The Calcineurin-FoxO-MuRF1 signaling pathway regulates myofibril integrity in cardiomyocytes.

Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a zebrafish ncx1 mutant, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase murf1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down murf1 activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the murf1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced murf1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated murf1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway

Involvement of zebrafish Na+,K+ ATPase in myocardial cell junction maintenance

Na+,K+ ATPase is an essential ion pump involved in regulating ionic concentrations within epithelial cells. The zebrafish heart and mind (had) mutation, which disrupts the α1B1 subunit of Na+,K+ ATPase, causes heart tube elongation defects and other developmental abnormalities that are reminiscent of several epithelial cell polarity mutants, including nagie oko (nok). We demonstrate genetic interactions between had and nok in maintaining Zonula occludens-1 (ZO-1)–positive junction belts within myocardial cells. Functional tests and pharmacological inhibition experiments demonstrate that Na+,K+ ATPase activity is positively regulated via an N-terminal phosphorylation site that is necessary for correct heart morphogenesis to occur, and that maintenance of ZO-1 junction belts requires ion pump activity. These findings suggest that the correct ionic balance of myocardial cells is essential for the maintenance of epithelial integrity during heart morphogenesis

The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear

In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized 'ear stones' (or otoliths) 1. Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization

Mitochondrial Ca(2+) uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity.

Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin\u27s rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity

Novel Neuroprotective Mechanisms of Memantine: Increase in Neurotrophic Factor Release from Astroglia and Anti-Inflammation by Preventing Microglial Activation

Memantine provides clinically relevant efficacy in patients with Alzheimer's disease and Parkinson’s diseases. In addition to blockade of N-methyl-D-aspartate receptor on neurons, memantine has neurotrophic and neuroprotective effects in in vivo and in vitro studies, however, the mechanism underlying these effects remains unclear. To address this question, primary midbrain neuron-glia cultures and reconstituted cultures were used, and lipopolysaccharide (LPS), an endotoxin from bacteria, was used to produce inflammation-mediated dopaminegic neuronal death. Here, we show that memantine exerted both potent neurotrophic and neuroprotective effects on dopaminergic neurons in rat neuron-glia cultures. The neurotrophic effect of memantine was glia-dependent, since memantine failed to show any positive effect on dopaminergic neurons in neuron-enriched cultures. More specifically, it appears that astroglia, not microglia, are the source of the memantine-elicited neurotrophic effects through the increased production of GDNF. Mechanistic studies revealed that GDNF upregulaton was associated with histone hyperacetylation by inhibiting the cellular histone deacetylase activity. In addition, memantine also displays neuroprotective effects against LPS-induced dopaminergic neuronal damage through its inhibition of microglia over-activation revealed by both OX-42 immunostaining and by the reduction of pro-inflammatory factors production such as extracelluar superoxide anion, intracellular reactive oxygen species, nitric oxide, prostaglandin E2, and tumor necrosis factor-α. These results suggest that memantine therapy for neurodegenerative diseases acts in part through alternative novel mechanisms by reducing microglia-associated inflammation and stimulating the release of neurotrophic factors from astroglia

Mutation in utp15 Disrupts Vascular Patterning in a p53-Dependent Manner in Zebrafish Embryos

Angiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning.In a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes.Taken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos

A structural and functional analysis of thec-ki-ras promoter and its binding proteins

The essential role of the ras genes in coupling receptors to intracellular signals required for cell growth and differentiation has been well established. Mutations in the coding region of the ras genes and the elevation of normal ras proteins have been found to cause tumor formation, indicating the quantitative and qualitative importance of the ras genes. The function of the ras proteins has been extensively studied. However, little is known about the regulation of ras gene expression. We used the mouse c-ki-ras gene as a model to investigate the transcriptional control of ras genes. Using a series of internal deletion mutants of the c-ki-ras promoter, two regulatory elements were identified. One has a negative effect while the other has a positive effect on c-ki-ras expression. The nucleotide substitution experiment and the structural analysis suggest that formation of the unusual triple-helix structure (H-DNA) in the homopurine-homopyrimidine region (CT-repeat) of the c-ki-ras promoter has a negative effect on its expression. Three c-ki-ras promoter binding proteins were isolated by southwestern screening. 101b binds the sequence which is identical between the mouse and the human c-ki-ras promoter. L1 binds the single-stranded region of H-DNA. KRZ, a zinc finger protein, binds the double-stranded CT-repeat. Together with the studies on c-myc gene expression, we proposed that the binding of 101b and KRZ activates while the binding of L1 inactivates c-ki-ras expression. Two forms of KRZ, KRZ1 and KRZ2, containing different numbers of zinc-finger motifs and the very C-terminal sequence were generated by alternative splicing. KRZ1 activates while KRZ2 represses the expression of c-ki-ras. Furthermore, KRZ1 is the predominant form in mouse tissue which is consistent with the fact that the tissues have more KRZ RNA containing more c-ki-ras transcripts. In summary, our results suggest that the promoter sequence of c-ki-ras, the secondary structure of this promoter, the interactions between promoter and its binding proteins, and the competition between the binding proteins are crucial for c-ki-ras expression

Mutation in utp15 disrupts vascular patterning in a p53-dependent manner in zebrafish embryos.

BackgroundAngiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning.Methodology and principal findingsIn a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes.Conclusions and significanceTaken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos

Tbx20 drives cardiac progenitor formation and cardiomyocyte proliferation in zebrafish

Tbx20 is a T-box transcription factor that plays essential roles in the development and maintenance of the heart. Although it is expressed by cardiac progenitors in all species examined, an involvement of Tbx20 in cardiac progenitor formation in vertebrates has not been previously described. Here we report the identification of a zebrafish tbx20 mutation that results in an inactive, truncated protein lacking any functional domains. The cardiac progenitor population is strongly diminished in this mutant, leading to the formation of a small, stretched-out heart. We found that overexpression of Tbx20 results in an enlarged heart with significantly more cardiomyocytes. Interestingly, this increase in cell number is caused by both enhanced cardiac progenitor cell formation and the proliferation of differentiated cardiomyocytes, and is dependent upon the activity of Tbx20's T-box and transcription activation domains. Together, our findings highlight a previously unappreciated role for Tbx20 in promoting cardiac progenitor formation in vertebrates and reveal a novel function for its activation domain in cardiac cell proliferation during embryogenesis