Non-coding RNAs in endothelial cell signalling and hypoxia during cardiac regeneration

Heart failure (HF) as a result of myocardial infarction (MI) is the leading cause of death worldwide. In contrast to the adult mammalian heart, which has low regenerative capacity, newborn mammalian and zebrafish hearts can completely regenerate after injury. Cardiac regeneration is considered to be mediated by proliferation of pre-existing cardiomyocytes (CMs) mainly located in a hypoxic niche. To find new therapies to treat HF, efforts are being made to understand the molecular pathways underlying the regenerative capacity of the heart. However, the multicellularity of the heart is important during cardiac regeneration as not only CM proliferation but also the restoration of the endothelium is imperative to prevent progression to HF. It has recently come to light that signalling from non-coding RNAs (ncRNAs) and extracellular vesicles (EVs) plays a role in the healthy and the diseased heart. Multiple studies identified differentially expressed ncRNAs after MI, making them potential therapeutic targets. In this review, we highlight the molecular interactions between endothelial cells (ECs) and CMs in cardiac regeneration and when the heart loses its regenerative capacity. We specifically emphasize the role of ncRNAs and cell-cell communication via EVs during cardiac regeneration and neovascularisation.     

Identifying the Evolutionary Building Blocks of the Cardiac Conduction System

The endothermic state of mammals and birds requires high heart rates to accommodate the high rates of oxygen consumption. These high heart rates are driven by very similar conduction systems consisting of an atrioventricular node that slows the electrical impulse and a His-Purkinje system that efficiently activates the ventricular chambers. While ectothermic vertebrates have similar contraction patterns, they do not possess anatomical evidence for a conduction system. This lack amongst extant ectotherms is surprising because mammals and birds evolved independently from reptile-like ancestors. Using conserved genetic markers, we found that the conduction system design of lizard (Anolis carolinensis and A. sagrei), frog (Xenopus laevis) and zebrafish (Danio rerio) adults is strikingly similar to that of embryos of mammals (mouse Mus musculus, and man) and chicken (Gallus gallus). Thus, in ectothermic adults, the slow conducting atrioventricular canal muscle is present, no fibrous insulating plane is formed, and the spongy ventricle serves the dual purpose of conduction and contraction. Optical mapping showed base-to-apex activation of the ventricles of the ectothermic animals, similar to the activation pattern of mammalian and avian embryonic ventricles and to the His-Purkinje systems of the formed hearts. Mammalian and avian ventricles uniquely develop thick compact walls and septum and, hence, form a discrete ventricular conduction system from the embryonic spongy ventricle. Our study uncovers the evolutionary building plan of heart and indicates that the building blocks of the conduction system of adult ectothermic vertebrates and embryos of endotherms are similar.     

Nucleated red blood cells participate in myocardial regeneration in the toad Bufo Gargarizan Gargarizan

Heart regeneration is negligible in humans and mammals but remarkable in some ectotherms. Humans and mammals lack nucleated red blood cells (NRBCs), while ectotherms have sufficient NRBCs. This study used Bufo gargarizan gargarizan, a Chinese toad subspecies, as a model animal to verify our hypothesis that NRBCs participate in myocardial regeneration. NRBC infiltration into myocardium was seen in the healthy toad hearts. Heart needle-injury was used as an enlarged model of physiological cardiomyocyte loss. It recovered quickly and scarlessly. NRBC infiltration increased during the recovery. Transwell assay was done to in vitro explore effects of myocardial injury on NRBCs. In the transwell system, NRBCs could infiltrate into cardiac pieces and could transdifferentiate toward cardiomyocytes. Heart apex cautery caused approximately 5% of the ventricle to be injured to varying degrees. In the mildly to moderately injured regions, NRBC infiltration increased and myocardial regeneration started soon after the inflammatory response; the severely damaged region underwent inflammation, scarring, and vascularity before NRBC infiltration and myocardial regeneration, and recovered scarlessly in four months. NRBCs were seen in the newly formed myocardium. Enzyme-linked immunosorbent assay and Western blotting showed that the levels of tumor necrosis factor-α, interleukin- 1β, 6, and11, cardiotrophin-1, vascular endothelial growth factor, erythropoietin, matrix metalloproteinase- 2 and 9 in the serum and/or cardiac tissues fluctuated in different patterns during the cardiac injury-regeneration. Cardiotrophin-1 could induce toad NRBC transdifferentiation toward cardiomyocytes in vitro. Taken together, the results suggest that the NRBC is a cell source for cardiomyocyte renewal/regeneration in the toad; cardiomyocyte loss triggers a series of biological processes, facilitating NRBC infiltration and transition to cardiomyocytes. This finding may guide a new direction for improving human myocardial regeneration.     

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.     

Regenerative medicine for the treatment of spinal cord injury: more than just promises?

