult mammalian cardiomyocyte proliferation

In recent years, there has been a dramatic increase in research aimed at regenerating the mammalian heart by promoting endogenous cardiomyocyte proliferation. Despite many encouraging successes, it remains unclear if we are any closer to achieving levels of mammalian cardiomyocyte proliferation for regeneration as seen during zebrafish regeneration. Furthermore, current cardiac regenerative approaches do not clarify whether the induced cardiomyocyte proliferation is an epiphenomena or responsible for the observed improvement in cardiac function. Moreover, due to the lack of standardized protocols to determine cardiomyocyte proliferation in vivo, it remains unclear if one mammalian regenerative factor is more effective than another. Here, we discuss current methods to identify and evaluate factors for the induction of cardiomyocyte proliferation and challenges therein. Addressing challenges in evaluating adult cardiomyocyte proliferation will assist in determining 1) which regenerative factors should be pursued in large animal studies; 2) if a particular level of cell cycle regulation presents a better therapeutic target than another (e.g., mitogenic receptors vs. cyclins); and 3) which combinatorial approaches offer the greatest likelihood of success. As more and more regenerative studies come to pass, progress will require a system that not only can evaluate efficacy in an objective manner but can also consolidate observations in a meaningful way.

Heart regeneration

Regenerating an injured heart holds great promise for millions of patients suffering from heart diseases. Since the human heart has very limited regenerative capacity, this is a challenging task. Numerous strategies aiming to improve heart function have been developed. In this review we focus on approaches intending to replace damaged heart muscle by new cardiomyocytes. Different strategies for the production of cardiomyocytes from human embryonic stem cells or human induced pluripotent stem cells, by direct reprogramming and induction of cardiomyocyte proliferation are discussed regarding their therapeutic potential and respective advantages and disadvantages. Furthermore, different methods for the transplantation of pluripotent stem cell-derived cardiomyocytes are described and their clinical perspectives are discussed. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Acute inflammation stimulates a regenerative response in the neonatal mouse heart.

Cardiac injury in neonatal 1-day-old mice stimulates a regenerative response characterized by reactive cardiomyocyte proliferation, which is distinguished from the fibrotic repair process in adults. Acute inflammation occurs immediately after heart injury and has generally been believed to exert a negative effect on heart regeneration by promoting scar formation in adults; however, little is known about the role of acute inflammation in the cardiac regenerative response in neonatal mice. Here, we show that acute inflammation induced cardiomyocyte proliferation after apical intramyocardial microinjection of immunogenic zymosan A particles into the neonatal mouse heart. We also found that cardiac injury-induced regenerative response was suspended after immunosuppression in neonatal mice, and that cardiomyocytes could not be reactivated to proliferate after neonatal heart injury in the absence of interleukin-6 (IL-6). Furthermore, cardiomyocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3), the major downstream effector of IL-6 signaling, decreased reactive cardiomyocyte proliferation after apical resection. Our results indicate that acute inflammation stimulates the regenerative response in neonatal mouse heart, and suggest that modulation of inflammatory signals might have important implications in cardiac regenerative medicine.

Abstract 13: Hippo Signaling Deletion During Heart Failure Reverses Functional Decline

The heart has long been thought of as a static organ incapable of repair. Recent findings have challenged this view of the heart, and have demonstrated that mature cardiomyocytes are capable of re-entering the cell-cycle. However, there is still paucity in understanding endogenous mechanisms preventing cardiomyocyte self-renewal. Our approach is to apply developmental mechanisms of cardiomyocyte cell-cycle control to the damaged heart by altering the Hippo signaling pathway. During development Hippo signaling regulates intrinsic organ size. The core mammalian Hippo pathway includes the Ste20-like serine/threonine kinases Mst1 and Mst2, homologous to the Drosophila Hippo kinase. A subsequent kinase cascade leads to the phosphorylation of the transcription factor Yap. Phosphorylated Yap is sequestered in the cytoplasm, thus preventing transcriptional activity. We previously demonstrated Hippo signaling controls cardiomyocyte proliferation during development to restrain heart size. Additionally, using both the Apex resection (AR) and LAD-ligation (MI) models of cardiac damage, in a Hippo signaling deletion mouse, we demonstrated cardiac regeneration. Indicated by preserved cardiac function and reduced fibrotic scar formation. Additionally, these hearts display cardiomyocyte proliferation as marked by EDU incorporation, pHH3, AurkB, and Ki67 staining. We are taking a new approach to determine the effect of Hippo signaling deletion on the failing heart, by inducing Hippo deletion after fibrotic scar formation has already occurred. To Thus far our results indicate functional recovery of the failing heart only after inducible deletion of Hippo signaling. Consistent with our previous data, preliminary results indicate adult cardiomyocytes after Hippo deletion re-enter the cell cycle. By altering Hippo signaling during heart failure and subsequently inducing cardiomyocyte proliferation we have established recovery of cardiac function. These results will greatly advance strategies to induce cardiac repair.

Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window.

Therapies developed for adult patients with heart failure have been shown to be ineffective in pediatric clinical trials, leading to the recognition that new pediatric-specific therapies for heart failure must be developed. Administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates regeneration of heart muscle cells (cardiomyocytes) in adult mice. Because proliferation-competent cardiomyocytes are more abundant in growing mammals, we hypothesized that administration of rNRG1 during the neonatal period might be more effective than in adulthood. If so, neonatal rNRG1 delivery could be a new therapeutic strategy for treating heart failure in pediatric patients. To evaluate the effectiveness of rNRG1 administration in cardiac regeneration, newborn mice were subjected to cryoinjury, which induced myocardial dysfunction and scar formation and decreased cardiomyocyte cell cycle activity. Early administration of rNRG1 to mice from birth to 34 days of age improved myocardial function and reduced the prevalence of transmural scars. In contrast, administration of rNRG1 from 4 to 34 days of age only transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). We also assessed the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease who were less than 6 months of age. Our results identify an effective time period within which to execute rNRG1 clinical trials in pediatric patients for the stimulation of cardiomyocyte regeneration.

Recombinant neuregulin 1 does not activate cardiomyocyte DNA synthesis in normal or infarcted adult mice.

ObjectivesNeuregulin 1 signaling plays an important role in cardiac trabecular development, and in sustaining functional integrity in adult hearts. Treatment with neuregulin 1 enhances adult cardiomyocyte differentiation, survival and/or function in vitro and in vivo. It has also been suggested that recombinant neuregulin 1β1 (NRG1β1) induces cardiomyocyte proliferation in normal and injured adult hearts. Here we further explore the impact of neuregulin 1 signaling on adult cardiomyocyte cell cycle activity.Methods and ResultsAdult mice were subjected to 9 consecutive daily injections of recombinant NRG1β1 or vehicle, and cardiomyocyte DNA synthesis was quantitated via bromodeoxyuridine (BrdU) incorporation, which was delivered using mini-osmotic pumps over the entire duration of NRG1β1 treatment. NRG1β1 treatment inhibited baseline rates of cardiomyocyte DNA synthesis in normal mice (cardiomyocyte labelling index: 0.019±0.005% vs. 0.003±0.001%, saline vs. NRG1β1, P<0.05). Acute NRG1β1 treatment did result in activation of Erk1/2 and cardiac myosin regulatory light chain (down-stream mediators of neuregulin signalling), as well as activation of DNA synthesis in non-cardiomyocytes, validating the biological activity of the recombinant protein. In other studies, mice were subjected to permanent coronary artery occlusion, and cardiomyocyte DNA synthesis was monitored via tritiated thymidine incorporation which was delivered as a single injection 7 days post-infarction. Daily NRG1β1 treatment had no impact on cardiomyocyte DNA synthesis in the infarcted myocardium (cardiomyocyte labelling index: 0.039±0.011% vs. 0.027±0.021%, saline vs. NRG1β1, P>0.05).SummaryThese data indicate that NRG1β1 treatment does not increase cardiomyocyte DNA synthesis (and consequently does not increase the rate of cardiomyocyte renewal) in normal or infarcted adult mouse hearts. Thus, any improvement in cardiac structure and function observed following neuregulin treatment of injured hearts likely occurs independently of overt myocardial regeneration.

