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Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, reactive oxygen species homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations ( hbaa + Mac and timp4.3 + Mac 3) were enriched only in regenerative hearts and barely recovered after +1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 d (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and extracellular matrix remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration., Competing Interests: KW, IL, KC, KL, KL, TK, YC, KY, SL No competing interests declared, (© 2023, Wei et al.)

Background: Adult mammalian heart loses regeneration ability following ischemic injury due to the loss of cardiomyocyte mitosis. However, the molecular mechanisms underlying the post-mitotic nature of cardiomyocytes remain largely unknown.Objectives: The purpose of this study was to define the essential role of long noncoding ribonucleic acids (lncRNAs) in heart regeneration during postnatal and adult injury.Methods: Myh6-driving cardiomyocyte-specific lncRNA-CAREL transgenic mice and adenovirus-mediated in vivo silencing of endogenous CAREL were used in this study. The effect of CAREL on cardiomyocyte replication and heart regeneration after apical resection or myocardial infarction was assessed by detecting mitosis and cytokinesis.Results: An lncRNA CAREL was found significantly up-regulated in cardiomyocytes from neonatal mice (P7) in parallel with loss of regenerative capacity. Cardiac-specific overexpression of CAREL in mice reduced cardiomyocyte division and proliferation and blunted neonatal heart regeneration after injury. Conversely, silencing of CAREL in vivo markedly promoted cardiac regeneration and improved heart functions after myocardial infarction in neonatal and adult mice. CAREL acted as a competing endogenous ribonucleic acid for miR-296 to derepress the expression of Trp53inp1 and Itm2a, the target genes of miR-296. Consistently, overexpression of miR-296 significantly increased cardiomyocyte replication and cardiac regeneration after injury. Decline of cardiac regenerative ability in CAREL transgenic mice was also rescued by miR-296. A short fragment containing the conserved sequence of CAREL reduced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes as the full-length CAREL.Conclusions: LncRNA CAREL regulates cardiomyocyte proliferation and heart regeneration in postnatal and adult heart after injury by acting as a competing endogenous ribonucleic acid on miR-296 that targets Trp53inp1 and Itm2a. [ABSTRACT FROM AUTHOR]

Objective: We sought to define the intrinsic stem cell capacity in pediatric heart lesions, and the effects of diagnosis and of age, in order to inform evidence-based use of potential autologous stem cell sources for regenerative medicine therapy. Methods: Ventricular explants derived from patients with hypoplastic left heart syndrome (HLHS), tetralogy of Fallot (TF), dilated cardiomyopathy (DCM) and ventricular septal defect (VSD) were analyzed following standard in vitro culture conditions, which yielded cardiospheres (C-spheres), indicative of endogenous stem cell capacity. C-sphere counts generated per 5 mm3 tissue explant and the presence of cardiac progenitor cells were correlated to patient age, diagnosis and echocardiographic function. Results: Cardiac explants from patients less than one year of age with TF and DCM robustly generated c-kit- and/or vimentin-positive cardiac mesenchymal cells (CMCs), populating spontaneously forming C-spheres. Beyond one year of age, there was a marked reduction or absence of cardiac explant-derivable cardiac stem cell content in patients with TF, VSD and DCM. Stem cell content in HLHS and DCM strongly correlated to the echocardiographic function in the corresponding ventricular chamber, with better echocardiographic function correlating to a more robust regenerative cellular content. Conclusions: We conclude that autologous cardiomyogenic potential in pediatric heart lesions is robust during the first year of life and uniformly declines thereafter. Depletion of stem cell content occurs at an earlier age in HLHS with the onset of ventricular failure in a chamber-specific pattern that correlates directly to ventricular dysfunction. These data suggest that regenerative therapies using autologous cellular sources should be implemented in the neonatal period before the potentially rapid onset of single ventricle failure in HLHS or the evolution of biventricular failure in DCM. [ABSTRACT FROM AUTHOR]