Spinal cord injury triggers a complex set of events that lead to tissue healing without the restoration of normal function due to the poor regenerative capacity of the spinal cord. Nevertheless, current knowledge about the intrinsic regenerative ability of central nervous system axons, when in a supportive environment, has made the prospect of treating spinal cord injury a reality. Among the range of strategies under investigation, cell‐based therapies offer the most promising results, due to the multifactorial roles that these cells can fulfil. However, the best cell source is still a matter of debate, as are clinical issues that include the optimal cell dose as well as the timing and route of administration. In this context, the role of biomaterials is gaining importance. These can not only act as vehicles for the administered cells but also, in the case of chronic lesions, can be used to fill the permanent cyst, thus creating a more favourable and conducive environment for axonal regeneration in addition to serving as local delivery systems of therapeutic agents to improve the regenerative milieu. Some of the candidate molecules for the future are discussed in view of the knowledge derived from studying the mechanisms that facilitate the intrinsic regenerative capacity of central nervous system neurons. The future challenge for the multidisciplinary teams working in the field is to translate the knowledge acquired in basic research into effective combinatorial therapies to be applied in the clinic.     

CUGBP1, a crucial factor for heart regeneration in mice

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

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

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

Regulation of cardiomyocyte proliferation during development and regeneration

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

Cardiac regeneration: epicardial mediated repair

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

Structure and hibernation-associated expression of the transient receptor potential vanilloid 4 channel (TRPV4) mRNA in the Japanese grass lizard (Takydromus tachydromoides)

Animals possess systems for sensing environmental temperature using temperature-sensitive ion channels called transient receptor potential channels (TRPs). Various TRPs have been identified and characterized in mammals. However, those of ectotherms, such as reptiles, are less well studied. Here, we identify the V subfamily of TRP (TRPV) in two reptile species: Japanese grass lizard (Takydromus tachydromoides) and Japanese four-lined ratsnake (Elaphe quadrivirgata). Phylogenetic analysis of TRPVs indicated that ectothermic reptilian TRPVs are more similar to those of endothermic chicken and mammals, than to other ectotherms, such as frog and fish. Expression analysis of TRPV4 mRNA in the lizard showed that its expression in tissues and organs is specifically controlled in cold environments and hibernation. The mRNA was ubiquitously expressed in seven tissues/organs examined. Both cold-treatment and hibernation lowered TRPV4 expression, but in a tissue/organ-specific manner. Cold-treatment reduced TRPV4 expression in tongue and muscle, while in hibernation it was reduced more widely in brain, tongue, heart, lung, and muscle. Interestingly, however, levels of TRPV4 mRNA in the skin remained unaffected after entering hibernation and cold-treatment, implying that TRPV4 in the skin may act as an environmental temperature sensor throughout the reptilian life cycle, including hibernation. This is the first report, to our knowledge, to describe reptilian TRPV4 in relation to hibernation.     

Endotherms,ectotherms, and mitochondrial genome-size variation

The patterns of mitochondrial genomesize variation were investigated in endothermic and ectothermic species to examine the role that thermal habit might play in the evolution of animal mitochondrial DNA (mtDNA). Data on mtDNA size (the modal, largest, and smallest mtDNA reported within a species), the percent variation in mtDNA size (the difference in size between the largest and smallest mtDNAs divided by the model genome size for a given species), and the frequency of heteroplasmic individuals (those carrying more than one mtDNA length variant) were tabulated from the literature. Endotherms showed significantly less variation in mtDNA size and tended to have smaller mtDNAs than ectotherms. Further comparisons between endothermic and ectothermic vertebrates revealed that the largest genome and the percent variation in genome size were significantly smaller in the former than the latter. There was no difference between endothermic and ectotherms in the frequency of heteroplasmy. These data are discussed in light of two hypotheses: (1) more intense directional and purifying selection for small genome size in the cytoplasms of species with higher metabolic rates and (2) reduced mutation pressures generating mtDNA size variants in endotherms relative to those in ectotherms. The general trends are consistent with the selection hypothesis but in certain species mtDNA size variation appears to be governed by mutational pressures. To test these competing hypotheses further, comparative studies are proposed where mitochondrial genome size is quantified in sister taxa and tissue types with very different metabolic rates.     

构建斑马鱼心脏损伤-再生模型的手术方法

斑马鱼心脏再生是近年来心血管再生医学研究的新热点之一, 也是以斑马鱼为模式进行脊椎动物遗传发育研究的一个新的重要方向。通过了解斑马鱼成体心脏再生的过程和研究其分子和细胞机制有可能为诱导哺乳动物成体心脏再生、治疗心肌梗塞等人类心脏疾病提供理论依据。文章主要介绍通过简单的手术切除成体斑马鱼约20%心室造成成体心脏损伤、诱导心脏再生的操作方法与经验。其基本流程主要包括麻醉成鱼、在体视镜下用尖镊撕开斑马鱼心脏腹面的皮肤和心包膜以暴露心脏、用剪刀切除心尖区域的部分心室。这种方法的手术成功率可达90%以上, 操作简便且重复性好, 是目前研究斑马鱼成体心脏损伤-再生的最常用的方法。     

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

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

BERGMANN SIZE CLINES: A SIMPLE EXPLANATION FOR THEIR OCCURRENCE IN ECTOTHERMS

In general ectothermic organisms grow larger at both lower temperatures and higher latitudes. Adult size in the soil nematode Caenorhabditis elegans reared at 10°C was approximately 33% greater than worms grown at 25°C. Nematode egg size and fish red blood cell size showed similar size increases at lower temperatures. These results indicate that body size differences in many ectotherms may simply be a consequence of developmental processes that cause cells to grow larger at lower temperatures. This would provide a general explanation for the increased size of ectotherms at lower temperatures independent of species-specific ecology.     

