Impacts of Deepwater Horizon Crude Oil on Mahi-Mahi (Coryphaena

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Ecotoxicology and Human Environmental Health

Impacts of Deepwater Horizon crude oil on mahimahi (Coryphaena hippurus) heart cell function Rachael M. Heuer, Gina L.J. Galli, Holly A. Shiels, Lynne A. Fieber, Georgina K. Cox, Edward Mager, John D. Stieglitz, Daniel D. Benetti, Martin Grosell, and Dane A. Crossley Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03798 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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Environmental Science & Technology

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TITLE

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Impacts of Deepwater Horizon crude oil on mahi-mahi (Coryphaena hippurus) heart cell

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function

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AUTHOR NAMES

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Rachael M. Heuer*†§, Gina L.J. Galli‡, Holly A. Shiels‡, Lynne A. Fieber§, Georgina K.

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Cox§⊥, Edward M. Mager§†, John D. Stieglitz♯, Daniel D. Benetti♯, Martin Grosell§, Dane

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A. Crossley II†

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AUTHOR ADDRESSES

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†Department

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Street, Denton Texas 76203, USA

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‡University

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University of Manchester, Core Technology Facility, Grafton Street, Manchester M13

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9PL, UK

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§Department

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Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, Florida 33149,

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USA

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⊥Department

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Ontario, Canada, N1G 2W1

of Biological Sciences, University of North Texas, 1511 W. Sycamore

of Manchester, Faculty of Biology, Medicine and Health Sciences, The

of Marine Biology and Ecology, University of Miami Rosenstiel School of

of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph,

1 ACS Paragon Plus Environment


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♯Department

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of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, Florida

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33149, USA

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of Marine Ecosystems and Society, University of Miami Rosenstiel School

ABSTRACT

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Deepwater Horizon crude oil is comprised of polycyclic aromatic hydrocarbons that

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cause a number of cardiotoxic effects in marine fishes across all levels of biological

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organization and at different life stages. Although cardiotoxic impacts have been widely

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reported, the mechanisms underlying these impairments in adult fish remain

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understudied. In this study, we examined the impacts of crude oil on cardiomyocyte

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contractility and electrophysiological parameters in freshly isolated ventricular

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cardiomyocytes from adult mahi-mahi (Coryphaena hippurus). Cardiomyocytes directly

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exposed to oil exhibited reduced contractility over a range of environmentally-relevant

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concentrations (2.8-12.9 g l-1 ∑PAH). This reduction in contractility was most

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pronounced at higher stimulation frequencies, corresponding to the upper limits of

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previously measured in situ mahi heart rates. To better understand the mechanisms

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underlying impaired contractile function, electrophysiological studies were performed,

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which revealed oil exposure prolonged cardiomyocyte action potentials and disrupted

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potassium cycling (9.9-30.4 g l-1 ∑PAH). This study is the first to measure cellular

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contractility in oil-exposed cardiomyocytes from a pelagic fish. Results from this study

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contribute to previously observed impairments to heart function and whole-animal

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exercise performance in mahi, underscoring the advantages of using an integrative

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approach in examining mechanisms of oil-induced cardiotoxicity in marine fish.

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INTRODUCTION The Deepwater Horizon oil spill (2010) was the largest offshore marine oil spill in

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U.S. history, releasing more than 3 million barrels of crude oil into the Gulf of Mexico

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over 87 days1-3. The location and timing of the Deepwater Horizon spill overlapped with

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the habitat utilization patterns of many important pelagic fish species in the Gulf of

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Mexico, including Atlantic bluefin tuna, yellowfin tuna, and mahi-mahi4-7. This is

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important because the polycyclic aromatic hydrocarbons (PAHs) present in crude oil

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have been demonstrated to impair cardiac function in marine fish, both across species

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and across life stages8-15.

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Over the last decade, the mahi-mahi (Coryphaena hippurus: referred to herein as

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“mahi”) has served as a valuable model for pelagic fish, facilitating studies on the

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impacts of oil exposure across levels of biological organization. Mahi exposed to oil

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have compromised swim performance, including reductions to maximal sustained

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swimming speed16, 17 and reduced maximal metabolic rate17. Further examination of

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heart function revealed that short-term oil exposure (24 hours) was found to reduce

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stroke work, stroke volume, and cardiac output in an in situ preparation12.

