Chris Jopling

Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation

Although mammalian hearts show almost no ability to regenerate, there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response. In contrast to mammals, several non-mammalian vertebrate species are able to regenerate their hearts, including the zebrafish, which can fully regenerate its heart after amputation of up to 20% of the ventricle. To address directly the source of newly formed cardiomyocytes during zebrafish heart regeneration, we first established a genetic strategy to trace the lineage of cardiomyocytes in the adult fish, on the basis of the Cre/lox system widely used in the mouse. Here we use this system to show that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes. Furthermore, we show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure, detachment from one another and the expression of regulators of cell-cycle progression. Specifically, we show that the gene product of polo-like kinase 1 (plk1) is an essential component of cardiomyocyte proliferation during heart regeneration. Our data provide the first direct evidence for the source of proliferating cardiomyocytes during zebrafish heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process.

Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration

The ultimate goal of regenerative medicine is to replace lost or damaged cells. This can potentially be accomplished using the processes of dedifferentiation, transdifferentiation or reprogramming. Recent advances have shown that the addition of a group of genes can not only restore pluripotency in a fully differentiated cell state (reprogramming) but can also induce the cell to proliferate (dedifferentiation) or even switch to another cell type (transdifferentiation). Current research aims to understand how these processes work and to eventually harness them for use in regenerative medicine.

Hypoxia Induces Myocardial Regeneration in Zebrafish

Background— Hypoxia plays an important role in many biological/pathological processes. In particular, hypoxia is associated with cardiac ischemia. which, although initially inducing a protective response, will ultimately lead to the death of cardiomyocytes and loss of tissue, severely affecting cardiac functionality. Although myocardial damage/loss remains an insurmountable problem for adult mammals, the same is not true for adult zebrafish, which are able to completely regenerate their heart after extensive injury. Myocardial regeneration in zebrafish involves the dedifferentiation and proliferation of cardiomyocytes to replace the damaged/missing tissue; at present, however, little is known about what factors regulate this process. Methods and Results— We surmised that ventricular amputation would lead to hypoxia induction in the myocardium of zebrafish and that this may play a role in regulating the regeneration of the missing cardiac tissue. Using a combination of O2 perturbation, conditional transgenics, in vitro cell culture, and microarray analysis, we found that hypoxia induces cardiomyocytes to dedifferentiate and proliferate during heart regeneration in zebrafish and have identified a number of genes that could play a role in this process. Conclusion— These results indicate that hypoxia plays a positive role during heart regeneration, which should be taken into account in future strategies aimed at inducing heart regeneration in humans.

Piezo1 plays a role in erythrocyte volume homeostasis

Mechanosensitivity is an inherent property of virtually all cell types, allowing them to sense and respond to physical environmental stimuli. Stretch-activated ion channels represent a class of mechanosensitive proteins which allow cells to respond rapidly to changes in membrane tension; however their identity has remained elusive. The piezo genes have recently been identified as a family of stretch-activated mechanosensitive ion channels. We set out to determine the role of piezo1 during zebrafish development. Here we report that morpholino-mediated knockdown of piezo1 impairs erythrocyte survival without affecting hematopoiesis or differentiation. Our results demonstrate that piezo1 is involved in erythrocyte volume homeostasis, disruption of which results in swelling/lysis of red blood cells and consequent anemia.

Transcriptomics approach to investigate zebrafish heart regeneration

In mammals, after a myocardial infarction episode, the damaged myocardium is replaced by scar tissue with negligible cardiomyocyte proliferation. Zebrafish, in contrast, display an extensive regenerative capacity, as they are able to restore completely lost cardiac tissue after partial ventricular amputation. Although questions about the early signals that drive the regenerative response and the relative role of each cardiac cell type in this process still need to be answered, the zebrafish is emerging as a very valuable tool to understand heart regeneration and to devise strategies that may be of potential value to treat human cardiac disease. Here, we performed a genome-wide transcriptome profile analysis focusing on the early time points of zebrafish heart regeneration and compared our results with those of previously published data. Our analyses confirmed the differential expression of several transcripts and identified additional genes whose expression is differentially regulated during zebrafish heart regeneration. We validated the microarray data by conventional and/or quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). For a subset of these genes, their expression pattern was analyzed by in-situ hybridization and shown to be upregulated in the regenerating area of the heart. Our results offer new insights into the biology of heart regeneration in the zebrafish and, together with future experiments in mammals, may be of potential interest for clinical applications.

piezo2b Regulates Vertebrate Light Touch Response

The sense of touch allows an organism to detect and respond to physical environmental stimuli. Mechanosensitive proteins play a crucial role in this process by converting the mechanical cue into a biological response. Recently, the Piezo family of stretch-activated ion channels has been identified as genuine mechanosensitive proteins. We set out to determine whether any of these genes are involved in touch response during zebrafish development. In situ hybridization indicates that piezo2b is specifically expressed in a subset of neurons (Rohon–Beard cells) responsible for detecting light touch. Using morpholino-mediated knockdown, we specifically targeted piezo2b and determined that it is involved in mediating touch-evoked response.

Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts

This protocol describes how to isolate primary cardiomyocytes from adult zebrafish hearts and culture them for up to 4 weeks, thereby using them as an alternative to in vivo experiments. After collagenase digestion of the ventricle, cells are exposed to increasing calcium concentrations in order to obtain high-purity cardiomyocytes. The whole isolation process can be accomplished in 4–5 h. The culture conditions we established allow the cells to preserve their mature sarcomeric integrity and contractile properties. Furthermore, adult zebrafish cardiomyocytes in culture, similarly to zebrafish in vivo heart regeneration, undergo partial dedifferentiation and, in contrast to their mammalian counterparts, are able to proliferate. Our protocol enables the study of structural and functional properties in close-to-native cardiomyocytes and allows the application of in vitro techniques and assays that are not feasible to perform in living animals.

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.

Zebrafish as a model of cardiac disease.

The zebrafish has been rapidly adopted as a model for cardiac development and disease. The transparency of the embryo, its limited requirement for active oxygen delivery, and ease of use in genetic manipulations and chemical exposure have made it a powerful alternative to rodents. Novel technologies like TALEN/CRISPR-mediated genome engineering and advanced imaging methods will only accelerate its use. Here, we give an overview of heart development and function in the fish and highlight a number of areas where it is most actively contributing to the understanding of cardiac development and disease. We also review the current state of research on a feature that we only could wish to be conserved between fish and human; cardiac regeneration.

Illuminating Phenylazopyridines To Photoswitch Metabotropic Glutamate Receptors: From the Flask to the Animals

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity.