Download Evolutionary Novelties: How Fish Have Built a Heater Out of Muscle1

AMER. ZOOL., 31:726-742 (1991)
Evolutionary Novelties: How Fish Have
Built a Heater Out of Muscle1
BARBARA A. BLOCK
Department of Organismal Biology and Anatomy, The University of Chicago, Chicago, Illinois 60637
SYNOPSIS. The evolution of any complex morphology or physiological adaptation involves
the historical transformation of numerous interacting components from an ancestral to a
derived state. How such transformations occur are central to our understanding of how
novel morphologies arise. The rapid explosion of technology in the field of molecular biology
provides new tools that can be incorporated into studies examining the origin of novel
phenotypes. Molecular biological techniques can now be used to probe how changes in gene
expression result in pathways leading to novel or altered morphologies. The integration of
molecular approaches into problems in organismal biology provides a promising new direction for the analysis of form and function. Interdisciplinary studies, combining the resolving
power of molecular biology with the complex problems of organismal biology, will shed
new light on whole animal function and evolution.
ogists and organismal physiologists and
morphologists are bringing complementary
perspectives and talents to bear on the same
problems but in isolation from one another.
This is detrimental to progress because many
molecular biologists lack insight into the
nature of the environmental variation to
which genes are reacting, and most comparative physiologists and functional morphologists presently lack the technical
expertise to resolve unambiguously the
mechanisms by which environmental variation is transduced (Feder and Block, 1991).
Recent studies by organismal biologists have
attempted to bridge this gap by focusing on
how variation at the molecular and biochemical level results in changes in phenotypic expression at the organismal level
(Watt, 1983; Powers, 1987; Koehn et al,
1988; Crawford and Powers, 1989).
Cross-disciplinary training in molecular
biology and organismal biology should be
encouraged. Functional morphologists and
comparative physiologists should not avoid
reductionism, when such an approach can
lead to a fuller understanding of the mechanisms of pattern and process. Molecular
techniques may allow organismal biologists
to unravel questions that have not been
approachable by classical experimental protocols. Such an integrative approach will not
1
From the Symposium on Experimental Approaches replace the more traditional fields of study
to the Analysis of Form and Function presented at the of organismal biology. Rather the incorCentennial Meeting of the American Society of Zool- poration of a molecular approach will be
ogists, 27-30 December 1989, at Boston, Massachucomplementary and will expand the abilisetts.
INTRODUCTION
The goal of this paper is to examine how
molecular biology when integrated into
organismal biology can provide increasing
resolving power for studies of form and
function. I intend to illustrate the valuable
information and insights that are available
if we embrace molecular technology and
incorporate the tools of this field into organismal level problems. The benefits of a
reductionist approach will be illustrated by
examining an empirical case: How muscle
has been modified into a furnace in fish. In
this particular system the integration of cell
and molecular techniques have enabled an
analysis of structure and function at the
molecular level to shed new light on whole
animal function.
Research at the forefront of molecular
biology investigates the regulation of gene
expression, or more specifically how a given
change in the environment (cellular) initiates or halts the manufacture of a gene product. How organisms respond or adapt to
environmental change is of course, one of
the central paradigms of comparative physiology (Schmidt-Nielsen, 1972&; Prosser,
1986) and to some extent functional morphology. In many instances, molecular biol-
726
THERMOGENESIS IN MUSCLE
727
Gasterochisma melampus
Scomber scomber
Trichiurus lepturus
Acanthocybium solanderi
Scomberomorus cavalla
Thunnus thynnus
Thunnus albacares
Scomberomorus maculatus
-Xiphias gladius
Makaira nigricans
Istiophorus platypterus
Tetrapturus albidus
Tetrapturus audax
Makaira indica
Tetrapturus angustirostris
Scombrolabrax
Ruvettus pretiosus
FIG. 1. Relationships among scombroid fishes of the family Scombridae (tunas, mackerels) and billfishes
(martins, sailfish, spearfish and swordfish). This phylogeny is based on parsimony analysis of 600 bp from the
cytochrome b gene using R. pretiosus as the outgroup (Block and Stewart, 1990). Regional endothermy (square),
involving a brain heater has evolved independently two times (once within billfishes, Xiphiidae and Istiophoridae,
and once in the Scombridae). Whole body endothermy has only evolved within true tunas (circle). The molecular
results when combined with the morphological phylogenies (Collette et al, 1984), indicate that both Gasterochisma and the swordfish are more primitive members of the Scombroidei than the tunas. This suggests that
brain heaters and hence regional endothermy evolved earlier than the whole body endothermy characteristic of
tunas.
presence of endothermy is associated with
two distinct anatomical changes: (1) the
movement of the red oxidative muscle mass
internally, and (2) the insertion of a complex
ENDOTHERMY IN FISH
counter-current heat exchanger into the cirEndothermy in teleost fish is unusual and culation to the oxidative muscle mass to
has only been documented within one major reduce conductive and convective heat loss
assemblage of oceanic fishes, the Scombroi- (Kishinouye, 1923; Carey and Teal, 1966;
dei (mackerel, tuna, swordfish, marlin and Sharp and Dizon, 1978). More recently it
spearfish). The high heat capacity of the has become apparent that the monophyletic
aquatic environment coupled with an oxy- assemblage commonly known as billfishes
gen content that is Vw that of air, results (Xiphiidae and Istiophoridae) are also capain large heat losses when respiring via a gill. ble of maintaining elevated tissue temperAmong scombroid fishes, two distinct strat- atures. In billfishes, the body temperature
egies for elevating tissue temperatures have is allowed to fluctuate to varying degrees
evolved and both utilize skeletal muscle as depending upon the species (swordfish are
capable of regulating their body temperathe heat source (Fig. 1).
