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.     

Turning regenerative technologies into treatment to repair myocardial injuries

Regenerative therapies including stem cell treatments hold promise to allow curing patients affected by severe cardiac muscle diseases. However, the clinical efficacy of stem cell therapy remains elusive, so far. The two key roadblocks that still need to be overcome are the poor cell engraftment into the injured myocardium and the limited knowledge of the ideal mixture of bioactive factors to be locally delivered for restoring heart function. Thus, therapeutic strategies for cardiac repair are directed to increase the retention and functional integration of transplanted cells in the damaged myocardium or to enhance the endogenous repair mechanisms through cell-free therapies. In this context, biomaterial-based technologies and tissue engineering approaches have the potential to dramatically impact cardiac translational medicine. This review intends to offer some consideration on the cell-based and cell-free cardiac therapies, their limitations and the possible future developments.     

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.     

Expression of the cardiac actin gene in axolotl embryos

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.     

Regenerating cardiac cells: insights from the bench and the clinic

A major challenge in cardiovascular regenerative medicine is the development of novel therapeutic strategies to restore the function of cardiac muscle in the failing heart. The heart has historically been regarded as a terminally differentiated organ that does not have the potential to regenerate. This concept has been updated by the discovery of cardiac stem and progenitor cells that reside in the adult mammalian heart. Whereas diverse types of adult cardiac stem or progenitor cells have been described, we still do not know whether these cells share a common origin. A better understanding of the physiology of cardiac stem and progenitor cells should advance the successful use of regenerative medicine as a viable therapy for heart disease. In this review, we summarize current knowledge of the various adult cardiac stem and progenitor cell types that have been discovered. We also review clinical trials presently being undertaken with adult stem cells to repair the injured myocardium in patients with coronary artery disease.     

Functionalization of soft materials for cardiac repair and regeneration

Coronary artery disease is a leading cause of death in developed nations. As the disease progresses, myocardial infarction can occur leaving areas of dead tissue in the heart. To compensate, the body initiates its own repair/regenerative response in an attempt to restore function to the heart. These efforts serve as inspiration to researchers who attempt to capitalize on the natural regenerative processes to further augment repair. Thus far, researchers are exploiting these repair mechanisms in the functionalization of soft materials using a variety of growth factor-, ligand- and peptide-incorporating approaches. The goal of functionalizing soft materials is to best promote and direct the regenerative responses that are needed to restore the heart. This review summarizes the opportunities for the use of functionalized soft materials for cardiac repair and regeneration, and some of the different strategies being developed.     

An allometric analysis of oxygen consumption rate and cardiovascular function in the cockroach, Blaberus discoidalis.

The interrelationship of metabolic rate and cardiovascular function has been well documented in vertebrates through allometric analyses. However, similar studies are lacking in insects. Unlike vertebrates, the cardiovascular system of insects does not play a significant role in oxygen transport. A comparison of the interrelationship in insects and vertebrates might provide insight into the nature of the connection between metabolic rate and the cardiovascular system. Oxygen consumption, heart rate and heart dimensions were measured in the nymphs of the cockroach Blaberus discoidalis over a mass range of 0.03-5 g. Oxygen consumption rate scaled with an exponent of 0.83. The relationship between heart rate and body mass scaled negatively, however, it did not appear to be linear. Using measurements of heart widths, abdominal length and heart rate, stroke volume and cardiac output were estimated. Cardiac output appeared to scale linearly with an exponent of 0.85, which was not significantly different from the exponent observed for the rate of oxygen consumption. Thus, the observed similarity between the exponents for oxygen consumption rate and cardiac output in vertebrates also appears to be present in insects.     

