Deletion of Siah-interacting protein gene in <Emphasis Type="Italic">Drosophila</Emphasis> causes cardiomyopathy

Drosophila is a useful model organism in which the genetics of human diseases, including recent advances in identification of the genetics of heart development and disease in the fly, can be studied. To identify novel genes that cause cardiomyopathy, we performed a deficiency screen in adult Drosophila. Using optical coherence tomography to phenotype cardiac function in awake adult Drosophila, we identified Df(1)Exel6240 as having cardiomyopathy. Using a number of strategies including customized smaller deletions, screening of mutant alleles, and transgenic rescue, we identified CG3226 as the causative gene for this deficiency. CG3226 is an uncharacterized gene in Drosophila possessing homology to the mammalian Siah-interacting protein (SIP) gene. Mammalian SIP functions as an adaptor protein involved in one of the β-catenin degradation complexes. To investigate the effects of altering β-catenin/Armadillo signaling in the adult fly, we measured heart function in flies expressing either constitutively active Armadillo or transgenic constructs that block Armadillo signaling, specifically in the heart. While, increasing Armadillo signaling in the heart did not have an effect on adult heart function, decreasing Armadillo signaling in the fly heart caused the significant reduction in heart chamber size. In summary, we show that deletion of CG3226, which has homology to mammalian SIP, causes cardiomyopathy in adult Drosophila. Alterations in Armadillo signaling during development lead to important changes in the size and function of the adult heart.     

Disruption of Sarcoendoplasmic Reticulum Calcium ATPase Function in Drosophila Leads to Cardiac Dysfunction

Abnormal sarcoendoplasmic reticulum Calcium ATPase (SERCA) function has been associated with poor cardiac function in humans. While modifiers of SERCA function have been identified and studied using animal models, further investigation has been limited by the absence of a model system that is amenable to large-scale genetic screens. Drosophila melanogaster is an ideal model system for the investigation of SERCA function due to the significant homology to human SERCA and the availability of versatile genetic screening tools. To further the use of Drosophila as a model for examining the role of SERCA in cardiac function, we examined cardiac function in adult flies. Using optical coherence tomography (OCT) imaging in awake, adult Drosophila, we have been able to characterize cardiac chamber dimensions in flies with disrupted in Drosophila SERCA (CaP60A). We found that the best studied CaP60A mutant, the conditional paralytic mutant CaP60A^kum170, develops marked bradycardia and chamber enlargement that is closely linked to the onset of paralysis and dependent on extra cardiac CaP60A. In contrast to prior work, we show that disruption of CaP60A in a cardiac specific manner results in cardiac dilation and dysfunction rather than alteration in heart rate. In addition, the co-expression of a calcium release channel mutation with CaP60A ^kum170 is sufficient to rescue the cardiac phenotype but not paralysis. Finally, we show that CaP60A overexpression is able to rescue cardiac function in a model of Drosophila cardiac dysfunction similar to what is observed in mammals. Thus, we present a cardiac phenotype associated with Drosophila SERCA dysfunction that would serve as additional phenotyping for further large-scale genetic screens for novel modifiers of SERCA function.     

Adaptable doxycycline-regulated gene expression systems for Drosophila

We have engineered two new versions of the doxycycline (dox) inducible system for use in Drosophila. In the first system, we have used the ubiquitously expressed Drosophila actin5C promoter to express the Tet-Off transactivator (tTA) in all tissue. Induction of a luciferase target transgene begins 6 h after placing the flies on dox-free food. Feeding drug-free food to mothers results in universal target gene expression in their embryos. Larvae raised on regular food also show robust expression of a target reporter gene. In the second version, we have used the Gal4-UAS system to spatially limit expression of the transactivator. Dox withdrawal results in temporally- and spatially-restricted, inducible expression of luciferase in the adult head and embryo. Both the actin5C and Gal4-UAS versions produce more than 100-fold induction of luciferase in the adult, with virtually no leaky expression in the presence of drug. Reporter gene expression is also undetectable in larvae or embryos from mothers fed dox-containing food. Such tight control may be due to the incorporation of Drosophila insulator elements (SCS and SCS′) into the transgenic vectors. These systems offer a practical, effective alternative to currently available expression systems in the Drosophila research community.     

Characterization of a dorsal‐eye Gal4 Line in Drosophila

In Drosophila, the Gal4‐UAS system is used to drive ectopic gene expression in a tissue‐specific manner. In this system, transgenic flies expressing tissue specific Gal4 are crossed to a line in which the gene to be expressed is under the control of a Gal4‐responsive UAS sequence. The resulting progeny express the gene of interest in the pattern of the particular Gal4 line. Since a given UAS‐transgene can be driven by any Gal4 line, this system is predominantly limited by available Gal4 lines. Here we report the characterization of a novel line, DE‐Gal4, which in the eye is expressed in the dorsal compartment for the majority of development. Furthermore, we use functional tests to show that the DE‐Gal4 line is a useful tool with which to manipulate gene expression in half of the developing eye. genesis 48:3–7, 2010. © 2009 Wiley‐Liss, Inc.     

