Application of an automated analysis framework for pulsed-wave Doppler cardiac ultrasound measurements to generate reference data in adult zebrafish.

High-frequency cardiac ultrasound is the only well-established method to characterize in vivo cardiovascular function in adult zebrafish noninvasively. Pulsed-wave Doppler imaging allows measurements of blood flow velocities at well-defined anatomical positions, but the measurements and results obtained using this technique need to be analyzed carefully, taking into account the substantial baseline variability within one recording and the possibility for operator bias. To address these issues and to increase throughput by limiting hands-on analysis time, we have developed a fully automated processing pipeline. This framework enables the fast, unbiased analysis of all cardiac cycles in a zebrafish pulsed-wave Doppler recording of both atrioventricular valve flow as well as aortic valve flow without operator-dependent inputs. Applying this automated pipeline to a large number of recordings from wild-type zebrafish shows a strong agreement between the automated results and manual annotations performed by an experienced operator. The reference data obtained from this analysis showed that the early wave peak during ventricular inflow is lower for female compared with male zebrafish. We also found that the peaks of the ventricular inflow and outflow waves as well as the peaks of the regurgitation waves are all correlated positively with body surface area. In general, the presented reference data, as well as the automated Doppler measurement processing tools developed and validated in this study will facilitate future (high-throughput) cardiovascular phenotyping studies in adult zebrafish ultimately leading to a more comprehensive understanding of human (genetic) cardiovascular diseases.

Loss of zebrafish atp6v1e1b, encoding a subunit of vacuolar ATPase, recapitulates human ARCL type 2C syndrome and identifies multiple pathobiological signatures.

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases (v-ATPases) has been associated with various human diseases including heritable connective tissue disorders. Autosomal recessive (AR) cutis laxa (CL) type 2C syndrome is associated with genetic defects in the ATP6V1E1 gene and is characterized by skin wrinkles or loose redundant skin folds with pleiotropic systemic manifestations. The underlying pathological mechanisms leading to the clinical presentations remain largely unknown. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, N-glycosylation defects, hypotonia, and epidermal structural defects. These features are reminiscent of the phenotypic manifestations in ARCL type 2C patients. Our data demonstrates that loss of atp6v1e1b alters endo(lyso)somal protein levels, and interferes with non-canonical v-ATPase pathways in vivo. In order to gain further insights into the processes affected by loss of atp6v1e1b, we performed an untargeted analysis of the transcriptome, metabolome, and lipidome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid, fatty acid, and energy metabolism together with profound defects on mitochondrial respiration. Taken together, our results identify complex pathobiological effects due to loss of atp6v1e1b in vivo.

Loss-of-Function Variants in EFEMP1 Cause a Recognizable Connective Tissue Disorder Characterized by Cutis Laxa and Multiple Herniations.

Hereditary disorders of connective tissue (HDCT) compromise a heterogeneous group of diseases caused by pathogenic variants in genes encoding different components of the extracellular matrix and characterized by pleiotropic manifestations, mainly affecting the cutaneous, cardiovascular, and musculoskeletal systems. We report the case of a 9-year-old boy with a discernible connective tissue disorder characterized by cutis laxa (CL) and multiple herniations and caused by biallelic loss-of-function variants in EFEMP1. Hence, we identified EFEMP1 as a novel disease-causing gene in the CL spectrum, differentiating it from other HDCT.