Several requirements are necessary for ture by behavioral means) and the brain
endothermy, foremost is a large body size, temperature is elevated (Carey, 1982,1990;
a heat source, and a mechanism (heat Block, 1986, 1991). The basis for this
exchangers) within the circulation for con- regional endothermy in the brain and eye is
serving the heat. The bonitos and tunas the presence of a heat generating organ
(Scombridae) maintain elevated body tem- beneath the brain that is modified from
peratures by conserving heat generated in skeletal muscle.
In the billfishes, elevated tissue temperthe slow oxidative swimming musculature
that powers sustained cruising. In tunas the atures are primarily associated with the era-
ties of organismal biologists to address fundamental questions in morphological and
physiological research.
728
BARBARA A. BLOCK
1800
2400
0600
1200
Time of Day
FIG. 2. Temperature telemetry record from a swordfish, Xiphias gladius. Upper line is cranial temperature,
lower line is water temperature (from Carey, 1990).
0600
1200
nial cavity (Fig. 2). Warm brain tempera- animals (Eisenberg, 1983) the fish red
tures are directly associated with the bilateral myotomal fibers used for sustained swimmodification of the superior rectus muscle ming are extraordinarily oxidative. Table 1
into a heat generating tissue (Carey, 1982; compares data on fish muscle mitochonBlock, 1986). Large portions of this extra- drial volumes with other oxidative muscle
ocular muscle contain a novel type of mus- fiber types considered among the most aercle fiber, that is specialized for heat gener- obic in the animal kingdom. The aerobic
ation and lacks the contractile components capacity of the fish slow oxidative muscle
necessary for force production (Block, 1987; fibers, as indicated by the mitochondrial
Block and Franzini-Armstrong, 1988). At volume (30 to 45% of the cell volume), is
the base of the eye muscle there is a large closer to that of bird and insect flight muscounter-current heat exchanger formed from cle. Measurements of enzymatic activities
the carotid circulation that reduces heat loss of various oxidative enzymes indicate there
is a high potential for heat production in the
from the blood at the gill.
In teleost fish the maintenance of elevated red muscle of scombroid fishes (Gordon,
body temperature is dependent upon oxi- 1968;Guppy«a/., 1979;Suarezef al., 1986;
dative muscle as the source of heat. Why is Tullis et al., 1991). Similarly, spectrophored muscle central to endothermy in fish tometric determination of myoglobin conand how has skeletal muscle actually been centration in tuna and marlin red muscle
converted from a force generating fiber into indicates a two tofivefold higher myoglobin
a heat-generating fiber? In comparison to concentration than in flight muscle from
oxidative muscle fibers of many terrestrial birds or slow oxidative muscles from terTABLE 1. Mitochondrial volume of oxidative muscle cells.
Cell type
Pigeon pectoralis
Trout red muscle
Antarctic fish red muscle
Cicada tymbal muscle
Finch cardiac muscle
Mackerel red muscle
Insect flight muscle
Anchovy red muscle
Billfish heater cells
Mitochondrial volume
as % of cell volume
29%
31%
30%
33%
34%
36%
44%
45%
63%
Reference
James and Meek, 1979
Johnston and Moon, 1981
Londraville and Sidell, 1990
Josephson and Young, 1985
Bossen et al., 1978
Bone, 1978
Elder, 1975
Johnston and Moon, 1981
Block, 1991
THERMOGENESIS IN MUSCLE
restrial mammals (Schuder et al., 1979). The
muscles that power these large pelagic fish
through water, a medium of relatively high
viscosity, have a high metabolic potential
that underlies the evolution of endothermy
in scombroid fishes.
The evolution of endothermic mechanisms in teleost fish utilizing skeletal muscle
as the heat source provides insight into the
role of aerobic muscle for heat generation.
However, the ability of billfishes, tunas (and
some sharks) to raise their brain and eye
temperature is not due entirely to muscular
activity alone (Linthicum and Carey, 1972;
Block and Carey, 1985; Wolf et al., 1988).
Although muscle is the source of the heat
in the head region it is doubtful that the
extraocular muscles are utilized in sustained
activities in a similar manner to the muscles
that power slow swimming. Are there other
properties of oxidative muscle that contribute more to heat production purposes than
other fiber types? Does the pattern of innervation and activation, the regulation of the
calcium ATPase or calcium release channels, or the differences in the buffering of
calcium in red muscle make it more suitable
as a heat source? Are the mitochondria regulated similarly as in fast-glycolytic muscle?
Many of these questions are molecular level
problems that when explored in detail will
address why red muscle is warmer.
