Ina M. Berger

Site-specific methylation of Notch1 controls the amplitude and duration of the Notch1 response

Methylation of the active Notch cleavage product promotes its degradation to produce short periods of Notch signaling necessary for proper development. Turned on by cleavage; turned off by methylation and ubiquitination Notch signaling regulates several processes during development, and aberrant signaling contributes to human disease. The Notch receptor is proteolytically processed, releasing an intracellular fragment called NICD that translocates to the nucleus to regulate gene expression. Hein et al. found that NICD is methylated by the methyltransferase CARM1 at five conserved arginine residues within a domain required for gene regulatory activity. Methylated NICD was targeted for degradation. A mutant form of NICD that could not be methylated was more stable but biologically less active when expressed in frog or zebrafish embryos. Thus, control of Notch signaling involves cleavage to produce the transcriptional regulator and then methylation to target this irreversibly activated product for degradation. Physiologically, Notch signal transduction plays a pivotal role in differentiation; pathologically, Notch signaling contributes to the development of cancer. Transcriptional activation of Notch target genes involves cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), and NICD migration into the nucleus and assembly of a coactivator complex. Posttranslational modifications of the NICD are important for its transcriptional activity and protein turnover. Deregulation of Notch signaling and stabilizing mutations of Notch1 have been linked to leukemia development. We found that the methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1; also known as PRMT4) methylated NICD at five conserved arginine residues within the C-terminal transactivation domain. CARM1 physically and functionally interacted with the NICD-coactivator complex and was found at gene enhancers in a Notch-dependent manner. Although a methylation-defective NICD mutant was biochemically more stable, this mutant was biologically less active as measured with Notch assays in embryos of Xenopus laevis and Danio rerio. Mathematical modeling indicated that full but short and transient Notch signaling required methylation of NICD.

In-vivo characterization of human dilated cardiomyopathy genes in zebrafish

Due to lack of families suitable for linkage analysis and positional cloning most of the genetic causes of human dilated cardiomyopathy (DCM) are still unknown. To facilitate rapid identification and validation of novel DCM disease genes appropriate animal models are needed. To assess here for the first time whether the zebrafish is a suitable model organism to validate DCM candidate genes using antisense knock-down strategies, we inactivated in zebrafish known human DCM disease genes and then evaluated the resulting cardiac phenotypes. Consistently, knock-down of the here selected human DCM genes leads to severe heart failure with impairment of systolic cardiac function in zebrafish. Furthermore, gene-specific differences which are also seen in human DCM can be reliably reproduced in the zebrafish model. Our results indicate that the zebrafish is a suitable model organism to rapidly evaluate novel DCM disease genes in-vivo.

Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia

The heart’s rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2−/− hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2−/− hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.

Protein Kinase D2 Controls Cardiac Valve Formation in Zebrafish by Regulating Histone Deacetylase 5 Activity

Background— The molecular mechanisms that guide heart valve formation are not well understood. However, elucidation of the genetic basis of congenital heart disease is one of the prerequisites for the development of tissue-engineered heart valves. Methods and Results— We isolated here a mutation in zebrafish, bungee (bngjh177), which selectively perturbs valve formation in the embryonic heart by abrogating endocardial Notch signaling in cardiac cushions. We found by positional cloning that the bng phenotype is caused by a missense mutation (Y849N) in zebrafish protein kinase D2 (pkd2). The bng mutation selectively impairs PKD2 kinase activity and hence Histone deacetylase 5 phosphorylation, nuclear export, and inactivation. As a result, the expression of Histone deacetylase 5 target genes Krüppel-like factor 2a and 4a, transcription factors known to be pivotal for heart valve formation and to act upstream of Notch signaling, is severely downregulated in bungee (bng) mutant embryos. Accordingly, the expression of Notch target genes, such as Hey1, Hey2, and HeyL, is severely decreased in bng mutant embryos. Remarkably, downregulation of Histone deacetylase 5 activity in homozygous bng mutant embryos can rescue the mutant phenotype and reconstitutes notch1b expression in atrioventricular endocardial cells. Conclusions— We demonstrate for the first time that proper heart valve formation critically depends on Protein kinase D2-Histone deacetylase 5-Krüppel-like factor signaling.