Functional Morphology of the Heart in Fishes

The systemic heart of fishes consists of four chambers in series,the sinus venosus, atrium, ventricle, and conus or bulbus. Valvesbetween the chambers and contraction of all chambers exceptthe bulbus maintain a unidirectional blood flow through theheart. The heart is composed of typical vertebrate cardiac muscle,although there may be minor differences in the distributionof spontaneously active cells, the rate and nature of spreadof excitatory waves, and the characteristics of resting andaction potentials between different fish and other vertebrates.Cholinergic fibers innervate the heart, except in hagfish whichhave aneural hearts. Fish hearts lack sympathetic innervation.The level of vagal tone varies considerably, and is affectedby many factors. In some fish the heart is essentially aneural(without vagal tone) during exercise and may resemble an isolatedmammalian ventricle with increased venous return causing increasedcardiac output. There are many mechanisms that could increasevenous return in exercising fish. rß-adrenergic receptorshave been located on the hearts of some fish, and changing levelsof catecholamines may play a role in regulating cardiac activity.Changes in cardiac output in fish are normally associated withlarge changes in stroke volume and small cha-nges in heart rate.     

Non-coding RNAs in cardiomyocyte proliferation and cardiac regeneration: Dissecting their therapeutic values

Cardiovascular diseases are associated with high incidence and mortality, contribute to disability and place a heavy economic burden on countries worldwide. Stimulating endogenous cardiomyocyte proliferation and regeneration has been considering as a key to repair the injured heart caused by ischaemia. Emerging evidence has proved that non-coding RNAs participate in cardiac proliferation and regeneration. In this review, we focus on the observation and mechanism that microRNAs (or miRNAs), long non-coding RNAs (or lncRNAs) and circular RNA (or circRNAs) regulate cardiomyocyte proliferation and regeneration to repair a damaged heart. Furthermore, we highlight the potential therapeutic role of some non-coding RNAs used in stimulating CMs proliferation. Finally, perspective on the development of non-coding RNAs therapy in cardiac regeneration is presented.     

Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.     

Atrial natriuretic peptide in the granular cells of the snail heart

Cardiomyocytes of vertebrates combine contractile and endocrine functions. They synthesize and secrete atrial natriuretic peptide (ANP), which is localized in their specific granules. The presence of ANP has been shown in some tissues of invertebrates, including the heart of molluscs. We have studied localization of ANP in cells of the snail heart. METHOD: The atrial and ventricular tissues of the snail Helix pomatia were studied by electron microscope immunocytochemistry, using anti-ANP antibodies. ANP-immunoreactivity has been detected in granules of granular cells located on the luminal surface of the snail myocardium. These cells are abundant in the atrium being very rare in the ventricle. Granular cells at different stages of maturation were revealed. Immature granular cells have light granules of moderate size with homogeneous tight content, while mature granular cells are huge in size and all their granules are fused together. The material of these granules loosens up and almost completely fills up the cytoplasm. No ANP-immunoreactivity was observed in muscle cells or nerve fibers. A possible origin of granular cells from the cardiac endothelial cells is discussed. The molluscan heart, similar to that of vertebrates, is a bifunctional organ. However, contrary to the heart of vertebrates, in the molluscan heart contractile and endocrine functions are separated between different types of cells.     

The heart as a working model to explore themes and strategies for anoxic survival in ectothermic vertebrates

Most vertebrates die within minutes when deprived of molecular oxygen (anoxia), in part because of cardiac failure, which can be traced to an inadequate matching of cardiac ATP supply to ATP demand. Cardiac power output (PO; estimated from the product of cardiac output and central arterial pressure and an indirect measure of cardiac ATP demand) is directly related to cardiac ATP supply up to some maximal level during both normoxia (ATP supply estimated from myocardial O(2) consumption) and anoxia (ATP supply estimated from lactate production rates). Thus, steady state PO provides an excellent means to examine anoxia tolerance strategies among ectothermic vertebrates by indicating a matching of cardiac glycolytic ATP supply and demand. Here, we summarize in vitro measurements of PO data from rainbow trout, freshwater turtles and hagfishes to provide a reasonable benchmark PO of 0.7 mW g(-1) for maximum glycolytic potential of ectothermic hearts at 15 degrees C, which corresponds to a glycolytic ATP turnover rate of about 70 nmol ATP g(-1) s(-1). Using this benchmark to evaluate in vivo PO data for hagfishes, carps and turtles, we identify two cardiac survival strategies, which in conjunction with creative waste management techniques to reduce waste accumulation, allow for long-term cardiac survival during anoxia in these anoxia-tolerant species. Hagfish and crucian carp exemplify a strategy of evolving such a low routine PO that routine cardiac ATP demand lies within the range of the maximum cardiac glycolytic potential. Common carp and freshwater turtles exemplify an active strategy of temporarily and substantially decreasing cardiac and whole body metabolism so that PO is below maximum cardiac glycolytic potential during chronic anoxia despite being quite close to this potential under normoxia.     