The heater organ
Extraocular muscles in vertebrates are
complex muscles composed of many fiber
types. In all billfishes a unique type of muscle fiber that lacks the myofibrillar lattice
typical of most skeletal muscle cells is found
in the superior rectus muscles (Block, 1986;
Block and Franzini-Armstrong, 1988). A
similar fiber type is expressed in the lateral
rectus muscles of an unusual mackerel, Gasterochisma melampus. The heater phenotype is a modification of an oxidative fiber
type within the extraocular muscle (studies
thus far suggest either FOG or SO as the
fiber type giving rise to the heater phenotype). The oxidative capacity of the heater
cell as measured by examining the levels of
key metabolic enzymes is similar among the
biUfishes and highest in Gasterochisma, the
729
fish from the coldest oceans. Tullis et al.
(1991) have demonstrated that the aerobic
capacity of the heater cells in Gasterochisma
is the highest of all vertebrates.
Among the billfishes there is a gradient
of phenotypic expression of the heater cell
in the eye muscle which appears to be correlated with the thermal ecology of the species (Block, 1990). Species that experience
the coldest temperatures, such as the swordfish, Xiphias gladius, have more of the heater
phenotype expressed in the superior rectus
muscles and only a small remnant portion
of the superior rectus muscle remains. Billfishes which spend less of their time at great
depths (marlins, sailfish and spearfish) have
more of the eye muscle intact and less of
the heater fiber type expressed (Fig. 3A, B).
Phenotypic expression of the heat-generating cell type within the extraocular muscles
is most likely regulated so that conversion
of muscle to heater occurs in relation to how
much heat is required to meet the demands
of heat loss in the head region.
How FISH HAVE BUILT A FURNACE
OUT OF MUSCLE
Molecular plasticity
Muscle is a remarkable tissue for examining how molecular plasticity confers
diversity in physiological function. As functional morphologists we have long recognized the range of structural and physiological properties that give rise to skeletal
musclefiberdiversity. This diversity in fiber
types is due to suites of isoforms of structural and regulatory proteins being expressed
simultaneously. Thus fibers with fast contraction times have a fast myosin ATPase
capable of forming cross-bridges at high
rates, a fast calcium ATPase that rapidly
pumps calcium into the SR, distinct isoforms of calcium binding proteins such as
troponin and calsequestrin, increased
expression of the SR calcium release channel and numerous other characteristics that
together provide the physiological basis for
fast contraction times.
In vertebrate skeletal muscles, over 70%
of the fiber volume is packed with contractile filaments. Mitochondrial, lipid and gly-
730
BARBARA A. BLOCK
XIPHIAS
10
20
*
5
20
TIME, HOURS
MAKAIRA NIGRICANS
20-
en
60-
•4-1
Q
Time (hours)
FIG. 3. A. Depth record from a swordfish indicating the vertical migration pattern typical of these fish. Swordfish
pass through the thermocline and encounter large changes in water temperature at dusk and dawn (B). Depth
record from a blue marlin, Makaira nigricans. This depth record is typical for blue marlin off of Kona, Hawaii
(Block et al., 1991). Blue marlin make infrequent dives below the thermocline and do not encounter temperature
gradients as large as the swordfish. Swordfish have significantly larger heater organs than blue marlin. (Swordfish
record is from Carey, 1990.)
THERMOGENESIS IN MUSCLE
731
FIG. 4. Electron micrograph of a heater cell from a blue marlin. The modified muscle cell lacks contractile
filaments but is packed with mitochondria and the membranes that regulate calcium release and reuptake
(sarcoplasmic reticulum [arrows] and transverse tubules).
cogen volumes, vary depending upon the
fiber type (Eisenberg, 1983). The membranes that regulate the contraction-relaxation cycle, the sarcoplasmic reticulum (SR)
and transverse (T) tubule system usually
occupy less than 5% of the cell volume. Only
when speed and endurance are required does
the amount of SR in a muscle increase dramatically. In the heater cell (Fig. 4) organized contractile filaments are lacking and
the cell is instead packed with mitochondria
(55-70% of the cell volume depending upon
species), and smooth endoplasmic reticulum (25-30% of the cell volume). Structural,
biochemical, and molecular studies of the
membranes in the heater cell indicate that
they are equivalent to the SR and T system
of normal muscle (Block et ah, 1988a b;
Block, 1991).
To understand how fish have converted
skeletal muscle into a furnace we need to
know which molecular properties of a muscle fiber are best suited for generating heat.
The loss of the contractile filament fraction
of the heater cell takes the emphasis off the
cross-bridge cycle as the source of heat
(Block, 1987). Instead the hypertrophy of
the membrane volume in the heater cells
focuses attention on the energy dependent
process of calcium release and reuptake. At
732
BARBARA A. BLOCK
the heart of this process are two molecules,
the SR calcium ATPase or calcium pump,
and the SR calcium release channel.
In vertebrate skeletal muscle, contraction
and relaxation are regulated by the cytoplasmic calcium concentration, which in
turn is controlled by the SR and T tubule
membranes. The SR of skeletal muscle is a
highly organized intracellular membrane
system that plays a crucial role in calcium
uptake, release and storage. Several key proteins found in the SR membrane are required
for cycling of calcium in and out of the SR
and the particular isoform or quantity of
these proteins expressed in a given muscle
cell type determines the speed at which calcium can be removed or released from the
sarcoplasm. Calcium is pumped into the SR
by the Ca2+ ATPase which is the major protein constituent of the SR bilayer. This
enzyme uses energy derived from ATP
hydrolysis to generate significant calcium
ion gradients between the interior of the SR
and the muscle cell cytoplasm.
