Analysis of Drosophila cardiac hypertrophy by micro-computerized tomography for genetic dissection of heart growth mechanisms

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic and systolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

Multi-Targeting DKK1 and LRP6 Prevents Myeloma-Induced Bone Disease

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodeling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting their quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. Therapeutic strategies aimed at promoting bone formation are therefore required to overcome the loss of skeletal integrity and subsequent fractures in MM patients. Therapeutic agents that target the canonical Wnt signaling pathway, a potent regulator of bone formation, have the potential to address these skeletal complications, where they could rebuild lost bone and improve bone strength in affected individuals. We have demonstrated a novel anti-LRP6 agent, which potentiates Wnt signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. However, since MM patients present with both increased bone resorption and decreased bone formation, we hypothesised that combining anti-LRP6 with the bone anabolic anti-DKK1 (100mg/kg twice weekly intravenously) would lead to more robust improvements in bone structure than single treatment approaches. MicroCT analysis demonstrated a 74% increase in femoral trabecular bone volume per tissue volume (BV/TV) in naïve, non-tumour bearing mice given the combination treatment compared to control agents (p<0.0001). Mice injected with 5TGM1eGFP murine myeloma cells had a 34% reduction in femoral BV/TV compared to naïve controls (p<0.0001). Combination treatment drastically improved BV/TV in 5TGM1-bearing mice by 111% (p<0.0001), compared to control, and this improvement with the combination treatment strategy was 25% greater than anti-LRP6 single treatment approaches (p<0.001). MicroCT analysis in L4 lumbar vertebrae demonstrated similar bone structural changes in 5TGM1-bearing mice treated with the combination strategy. Consequently, this combination significantly improved resistance to fracture in L4 vertebrae in 5TGM1eGFP-bearing mice compared to their controls (p<0.001), and it provided greater protection against fracture compared to anti-LRP6 single agent treatment. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1eGFP-bearing mice treated with the combination strategy (p<0.001) compared to control. Importantly, tumour activity was not altered with either single or combination Wnt-promoting treatment strategies. This study defines a novel therapeutic strategy, which will reduce fractures and improve quality of life in patients with MM when used in combination with tumour-targeted treatments. Figure 1 Figure 1. Disclosures Cong: Novartis Institutes for Biomedical Research: Current Employment. Daley: Novartis Institutes of Biomedical Research: Current Employment.

A novel micro-computerized tomography method reveals Drosophila cardiac hypertrophy caused by upregulated store-operated calcium entry

ABSTRACTHeart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the mechanisms that underlie cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of the morphology and size of the Drosophila heart. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Ca2+ influx through Orai channels. Importantly, dysregulation of Ca2+ homeostasis in cardiomyocytes is a major driver of cardiac hypertrophy, but the underlying mechanisms are unclear. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

Sleeping with the enemy: unravelling the symbiotic relationships between the scale worm Neopolynoe chondrocladiae (Annelida: Polynoidae) and its carnivorous sponge hosts

The North Atlantic deep-water polynoid worm Neopolynoe chondrocladiae is involved in an exceptional symbiotic relationship with two hosts: the carnivorous sponges Chondrocladia robertballardi and Chondrocladia virgata. While this is an obligate symbiotic relationship, its real nature is unclear. We used a multidisciplinary approach to narrow down the type of symbiotic relationship between symbiont and hosts. Molecular connectivity analyses using COI and 16S suggest that N. chondrocladiae has high potential for dispersal, connecting sites hundreds of kilometres apart, likely aided by oceanographic currents. Microbial analyses on different anatomical parts of five Chondrocladia species suggest that the presence of the worm in C. robertballardi does not affect the microbiome of the sponge. MicroCT analysis on N. chondrocladiae show that it has dorsally oriented parapodia, which might prevent the worm from getting trapped in the sponge. A faecal pellet recovered from the worm suggests that the polynoid feeds on the crustacean prey captured by the sponge, something corroborated by our stable isotope analysis. Light and confocal microscopy images suggest that N. chondrocladiae elytra produce bioluminescence. We propose that the worm might use bioluminescence as a lure for prey (increasing the food available for both the sponge and the polynoid) and thus fuelling a mutualistic relationship.

