Tunas are the athletes of the fish world. They are endothermic, maintaining high
metabolic rates that are approximately 3-5 times those of other active teleosts (Brill and
Bushnell, 1991) and more like those of small mammals (Keen et al., 1991). The anatomy
of tuna allows them to keep their swimming muscles warm. Tunas have a retia mirabilia,
a system of countercurrent heat exchangers composed of a masses of intertwined arteries
and veins that prevent heat loss and shunt metabolic heat back to the tissues (Carey.
1973). The result of increased temperature in their muscles is increased power output of
the swimming muscles. While their swimming muscles are maintained at high
temperatures, their hearts are at the ambient temperature. Tuna sustain delivery of
oxygen to their tissues though a wide range of temperatures. The thermal niche of the
more tropical yellowfin tuna is between 12° and 28° C while that of a bluefin tuna is
between 2 and 30°C (Block, pers. comm).
Tuna hearts produce high cardiac outputs to sustain delivery of oxygen to their
swimming muscles. The maximum cardiac output of yellowfin tuna is 97.6 mls/kg/min
(Blank et al, 2001) whereas the maximum cardiac output of a rainbow trout
(Oncorhynchus mykiss) is 17.6 ml/min/kg. (Kiceniuk and Jones, 1977). The regulation of
cardiac output in tunas has been suggested to be primarily through heart rate (Farrell et al,
1992). Tunas have heart rates that exceed 120 bpm (Farrell and Jones, 1992) and have
been measured as high as 240 bpm (Kanwisher et al,1974). The heart rates of tunas
exceed those of other lower vertebrates. A high heart rate may be a prerequisite for high
cardiac output and concommitant oxygen delivery to their powerful swimming muscles.
Calcium cycling, which is necessary tension generation in the heart, is essential to
contraction and relaxation of the heart. The sarcoplasmic reticulum is an internal store
of calcium in muscle cells that plays an important role in cardiac muscle contractility by
regulating intracellular calcium concentration. (Fig. 1) (Tibbits et al, 1992). Release of
calcium from the SR through the ryanodine receptors initiates muscle contraction
whereas uptake of calcium by an SR CaATPase (SERCA) results in muscle relaxation.
Cardiac cells have a unique isoforms of these proteins (Brandle et al, 1986; Franck et al,
1998).
Mammals rely primarily on SR release and uptake of calcium during the
contraction and relaxation cycle (Tibbits et al, 1992). The primary reliance of SR stores
of calcium in mammals creates a shorter diffusion distance for calcium. This permits an
increased rate of relaxation of the heart, a higher heart rate, and thus, an increased cardiac
output. With an increased cardiac output, mammals and birds can deliver oxygen to their
tissues at a higher rate, sustaining their high metabolic rates. This suggests that the high
metabolic rates of mammals are correlated with the reliance on SR calcium cycling in
heart cells.
In contrast to mammals, in most fish, tension is developed primarily through
calcium derived from the extracellular space. Calcium entry into the cytoplasm is
mediated by the plasma membrane voltage gated channel, or dihydropuridine receptor
(DHPR) (Tibbits et al, 1992). Calcium is removed from the heart by a plasma membrane
calcium ATPase (PMCA) and the sodium-calcium exchanger during relaxation of the
heart. The sarcoplasmic reticulum is thought to play minor role in the cardiac
contraction and relaxation cycle. The decreased use of the SR in calcium cycling may
explain the lower cardiac outputs and metabolic rates of fishes.
Because tuna maintain the high heart rates necessary to sustain their high cardiac
outputs and metabolic rates, I hypothesized in this study that the tuna myocyte has an
internal SR with calcium ATPase and calcium release channels. A tuna that is able to
maintain a high metabolic rate may have an increased reliance on SR calcium cycling,
which permits a higher heart rate and cardiac output. Examination of the SR in teleosts
has been done mainly on salmonids (Tibbits et al, 1991); however, little work has been
done examining calcium cycling in tunas. It has been shown that skipjack tuna heart
derives a large portion of its calcium required for contraction from the release of calcium
from intracellular SR stores (Keen et al, 1992). Moreover, rates of relaxation and
contraction in yellowfin tuna hearts have been shown to be impaired by ryanodine, a
natural plant alkaloid that blocks SR calcium release, suggesting that SR calcium plays a
role in initiating contraction (Shiels and Farrell, 1999). In this study, we examined the
presence of sarcoplasmic reticulum with calcium ATPase and calcium release channel in
two species, the yellowfin tuna and bluefin tuna.