Proteins that cycle calcium
The Ca2+ ATPase of skeletal muscle SR
has been extensively characterized with
respect to structure and function. Brandl et
al. (1986) have demonstrated the existence
of four distinct Ca2+ ATPase isoforms which
differ in their efficiency (speed) of pumping
calcium into the SR. Two of these isoforms
are the products of a single gene and are
expressed only in fast-twitch muscle while
the other two isoforms are alternatively
spliced products of a second gene. One of
these isoforms is expressed in cardiac and
slow-twitch muscle while the other is
expressed entirely in nonmuscle cells and in
smooth muscle. Red muscle and cardiac SR
also contain a unique integral membrane
protein, phospholamban, which has an
important role in the regulation of calcium
uptake (James et al., 1989). The phosphorylation of phospholamban by cAMPdependent protein kinase enhances calcium
uptake by increasing the turnover rate of
the SR Ca2+ ATPase. Thus, a slower isoform
of the Ca2+ ATPase is expressed in tissues
with slower contraction kinetics along with
a modulating protein that can turn the slow
geared pump into a high gear pump.
Release of Ca2+ from the SR followed
depolarization of the transverse T-tubules.
Ca2+ release only occurs at morphological
sites where the T tubules and SR form junctions (Franzini-Armstrong et al., 1990). This
coupling of the depolarization and calcium
release events is termed excitation-contraction (EC) coupling. A molecular basis for
nervous control of muscle contraction has
recently been elucidated in great detail
(Block et al, 19886; Fleischer and Inui, 1989;
Takeshima et al., 1989; Zorzato et al., 1990).
A molecular bridge (Fig. 5) between the T
and SR membranes is composed of two
molecules that together regulate the release
of calcium: the T tubule "voltage sensor"
(dihydropyridine receptor) and the SR calcium release channel (ryanodine receptor).
The skeletal SR calcium release channel
is a large tetrameric protein (composed of
5,032 amino acid residues) that has an SR
membrane domain which forms the actual
calcium pore in the SR, and a cytoplasmic
domain which stretches across the 15 nm
gap between SR and T tubules (Fig. 5B). In
skeletal muscle, the signal for Ca2+ release
is initiated at the neuromuscular junction.
The action potential then spreads down the
T tubules and is presumed to initiate a calcium release event from SR by direct
mechanical coupling across the molecules
spanning the triad junction (Block et al.,
1988ft; Tanabe et al., 1988; Fleischer and
Inui, 1989). The exact mechanism of signaling between the T tubule voltage sensor
and the SR calcium release remains to be
determined. The leading hypothesis suggests that depolarization of the T results in
a conformational change in the voltage
sensing molecule (the dihydropyridine
receptor of T tubules) which directly gates
calcium release via the SR calcium release
channel. Such direct molecular coupling
between T and SR provides a precise means
for transducing the depolarization event into
a tightly regulated calcium release signal and
explains the precise coupling between membrane potential and tension in skeletal muscle. Alternative hypotheses invoke slower
mechanisms involving a possible chemical
THERMOGENESIS IN MUSCLE
733
transmitter (Vergara, 1985). Both calcium
release mechanisms, with their dramatically
different kinetics (fast and slow) may play
a role in different types of muscle fibers.
Structural and biochemical results indicate
there are two isoforms of the SR calcium
release channel protein sitting side by side
within the junctional membrane in birds,
reptiles and fish (Block et al., 1988a; Airey
et al., 1990). It is possible that each channel
complex is governed by slightly different
release properties. The chemical second
messenger hypothesis (Fig. 5B) provides a
mechanism for a more prolonged release of
calcium. The transmitter substance is
believed to be released from T tubular
membranes upon depolarization. Binding
of the transmitter to the cytoplasmic domain
of the SR calcium release channel results in
opening of the calcium release channel.
Cycling calcium for speed
How fast a muscle fiber can contract is
directly proportional to the SR and T tubule
membrane fraction in the muscle cell. This
hypertrophy of the membrane volume is due
entirely to the role of the membranes in
regulating the release and reuptake of calcium which in turn regulates the speed of
contraction. Fast contracting muscles not
only have more longitudinal SR rich in the
Ca2+ ATPase, but they also have more of
the morphologically recognizable junctional regions which house the proteins
involved in EC coupling (Eisenberg, 1983;
Franzini-Armstrong et al., 1990). In the fastest contracting muscle fibers, such as the
sound producing muscles of toadfish, cicadas and lobsters the membrane fraction is
amplified and occupies as much as 70% of
the cell volume at the expense of the myofibrillar components. The hypertrophy allows
for maximal packing of the SR calcium
pump and release channel (Block, personal
observation) providing the molecular components necessary for superfast contraction
speeds. Not surprisingly, superfast contraction entails a high metabolic cost. This is
due to the large ATP requirement for transporting calcium into the SR. Thus as speed
increases in muscle contraction there is a
FIG. 5. Model of the junctional region between T and
SR revealing the disposition of the two molecules, the
voltage sensor and SR calcium release channel found
in this region. The SR calcium release channel has a
four-fold symmetry with an intramembranous domain
as well as a large cytoplasmic domain. The voltage
sensor proteins of the T tubule also have a four-fold
distribution and line up with alternate release channels
(modified from Block et al., 19886). (B) Diagram of
the transmembrane topology and molecular architecture of the two signal molecules based on molecular
structure revealed from sequence data. The large "foot"
portion of the SR calcium release channel spans the
gap between SR and T tubule and presumably receives
a signal from the voltage sensor molecular that results
in opening and closing of the channel spanning the SR
bilayer. An alternative hypothesis (2) is that depolarization results in release of an inositol triphosphate
from the T tubule membrane which then binds to and
opens the SR calcium release channel (Vergara, 1985).
