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Despite considerable efforts carried out to develop stem/progenitor cell-based technologies aiming at replacing and restoring the cardiac tissue following severe damages, thus far no strategies based on adult stem cell transplantation have been demonstrated to efficiently generate new cardiac muscle cells. Intriguingly, dedifferentiation, and proliferation of pre-existing cardiomyocytes and not stem cell differentiation represent the preponderant cellular mechanism by which lower vertebrates spontaneously regenerate the injured heart. Mammals can also regenerate their heart up to the early neonatal period, even in this case by activating the proliferation of endogenous cardiomyocytes. However, the mammalian cardiac regenerative potential is dramatically reduced soon after birth, when most cardiomyocytes exit from the cell cycle, undergo further maturation, and continue to grow in size. Although a slow rate of cardiomyocyte turnover has also been documented in adult mammals, both in mice and humans, this is not enough to sustain a robust regenerative process. Nevertheless, these remarkable findings opened the door to a branch of novel regenerative approaches aiming at reactivating the endogenous cardiac regenerative potential by triggering a partial dedifferentiation process and cell cycle re-entry in endogenous cardiomyocytes. Several adaptations from intrauterine to extrauterine life starting at birth and continuing in the immediate neonatal period concur to the loss of the mammalian cardiac regenerative ability. A wide range of systemic and microenvironmental factors or cell-intrinsic molecular players proved to regulate cardiomyocyte proliferation and their manipulation has been explored as a therapeutic strategy to boost cardiac function after injuries. We here review the scientific knowledge gained thus far in this novel and flourishing field of research, elucidating the key biological and molecular mechanisms whose modulation may represent a viable approach for regenerating the human damaged myocardium.

During early postnatal heart development, the metabolic profile switches from glycolysis to fatty acid oxidation (FAO). At the same time, cardiomyocytes (CM) undergo profound maturation characterized by hypertrophic growth, cytoarchitectural remodeling, chromatin reconfiguration, and cell cycle withdrawal. The subsequent structural and functional alterations in mature CM indicate synergistic rewiring of transcriptional networks regulated by epigenetic mechanisms in combination with environmental and metabolic signals. Recent studies of cellular metabolism demonstrated that metabolites derived from various metabolic pathways function as cofactors or substrates for distinct epigenetic modifiers, thereby coupling chromatin-dependent gene regulation with the metabolic state. To gain a deeper insight into the interplay between epigenetic processes and metabolic pathways during cardiac development, maturation, and heart regeneration, I prevented metabolic maturation by cardiomyocyte-specific inactivation of CPT1B, a crucial enzyme for FAO. Cpt1b inactivation led to cardiomegaly and attenuated cardiac damage after myocardial injury due to augmented CM proliferation and enhanced resistance to ischemic injury. Interestingly, FAO inhibition after the loss of Cpt1b did not reduce the intracellular level of acetyl-CoA, which is an essential metabolite mainly produced by FAO, due to enhanced metabolic compensation from glucose and amino acids. In contrast, Cpt1b inactivation led to marked accumulation of αKG, caused by increased generation but decreased consumption. Excessive αKG was sensed by KDM5, leading to demethylation of cell-specific broad H3K4me3 peaks located in promoters of key genes driving cardiac development. Demethylation of H3K4me3 reduced expression of cardiac maturation genes, converting cardiomyocytes to a more immature, proliferation-competent state. Overall, the results obtained for the thesis uncover a complex interplay between epigenetics and metabolic pathways for regulating the maintenance of CM maturity and cell cycle arrest. The study identifies mitochondrial oxidative metabolism as an attractive target to treat heart failure. Manipulation of metabolic processes were found to alter epigenetic events in the nucleus, which was exploited to regenerate diseased hearts. Furthermore, my research uncovered that KDM5 senses metabolic cues to deactivate crucial cardiac maturation genes, which offers new perspectives to treat heart diseases.