Methods and Materials
SR isolation
Wild tunas were obtained in the Atlantic and Pacific oceans during routine
collections. Tissues were freeze clamped after the fish were caught on hook and line and
sacrificed by pithing. Additionally, tunas were maintained in captivity at the Tuna
Research and Conservation Center as described by Altrinham and Block, 1997
For purification of sarcoplasmic reticulum from the tuna hearts, between 3.5g and
30.0 g of liquid nitrogen freeze-clamped tissues from bluefin tuna (Thunnus thynnus) or
yellowfin tuna (Thunnus albacares) heart muscle was homogenized using a Tekmar
homogenizer in 10 volumes of ice-cold homogenization buffer containing 0.3 M sucrose
and 20 mM potassium-piperazine-N, N’-bis[2-ethanesulfonic acid (K-PIPES), pH 7.6,
and a protease inhibitor cocktail containing 1.0 M pepstatin A, 1.0 mM iodoacetamide,
0.1 mM phenylmethylesulfonylflouride (PMSF), 1.0 M leupeptin, 1.0 mM benzamidine,
0.1 M aprotinin, and 6.0 mg/ml trypsin inhibitor. The protease inhibitors were re-added to
each buffer at every step of the procedure. Äfter homogenization the slurry was
centrifuged for 20 minutes at 2600 g (4670 RPM) in a Sorval SL5OT rotor. The
supernatant was passed through two layers of cheesecloth, and the crude microsomes
pelleted by centrifugation at 100,000 g (35,000 RPM) for one hour in a Beckman Ti50.2.
The pellets were resuspended in the resuspension buffer containing 0.3 M sucrose and 5
mM potassium-piperazine-N, N'-bis[2-ethanesulfonic acid] (K-PIPES), pH 7.3. A
portion of the resulting microsome preparation was frozen in liquid N2 and stored at -
80°C until use. The remaining material was layered on top of discontinuous sucrose
gradients (2.0 ml 45%, 3.0 ml 35%, 3.0 ml 25%) containing 0.4 M KCl, 0.1 mM
NazEGTA, O.1 mM CaCl, and 5.0 mM K-PIPES, pH 6.8, and centrifuged 16 hours at
23,000 rpm in a Beckman SW41 rotor. Heavy SR (HSR) membrane vesicles were
recovered from the 35%-45% interface and pelleted at 100,000 g. Light SR (LSR)
membrane vesicles were recovered from the 30-35% interface and pelleted at 100,000 g.
Vesicles were resuspended in 200 mM sucrose and 5.0 mM K-PIPES, pH 7.0, frozen in
liquid N», and stored at -80°C until use.
Calcium ATPase (SERCA 2) antibody production and purification
Isoform specific antibodies were raised against a peptide of SERCA 2 by
Research Genetics Inc. (Huntsville, AL). The peptide corresponding to amino acids
(192-205) of rabbit Serca 2 were synthesized and used to immunize four rabbits. Serum
IgG purification was performed using ImmunoPure Affinity Pak protein A columns
following the instructions provided (Pierce, Rockford, IL). Two affinity columns were
made by immobilization of the 5 mg of the synthesized peptides provided by Research
Genetics using a EDC/Diaminodipropylamine Immobilization Kit and the included
protocol (Pierce, Rockford, IL). 1 ml of the IgG fractions from the protein A columns
followed by 2 ml of PBS (phosphate buffered saline) were applied to the appropriate
affinity column and incubated for 1 hour at room temperature. The columns were
washed with an additional 14 ml of PBS after which the antibodies were eluted with 0.1
M glycine pH 3.0. 1 ml fractions were collected and their absorbance at 280 nm was
monitored to determine the protein peak. Peak fractions were pooled, brought back to
neutral pH and stored at -80°C until use.
Gel Electrophoresis and Western Blots
Heart SR microsomes, HSR, and LSR fractions were run on 7.5% or 3-12% SDS
polyacrylamide gels according to the method of Laemmli, 1970. Gels were either stained
with Coomassie blue or blotted onto PVDF membranes which were subsequently blocked
in 5% nonfat milk, 0.2% Tween, and 0.02% NaAzide in PBS for 1 hour. Blots were
incubated in 1:750 SERCA 2 antibody overnight at room temperature. Blots were washed
3x in PBS and subsequently incubated with a goat anti-rabbit secondary antibody/alkaline
phosphatase conjugate diluted 1:1000 for 1.5 hours. Blots were developed with
BCIP/NBT.