734
BARBARA A. BLOCK
FIG. 6. Freeze fracture through a pancake-like stack
of sarcoplasmic reticulum membranes reveals the
numerous intramembranous particles which represent
the Ca2+ ATPase molecules. Pumps appear like pebbles
on the cytoplasmic leaflet (c) and are absent on the
luminal leaflet (1).
need for a higher mitochondrial volume to
supply the ATP to fuel the ATPase associated with uptake of calcium. This linkage
between speed of contraction and ATP utilization due to the calcium ATPase, is a
large component of the metabolic cost and
subsequent heat production in flying insects
and singing cicadas.
Cycling calcium for heat production
Biochemical analysis of the SR membrane fraction of the blue marlin heater cells
indicates that the most abundant protein in
the marlin heater is the Ca2+ ATPase (Block
and Franzini-Armstrong, 1988). The large
cytoplasmic head of the molecule protrudes
above the SR bilayer and can easily be visualized by freeze-fracture of the heater cells.
Figure 6 reveals the dense covering of 12
nm particles on the cytoplasmic leaflet of
the heater SR which correspond to the tightly
packed Ca2+ ATPase molecules. The presence of numerous mitochondria capable of
generating ATP, and a membrane system
loaded with a calcium activated ATPase,
has led to the hypothesis that "non-shivering thermogenesis" in the heater cell occurs
via a calcium-mediated thermogenic cycle
(Block, 1987; Block and Franzini-Armstrong, 1988). This hypothesis suggests that
neural regulation of calcium release results
in a thermogenic cycle associated with the
stimulatory effect of the calcium on both
the oxidative processes at the mitochondria
(either stimulation of dehydrogenases or
proton-calcium exchange) and the ATP
dependent activity of the SR calcium ATPase (Fig. 7).
At the center of the model for non-shiv-
Axon
SARCOPLASMIC RETICULUM
Ca + +
V Ca
FIG. 7. Excitation-thermogenic coupling in the heater cell. A nervous impulse stimulates thermogenesis via
the same molecular components found in the EC coupling pathway. Heat would be produced during prolonged
calcium release events due to cycling of calcium at the SR. The high mitochondria and myoglobin content
provides ample ATP and oxygen for the calcium mediated thermogenic cycle. Calcium may stimulate mitochondrial metabolism in heater cells and thus also contribute to heat generation.
THERMOGENESIS IN MUSCLE
ering thermogenesis is the regulation of calcium release. Excitation-thermogenic coupling whereby a nervous impulse results in
a calcium release event is the presumed
pathway for regulating heat production out
of muscle in the heads of billfish. To fully
understand how muscle has been converted
from a force producing cell into a heat producing cell we need to understand the role
of the molecules involved in regulating calcium release and reuptake. What remains
to be determined is how the proteins
involved in the traditional EC coupling
pathway have been modified or rearranged
in a novel pathway to bring about calcium
release in a manner that stimulates thermogenesis.
In normal muscle, firing of the motor
nerve supplying the muscle fiber leads to T
tubule depolarization, and a possible conformational change in the T tubule voltage
sensing molecule which then presumably
gates the opening of the SR calcium release
channel (Tanabe et al., 1988). The subsequent rise in cytoplasmic calcium initiates
cross-bridge cycling and force production.
The pumping of calcium back into the the
SR via the Ca2+ ATPase, and the lowering
of the cytoplasmic calcium concentration,
inhibits the interaction between the contractile filaments.
In the heater cell up to 30% of the cell
volume is packed with the SR membrane
system. The SR in turn is loaded with the
Ca2+ ATPase. Additionally, the neuromuscular junction and an extensive T tubule
network (the cable system that propagates
the action potential) is present in these cells
(Fig. 8). All of the components for neural
control are present in the heater cell, indicating that thermogenesis is mediated by the
same neuromuscular pathway found in normal skeletal muscle. Thus, firing of the oculomotor nerve would lead to T tubule depolarization and presumably a calcium release
event, much as it does in normal muscle.
The presence of calcium in the cytoplasm
would stimulate the hydrolysis of ATP and
heat production. The calcium not only stimulates the pump but most likely has a direct
effect on the electron transport chain of the
mitochondria perhaps by stimulating proton-calcium exchange. Heat production
735
would continue in the heater cell as long as
the motorneuron fired and an ensuing calcium release event occurred.
As in normal muscle, a controlled release
and reuptake of calcium requires that the
molecules (the SR calcium release channel
and T tubule voltage sensor) involved in
excitation-contraction coupling are present
in the heater cell. Experiments thus far have
revealed that the SR calcium release channel is present but in very low numbers (Block
et al, 1988a). In normal muscles, the calcium release channel is found in abundance
in fast-contracting fibers in junctional
regions between SR and T tubules. Ample
sites for calcium release naturally increase
the ability to saturate the cytoplasm with
calcium quickly. Slow contracting fibers
found in slow oxidative or tonic fiber bundles have fewer sites for calcium release and
thus activation times are longer.