In contrast to mammals, lower vertebrates are capable of extraordinary myocardial regeneration thanks to the ability of their cardiomyocytes to undergo transient dedifferentiation and proliferation. Somatic cells can be temporarily reprogrammed to a proliferative, dedifferentiated state through forced expression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM). Here, we aimed to induce transient reprogramming of mammalian cardiomyocytes in vitro utilising an OSKM-encoding non-integrating vector. Reprogramming factor expression in postnatal rat and mouse cardiomyocytes triggered rapid but limited cell dedifferentiation. Concomitantly, a significant increase in cell viability, cell cycle related gene expression and Ki67 positive cells was observed consistent with an enhanced cell cycle activation. The transient nature of this partial reprogramming was confirmed as cardiomyocyte-specific cell morphology, gene expression and contractile activity were spontaneously recovered by day 15 after viral transduction. This study provides the first evidence that adenoviral OSKM delivery can induce partial reprogramming of postnatal cardiomyocytes. Therefore, adenoviral mediated transient reprogramming could be a novel and feasible strategy to recapitulate the regenerative mechanisms of lower vertebrates., TK was supported by the Engineering and Physical Sciences Research Council (EPSRC) & Medical Research Council (MRC) Centre for Doctoral Training (CDT) in Regenerative Medicine (EP/L014904/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acute myocardial infarction is a leading cause of death. Unlike most adult mammals, zebrafish have the capability to almost fully regenerate their hearts after injury. In contrast, ischemic damage in adult human and mouse hearts usually results in scar tissue. mRNA-Sequencing (Seq) and miRNA-Seq analyses of heart regeneration in zebrafish over time showed that the process can be divided into three phases: the first phase represents dedifferentiation and proliferation of cells, the second phase is characterized by migration, and in the third phase cell signals indicate heart development and differentiation. The first two phases seem to share major similarities with tumor development and growth. To gain more insight into these similarities between cardiac regeneration and tumor development and growth, we used patient matched tumor normal (“healthy”) RNA-Seq data for several tumor entities from The Cancer Genome Atlas (TCGA). Subsequently, RNA data were processed using the same pipeline for both the zebrafish samples and tumor datasets. Functional analysis showed that multiple Gene Ontology terms (GO terms) are involved in both early stage cardiac regeneration and tumor development/growth across multiple tumor entities. These GO terms are mostly associated with cell cycle processes. Further analysis showed that orthologous genes are the same key players that regulated these changes in both diseases. We also observed that GO terms associated with heart development in the third late phase of cardiac regeneration are downregulated in the tumor entities. Taken together, our analysis illustrates similarities between cardiac remodeling and tumor progression.

The heart has a high-metabolic rate, and its "around-the-clock" vital role to sustain life sets it apart in a regenerative setting from other organs and appendages. The landscape of vertebrate species known to perform intrinsic heart regeneration is strongly biased toward ectotherms-for example, fish, salamanders, and embryonic/neonatal ectothermic mammals. It is hypothesized that intrinsic heart regeneration is exclusively limited to the low-metabolic hearts of ectotherms. The biomedical field of regenerative medicine seeks to devise biologically inspired regenerative therapies to diseased human hearts. Falsification of the ectothermy dependency for heart regeneration hypothesis may be a crucial prerequisite to meaningfully seek inspiration in established ectothermic regenerative animal models. Otherwise, engineering approaches to construct artificial heart components may constitute a more viable path toward regenerative therapies. A more strict definition of regenerative phenomena is generated and several testable sub-hypotheses and experimental avenues are put forward to elucidate the link between heart regeneration and metabolism. Also see the video abstract here https://youtu.be/fZcanaOT5z8.

The study of heart repair post-myocardial infarction has historically focused on the importance of cardiomyocyte proliferation as the major factor limiting adult mammalian heart regeneration. However, there is mounting evidence that a narrow focus on this one cell type discounts the importance of a complex cascade of cell–cell communication involving a whole host of different cell types. A major difficulty in the study of heart regeneration is the rarity of this process in adult animals, meaning a mammalian template for how this can be achieved is lacking. Here, we review the adult zebrafish as an ideal and unique model in which to study the underlying mechanisms and cell types required to attain complete heart regeneration following cardiac injury. We provide an introduction to the role of the cardiac microenvironment in the complex regenerative process and discuss some of the key advances using this in vivo vertebrate model that have recently increased our understanding of the vital roles of multiple different cell types. Due to the sheer number of exciting studies describing new and unexpected roles for inflammatory cell populations in cardiac regeneration, this review will pay particular attention to these important microenvironment participants.