Calcium Flourimetry
Sarcoplasmic reticulum calcium uptake in heart SR preparations was measured
using the calcium-sensitive dye fura-2 and a Shimadzu RF 5301
spectroflourophotometer. SR microsomes were added at a final concentration of 1.5
mg/ml to a cuvette containing 1.4 ml calcium transport buffer (95.0 mM KCl, 20 mM H¬
MOPS, 7.5 mM NaPyrophosphate), 5.0 mM creatine phosphate, 0.01 mg/ml creatine
phosphokinase and 1.5 uM fura-2. Extravesicular calcium was monitored as a ratio of
the fura-2 fluorescence emission intensity at 510 nm at excitation wavelengths 340 nm
and 380 nm. Calcium uptake was initiated with the addition of 2.5 mM MgATP and
allowed to proceed to equilibrium. Subsequential 15 nmol additions of CaClz were added
to the cuvette to load the vesicles. At the end of the experiment, 1 mM CaClz was added
followed by 20 mM EGTA.
Results
Purification of sarcoplasmic reticulum
The homogenate, microsomal, light sarcoplasmic reticulum (LSR) and heavy
sarcoplasmic reticulum (HSR) fractions of bluefin and yellowfin ventricle were run on a
3-12% SDS-PAGE that was stained with Coomassie blue (Fig. 2). The homogenate
retains contractile filaments, as evidenced by the band at the 207 kDa molecular weight
marker, myosin. In the microsomal, LSR, and HSR preparations, the band at 207 kDa is
less dense, demonstrating the removal of contractile proteins during the purification
process. Also in these fractions, there is an enrichment of a band at about 110 kDa,
which, from previous work, has been shown to be SERCA 2 (Brandl et al, 1986).
Western blot
To determine if the band at 1 1OkDa was SERCA 2, a Western blot analysis was
conducted using a SERCA 2 specific primary antibody (Fig. 3). A band appears at 110
kDa in yellowfin tuna atria and ventricles and bluefin tuna ventricles, confirming the
presence of SERCA 2 in these tissues.
Densitometric comparison of bluefin and yellowfin, atrium and ventricle SERCA 2
A band at 110kDa appears in all lanes in 3-12% SDS-PAGE comparing bluefin
and yellowfin atrium and ventricle microsomal fractions (Fig. 4). A thin band at about
500 kDA also appears on the stained gels, demonstrating the existence of the ryanodine
receptor in tuna heart. Previous work has demonstrated that the molecular weight of the
fish ryanodine receptor is 576 kDa (Franck et al, 1998).
Densitometry performed on the SDS gels using NIH Image suggests that tuna
atrium has more SERCA 2 than ventricle. The ratio of SERCA 2 band intensities
between bluefin atrium and bluefin ventricle is 2.5 + 0.7 (n-2). The ratio between
yellowfin atrium and ventricle is 1.5 + 0.2 (n—4). Densitometry also suggests that
yellowfin and bluefin tuna heart have similar amounts of SERCA 2. The ratio between
yellowfin atrium and bluefin atrium is 1.5 + 1.0 (n-2). The ratio between yellowfin
ventricle and bluefin ventricle is 0.9 + 0.2 (n-4).
Flourimetry
SR vesicles take up calcium when ATP is added to the cuvette containing BFT
fast twitch SR vesicles, creatine phosphate, creatine phosphokinase, mitochondrial
inhibitors and fura 2 (Fig. 5). At t =O, ATP is absent in the cuvette and the level of
calcium in the extravesicular space, as evidenced by the ratio of fluorescence at 340 nm
to 380 nm, is high. Upon addition of ATP, the amount of calcium in the extravesicular
space decreases as the SR sequesters calcium. Upon the addition of CaCl, the amount of
calcium in the extravesicular space quickly increases and then declines back to baseline
as the SERCA 2 pumps are activated. The rates of uptake of calcium by the SR can be
calculated given the known amount of calcium added. The experiment is repeated with
bluefin and yellowfin atria and ventricles. (Fig. 6) Bluefin fast twitch muscle SR
sequesters calcium at a rate of 3.2 nmol Ca“/mg/s. Bluefin tuna slow twitch muscle SR
sequesters calcium at a slower rate of 0.094 nmol Ca“/mg/s. Bluefin atrium SR
sequesters calcium at a rate of 0.020 nmol/mg/s and bluefin ventricle SR sequesters
calcium at a rate of 0.014 nmol/mg/s. Yellowfin atrium and ventricle sequester calcium
at an identical rate of 0.012 nmol/mg/s (Table 1).