The low expression of the ryanodine
receptor is perhaps puzzling; however, it may
be an important functional distinction
between operating a muscle for heat generation rather than force production. There
is a temporal difference between initiating
a synchronous contraction event throughout a fast-contracting muscle fiber and turning on a furnace. The presence of numerous
calcium channels (and the ability to rapidly
saturate the cytoplasm with calcium) is an
indication of speed, while having few channels indicates that events occurring in the
modified muscle cell proceed slowly. In
redesigning a muscle fiber for heat production purposes, what appears to have been
retained is the ability to turn on thermogenesis via the same nerve to muscle pathway found in all skeletal muscles. This allows
for a control of thermogenesis based on the
same calcium release and reuptake pathway
inherent in skeletal muscle. Regulation of
these molecules may be different in the
heater organ but their presence indicates that
in going from a force producing cell to a
heat producing cell, a major rewiring has
not occurred. Instead, one can postulate that
the differences are associated with molecular regulation of either the voltage sensor,
the SR calcium release channel or the SR
calcium ATPase. One hypothesis is that
there may be molecular differences between
736
BARBARA A. BLOCK
FIG. 8. Golgi-stained thick sections of muscle (a) and heater (b) reveal the disposition of the T tubule network
in both of these cells. In muscle the tubules course transversely between the myofibrils whereas in the heater
cells the T tubules take a circular route around the numerous mitochondria in the cytoplasm. In both cells the
T network acts as an electrical cable system propagating the action potential throughout the cell. N, nucleus.
the SR calcium release channel in the heater
and normal muscle and that stimulation
leads to locking open of the heater calcium
channel (similar to the effects of low ryano-
dine concentrations on this channel [Meissner, 1986]). A channel with prolonged open
states would result in a continuous leak of
calcium and constant stimulation of the
THERMOGENESIS IN MUSCLE
thermogenic cycle. An intriguing parallel
exists in mammalian muscle. The site of a
mutation in a fatal heat producing muscle
disease has recently been mapped to a chromosomal location close to the SR calcium
release channel indicating that leaky SR
membranes may also be responsible for
intense heat production in mammalian
muscles (MacLennan et al., 1990).
In summary, structural and biochemical
results indicate that a heater cell is a modified muscle fiber with a hypertrophy of the
SR, T and mitochondrial volumes along
with a loss of the contractile filaments. This
cell has an enormous potential to oxidize
both lipid and carbohydrate (Tullis et al.,
1991). Neural control of the heater has been
retained as evidenced by the extensive T
system and the presence of neuromuscular
junctions. The components for excitationthermogenic coupling are present in the
heater cell and represent a controllable neural pathway for initiating calcium release
and reuptake resulting in heat production.
Release of calcium in the modified muscle
cell does not result in contraction due to the
absence of actin, myosin and troponin. Calcium will however stimulate the futile
pumping of calcium back into the SR at the
expense of ATP and have a direct effect on
the rate of oxidative processes occurring at
the mitochondrion. The high mitochondrial
volume and myoglobin content are indicative of high oxidative capacity in this tissue, indicating that heat production could
continue for long periods of time. Sustained
performance of the heater is clearly necessary given that telemetry experiments with
free-swimming swordfish indicate that these
fish diumally dive to great depths and spend
up to 12 hr in 6°C water while the brain
temperature remains elevated (see Fig. 3;
Carey, 1982, 1990).
MOLECULAR APPROACHES
The aim below is to outline how molecular techniques can be used to address in
greater detail some of the fundamental
questions raised above. Although the focus
in the present paper is to address one specific problem, approaches such as this could
be applied to many different morphological
or physiological studies.
737
Molecular phylogenetic techniques
An explosion of new phylogenies utilizing
direct sequencing of mtDNA, and in particular cytochrome b, have provided a concrete new approach for resolving questions
of historical context (Kocher et al., 1989).
This molecular revolution of systematics
provides a new tool for functional morphologists and physiologists. It is now possible to determine direct ancestry within a
clade using molecules in combination with
norphology. The use of the polymerase chain
reaction and direct sequencing of mtDNA
has recently been used to clarify the relationships within scombroidfishesin an effort
to determine how many times endothermy
has evolved within teleost fishes (Fig. 1;
Block and Stewart, 1990). The molecular
phylogeny indicates endothermy has
evolved three separate times within the
scombroid fishes. The molecular phylogeny, when combined with morphology,
indicates that regional endothermy in the
swordfish, Xiphias gladius and in the scombrid, Gasterochisma, is more primitive to
the whole body endothermy characteristic
of the tunas. Thus, this suggests that selection for niche expression (brain heaters allow
swordfish to forage in the cold waters
beneath the thermocline) preceded warm
muscles (as in tunas).
Excitation-thermogenic coupling in muscle
When a fish dives into cooler waters how
is the thermogenesis initiated beneath the
brain? In heater cells, as in normal muscle
fibers, the cell is most likely activated by
the firing of a nerve and the coupling of the
depolarization event to calcium release
within the cell. To demonstrate that excitation-thermogenic coupling occurs in the
modified muscle cells utilizing a molecular
pathway similar to the EC coupling pathway
in muscle it is necessary to isolate the molecules involved (the voltage sensor and SR
calcium release channel) and determine their
structure and function in the heater cells.
