Oil Exposure Impairs In Situ Cardiac Function in Response to Î²

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Oil exposure impairs in situ cardiac function in response to #-adrenergic stimulation in Cobia (Rachycentron canadum) Georgina Kimberly Cox, Dane A Crossley, John D. Stieglitz, Rachael M Heuer, Daniel D. Benetti, and Martin Grosell Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03820 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 17, 2017

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Environmental Science & Technology

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Oil exposure impairs in situ cardiac function in response to β-adrenergic stimulation

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in Cobia (Rachycentron canadum)

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Georgina K Cox,*,δ Dane A. Crossley IIϒ, John D. Stieglitzφ, Rachael M. Heuerδ, Daniel D.

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Benettiφ and Martin Grosellδ

7 8 9 10

δ Department

of Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric

Sciences, University of Miami, Miami, Florida 33149-1098, United States

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ΥUniversity

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Denton, Texas 76203 United States

of North Texas, Department of Biological Sciences, 1155 Union Circle

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φDepartment

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Atmospheric Sciences, University of Miami, Miami, Florida 33149-1098, United States

of Marine Ecosystems and Society , Rosenstiel School of Marine and

17 18 19 20 21 22 23 Corresponding Author: Georgina K. Cox Phone: 305 421 4366 Email: [email protected]

ACS Paragon Plus Environment


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Abstract Aqueous crude oil spills expose fish to varying concentrations of dissolved

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polycyclic aromatic hydrocarbons (PAHs), which can have lethal and sub-lethal effects. The

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heart is particularly vulnerable in early life stages, as PAH toxicity causes developmental

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cardiac abnormalities and impaired cardiovascular function. However, cardiac responses of

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juvenile and adult fish to acute oil exposure remain poorly understood. We sought to assess

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cardiac function in a pelagic fish species, the cobia (Rachycentron canadum), following

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acute (24 hr) exposure to two ecologically relevant levels of dissolved PAH’s. Cardiac

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power output (CPO) was used to quantify cardiovascular performance using an in situ heart

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preparation. Cardiovascular performance was varied using multiple concentrations of the

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β-adrenoceptor agonist isoproterenol (ISO) and by varying afterload pressures. Oil

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exposure adversely affected CPO with control fish achieving maximum CPO’s (4 mW g-1

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Mv) greater than that of oil-exposed fish (1 mW g-1 Mv) at ISO concentrations of 1 x 10-6 M.

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However, the highest concentration of ISO (1 x 10-5 M) rescued cardiac function. This

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indicates an interactive effect between oil-exposure and β-adrenergic stimulation and

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suggests if animals achieve very large increases in β-adrenergic stimulation it could play a

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compensatory role that may mitigate some adverse effects of oil-exposure in vivo.

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Introduction

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Exploration and removal of subaqueous oil has lead to >10 very large (>90,000

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tonnes) ocean spills since 1980. The largest in US history, the Deepwater Horizon oil spill in

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2010, discharged 627,000 tonnes of oil into the Gulf of Mexico off the south-eastern coast of

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Louisiana1 impacting the ecosystem. Exposure to dissolved polycyclic aromatic

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hydrocarbons (PAHs), the toxic components of oil, causes lethality and numerous sub-

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lethal effects in aquatic organisms2. The cardiovascular system, the heart in particular, has

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been shown to be particularly sensitive to PAH toxicity in fish3,4. Exposure to PAHs at the

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embryonic phase results in developmental abnormalities such as pericardial edemas,

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abnormal heart looping, and the impairment of cardiac neural crest cells5–9. Surviving

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embryos show reductions in cardiac function in the form of arrhythmias, reduced heart

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rates, and compromised contractility10–12 ultimately resulting in reduced cardiac output 13.

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Even when surviving embryos are raised in clean seawater following brief exposure during

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embryonic development, there are abnormalities detected in overall heart morphology and

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reductions in swim performance are detected in later life stages 8,14.

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Comparatively less is known about cardiovascular responses to acute oil exposure

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in juvenile and adult fish. It has been proposed that the reduced swimming performance in

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juvenile and adult mahi-mahi (Coryphaena hippurus) exposed to PAH’s 8,15 can be

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attributed to reductions in cardiovascular performance. Indeed, reductions in stroke

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volume, contractility, and cardiac power output in mahi-mahi have been reported from in

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situ heart preparations conducted on anaesthetized fish16. While these results show that oil

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exposure compromises routine cardiovascular function, the consequences of oil exposure



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on maximal cardiac function, relevant for swim performance, have not been assessed and

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the depth to which cardiac function is compromised when demands on cardiac

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performance increase remains unanswered.