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Although cardiotoxic effects of oil have been widely reported, few studies have

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examined the cardiac cellular mechanisms underlying observed whole animal and

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organ-level impacts18, 19. Cardiomyocyte function is dependent on excitation-contraction

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coupling which consists of three key events: (1) The generation of an action potential,

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(2) followed by a mobilization of calcium from extracellular and intracellular sources, (3)

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which initiates cross-bridge formation and contraction at the myofilaments20, 21.

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Previous studies examining the impacts of oil on cardiomyocytes from Pacific bluefin



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tuna, yellowfin tuna, and Pacific mackerel, have exclusively focused on the first two

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components of excitation-contraction coupling18, 19. These studies have demonstrated

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that PAH exposure causes prolonged action potentials, diminished calcium transients,

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disruptions to sarcoplasmic reticular calcium cycling, and a reduction of the potassium

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current (IKr) necessary for repolarization18, 19. While these findings inferred reduced

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cardiomyocyte contractility, direct measurements of contractility following oil exposure

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have not previously been examined in any fish.

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Accordingly, the first objective of this study was to examine mahi cardiomyocyte

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contractility over a range of environmentally-relevant oil exposures, with the expectation

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that oil would cause a dose-dependent decrease in isolated cardiomyocyte contractile

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function. The second objective was to examine the impacts of oil on contractility over a

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range of stimulation frequencies representative of heart rates measured in whole-organ

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in situ studies on mahi12, with the expectation that oil would exacerbate impaired

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contractile function at higher stimulation frequencies. The third objective was to quantify

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electrophysiological properties of mahi cardiomyocytes over a range of oil

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concentrations to gain mechanistic insight into the oil-induced disruptions to excitation-

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contraction coupling.

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MATERIALS AND METHODS Experimental animals Adult mahi (F1 generation) (n=10-15) ranging from 0.3-0.91 kg (Supporting

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Information, SI, Table S1) were raised from wild-caught broodstock and maintained in

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two 3000L fiberglass tanks at the University of Miami Experimental Hatchery (UMEH)22.

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Fish were maintained on flow-through seawater (25-29°C), and fed daily with a mix of 4 ACS Paragon Plus Environment

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squid, mackerel, and silversides. All animal care and experimental procedures were

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approved by the University of Miami’s Institutional Animal Care and Use Committee

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(IACUC protocol 15-019).

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Ventricular cardiomyocyte isolation

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Myocyte isolations were performed as in previous studies23, 24. Mahi were

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stunned by a blow to the head then euthanized by pithing, and the heart (n=10-15; SI

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Table S1) was extracted and retrograde perfused with isolation solution (SI Table S2)

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for 10 minutes, followed by perfusion with proteolytic enzymes (Trypsin Type 1X 0.5 mg

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ml-1, Collagenase Type 1A 0.37-0.43 mg ml-1; Sigma-Aldrich) and fatty-acid free bovine

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serum albumin (0.75 mg ml-1; Sigma-Aldrich) for 10-25 minutes. The ventricle was then

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transferred into fresh isolation solution and triturated to free individual cardiomyocytes.

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Cardiomyocytes were stored in fresh isolation solution up to 8 hours following isolation.

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Oiled saline preparation and PAH analysis

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Surface oil collected from the Deepwater Horizon oil spill (Sample ID: OFS-

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20100719-Juniper-001: 00979, 00919) was transferred to the University of Miami under

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chain of custody and used to prepare all high energy water accommodated fractions

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(HEWAF). To prepare HEWAF saline needed for dilutions, 1 g of oil was added to 1 liter

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of control extracellular saline (referred to herein as “saline,” SI Table S2) at room

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temperature, blended in a Waring CB15 industrial blender (Torrington, CT) for 30

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seconds at low speed, and transferred to 1-liter separatory funnel for 1 hour. The

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bottom 90% of this mixture was used as HEWAF saline. Unfiltered dilutions of HEWAF

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salines were used for Objective 3. For Objectives 1 and 2, each HEWAF saline was

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filtered through two stacked 0.3 m 90 mm glass fiber filters with a vacuum pump under



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low suction25 prior to preparing dilutions. Filtering was necessary to prevent oil micro-

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droplets from interfering with the IonOptix imaging system.

