斑马鱼心脏再 z-bo 2020-11-03 332

　　Abstract: Heart repair studies after myocardial infarction have found that myocardial cell proliferation is the main factor limiting the regeneration of the adult mammalian heart. However, more and more evidence shows that the narrow focus on this cell type diminishes the importance of the complex cascade of cell communication involving a whole set of different cell types. One of the main difficulties in studying heart regeneration is that this process is very rare in adult animals. We reviewed the adult zebrafish as an ideal and unique model in which the underlying mechanisms and cell types required for complete heart regeneration after heart injury were studied. We introduced the role of the cardiac microenvironment in the complex regeneration process and discussed some of the key advances in using this in vivo vertebrate model. These advances have recently increased our understanding of the important role of many different cell types. Since a large number of exciting studies describe the new and unexpected role of inflammatory cells in heart regeneration, this review will pay special attention to these important microenvironmental participants.

　　Introduction: Cardiovascular diseases and myocardial infarction (MI) are still the major health burdens globally, and their relationship with the prevalence of obesity and increasingly unhealthy lifestyles means that their incidence is unlikely to decline in the next few years. Myocardial infarction is usually secondary to coronary heart disease, which can lead to the loss of a large number of cardiomyocytes (CM), which may be as high as 1 billion cells, and is often accompanied by fibrotic tissue and scar formation, which severely restricts the functional capacity of the heart and leads to heart failure. The regeneration of mammalian models is limited to early developmental stages. Current treatment options for cardiovascular disease range from drugs to transplants. However, it is inevitable that each strategy has related disadvantages. For a long time, stimulating the production of new CMs through stem cells or cell replacement therapy has been the ultimate goal. CM-centric therapies have had limited success. These methods are too narrow and focus too much on a single cell type. In order to develop a method that may stimulate endogenous regeneration or provide additional support for CM alternative strategies, a microenvironment that promotes regeneration must also be used to expand the scope to include various non-cardiomyocytes and extracellular environments. In order to study this complexity, it seems necessary to ask for a model system that can provide both natural regeneration capabilities and a complete in vivo system. Zebrafish can (almost only) meet these criteria. Surgical resection of the apex of the ventricle has been used for nearly two decades to study the mechanisms involved in the regeneration of adult zebrafish heart tissue. In addition, genetic cell ablation models have been used to reduce the number of CMs and trigger regeneration. Recently, the development of cryodamage models places liquid nitrogen cooled probes on the ventricles to induce local cell death, which provides a new perspective to observe the resulting regeneration and is more representative of the cell damage caused by MI. Regardless of which injury model is chosen, the study of the complex injury response in this model has provided a wealth of information about the cellular and molecular mechanisms required to rebuild the heart after injury, and there is increasing emphasis on the need to extend the scope from CM to these cells. Microenvironment.

　　Current treatment prospects: Although the structure of the adult mammalian heart is relatively simple, the repair is complicated, and so far it has not been possible to achieve regeneration. Since regeneration is currently impossible, and heart transplantation is also limited by practical considerations (not only lack of donors, but also related surgical complexity), the focus is on treatment rather than cure, especially preventing the development of ischemic heart disease. Heart failure. Cardioprotective treatment options can improve blood supply (such as thrombolysis or bypass surgery for revascularization), while pharmacological interventions can slow heart remodeling (such as ACE inhibitors and beta blockers), and Patients with more advanced heart failure benefit from mechanical support therapy (such as a left ventricular assist device or cardiac resynchronization therapy). Despite a wide range of therapeutic interventions, ischemic heart failure and its associated adverse cardiac remodeling remain a major challenge for global health services. Cell replacement therapy aims to directly solve the problem of insufficient CM proliferation, and CM proliferation is widely regarded as a limiting factor for heart regeneration. These treatments include culturing a large number of cells in vitro and injecting them directly into the injured heart, showing promise in many preclinical settings. However, the results of this relatively new strategy are often inconsistent and fail to achieve significant improvements in cardiac function. Animal model studies have revealed worryingly common side effects, including arrhythmia and tachycardia. Studies have found that direct chemical stimulation of the innate immune system produces results comparable to previously reported cardiac ischemic damage repair cell therapy. The beneficial effects of stem cell therapy are mainly mediated by the acute regional accumulation of specific macrophage populations and the inflammatory wound healing response triggered by cell transplantation, rather than the direct proliferation of transplanted cells. These findings are also consistent with a new research trend to further clarify the role of immune response in heart repair and regeneration.