Discussion
In this study, I have shown the presence of proteins associated with the
sarcoplasmic reticulum in tuna atrial and ventricle tissues. I have examined the content
of SERCA 2 and found it slightly higher in the atrium in both tunas. The results may
indicate that the high metabolic rates of the tunas are correlated with the presence of
sarcoplasmic reticulum and the machinery for calcium cycling.
In mammalian and avian lineages, the metabolic rates require high cardiac
outputs, which are enhanced by the short diffusion distance for calcium from the internal
SR stores to the contractile filaments. This allows for an increased relaxation rate, heart
rate, cardiac output, and delivery of oxygen to the demanding muscles. In the classic
view of fish, tension generation relies primarily on extracellular calcium, thus
lengthening the diffusion distance and decreasing fish heart rates. Tunas are powerful,
highly metabolic, endothermic fishes that maintain high muscle temperatures through a
wide range of ambient temperatures. The heart, at ambient temperature, somehow fulfills
the high oxygen demand of the working tuna muscle. The use of an internal specialized
structure, such as the SR, could explain how tuna maintain a cardiac output high enough
to supply oxygen to the swimming muscles.
Tuna atrium may have more SERCA 2 than ventricle. Densitometry shows that
the ratio of SERCA 2 in bluefin atrium to bluefin ventricle is 2.5 + 0.7 (n-2), which
correlates with the faster rates of calcium uptake in bluefin atrium than bluefin ventricle.
Bluefin atrium SR sequesters calcium at a rate of 0.020 nmol/mg/s and bluefin ventricle
sequesters calcium at a rate of 0.014 nmol/mg/s. Moreover, the ratio between yellowfin
atrium and ventricle is 1.5 + 0.2 (n—4). The ratio between yellowfin atrium and ventricle,
which is not as high as the ratio between bluefin atrium and ventricle, is reflected in rates
of uptake; yellowfin atrium and ventricle SR sequester calcium at an identical rate of
0.012 nmol/mg/s. The calcium uptake assay may not be sensitive enough to detect
differences in rates of uptake in yellowfin heart SR.
The greater amount of SERCA 2 in tuna atrium and ventricle, and the faster rate
of uptake by the bluefin atrium than ventricle, can be explained by the roles of the
different chambers in the cardiac cycle. The atrium contracts and ejects its volume of
blood into the ventricle; the ventricle subsequently contracts and must sustain its
contraction against a load. While the ventricle must be able to sustain its contraction, the
atrium must contract and relax quickly before the ventricle contracts. The speed of
contraction of the atrium is critical during cardiac contraction whereas the force
generated in the ventricle is critical. An increased amount of SERCA 2 in atrium may
permit faster uptake of calcium into the SR and faster relaxation rates.
There is not a substantial difference in the amount of SERCA 2 between bluefin
and yellowfin hearts. Densitometry shows that the ratio of yellowfin atrium and bluefin
atrium is 1.5 + 1.0 (n=2). The ratio between yellowfin ventricle and bluefin ventricle is
0.9 +0.2 (n=4). The similar amounts of SERCA 2 between the two species of tuna can
be explained by the following: Both bluefin and yellowfin tuna live in warm waters for
part of the year. This period, where muscles are warm and demand an abundance of
oxygen, sets the maximum limit of oxygen delivery to the tissues. For the heart to
deliver oxygen to warm, highly demanding swimming muscles at 30°C, both yellowfin
and bluefin tuna hearts must have high frequency contractions. Thus, both species have
the calcium cycling machinery necessary to deliver these high frequency contractions;
both species are equipped with high amounts of SERCA 2. How bluefin tuna can
maintain their high metabolic rates and high cardiac outputs to sustain their aerobic
performance necessitates fürther research. Perhaps the SERCA pumps are at a high
concentration in cold waters and are pumping just as much calcium into the SR in cold
waters as they do in warm waters. Other proteins in the calcium cycle may be
responsible for high bluefin tuna cardiac outputs through a wide range of temperatures.