Thyroid hormone does not induce maturation of embryonic chicken cardiomyocytes in vitro.

Fetal cardiac growth in mammalian models occurs primarily by cell proliferation (hyperplasia). However, most cardiomyocytes lose the ability to proliferate close to term and heart growth continues by increasing cell size (hypertrophy). In mammals, the thyroid hormone triiodothyronine (T3) is an important driver of this process. Chicken cardiomyocytes, however, keep their proliferating ability long after hatching but little information is available on the mechanisms controlling cell growth and myocyte maturation in the chicken heart. Our aim was to study the role of T3 on proliferation and differentiation of embryonic chicken cardiomyocytes (ECCM), enzymatically isolated from 19‐day‐old embryos and to compare the effects to those of insulin‐like growth factor‐1 (IGF‐1) and phenylephrine (PE). Hyperplasia was measured using a proliferation assay (MTS) and hypertrophy/multinucleation was analyzed morphologically by phalloidin staining of F‐actin and nuclear staining with DAPI. We show that IGF‐1 induces a significant increase in ECCM proliferation (30%) which is absent with T3 and PE. PE induced both hypertrophy (61%) and multinucleation (41%) but IGF‐1 or T3 did not. In conclusion, we show that T3 does not induce maturation or proliferation of cardiomyocytes, while IGF‐1 induces cardiomyocyte proliferation and PE induces maturation of cardiomyocytes.

Abstract 20492: Mlf1 Regulates Myocyte Proliferation in Postnatal Hearts

Lower vertebrates, such as newt and zebrafish, retain a robust cardiac regenerative capacity following injury. Although adult mammals lack this cardiac regenerative potential, there is ample interest in understanding how heart regeneration occurs, and to reawaken this process in adult humans. Recently, we showed that mice are capable of regenerating their hearts shortly after birth following injury. This regenerative response is associated with robust proliferation of cardiomyocytes without significant hypertrophy or fibrosis. However, this regenerative capacity is lost by 7 days postnatally, coinciding with cell cycle arrest. In an effort to determine the mechanism of cardiomyocytes cell cycle arrest after the first week of life, we performed a gene array after cardiac injury at multiple post-natal time points. This enabled us to identify a number of transcription factors that are differentially expressed during this postnatal window. We recently reported that one of these transcription factors Meis1 regulates postnatal cell cycle arrest of cardiomyocytes. Furthermore, Myeloid leukemia factor 1 (Mlf1), a bhlh transcription factor that has not been previously studied in the heart has similar dysregulated pattern following injury. Our preliminary data with in-vitro knockdown of Mlf1 in cardiomyocyte resulted in 2-fold increase in cardiomyocyte proliferation. Furthermore, immunohistochemistry results indicated that the endogenous expression and nuclear localization of Mlf1 in the post-natal cardiomyocytes coincides with cell cycle arrest. To explore this pattern, we generated a cardiomyocyte-specific Mlf1 knockout mouse, and showed that loss of Mlf1 results in robust cardiomyocyte proliferation in postnatal hearts (P14). Additionally, we confirmed previous reports that Mlf1 regulates p53 and induces cell cycle arrest by induction of CDK inhibitors like p21 and p57 in these Mlf1 KO mice. This suggests a role of Mlf1 in promoting reactivation of injured myocardium through induction of cardiomyocyte proliferation. These findings will further provide evidences of molecular mechanisms involved in the dormant regenerative capacity in adult mammals that can be a potential target of therapeutic approaches.