　　Myocardial cell proliferation: In recent years, insufficient proliferation capacity of CMs has become the main limiting factor for heart regeneration after heart injury/myocardial infarction. In the heart of mature mammals, the proliferation of CM is poor, and the number of progenitor cells is small. After birth, the heart is mainly enlarged by myocardial hypertrophy. Considering its regenerative potential, the composition of adult cardiac muscle is worth noting. Although about 90% of myocardial tissue is composed of CMs, these cells only account for 30% of the total number of cells, and the rest are mainly composed of endothelial cells (43%), fibroblasts (20%) and white blood cells (about 7%). Approximately 1% are CM progenitor cells (or stem cells), plus the fact that most adult CMs are in a quiescent state, which is considered to be the fact that the regenerative ability of adult human heart is very low after injury. Considering that the cell damage after myocardial infarction may be in the range of 25% cell loss, it is obvious that normal turnover and replacement operations have obvious defects and are not enough to restore the functional myocardium after infarction. However, if only 30% of the total number of cardiomyocytes are CMs, then narrowing our focus on regenerative capacity to this single cell type appears to be limiting. Although there is no robust regeneration in the heart of adult mammals, varying degrees of cardiac regeneration have been observed in newborn mammals. The heart of mice and pigs can be effectively regenerated, provided that the injury occurs in the first two days after birth and the degree of injury is not too great (<15%). Damage caused by up to 20% of the ventricular cell death (calculated by area) seems to be well tolerated and completely eliminated within 60 days after injury, but increasing the damage area by 5% seems to make the zebrafish heart’s regenerative capacity exceed its limit. Even at 130 dpi, the scar cannot be completely eliminated. In addition, a recent report indicated that repeated freezing damage limits the regenerative capacity of adult zebrafish. Interestingly, CM proliferation will be activated after each injury (although this proliferation efficiency will decrease over time), but each injury will intensify collagen deposition, and scar removal will gradually fail, which further indicates that the regeneration capacity is limited. The zebrafish injury model also helps to elucidate new sources of cardiomyocytes in a regenerative environment. Several studies have used lineage tracing methods to show that the existing mature CM is the source of new myocardium, and stem/progenitor cells are not significantly involved in this process. From the morphological point of view, the dividing zebrafish CMs change their contraction state in a way similar to the structural changes that promote the proliferation of mouse cells. There is some CM proliferation in adult mammals, which may be caused by existing CMs (such as zebrafish); however, this occurs much less frequently, and the characteristics of CMs that maintain this proliferation capacity have not yet been Absolutely sure. In recent years, there is evidence that ploidy is an important factor. Most zebrafish cytoplasmic male sterile lines are mononuclear and diploid; however, these characteristics are lost in mammalian cells, which become binuclear or polyploid soon after birth. This hypothetical relationship is further confirmed, because the prevalence of mononuclear diploid CMs has been shown to be related to the functional recovery and CM proliferation after coronary artery ligation in mice, and CM ploidy can usually predict the regeneration potential of vertebrates. Recent studies by Gonzalez-Rosa et al. have shown the importance of ploidy, because the experimental polyploidization of zebrafish male sterile lines is sufficient to inhibit the proliferation potential in this highly regenerative model, and a large number of diploids are required CM to support regeneration.

　　Zebrafish as a model for repair and regeneration: Many advantages of zebrafish, such as its high fecundity, external fertilization and transparency of developing larvae, are the basis for its use as a valuable model system for vertebrate developmental biology. However, 70% of the genes encoding humans are related to genes found in zebrafish, and 84% of disease-related genes are zebrafish equivalent, which means that they have become important models of human diseases. In addition to these well-known characteristics, the key attribute of zebrafish in this study is that they have almost unique ability to completely regenerate the heart after injury, thereby providing a cellular and molecular map for the process from repair to regeneration. As mentioned above, three main heart injury models have been described in adult zebrafish: (1) cardiac resection, (2) cryo-injury and (3) genetic ablation of CM. It is worth noting that after heart injury, such as myocardial infarction (human) or freezing injury (zebrafish), the initial stages from injury to repair and scar formation are conservative, which further strengthens zebrafish as a powerful The value of the tool to understand the limiting factors that prevent mammalian regeneration. Both human and zebrafish repair involves an initial inflammatory phase (defined as the recruitment of immune cells and phagocytosis to remove cell debris), followed by the repair phase, which is characterized by the deposition of collagen and other extracellular matrix (ECM) components and The formation of scars. In the human body, this collagen matrix can form mature scars that cannot be eliminated. However, in zebrafish, discarded collagen is quickly remodeled and replaced by new heart muscle.

