斑马鱼心脏再生 z-bo 2020-10-22 894

　　Human myocardial infarction will lead to ventricular remodeling and eventually heart failure, which is one of the main causes of death worldwide. Compared with adult mammalian hearts, zebrafish model organisms have significant regenerative capacity, which provides the possibility to study the basis of natural regeneration. Here, we summarize the latest insights into the cellular and molecular mechanisms of zebrafish heart regeneration.

　　Introduction: Cardiovascular disease is still the main cause of death worldwide. It is expected that the burden of cardiomyopathy will increase greatly in the future. Myocardial infarction is caused by the formation of atherosclerotic plaque and the blockage of the coronary arteries. The blockage of the coronary arteries cannot deliver nutrients and oxygen to the heart muscle, resulting in the death of millions of cardiomyocytes. Replacing damaged tissue with non-contractile scar tissue can protect the heart from rupture, but will eventually lead to poor heart remodeling and heart failure. For decades, the myocardium of adult mammals has been considered to be a post-mitotic tissue, with little regenerative capacity. The growth of the heart after birth is mainly the result of cardiomyocyte hypertrophy. In the absence of cytokinesis, additional DNA synthesis produces mononuclear polyploid and binuclear diploid cardiomyocytes in humans and mice, respectively. It is worth noting that the heart of newborn mice can regenerate in a short time after birth. Interestingly, a case of neonatal functional recovery after severe myocardial infarction was reported. Therefore, it is of interest to explore how other species maintain heart regeneration during their life cycle. The zebrafish is one of the most relevant models for studying regenerative biology, because zebrafish have an amazing ability to regenerate most organs and tissues (including the heart). The biological response of zebrafish to heart injury requires the coordinated participation of multiple cell types, which involve many molecular mechanisms, which ultimately lead to the regeneration of damaged tissues. Here, we summarize the latest findings of heart regeneration in adult zebrafish.

　　Zebrafish heart regeneration: The zebrafish heart has many similarities with the mammalian heart in terms of morphology, cell composition, genetic regulation and embryonic development. During development, the cardiac progenitor cells in the first heart region initially form primitive heart tubes. Fusion of cardiac progenitor cells from the second heart region to the vein and arterial poles, the structure elongates and loops to form a two-chamber embryonic heart. The adult myocardium is arranged by an endocardial layer facing the cavity and covered by an epicardial layer. The zebrafish heart has two chambers, and the single atrium and ventricle are connected by atrioventricular valves. Blood enters the heart through the atria, is pumped in from the ventricles, and ejected into the circulation through the bulbar artery (a prominent outflow tract). The myocardium can be divided into three layers: the inner trabecular layer, the primitive layer and the outer cortical layer.

　　A groundbreaking study conducted by Poss and colleagues showed that after removing 20% of the adult zebrafish ventricles, the lost myocardium was replaced by a new function of myocardium, and regeneration was achieved through a process with almost no scars. Later, further cardiac injury models were developed, including ventricular cryodamage and genetic ablation. Ventricular freezing injury induces cell death by rapidly freezing part of the ventricle. Hearts damaged by low temperature can also regenerate, but the regeneration is accompanied by a temporary fibrotic scar deposition, which is finally resolved. The third major damage model, gene ablation of cardiomyocytes, is currently based on the induction and tissue-specific expression of diphtheria toxin A or nitroreductase, which is a prodrug that can transform metronidazole into cells Enzymes that cause cell death due to toxic metabolites. These and other methods have been widely used to study the heart regeneration mechanism of zebrafish.

　　The cell source of regenerating myocardium: Regarding heart regeneration, a central question that needs to be solved is: Where do the new myocardial cells come from? The current consensus is that newly formed cardiomyocytes are derived from pre-existing differentiated cardiomyocytes. This hypothesis is strongly supported by the lineage tracking study of Cre-lox technology. In this study, the cardiomyocytes of the uninjured heart were irreversibly labeled with the cardiomyocyte-specific promoter cmlc2 (myl7). Myl7 is expressed in myocardial progenitor cells in the premesoderm before cardiac circulation. Therefore, not only fully differentiated cardiomyocytes express myl7. Some cardiomyocytes, mainly located under the epicardium and near the edge of the injury, reactivate the expression of gata4 and ctgfa gene regulatory regions after injury. The expression of Sox10 can mark a portion of cardiomyocytes in embryonic and adult zebrafish hearts. These cells preferentially proliferate and contribute to myocardial regeneration after heart injury. These findings may represent the contribution of neural crest-derived cardiomyocytes to heart regeneration, or the activation of specific neural crest genetic features in certain proliferating cardiomyocytes. In short, the extent to which some cardiomyocytes show high regenerative capacity, and which specific cell and transcriptome changes are involved in this process, require further research. There is evidence that cardiomyocytes can partially change their fate during regeneration and rebuild different myocardium. For example, atrial cardiomyocytes can be used to compensate for the ablation of embryonic ventricular cardiomyocytes. In addition, clonal analysis of excised ventricles showed that cortical cardiomyocytes help regenerate the cortical layer. Recently, trabecular cardiomyocytes also showed regeneration of the cortex, which revealed a certain degree of plasticity of the cardiomyocytes. Whether cortical cardiomyocytes help regenerate trabeculae is currently unclear. Interestingly, in a heart damaged by hypothermia, the original layer of the myocardium is not regenerated. This observation and the finding that the regenerated cortical layer in the excised and frostbited heart still remains thickened and the ventricular wall contractility is not completely rebuilt, suggesting that zebrafish's myocardial regeneration is not fully achieved. For a long time, the discovery that adult cardiomyocytes in zebrafish are mainly diploid has been considered as a possible explanation for its high proliferation potential. Polyploidy of non-regenerative cardiomyocytes is more common than regenerative species and represents an obstacle to proliferation. In fact, the polyploidization of cardiomyocytes is related to the loss of mouse heart regeneration and repair. Genetic models indicate that the increase in cardiomyocyte ploidy will reduce the zebrafish’s ability to regenerate the heart, indicating that ploidy plays a key role in this process.