Background: The adult mammalian heart is incapable of meaningful functional recovery after injury, and thus promoting heart regeneration is 1 of the most important therapeutic targets in cardiovascular medicine. In contrast to the adult mammalian heart, the neonatal mammalian heart is capable of regeneration after various types of injury. Since the first report in 2011, a number of groups have reported their findings on neonatal heart regeneration. The current review provides a comprehensive analysis of heart regeneration studies in neonatal mammals conducted to date, outlines lessons learned, and poses unanswered questions.Methods: We performed a PubMed search using the keywords "neonatal" and "heart" and "regeneration." In addition, we assessed all publications that cited the first neonatal heart regeneration reports: Porrello et al, Science, Feb 2011 for apical resection injury; Porrello et al, PNAS, Dec 2012 for coronary ligation injury; and Mahmoud et al, Nature Methods, Jan 2014 for surgical methodology. Publications were examined for surgical models used, timing of surgery, and postinjury assessment including anatomic, histological, and functional assessment, as well as conclusions drawn.Results: We found 30 publications that performed neonatal apical resection, 19 publications that performed neonatal myocardial infarction by coronary artery ligation, and 6 publications that performed cryoinjury using liquid nitrogen-cooled metal probes. Both apical resection and ischemic infarction injury in neonatal mice result in a robust regenerative response, mediated by cardiomyocyte proliferation. On the other hand, several reports have demonstrated that cryoinjury is associated with incomplete heart regeneration in neonatal mice. Not surprisingly, several studies suggest that injury size, as well as surgical and histological techniques, can strongly influence the observed regenerative response and final conclusions. Studies have utilized these neonatal cardiac injury models to identify factors that either inhibit or stimulate heart regeneration.Conclusions: Overall, there is consensus that both apical resection and coronary ligation injuries during the first 2 days of life result in heart regeneration in neonatal mammals, whereas cryoinjury was not associated with a similar regenerative response. This regenerative response is mediated by proliferation of preexisting cardiomyocytes, and is modifiable by injury size and surgical technique, as well as metabolic, immunologic, genetic, and environmental factors. [ABSTRACT FROM AUTHOR]

The ability to conditionally inactivate genes is instrumental for fine genetic analysis of all biological processes, but is especially important for studies of biological events, such as regeneration, which occur late in ontogenesis or in adult life. We have constructed and tested a fully conditional gene trap vector, and used it to inactivate tbx5a in the cardiomyocytes of larval and adult zebrafish. We observe that loss of tbx5a function significantly impairs the ability of zebrafish hearts to regenerate after ventricular resection, indicating that Tbx5a plays an essential role in the transcriptional program of heart regeneration. [ABSTRACT FROM AUTHOR]

In 2002, Ken Poss discovered that zebrafish, at that time an emerging vertebrate model organism in basic research, were able to regenerate their heart upon resection of the ventricular apex. This finding set in motion a new field of research on heart regeneration, which has recently expanded in other model organisms including mammals. We interviewed Ken Poss to find out more about his motivation and vision for the future of tissue regeneration research. [ABSTRACT FROM AUTHOR]

In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (co/1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation. [ABSTRACT FROM AUTHOR]

Atrial natriuretic factor and brain natriuretic peptide are two important biomarkers in clinical cardiology. These two natriuretic peptide hormones are encoded by the paralogous genes Nppa and Nppb, which are evolutionary conserved. Both genes are predominantly expressed by the heart muscle during the embryonic and fetal stages, and in particular Nppa expression is strongly reduced in the ventricles after birth. Upon cardiac stress, Nppa and Nppb are strongly upregulated in the ventricular myocardium. Much is known about the molecular and physiological ques inducing Nppa and Nppb expression; however, the transcriptional regulatory mechanisms of the Nppa-Nppb cluster in vivo has proven to be quite complex and is not well understood. In this review, we will provide recent insights into the dynamic and complex regulation of Nppa and Nppb during heart development and hypertrophy, and the association of this gene cluster with the cardiomyocyte-intrinsic program of heart regeneration. [ABSTRACT FROM AUTHOR]