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In salmonids, in vivo cardiac output can increase 90 to 300% during swimming

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through significant increases in heart rate and stroke volume17,18. Cardiac power output

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(CPO), the product of pressure and flow generation, likewise shows significant increases in

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response to exercise in vivo and increases in cardiac afterload pressures in situ19,20. These

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increases in cardiac performance are often accomplished, in part, via β-adrenergic

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stimulation, controlled through the release of intrinsic stores of catecholamines21,22.

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Catecholamines are released during bouts of physiological stress (such as intense exercise)

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and significantly increase cardiac performance via positive inotropic (force) and

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chronotropic (rate) effects by binding to β -adrenergic receptors in the heart 21.

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Cardiovascular responses to β-adrenergic stimulation are critical for the regulation of

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cardiac function, and thus convective oxygen transport, in response to increasing energy

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demands. In this study we test the hypotheses that oil exposure impairs cardiac power

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output and that β-adrenergic stimulation mitigates adverse cardiac effects from oil

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exposure. To test these hypotheses we quantified in situ routine and maximal cardiac

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performance in oil exposed cobia (Rachycentron canadum) and determined if β-adrenergic

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stimulation protects cardiac function following oil exposure. Wild cobia are highly migratory

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and were very likely affected by the spill. Hatchery raised fish that were used in this study were un-

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affected but were used as a model for the wild population.

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Material and methods

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Experiments were conducted at the University of Miami Experimental Hatchery

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(UMEH, Miami, FL, USA) from October 1st to December 10th 2016. Animal housing and

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experimental procedures were approved by the University of Miami’s Institutional Animal

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Care and Use Committee (IACUC Protocol number 15-019) and followed all applicable laws

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and regulations.

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Fish

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Prior to experimentation hatchery raised cobia (Rachycentron canadum, n= 24) of

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both sexes (0.22 ± 0.01 kg body mass; range 0.14 -0.38 kg) were housed in several 4.5 m3

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outdoor cylindrical fiberglass tanks supplied with filtered flow-through seawater (24-

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30°C). Fish were exposed to a natural photoperiod, though tanks were shaded to protect

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from direct sunlight. Fish were fed daily with a mixture of squid, mackerel, sardines, and

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pelletized diets but fasted for at least 24 hr prior to experimental use.

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Experimental Conditions

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Individual cobia were removed directly from holding tanks and then held for 24 hr in

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covered, oxygenated, static 300L cylindrical tanks. Fish in the static tanks were exposed to

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either control seawater (n = 8), oiled seawater containing 10% of a high energy water

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accommodated fraction of crude oil (HEWAF, n = 8), or oiled seawater containing 20%



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HEWAF (n = 8). These HEWAF concentrations were chosen based on reported levels

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following the oil spill23,24. HEWAF solution preparation has been described previously by

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Mager et al., 2014 8. The HEWAF mixture was added to the experimental tanks within 24

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hours of preparation. The crude oil used to make the HEWAF in this study was collected via

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skimming from surface waters by British Petroleum on July 29, 2010 at the site of the

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Deepwater Horizon oil spill and subsequently transferred under chain of custody to the

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University of Miami (sample ID: OFS-20100719-Juniper-001 A0087P & A00919).

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Temperature in both control and oiled static tanks was maintained at 26± 1 ˚C using a

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1000-watt submersible heating system (Innovative Heat Concepts QDPTY1-1, Homestead,

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FL, USA). Oxygen was supplied at a flow rate of ~1 l·min-1. A small submersible aquarium

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pump (Rio©+ 800, Technological Aquatic Associated Manufacturing, Camarillo, CA USA)

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ensured adequate water circulation.

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Water quality measurements in the static tanks were taken at the beginning and end

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of the 24 hr exposure period. These measurements included temperature and dissolved

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oxygen (ProODO, YSI, Inc., Yellow Springs, OH), pH (PHC3005, Radiometer, France), salinity

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(Pentair Aquatic Ecosystems, Apopka, FL), and total ammonia. Total ammonia analyses

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were conducted by colorimetric assay25. Additionally, initial and final water samples were

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collected for total (∑) PAH analysis (S.I. table 1). The percent distribution of individual

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PAH’s was similar among all oil exposures (S.I. fig. 1) and similar to the profiles found in the

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Gulf of Mexico during the spill. Reported ΣPAH values represent the sum of 50 PAH

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analytes, selected by the Environmental Protection Agency based on individual toxicity and

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concentration. Samples were analyzed by using gas chromatography and selective ionic


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monitoring mass spectrometry within seven days of collection by ALS Environmental (ALS

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Environmental, Kelso, WA, USA).