Quantitative analysis of airway obstruction in lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease with progressive pulmonary function loss caused by progressively proliferating LAM cells. The degree of airway obstruction has not been well investigated within the pathogenesis of LAM.Using a combination of ex vivo computed tomography (CT), microCT and histology, the site and nature of airway obstruction in LAM explant lungs was compared with matched control lungs (n=5 each). The total number of airways per generation, total airway counts, terminal bronchioles number and surface density were compared in LAM versus control.Ex vivo CT analysis demonstrated a reduced number of airways from generation 7 on (p<0.0001) in LAM compared with control, whereas whole-lung microCT analysis confirmed the three- to four-fold reduction in the number of airways. Specimen microCT analysis further demonstrated a four-fold decrease in the number of terminal bronchioles (p=0.0079) and a decreased surface density (p=0.0079). Serial microCT and histology images directly showed the loss of functional airways by collapse of airways on the cysts and filling of the airway by exudate.LAM lungs show a three- to four-fold decrease in the number of (small) airways, caused by cystic destruction which is the likely culprit for the progressive loss of pulmonary function.

Extensive Recontouring of the Femoral Head with Osteochondral Allografting: A Case Report with Histological and MicroCT Analysis

Morphological abnormalities such as cam deformity or growth disturbances can have a detrimental effect on the smooth function of the hip joint. This case reports an attempt to salvage the hip joint of a young patient with a posttraumatic growth disturbance of the femoral head using a fresh osteochondral allograft. This treatment has been used very rarely in the femoral head due to the presumed tenuous blood supply of the head and the perceived risk of nonunion or progressive avascular necrosis. The patient in this case had persistent pain and mechanical symptoms leading to hip replacement. A detailed analysis of the retrieved femoral head demonstrated durability and healing of the grafts based on gross inspection, histology of bone and cartilage, and microCT analysis. This case is the first report to our knowledge of a detailed histological and radiographic analysis of the fate of osteochondral allografts of the femoral head. We hope that this case provides justification for the use of osteochondral allografts of the femoral head for other indications such as femoral head fractures, avascular necrosis, and benign epiphyseal tumors of the femoral head in an effort to avoid arthroplasty in young patients. The authors have obtained the patient’s informed written consent for print and electronic publication of the case report.

Airway morphometry in COPD with bronchiectasis: a view on all airway generations

The pathophysiological processes underlying bronchiectasis in chronic obstructive pulmonary disease (COPD) are not understood. In COPD, both small and large airways are progressively lost. It is currently not known to what extent the different airway generations of patients with COPD and bronchiectasis are involved.COPD explant lungs with bronchiectasis were compared to COPD explant lungs without bronchiectasis and unused donor lungs as controls. In order to investigate all airway generations, a multimodal imaging approach using different resolutions was conducted. Per group, five lungs were frozen (n=15) and underwent computed tomography (CT) imaging for large airway evaluation, with four tissue cores per lung imaged for measurements of the terminal bronchioles. Two additional lungs per group (n=6) were air-dried for lobar microCT images that allow airway segmentation and three-dimensional quantification of the complete airway tree.COPD lungs with bronchiectasis had significantly more airways compared to COPD lungs without bronchiectasis (p<0.001), with large airway numbers similar to control lungs. This difference was present in both upper and lower lobes. Lack of tapering was present (p=0.010) and larger diameters were demonstrated in lower lobes with bronchiectasis (p=0.010). MicroCT analysis of tissue cores showed similar reductions of tissue percentage, surface density and number of terminal bronchioles in both COPD groups compared to control lungs.Although terminal bronchioles were equally reduced in COPD lungs with and without bronchiectasis, significantly more large and small airways were found in COPD lungs with bronchiectasis.

Spatially restricted dental regeneration drives pufferfish beak development

Vertebrate dentitions are extraordinarily diverse in both morphology and regenerative capacity. The teleost order Tetraodontiformes exhibits an exceptional array of novel dental morphologies, epitomized by constrained beak-like dentitions in several families, i.e., porcupinefishes, three-toothed pufferfishes, ocean sunfishes, and pufferfishes. Modification of tooth replacement within these groups leads to the progressive accumulation of tooth generations, underlying the structure of their beaks. We focus on the dentition of the pufferfish (Tetraodontidae) because of its distinct dental morphology. This complex dentition develops as a result of (i) a reduction in the number of tooth positions from seven to one per quadrant during the transition from first to second tooth generations and (ii) a dramatic shift in tooth morphogenesis following the development of the first-generation teeth, leading to the elongation of dental units along the jaw. Gene expression and 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) lineage tracing reveal a putative dental epithelial progenitor niche, suggesting a highly conserved mechanism for tooth regeneration despite the development of a unique dentition. MicroCT analysis reveals restricted labial openings in the beak, through which the dental epithelium (lamina) invades the cavity of the highly mineralized beak. Reduction in the number of replacement tooth positions coincides with the development of only four labial openings in the pufferfish beak, restricting connection of the oral epithelium to the dental cavity. Our data suggest the spatial restriction of dental regeneration, coupled with the unique extension of the replacement dental units throughout the jaw, are primary contributors to the evolution and development of this unique beak-like dentition.