Zebrafish models in cardiac development and congenital heart birth defects

The zebrafish has become an ideal vertebrate animal system for investigating cardiac development due to its genetic tractability, external fertilization, early optical clarity and ability to survive without a functional cardiovascular system during development. In particular, recent advances in imaging techniques and the creation of zebrafish transgenics now permit the in vivo analysis of the dynamic cellular events that transpire during cardiac morphogenesis. As a result, the combination of these salient features provides detailed insight as to how specific genes may influence cardiac development at the cellular level. In this review, we will highlight how the zebrafish has been utilized to elucidate not only the underlying mechanisms of cardiac development and human congenital heart diseases (CHDs), but also potential pathways that may modulate cardiac regeneration. Thus, we have organized this review based on the major categories of CHDs-structural heart, functional heart, and vascular/great vessel defects, and will conclude with how the zebrafish may be further used to contribute to our understanding of specific human CHDs in the future.     

8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5⁺ cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits.     

The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion

Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.     

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see ¹). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel² or sapje³) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes⁴. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy⁵. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally¹. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo⁶, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells⁷.Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail^{8, 9}. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish^{10, 11}.     

Muscle contraction controls skeletal morphogenesis through regulation of chondrocyte convergent extension

Convergent extension driven by mediolateral intercalation of chondrocytes is a key process that contributes to skeletal growth and morphogenesis. While progress has been made in deciphering the molecular mechanism that underlies this process, the involvement of mechanical load exerted by muscle contraction in its regulation has not been studied. Using the zebrafish as a model system, we found abnormal pharyngeal cartilage morphology in both chemically and genetically paralyzed embryos, demonstrating the importance of muscle contraction for zebrafish skeletal development. The shortening of skeletal elements was accompanied by prominent changes in cell morphology and organization. While in control the cells were elongated, chondrocytes in paralyzed zebrafish were smaller and exhibited a more rounded shape, confirmed by a reduction in their length-to-width ratio. The typical columnar organization of cells was affected too, as chondrocytes in various skeletal elements exhibited abnormal stacking patterns, indicating aberrant intercalation. Finally, we demonstrate impaired chondrocyte intercalation in growth plates of muscle-less Sp(d) mouse embryos, implying the evolutionary conservation of muscle force regulation of this essential morphogenetic process.Our findings provide a new perspective on the regulatory interaction between muscle contraction and skeletal morphogenesis by uncovering the role of muscle-induced mechanical loads in regulating chondrocyte intercalation in two different vertebrate models.     

Heart and soul/PRKCi and nagie oko/Mpp5 regulate myocardial coherence and remodeling during cardiac morphogenesis

Organ morphogenesis requires cellular shape changes and tissue rearrangements that occur in a precisely timed manner. Here, we show that zebrafish heart and soul (Has)/protein kinase C iota (PRKCi) is required tissue-autonomously within the myocardium for normal heart morphogenesis and that this function depends on its catalytic activity. In addition, we demonstrate that nagie oko (Nok) is the functional homolog of mammalian protein associated with Lin-seven 1 (Pals1)/MAGUK p55 subfamily member 5 (Mpp5), and we dissect its earlier and later functions during myocardial morphogenesis. Has/PRKCi and Nok/Mpp5 are required early for the polarized epithelial organization and coherence of myocardial cells during heart cone formation. Zygotic nok/mpp5 mutants have later myocardial defects, including an incomplete heart tube elongation corresponding with a failure of myocardial cells to correctly expand in size. Furthermore, we show that nok/mpp5 acts within myocardial cells during heart tube elongation. Together, these results demonstrate that cardiac morphogenesis depends on the polarized organization and coherence of the myocardium, and that the expansion of myocardial cell size contributes to the transformation of the heart cone into an elongated tube.     

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.     

Effect of Metabolic Syndrome on Mitsugumin 53 Expression and Function

Metabolic syndrome is a cluster of risk factors, such as obesity, insulin resistance, and hyperlipidemia that increases the individual’s likelihood of developing cardiovascular diseases. Patients inflicted with metabolic disorders also suffer from tissue repair defect. Mitsugumin 53 (MG53) is a protein essential to cellular membrane repair. It facilitates the nucleation of intracellular vesicles to sites of membrane disruption to create repair patches, contributing to the regenerative capacity of skeletal and cardiac muscle tissues upon injury. Since individuals suffering from metabolic syndrome possess tissue regeneration deficiency and MG53 plays a crucial role in restoring membrane integrity, we studied MG53 activity in mice models exhibiting metabolic disorders induced by a 6 month high-fat diet (HFD) feeding. Western blotting showed that MG53 expression is not altered within the skeletal and cardiac muscles of mice with metabolic syndrome. Rather, we found that MG53 levels in blood circulation were actually reduced. This data directly contradicts findings presented by Song et. al that indict MG53 as a causative factor for metabolic syndrome (Nature 494, 375-379). The diminished MG53 serum level observed may contribute to the inadequate tissue repair aptitude exhibited by diabetic patients. Furthermore, immunohistochemical analyses reveal that skeletal muscle fibers of mice with metabolic disorders experience localization of subcellular MG53 around mitochondria. This clustering may represent an adaptive response to oxidative stress resulting from HFD feeding and may implicate MG53 as a guardian to protect damaged mitochondria. Therapeutic approaches that elevate MG53 expression in serum circulation may be a novel method to treat the degenerative tissue repair function of diabetic patients.     

Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish

The heart maintains structural and functional integrity during years of continual contraction, but the extent to which new cell creation participates in cardiac homeostasis is unclear. Here, we assessed cellular and molecular mechanisms of cardiac homeostasis in zebrafish, which display indeterminate growth and possess an unusual capacity to regenerate after acute cardiac injury. Lowering fish density in the aquarium triggered rapid animal growth and robust cardiomyocyte proliferation throughout the adult ventricle, greater than that observed during slow animal growth or size maintenance. Rapid animal growth also induced strong expression of the embryonic epicardial markers raldh2 (aldh1a2) and tbx18 in adult epicardial tissue. Pulse-chase dye labeling experiments revealed that the epicardium recurrently contributes cells to the ventricular wall, indicating an active homeostatic process. Inhibition of signaling by Fibroblast growth factors (Fgfs) decreased this epicardial supplementation of the ventricular wall in growing zebrafish, and led to spontaneous ventricular scarring in animals maintaining cardiac size. Our results demonstrate that the adult zebrafish ventricle grows and is maintained by cardiomyocyte hyperplasia, and that epicardial cells are added to the ventricle in an Fgf-dependent fashion to support homeostasis.     

Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues. Tucker and Ustyugov contributed equally to this work.     

Biological approaches to improve skeletal muscle healing after injury and disease

Skeletal muscle injury and repair are complex processes, including well‐coordinated steps of degeneration, inflammation, regeneration, and fibrosis. We have reviewed the recent literature including studies by our group that describe how to modulate the processes of skeletal muscle repair and regeneration. Antiinflammatory drugs that target cyclooxygenase‐2 were found to hamper the skeletal muscle repair process. Muscle regeneration phase can be aided by growth factors, including insulin‐like growth factor‐1 and nerve growth factor, but these factors are typically short‐lived, and thus more effective methods of delivery are needed. Skeletal muscle damage caused by traumatic injury or genetic diseases can benefit from cell therapy; however, the majority of transplanted muscle cells (myoblasts) are unable to survive the immune response and hypoxic conditions. Our group has isolated neonatal skeletal muscle derived stem cells (MDSCs) that appear to repair muscle tissue in a more effective manner than myoblasts, most likely due to their better resistance to oxidative stress. Enhancing antioxidant levels of MDSCs led to improved regenerative potential. It is becoming increasingly clear that stem cells tissue repair by direct differentiation and paracrine effects leading to neovascularization of injured site and chemoattraction of host cells. The factors invoked in paracrine action are still under investigation. Our group has found that angiotensin II receptor blocker (losartan) significantly reduces fibrotic tissue formation and improves repair of murine injured muscle. Based on these data, we have conducted a case study on two hamstring injury patients and found that losartan treatment was well tolerated and possibly improved recovery time. We believe this medication holds great promise to optimize muscle repair in humans. (Part C) 96:82–94, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Effect of therapeutic pulsed ultrasound on parameters of oxidative stress in skeletal muscle after injury

Contusion injuries are a very common form of both athletic and non-athletic injury, that effect muscle function. Treatments to augment the normal repair and regeneration processes are important for a wide variety of patients. Therapeutic ultrasound has been claimed to promote tissue repair, especially by enhancing cell proliferation and protein synthesis. The present study aimed to investigate the effect of therapeutic pulsed ultrasound (TPU) on parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBARS), protein carbonyl content and the activities of antioxidant enzymes, catalase and superoxide dismutase (SOD), in skeletal muscle after injury. Wistar rats were submitted to an animal model of muscle (gastrocnemius) laceration. TPU was used once a day. One, three or five days after muscle laceration, the animals were killed by decapitation and oxidative stress parameters were evaluated. Serum CK levels were increased in muscle-injured animals, indicating that the laceration animal model was successful. TBARS were not altered after muscle injury, when compared to the sham group. Protein carbonyl content was increased after muscle laceration. Catalase and SOD activities were increased 1 day after muscle injury and not altered at days 3 and 5. TPU decreased TBARS levels after muscle laceration when compared to injured muscle animals without treatment. Protein carbonyl content evaluation presented similar results. It is tempting to speculate that TPU seems to protect the tissue from oxidative injury. TPU diminished catalase and SOD activities, especially on the first day following muscle laceration.     

Regulation of zebrafish heart regeneration by miR-133

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation.     

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.     

Regeneration of amputated zebrafish fin rays from de novo osteoblasts

Determining the cellular source of new skeletal elements is critical for understanding appendage regeneration in amphibians and fish. Recent lineage-tracing studies indicated that zebrafish fin ray bone regenerates through the dedifferentiation and proliferation of spared osteoblasts, with limited if any contribution from other cell types. Here, we examined the requirement for this mechanism by using genetic ablation techniques to destroy virtually all skeletal osteoblasts in adult zebrafish fins. Animals survived this injury and restored the osteoblast population within 2 weeks. Moreover, amputated fins depleted of osteoblasts regenerated new fin ray structures at rates indistinguishable from fins possessing a resident osteoblast population. Inducible genetic fate mapping confirmed that new bone cells do not arise from dedifferentiated osteoblasts under these conditions. Our findings demonstrate diversity in the cellular origins of appendage bone and reveal that de novo osteoblasts can fully support the regeneration of amputated zebrafish fins.