Srčno–žilne bolezni so v razvitem delu sveta in tudi v Sloveniji že desetletja najpogostejši vzrok obolevnosti in umrljivosti odraslih. Če je ob srčnem infarktu pretok krvi prekinjen dlje kot 20-30 minut, so posledice ireverzibilne, saj srčne celice odmrejo. Naravno prisotna regenerativna sposobnost človekovega srčnega mišičnega tkiva je bistveno preslaba, da bi lahko kompenzirala obsežne izgube celic. S celično terapijo se prvič, če izvzamemo presaditve srca, lotevamo reševanja dejanskega vzroka in odpravljanja težav ne le lajšanja simptomov. Glavni namen te diplomske naloge je opis različnih celičnih tipov in njihova ustreznost za regeneracijo srca, nekaj pozornosti pa namenim nadaljnjim izzivom, ki jih bo potrebno rešiti preden bo celična terapija postala učinkovita in razširjena terapija za regeneracijo srca po srčni kapi. Za regeneracijo srca s celično terapijo so primerni različni donorski celični tipi – od somatskih matičnih (predniških) celic pa vse do induciranih pluripotentnih matičnih celic in transdiferenciranih celic. Idealnega celičnega tipa, ki bi bil primeren za vse paciente verjetno ni. Nadaljnje študije se bodo verjetno vse bolj usmerjale v uporabo programiranih celic ter čedalje manj v uporabo alogenih (matičnih) celic. Poleg ustreznega izbora donorskih celic je za višjo učinkovitost celične terapije potrebno izboljšati metode vnosa celic, njihovo preživetje in vraščanje v tkivo. Cardiovascular diseases have been the most common cause of morbidity and mortality in adults in the developed part of the world and in Slovenia for decades. If the blood flow is interrupted for more than 20-30 minutes during a heart attack, the consequences are irreversible, as the heart cells die. The naturally present regenerative capacity of human cardiac muscle tissue is too weak to be able to compensate for extensive tissue loss. Cell therapy enables for the first time, with the exception of heart transplants, we to tackle and resolve the actual cause instead of just relieving symptoms. The main purpose of this thesis is to describe the different cell types and their suitability for heart regeneration, and further challenges that will need to be addressed before heart regeneration cell therapy becomes effective and widespread therapy for heart regeneration after heart attack. Various donor cell types are suitable for heart regeneration with cell therapy - from somatic stem (progenitor) cells, to induced pluripotent stem cells and transdifferentiated cells. There is probably no ideal cell type that would be suitable for all patients. Further studies are likely to focus more on the use of programmed cells. In addition to the suitable selection of donor cells, the methods of cell delivery, cell survival, and tissue growth need to be improved to increase the effectiveness of heart regeneration cell therapy.

Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modulates the Stat3 methylation and phosphorylation activity. Inhibition of the KMT activity of Smyd2 reduces phosphorylated Stat3 at cardiac wounds, suppressing the elevated CM proliferation in injured grl mutant hearts. Our findings establish an injury-specific transcriptional repression program in governing CM renewal during heart regeneration, providing a potential strategy whereby silencing Grl repression at local regions might empower regeneration capacity to the injured mammalian heart., Highlighted Article: Novel mechanisms of the Grl-Smyd2 network govern vertebrate CM renewal and heart regeneration, which might be relevant in developing strategies for regeneration interventions in humans.