F-Box and Leucine-Rich Repeat Protein 22 Is a Cardiac-Enriched F-Box Protein That Regulates Sarcomeric Protein Turnover and Is Essential for Maintenance of Contractile Function In Vivo

Rationale: The emerging role of the ubiquitin–proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. However, little is known about cardiac components of this machinery. Objective: We sought to determine whether molecules exist that control turnover of cardiac-specific proteins. Methods and Results: Using a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins, we identified F-box and leucine-rich repeat protein 22 (Fbxl22). Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as quantitative reverse-transcriptase polymerase chain reaction on a human cDNA library. Immunocolocalization experiments in neonatal and adult rat ventricular cardiomyocytes as well as murine heart tissue located Fbxl22 to the sarcomeric z-disc. To detect cardiac protein interaction partners, we performed a yeast 2-hybrid screen using Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with S-phase kinase-associated protein 1 and Cullin1, 2 critical components of SCF (Skp1/Cul1/F-box) E3- ligases. Moreover, we identified several potential substrates, including the z-disc proteins &agr;-actinin and filamin C. Consistently, in vitro overexpression of Fbxl22-mediated degradation of both substrates in a dose-dependent fashion, whereas proteasome inhibition with MG-132 markedly attenuated degradation of both &agr;-actinin and filamin C. Finally, targeted knockdown of Fbxl22 in rat cardiomyocytes as well as zebrafish embryos results in the accumulation of &agr;-actinin associated with severely impaired contractile function and cardiomyopathy in vivo. Conclusions: These findings reveal the previously uncharacterized cardiac-specific F-box protein Fbxl22 as a component of a novel cardiac E3 ligase. Fbxl22 promotes the proteasome-dependent degradation of key sarcomeric proteins, such as &agr;-actinin and filamin C, and is essential for maintenance of normal contractile function in vivo.

Fbxl22, A Cardiac-Enriched F-Box Protein, Regulates Sarcomeric Protein Turnover and is Essential for Maintenance of Contractile Function In Vivo

Rationale: The emerging role of the ubiquitin–proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. However, little is known about cardiac components of this machinery. Objective: We sought to determine whether molecules exist that control turnover of cardiac-specific proteins. Methods and Results: Using a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins, we identified F-box and leucine-rich repeat protein 22 (Fbxl22). Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as quantitative reverse-transcriptase polymerase chain reaction on a human cDNA library. Immunocolocalization experiments in neonatal and adult rat ventricular cardiomyocytes as well as murine heart tissue located Fbxl22 to the sarcomeric z-disc. To detect cardiac protein interaction partners, we performed a yeast 2-hybrid screen using Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with S-phase kinase-associated protein 1 and Cullin1, 2 critical components of SCF (Skp1/Cul1/F-box) E3- ligases. Moreover, we identified several potential substrates, including the z-disc proteins &agr;-actinin and filamin C. Consistently, in vitro overexpression of Fbxl22-mediated degradation of both substrates in a dose-dependent fashion, whereas proteasome inhibition with MG-132 markedly attenuated degradation of both &agr;-actinin and filamin C. Finally, targeted knockdown of Fbxl22 in rat cardiomyocytes as well as zebrafish embryos results in the accumulation of &agr;-actinin associated with severely impaired contractile function and cardiomyopathy in vivo. Conclusions: These findings reveal the previously uncharacterized cardiac-specific F-box protein Fbxl22 as a component of a novel cardiac E3 ligase. Fbxl22 promotes the proteasome-dependent degradation of key sarcomeric proteins, such as &agr;-actinin and filamin C, and is essential for maintenance of normal contractile function in vivo.

Reconstitution of defective protein trafficking rescues Long-QT syndrome in zebrafish

Inherited cardiac arrhythmias are caused by genetic defects in ion channels and associated proteins. Mutations in these channels often do not affect their biophysical properties, but rather interfere with their trafficking to the cell membrane. Accordingly, strategies that could reroute the mutated channels to the membrane should be sufficient to restore the electrical properties of the affected cells, thereby suppressing the underlying arrhythmia. We identified here both, embryonic and adult zebrafish breakdance (bre) as a valuable model for human Long-QT syndrome. Electrocardiograms of adult homozygous bre mutants exhibit significant QT prolongation caused by delayed repolarization of the ventricle. We further show that the bre mutation (zERG(I59S)) disrupts ERG protein trafficking, thereby reducing the amount of active potassium channels on the cell membrane. Interestingly, improvement of channel trafficking by cisapride or dimethylsulfoxid is sufficient to reconstitute ERG channels on the cell membrane in a manner that suffices to suppress the Long-QT induced arrhythmia in breakdance mutant zebrafish. In summary, we show for the first time that therapeutic intervention can cure protein trafficking defects and the associated cardiac arrhythmia in vivo.