Abstract 15973: miRNA-Hippo Pathway Plays a Critical Role in the Cardiac Conduction System

The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats and the CCS defects can give rise to cardiac arrhythmia, a leading cause for morbidity worldwide. Given the poor self-repair potential in the adult human CCS, it is critical to elucidate the molecular mechanisms limiting the CCS regeneration to facilitate developing efficient cardiovascular therapies. MicroRNAs (miRs) are small non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster can induce cardiomyocyte proliferation and regeneration. Hippo signaling, an ancient organ size control pathway, represses cardiomyocyte proliferation and regeneration. Here we found that both miR-17-92 and Hippo signaling were active in the CCS. Specific disruption of either miR-17-92 or Hippo signaling in the CCS gave rise to cardiac arrhythmias in mice. Notably, miR-17-92 regulates Hippo signaling through directly repressing Lats2, a core Hippo pathway component. In miR-17-92 null mutant hearts, up-regulated Lats2 led to increased Hippo pathway activity. Moreover, we performed chromatin immunoprecipitation deep sequencing (ChIP-Seq) using Yap antibody, the Hippo signaling effector, which data suggested that Hippo signaling regulates genes involved in the CCS homeostasis. Together, our data indicate a novel miR-Hippo genetic pathway plays critical function in the CCS.

How to make a cardiomyocyte.

During development, cardiogenesis is orchestrated by a family of heart progenitors that build distinct regions of the heart. Each region contains diverse cell types that assemble to form the complex structures of the individual cardiac compartments. Cardiomyocytes are the main cell type found in the heart and ensure contraction of the chambers and efficient blood flow throughout the body. Injury to the cardiac muscle often leads to heart failure due to the loss of a large number of cardiomyocytes and its limited intrinsic capacity to regenerate the damaged tissue, making it one of the leading causes of morbidity and mortality worldwide. In this Primer we discuss how insights into the molecular and cellular framework underlying cardiac development can be used to guide the in vitro specification of cardiomyocytes, whether by directed differentiation of pluripotent stem cells or via direct lineage conversion. Additional strategies to generate cardiomyocytes in situ, such as reactivation of endogenous cardiac progenitors and induction of cardiomyocyte proliferation, will also be discussed.

Cover Image

Background: The zebrafish heart regenerates after various severe injuries. Common processes of heart regeneration are cardiomyocyte proliferation, activation of epicardial tissue, and neovascularization. In order to further characterize heart regeneration processes, we introduced milder injuries and compared responses to those induced by ventricular apex resection, a widely used injury method. We used scratching of the ventricular surface and puncturing of the ventricle with a fine tungsten needle as injury‐inducing techniques. Results: Scratching the ventricular surface induced subtle cardiomyocyte proliferation and responses of the epicardium. Endothelial cell accumulation was limited to the surface of the heart. Ventricular puncture induced cardiomyocyte proliferation, endocardial and epicardial activation, and neo‐vascularization, similar to the resection method. However, the degree of the responses was milder, correlating with milder injury. Sham operation induced epicardial aldh1a2 expression but not tbx18 and WT1. Conclusions: Puncturing the ventricle induces responses equivalent to resection at milder degrees in a shorter time frame and can be used as a simple injury model. Scratching the ventricle did not induce heart regeneration and can be used for studying wound responses to epicardium. Developmental Dynamics 243:1477–1486, 2014. © 2014 Wiley Periodicals, Inc.

Abstract 260: A miRNA-Hippo Pathway Promotes Cardiac Conduction System Regeneration

The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats and CCS defects can give rise to cardiac arrhythmia, a leading cause for morbidity worldwide. Given the poor self-repair potential in the adult human CCS, it is critical to elucidate the molecular mechanisms limiting CCS regeneration to facilitate developing efficient cardiovascular therapies. microRNAs (miRs) are small non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster can induce cardiomyocyte proliferation and regeneration. Hippo signaling, an ancient organ size control pathway, represses cardiomyocyte proliferation and regeneration. Here we found that both miR-17-92 and Hippo signaling were active in CCS. Disruption of either miR-17-92 or Hippo signaling in heart gave rise to cardiac arrhythmias in mice. Notably, miR-17-92 regulates Hippo signaling through repressing Lats2, a core Hippo pathway component. In miR-17-92 null mutant hearts, up-regulated Lats2 led to increased Hippo pathway activity. Moreover, we performed chromatin immunoprecipitation deep sequencing (ChIP-Seq) using YAP, the Hippo signaling effector, which suggested that Hippo signaling regulates genes involved in CCS homeostasis. Together, we propose a novel miR-Hippo genetic pathway that promotes CCS regeneration.