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Samples for ∑PAH analysis were taken immediately after HEWAF was made

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and sent to ALS Environmental (Kelso, WA) for analysis by gas chromatography/mass

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spectrometry with selective ion monitoring (GC/MS-SIM; based on EPA method 8270D).

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Reported ∑PAH values represent the sum of 50 selected PAH analytes dominated by 3-

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ring PAHs (SI Figures S1-S4). In a subset of samples (n=4), unfiltered and filtered PAH

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analyses were performed from the same HEWAF saline to compare ∑PAH

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concentration and PAH composition (SI Figures S5-S7; “HEWAF salines”)

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Cardiomyocyte oil exposure

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PAH concentrations corresponding to HEWAF dilutions are summarized in Table

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1. Filtered HEWAF saline was diluted to nominal concentrations of 10, 20, or 40% in

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control saline for Objective 1, and based on these findings, a 10% filtered HEWAF was

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chosen for Objective 2. Since HEWAF saline was unfiltered for Objective 3, dilutions of

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1, 2, and 5% were used. Validation of nominal dilutions (Table 1) were obtained using

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an established fluorescence method26 as described in Esbaugh et al 201625 (SI,

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“Fluorescence measurements”). Samples for fluorescence in Objectives 1 and 2 were

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collected directly from the microscope stage to calculate dilutions for each cell. In a

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small subset of volume-limited samples, nominal dilutions were used. In Objective 3,

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samples were collected directly from HEWAF saline nominal dilutions before and after

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they had passed through the perfusion chamber and measured using fluorescence.

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Procedures for cleaning and testing the experimental stage after oil exposure can be

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found in SI section “Microscope stage cleaning.”


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Experimental protocols: Objectives 1 and 2

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Sarcomere length and other kinetic properties of mahi cardiomyocyte shortening

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were recorded in real-time using an IonOptix Myocyte Contractility system comprised of

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a perfusion chamber (FHDRCC1, IonOptix) mounted to an inverted microscope (Motic

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AE31) attached to a specialized fast digitizing dimensioning camera (MyoCam-S,

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IonOptix). The perfusion chamber was instrumented with electrodes connected to a

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MyoPacer stimulator (IonOptix) for field stimulation to induce myocyte shortening. The

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average sarcomere length was determined using SarcLens within IonWizard 6.0

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software, which uses the A- and I- bands as a markers of sarcomere spacing within a

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user-defined region of interest and applies a fast Fourier transform to determine the

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mean spacing frequency. IonOptix software was used to analyze sarcomere shortening

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(our index of cell contractility) in addition to the other kinetic properties of mahi

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cardiomyocyte shortening including departure velocity, return velocity, time to peak

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contraction (50 and 70%), time to recovery (50% and 70%), time at maximal rate of

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departure, and time at maximal rate of return.

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To obtain an individual cell recording, a small set of cardiomyocytes were gently

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transferred to the perfusion chamber and allowed to settle on the bottom for 5-10

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minutes prior to perfusion with control saline (SI Table S2). Then, a randomly selected

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healthy cell was chosen for an individual cell recording. To assess changes in

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sarcomere shortening at different oil concentrations (Objective 1), cardiomyocytes were

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stimulated at 0.5 Hz and perfused with control saline followed by either an identical

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control saline or dilutions of HEWAF saline (10, 20, or 40%; Table 1). During Objective

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2, all cardiomyocytes were stimulated at 0.5 Hz and perfused with control saline and



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followed by a switch to either an identical control saline or a 10% filtered HEWAF saline

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dilution at the same frequency (0.5Hz; Table 1). After 1-2 minutes following the saline

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switch, cardiomyocytes were stimulated at progressively increasing frequencies (1.5,

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2.0, 2.5, 3.0 Hz, 12 times/frequency) that represented a range of heart rates reported in

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anesthetized mahi in an in situ heart preparation (~100-180 bpm)12. The average of the

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middle 6 contractions from each frequency were used for analysis27. Cardiomyocytes

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were then returned to 0.5 Hz to assess whether or not the cell experienced reduced

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performance over the course of the experiment due to repeated stimulation.

