Christine Fink

Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes.

A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO(2) incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length x width x thickness, 11 x 6 x 0.4 mm) was 0.51 mN at length of maximal force development (L(max)) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force-length and a negative force-frequency relation, high sensitivity to calcium (EC(50) 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of beta-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement.

Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement

To examine the influence of chronic mechanical stretch on functional behavior of cardiac myocytes, we reconstituted embryonic chick or neonatal rat cardiac myocytes to a 3‐dimensional engineered heart tissue (EHT) by mixing freshly isolated cells with neutralized collagen I and culturing them between two Velcro‐coated silicone tubes, held at a fixed distance with a metal spacer. After 4 days, EHTs were subjected to a phasic unidirectional stretch for 6 days in serum‐containing medium. Compared to unstretched controls, RNA/DNA and protein/cell ratios increased by 100% and 50%, respectively. ANF mRNA and a‐sarcomeric actin increased by 98% and 40%, respectively. Morphologically, stretched EHTs exhibited improved organization of cardiac myocytes into parallel arrays of rod‐shaped cells, increased cell length and width, longer myofilaments, and increased mitochondrial density. Thus, stretch induced phenotypic changes, generally referred to as hypertrophy. Concomitantly, force of contraction was two‐ to fourfold higher both under basal conditions and after stimulation with calcium or the β‐adrenergic agonist isoprenaline. Contraction kinetics were accelerated with a 14–44% decrease in twitch duration under all those conditions. In summary, we have developed a new in vitro model that allows morphological, molecular, and functional consequences of stretch to be studied under defined conditions. The main finding was that stretch of EHTs induced cardiac myocyte hypertrophy, which was accompanied by marked improvement of contractile function.—Fink, C., Ergün, S., Kralisch, D., Remmers, U., Weil, J., Eschenhagen, T. Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. FASEB J. 14, 669–679 (2000)

Expression analysis of octopamine and tyramine receptors in Drosophila

The monoamines octopamine and tyramine, which are the invertebrate counterparts of epinephrine and norepinephrine, transmit their action through sets of G protein-coupled receptors. Four different octopamine receptors (Oamb, Octß1R, Octß2R, Octß3R) and 3 different tyramine receptors (TyrR, TyrRII, TyrRIII) are present in the fruit fly Drosophila melanogaster. Utilizing the presumptive promoter regions of all 7 octopamine and tyramine receptors, the Gal4/UAS system is utilized to elucidate their complete expression pattern in larvae as well as in adult flies. All these receptors show strong expression in the nervous system but their exact expression patterns vary substantially. Common to all octopamine and tyramine receptors is their expression in mushroom bodies, centers for learning and memory in insects. Outside the central nervous system, the differences in the expression patterns are more conspicuous. However, four of them are present in the tracheal system, where they show different regional preferences within this organ. On the other hand, TyrR appears to be the only receptor present in the heart muscles and TyrRII the only one expressed in oenocytes. Skeletal muscles express octß2R, Oamb and TyrRIII, with octß2R being present in almost all larval muscles. Taken together, this study provides comprehensive information about the sites of expression of all octopamine and tyramine receptors in the fruit fly, thus facilitating future research in the field.

The octopamine receptor octß2R is essential for ovulation and fertilization in the fruit fly Drosophila melanogaster.

The biogenic monoamine octopamine is essential for ovulation and fertilization in insects. Release of this hormone from neurons in the thoracoabdominal ganglion triggers ovulation and sperm release from the spermathecae. Here we show that the effects of octopamine on ovulation are mediated by at least two different octopamine receptors. In addition to the Oamb receptor that is present in the epithelium of the oviduct, the octß2R receptor is essential for ovulation and fertilization. Octß2R is widely expressed in the female reproductive tract. Most prominent is expression in the oviduct muscle and the spermathecae. Animals deficient in expression of the receptor show a severe egg-laying defect. The corresponding females have a much larger ovary that is caused by egg retention in the ovary. Moreover, the very few laid eggs are not fertilized, indicating problems in the process of sperm delivery. We assume that octß2R acts in a similar way as ß2-adrenoreceptors in smooth muscles, were activation of this receptor induces an increase in cAMP levels that lead to relaxation of the muscle. Taken together, our findings show that octopaminergic control of ovulation and fertilization is more complex than anticipated and that various receptors located in different cells act together to enable a well-orchestrated activity of the female reproductive system in response to copulation.

Noninvasive Analysis of Microbiome Dynamics in the Fruit Fly Drosophila melanogaster

ABSTRACT The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.

Octopamine controls starvation resistance, life span and metabolic traits in Drosophila

The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. We show that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. We inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes.

