Distinct effects of inflammation on preconditioning and regeneration of the adult zebrafish heart

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

Preconditioning boosts regenerative programmes in the adult zebrafish heart

During preconditioning, exposure to a non-lethal harmful stimulus triggers a body-wide increase of survival and pro-regenerative programmes that enable the organism to better withstand the deleterious effects of subsequent injuries. This phenomenon has first been described in the mammalian heart, where it leads to a reduction of infarct size and limits the dysfunction of the injured organ. Despite its important clinical outcome, the actual mechanisms underlying preconditioning-induced cardioprotection remain unclear. Here, we describe two independent models of cardiac preconditioning in the adult zebrafish. As noxious stimuli, we used either a thoracotomy procedure or an induction of sterile inflammation by intraperitoneal injection of immunogenic particles. Similar to mammalian preconditioning, the zebrafish heart displayed increased expression of cardioprotective genes in response to these stimuli. As zebrafish cardiomyocytes have an endogenous proliferative capacity, preconditioning further elevated the re-entry into the cell cycle in the intact heart. This enhanced cycling activity led to a long-term modification of the myocardium architecture. Importantly, the protected phenotype brought beneficial effects for heart regeneration within one week after cryoinjury, such as a more effective cell-cycle reentry, enhanced reactivation of embryonic gene expression at the injury border, and improved cell survival shortly after injury. This study reveals that exposure to antecedent stimuli induces adaptive responses that render the fish more efficient in the activation of the regenerative programmes following heart damage. Our results open a new field of research by providing the adult zebrafish as a model system to study remote cardiac preconditioning

Ciliary neurotrophic factor stimulates cardioprotection and the proliferative activity in the adult zebrafish heart

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

A dual epimorphic and compensatory mode of heart regeneration in zebrafish

Zebrafish heart regeneration relies on the capacity of cardiomyocytes to proliferate upon injury. To understand the principles of this process after cryoinjury-induced myocardial infarction, we established a spatio-temporal map of mitotic cardiomyocytes and their differentiation dynamics. Immunodetection of phosphohistone H3 and embryonic ventricular heavy chain myosin highlighted two distinct regenerative processes during the early phase of regeneration. The injury-abutting zone comprises a population of cardiac cells that reactivates the expression of embryo-specific sarcomeric proteins and it displays a 10-fold higher mitotic activity in comparison to the injury-remote zone. The undifferentiated cardiomyocytes resemble a blastema-like structure between the original and wound tissues. They integrate with the fibrotic tissue through the fibronectin-tenascin C extracellular matrix, and with the mature cardiomyocytes through upregulation of the tight junction marker, connexin 43. During the advanced regenerative phase, the population of undifferentiated cardiomyocytes disperses within the regenerating myocardium and it is not detected after the termination of regeneration. Although the blastema represents a transient landmark of the regenerating ventricle, the remaining mature myocardium also displays an enhanced mitotic index when compared to uninjured hearts. This suggests an unexpected contribution of a global proliferative activity to restore the impaired cardiac function. Based on these findings, we propose a new model of zebrafish heart regeneration that involves a combination of blastema-dependent epimorphosis and a compensatory organ-wide response

Global Transcriptional Programs in Peripheral Nerve Endoneurium and DRG Are Resistant to the Onset of Type 1 Diabetic Neuropathy in Ins2Akita/+ Mice

While the morphological and electrophysiological changes underlying diabetic peripheral neuropathy (DPN) are relatively well described, the involved molecular mechanisms remain poorly understood. In this study, we investigated whether phenotypic changes associated with early DPN are correlated with transcriptional alterations in the neuronal (dorsal root ganglia [DRG]) or the glial (endoneurium) compartments of the peripheral nerve. We used Ins2Akita/+ mice to study transcriptional changes underlying the onset of DPN in type 1 diabetes mellitus (DM). Weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Ins2Akita/+ and control mice during the first three months of life in order to determine the onset of DPN. Based on this phenotypic characterization, we performed gene expression profiling using sciatic nerve endoneurium and DRG isolated from pre-symptomatic and early symptomatic Ins2Akita/+ mice and sex-matched littermate controls. Our phenotypic analysis of Ins2Akita/+ mice revealed that DPN, as measured by reduced MNCV, is detectable in affected animals already one week after the onset of hyperglycemia. Surprisingly, the onset of DPN was not associated with any major persistent changes in gene expression profiles in either sciatic nerve endoneurium or DRG. Our data thus demonstrated that the transcriptional programs in both endoneurial and neuronal compartments of the peripheral nerve are relatively resistant to the onset of hyperglycemia and hypoinsulinemia suggesting that either minor transcriptional alterations or changes on the proteomic level are responsible for the functional deficits associated with the onset of DPN in type 1 DM

Collagen XII contributes to epicardial and connective tissues in the Zebrafish heart during ontogenesis and regeneration

Zebrafish heart regeneration depends on cardiac cell proliferation, epicardium activation and transient reparative tissue deposition. The contribution and the regulation of specific collagen types during the regenerative process, however, remain poorly characterized. Here, we identified that the non-fibrillar type XII collagen, which serves as a matrix-bridging component, is expressed in the epicardium of the zebrafish heart, and is boosted after cryoinjury-induced ventricular damage. During heart regeneration, an intense deposition of Collagen XII covers the outer epicardial cap and the interstitial reparative tissue. Analysis of the activated epicardium and fibroblast markers revealed a heterogeneous cellular origin of Collagen XII. Interestingly, this matrix-bridging collagen co-localized with fibrillar type I collagen and several glycoproteins in the post-injury zone, suggesting its role in tissue cohesion. Using SB431542, a selective inhibitor of the TGF-β receptor, we showed that while the inhibitor treatment did not affect the expression of collagen 12 and collagen 1a2 in the epicardium, it completely suppressed the induction of both genes in the fibrotic tissue. This suggests that distinct mechanisms might regulate collagen expression in the outer heart layer and the inner injury zone. On the basis of this study, we postulate that the TGF-β signaling pathway induces and coordinates formation of a transient collagenous network that comprises fibril-forming Collagen I and fiber-associated Collagen XII, both of which contribute to the reparative matrix of the regenerating zebrafish heart

Collagen XII distribution correlates with the activated epicardium and fibroblast-like cells.

