Physiological Responses to Swimming-Induced Exercise in the Adult Zebrafish Regenerating Heart.
ABSTRACT: Exercise promotes a set of physiological responses known to provide long-term health benefits and it can play an important role in cardioprotection. In the present study, we examined cardiac responses to exercise training in the adult zebrafish and in the context of cardiac regeneration. We found that swimming-induced exercise increased cardiomyocyte proliferation and that this response was also found under regenerating conditions, when exercise was performed either prior to and after ventricular cryoinjury (CI). Exercise prior to CI resulted in a mild improvement in cardiac function and lesion recovery over the non-exercise condition. Transcriptomic profiling of regenerating ventricles in cryoinjured fish subjected to exercise identified genes possibly involved in the cardioprotective effects of exercise and that could represent potential targets for heart regeneration strategies. Taken together, our results suggest that exercise constitutes a physiological stimulus that may help promote cardiomyogenic mechanisms of the vertebrate heart through the induction of cardiomyocyte proliferation. The zebrafish exercise model may be useful for investigating the potential cardioprotective effects of exercise in teleost fish and to contribute to further identify and develop novel avenues in basic research to promote heart regeneration.
Project description:Exercise promotes a set of physiological adaptations known to provide long-term health benefits and it can play an important role in cardioprotection. However, the mechanisms underlying exercise-induced cardiac adaptation are not fully understood. In the present study, we examined cardiac adaptive responses to exercise training in the adult zebrafish and in the context of cardiac regeneration.We found that swimming-induced exercise increased cardiomyocyte proliferation and that this response was also found under regenerating conditions, when exercise was performed either prior to and after ventricular cryoinjury (CI). Exercise prior to CI resulted in a mild improvement in cardiac function and lesion recovery over the non-exercise condition.Transcriptomic profiling of regenerating ventricles in cryoinjured fish subjected to exercise revealed a differential regulation of genes related toregeneration as well as other biological processes potentially involved in the exercise response. Taken together, these results suggest that exercise constitutes a physiological stimulus thatmay help promote cardiomiogenic mechanisms of the vertebrate heart and that the zebrafish exercise model may be useful for evaluating the potential cardioprotective effects of exercise. Overall design: mRNA profiles of adult zebrafish regenerating ventricles at 7 and 14 days post injury that were exercising or not, were compared.
Project description:Unlike mammals, adult zebrafish can regenerate their hearts after injury via proliferation of cardiomyocytes. The cell-cycle entry of zebrafish cardiac cells can also be stimulated through preconditioning by thoracotomy, a chest incision without myocardial damage. To identify effector genes of heart preconditioning, we performed transcriptome analysis of ventricles from thoracotomized zebrafish. This intervention led to enrichment of cardioprotective factors, epithelial-to-mesenchymal transition genes, matrix proteins and components of LIFR/gp130 signaling. We identified that inhibition of the downstream signal transducer of the LIFR/gp130 pathway through treatment with Ruxolitinib, a specific JAK1/2 antagonist, suppressed the cellular effects of preconditioning. Activation of LIFR/gp130 signaling by a single injection of the ligand Cilliary Neurotrophic Factor, CNTF, was sufficient to trigger cardiomyocyte proliferation in the intact heart. In addition, CNTF induced other pro-regenerative processes, including expression of cardioprotective genes, activation of the epicardium, enhanced intramyocardial Collagen XII deposition and leucocyte recruitment. These effects were abrogated by the concomitant inhibition of the JAK/STAT activity. Mutation of the cntf gene suppressed the proliferative response of cardiomyocytes after thoracotomy. In the regenerating zebrafish heart, CNTF injection prior to ventricular cryoinjury improved the initiation of regeneration via reduced cell apoptosis and boosted cardiomyocyte proliferation. Our findings reveal the molecular effectors of preconditioning and demonstrate that exogenous CNTF exerts beneficial regenerative effects by rendering the heart more resilient to injury and efficient in activation of the proliferative programs.
Project description:<h4>Rationale</h4>The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur.<h4>Objective</h4>To identify transcription factor regulators of the chromatin landscape, which promote cardiomyocyte regeneration in zebrafish, and investigate their function.<h4>Methods and results</h4>Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that activator protein-1 (AP-1) binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the <i>fbxl22</i> and <i>ilk</i> loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the <i>AP-1</i> family members <i>Junb</i> and <i>Fosl1</i> can promote changes in mammalian cardiomyocyte behavior in vitro.<h4>Conclusions</h4>AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.