To pull out the proteins from the heater
organ for investigation, polyclonal antibodies with binding sites to conserved regions
of the proteins of interest are used as probes.
DNA hybridization techniques along with
the antibodies can be used to directly local-
738
BARBARA A. BLOCK
ize the molecules within the heater cell. The
recent discovery suggesting that a point
mutation results in the hypersensitivity of
the malignant hyperthermia SR calcium
release channel of muscle to calcium agonists provides a strong lead that the heater
cell may also have a mutation in this large
protein which effects the channel opening
and ultimately calcium release (Mickelson
et al, 1988; MacLennan et al, 1990). The
low number of SR calcium release channels
in heater strongly suggests that the large
conductance channels must have prolonged
channel opening periods. Structure and thus
function at the molecular level of the channel in heater has most likely been modified.
This has been shown to be the case in malignant hyperthermia and research efforts are
presently focused on cloning the complementary DNA to compare the heater SR
calcium release channel with the normal fish
muscle SR calcium release channel.
Muscle to heater trajectory
Understanding which fiber type is giving
rise to the heater phenotype is the first step
toward identifying how one goes from a
muscle cell to a heater cell. To examine in
more detail the transformation of a red
muscle fiber into a heater cell, we need to
understand what is being expressed in a
muscle cell vs. a heater cell. It is possible
with the relatively easy methods for isolating RNA to approach this question (Chomczynski and Sacchi, 1987). To begin investigating how much gene expression in a
normal muscle cell is altered to give rise to
a heater cell, the first step is to examine the
difference between a presumptive ancestral
fiber (red oxidative) and a heater cell. From
total RNA, messenger RNA is isolated and
can be used to examine expression levels of
proteins found in muscle. Construction of
a complementary DNA library of the heater
cell combined with subtraction hybridization permits analysis of sequences shared in
common with a muscle fiber and heater cell.
Levels of expression of actin, myosin and
other myofibrillar proteins along with SR
proteins can readily be determined using
standard molecular techniques (Maniatis,
1982).
With the cDNA library to the heater cell,
many questions raised above can be
addressed. Both the origin of fiber type and
the subsequent questions concerning the
Ca2+ ATPase and SR calcium release channel can be examined in detail. For example,
as mentioned above there are four distinct
Ca2+ ATPase isoforms that have been cloned
or sequenced. Using cDNA probes constructed from conserved portions of the Ca2+
ATPase genes prepared from the sequences
reported in the literature, one can screen the
heater library and distinguish which isoform of the Ca2+ ATPase is present in the
heater. If the slow isoform was present, this
would be a positive indication that the heater
phenotype is a result of a slow oxidative
fiber transforming into the heater cell.
An additional question concerning regulation of ATP turnover in red muscle tissues
in general and the heater in particular is to
determine if phospholamban, a regulatory
protein involved in ATP turnover, is
expressed. Phospholamban serves as a
modulator of SR Ca2+ ATPase activity, and
in the nonphosphorylated state it is thought
to inhibit SR Ca2+ ATPase activity by raising the K™ for calcium (James et al., 1989).
This inhibition is relieved when phospholamban is phosphorylated following B-adrenergic stimulation by a cAMP-dependent
protein kinase. The phosphorylated form
significantly increased SR Ca2+ uptake by
augmenting the turnover rate of the Ca2+
ATPase. Enhanced ATP turnover also is
indicative of higher levels of heat production. Excessive levels of phospholamban
expressed in the heater cell would be indicative of a possible role for this molecule in
the heat production cycle of the heater cell.
The presence of a cDNA library allows for
determination by screening for the presence
of this regulatory protein.
At a functional level, the new information
gained by molecular experiments would be
advantageous for addressing the basic question of where does the heat come from. Heat
production in the heater cell is either due
to an altered Ca2+ release channel with prolonged open states or, alternatively it is the
Ca2+ ATPase that is altered and is somehow
associated with enhanced ATP turnover or
unusual regulation. As outlined above, the
THERMOGENESIS IN MUSCLE
release channel hypothesis can be studied
by examining the physiological release
properties of the channel isolated from
heater tissue or by cloning and sequencing.
The large size of this channel (it is composed
of four monomeric subunits of relative
molecular mass > 500,000) would make
cloning the fish heater SR channel (from the
cDNA) a difficult task. However availability
of the sequence would allow comparison
with the receptor isolated and sequenced
from mammalian skeletal and cardiac muscle (Takeshima et al., 1989; Zorzato et al.,
1990; Otsu et al, 1990) and could provide
intriguing comparative data if there are
indications of physiological differences.
Mapping the pathway from ancestral to
derived state
To directly examine what type of presumptive muscle fiber is giving rise to the
novel heater phenotype a direct approach is
to identify which isoforms of muscle specific proteins are being expressed in the
heater cell. One methodological approach is
to identify which mRNA transcripts are
expressed in the heater cell. Alternatively,
polyclonal antibodies raised against conserved proteins with fiber specific distribution, such as the calcium ATPase or the
contractile proteins, can be used as markers
(Jorgenson and Jones, 1986). Immunoblotting techniques using fiber type specific
probes for myofibrillar or SR proteins can
also be used to determine which isoform of
a protein is expressed in the heater cell. In
the heater tissue, western blotting (Towbin
etai, 1979; Burnett el al., 1981) has revealed
only slow oxidative isoforms of proteins in
the heater cell, providing a strong evidence
of a link with this fiber type.