The cellular basis for enhanced volume-modulated cardiac output in fish hearts

During vertebrate evolution there has been a shift in the way in which the heart varies cardiac output (the product of heart rate and stroke volume). While mammals, birds, and amphibians increase cardiac output through large increases in heart rate and only modest increases (approximately 30%) in stroke volume, fish and some reptiles use modest increases in heart rate and very large increases in stroke volume (up to 300%). The cellular mechanisms underlying these fundamentally different approaches to cardiac output modulation are unknown. We hypothesized that the divergence between volume modulation and frequency modulation lies in the response of different vertebrate myocardium to stretch. We tested this by progressively stretching individual cardiac myocytes from the fish heart while measuring sarcomere length (SL), developed tension, and intracellular Ca2+ ([Ca2+]i) transients. We show that in fish cardiac myocytes, active tension increases at SLs greater than those previously demonstrated for intact mammalian myocytes, representing a twofold increase in the functional ascending limb of the length-tension relationship. The mechanism of action is a length-dependent increase in myofilament Ca2+ sensitivity, rather than changes in the [Ca2+]i transient or actin filament length in the fish cell. The capacity for greater sarcomere extension in fish myocardium may be linked to the low resting tension that is developed during stretch. These adaptations allow the fish heart to volume modulate and thus underpin the fundamental difference between the way fish and higher vertebrates vary cardiac output.     

利用果蝇心力衰竭模型筛选果蝇第2号染色体缺失系

果蝇心脏一直以来都是研究心血管发育的极好模型,许多控制心脏分化和特化的调控基因和信号途径从果蝇到哺乳动物都是保守的.由于近年心力衰竭的发病率不断升高,我们最近又建立了果蝇心力衰竭模型用于大规模筛选和鉴定心力衰竭的相关基因.在这个模型中,适龄的成体果蝇被整齐排列在导电的载玻片上,通过电极短暂刺激30s,使果蝇的心跳频率由正常的3Hz增加到6Hz,停止后检测果蝇心率恢复情况,不能恢复正常心跳频率或出现纤维性震颤的果蝇视为心力衰竭.该模型可以在短期内大规模筛选到与心力衰竭相关的基因.利用此心力衰竭模型,我们筛选了164个果蝇2号染色体缺失系,获得33个候选缺失系.这些候选缺失系的心衰率要么与野生型品系相比差异显著,要么与tinman或panier突变系相比差异显著,提示这些缺失系中可能含有与心力衰竭相关的调控基因.     

Improving regenerating potential of the heart after myocardial infarction: Factor-based approach

The emerging evidence that the heart has the potential to regenerate, albeit not ideally, has stimulated considerable interest in the field of cardiac regenerative medicine. Several lines of research demonstrated that factor-based therapy is feasible and effective, whether it is used independently or as an adjunct to cell therapy. The ultimate goal of the factor-based approach is to improve the regenerating potential of the heart as a means to treat patients with cardiovascular disease. This article reviews recent approaches involving factor-based therapy for cardiac repair and regeneration including some of the advantages of this type of therapy as well as some of the hurdles that must be overcome before this therapeutic approach becomes a standard part of clinical medicine.     