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Surgical Procedures and cardiovascular parameters

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Following the 24 hour exposure in the static tanks, each fish was transferred to a 12

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L plastic tank containing oxygenated seawater with 200 mg·l-1 tricaine methane sulphonate

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(MS-222, Sigma-Aldrich, St. Louis, MO) buffered to neutral pH with 100 mg·l-1 sodium

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bicarbonate (NaHCO3, Sigma-Aldrich, St. Louis, MO) for anesthesia. Once the righting reflex

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was lost, the fish was transferred to a custom surgical table and placed ventral side up. A

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plastic tube was placed in the mouth of the fish to perfuse the gills at a rate of 5 l·min-1,

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with oxygenated seawater pumped (Rio©+ 800, Technological Aquatic Associated

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Manufacturing, Camarillo, CA USA) from a reservoir containing a surgical anesthetic

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concentration (100 mg·L-1). The in situ perfused heart preparation with an intact

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pericardium has been previously described21,26. Briefly, a hepatic vein leading into the

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heart was cannulated and all other (set at 5 cm) s to the heart were tied off. This allowed

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for control of flow rate, input pressure, and chemical composition of the input saline that

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flowed through the heart. Perfusing saline was composed of (in mmol L-1) 181 NaCl, 7 KCl,

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1.1 MgSO4 7H2O, 1.9 CaCl2 2H2O, 5.6 glucose, 8.8 HEPES Free Acid, and 18.5 HEPES Na Salt

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(Sigma-Aldrich). The outflow tract of the heart was then cannulated and connected to a

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flow probe in line with an afterload column (Transonic Systems Inc., Ithaca, NY, USA)

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allowing direct measurement of cardiac output (ml·min-1), heart rate (bpm) and stroke

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volume (ml· min-1 ·beat-1). Adjusting the height of the output cannula controlled output



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(afterload) pressure. Input and output pressures are measured with a pressure transducer

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(ADinstruments model MLT0699, CO, USA) calibrated against a static water column. Total

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output pressure was calculated as the output pressure minus the input pressure.

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Resistance of the cannula was accounted for in the final calculations of total output

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pressure. CPO was calculated as the product of cardiac output (ml·sec-1) and total output

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pressure (with a conversion factor of 0.0981 to convert to ml cm H2O·sec-1 to mW, 1kPa =

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10.2 cm H2O) divided by ventricular mass (g). All data was recorded using a Power Lab

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unit (ADInstruments, Castle Hill, Australia) connected to a laptop computer running

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LabChart Pro software (v. 7.3.7 ADInstruments, CO USA) for subsequent analysis.

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Assessment of cardiac performance

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Following instrumentation the preparation was connected to a temperature controlled

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(26± 1 ˚C) saline reservoir that contained 1 x 10-8 M concentration of isoproterenol and

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allowed to stabilize for 20 min with output pressure set at 1 kPa (fig. 1). ISO was used

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instead of the catecholamines adrenalin or noradrenalin because it is more stable and has a

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similar affinity for β-adrenoceptors27. ISO concentrations were selected based on published

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values of ISO and adrenaline in trout and tuna 28–30. To assess maximal cardiac performance

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a “max test” was performed. During this test, output pressure was raised in 1 kPa

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increments. Cardiac function was allowed to stabilize at each increment up to 5 kPa. If

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cardiac output dropped by > 15% before a 5 kPa output pressure was reached the max test

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was terminated and output pressure was returned to 1 kPa. Each assessment of maximal

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cardiac performance lasted ~ 8 min. Following this, the input saline was changed to a 10-7


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M ISO concentration, allowed to recover for 15 min, and then the max test was repeated.

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The procedure was then repeated at ISO concentrations of 10-6 M and 10-5 M. The 10-8 M

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ISO concentration was insufficient to elicit routine cardiovascular function in several of the

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control and HEWAF 10% fish during the rest period as indicated by abnormal flow traces

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and arrhythmias. Fish showing abnormal flow traces or arrhythmias were immediately

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switched to a 10-7 M ISO concentration. As such the analysis of cardiovascular function in

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response to oil exposure and β-adrenergic stimulation was assessed at concentrations of

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10-7 to 10-5 M ISO, where cardiac function in all control fish was supported.

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Analysis of cardiovascular data

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Linear mixed effects models were used to assess the effect of oil exposure and 3 ISO

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treatments and the interaction between these factors. The 10-8 M ISO treatment was

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excluded as not all control fish could operate at this level of β-adrenergic stimulation. Oil

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exposure and ISO treatment were treated as fixed effects and individual as random effect

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(e.g. lme(response ~ oil exposure * ISO treatment, random = individual)) (‘lmer’ package,

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R). We assessed the significance of each fixed effect and the interaction between them

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using a type III ANOVA. Significance was set at p ≤ 0.05 and data is reported as mean ± SEM.