Soon after birth, the regenerative capacity of the mammalian heart is lost, cardiomyocytes withdraw from the cell cycle and demonstrate a minimal proliferation rate. Despite improved treatment and reperfusion strategies, the uncompensated cardiomyocyte loss during injury and disease results in cardiac remodeling and subsequent heart failure. The promising field of regenerative medicine aims to restore both the structure and function of damaged tissue through modulation of cellular processes and regulatory mechanisms involved in cardiac cell cycle arrest to boost cardiomyocyte proliferation. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are functional RNA molecules with no protein-coding function that have been reported to engage in cardiac regeneration and repair. In this review, we summarize the current understanding of both the biological functions and molecular mechanisms of ncRNAs involved in cardiomyocyte proliferation. Furthermore, we discuss their impact on the structure and contractile function of the heart in health and disease and their application for therapeutic interventions.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. In recent years, researchers are attracted to the use of cell therapy based on stem cell and progenitor cells, which has been a promising strategy for cardiac repair after injury. However, conducted research using intracoronary or intramyocardial transplantation of various types of stem/progenitor cells as a cell suspension showed modest efficiency. This is due to the low degree of integration and cell survival after transplantation. To overcome these limitations, the concept of the use of multicellular spheroids modeling the natural microenvironment of cells has been proposed, which allows maintaining their viability and therapeutic properties. It is of great interest to use so-called cardial spheroids (cardiospheres) spontaneously forming three-dimensional structures under low-adhesive conditions, consisting of a heterogeneous population of myocardial progenitor cells and extracellular matrix proteins. This review presents data on methods for creating cardiospheres, directed regulation of their properties and reparative potential, as well as the results of preclinical and clinical studies on their use for the treatment of heart diseases.Сердечно-сосудистые заболевания являются основной причиной заболеваемости и смертности населения во всем мире. В последние годы внимание исследователей привлечено к использованию клеточной терапии на основе трансплантации стволовых клеток и клеток-предшественников, что явилось многообещающей стратегией восстановления сердца после повреждения. Однако проведенные исследования с использованием внутрикоронарной или внутримиокардиальной трансплантации разных типов стволовых/прогениторных клеток в виде клеточной суспензии показали весьма умеренную эффективность. Это обусловлено низкой степенью интеграции и выживаемости клеток после трансплантации. Для преодоления этих ограничений предложена концепция использования многоклеточных сфероидов, моделирующих естественное микроокружение клеток, что позволяет поддерживать их жизнеспособность и терапевтические свойства. Большой интерес представляет использование так называемых кардиальных сфероидов (кардиосфер) самопроизвольно формирующихся в низкоадгезивных условиях трехмерных структур, состоящих из гетерогенной популяции прогениторных клеток миокарда и белков внеклеточного матрикса. В обзоре представлены данные о способах создания кардиосфер, направленной регуляции их свойств и репаративного потенциала, а также результаты доклинических и клинических исследований по их применению для лечения заболеваний сердца.

Aims Cell therapy regenerative potential is hindered by cell access to the infarct zone. We studied function recovery at the scar zone and its impact in global left ventricular function after intracoronary injection of haematopoietic precursor cells. Methods and results Haematopoietic precursor cells were obtained by blood apheresis in patients with an old myocardial infarction, and the presence of CD34+ and CD133+ cells was quantified. Left ventricular function, volumes, and infarct zone segmental motion were measured by magnetic resonance imaging (MRI) and echo left ventricular segmental strain (LVSS). The aphaeresis product was administered to 20 patients in the coronary artery responsible for the myocardial infarction. High cell yield in blood aphaeresis product allowed us to inject a high number of cells in most patients. Three patients were excluded because of insufficient CD133+ cell number, and one more patient was excluded because of artefacts in MRI images. The remaining 16 patients were compared with 16 controls. After 1 year, infarct zone reduction was related to the number of CD133+ (R = 0.53;P %3C 0.05) and CD34+ (R = 0.63;P %3C 0.01) cells injected. The number of CD133+ cells injected was also related to an improvement in LVSS (R = 0.62;P %3C 0.01). In turn, scar zone reduction was related to an improvement in LVSS (R = 0.64;P %3C 0.01). End-diastolic volume showed a reduction at follow-up in the treated group when compared with control patients. MRI infarct area segments systolic thickness increase improved after treatment in treated patients [expressed as median (interquartile range)] [0.42 (-0.38 to 1.14) vs. 1.06 (-0.10 to 2.12) mm; P %3C 0.01], but not in controls [2.02 (0.75 to 3.4) vs. 1.91 (0.77-3.17) mm; P = not significant (n.s.)]. In cell therapy patients, the borders of the infarct zone, but not the core, showed a significant recovery [proximal rim: 0.48 (-0.18 to 1.33) vs. 1.07 (0.22-2.40) mm; P %3C 0.05, distal rim: 0.75 (0.26-1.40) vs. 1.76 (0.65-2.86) mm; P %3C 0.05, and core: 0.36 (-0.33 to 1.20) vs. 0.60 (-0.18 to 1.62) mm; P = n.s.]. That improvement was not observed in the control group [proximal rim: 1.20 (0.33-2.53) vs. 0.82 (-0.13 to 1.65) mm; P = n.s., distal rim: 1.24 (0.80-1.72) vs. 0.96 (0.19-1.81) mm; P = n.s., and core: 0.30 (-0.42 to 1.64) vs. 0.07 (-0.60 to 1.20) mm; P = n.s.]. Only small size infarcts showed a complete recovery in the cell therapy patients [systolic thickness increase post-treatment increment in infarcts 6 segments affected: 0.28 (-0.19 to 0.71) vs. -1.21 (-2.60 to -0.53) mm; P %3C 0.01]. Conclusions Intracoronary injection of peripheral blood-derived haematopoietic precursor cells produces a complete recovery of the borders and partial regeneration of the infarct core, which is directly related to the number of CD133+ and CD34+ cells injected. Cell therapy infarct zone regeneration prevents ventricular remodelling by preserving segmental contractility and halting left ventricular dilatation., This research work was funded by Grupo Quiron Salud Hospitals, Spain.

Injury to the newborn mouse heart is efficiently regenerated, but this capacity is lost by one week after birth. We found that IGF2, an important mitogen in heart development, is required for neonatal heart regeneration. IGF2 originates from the endocardium/endothelium and is transduced in cardiomyocytes by the insulin receptor. Following injury on postnatal day 1, absence of IGF2 abolished injury-induced cell cycle entry during the early part of the first postnatal week. Consequently, regeneration failed despite the later presence of additional cell cycle-inducing activities 7 days following injury. Most cardiomyocytes transition from mononuclear diploid to polyploid during the first postnatal week. Regeneration was rescued in Igf2-deficient neonates in three different contexts that elevate the percentage of mononuclear diploid cardiomyocytes beyond postnatal day 7. Thus, IGF2 is a paracrine-acting mitogen for heart regeneration during the early postnatal period, and IGF2-deficiency unmasks the dependence of this process on proliferation-competent mononuclear diploid cardiomyocytes.

Myocardial infarction (MI) is a worldwide condition that affects millions of people. This is mainly caused by the adult human heart lacking the ability to regenerate upon injury, whereas zebrafish have the capacity through cardiomyocyte proliferation to fully regenerate the heart following injury such as apex resection (AR). But a systematic overview of the methods used to evidence heart regrowth and regeneration in the zebrafish is lacking. Herein, we conducted a systematical search in Embase and Pubmed for studies on heart regeneration in the zebrafish following injury and identified 47 AR studies meeting the inclusion criteria. Overall, three different methods were used to assess heart regeneration in zebrafish AR hearts. 45 out of 47 studies performed qualitative (37) and quantitative (8) histology, whereas immunohistochemistry for various cell cycle markers combined with cardiomyocyte specific proteins was used in 34 out of 47 studies to determine cardiomyocyte proliferation qualitatively (6 studies) or quantitatively (28 studies). For both methods, analysis was based on selected heart sections and not the whole heart, which may bias interpretations. Likewise, interstudy comparison of reported cardiomyocyte proliferation indexes seems complicated by distinct study designs and reporting manners. Finally, six studies performed functional analysis to determine heart function, a hallmark of human heart injury after MI. In conclusion, our data implies that future studies should consider more quantitative methods eventually taking the 3D of the zebrafish heart into consideration when evidencing myocardial regrowth after AR. Furthermore, standardized guidelines for reporting cardiomyocyte proliferation and sham surgery details may be considered to enable inter study comparisons and robustly determine the effect of given genes on the process of heart regeneration.

