Download Temperature dependence of cardiac performance in the lobster

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The Journal of Experimental Biology 209, 1024-1034
Published by The Company of Biologists 2006
doi:10.1242/jeb.02082
Temperature dependence of cardiac performance in the lobster Homarus
americanus
Mary Kate Worden1,*, Christine M. Clark1, Mark Conaway2 and Syed Aman Qadri1
1
Department of Neuroscience and 2Division of Biostatistics and Epidemiology, University of Virginia, PO 801392,
Charlottesville, VA 22908, USA
*Author for correspondence (e-mail: [email protected])
Accepted 9 November 2005
Summary
The lobster Homarus americanus inhabits ocean waters
the temperature effects on the cardiac ganglion. In
that vary in temperature over a 25°C range, depending on
contrast to earlier reports suggesting that the strength and
the season and water depth. To investigate whether the
the frequency of the lobster heartbeat are positively
lobster heart functions effectively over a wide range of
correlated, we observe no consistent relationship between
these parameters as they change as a function of
temperatures we examine the temperature dependence of
temperature. Stroke volume decreases as a function of
cardiac performance of isolated lobster hearts in vitro. In
temperature. However, the opposing temperatureaddition, we examined whether modulation of the heart by
dependent increase in heart rate partially compensates to
serotonin depends on temperature. The strength of the
produce a relationship between cardiac output and
heartbeat strongly depends on temperature, as isolated
temperature in which cardiac output is maximal at 10°C
hearts are warmed from 2 to 22°C the contraction
and significantly decreases above 20°C. Serotonin
amplitude decreases by greater than 60%. The rates of
potentiates contraction amplitude and heart rate in a
contraction and relaxation of the heart are most strongly
temperature-independent manner. Overall, our results
temperature dependent in the range from 2 to 4°C but
show that although the parameters underlying cardiac
become
temperature
independent
at
warmer
performance show different patterns of temperature
temperatures. Heart rates increase as a function of
dependence, cardiac output remains relatively constant
temperature both in isolated hearts and in intact animals,
over most of the wide range of environmental
however hearts in intact animals beat faster in the
temperature range of 12–20°C. Interestingly, acute Q10
temperatures the lobster inhabits in the wild.
values for heart rate are similar in vivo and in vitro over
most of the temperature range, suggesting that
temperature dependence of heart rate arises mainly from
Key words: temperature, heart, lobster, Homarus americanus.
Introduction
The North American lobster Homarus americanus inhabits
both the deep ocean waters and the coastal shallows and
estuaries. Water temperature in its habitat varies over a range
of 25°C (Lawton and Lavalli, 1995). Like other poikilotherms,
lobsters are unable to regulate their own body temperature.
However, they apparently detect changes in water temperature
with great resolution, as they exhibit altered cardiac activity
when temperature shifts by less than 0.5°C (Jury and Watson,
2000). Significant changes in environmental temperature pose
an obvious physiological challenge to the lobster: how can its
organs sustain appropriate performance, as each element of the
system (including the ion channels, cell membranes, neural
circuits and contractile machinery) is likely to depend on
temperature in a different way? In the wild, lobsters undergo
seasonal migrations (Campbell, 1986; Campbell, 1989;
Campbell and Stasko, 1986; Cooper and Uzmann, 1971; Ellis,
2001; Ennis, 1984; Estrella and Morrissey, 1997; Watson et
al., 1999). These migrations are thought to enable the lobsters
to reach or remain at water temperatures favorable for growth,
development and molting, all of which become compromised
below 10°C (reviewed by Ennis, 1995; Waddy et al., 1995).
However, more recent data provide direct evidence that
some lobster populations do not escape the extremes of winter.
Temperature probes affixed to Gulf of Maine lobsters record
extremely cold (0–6°C) water temperatures throughout six
months of winter and early spring (Cowan, 2004). Under
laboratory conditions, lobsters exposed to thermal gradients
show a preference for significantly warmer water temperatures,
in the range 13–20°C, and avoid water at higher temperatures
(Crossin et al., 1998; Reynolds and Casterlin, 1979). In the
wild, relatively warm temperatures are most likely to be found
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Lobster heart and temperature 1025
temperature. In addition, serotonin modulates the strength and
frequency of the lobster heartbeat in a manner that is
independent of temperature.
Temperature (°C)
24
20
16
12
11
Ju
13 ly
Ju
15 ly
Ju
17 l y
Ju
19 ly
Ju
21 l y
Ju
23 ly
Ju
ly
8
Summer 2002
Fig.·1. Hourly water temperatures recorded at a lobster trap at a depth
of 7 fathoms (12.8·m). Data courtesy of James Manning of the
National Oceanographic and Atmospheric Association and the
Environmental
Monitors
on
Lobster
Traps
project
(http://www.emolt.org).
in shallow waters in summer months. Water temperature in
shallow water is strongly affected by winds and tides.
Temperature swings of greater than 12°C during the daily tides
have been recorded at the level of the lobster traps (Fig.·1)
(Manning, in press). Thus, lobsters face thermal shifts not only
throughout the seasons but on shorter time scales, potentially
even as they move in and out of thermoclines on the ocean
floor.
In this study we explore how the cardiac system of the
lobster responds to temperature changes in the physiological
range. Because crustacean hearts are regulated by
physiological
inputs
(neural,
neurohormonal
and
chemosensory) as well as environmental influences (oxygen
levels and salinity) (McMahon, 1999), it is also of interest to
know whether temperature affects the response of the heart to
other modulatory factors. We address this question by testing
whether modulation of the cardiac performance by serotonin
is temperature dependent. Serotonin is a neurohormone
localized in and released from the pericardial organs in the
lobster (Cooke, 1966; Pulver and Marder, 2002). It has been
shown previously that serotonin modulates lobster hearts both
in vivo and in vitro (Battelle and Kravitz, 1978; Guirguis and
Wilkens, 1995; Wilkens and Kuramoto, 1998; Wilkens et al.,
1996). Whether these modulatory effects might depend on
temperature is unknown.