Comparative and scaling aspects of heart and body weights with reference to blood supply of cardiac fibers

Relative heart weight (RHW) differs in vertebrates with the ratio 1:20 between extremes (bottom bound fishes--Pleuronectidae--and birds). When plotting heart weight (HW) against body weight (BW) one obtains channels which contain not only vertebrates of the same classes (poikilotherms, small and big mammals and birds) but also animals belonging to different classes: tuna fish data are located in the "small mammalian channel" together with data of large tropical snakes while large mammals (upwards 4000 g) belong to the "bird channel". Reasons for such groupings are not clear and physical activity seems not to be the only reason. When comparing active and non active vertebrates one finds that the RHW is as a rule greater in physically more active poikilotherms and homoiotherms. The RHW is also higher in wild than in domesticated forms the differences appearing after weaning (wild vs laboratory rat). In spongy type of myocardium the growth of cardiac fibers results in restriction of the blood flow through lacunae and the contact between endothelial cells lining growing strands of musculature probably provokes formation of capillaries. The appearance of mixed type of myocardium (outer compact and inner spongy compartments) is not bound to the water to land transition since it occurs also in some fishes; it does not occur or is rare in amphibia and is frequent in reptiles. The compact outer layer comprises a different proportion of the cardiac wall volume (5-73%). Metabolic differences were described between cardiac cells in compact and spongy compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac survival in anoxia-tolerant vertebrates: An electrophysiological perspective

Certain vertebrates, such as freshwater turtles of the genus Chrysemys and Trachemys and crucian carp (Carassius carassius), have anoxia-tolerant hearts that continue to function throughout prolonged periods of anoxia (up to many months) due to successful balancing of cellular ATP supply and demand. In the present review, we summarize the current and limited understanding of the cellular mechanisms underlying this cardiac anoxia tolerance. What emerges is that cold temperature substantially modifies cardiac electrophysiology to precondition the heart for winter anoxia. Intrinsic heart rate is slowed and density of sarcolemmal ion currents substantially modified to alter cardiac action potential (AP) characteristics. These changes depress cardiac activity and reduce the energetic costs associated with ion pumping. In contrast, anoxia per se results in limited changes to cardiac AP shape or ion current densities in turtle and crucian carp, suggesting that anoxic modifications of cardiac electrophysiology to reduce ATP demand are not extensive. Additionally, as knowledge of cellular physiology in non-mammalian vertebrates is still in its infancy, we briefly discuss the cellular defense mechanisms towards the acidosis that accompanies anoxia as well as mammalian cardiac models of hypoxia/ischemia tolerance. By examining if fundamental cellular mechanisms have been conserved during the evolution of anoxia tolerance we hope to have provided a framework for the design of future experiments investigating cardiac cellular mechanisms of anoxia survival.     

利用果蝇模型研究人类心脏早期发育的分子机理(英文)

近年来 ,果蝇心脏特化的遗传机制已初步研究清楚 ,但控制人类心脏早期发育的基因尚待鉴定。因为调控果蝇和脊椎动物早期心脏细胞命运定型的途径具有保守性 ,果蝇是一种探讨人类心脏早期发育的分子机理的理想动物模式。为此目的 ,我们采用P转座子和EMS诱变技术建立了约 3 0 0 0个隐性致死基因平衡系。通过心脏前体细胞特异性抗体免疫组化筛选 ,我们检出 2 0 0余个表现心脏突变表型的平衡致死系。我们进一步利用RNAi技术对一些基因的功能进行了初步的研究 ,证明这些基因表现RNAi的突变表型 ,该类突变表型与基因突变时表现的表型相似 ,即心管呈缺陷型或无心脏前体细胞形成。利用果蝇和人类基因组计划获得的成果 ,我们从果蝇心脏侯选基因中初步克隆和鉴定了 5 0个人类同源基因 ,其中 2 0个是新基因。Northen印迹分析表明 ,一部分人类基因在心脏组织中有表达 ,从而为研究这些基因在人类心脏早期发育中的作用提供了信息。目前 ,我们正在建立转基因果蝇 ,以此为模型研究这些基因是否对心肌细胞发生或心肌功能起调控作用。产生心肌细胞突变类型的基因如果类似于人类心脏病综合症 ,则可以作为人类心脏疾病侯选基因作进一步的分析。