Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

Technische Universität Dresden

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3 beta under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3 beta diminished hypertrophy in response to chronic beta-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3 beta as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3 beta activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo.

The heart responds to stress signals by hypertrophic growth, which is accompanied by activation of the MEF2 transcription factor and reprogramming of cardiac gene expression. We show here that class II histone deacetylases (HDACs), which repress MEF2 activity, are substrates for a stress-responsive kinase specific for conserved serines that regulate MEF2-HDAC interactions. Signal-resistant HDAC mutants lacking these phosphorylation sites are refractory to hypertrophic signaling and inhibit cardiomyocyte hypertrophy. Conversely, mutant mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals and exhibit stress-dependent cardiomegaly. Thus, class II HDACs act as signal-responsive suppressors of the transcriptional program governing cardiac hypertrophy and heart failure.

Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy.

Postnatal cardiac myocytes respond to stress signals by hypertrophic growth and activation of a fetal gene program. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy, and mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals. To further define the roles of HDACs in cardiac hypertrophy, we analyzed the effects of HDAC inhibitors on the responsiveness of primary cardiomyocytes to hypertrophic agonists. Paradoxically, HDAC inhibitors imposed a dose-dependent blockade to hypertrophy and fetal gene activation. We conclude that distinct HDACs play positive or negative roles in the control of cardiomyocyte hypertrophy. HDAC inhibitors are currently being tested in clinical trials as anti-cancer agents. Our results suggest that these inhibitors may also hold promising clinical value as therapeutics for cardiac hypertrophy and heart failure.

Dose-dependent blockade to cardiomyocyte hypertrophy by histone deacetylase inhibitors.

Although beta-adrenergic receptor (AR) blockade therapy is beneficial in the treatment of heart failure, little is known regarding the transcriptional mechanisms underlying this salutary action.

Common genomic response in different mouse models of beta-adrenergic-induced cardiomyopathy.

The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl--coupled betaine/gamma-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.

Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression.

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3beta (GSK-3beta). Alternatively, receptor tyrosine kinase or certain G protein-coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3beta, via protein kinase B/Akt and mTOR/p70(s6k) pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3beta on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.

Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore.

In response to stress signals, postnatal cardiomyocytes undergo hypertrophic growth accompanied by activation of a fetal gene program, assembly of sarcomeres, and cellular enlargement. We show that hypertrophic signals stimulate the expression and transcriptional activity of myocardin, a cardiac and smooth muscle-specific coactivator of serum response factor (SRF). Consistent with a role for myocardin as a transducer of hypertrophic signals, forced expression of myocardin in cardiomyocytes is sufficient to substitute for hypertrophic signals and induce cardiomyocyte hypertrophy and the fetal cardiac gene program. Conversely, a dominant-negative mutant form of myocardin, which retains the ability to associate with SRF but is defective in transcriptional activation, blocks cardiomyocyte hypertrophy induced by hypertrophic agonists such as phenylephrine and leukemia inhibitory factor. Myocardin-dependent hypertrophy can also be partially repressed by histone deacetylase 5, a transcriptional repressor of myocardin. These findings identify myocardin as a nuclear effector of hypertrophic signaling pathways that couples stress signals to a transcriptional program for postnatal cardiac growth and remodeling.

Myocardin induces cardiomyocyte hypertrophy.

Mutations in myosin heavy chain (MyHC) can cause hypertrophic cardiomyopathy (HCM) that is characterized by hypertrophy, histopathology, contractile dysfunction, and sudden death. The signaling pathways involved in the pathology of HCM have not been elucidated, and an unresolved question is whether blocking hypertrophic growth in HCM may be maladaptive or beneficial. To address these questions, a mouse model of HCM was crossed with an antihypertrophic mouse model of constitutive activated glycogen synthase kinase-3beta (caGSK-3beta). Active GSK-3beta blocked cardiac hypertrophy in both male and female HCM mice. However, doubly transgenic males (HCM/GSK-3beta) demonstrated depressed contractile function, reduced sarcoplasmic (endo) reticulum Ca(2+)-ATPase (SERCA) expression, elevated atrial natriuretic factor (ANF) expression, and premature death. In contrast, female HCM/GSK-3beta double transgenic mice exhibited similar cardiac histology, function, and survival to their female HCM littermates. Remarkably, dietary modification from a soy-based diet to a casein-based diet significantly improved survival in HCM/GSK-3beta males. These findings indicate that activation of GSK-3beta is sufficient to limit cardiac growth in this HCM model and the consequence of caGSK-3beta was sexually dimorphic. Furthermore, these results show that blocking hypertrophy by active GSK-3beta in this HCM model is not therapeutic.

Blocking cardiac growth in hypertrophic cardiomyopathy induces cardiac dysfunction and decreased survival only in males.

Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet (smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.

Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration.

