David Hassel

A dual role for ErbB2 signaling in cardiac trabeculation.

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.

Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy

Z-disks, the mechanical integration sites of heart and skeletal muscle cells, link anchorage of myofilaments to force reception and processing. The key molecules that enable the Z-disk to persistently withstand the extreme mechanical forces during muscle contraction have not yet been identified. Here we isolated nexilin (encoded by NEXN) as a novel Z-disk protein. Loss of nexilin in zebrafish led to perturbed Z-disk stability and heart failure. To evaluate the role of nexilin in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found several mutations in NEXN associated with the disease. Nexilin mutation carriers showed the same cardiac Z-disk pathology as observed in nexilin-deficient zebrafish. Expression in zebrafish of nexilin proteins encoded by NEXN mutant alleles induced Z-disk damage and heart failure, demonstrating a dominant-negative effect and confirming the disease-causing nature of these mutations. Increasing mechanical strain aggravated Z-disk damage in nexilin-deficient skeletal muscle, implying a unique role of nexilin in protecting Z-disks from mechanical trauma.

Cardiac Myosin Light Chain-2. A Novel Essential Component of Thick-Myofilament Assembly and Contractility of the Heart

Although it is well known that mutations in the cardiac regulatory myosin light chain-2 (mlc-2) gene cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of MLC-2 in the heart are only poorly understood. We have isolated a mutation in zebrafish, tell tale heart (telm225), which selectively perturbs contractility of the embryonic heart. By positional cloning, we identified tel to encode the zebrafish mlc-2 gene. In contrast to mammals, zebrafish have only 1 cardiac-specific mlc-2 gene, which we find to be expressed in atrial and ventricular cardiomyocytes during early embryonic development, but also in the adult heart. Accordingly, loss of zMLC-2 function cannot be compensated for by upregulation of another mlc-2 gene. Surprisingly, ultrastructural analysis of tel cardiomyocytes reveals complete absence of organized thick myofilaments. Thus, our findings provide the first in vivo evidence that cardiac MLC-2 is required for thick-filament stabilization and contractility in the vertebrate heart.

Deficient Zebrafish Ether-à-Go-Go-Related Gene Channel Gating Causes Short-QT Syndrome in Zebrafish Reggae Mutants

Background— Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking. Methods and Results— In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go-related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish. Conclusions— With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.

MicroRNA-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting Vascular Endothelial Growth Factor Signaling

Rationale: Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood. Objective: To identify microRNAs that regulate angiogenesis. Methods and Results: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling. Conclusions: Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.

A Single Serine in the Carboxyl Terminus of Cardiac Essential Myosin Light Chain-1 Controls Cardiomyocyte Contractility In Vivo

Although it is well known that mutations in the cardiac essential myosin light chain-1 (cmlc-1) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan (laz), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195–deficient cMLC-1 (cMLC-1S195A) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1S195D) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility.

Regulation of β1 Integrin-Klf2-Mediated Angiogenesis by CCM Proteins

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor β1 integrin and occurs in the absence of blood flow. Downregulation of β1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a β1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

Orai1 deficiency leads to heart failure and skeletal myopathy in zebrafish

Mutations in the store-operated Ca2+ entry pore protein ORAI1 have been reported to cause myopathies in human patients but the mechanism involved is not known. Cardiomyocytes express ORAI1 but its role in heart function is also unknown. Using reverse genetics in zebrafish, we demonstrated that inactivation of the highly conserved zebrafish orthologue of ORAI1 resulted in severe heart failure, reduced ventricular systolic function, bradycardia and skeletal muscle weakness. Electron microscopy of Orai1-deficient myocytes revealed progressive skeletal muscle instability with loss of myofiber integrity and ultrastructural abnormalities of the z-disc in both skeletal and cardiac muscle. Isolated Orai1-deficient cardiomyocytes showed loss of the calcineurin-associated protein calsarcin from the z-discs. Furthermore, we found mechanosignal transduction was affected in Orai1-depleted hearts, indicating an essential role for ORAI1 in establishing the cardiac signaling transduction machinery at the z-disc. Our findings identify ORAI1 as an important regulator of cardiac and skeletal muscle function and provide evidence linking ORAI1-mediated calcium signaling to sarcomere integrity and cardiomyocyte function.

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.

Selective noradrenaline reuptake inhibitor atomoxetine directly blocks hERG currents

Background and purpose:  Atomoxetine is a selective noradrenaline reuptake inhibitor, recently approved for the treatment of attention‐deficit/hyperactivity disorder. So far, atomoxetine has been shown to be well tolerated, and cardiovascular effects were found to be negligible. However, two independent cases of QT interval prolongation, associated with atomoxetine overdose, have been reported recently. We therefore analysed acute and subacute effects of atomoxetine on cloned human Ether‐à‐Go‐Go‐Related Gene (hERG) channels.