Conditional UAS-targeted repression in Drosophila

The Gal4–UAS enhancer trap system is useful for driving gene expression in various tissues. A new tool that extends Gal4 technology is described here. A fusion protein containing the Gal4 binding domain and the repression domain of the isolator suppressor of hairy wing was placed under the control of a heat shock-inducible promoter. The construct mediates the conditional repression of genes located downstream of a UAS sequence. The repressive effects of the chimeric protein on fasII gene expression were tested by western-blot analysis and in brain sections of adult Drosophila. Owing to the increasing number of Gal4 and UAS transgenic lines, this versatile system will facilitate the study of gene function.     

Inducible and myocyte-specific inhibition of PKCalpha enhances cardiac contractility and protects against infarction-induced heart failure

Mice null for the gene encoding protein kinase Calpha (Prkca), or mice treated with pharmacologic inhibitors of the PKCalpha/beta/gamma isoforms, show an augmentation in cardiac contractility that appears to be cardioprotective. However, it remains uncertain if PKCalpha itself functions in a myocyte autonomous manner to affect cardioprotection in vivo. Here we generated cardiac myocyte-specific transgenic mice using a tetracycline-inducible system to permit controlled expression of dominant negative PKCalpha in the heart. Consistent with the proposed function of PKCalpha, induction of dominant negative PKCalpha expression in the adult heart enhanced baseline cardiac contractility. This increase in cardiac contractility was associated with a partial protection from long-term decompensation and secondary dilated cardiomyopathy after myocardial infarction injury. Similarly, Prkca null mice were also partially protected from infarction-induced heart failure, although the area of infarction injury was identical to controls. Thus, myocyte autonomous inhibition of PKCalpha protects the adult heart from decompensation and dilated cardiomyopathy after infarction injury in association with a primary enhancement in contractility.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Arrhythmia caused by a Drosophila tropomyosin mutation is revealed using a novel optical coherence tomography instrument

Background

Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows in vivo measurement of cardiac phenotypes and a detecting instrument capable of recording multiple phenotype parameters.

Methodology and Principal Findings

With a simple heart, a transparent body surface at larval stages and available genetic tools we chose Drosophila melanogaster as our model organism and developed for it a dual en-face/Doppler optical coherence tomography (OCT) instrument capable of recording multiple aspects of heart activity, including heart contraction cycle dynamics, ostia dynamics, heartbeat rate and rhythm, speed of heart wall movement and light reflectivity of cardiomyocytes in situ. We applied this OCT instrument to a model of Tropomyosin-associated DCM established in adult Drosophila. We show that DCM pre-exists in the larval stage and is accompanied by an arrhythmia previously unidentified in this model. We also detect reduced mobility and light reflectivity of cardiomyocytes in mutants.

Conclusion

These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity in genetic models for heart disease in Drosophila.     

Human Matrix Attachment Regions Insulate Transgene Expression from Chromosomal Position Effects in Drosophila melanogaster

Germ line transformation of white⁻ Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs′ elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and α1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.     

Characterization of Sgo1 expression in developing and adult mouse

SGO1 has been characterized in its function in correct cell division and its role in centrosome cohesion in the nucleus. However, its organ-specific maturation-related expression pattern in vivo remains largely uncharacterized. Here, we show clear SGO1 expression in post-developmental neuronal cells and cytoplasmic localisation in nucleated cells with a transgenic mice model and immunohistochemistry of wild type mice. We demonstrate extranuclear expression of Sgo1 in the developing heart and gut, which have been shown to be dysregulated in humans with homozygous SGO1 mutation. Additionally, we show Sgo1 expression in select population of retinal cells in developing and post-developmental retina. Our expression analysis strongly suggests that the function of SGO1 goes beyond its well characterized role in cell division.     

SIFamide and SIFamide Receptor Define a Novel Neuropeptide Signaling to Promote Sleep in Drosophila

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.     