　　Human and zebrafish heart repair. The repair/regeneration stage after human and zebrafish heart injury indicates that the initial inflammatory response is similar to the scar response, but the final stage is different. Humans show persistent scar tissue and poor CMs renewal. The zebrafish myocardial infarction model (frozen injury as shown in the figure) shows the regeneration stage of scar healing and CM proliferation, and finally restores healthy myocardium.

　　Freezing injury leads to the death of ventricular wall cells (apoptotic cells can also be detected in the coronary artery lumen), the peak value is about 4dpi, and it gradually decreases to less than 0.5% at 60 dpi. This peak of apoptosis is accompanied by the initial inflammatory response and the start of new blood vessels, while the existing coronary blood vessels sprout into the injured area. At 4 dpi, extensive fibrin accumulation in the damaged area can also be seen, but most of it disappears at 14-21 dpi. Extensive CM (and other cell) proliferation was observed during the initial stages of the injury response, peaking in the first week. By 21dpi, the blood vessel coverage in the injured area was completed, and the blood vessel reconstruction in the injured area was so rapid that the blood vessels between the control group and the injured heart could not be distinguished only 40 days after the injury. The deposition, remodeling and maturation of collagen-rich scars are the final stages of the repair process in mammals; however, the tissues in zebrafish dissolve very quickly, and the scar removal and regeneration of heart tissues are completed within 60-130 dpi , It depends on the size of the initial damage. Interestingly, the comparative analysis of freezing injury and resection injury response showed that the degree of apoptosis, inflammation and scarring were different, further highlighting the difference between injuries. This regenerative ability of the adult body system provides unparalleled opportunities to study the molecular and cellular processes of regeneration in a complete environment.

　　Recently, people have begun to focus on inflammation as a key aspect of the regeneration process. Zebrafish have many innate and adaptive immune cell populations similar to mammals, and similar proportions can be found in healthy and injured zebrafish hearts. Our group and others have described the systemic recruitment and expansion of innate and adaptive immune cell populations, and have begun to reveal their role in the characteristic stages of the adult zebrafish heart repair and regeneration schedule. This indicates that there is a direct similarity between zebrafish and mammalian heart repair. Timely induction and elimination of inflammatory response is essential for the normal regeneration of zebrafish heart and regulating many other aspects of repair, including revascularization, CM proliferation and scarring. Deposition and subsidence. Recent observations have also shown that if there is no strict inflammatory response regulation, regardless of the presence of proliferating cells and ECM, excessive immune cell aggregation will inhibit regeneration. This suggests that inflammatory cells play a key role in coupling CM proliferation and scar elimination, both of which are essential for effective regeneration results. A comprehensive description of the immune cell types of zebrafish and mammals in the process of repair and regeneration, as well as a comprehensive understanding of the differences between zebrafish and mammals, are critical to understanding their different roles after injury. Here, we introduce the main immune cell types that have been studied during heart regeneration.

　　Granulocytes: Granulocytes are a key component of the innate immune system, including neutrophils and eosinophils, and they have begun to clarify their role and dynamics after adult zebrafish heart injury. Neutrophils are the "first responders" of tissue damage. They are quickly mobilized and recruited into the heart injured by low temperature from 6 hours after injury (hpi). The number of neutrophils peaks in the first 24 hours, during which time they are the main factor that promotes inflammation repair. In addition to the phagocytosis of necrotic tissue, the recruited neutrophils also secrete pro-inflammatory mediators, reactive oxygen species (ROS) and cytokines. These substances recruit additional immune cells to promote myofibroblast differentiation and angiogenesis. Late regeneration is crucial. However, the release of ROS by neutrophils and infiltrating monocytes can cause further tissue necrosis and scar formation. In the heart injury model, neutrophil retention can prolong the inflammatory period, leading to delayed scar regression and reduced CM proliferation. Therefore, stopping the neutrophil response in a timely manner is critical to the outcome of zebrafish regeneration. Eosinophils also rapidly accumulate in the injured zebrafish heart and increase from 7-21dpi. Nevertheless, functional studies of this cell type in zebrafish have not yet been conducted, so further studies are needed to clarify that they are in coordination Role in repair and regeneration.