　　An important exploration is to identify endogenous and exogenous molecules and environmental stimuli that induce cardiomyocyte proliferation. Tyrosine protein kinase receptor Erbb2 is one of the main mediators in the process of myocardial regeneration. One of its ligands, neuregulin 1 (Nrg1), is a powerful cardiomyocyte mitogen that is rapidly induced in perivascular cells during heart regeneration. The Erbb2 signal also acts on the downstream effect cascade involved in vitamin D or hemodynamics in the process of cardiomyocyte proliferation. In proliferating cardiomyocytes, Erbb2 signaling mediates the transition from oxidative phosphorylation to glycolysis-based metabolism. Erbb2 signal is also related to heart regeneration in newborn mice. Other signaling pathways that affect cardiomyocyte proliferation have been identified, including PPARδ and vegfaa. Whether these also interact with the Erbb2 signaling pathway is unclear. Extensive epigenetic remodeling precedes the regeneration response of cardiomyocytes. H3K27me3-mediated epigenetic silencing inhibits sarcomere and cytoskeleton genes is a prerequisite for re-entering the cell cycle. As revealed by histone H3.3 analysis, specific enhancers are activated during the injury response. In addition, it has been shown that transient cell membrane fusion in cardiomyocytes plays a role in myocardial regeneration. Other factors that work at the body level also affect the proliferation of cardiomyocytes, including exercise caused by swimming and cardiac preconditioning. In short, strict timing and conditioning of mitotic signals is essential to promote cardiomyocyte proliferation and heart regeneration.

　　Immune system response: After heart injury, there is an initial pro-inflammatory period, in which necrotic cells trigger the activation and infiltration of immune cells. These cells from inside and outside the heart remove debris and dead cells and reshape the extracellular matrix (ECM). Several immune cell types participate in this process in a coordinated time and space. For example, an increase in neutrophil survival or ablation of Treg cells can lead to a decrease in organ regeneration. In mammals, macrophages in the heart are the most abundant immune cell population in the heart, most of which come from the yolk sac. The depletion of macrophages leads to impaired heart regeneration in newborn mice and zebrafish. For example, stimulation of Toll-like receptors in medaka can promote immune cell recruitment, neovascularization, neutrophil clearance, cardiomyocyte proliferation, and scar healing. On the other hand, delayed recruitment of macrophages in zebrafish leads to neovascularization, neutrophil clearance, cardiomyocyte proliferation, and scar regression. In general, precise control of the time and space of inflammation is essential for heart regeneration. However, the identification of other immune cell types and specific subgroups involved in the regeneration of the zebrafish heart remains to be fully explored.

　　Heart endothelium, nerve and lymphatic system: The heart endothelium is composed of two structures: coronary artery endothelium and endocardial endothelium. 15 hours after injury, angiogenesis buds can be seen infiltrating the damaged tissue. Overexpression of the dominant negative isoform of vegfaa inhibits this process, reduces the proliferation of cardiomyocytes, and prevents heart regeneration. The proliferation peak of endocardial cells around the injured tissue appeared between 3～5dpi, and the peak of myocardial cell proliferation appeared before 7dpi. The involvement of Notch and Wnt signaling in endocardial cells has been described. In addition to its function in oxygenation and nutrient delivery, regenerated coronary arteries can also be used as a scaffold for myocardial cells to rebuild damaged areas, and the epicardial Cxcl12 / Cxcr4 signal transduction axis plays an important role in this process.

　　Heart innervation also affects the regeneration process. The hypoinnervation of the adult zebrafish heart leads to a decrease in the proliferation potential of cardiomyocytes, leading to loss of heart regeneration. Although the role of the lymphatic system in the regenerative environment has long been a mystery, recent studies have shown that it is important in draining and clearing inflammatory cells from damaged myocardium. Overall, these results establish the important role of endocardium, coronary artery endothelium, nerve and lymphatic system as signal sources and physical stents to support and promote heart regeneration. In the process of heart regeneration, epicardial and epicardial-derived cells are involved in the production of perivascular cells and fibroblasts. These cells and fibroblasts play an important role in the deposition and remodeling of scars. In fact, gene ablation of tcf21+ epicardial cells will reduce the proliferation of cardiomyocytes. Interestingly, epicardial cells secrete extracellular matrix, allowing them to migrate on the surface of the heart. The epicardium secretes nutritional factors that are important for heart regeneration, including mitotic signals such as neuregulin 1.

　　Fibroblasts are the main source of collagen and other ECM proteins after heart injury. In the scar ablation stage, some myocardial-derived fibroblasts were inactivated. In addition, cell senescence has been observed in the damaged part of zebrafish, and it may be necessary to properly balance senescent cells to promote heart regeneration. Studies on newborn mice have shown that fibroblast senescence is required for heart regeneration, which needs to be confirmed in a zebrafish model.

　　Outlook: We have a better understanding of how different heart structures promote heart regeneration. It is also understood that some cellular and molecular mechanisms between zebrafish and newborn mice are conserved. In addition, zebrafish has also proven to be a good model for studying heart valve regeneration. Cross-species analysis to define which results have transformative value will be an important step to clarify the complex process of heart regeneration in the future.