Project description:<b><i>Aims:</i></b> Nitrite is reduced to nitric oxide (NO) under physiological and pathological hypoxic conditions to modulate angiogenesis and improve ischemia<b>-</b>reperfusion injury. Although adult mammals lack the ability to regenerate the heart after injury, this is preserved in neonates and efforts to reactivate this process are of great interest. Unlike mammals, the adult zebrafish maintain the innate ability to regenerate their hearts after injury, providing an important model to study cardiac regeneration. We thus explored the effects of physiological levels of nitrite on cardiac and fin regeneration and downstream cellular and molecular signaling pathways in response to amputation and cryoinjury. <b><i>Results:</i></b> Nitrite treatment of zebrafish after ventricular amputation or cryoinjury to the heart in hypoxic water (?3 parts per million of oxygen) increases cardiomyocyte proliferation, improves angiogenesis, and enhances early recruitment of thrombocytes, macrophages, and neutrophils to the injury. When tested in a fin regeneration model, neutrophil recruitment to the injury site was found to be dependent on NO. <b><i>Innovation:</i></b> This is the first study to evaluate effects of physiological levels of nitrite on cardiac regeneration in response to cardiac injury, with the observation that nitrite in water accelerates zebrafish heart regeneration. <b><i>Conclusion:</i></b> Physiological and therapeutic levels of nitrite increase thrombocyte, neutrophil, and macrophage recruitment to the heart after amputation and cryoinjury in zebrafish, resulting in accelerated cardiomyocyte proliferation and angiogenesis. Translation of this finding to mammalian models of injury during early development may provide an opportunity to improve outcomes during intrauterine fetal or neonatal cardiac surgery.
Project description:During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from preexisting ones. It is unclear whether the heart is composed of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identify a subset of preexistent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded after cryoinjury. Genetic ablation of sox10+ cardiomyocytes impairs cardiac regeneration, revealing that these cells play a role in heart regeneration.
Project description:After myocardial infarction in humans, lost cardiomyocytes are replaced by an irreversible fibrotic scar. In contrast, zebrafish hearts efficiently regenerate after injury. Complete regeneration of the zebrafish heart is driven by the strong proliferation response of its cardiomyocytes to injury. Here we show that, after cardiac injury in zebrafish, telomerase becomes hyperactivated, and telomeres elongate transiently, preceding a peak of cardiomyocyte proliferation and full organ recovery. Using a telomerase-mutant zebrafish model, we found that telomerase loss drastically decreases cardiomyocyte proliferation and fibrotic tissue regression after cryoinjury and that cardiac function does not recover. The impaired cardiomyocyte proliferation response is accompanied by the absence of cardiomyocytes with long telomeres and an increased proportion of cardiomyocytes showing DNA damage and senescence characteristics. These findings demonstrate the importance of telomerase function in heart regeneration and highlight the potential of telomerase therapy as a means of stimulating cell proliferation upon myocardial infarction.
Project description:The adult heart is able to activate cardioprotective programmes and modifies its architecture in response to physiological or pathological changes. While mammalian cardiac remodelling often involves hypertrophic expansion, the adult zebrafish heart exploits hyperplastic growth. This capacity depends on the responsiveness of zebrafish cardiomyocytes to mitogenic signals throughout their entire life. Here, we have examined the role of inflammation on the stimulation of cell cycle activity in the context of heart preconditioning and regeneration. We used thoracotomy as a cardiac preconditioning model and cryoinjury as a model of cardiac infarction in the adult zebrafish. First, we performed a spatio-temporal characterization of leucocytes and cycling cardiac cells after thoracotomy. This analysis revealed a concomitance between the infiltration of inflammatory cells and the stimulation of the mitotic activity. However, decreasing the immune response using clodronate liposome injection, PLX3397 treatment or anti-inflammatory drugs surprisingly had no effect on the re-entry of cardiac cells into the cell cycle. In contrast, reducing inflammation using the same strategies after cryoinjury strongly impaired cardiac cell mitotic activity and the regenerative process. Taken together, our results show that, while the immune response is not necessary to induce cell-cycle activity in intact preconditioned hearts, inflammation is required for the regeneration of injured hearts in zebrafish.
Project description:Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.
Project description:Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.
Project description:Heart regeneration offers a novel therapeutic strategy for heart failure. Unlike mammals, lower vertebrates such as zebrafish mount a strong regenerative response following cardiac injury. Heart regeneration in zebrafish occurs by cardiomyocyte proliferation and reactivation of a cardiac developmental program, as evidenced by induction of gata4 regulatory sequences in regenerating cardiomyocytes. Although many of the cellular determinants of heart regeneration have been elucidated, how injury triggers a regenerative program through dedifferentiation and epicardial activation is a critical outstanding question. Here, we show that NF-κB signaling is induced in cardiomyocytes following injury. Myocardial inhibition of NF-κB activity blocks heart regeneration with pleiotropic effects, decreasing both cardiomyocyte proliferation and epicardial responses. Activation of gata4 regulatory sequences is also prevented by NF-κB signaling antagonism, suggesting an underlying defect in cardiomyocyte dedifferentiation. Our results implicate NF-κB signaling as a key node between cardiac injury and tissue regeneration.