Crude Oil Impairs Cardiac Excitation-Contraction Coupling in Fish

Abstract

Expression was evaluated 7-10 days later. CHR2 and ARCH were excited with the 488nm and 514nm laser lines from an argon laser. Light power was controlled with an acousto-optic modulator, and focused onto selected areas (10-20mm) of the fibers via optic fibers. TPLSM imaging showed that CHR2 and ARCH were targeted to t-tubules and surface membranes. In fibers expressing CHR2, 1-3ms light pulses of increasing power elicited graded transient depolarizations until an action potential (AP) was triggered. The amplitude and FDHM of light-triggered APs were similar to those in response to current pulses. At all powers used, illuminating non-transfected fibers had no effect. Repetitive (<10Hz) pulsed illuminations elicited trains of APs with no failures. Failures at higher frequencies could be reduced by increasing the muscle fiber's input resistance (e.g. by blocking the chloride conductance with 9-AC), and/or increasing the illumination power. In voltage-clamped fibers, 40ms light pulses elicited slowly decaying inward currents with amplitudes graded with the applied illumination power. When fibers expressing ARCH were illuminated (at 514nm) otherwise suprathreshold current pulses became ineffective to trigger APs. Our results demonstrate that optogenetic tools can be used to excite or depress the excitability of adult skeletal muscle fibers. Since stem cell-derived cardiomyocytes will potentially play an important role in safety evaluations and drug screening in the future, we investigated cells from different stem cell suppliers with a new label-free technique based on impedance analysis. The used platform, called the CardioExcyte 96, allows precise impedance measurements at a time resolution of 1 ms and generates 96 recordings in parallel. Stem cells from GE Healthcare, Axiogenesis and Cellectis were successfully tested and data will be compared. Dose response curves or single-point screening experiments of several drugs (e.g. hERG and Cav1.2 blockers) were collected and will be presented. The capability of the data analysis software of the CardioExcyte 96 platform allows for a time effective analysis of data sets due to it's features and easeof-use. The presented data will show that the CardioExcyte 96 and the corresponding CardioExcyte Control software can be used efficiently to predict drug-induced arrhythmia for cardiac risk assessment. 3703-Pos Board B431The use of focused-ion-beam milling (FIB) to thin vitreously frozen cells for application of cryo-electron tomography is an emerging technology. However, successful application of cryo-FIB milling and tomography to frozen biological tissue has, to our knowledge, not been reported. This is because bulk tissue cannot be effectively frozen by simple plunge freezing of EM grids, but rather must be prepared by high-pressure freezing, which requires extra steps. The cryo-transfers that are required at each step of the process can cause devitrification, excessive frost contamination and mechanical damage, consequently resulting in unacceptably high failure rates. To ad...

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish

“…Also important is the interaction of individual components of complex PAH mixtures with multiple cellular targets in the fish heart (17-19, 21, 43), each of which may vary across species with different cardiovascular physiology. Crude oil HEWAFs similar to those used here were shown to block key voltage-gated potassium and calcium ion channels controlling excitation-contraction coupling in fish cardiomyocytes (22), and the precise impacts on cardiac function may depend on the relative levels of these targets in different species. In addition, results from studies on alkylated phenanthrenes and more weathered crude oil mixtures enriched with higher levels of alkylated tricyclic PAHs suggest mechanisms of cardiotoxicity that are downstream of aryl hydrocarbon receptor (AHR) activation in cardiac muscle cells (21,43).…”

“…Exposure of fish embryos to PAH mixtures derived from crude oil slows the heartbeat (bradycardia) and reduces contractility (17,(19)(20)(21). The underlying mechanism was recently shown to be blockade of key potassium and calcium ion channels involved in cardiac excitation-contraction coupling (22). These…”

Physical and Chemical Characterization of Crude Oil-Water Mixtures: Understanding the Effects of Interfacial Process to Chemical Bioavailability

“…The TPAH concentrations for all preparations for all the oils were compared to the most extensive compilation ( Several published studies have utilized the HEWAF methods and applied large dilution factors in order to reach potential environmentally realistic exposure to PAH concentrations similar to water samples collected from the GOM (Brette et al, 2014;Mager et al, 2014). Impaired cardiac responses on embryo-larval fish stages were found at 0.2% CTC HEWAF dilutions (1.2 µg/L TPAH) and swimming performance changes on juvenile Mahi-Mahi were reported for 2% CTC HEWAF dilutions (30 µg/L TPAH) .…”

“…Recently, new methods for the preparation of high-energy water accommodated fractions (HEWAFs) which consist of the formation of oil containing exposure media in the absence of chemical dispersants for both source and weathered oils have been described (Brette et al, 2014;Brewton et al, 2013;Incardona et al, 2013;Jung et al, 2013;Mager et al, 2014). These methods, invariably involve turbulent mixing of oil in seawater followed by a settling period to remove resurfacing oil droplets from the exposure media.…”

“…These methods, invariably involve turbulent mixing of oil in seawater followed by a settling period to remove resurfacing oil droplets from the exposure media. The HEWAF preparations using a commercial blender do provide the opportunity to create droplet-containing exposure media with weathered oils that are not easily dispersible but its implementation and application are highly dependent in its proper standardization and characterization since most reports in the literature do not seem to reflect a single protocol with respect to basic parameters such as mixing times or oil loadings as reflected by the oil to water ratios (Brette et al, 2014;Incardona et al, 2013;Mager et al, 2014).…”