Present address: Department of Biochemistry, Memorial University of New-
foundland, St. John’s, Newfoundland A1C 5S7, Canada.†Present address: Biological Sciences, University of Alberta, Edmonton, Alberta
T59 2E9, Canada.‡Corresponding author; e-mail: wdriedzic@mun.ca.
Physiological and Biochemical Zoology 80(5):542—550. 2007. !2007 by The
University of Chicago. All rights reserved. 1522-2152/2007/8005-0702$15.00
DOI: 10.1086/520129
Jason R. Treberg1,*
Tyson J. MacCormack1,†
Johanne M. Lewis1
Vera M. F. Almeida-Val2
Adalberto L. Val2
William R. Driedzic1,‡
1Ocean Sciences Centre, Memorial University of
Newfoundland, St. John’s, Newfoundland A1C 5S7, Canada;2Laboratory for Ecophysiology and Molecular Evolution,
Instituto Nacional de Pesquisas da Amazoˆnia, Alameda
Cosme Ferreira, 1756 69083-000 Manaus, Amazonas, Brazil
Accepted 4/25/2007; Electronically Published 7/13/2007
ABSTRACT
Armored catfish (Liposarcus pardalis), indigenous to the Am-
azon basin, have hearts that are extremely tolerant of oxygen
limitation. Here we test the hypothesis that resistance to hyp-
oxia is associated with increases in binding of selected glycolytic
enzymes to subcellular fractions. Preparations of isolated ven-
tricular sheets were subjected to 2 h of either oxygenated or
hypoxic (via nitrogen gassing) treatment during which time the
muscle was stimulated to contract. The bathing medium con-
tained 5 mM glucose and was maintained at 25“C. Initial ex-
periments revealed increases in anaerobic metabolism. There
was no measurable decrease in glycogen level; however, hypoxic
treatment led to a twofold increase in heart glucose and a 10-
fold increase in lactate content. It is suggested that the increase
in heart glucose content is a result of an enhanced rate of
facilitated glucose transport that exceeds the rate of phos-
phorylation of glucose. Further experiments assessed activities
of metabolic enzymes in crude homogenates and subsequently
tracked the degree of enzyme binding associated with subcel-
lular fractions. Total maximal activities of glycolytic enzymes
(hexokinase [HK], phosphofructokinase [PFK], aldolase, py-
ruvate kinase, lactate dehydrogenase), and a mitochondrial
marker, citrate synthase, were not altered with the hypoxic
treatment. A substantial portion (≥50%) of HK is permanently
bound to mitochondria, and this level increases under hypoxia.
The amount of HK that is bound to the mitochondrial fraction
is at least fourfold higher in hearts of L. pardalis than in rat
hearts. Hypoxia also resulted in increased binding of PFK to a
particulate fraction, and the degree of binding is higher in
hypoxia-tolerant fish than in hypoxia-sensitive mammalian
hearts. Such binding may be associated with increased glycolytic
flux rates through modulation of enzyme-specific kinetics. The
binding of HK and PFK occurs before any significant decrease
in glycogen level.
Introduction
Water in the Amazon basin is often severely hypoxic on either
an annual or a diurnal cycle (Kramer et al. 1978; Val 1996).
The armored catfish (Liposarcus pardalis), which is indigenous
to this region, has an exceptional tolerance to environmental
hypoxia through both air-breathing mechanisms and anaerobic
metabolism. Heart rate is maintained in water of less than 10%
normal oxygen level and at 28“C even when fish are denied
access to the surface (MacCormack et al. 2003a). Under these
conditions of severe hypoxia, blood glucose, heart glucose, and
heart lactate all increase, whereas heart glycogen decreases
(MacCormack et al. 2006). Consistent with these in vivo find-
ings, isolated ventricular preparations from L. pardalis sustain
high levels of performance under cyanide poisoning (Bailey et
al. 1999), recover fully from 30 min of anoxia, and, on recovery
after 2 h of anoxia, exhibit the same level of force development
as aerobic controls (MacCormack et al. 2003b). The heart of
this species is therefore an excellent model system in which to
investigate sustained metabolism under hypoxia.
Under conditions of oxygen limitation a number of glycolytic
enzymes show increased binding to subcellular components
(Brooks and Storey 1995). The degree of increased binding in
hypoxia-tolerant and hypoxia-sensitive hearts requires further
study before generalities may be reached. In hypoxia-sensitive
rat hearts, subjecting isolated preparations to ischemic condi-
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Increased Glucose and Enzyme Binding in Catfish Hypoxic Heart 543
tions resulted in increased binding of hexokinase (HK) to mi-
tochondria (Zuurbier et al. 2005) and increased binding to a
particulate fraction for phosphofructokinase (PFK) and aldol-
ase (ALD) but not pyruvate kinase (PyK) or lactate dehydro-
genase (LDH; Clarke et al. 1984). Comparable studies have been
conducted with hearts from hypoxia-tolerant fish. Heart prep-
arations from American eel displayed increases in anoxia-stim-
ulated glucose uptake but no increase in HK binding to mi-
tochondria (Rodnick et al. 1997), and in goldfish held in anoxic
water, there was increased binding to the particulate fraction
for PFK, ALD, and PyK but not LDH or HK (Duncan and
Storey 1991). The available literature therefore suggests that
although hypoxia/ischemia-associated binding of HK to mi-
tochondria may be important in mammalian hearts, it is less
so in anoxia-tolerant fish hearts, whereas binding of PFK and
ALD may be a common response. This contention is addressed
in this experiment. All of the previous studies, related to enzyme
binding under oxygen-limiting conditions in heart, are deficient
in measurements of glycogen and lactate, and so the temporal
relationship between enzyme binding, initiation of glycogen-
olysis, and lactate production (i.e., the end point of glycolysis/
glycogenolysis) remains unknown.
