A Comparative Study of Embedded and Anesthetized Zebrafish in vivo on Myocardiac Calcium Oscillation and Heart Muscle Contraction

Abstract

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

In parallelinterconnectivity of the dorsal longitudinal anastomotic vessels requires both VEGF signaling and circulatory flow

“…S4, and data not shown), indicating that circulation promotes DLAV plexus morphogenesis. We next abrogated circulatory flow on demand by stopping the heartbeat with the action potential blocker tricaine (Muntean et al, 2010) or the myosin ATPase inhibitor BDM/2,3-butanedione 2-monoxime (Forster et al, 2004), as in Nicoli et al (Nicoli et al, 2010). Surprisingly, we found that post-DLAV formation (32-48 hpf) circulatory flow abrogation with either tricaine or BDM prevented, rather than impaired, DLAV plexus morphogenesis in WT embryos.…”

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

“…Zebrafish (Danio rerio) adults and embryos were kept at 28.5°C and were handled according to established protocols (Nüsslein-Volhard and Dahm, 2002) and in accordance with EU directive 2011/63/EU as well as the German Animal Welfare Act. Transgenic lines Tg(kdrl:GFP) (Jin et al, 2005) and Tg(myl7:GFP) (Huang et al, 2003) were used.…”

“…Low-melting-point agarose (Sigma) and methylcellulose (Sigma) were diluted in E3 (Nüsslein-Volhard and Dahm, 2002). For embedding in 1.5% (w/v) agarose, embryos were placed in liquid agarose, drawn into a glass capillary with Teflon-coated plungers (BRAND) and extruded after polymerization.…”

“…Tricaine concentrations ranging from 100 mg/l (Tsutsui et al, 2010) up to 750 mg/l (Truong et al, 2011) have been used as sedative, although as little as 30 mg/l Tricaine can influence embryonic development (Kimmel et al, 1995). Tricaine suppresses the contraction of not only skeletal muscles but also of smooth and cardiac muscles, resulting in reduced heart rate (Muntean et al, 2010) and affecting developmental processes that are influenced by hemodynamics (Culver and Dickinson, 2010).…”

Monosodium Glutamate Exposure at Early Developmental Stage Increases Apoptosis and Stereotypic Behavior Risks on Zebrafish (Danio Rerio) Larvae

“…Intracellular Ca 2+ level was determined in 72hpf embryos, post-treatment, incubated in 60µg/mL Fura-2AM (Sigma Aldrich, Cat# 47989) containing 2.5% DMSO in embryo medium for 30min at room temperature (Muntean et al, 2010). After washing embryos were mounted in 3% methylcellulose (Sigma Aldrich) and positioned for confocal laser scanning microscopic imaging (Olympus IX81 and FluoViewFV1000).…”