The identification of stem cells and growth factors as well as the development of biomaterials hold great promise for regenerative medicine applications. However, the therapeutic efficacy of regenerative therapies can be greatly influenced by the host immune system, which plays a pivotal role during tissue repair and regeneration. Therefore, understanding how the immune system modulates tissue healing is critical to design efficient regenerative strategies. While the innate immune system is well known to be involved in the tissue healing process, the adaptive immune system has recently emerged as a key player. T-cells, in particular, regulatory T-cells (Treg), have been shown to promote repair and regeneration of various organ systems. In this review, we discuss the mechanisms by which Treg participate in the repair and regeneration of skeletal and heart muscle, skin, lung, bone, and the central nervous system. [ABSTRACT FROM AUTHOR]

The neonatal mouse heart has the remarkable capacity to regenerate lost myocardium within the first week of life. Neonatal cardiomyocytes re-express fetal genes that control cell proliferation after injury to promote regeneration. The loss of regenerative capacity of the heart one week after birth coincides with repression of a fetal transcriptional program coordinated by epigenetic regulators. The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) is a repressor of fetal cardiac transcriptional programs and suppresses cardiomyocyte cell proliferation, suggesting a potential function in heart regeneration. However, it was recently demonstrated that Ezh2 is dispensable for heart regeneration in the neonatal heart. Here, we provide evidence supporting this finding and demonstrate that Ezh2 deficiency does not affect regeneration of the neonatal heart. We inactivated Ezh2 in differentiating embryonic cardiomyocytes, which led to depletion of histone H3 trimethylated at lysine 27 (H3K27me3). Ezh2 deficiency in cardiomyocytes did not affect clearance of the fibrotic scar in myocardial infarction (MI) and apical resection models of cardiac injury at post-natal day 1 (P1). Similarly, cardiomyocyte-specific loss of Ezh2 did not affect fibrotic scar size after MI or apical resection at P7, suggesting that it does not extend the regenerative time window. Our results demonstrate that Ezh2 is not required for innate neonatal cardiac regeneration. [ABSTRACT FROM AUTHOR]

Background: Application of cardiac stem cells combined with biomaterial scaffold is a promising therapeutic strategy for heart repair after myocardial infarction. However, the optimal cell types and biomaterials remain elusive. Methods: In this study, we seeded Isl1+ embryonic cardiac progenitor cells (CPCs) into decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) to assess the therapeutic potential of Isl1+ CPCs and the biocompatibility of SIS-ECM with these cells. Results: We observed that SIS-ECM supported the viability and attachment of Isl1+ CPCs. Importantly, Isl1+ CPCs differentiated into cardiomyocytes and endothelial cells 7 days after seeding into SIS-ECM. In addition, SIS-ECM with CPC-derived cardiomyocytes showed spontaneous contraction and responded to β-adrenergic stimulation. Next, patches of SIS-ECM seeded with CPCs for 7 days were transplanted onto the outer surface of infarcted myocardium in mice. Four weeks after transplantation, the patches were tightly attached to the surface of the host myocardium and remained viable. Transplantation of patches improved cardiac function, decreased the left ventricular myocardial scarring area, and reduced fibrosis and heart failure. Conclusions: Transplantation of Isl1+ CPCs seeded in SIS-ECM represents an effective approach for cell-based heart therapy. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is caused by the occlusion of a coronary artery due to underlying atherosclerosis complicated by localized thrombosis. The blockage of blood flow leads to cardiomyocyte (CM) death in the infarcted area. Adult mammalian cardiomyocytes have little capacity to proliferate in response to injury; however, some pathways active during embryogenesis and silent during adult life are recruited in response to tissue injury. One such example is hedgehog (Hh) signaling. Hh is involved in the embryonic development of the heart and coronary vascular system. Pathological conditions including ischemia activate Hh signaling in adult tissues. This review highlights the involvement of Hh signaling in ischemic tissue regeneration with a particular emphasis on heart regeneration and discusses its potential role as a therapeutic agent. [ABSTRACT FROM AUTHOR]