Deux ma&icirc;tres mots de r&eacute;g&eacute;n&eacute;ration &eacute;pimorphose vert&eacute;br&eacute;s sont une augmentation significative de la prolif&eacute;ration des cellules quiescentes normalement et une r&eacute;activation des g&egrave;nes qui sont actifs durant le d&eacute;veloppement embryonnaire. On ignore ce que les d&eacute;terminants mol&eacute;culaires qui r&eacute;gissent ces &eacute;v&eacute;nements sont et comment ils sont coordonn&eacute;s. Poisson z&egrave;bre ont la capacit&eacute; de r&eacute;g&eacute;n&eacute;rer plusieurs structures compos&eacute;s de r&eacute;guler la prolif&eacute;ration cellulaire et la transcription du g&egrave;ne. Les auteurs rapportent que fam53b/simplet (smp) r&eacute;gule la prolif&eacute;ration cellulaire et la transcription de g&egrave;nes sp&eacute;cifiques. Hybridation in situ et des exp&eacute;riences de RT-PCR quantitatives a montr&eacute; qu&#39;amputation des nageoires et des coeurs de poisson-z&egrave;bre a entra&icirc;n&eacute; une forte r&eacute;gulation du g&egrave;ne smp. Dans la r&eacute;g&eacute;n&eacute;ration de nageoire adulte, expression de smp est rest&eacute;e forte dans le m&eacute;senchyme distal qui par la suite &eacute;largi pour les couches basales de l&#39;&eacute;piderme distal et distale tip &eacute;pith&eacute;lium. Morpholino knockdown de smp r&eacute;duit l&#39;excroissance r&eacute;g&eacute;n&eacute;ratrice en diminuant la prolif&eacute;ration cellulaire telle que mesur&eacute;e par la phosphorylation BrdU incorporation et histone H3. En outre, smp knockdown a augment&eacute; l&#39;expression de msxb, msxc et Chut, ainsi que la formation ult&eacute;rieure des os ectopique. Pris ensemble, ces donn&eacute;es indiquent une exigence pour smp dans la r&eacute;g&eacute;n&eacute;ration de la fin gr&acirc;ce au contr&ocirc;le de la prolif&eacute;ration cellulaire, la r&eacute;gulation de g&egrave;nes sp&eacute;cifiques et la structuration de l&#39;OS.

Simplet contr&ocirc;les cellulaires la prolif&eacute;ration et la transcription de g&egrave;nes au cours de la r&eacute;g&eacute;n&eacute;ration caudale chez le poisson z&egrave;bre.

Due caratteristiche dei vertebrati epimorphic rigenerazione sono un significativo aumento della proliferazione delle cellule normalmente quiescente e una riattivazione di geni che sono attivi durante lo sviluppo embrionale. Non &egrave; chiaro quali sono i determinanti molecolari che regolano questi eventi e come essi sono coordinati. Zebrafish hanno la capacit&agrave; di rigenerare le diverse strutture di composte che regolano la proliferazione cellulare e la trascrizione del gene. Segnaliamo che fam53b/simplet (smp) regola la proliferazione cellulare e la trascrizione di geni specifici. Ibridazione in situ e gli esperimenti di RT-PCR quantitativi hanno mostrato che amputazione di zebrafish cuori e pinne ha portato a forti su-regolazione del gene smp. In rigenerante pinna adulti, espressione smp &egrave; rimasto forte nel mesenchima distale che successivamente esteso agli strati basali dell&#39;epidermide distale e distale punta dell&#39;epitelio. Oligonucleotides atterramento di smp ridotto rigenerativa escrescenza diminuendo la proliferazione cellulare come misurato dalla fosforilazione di BrdU incorporazione e istone H3. Inoltre, smp atterramento aumentato l&#39;espressione di msxb, msxc e lo shh, cos&igrave; come la successiva formazione di osso ectopico. Presi insieme, questi dati indicano un requisito per smp in rigenerazione pinna attraverso il controllo della proliferazione cellulare, la regolazione dei geni specifici e patterning osseo adeguato.

Simplet controlli delle cellule di proliferazione e la trascrizione del gene durante la rigenerazione di zebrafish pinna caudale.

There are several animal model organisms that have the ability to regenerate severe injuries by stimulating local cells to restore damaged and lost organs and appendages. In this chapter, we will describe how various vertebrate animals regenerate different structures (central nervous system, heart and appendages) as well as detail specific cellular and molecular features concerning the regeneration of these structures.

Vertebrates that regenerate as models for guiding stem cels.

Vertebrates develop organs and appendages in a proportionally coordinated manner, and animals that regenerate them do so to the same dimensions as the original structures. Coordinated proportional growth involves controlled regulation between allometric and isometric growth programs, but it is unclear what executes this control. We show that calcineurin inhibition results in continued allometric outgrowth of regenerating fins beyond their original dimensions. Calcineurin inhibition also maintains allometric growth of juvenile fins and induces it in adult fins. Furthermore, calcineurin activity is low when the regeneration rate is highest, and its activity increases as the rate decreases. Growth measurements and morphometric analysis of proximodistal asymmetry indicate that calcineurin inhibition shifts fin regeneration from a distal growth program to a proximal program. This shift is associated with the promotion of retinoic acid signaling. Thus, we identified a calcineurin-mediated mechanism that operates as a molecular switch between position-associated isometric and allometric growth programs.

Christopher L. Antos

DFG-Center for Regenerative Therapies Dresden

Technische Universität Dresden

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Christopher L. Antos

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Technische Universität Dresden

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

DFG-Center for Regenerative Therapies Dresden