Thermal biology and mate acquisition in ectotherms

Many elements of the behavioural repertoire of ectothermic animals depend on body temperature. Under differing thermal conditions, behaviours in insects, reptiles and other terrestrial ectotherms may therefore vary widely, and in any given thermal regime there may be simple physical and physiological character differences between individuals that lead to a predictable variation of behaviour. Where mating behaviour patterns are involved, digferential fitness may result. Recent studies show that the interactions of physiology with behavioural ecology should be a fruitful area for future research.     

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

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

Cardiac regeneration in non-mammalian vertebrates

The heart is a robust organ, capable of pumping nutrients and transferring oxygen throughout the body via a network of capillaries, veins and arteries, for the entirety of a human's life. However, the fragility of mammalian hearts is also evident when it becomes damaged and parts of the organ fail to function. This is due to the fact that rather than replenishing the damaged areas with functional cellular mass, fibrotic scar tissue is the preferred replacement, resulting in an organ with functional deficiencies. Due to the mammalian hearts incapability to regenerate following damage and the ever-increasing number of people worldwide suffering from heart disease, tireless efforts are being made to discover ways of inducing a regenerative response in this most important organ. One such avenue of investigation involves studying our distantly related non-mammalian vertebrate cousins, which over the last decade has proved to us that cardiac regeneration is possible. This review will highlight these organisms and provide insights into some of the seminal discoveries made in the heart regeneration field using these amazing chordates.     

Spiked-in Pulsed in Vivo Labeling Identifies a New Member of the CCN Family in Regenerating Newt Hearts

The newt Notophthalmus viridescens , which belongs to the family of salamanders (Urodela), owns remarkable regenerative capacities allowing efficient scar-free repair of various organs including the heart. Salamanders can regrow large parts of the myocardium unlike mammals, which cannot replace lost cardiomyocytes efficiently. Unfortunately, very little is known about the molecules and the regulatory circuits facilitating efficient heart regeneration in newts or salamanders. To identify proteins that are involved in heart regeneration, we have developed a pulsed SILAC-based mass spectrometry method based on the detection of paired peptide peaks after (13)C(6)-lysine incorporation into proteins in vivo. Proteins were identified by matching mass spectrometry derived peptide sequences to a recently established normalized newt EST library. Our approach enabled us to identify more than 2200 nonredundant proteins in the regenerating newt heart. Because of the pulsed in vivo labeling approach, accurate quantification was achieved for 1353 proteins, of which 72 were up- and 31 down-regulated with a (|log 2 ratio| > 1) during heart regeneration. One deregulated member was identified as a new member of the CCN protein family, showing a wound specific activation. We reason that the detection of such deregulated newt-specific proteins in regenerating hearts supports the idea of a local evolution of tissue regeneration in salamanders. Our results significantly improve understanding of dynamic changes in the complex protein network that underlies heart regeneration and provides a basis for further mechanistic studies.     

Myocardial cell death and regeneration during progression of cardiac hypertrophy to heart failure

Cardiac hypertrophy and ensuing heart failure are among the most common causes of mortality worldwide, yet the triggering mechanisms for progression of hypertrophy to failure are not fully understood. Tissue homeostasis depends on proper relationships between cell proliferation, differentiation, and death and any imbalance between them results in compromised cardiac function. Recently, we developed a transgenic (Tg) mouse model that overexpress myotrophin (a 12-kDa protein that stimulates myocyte growth) in heart resulting in hypertrophy that progresses to heart failure. This provided us an appropriate model to study the disease process at any point from initiation of hypertrophy end-stage heart failure. We studied detailed apoptotic signaling and regenerative pathways and found that the Tg mouse heart undergoes myocyte loss and regeneration, but only at a late stage (during transition to heart failure). Several apoptotic genes were up-regulated in 9-month-old Tg hearts compared with age-matched wild type or 4-week-old Tg hearts. Cardiac cell death during heart failure involved activation of Fas, tumor necrosis factor-alpha, and caspases 9, 8, and 3 and poly(ADP-ribose) polymerase cleavage. Tg mice with hypertrophy associated with compromised function showed significant up-regulation of cyclins,cyclin-dependent kinases (Cdks), and cell regeneration markers in myocytes. Furthermore, in human failing and nonfailing hearts, similar observations were documented including induction of active caspase 3 and Ki-67 proteins in dilated cardiomyopathic myocytes. Taken together, our data suggest that the stress of extensive myocardial damage from longstanding hypertrophy may cause myocytes to reenter the cell cycle. We demonstrate, for the first time in an animal model, that cell death and regeneration occur simultaneously in myocytes during end-stage heart failure, a phenomenon not observed at the onset of the disease process.