Intestinal FoxO signaling is required to survive oral infection in Drosophila

The intestinal immune system is tailored to fight pathogens effectively while tolerating the indigenous microbiota. Impairments of this homeostatic interaction may contribute to the etiology of various diseases including inflammatory bowel diseases. However, the molecular architecture underlying this complex regulatory interaction is not well understood. Here, we show that the fruit fly Drosophila melanogaster has a multilayered intestinal immune system that ensures strictly localized antimicrobial responses. Enterocytes, a major cell population of the intestine, produced antimicrobial peptides (AMPs) in a FoxO- but not NF-κB-dependent manner. Consequently, animals impaired in FoxO-mediated signaling had a significantly lowered resistance to intestinal infections; they were unable to increase the expression of AMP genes and males showed an increased bacterial load in response to an infection. Conventional innate immune signaling converging onto NF-κB activation was operative in only a few regions of the intestine, comprising the proventriculus, copper cells, and intestinal stem cells. Taken together, our results imply that danger-mediated as well as conventional innate immune signaling constitute modules that contribute to the fruit fly’s intestinal immune system. We propose that this special architecture ensures localized and efficient antimicrobial responses against invasive pathogens while preserving the microbiota.

Drosophila as a Model to Study Metabolic Disorders

Metabolic disorders including obesity and diabetes are among the most relevant lifestyle diseases. They show a dramatically increasing incidence espe- cially in industrialized countries. Although these diet-induced morbidities reached pandemic dimensions, our knowledge about the underlying mechanisms is sur- prisingly sparse. Simple model organisms including the fruit fly Drosophila melanogaster might fill this gap, inasmuch as they allow complementary scientific approaches enhancing our understanding regarding the pathomechanisms under- lying these diseases. Based on the armamentarium of methods available to tailor disease models in the fly, very instructive information about diabetes or the effects of high-fat diets on heart ageing and dysfunction have been generated. In addition, genome wide approaches already have provided us with almost complete sets of genes relevant for fat storage defects or heart dysfunction.

Transcriptional regionalization of the fruit fly's airway epithelium.

Although airway epithelia are primarily devoted to gas exchange, they have to fulfil a number of different tasks including organ maintenance and the epithelial immune response to fight airborne pathogens. These different tasks are at least partially accomplished by specialized cell types in the epithelium. In addition, a proximal to distal gradient mirroring the transition from airflow conduction to real gas exchange, is also operative. We analysed the airway system of larval Drosophila melanogaster with respect to region-specific expression in the proximal to distal axis. The larval airway system is made of epithelial cells only. We found differential expression between major trunks of the airways and more distal ones comprising primary, secondary and terminal ones. A more detailed analysis was performed using DNA-microarray analysis to identify cohorts of genes that are either predominantly expressed in the dorsal trunks or in the primary/secondary/terminal branches of the airways. Among these differentially expressed genes are especially those involved in signal transduction. Wnt-signalling associated genes for example are predominantly found in secondary/terminal airways. In addition, some G-protein coupled receptors are differentially expressed between both regions of the airways, exemplified by those activated by octopamine or tyramine, the invertebrate counterparts of epinephrine and norepinephrine. Whereas the OAMB is predominantly found in terminal airway regions, the oct3βR has higher expression levels in dorsal trunks. In addition, we observed a significant association of both, genes predominantly expressed in dorsal trunks or in primary to terminal branches branches with those regulated by hypoxia. Taken together, this observed differential expression is indicative for a proximal to distal transcriptional regionalization presumably reflecting functional differences in these parts of the fly’s airway system.

The Role of Monoaminergic Neurotransmission for Metabolic Control in the Fruit Fly Drosophila Melanogaster

Hormones control various metabolic traits comprising fat deposition or starvation resistance. Here we show that two invertebrate neurohormones, octopamine (OA) and tyramine (TA) as well as their associated receptors, had a major impact on these metabolic traits. Animals devoid of the monoamine OA develop a severe obesity phenotype. Using flies defective in the expression of receptors for OA and TA, we aimed to decipher the contributions of single receptors for these metabolic phenotypes. Whereas those animals impaired in octß1r, octß2r and tar1 share the obesity phenotype of OA-deficient (tβh-deficient) animals, the octß1r, octß2r deficient flies showed reduced insulin release, which is opposed to the situation found in tβh-deficient animals. On the other hand, OAMB deficient flies were leaner than controls, implying that the regulation of this phenotype is more complex than anticipated. Other phenotypes seen in tβh-deficient animals, such as the reduced ability to perform complex movements tasks can mainly be attributed to the octß2r. Tissue-specific RNAi experiments revealed a very complex interorgan communication leading to the different metabolic phenotypes observed in OA or OA and TA-deficient flies.