Myocardial infarction afflicts close to three quarters of a million Americans annually, resulting in reduced heart function, arrhythmia, and frequently death. Cardiomyocyte death reduces the heart's pump capacity while the deposition of a non-conductive scar incurs the risk of arrhythmia. Direct cardiac reprogramming emerged as a novel technology to simultaneously reduce scar tissue and generate new cardiomyocytes to restore cardiac function. This technology converts endogenous cardiac fibroblasts directly into induced cardiomyocyte-like cells using a variety of cocktails including transcription factors, microRNAs, and small molecules. Although promising, direct cardiac reprogramming is still in its fledging phase, and numerous barriers have to be overcome prior to its clinical application. This review discusses current findings to optimize reprogramming efficiency, including reprogramming factor cocktails and stoichiometry, epigenetic barriers to cell fate reprogramming, incomplete conversion and residual fibroblast identity, requisite growth factors, and environmental cues. Finally, we address the current challenges and future directions for the field. [ABSTRACT FROM AUTHOR]

Despite the extensive use of zebrafish as a model organism in developmental biology and regeneration research, genetic techniques enabling conditional analysis of gene function are limited. In this study, we generated Zwitch, a Cre-dependent invertible gene-trap cassette, enabling the establishment of conditional alleles in zebrafish by generating intronic insertions via in vivo homologous recombination. To demonstrate the utility of Zwitch, we generated a conditional sonic hedgehog a (shha) allele. Homozygous shha mutants developed normally; however, shha mutant embryos globally expressing Cre exhibited strong reductions in endogenous shha and shha target gene mRNA levels and developmental defects associated with null shha mutations. Analyzing a conditional shha mutant generated using an epicardium-specific inducible Cre driver revealed unique roles for epicardium-derived Shha in myocardial proliferation during heart development and regeneration. Zwitch will extend the utility of zebrafish in organ development and regeneration research and might be applicable to other model organisms.

Abstract Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non‐myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere‐derived cells amplifies innate cardioblast‐mediated tissue regeneration, in part through the secretion of stromal cell‐derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.

Heart failure is a costly and deadly disease, affecting over 23 million patients worldwide, half of which die within 5 years of diagnosis. The pathophysiological basis of heart failure is the inability of the adult heart to regenerate lost or damaged myocardium. Although limited myocyte turnover does occur in the adult heart, it is insufficient for restoration of contractile function1-6. In contrast to lower vertebrates which can regenerate their myocardium through cardiomyocyte proliferation,7-13, adult mammalian heart cardiomyogenesis very limited1-5. Studies in the late 90s elegantly demonstrated that mammalian cardiomyocytes continue to divide for a few days after birth 14-16, only to undergo permanent cell cycle arrest shortly thereafter. Recently, we demonstrated that resection of up to 15% of the apex of the left ventricle of postnatal day 1 (P1) mice results in complete regeneration within 21 days following injury, without significant fibrosis and cardiac dysfunction17. Moreover, we described a similar regenerative response following ischemic myocardial infarction 18. This response was well characterized by robust cardiomyocyte proliferation, with gradual restoration of normal cardiac morphology and function. In addition to the histological evidence of proliferating myocytes, genetic fate-mapping studies confirmed that the majority of newly formed cardiomyocytes are derived from proliferation of preexisting cardiomyocytes17. Intriguingly, this regenerative capacity is lost by P7, after which injury results in the cardiomyocyte hypertrophy and scar-formation, which coincides with binucleation and cell cycle exit of cardiomyocytes 14, 19. An important approach to understanding the loss of regenerative ability of the mammalian heart is to first consider why, and not only how, this happens. The regenerative ability of the early postnatal heart following resection or ischemic infarction involves regeneration of the lost myocardium and vasculature with restoration of normal myocardial thickness and architecture, and long-term functional recovery. Why would the heart permanently forego such a remarkable regenerative program shortly after birth? The answer may lie in within the fundamental principal of evolutionary tradeoff.