FLP Recombinase-Mediated Induction of Cu/Zn-Superoxide Dismutase Transgene Expression Can Extend the Life Span of Adult Drosophila melanogaster Flies

Yeast FLP recombinase was used in a binary transgenic system (“FLP-OUT”) to allow induced overexpression of catalase and/or Cu/Zn-superoxide dismutase (Cu/ZnSOD) in adult Drosophila melanogaster. Expression of FLP recombinase was driven by the heat-inducible hsp70 promoter. Once expressed, FLP catalyzed the rearrangement and activation of a target construct in which expression of catalase or Cu/ZnSOD cDNAs was driven by the constitutive actin5C promoter. In this way a brief heat pulse (120 or 180 min, total) of young adult flies activated transgene expression for the rest of the life span. FLP-OUT allows the effects of induced transgene expression to be analyzed in control (no heat pulse) and experimental (heat pulse) populations with identical genetic backgrounds. Under the conditions used, the heat pulse itself always had neutral or slightly negative effects on the life span. Catalase overexpression significantly increased resistance to hydrogen peroxide but had neutral or slightly negative effects on the mean life span. Cu/ZnSOD overexpression extended the mean life span up to 48%. Simultaneous overexpression of catalase with Cu/ZnSOD had no added benefit, presumably due to a preexisting excess of catalase. The data suggest that oxidative damage is one rate-limiting factor for the life span of adult Drosophila. Finally, experimental manipulation of the genetic background demonstrated that the life span is affected by epistatic interactions between the transgene and allele(s) at other loci.     

Metabolic Stress Responses in Drosophila Are Modulated by Brain Neurosecretory Cells That Produce Multiple Neuropeptides

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.     

ndufa7 plays a critical role in cardiac hypertrophy

Cardiac hypertrophy is a common pathological change in patients with progressive cardiac function failure, which can be caused by hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or arterial hypertension. Despite years of study, there is still limited knowledge about the underlying molecular mechanisms for cardiac hypertrophy. NDUFA7, a subunit of NADH:ubiquinone oxidoreductase (complex I), has been reported to be a novel HCM associated gene. However, the biological role of NDUFA7 in heart remains unknown. In this study, we found that NDUFA7 exhibited high expression in the heart, and its level was significantly decreased in mice model of cardiac hypertrophy. Moreover, we demonstrated that ndufa7 knockdown in developing zebrafish embryos resulted in cardiac development and functional defects, associated with increased expression of pathological hypertrophy biomarkers nppa (ANP) and nppb (BNP). Mechanistic study demonstrated that ndufa7 depletion promoted ROS production and calcineurin signalling activation. Moreover, NDUFA7 depletion contributed to cardiac cell hypertrophy. Together, these results report for the first time that ndufa7 is implicated in pathological cardiac hypertrophy.     

Identification of neurons responsible for feeding behavior in the Drosophila brain

Drosophila melanogaster feeds mainly on rotten fruits,which contain many kinds of sugar.Thus,the sense of sweet taste has evolved to serve as a dominant regulator and driver of feeding behavior.Although several sugar receptors have been described,it remains poorly understood how the sensory input is transformed into an appetitive behavior.Here,we used a neural silencing approach to screen brain circuits,and identified neurons labeled by three Gal4 lines that modulate Drosophila feeding behavior.These three Gal4 lines labeled neurons mainly in the suboesophageal ganglia(SOG),which is considered to be the fly’s primary taste center.When we blocked the activity of these neurons,flies decreased their sugar consumption significantly.In contrast,activation of these neurons resulted in enhanced feeding behavior and increased food consumption not only towards sugar,but to an array of food sources.Moreover,upon neuronal activation,the flies demonstrated feeding behavior even in the absence of food,which suggests that neuronal activation can replace food as a stimulus for feeding behavior.These findings indicate that these Gal4-labeled neurons,which function downstream of sensory neurons and regulate feeding behavior towards different food sources is necessary in Drosophila feeding control.     

人参皂甙Rb1抑制扩张型心肌病发生中的HB-EGF表达和纤维化

目的HB-EGF过表达可促进心肌纤维化及心肌细胞凋亡,本文研究人参皂甙Rb1对cTnT^R141W转基因扩张型心肌病小鼠发病过程中的HB-EGF表达和心肌纤维化的影响。方法将cTnT^R141W转基因小鼠随机分为模型组和人参皂甙Rb1组（70 mg/kg/d）,连续给药7个月,取野生型小鼠作为对照组。用Kaplan-Meier法进行生存分析。心脏超声检测心功能及心脏几何构型。计算心重指数。光镜观察心肌细胞及间质变化。Western blot检测心脏HB-EGF,pSTAT3表达水平。结果Rb1长期给药能显著改善该模型的心功能和心脏几何构型,将死亡率降低50%。Rb1治疗组心重指数降低11.3%（P〈0.05）,光镜观察显示Rb1能减轻心肌细胞排列紊乱以及间质纤维化。Western blot结果显示Rb1能够显著降低模型中的HB-EGF及pSTAT3的表达。结论Rb1抑制心肌病发生中的HB-EGF表达及抑制下游信号pSTAT的激活,并改善扩张型心肌病模型的心功能及心脏重构。