Mammalian hearts have regenerative potential restricted to early neonatal stage and lost within seven days after birth. Carbohydrates exclusive to cardiac neonatal tissue may be key regulators of regenerative potential. Although cell surface and extracellular matrix glycosylation are known modulators of tissue and cellular function and development, variation in cardiac glycosylation from neonatal tissue to maturation has not been fully examined. In this study, glycosylation of the adult rat cardiac ventricle showed no variability between the two strains analysed, nor were there any differences between the glycosylation of the right or left ventricle using lectin histochemistry and microarray profiling. However, in the Sprague-Dawley strain, neonatal cardiac glycosylation in the left ventricle differed from adult tissues using mass spectrometric analysis, showing a higher expression of high mannose structures and lower expression of complex N-linked glycans in the three-day-old neonatal tissue. Man6GlcNAc2 was identified as the main high mannose N-linked structure that was decreased in adult while higher expression of sialylated N-linked glycans and lower core fucosylation for complex structures were associated with ageing. The occurrence of mucin core type 2 O-linked glycans was reduced in adult and one sulfated core type 2 O-linked structure was identified in neonatal tissue. Interestingly, O-linked glycans from mature tissue contained both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), while all sialylated N-linked glycans detected contained only Neu5Ac. As glycans are associated with intracellular communication, the specific neonatal structures found may indicate a role for glycosylation in the neonatal associated regenerative capacity of the mammalian heart. New strategies targeting tissue glycosylation could be a key contributor to achieve an effective regeneration of the mammalian heart in pathological scenarios such as myocardial infarction.

Various strategies have been applied to replace the loss of cardiomyocytes in order to restore reduced cardiac function and prevent the progression of heart disease. Intensive research efforts in the field of cellular reprogramming and cell transplantation may eventually lead to efficient in vivo applications for the treatment of cardiac injuries, representing a novel treatment strategy for regenerative medicine. Modulation of cardiac transcription factor (TF) networks by chemical entities represents another viable option for therapeutic interventions. Comprehensive screening projects have revealed a number of molecular entities acting on molecular pathways highly critical for cellular lineage commitment and differentiation, including compounds targeting Wnt- and transforming growth factor beta (TGF beta)-signaling. Furthermore, previous studies have demonstrated that GATA4 and NKX2-5 are essential TFs in gene regulation of cardiac development and hypertrophy. For example, both of these TFs are required to fully activate mechanical stretch-responsive genes such as atrial natriuretic peptide and brain natriuretic peptide (BNP). We have previously reported that the compound 3i-1000 efficiently inhibited the synergy of the GATA4-NKX2-5 interaction. Cellular effects of 3i-1000 have been further characterized in a number of confirmatory in vitro bioassays, including rat cardiac myocytes and animal models of ischemic injury and angiotensin II-induced pressure overload, suggesting the potential for small molecule-induced cardioprotection.

Mammalian cardiomyocytes exit the cell cycle shortly after birth. As a result, an occurrence of coronary occlusion-induced myocardial infarction often results in heart failure, postinfarction LV dilatation, or death, and represents one of the most significant public health morbidities worldwide. Interestingly however, the hearts of neonatal pigs have been shown to regenerate following an acute myocardial infarction (MI) occuring on postnatal day 1 (P1); a recovery period which is accompanied by an increased expression of markers for cell-cycle activity, and suggests that early postnatal myocardial regeneration may be driven in part by the MI-induced proliferation of pre-existing cardiomyocytes. In this study, we identified signaling pathways known to regulate the cell cycle, and determined of these, the pathways persistently upregulated in response to MI injury. We identified five pathways (mitogen associated protein kinase [MAPK], Hippo, cyclic [cAMP], Janus kinase/signal transducers and activators of transcription [JAK-STAT], and Ras) which were comprehensively upregulated in cardiac tissues collected on day 7 (P7) and/or P28 of the P1 injury hearts. Several of the initiating master regulators (e.g., CSF1/CSF1R, TGFB, and NPPA) and terminal effector molecules (e.g., ATF4, FOS, RELA/B, ITGB2, CCND1/2/3, PIM1, RAF1, MTOR, NKF1B) in these pathways were persistently upregulated at day 7 through day 28, suggesting there exists at least some degree of regenerative activity up to 4 weeks following MI at P1. Our observations provide a list of key regulators to be examined in future studies targeting cell-cycle activity as an avenue for myocardial regeneration.