Isolated ventricular preparations from L. pardalis were sub-
jected to severe hypoxia. Glycogen utilization/lactate produc-
tion and heart glucose level were assessed along with binding
of glycolytic enzymes to a general particulate fraction. Fur-
thermore, we tested the hypothesis that HK binding to mito-
chondria increases under hypoxia via protocols previously util-
ilized in studies with rat hearts (Zuurbier et al. 2005). The most
important findings are that glucose levels increase even at con-
stant levels of extracellular glucose, a high proportion of HK
is bound to mitochondria and increases under hypoxia, and
binding of PFK to a particulate fraction occurs to a greater
extent in fish hearts than in rat hearts.
Material and Methods
Animals
Armored catfish (Liposarcus pardalis) with the common name
acari-bodo« were purchased from an aquaculture facility (Am-
azon Fish, Itacoatiara Road, Manaus, Brazil). They were held
at the Laboratory for Ecophysiology and Molecular Evolution,
Instituto Nacional de Pesquisas da Amazoˆnia, Manaus, Brazil,
in aerated well water.
Ventricular Sheet Preparation
Metabolism was assessed in ventricular sheet preparations. Fish
were killed by a sharp blow to the head, and the hearts were
immediately excised and placed in oxygenated medium. The
bathing medium consisted of 125 mM NaCl, 3.0 mM KCl, 1.0
mM MgSO4, 1.5 mM CaCl2, 0.18 mM NaH2PO4, 3.12 mM
Na2HPO4, and 5.0 mM glucose. The medium was initially
gassed with 100% O2, and the pH was set to 7.8 at 25“C. Bathing
medium used for the initial isolation was maintained at room
temperature (∼25“C) to avoid rapid cooling effects. The ven-
tricle was dissected free of the bulbous arteriosus and atrium
and then splayed open via a longitudinal cut through the dorsal
wall. Ventricle sheets were pinned open with suture needles to
expose the inner trabeculae and bathed in 20 mL of medium
in watch glass vessels. The medium was gassed either with
oxygen or with nitrogen, which created a hypoxic environment,
for a period of 2 h, after which tissue samples were assayed.
Gassing was vigorous enough to facilitate mixing of medium
in the area of the tissue preparation. The Po2 in the area of
heart in the hypoxic preparations was not determined, but it
was low enough to result in substantial increases in lactate
accumulation in the tissue (see “Results”). Liposarcus pardalis
appears to be devoid of coronary arteries, and so oxygen and
nutrient delivery occurs normally across the trabeculae; thus,
the ventricular sheet preparation should not be diffusion lim-
ited, although we cannot confirm this because the preparations
were contracting primarily in an isometric mode, thus mini-
mizing mixing in the area of the trabeculae. Therefore, ven-
tricular sheets were bathed with medium gassed with 100% O2
to ensure maximum O2 availability in the control preparations.
It is recognized that high levels of O2 may have had detrimental
effects through the generation of reactive oxygen species (ROS)
and subsequent tissue damage (Lushchak et al. 2005); however,
in the context of the current experiments, we wanted to min-
imize any activation of anaerobic metabolism in the control
preparations and thus elected to use 100% O2. The metabolic
status of the isolated preparation was similar to that of intact
fish (see “Results”), thus minimizing concern over any negative
impact of ROS. Also, L. pardalis is routinely exposed to hy-
peroxia (MacCormack et al. 2003a); therefore, this species may
be more resistant to ROS generation than would otherwise be
expected.
The pins that maintained the ventricular sheets in an open
position also served as stimulating electrodes. The ventricular
sheets received a stimulus of 24 beats per minute and 200-ms
duration from either a Grass SD stimulator or the stimulator
output of a MacLab/2E. Voltage at the electrodes was typically
in the order of 1 V. In most cases, contractions were apparent
even under hypoxia; however, as there was no resistance to
contract against, shortening may have been minimal, and a lack
of macroscopically visible shortening is not an indicator of
viability per se. Biochemical assessment of the intactness of
mitochondria suggested high viability of all preparations even
after 2 h of extended hypoxia (see “Results”), and previous
findings showing mechanical integrity of ventricular strips from
L. pardalis under NaCN poisoning or hypoxia supported the
use of the ventricular sheet preparation in this study (Bailey et
al. 1999; MacCormack et al. 2003b). Following the incubation
period, the tissue was removed and blotted dry before further
analysis. Experimental temperature was 25“C.
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Draw a flow chart of metabolism process of catfish according to the findings of article.
Flow chart should be strictly based on metabolism and findings of the research article attached.