<p>(A-F) Analysis of transversal heart sections at 14 dpci. (A and D) AFOG staining of the sections used for immunostaining. (B and C) Raldh2 expression (red) demarcates the activated epicardium and endocardium. (C’) Col XII (green) and Raldh2 are colocalized in the intact epicardium (epicard). (C”) Raldh2-positive cells invade the post-cryoinjured area that is labeled by Col XII expression. Cardiac muscle is detected by Tropomyosin antibody staining (blue). N = 5. (E and F) Triple antibody staining against Col XII (green), intermediate filament Vimentin (blue) and alpha-Smooth Muscle Actin (αSMA; red). (F’ and F”) αSMA- and Vimentin-positive cells are non-overlapping cell populations in the epicardium and post-cryoinjured area. Both of them are associated with Col XII-labeled fibrils. N = 6.</p

Developmental dynamics of Col XII expression in the zebrafish heart.

<p>(A-D) Longtudinal sections of the zebrafish heart after double immunostaining against Col XII (green) and Tropomyosin (red), with DAPI contrastain (blue). dpf, days post-fertilization. Three chambers of the zebrafish heart: v, ventricle; a atrium; b.a., bulbus arteriosus (non-muscular structure). N ≥ 5. (A) At 3 dpf, embryos express Col XII in the pericardium, but not in the heart. The pericardial fibers seem to invade the surface of the heart. (B) At 14 dpf, the three chambers of the larval heart are surrounded by Col XII-positive fibrils within 10 μm of outer myocardial layer (white bar). (C) At 30 dpf, the juvenile fish heart contains a thickened myocardium (red), but the size of Col XII-positive layer remains unaltered. (D) At 120 days post fertilization, young adult fish maintain Col XII-labeled fibers along the heart circumference in a pattern similar to the one seen at the larval stage.</p

Col XII is expressed in the epicardium of the adult zebrafish heart.

<p>(A) Aniline blue staining of a ventricle transversal section visualizes collagen (blue). Framed areas encompass parts with the atrio-ventricular valve (A/V-Valve) and ventricular wall (V-Wall). The thickness of the compact myocardial layer is depicted as a bar in this and subsequent panels. Ep, epicardium; CoM, compact myocardium; TrM, trabecular myocardium. N = 5. (B-D) <i>In -situ</i> hybridization of ventricle sections detected by a color reaction (purple). Probe names are to the left. Framed areas encompass the parts that are enlarged in the panels to the right. N ≥ 4. (E) Superposition of a bright-field image with <i>in -situ</i> hybridization using <i>col12a1b</i> probe (purple) and fluorescent immunodetection of muscle protein Tropomyosin, TPM (red). <i>col12a1b</i> is expressed in the epicardium that is located externally from the myocardial border (dashed line). A few <i>col12a1b</i>-expressing cells are Tropomyosin-negative (arrows) and are interspersed within the compact myocardium (the thickness of the compact myocardium is indicated with a white bar). N = 4. (F) Immunofluorescence with anti-Tropomyosin (blue) to label cardiomyocytes and anti-Col XII (green) of transgenic fish <i>wt1a(-6</i>.<i>8kb)</i>:<i>GFP</i> (red), which labels cardiac subepicardial fibroblasts (white arrows) located mainly along the junction between the outer compact myocardium (white bar) and inner trabecular myocardium. N = 4. (G, H) Aniline blue, acid Fuchsin, Orange G (AFOG) staining detects collagen (blue) in bulbus arteriosus (G, longitudinal heart section) and the leaflets of the atrioventricular valve (H, transversal heart section). N = 6. (I, J) Triple immunofluorescence staining against Col XII (green), Col Iα (red) and Tropomyosin (blue) of the structures shown in above panels. (I’) Col XII is detected on the myocardial surface. (I”) In the bulbus arteriosus, Col Iα fibers are in the interstitium, while Col XII is restricted to its surface. (J’) The atrio-ventricular connection displays Col Iα, but little Col XII in the valve leaflets. N = 6. (A’, B’, C’, D’, E’, F’) Higher magnifications of the framed areas shown in images that are labeled with the same letter without prime symbol. The same rule applies to all subsequent figures.</p

Schematic representation of distinct effects of TGF-β inhibition on Col XII and Col Iα deposition in the epicardium and the fibrotic tissue during zebrafish heart regeneration.

<p>Illustrations of longitudinal heart sections at 14 dpci in normal conditions (left side) and after inhibition of the TGF-β signaling pathway (right side). The uninjured part of the heart displays the presence of Col XII along the heart surface, while Col Iα is expressed in the bulbus arteriosus and in the atrio-ventricular valves. The injured myocardial wall heals by enhanced Col XII deposition along the outer margin of the wound, forming a Col XII-rich epicardial cap. The inner part of the damaged myocardium is replaced with fibrotic tissue that contains <i>tgf-β</i>-expressing cells. The activity of this pathway stimulates deposition of fibrillar Col Iα and fibril-associated Col XII in the fibrotic tissue, but it is not required for the formation of the epicardial cap. The provisional matrix maintains the organ function during the regenerative process, until its completion at 30 dpci.</p