Coronary artery disease is the most common form of cardiovascular diseases, resulting in the loss of cardiomyocytes (CM) at the site of ischemic injury. To compensate for the loss of CMs, cardiac fibroblasts quickly respond to injury and initiate cardiac remodeling in an injured heart. In the remodeling process, cardiac fibroblasts proliferate and differentiate into myofibroblasts, which secrete extracellular matrix to support the intact structure of the heart, and eventually differentiate into matrifibrocytes to form chronic scar tissue. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate this pathologic remodeling and replace the cardiac fibrotic scar with myocardium in situ. Since the first discovery in 2010, many progresses have been made to improve the efficiency and efficacy of reprogramming by understanding the mechanisms and signaling pathways that are activated during direct cardiac reprogramming. Here, we overview the development and recent progresses of direct cardiac reprogramming and discuss future directions in order to translate this promising technology into an effective therapeutic paradigm to reverse cardiac pathological remodeling in an injured heart.

Stimulating cardiomyocyte regeneration after an acute injury remains the central goal in cardiovascular regenerative biology. While adult mammals respond to cardiac damage with deposition of rigid scar tissue, adult zebrafish and salamander unleash a regenerative program that culminates in new cardiomyocyte formation, resolution of scar tissue, and recovery of heart function. Recent studies have shown that immune cells are key to regulating pro-inflammatory and pro-regenerative signals that shift the injury microenvironment toward regeneration. Defining the genetic regulators that control the dynamic interplay between immune cells and injured cardiac tissue is crucial to decoding the endogenous mechanism of heart regeneration. In this review, we discuss our current understanding of the extent that macrophage and regulatory T cells influence cardiomyocyte proliferation and how microRNAs (miRNAs) regulate their activity in the injured heart.

Heart diseases are the leading cause of death for the vast majority of people around the world, which is often due to the limited capability of human cardiac regeneration. In contrast, zebrafish have the capacity to fully regenerate their hearts after cardiac injury. Understanding and activating these mechanisms would improve health in patients suffering from long-term consequences of ischemia. Therefore, we monitored the dynamic transcriptome response of both mRNA and microRNA in zebrafish at 1–160 days post cryoinjury (dpi). Using a control model of sham-operated and healthy fish, we extracted the regeneration specific response and further delineated the spatio-temporal organization of regeneration processes such as cell cycle and heart function. In addition, we identified novel (miR-148/152, miR-218b and miR-19) and previously known microRNAs among the top regulators of heart regeneration by using theoretically predicted target sites and correlation of expression profiles from both mRNA and microRNA. In a cross-species effort, we validated our findings in the dynamic process of rat myoblasts differentiating into cardiomyocytes-like cells (H9c2 cell line). Concluding, we elucidated different phases of transcriptomic responses during zebrafish heart regeneration. Furthermore, microRNAs showed to be important regulators in cardiomyocyte proliferation over time.

The epicardium, a mesothelial layer that envelops vertebrate hearts, has become a therapeutic target in cardiac repair strategies because of its vital role in heart development and cardiac injury response. Epicardial cells serve as a progenitor cell source and signaling center during both heart development and regeneration. The importance of the epicardium in cardiac repair strategies has been reemphasized by recent progress regarding its requirement for heart regeneration in zebrafish, and by the ability of patches with epicardial factors to restore cardiac function following myocardial infarction in mammals. The live surveillance of epicardial development and regeneration using zebrafish has provided new insights into this topic. In this review, we provide updated knowledge about epicardial development and regeneration.

Functional coronary circulation is essential for a healthy heart in warm-blooded vertebrates, and coronary diseases can have a fatal consequence. Despite the growing interest, the knowledge about the coronary vessel development and the roles of new coronary vessel formation during heart regeneration is still limited. It is demonstrated that early revascularization is required for efficient heart regeneration. In this comprehensive review, we first describe the coronary vessel formation from an evolutionary perspective. We further discuss the cell origins of coronary endothelial cells and perivascular cells and summarize the critical signaling pathways regulating coronary vessel development. Lastly, we focus on the current knowledge about the molecular mechanisms regulating heart regeneration in zebrafish, a genetically tractable vertebrate model with a regenerative adult heart and well-developed coronary system.