　　Macrophages: The importance of different macrophage functions in wound healing has been well described in repairing and regenerative organisms. This includes the phagocytic and pro-inflammatory activity at the initial stage of repair, as well as the reversal of the inflammatory environment to a steady state state. The functional scope of macrophages is also expanding. Recent publications have identified new roles of macrophages in wound angiogenesis, cardiac steady-state electrical conductivity, and collagen deposition after cardiac injury in mouse and zebrafish models. Macrophages are roughly divided into "M1" or "M2" according to their function and activation status. M1 macrophages are considered to be pro-inflammatory and antimicrobial, and M2 macrophages are considered to promote repair, in tissue reconstruction, immune regulation , Matrix deposition and phagocytosis play a role. However, it is becoming increasingly clear that the diverse and sometimes surprising roles of cardiac macrophages are coordinated by subpopulations of highly heterogeneous phenotypes, transcription profiles, and activation states, which are more than traditional The M1 or M2 classification is more instantaneous.

　　Lymphocytes: between adaptive immune system and regeneration

　　The relationship has always been the focus of controversy, because organisms with more advanced immune systems generally reduce their ability to regenerate. Therefore, the role of neutrophils and macrophages in wound healing and regeneration has been extensively studied, but the role of adaptive immune cells is less clear, especially in heart regeneration. However, it has become increasingly clear that many regenerative organisms, including zebrafish, possess a complex adaptive immune system, which proves the necessity of studying these cells in the regeneration process. The lymphocytes of the adaptive immune system, such as T cells, coordinate antigen-specific immunity. There are many subgroups of T cells with different functions, such as tumor and virus immunity, cytokine secretion, and establishment of humoral response. Another subgroup, CD4+ FOXP3+ T regulatory cells (Tregs) are important for suppressing inflammation by secreting anti-inflammatory cytokines such as IL-10. In mouse myocardial infarction models, these T cell populations have been shown to have different and opposite effects, with known functions in wound healing, inflammation, and CM quantity regulation. Therefore, it is necessary to further study the response of T cells to heart injury.

　　The composition and function of extracellular matrix: The formation of unresolved mature scars mainly inhibits the heart function of mammals after myocardial infarction. Therefore, the composition of ECM and the cells that regulate it should also be regarded as an important role in the regeneration process. Cardiac extracellular matrix (ECM) is a complex protein network, mainly composed of collagen, with a tissue surrounding CMs (intramuscular muscle layer), defining the main myocardial tissue bundle (sarcolemma) and enveloping the entire myocardium. It surrounds the heart, provides a scaffold and maintains the tissue structure. In mammals and zebrafish, cardiac fibroblasts are the main contributor to ECM. Under normal circumstances, mesenchymal fibroblasts respond quickly to stimuli such as injury or hypoxia, re-enter the cell cycle, synthesize ECM protein, regulate intercellular communication, and produce activated fibroblasts and myofibroblasts. In mammals, the cardiac ECM is believed to be mainly composed of collagen I (>85%), and other collagens, fibronectin, glycosaminoglycans (GAG) and proteoglycans constitute the rest of the matrix. Additional information about the composition of ECM can be obtained from immunoassay analysis. These test results indicate the presence of several matrix proteins during the regeneration of the zebrafish heart, including structural proteins such as non-fibrillar collagen XII and fibrillar collagen I. The decellularized cardiomyocyte extracellular matrix of zebrafish has a significant proliferation effect on human heart precursor cells and can make the mouse heart tissue endogenously regenerate after myocardial infarction. These obvious differences in the extracellular matrix of zebrafish and mammals highlight its importance in mediating regeneration and prove the value of continuing to study the exact way in which ECM components are produced and broken down.

　　Conclusion: This article reviews the advantages of zebrafish models in the study of heart regeneration, with particular emphasis on the role of different cell types in the cardiac microenvironment. Through the study of zebrafish and its extraordinary natural regeneration ability, many of these effects have been discovered. The restoration of the number of CM is critical to the regeneration and functional recovery of the heart, but studies of zebrafish and other regeneration models have revealed that the signals and other functions transmitted by immune cells, epicardial cells, nerves, fibroblasts, and endothelial cells are important Supporting this regenerative result is crucial. Future treatments need to protect or replace these other cell types and CM to be effective.