Instead of increasing heart rates to maintain a high cardiac output to deliver
oxygen to their swimming muscles, perhaps bluefin tuna rely on an increased stroke
volume to supply their high cardiac outputs. This could be caused by the surface
membrane voltage gated channels (DHPR), which may let calcium into the myocyte for a
longer period of time. An increased flow of calcium into the cell would sustain the
contraction and increase the contractile power of the heart. Investigation into the role of
other calcium cycling proteins may provide insight into the ability of bluefin tuna to
maintain their cardiac outputs in both cold and warm waters.
Calcium uptake assays show that bluefin white muscle SR sequesters calcium
more quickly than slow twitch or heart muscle. There are more SERCA pumps in white
muscle than red muscle. White muscle also has the SERCA 1 isoform of the pump while
red muscle and cardiac cells have the SERCA 2 isoform (Brandl et al, 1986). The white
muscle in tuna swimming muscle must contract quickly to generate the power necessary
to for tuna to swim at exceptionally high speeds. Red muscle and cardiac muscle cells,
on the other hand, contract more slowly with their smaller diameter and their high
concentration of mitochondria. Although uptake of calcium by the SR in tuna hearts is
slow, the uptake is ATP mediated, suggesting that tuna hearts use SERCA 2 as a means
of removing calcium from the cytoplasm.
Conclusions
The existence of SR in tuna cardiac cells is a factor in explaining the high cardiac
outputs and metabolic rates of tuna. Calcium cycling in the SR is crucial in determining
rates of contraction and relaxation. Tunas, the athletes of the fish world, have the ability
to coordinate the release and uptake of calcium by the SR and deliver oxygen to their
working muscles in a wide range of ambient temperatures. How tunas evolved this
extraordinary ability and the physiological mechanisms that allow them to do so is an
interesting question. The uptake of calcium by SERCA 2 hints at the paramount role of
the SR in contributing to their high metabolic rates. Further research possibilities include
an investigation of the role of other calcium cycling proteins, such as DHPR, the
ryanodine receptor, and myosin ATPase, which may contribute to the high cardiac
outputs of tuna.
Acknowledgements
Towe an incredible amount of gratitude to my advisor, Barbara Block, for her
dedication and excitement about my project. I thank her for sparking my interest in
research and for contributing to my critical thinking on this project. l’ve learned a little
bit about what science is about and couldn’t have done it without her. I had never
thought as tuna as amazing organisms until I met Barb! I also am so grateful to Jeffery
Morrissette for the amount of work and time he invested in this project. I thank him for
answering all my questions and being a wonderful teacher and I could not have done this
without him. 1 want to thank Jason Blank for also being an incredible teacher and for his
willingness to drop everything to help me. I thank him for furthering my excitement
about physiology. l’ve learned so much about being in a lab, research, and science from
these two gentlemen. l’d also owe a special thanks to everyone in the Tuna Research and
Conservation Center for their wonderful help.
12
Literature Cited
Blank, J. et al. 2001. Effects of temperature, adrenaline and calcium on the hearts of
yellowfin tuna (Thunnus albacares). Tuna Research and Conservation Center. Stanford
University, Hopkins Marine Station, Oceanview Blyd, Pacific Grove, CA 93950.
Brandl, C. J. et al. 1986. Two Ca2+ATPase genes: homologies and mechanistic
implications of deduced amino acid sequences. Cell 44: 597-607.
Brill, R.W. and Bushnell, P.G. 1991. Metabolic and cardiac scope of high energy
demand teleosts, the tunas. Can. J. Zool 69: 2002-2009
Carey, Francis G. Feb 1973. Fish with warm bodies. Sci. Am. 228 No 2.
Farrell A.P. and Jones, D.R. 1992. The heart. pp.1-88 in Fish Physiology, Vol XIIA,
Academic Press, San Diego, CA.
Franck , J. et al. 1998. Cloning and characterization of fiber type-specific ryanodine
receptor isoforms in skeletal muscles of fish. American Physiological Society
Kanwisher, J. et al. (1974). Acoustic telemetry from fish. Fish. Bull., U.S. 72: 251-255.
Keen, J. E. et al. 1992. Cardiac physiology in tunas II. Effect of ryanodine, calcium and
adrenaline on force-frequency relationships in atrial strips from skipjack tuna,
Katsuwonus pelamis. Can J. Zool. 70: 1211-1217.
Kiceniuk, J.W. and Jones, D.R. 1977. The oxygen transport system in trout (Salmo
gairdneri) during sustained exercise. J. Exp. Biol. 69: 247-260.
Korsemeyer, K. E. et al. 1977. Oxygen transport and cardiovascular responses to
exercise in the yellowfin tuna Thunnus albacares. J. Exp. Biol. 200: 1987-1997.
