Dataset Information

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

ABSTRACT: The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

SUBMITTER: Hou JH

PROVIDER: S-EPMC4161048 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

Hou Jennifer H JH Kralj Joel M JM Douglass Adam D AD Engert Florian F Cohen Adam E AE

Frontiers in physiology 20140911

The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a hi ...[more]

PMID: 25309445

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Project description:Pulsed focused ultrasound (pFUS) technology is being developed for clinical neuro/immune modulation and regenerative medicine. Biological signal transduction of pFUS forces can require mechanosensitive or voltage-gated plasma membrane ion channels. Previous studies suggested pFUS is capable of activating either channel type, but their mechanistic relationship remains ambiguous. We demonstrated pFUS bioeffects increased mesenchymal stem cell tropism (MSC) by altering molecular microenvironments through cyclooxygenase-2 (COX2)-dependent pathways. This study explored specific relationships between mechanosensitive and voltage-gated Ca2+ channels (VGCC) to initiate pFUS bioeffects that increase stem cell tropism. Methods: Murine kidneys and hamstring were given pFUS (1.15 or 1.125 MHz; 4MPa peak rarefactional pressure) under ultrasound or magnetic resonance imaging guidance. Cavitation and tissue displacement were measure by hydrophone and ultrasound radiofrequency data, respectively. Elastic modeling was performed from displacement measurements. COX2 expression and MSC tropism were evaluated in the presence of pharmacological ion channel inhibitors or in transient-receptor-potential-channel-1 (TRPC1)-deficient mice. Immunohistochemistry and co-immunoprecipitation examined physical channel relationships. Fluorescent ionophore imaging of cultured C2C12 muscle cells or TCMK1 kidney cells probed physiological interactions. Results: pFUS induced tissue deformations resulting in kPa-scale forces suggesting mechanical activation of pFUS-induced bioeffects. Inhibiting VGCC or TRPC1 in vivo blocked pFUS-induced COX2 upregulation and MSC tropism to kidneys and muscle. A TRPC1/VGCC complex was observed in plasma membranes. VGCC or TRPC1 suppression blocked pFUS-induced Ca2+ transients in TCMK1 and C2C12 cells. Additionally, Ca2+ transients were blocked by reducing transmembrane Na+ potentials and observed Na+ transients were diminished by genetic TRPC1 suppression. Conclusion: This study suggests that pFUS acoustic radiation forces mechanically activate a Na+-containing TRPC1 current upstream of VGCC rather than directly opening VGCC. The electrogenic function of TRPC1 provides potential mechanistic insight into other pFUS techniques for physiological modulation and optimization strategies for clinical implementation.

Dataset Information

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

Publications

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

Similar Datasets

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