Can we learn more about the trajectory
of transformation from muscle into heater?
The first step is to probe the transitional
regions (areas where muscle fibers actually
appear to be differentiating into heater in
adult fish) of the superior rectus muscles
with polyclonal or cDNA probes for various
myosin isoforms to determine exactly what
fiber types are transforming. Once this has
been established it might be useful exploring
how such a transformation might occur.
739
Knowing what we do about muscle plasticity, one would have to guess that either thyroid hormone or nervous innervation, or
both of these components working in concert, are somehow influencing differentiation of an oxidative fiber type in the billfish
eye muscles. One experimental approach to
understanding this plasticity of expression
in the billfish would be to establish cultures
of the presumptive oxidative muscle and
heater cells and experimentally manipulate
gene expression under various environmental perturbations (hormonal and temperature).
An alternative approach to examining
plasticity in billfish red muscle would be to
try to understand in a much more accessible
system (mouse or chicken), how plastic is
the phenotypic expression of a muscle fiber
under various hormonal and temperature
regimes. Studies such as this are currently
underway in several laboratories (Van Hardeveld and Clausen, 1986) and the results
for comparative physiologists and morphologists are rather fascinating. Several
groups have demonstrated that, in response
to thyroid hormone, the expression of energy
utilizing pumps increases dramatically
within two hours time (Gick et al., 1988;
Rohrer and Dillman, 1988). Thus, in terms
of increasing heat production capacity,
increased loading of pumps into the membranes would result in increased ATP
hydrolysis. Thyroid hormone also influences expression of other energy utilizing
ATPases and partially explains the increased
energy utilization associated with this hormone. The influence of this hormone on
increasing the leakiness of membranes such
as the SR in muscle is relatively unexplored.
WHY RED IS HOTTER
Exploring how things work at the molecular level can provide significant information beyond the specialized problem being
examined. The new perspective allowed by
this level of investigation not only provides
additional understanding of a mechanistic
model, but more importantly it provides a
new perspective for examining important
organismal questions. In the case of the
heater organ in billfish, is it possible to take
information concerning how heat is gener-
740
BARBARA A. BLOCK
ated from this specialized system and frame
a broader set of questions involving the evolution of endothermy and thermogenesis in
general? Can we address the question of why
red muscle is so important for endothermic
purposes? Is non-shivering thermogenesis a
widespread phenomenon in endotherms?
And, is excitation-thermogenic coupling or
calcium-mediated thermogenesis involved
not only in keeping fish eyes warm but in
maintaining elevated body temperatures in
birds and mammals?
Leaky membranes and ion pumps have
long been implicated as having a major role
in the transition from ectotherms to endotherms (Edelman, 1976; Else and Hurlburt,
1987). The SR of muscle provides one
example of how a controllable leak results
in increased energy utilization and higher
levels of heat production. One question that
should be explored in all animals is how
tight is the excitation-contraction coupling
pathway, in terms of calcium leakage, in
different groups of animals. Is there calcium
cycling in SR going on all the time? This is
presently being explored in mammals (Van
Hardeveld and Clausen, 1986; Simonides
and Van Hardeveld, 1986), and it remains
plausible that the mechanism for heat generation within the eye muscles of billfish is
just an amplified energy consuming leak that
occurs in the precursor muscle fiber type in
many other animals. Physiologists for many
years have been aware of the effects of thyroid hormone on whole body metabolism,
but only recently is the activity of this hormone being elucidated at the molecular level.
As we learn more about the direct role of
this hormone in regulating transcription and
translational rates of energy consuming proteins, it's up to the organismal biologist to
fit this into a context relating to endothermy. As mentioned above, two recent
reports demonstrate that thyroid hormone
has an effect on gene expression of both the
SR Ca2+ ATPase and the sodium potassium
ATPase. Thus, mammals exposed to T3
almost immediately begin adding more
energy consuming units to their membranes
(Gick et al, 1988; Rohrer and Dillmann,
1988). One interesting implication of this
result for organismal biologists is that one
nowhas a molecular tool for looking at ecto-
thermic vertebrates and determining if there
is a similar response of thyroid hormone
and gene expression of energy consuming
pumps in response to cold.
CONCLUSION
Studying whole organisms and the origin
of complex traits is a challenging task. The
fields of comparative physiology and functional morphology have taken biologists into
the realm of understanding how animals
work. Molecular biology is refining the level
at which we understand how lower level
variation produces changes in organismal
function. If we as organismal biologists are
able to absorb and integrate this exciting
explosion of molecular and cellular biological techniques into our fields we will certainly gain new insights. As organismal biologists we are in a unique position. Because
we are interested in comprehending the evolution of complex organisms we are better
able to grasp the tools of molecular biology
and to search for solutions to problems that
have a more integrative message. It will be
the rare molecular biologist who reaches into
our realm; thus it is up to organismal biologists to reach into theirs and take our broad
array of questions with us.
ACKNOWLEDGMENTS
The manuscript was enhanced by discussions and advice from M. E. Feder and the
advice of two anonymous reviewers. This
work was supported by NIH grant AR
40246-01 and NSF grant DCB-8958225.
Symposium support was provided by a
National Science Foundation grant (BSR8904370) to J. Hanken and M. H. Wake.
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