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Experimental protocols: Objective 3

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A sample of cardiomyocytes were placed in a recording chamber (RC-24,

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Warner Instruments) and superfused with control solution at room temperature (SI

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Table S2). Ventricular action potentials (APs), IKr (delayed rectifier current), and IK1

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(inward rectifier current responsible for setting the membrane potential) were measured

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under control conditions followed by increasing dilutions of unfiltered HEWAF saline: 1,

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2 and 5% (Table 1). Cardiomyocytes were then returned to control conditions to assess

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recovery from oil exposure.

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Experiments were performed using Molecular Devices Axopatch 200A and 200B

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amplifiers connected to computers using 1140A A-to-D converters. Pipettes had a

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resistance of 4.3 ± 0.2 MΩ when filled with pipette solution. Pipette solutions for K+

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channel and action potential measurements are reported in SI Table S2. Junction

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potentials were zeroed prior to seal formation. Pipette capacitance was compensated

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after formation of a GΩ seal. Mean series resistance was 5.2 ± 0.8 MΩ. Membrane

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capacitance was measured using the calibrated capacity compensation circuit of the


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Axopatch amplifier. Signals were low-pass filtered using the 4-pole low pass Bessel

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filter at a frequency of 2 kHz and were then analyzed off-line using pClamp 10.0

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software (Axon Instruments). To estimate cardiomyocyte surface area and normalize

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current density, total membrane capacitance (Cm) was recorded in each cardiomyocyte

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tested.

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Action potential and K+ channel recordings

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Action potentials from isolated cardiomyocytes were recorded in current clamp

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mode of the Warner patch clamp amplifier. Action potentials were elicited by 2 ms

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depolarizing voltage pulses (0.1-0.7 mV). K+ channel recordings were obtained by

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adaptation of protocols previously designed by Vornanen and colleagues28, 29.

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IKr and IK1 were isolated via pharmacological inhibition by blocking Na+, Ca2+, and ATP-

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sensitive K+ channels with tetrodotoxin (0.5µM), nifedipine (10 µM), and glibenclamide

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(10 µM), respectively29.

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In preliminary experiments, the voltage eliciting maximum IKr tail currents was

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assessed by a depolarizing 4 sec pre-pulse from 80mV to +60 mV followed by a 3

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second test pulse between -100 mV and +40 mV. Similar to results obtained from a

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previous study29, the maximum IKr tail current occurred at -20 mV. Therefore, when

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measuring the current-voltage relationship for IKr, -20 mV was applied as the test pulse

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voltage.

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Current-voltage relationship for IKr was measured as the tail current at -20 mV

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following a series of pre-pulses between -80 and +80 mV in the absence and presence

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of the specific IKr inhibitor, E-4031 (2 μM). IKr was measured as an E-4031-sensitive

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current. Following blockade of IKr, IK1 was measured by repolarizing voltage ramps



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(1sec) from 30 mV to -120 mV with 5 sec intervals, in the presence and absence of

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BaCl2 (0.5 mM). IK1 was then determined as the barium (Ba2+) sensitive difference in

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current.

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Statistical Analyses

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In Objective 1, the percent change in sarcomere shortening between saline 1

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(control) and saline 2 (either control, 10%, 20% or 40% HEWAF saline) was assessed

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using a one-way ANOVA. All other parameters were statistically assessed using paired-

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t-tests or signed-rank tests with Bonferonni corrections comparing raw values from the

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first and second saline (SI Table S5). Absolute values used to calculate percent

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changes are reported in SI Table S3 and Table S4 for Objective 1 and Objective 2,

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respectively. In Objective 2, the percent of sarcomere shortening during control saline 1

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was set at 100% at 0.5 Hz, and all shortening values at subsequent frequencies were

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expressed as a percent decline from this original value. Transformed (squared) percent

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values were assessed using a two-way repeated measures ANOVA, using frequency

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and treatment as factors. All other biophysical parameters were also assessed using a

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two-way repeated measures ANOVA, using frequency and treatment as factors (SI

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Table S6). Holm-Sidak post-hoc tests were performed following two-way repeated

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measures ANOVAs to assess treatment differences at specific frequencies. For

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Objective 3, differences in AP characteristics and IKr and IK1 density as a result of oil

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exposure were assessed with a one-way ANOVA followed by a Student-Neuman-Keuls

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post-hoc test to identify all pair-wise differences. All data are reported as means ±

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s.e.m. and were considered statistically significant at P

Impacts of Deepwater Horizon Crude Oil on Mahi-Mahi (Coryphaena

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