Cardiac regeneration post-injury is a tantalizing feature of many lower vertebrates such as fishes and urodeles, but absent in adult humans. Restoration of pumping function is a key endpoint of cardiac regeneration, but very little is known about the biomechanical remodeling process. Here, we quantify and compare the evolution of cellular composition and mechanical stiffness of the zebrafish ventricular myocardium during maturation and following cryoinjury during regeneration to better understand the dynamics of biomechanical remodeling during these two processes. With increasing age, normal myocardial trabecular density and cardiomyocyte fraction increased, while non-myocyte cell fractions decreased. Cell density remained constant during maturation. Cardiomyocyte sarcomeres shortened to a minimum reached at 7.5 months of age, but lengthened with additional age. Concomitantly, ventricular wall stiffness increased up until 7.5 months before plateauing with additional age. Endothelial, myofibroblast/smooth muscle, and cardiomyocyte cell fractions were disrupted following cryoinjury, but were progressively restored to age-specific natural norms by 35 days post infarct (DPI). Infarcted myocardium stiffened immediately following cryoinjury and was a 100-fold greater than non-infarcted tissue by 3 DPI. By 14 DPI, stiffness of the infarcted myocardium had fallen below that of 0 DPI and had completely normalized by 35 DPI. Interestingly, cardiomyocyte sarcomere length increased until 14 DPI, but subsequently shortened to lengths below age-specific natural norms, indicating recovery from a volume overloaded condition. These observations are consistent with the view that regenerating myocardium requires biomechanical stimulation (e.g. strain) to rescue from a volume overloaded condition. Intriguingly, the biomechanical progression of the infarcted adult myocardial wall mirrors that of normal remodeling during aging. The biomechanical progression of the infarcted myocardium targets the values of age-specific norms despite a large divergence in initial conditions. These findings identify a novel biomechanical control of heart regeneration that may orchestrate cellular and tissue level remodeling responses.

Cardiac regeneration, that is, restoration of the original structure and function in a damaged heart, differs from tissue repair, in which collagen deposition and scar formation often lead to functional impairment. In both scenarios, the early-onset inflammatory response is essential to clear damaged cardiac cells and initiate organ repair, but the quality and extent of the immune response vary. Immune cells embedded in the damaged heart tissue sense and modulate inflammation through a dynamic interplay with stromal cells in the cardiac interstitium, which either leads to recapitulation of cardiac morphology by rebuilding functional scaffolds to support muscle regrowth in regenerative organisms or fails to resolve the inflammatory response and produces fibrotic scar tissue in adult mammals. Current investigation into the mechanistic basis of homeostasis and restoration of cardiac function has increasingly shifted focus away from stem cell-mediated cardiac repair towards a dynamic interplay of cells composing the less-studied interstitial compartment of the heart, offering unexpected insights into the immunoregulatory functions of cardiac interstitial components and the complex network of cell interactions that must be considered for clinical intervention in heart diseases.

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.

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., Author summary Heart disease is one of the most common causes of death in all developed countries. While zebrafish cardiomyocytes are able to proliferate throughout adulthood, mammalian cardiomyocytes lose this ability during early development, and therefore are not capable to replace and renew cardiomyocytes after injury. The underlying mechanisms of cardiomyocyte proliferation are still not completely resolved. Understanding how zebrafish cardiomyocytes preserve their proliferating state, would be a valuable information to foster cardiac regeneration, e.g. after myocardial infarction in patients. Knowledge of the signaling pathways that need to be activated, or deactivated in order to induce cardiomyocyte proliferation after acute or chronic injury will pave the way for the development of genetic and/or pharmacological treatment options. In an ENU-mutagenesis screen, we identified the zebrafish mutant baldrian, which shows reduced embryonic cardiomyocyte proliferation. As genetic cause of the observed phenotype, we identified a missense mutation in the hdac1 gene. By treatment of heart-injured adult fish with the HDAC1 inhibitor Mocetinostat, we were able to show a reduced rate of cardiomyocyte proliferation also in the adult zebrafish heart in vivo, suggesting a role of Hdac1 in embryonic heart growth and adult regenerative cardiomyocyte proliferation in zebrafish.