Shiels, H. A. and Farrell, A. P. 1999. The sarcoplasmic reticulum plays a major role in
isometric contraction in atrial muscle of yellowfin tuna. J. Exp. Biol. 202: 881-890.
Tibbits, G. F. et al. 1991. Calcium transport and the regulation of cardiac contractility in
telesosts: a comparison with higher vertebrates. Can. J. Zool. 69: 2014-2019.
Tibbits, G. F. et al. 1992. Excitation-contraction coupling in the teleost heart. pp. 267-
304 in Fish Physiology Vol XIIA, Academic Press, San Diego, CA.
Table I. Rate of calcium uptake in BFT fast twitch and slow twitch muscle and BFT and
yFT atrium and ventricle SR vesicles. 75 nmol of CaClz were added to BFT fast twitch
SR vesicles. The concentration of protein was 0.5 mg/ml. 15 nmol of calcium were
added to BFT slow twitch muscle and BFT and YFT atrium and ventricle SR vesicles.
The concentration of these proteins was 1.5 mg/ml. Ti/ is the amount of time required
for the SR to take up half the amount of calcium added.
Type of Muscle
Ca Uptake (nmollmgs
Tia (seconds)
BFT Fast Twitch Muscle
3.2
15.7
0.094
BFT Slow Twitch Muscle
105.9
BFT Atrium
0.020
196.1
227
231.
BFT Ventricle
0.014
YFTAtrium
0.012
YFT Ventricle
296.1
0.012
14
Figure Legend
Fig 1. The heart cell. Calcium from the extracellular space enters the heart cell through a
voltage-gated channel, DHPR. This causes calcium induced calcium release by the
ryanodine receptors in the SR membrane. Tension can then be generated by the
contractile filaments. During relaxation of the heart cell, calcium is removed by plasma
membrane calcium ATPase (PMCA), the sodium-calcium exchanger, and SR calcium
ATPase (SERCA). (from Tibbits et al, 1992).
Fig 2. Purification of sarcoplasmic reticulum. A 3-12% SDS gel was loaded with
different fractions of the SR isolation preparation in YFT and BFT ventricle. The
homogenate contains the contractile filaments. Microsomes, light SR
and heavy SR fractions are enriched in SR.
Fig 3. Western blot with a SERCA 2-specific antibody. Tuna hearts express SERCA 2.
Fig. 2A indicates that BFT and YFT ventricle have SERCA 2, as evidenced by the bands
at 110 kDa. Fig. 2B indicates that YFT atrium and ventricle have SERCA 2, as
evidenced by the bands just below the 118 kDa molecular weight marker.
Fig. 4. A comparison between bluefin and yellowfin atrium and ventricle. SERCA 2
(110 kDa), myosin (200 kDa), and ryanodine receptor (576 kDa) can be
visualized on this 3-12% SDS gel. Tuna hearts have SERCA 2.
Fig 5. Calcium uptake by BFT fast twitch muscle SR. Uptake of calcium by BT fast
twitch muscle SR is ATP mediated. The amount of calcium in the extravesicular space
decreases when ATP is added and the SR sequesters calcium. When CaCl» is added, the
extravesicular calcium increases is followed by an immediate sequestration of calcium by
the SR.
Fig. 6. Calcium uptake in BFT atrium and ventricle. BFT atrium (2A) and ventricle (2B)
SR sequester calcium in the presence of ATP when CaCl, is added.
S1
co co
6
ADP 3N0
T-TUBULE
AT
SR
SR C

Ve
—
—
—
—
—
—
COR CNOICOX DRNOR
Fig. 1
17
7 kDa
8 kDa-
BFT Ventricle


Fig. 2
YFT Ventricle
4

Serca 2
A.
BFT Ventricle
110 kDa
B.
118 kDa
Fig. 3
YFT Ventricle

YFT
207 kDa -
118 kDa-
BFT YFT


Fig. 4
RyR
Myosin
Serca 2
0
2.5 mM MgATP

75.0 nmols CaCl, X 5


5 UM A23187 —
200 400 600 800 1000 1200 1400
Time (seconds)
Fig. 5
B.
1

15O nmols Ca 12



2.5 mM MgATP
200 400
600 800 1000 12
Time (seconds)
15.0 nmols CaCl, x2


2.5 mM MgATP
200 400
600 800 1000 1200 1400
Time (seconds)
5.