The salamander axolotl is capable of complete regeneration of amputated heart tissue. However, non-invasive imaging tools for assessing its cardiac function were so far not employed. In this study, cardiac magnetic resonance imaging is introduced as a non-invasive technique to image heart function of axolotls. Three axolotls were imaged with magnetic resonance imaging using a retrospectively gated Fast Low Angle Shot cine sequence. Within one scanning session the axolotl heart was imaged three times in all planes, consecutively. Heart rate, ejection fraction, stroke volume and cardiac output were calculated using three techniques: (1) combined long-axis, (2) short-axis series, and (3) ultrasound (control for heart rate only). All values are presented as mean ± standard deviation. Heart rate (beats per minute) among different animals was 32.2±6.0 (long axis), 30.4±5.5 (short axis) and 32.7±4.9 (ultrasound) and statistically similar regardless of the imaging method (p > 0.05). Ejection fraction (%) was 59.6±10.8 (long axis) and 48.1±11.3 (short axis) and it differed significantly (p = 0.019). Stroke volume (μl/beat) was 133.7±33.7 (long axis) and 93.2±31.2 (short axis), also differed significantly (p = 0.015). Calculations were consistent among the animals and over three repeated measurements. The heart rate varied depending on depth of anaesthesia. We described a new method for defining and imaging the anatomical planes of the axolotl heart and propose one of our techniques (long axis analysis) may prove useful in defining cardiac function in regenerating axolotl hearts. [ABSTRACT FROM AUTHOR]

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are members of a non-coding RNA family. They act as negative regulators of protein translation by affecting messenger RNA (mRNA) stability; they modulate numerous signaling pathways and cellular processes, and are involved in cell-to-cell communication. Thus, studies on miRNAs offer an opportunity to improve our understanding of complex biological mechanisms. In the cardiovascular system, miRNAs control functions of various cells, such as cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts. The pivotal role of miRNAs in the cardiovascular system provides a new perspective on the pathophysiology of disorders like myocardial infarction, hypertrophy, fibrosis, heart failure, arrhythmia, inflammation and atherosclerosis. MiRNAs are differentially expressed in diseased tissue and can be released into circulation. Manipulation of miRNA activity may influence the course of a disease. Therefore, miRNAs have become an active field of research for developing new diagnostic and therapeutic tools. This review discusses emerging functions of miRNAs in cardiogenesis, heart regeneration and the pathophysiology of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Replacing dying or diseased cells of a tissue with new ones that are converted from patient's own cells is an attractive strategy in regenerative medicine. In vivo reprogramming is a novel strategy that can circumvent the hurdles of autologous/allogeneic cell injection therapies. Interestingly, studies have demonstrated that direct injection of cardiac transcription factors or specific miRNAs into the infarct border zone of murine hearts following myocardial infarction converts resident cardiac fibroblasts into functional cardiomyocytes. Moreover, in vivo cardiac reprogramming not only drives cardiac tissue regeneration, but also improves cardiac function and survival rate after myocardial infarction. Thanks to the influence of cardiac microenvironment and the same developmental origin, cardiac fibroblasts seem to be more amenable to reprogramming toward cardiomyocyte fate than other cell sources (e.g. skin fibroblasts). Thus, reprogramming of cardiac fibroblasts to functional induced cardiomyocytes in the cardiac environment holds great promises for induced regeneration and potential clinical purposes. Application of small molecules in future studies may represent a major advancement in this arena and pharmacological reprogramming would convey reprogramming technology to the translational medicine paradigm. This study reviews accomplishments in the field of in vitro and in vivo mouse cardiac reprogramming and then deals with strategies for the enhancement of the efficiency and quality of the process. Furthermore, it discusses challenges ahead and provides suggestions for future research. Human cardiac reprogramming is also addressed as a foundation for possible application of in vivo cardiac reprogramming for human heart regeneration in the future. [ABSTRACT FROM AUTHOR]

Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is the leading cause of death worldwide. Compared to other organs such as the liver, the adult human heart lacks the capacity to regenerate on a macroscopic scale after injury. As a result, myocardial infarctions are responsible for approximately half of all cardiovascular related deaths. In contrast, the zebrafish heart regenerates efficiently upon injury through robust myocardial proliferation. Therefore, deciphering the mechanisms that underlie the zebrafish heart's endogenous regenerative capacity represents an exciting avenue to identify novel therapeutic strategies for inducing regeneration of the human heart. This review provides a historical overview of adult zebrafish heart regeneration. We summarize 15 years of research, with a special focus on recent developments from this fascinating field. We discuss experimental findings that address fundamental questions of regeneration research. What is the origin of regenerated muscle? How is regeneration controlled from a genetic and molecular perspective? How do different cell types interact to achieve organ regeneration? Understanding natural models of heart regeneration will bring us closer to answering the ultimate question: how can we stimulate myocardial regeneration in humans? [ABSTRACT FROM AUTHOR]

Models of cardiac repair are needed to understand mechanisms underlying failure to regenerate in human cardiac tissue. Such studies are currently dominated by the use of zebrafish and mice. Remarkably, it is between these two evolutionary separated species that the adult cardiac regenerative capacity is thought to be lost, but causes of this difference remain largely unknown. Amphibians, evolutionary positioned between these two models, are of particular interest to help fill this lack of knowledge. We thus developed an endoscopy-based resection method to explore the consequences of cardiac injury in adult Xenopus laevis. This method allowed in situ live heart observation, standardised tissue amputation size and reproducibility. During the first week following amputation, gene expression of cell proliferation markers remained unchanged, whereas those relating to sarcomere organisation decreased and markers of inflammation, fibrosis and hypertrophy increased. One-month post-amputation, fibrosis and hypertrophy were evident at the injury site, persisting through 11 months. Moreover, cardiomyocyte sarcomere organisation deteriorated early following amputation, and was not completely recovered as far as 11 months later. We conclude that the adult Xenopus heart is unable to regenerate, displaying cellular and molecular marks of scarring. Our work suggests that, contrary to urodeles and teleosts, with the exception of medaka, adult anurans share a cardiac injury outcome similar to adult mammals. This observation is at odds with current hypotheses that link loss of cardiac regenerative capacity with acquisition of homeothermy. [ABSTRACT FROM AUTHOR]

In the field of heart regeneration, the proliferative potential of cardiomyocytes in postnatal mice is under intense investigation. However, solely relying on immunostaining of proliferation markers, the long-term proliferation dynamics and potential of the cardiomyocytes cannot be readily addressed. Previously, we found that a p53 promoter-driving reporter predominantly marked the proliferating lineages in mice. Here, we established a p53-based genetic tracing system to investigate postnatal cardiomyocyte proliferation and heart regeneration. By selectively tracing proliferative cardiomyocytes, a differential pattern of clonal expansion in p53+ cardiac myocytes was revealed in neonatal, adolescent and adult stages. In addition, the percentage of p53+ lineage cardiomyocytes increased continuously in the first month. Furthermore, these cells rapidly responded to heart injury and greatly contributed to the replenished myocardium. Therefore, this study reveals complex proliferating dynamics in postnatal cardiomyocytes and heart repair, and provides a novel genetic tracing strategy for studying postnatal cardiac turnover and regeneration. [ABSTRACT FROM AUTHOR]