Overlapping and Opposing Functions of G Protein-coupled Receptor Kinase 2 (GRK2) and GRK5 during Heart Development

Background: GRK2 and GRK5 differentially control function and morphology of the adult heart. Results: We found that GRK2 and GRK5 distinctly govern myocardial development and function. Conclusion: GRK2 and GRK5 function during heart development. Significance: We found a differential impact of GRKs on embryonic development and adult physiology of the heart. G protein-coupled receptor kinases 2 (GRK2) and 5 (GRK5) are fundamental regulators of cardiac performance in adults but are less well characterized for their function in the hearts of embryos. GRK2 and -5 belong to different subfamilies and function as competitors in the control of certain receptors and signaling pathways. In this study, we used zebrafish to investigate whether the fish homologs of GRK2 and -5, Grk2/3 and Grk5, also have unique, complementary, or competitive roles during heart development. We found that they differentially regulate the heart rate of early embryos and equally facilitate heart function in older embryos and that both are required to develop proper cardiac morphology. A loss of Grk2/3 results in dilated atria and hypoplastic ventricles, and the hearts of embryos depleted in Grk5 present with a generalized atrophy. This Grk5 morphant phenotype was associated with an overall decrease of early cardiac progenitors as well as a reduction in the area occupied by myocardial progenitor cells. In the case of Grk2/3, the progenitor decrease was confined to a subset of precursor cells with a committed ventricular fate. We attempted to rescue the GRK loss-of-function heart phenotypes by downstream activation of Hedgehog signaling. The Grk2/3 loss-of-function embryos were rescued by this approach, but Grk5 embryos failed to respond. In summary, we found that GRK2 and GRK5 control cardiac function as well as morphogenesis during development although with different morphological outcomes.

Loss of dihydrolipoyl succinyltransferase (DLST) leads to reduced resting heart rate in the zebrafish

The genetic underpinnings of heart rate regulation are only poorly understood. In search for genetic regulators of cardiac pacemaker activity, we isolated in a large-scale mutagenesis screen the embryonic lethal, recessive zebrafish mutant schneckentempo (ste). Homozygous ste mutants exhibit a severely reduced resting heart rate with normal atrio-ventricular conduction and contractile function. External electrical pacing reveals that defective excitation generation in cardiac pacemaker cells underlies bradycardia in ste−/− mutants. By positional cloning and gene knock-down analysis we find that loss of dihydrolipoyl succinyltransferase (DLST) function causes the ste phenotype. The mitochondrial enzyme DLST is an essential player in the citric acid cycle that warrants proper adenosine-tri-phosphate (ATP) production. Accordingly, ATP levels are significantly diminished in ste−/− mutant embryos, suggesting that limited energy supply accounts for reduced cardiac pacemaker activity in ste−/− mutants. We demonstrate here for the first time that the mitochondrial enzyme DLST plays an essential role in the modulation of the vertebrate heart rate by controlling ATP production in the heart.

Tbx20 Is an Essential Regulator of Embryonic Heart Growth in Zebrafish.

The molecular mechanisms that regulate cardiomyocyte proliferation during embryonic heart growth are not completely deciphered yet. In a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified the recessive embryonic-lethal zebrafish mutant line weiches herz (whz). Homozygous mutant whz embryos display impaired heart growth due to diminished embryonic cardiomyocyte proliferation resulting in cardiac hypoplasia and weak cardiac contraction. By positional cloning, we found in whz mutant zebrafish a missense mutation within the T-box 20 (Tbx20) transcription factor gene leading to destabilization of Tbx20 protein. Morpholino-mediated knock-down of Tbx20 in wild-type zebrafish embryos phenocopies whz, indicating that the whz phenotype is due to loss of Tbx20 function, thereby leading to significantly reduced cardiomyocyte numbers by impaired proliferation of heart muscle cells. Ectopic overexpression of wild-type Tbx20 in whz mutant embryos restored cardiomyocyte proliferation and heart growth. Interestingly, ectopic overexpression of Tbx20 in wild-type zebrafish embryos resulted, similar to the situation in the embryonic mouse heart, in significantly reduced proliferation rates of ventricular cardiomyocytes, suggesting that Tbx20 activity needs to be tightly fine-tuned to guarantee regular cardiomyocyte proliferation and embryonic heart growth in vivo.