Dataset Information

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies.

ABSTRACT: Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

SUBMITTER: Singareddy SS

PROVIDER: S-EPMC9764968 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies.

Singareddy Soma S SS McClenaghan Conor C Roessler Helen I HI Tryon Robert R Nichols Colin G CG

Journal of visualized experiments : JoVE 20220209 180

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed ...[more]

PMID: 35225267

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Project description:ATP-sensitive potassium channels (KATP channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, KATP channels are activated during ischaemia and potentially during adrenergic stimulation. In the vasculature, they are normally active at a low level, reducing vascular tone, but the ubiquitous nature of these channels leads to complex and poorly understood channelopathies as a result of gain- or loss-of-function mutations. Zebrafish (ZF) models of these channelopathies may provide insights to the link between molecular dysfunction and complex pathophysiology, but this requires understanding the tissue dependence of channel activity and subunit specificity. Thus far, direct analysis of ZF KATP expression and functional properties has only been performed in pancreatic β-cells. Using a comprehensive combination of genetically modified fish, electrophysiology and gene expression analysis, we demonstrate that ZF cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular KATP channels, ZF channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. The results provide a first characterization of the molecular properties of fish KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome. KEY POINTS: Zebrafish cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular KATP channels, zebrafish channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. We provide a first characterization of the molecular properties of fish KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome.

Project description:Most teleost fish propel themselves with lateral body waves powered by their axial muscles. These muscles also power suction feeding through rapid expansion of the mouth cavity. They consist of muscle segments (myomeres), separated by connective tissue sheets (myosepts). In adult teleosts, the fast axial muscle fibres follow pseudo-helical trajectories, which are thought to distribute strain (relative fibre length change) approximately evenly across transverse sections during swimming, thereby optimizing power generation. To achieve strain equalization, a significant angle to the longitudinal axis on the frontal plane (azimuth) is necessary near the medial plane, increasing strain. Additionally, a deviation from longitudinal orientation on the sagittal plane (elevation) is required laterally to decrease strain. Despite several detailed morphological studies, our understanding of muscle-fibre orientations in the entire axial musculature of fish remains incomplete. Furthermore, most research has been done in post-larval stages, leaving a knowledge gap regarding the changing axial muscle architecture during larval development. Larval fish exhibit different body size, body shape and swimming kinematics compared to adults. They experience relatively high viscous forces, requiring higher tail-beat amplitudes to overcome increased drag. Additionally, larval fish swim with higher tail-beat frequencies. Histological studies have shown that in larval fish, muscle fibres in the anal region transition from an almost longitudinal orientation to a pseudo-helical pattern by 3 dpf (days post-fertilization). However, these studies were limited to a few sections of the body and were prone to shrinkage and tissue damage. Here, we introduce a novel methodology for quantifying muscle-fibre orientations along the entire axial muscles. We selected 4 dpf larval zebrafish for our analyses, a stage where larvae are actively swimming but not yet free-feeding. High-resolution confocal 3D scans were obtained from four genetically modified zebrafish expressing green fluorescent protein in fast muscle fibres. Fluorescence variation allowed segmentation of individual muscle fibres, which were then converted to fish-bound coordinates by correcting for the fish's position and orientation in the scan, and normalized to pool results across individuals. We show that at 4 dpf, muscle-fibre trajectories exhibit a helical pattern tapering towards the tail. Average fibre angles decrease from anterior to posterior, with azimuth varying over the dorsoventral axis and elevation varying over the mediolateral axis. Notably, only the anteriormost 20% of the body displayed higher azimuth angles near the medial plane. Angles between neighbouring fibres were substantial, particularly at the rim of the epaxial and hypaxial muscles. The revealed muscle-fibre architecture at this age presumably contributes to the swimming performance of these larvae, but that swimming performance is probably not the only driving factor for the fibre pattern. Our methodology offers a promising avenue for exploring muscle-fibre orientations across ontogenetic series and provides a foundation for in-depth functional studies on the role of muscle architecture in facilitating swimming performance of larval fish.

Dataset Information

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies.

Publications

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies.

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