The secretedWnt signaling molecules are essential to the coordination of cell-fate decision making in multicellular organisms. In adult animals, the secreted Wnt proteins are critical for tissue regeneration and frequently contribute to cancer. Small molecules that disable the Wnt acyltransferase Porcupine (Porcn) are candidate anticancer agents in clinical testing. Here we have systematically assessed the effects of the Porcn inhibitor (WNT-974) on the regeneration of several tissue types to identify potentially unwanted chemical effects that could limit the therapeutic utility of such agents. An unanticipated observation from these studies is proregenerative responses in heart muscle induced by systemic chemical suppression of Wnt signaling. Using in vitro cultures of several cell types found in the heart, we delineate theWnt signaling apparatus supporting an antiregenerative transcriptional program that includes a subunit of the nonfibrillar collagen VI. Similar to observations seen in animals exposed to WNT-974, deletion of the collagen VI subunit, COL6A1, has been shown to decrease aberrant remodeling and fibrosis in infarcted heart tissue. We demonstrate that WNT-974 can improve the recovery of heart function after left anterior descending coronary artery ligation by mitigating adverse remodeling of infarcted tissue. Injured heart tissue exposed to WNT-974 exhibits decreased scarring and reduced Col6 production. Our findings support the development of Porcn inhibitors as antifibrotic agents that could be exploited to promote heart repair following injury. [ABSTRACT FROM AUTHOR]

Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. Key message: [ABSTRACT FROM AUTHOR]

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-sh RNA ( 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. [ABSTRACT FROM AUTHOR]

Cardiac inflammation is considered by many as the main driving force in prolonging the pathological condition in the heart after myocardial infarction. Immediately after cardiac ischemic injury, neutrophils are the first innate immune cells recruited to the ischemic myocardium within the first 24 h. Once they have infiltrated the injured myocardium, neutrophils would then secret proteases that promote cardiac remodeling and chemokines that enhance the recruitment of monocytes from the spleen, in which the recruitment peaks at 72 h after myocardial infarction. Monocytes would transdifferentiate into macrophages after transmigrating into the infarct area. Both neutrophils and monocytes-derived macrophages are known to release proteases and cytokines that are detrimental to the surviving cardiomyocytes. Paradoxically, these inflammatory cells also play critical roles in repairing the injured myocardium. Depletion of either neutrophils or monocytes do not improve overall cardiac function after myocardial infarction. Instead, the left ventricular function is further impaired and cardiac fibrosis persists. Moreover, the inflammatory microenvironment created by the infiltrated neutrophils and monocytes-derived macrophages is essential for the recruitment of cardiac progenitor cells. Recent studies also suggest that treatment with anti-inflammatory drugs may cause cardiac dysfunction after injury. Indeed, clinical studies have shown that traditional ant-inflammatory strategies are ineffective to improve cardiac function after infarction. Thus, the focus should be on how to harness these inflammatory events to either improve the efficacy of the delivered drugs or to favor the recruitment of cardiac progenitor cells. [ABSTRACT FROM AUTHOR]

There is a variety of mechanisms(s) factor(s) that may influence stem cell therapies for heart regeneration. Among the best candidates for stem cell source are: mesenchymal stem cells (also those isolated from adipose tissue), cardiac cell progenitors (CPC) and descendants of iPSC cells. iPSC/s can be potentially beneficial although their pluripotent induction has been still in question due to: low propagation efficacy, danger of genomic integration/instability, biological risk of current vector system teratoma formation etc. which have been discussed in this review. Optimization protocols are required in order to enhance stem cells resistance to pathological conditions that they may encounter in pathological organ and to increase their retention. Combination between gene transfer and stem cell therapy is now more often used in pre-clinical studies with the prospect of subsequent clinical trials. Complementary substances have been contemplated to support stem cell viability (mainly anti-inflammatory and anti- apoptotic agents), which have been tested in animal models with promising results. Integration of nanotechnology both for efficient stem cell imaging as well as with the aim to provide cell supporting scaffolds seem to be inevitable for further development of cellular therapies. The whole organ (heart) reconstruction as well as biodegradable scaffolds and scaffold-free cell sheets have been also outlined. [ABSTRACT FROM AUTHOR]

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 μm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.