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Results and Discussion

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In this study we sought to determine if β-adrenergic stimulation could offset the documented reduced cardiac performance observed following oil exposure in other



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teleosts 31. An interactive effect was detected between oil exposures and ISO concentration

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as evident in the 20% HEWAF treatment group’s reduced ability to respond to β-adrenergic

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stimulation (S.I. table 2). As previously shown for other fish species β-adrenergic

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stimulation increased the power generating capacity of the heart in control cobia30,32–34 and

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this was also found for the HEWAF exposed treatment groups.

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Direct effects of oil on cardiovascular function

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Exposure to PAH’s has been shown to adversely affect cardiovascular function in

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fish at early life stages, juveniles and adults 8,15,16. Similar to other studies utilizing

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Deepwater Horizon oil, HEWAF exposures in this study had a high proportion of 3 ring

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PAH’s (S.I. fig. 2), which have been shown to produce the strongest negative effects35. The

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percent distribution of individual PAHs was similar among all oil exposures (S.I. fig. 1).

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Geometric means for the 10% and 20% HEWAF’s are 14.1 ± 1.4 ug L-1 and 21.6 ± 3.6 ug L-1,

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respectively.

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Previous work from our group has shown that acute exposure to HEWAF’s

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negatively impacts in situ cardiovascular function through reductions in stroke volume and

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cardiac output in mahi-mahi, a close relative of the cobia 16. In contrast, we found

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significant direct effects on cardiac performance in cobia acutely exposed to the 20%

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HEWAF only at the 10-6M ISO concentration (Fig 3). However, fewer hearts from oil

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exposed individuals were able to maintain cardiac output at high afterload pressures

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compared to control individuals at the lower ISO concentrations and failed to complete the

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full max test (afterload pressures of 50 cmH2O; Fig 2A-D). In comparison, the majority of


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control fish were able to maintain cardiac output at afterload pressures of 5 kPa. At the 10-7

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M ISO concentration, only one of the 20% HEWAF hearts was able to complete the full max

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test (Fig 2B). Although half of the 10% HEWAF group were able to reach 5 kPa at the 10-7 M

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ISO concentration, two of these hearts were unable to recover from the maximal exertion

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(Figure 2A, B). At the 10-6 M ISO concentration, oil exposure continued to affect the number

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of individual hearts capable of maintaining flow generation in the face of increasing output

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pressures (Fig 2B). At 10-5 M ISO concentration, however, all fish tested were able to

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maintain cardiac output at pressures up to 5 kPa (Fig 2C). The context dependent nature of

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the effects of oil exposure we observed suggests that, although the impacts of oil exposure

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are considerable, they are not pervasive across physiological parameters. This supports

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data found previously for European bass and trout exposed to crude oil36,37. This data also

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suggests that adverse effects of oil exposure may only be visible under moderate levels of

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physiological stress.

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The effect of β -stimulation on cardiac performance

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At a cellular level oil exposure reduces cardiac function by adversely affecting

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excitation-contraction coupling, blocking of potassium channels, and disrupting calcium

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cycling 10. As the binding of β-adrenoreceptor agonists, such as adrenaline and

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noradrenalin, increases cardiac function in teleost fish species 30,32,33 through increased

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calcium influx via sarcolemma-bound L-type channels38, we hypothesized that β-

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stimulation could improve cardiac function in oil-exposed fish. Dose-response curves

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conducted on tuna species indicate an operational range of 10-8 to 10-5 M 26 of β-



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stimulation, which is consistent with in vivo measurements of adrenaline levels in tuna39.

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The typical range for circulating adrenalin in stressed rainbow trout is typically lower,

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from 10-9 M to 10-7 M concentrations28,30,40,41. The concentration of circulating

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catecholamines is unknown for cobia but the 10-5 M concentration may lie above what is

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physiologically relevant for this species. Further it should be acknowledged that the

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myocardium of trout is more sensitive to ISO compared to adrenaline30. Our data illustrate

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that concentrations greater than 10-8 M ISO are required to produce routine cardiac

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function of in situ cobia hearts, which is similar to previous findings in other highly athletic

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fish33. Fitting with expectations, β -stimulation above this threshold had strong and

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significant direct effects on all metrics of cardiac performance, including stroke volume,

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heart rate, and CPO as it does in other teleost fish. We found a significant effect of ISO on

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heart rate (F2,37=24.05, p

Oil Exposure Impairs In Situ Cardiac Function in Response to Î²

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