Here we characterize the effects of temperature on cardiac
performance in the lobster hearts by measuring the strength and
frequency of the heartbeat and the cardiac output of the heart
in vitro. For comparison, we examine the temperature
dependence of heart rates in intact animals. In addition, we test
whether the modulation of in vitro cardiac activity by serotonin
depends on temperature. The results demonstrate that multiple
parameters of cardiac performance depend on temperature, and
that these temperature dependencies interact to produce a
relatively constant relationship between cardiac output and
Materials and methods
In vitro experiments
Lobsters (Homarus americanus Milne-Edwards 1837) were
obtained from commercial sources and kept in artificial
seawater at 5°C. To minimize the potentially confounding
effects of seasonal variability, animals were maintained for a
month or longer at 5°C before being used in experiments.
Lobsters were anesthetized on ice, after which the heart was
isolated for in vitro studies as described previously (Worden
et al., 1995). Briefly, a small section of the dorsal thoracic
carapace was removed with the heart attached and surrounding
tissues were dissected away. The alary ligaments and muscles
were left intact, providing stretch to the mechanoreceptors
(Cooke, 2002). The preparation was pinned in a recording
chamber ventral side up and submerged in 60·mls of lobster
saline (462·mmol·l–1 NaCl, 16·mmol·l–1 KCl, 26·mmol·l–1
CaCl2, 8·mmol·l–1 MgCl2, 11·mmol·l–1 glucose, 10·mmol·l–1
Hepes, pH·7.4). The interior of the recording chamber was
encircled by several loops of Tygon tubing through which
refrigerated coolant (antifreeze:water in a 50:50 mix) was
pumped by a refrigerated circulator (Isotemp model 1016P,
Fisher Scientific, Pittsburgh, PA, USA) to control bath
temperature in the range 2–25°C.
The organization of the lobster heart has been reviewed
elsewhere (McMahon, 1995). Briefly, hemolymph enters the
single chamber of the lobster heart through paired ostia and is
pumped out through seven arteries. Isolated hearts continue to
beat because of rhythmic neural output from the cardiac
ganglion, located on the dorsal inner wall of the heart. Following
isolation of the heart, the sternal artery was cannulated and
perfused with temperature-controlled lobster saline at
3.5·ml·min–1 to maintain stretch. In some experiments the
antennal arteries were tied off with 6.0 surgical silk and attached
to a tension transducer (Grass Instruments, West Warwick, RI,
USA; model FT-03) and amplifier (CyberAmp model 320; Axon
Instruments, Union City, CA, USA) to record contractions of the
heart during each heartbeat. Contractions were measured as
force (g). In some experiments cardiac output out via the sternal
artery was measured ultrasonically (in ml·min–1) using an inline
flow transducer (Model T206, Transonic Systems, Ithaca, NY,
USA). In these experiments the cannula was led back into the
water at zero afterload and saline was perfused through the heart
through one of the ostia. Temperature in the bath was continually
monitored. All physiological signals were recorded on a video
cassette recorder (VCR) and digitized by an analog to digital
converter using pClamp software (Digidata1200 A-D converter
and pClamp software from Axon Instruments-Molecular
Devices, Union City, CA, USA).
In each experiment, the parameters of cardiac function were
measured as the temperature was changed from 2 to 22°C at a
rate of 0.9°C·min–1. In some experiments, the temperature was
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1026 M. K. Worden and others
In vivo experiments
To record cardiac activity in vivo two holes were drilled on
the midline of the dorsal carapace for chronic implantation of
electrodes. The holes were located 1·cm and 4·cm rostral to the
posterior edge of the carapace. Analysis of the location of the
holes by later dissection showed that they were in positions
rostral and caudal to the perimeter of the underlying heart. Two
wires insulated to within 0.5·cm of their tips were inserted into
the holes, fixed in position with cryanoacrylate glue and duct
tape, and connected to an impedance converter (UFI model
2991). The DC and AC outputs of the impedance converter
were connected to a VCR as well as an analog to digital
converter to record data with pClamp software. Animals were
implanted at least 48·h before data were collected.
To record changes in cardiac activity as a function of the
environmental temperature the animals were placed in a
wire mesh cylinder surrounded by coils of tubing through
which coolant was pumped by a refrigerated circulator. The
cylinder was then submerged in an insulated chamber
(23·cm⫻18·cm⫻16·cm) filled with 3.5·l of seawater (Crystal
Sea Salt, Marine Enterprises International, Baltimore, MD,
USA) until the dorsal carapace was 2–3·cm below the surface
of the water. The temperature of the seawater bath was
regulated by adjusting the refrigerated circulator. The bath
temperature in the chamber was continually monitored by a
temperature probe fixed in position over the dorsal carapace
between the two recording electrodes. In all experiments the
animals were acclimated to 2°C for at least 30·min before data
were collected, during which time heart rate was monitored.
During each experiment the temperature of the seawater was
warmed from 2 to 22°C over a period of approximately
40–50·min.
To verify whether the temperature within the thoracic cavity
of a living animal approximated the temperature of the
surrounding water we repeated the temperature protocol while
measuring the temperature within the pericardial sinus. The
temperature of the pericardial sinus becomes isothermal with
the external temperature within the first 5–10·min of the 30·min
acclimation period at 2°C. Fig.·2 shows that the temperature
within the pericardial sinus rises as external water temperature
warms. In three experiments of this type the difference between
internal and external temperature was 1.31±0.42°C (mean ±
s.d.) over the temperature range from 2 to 22°C, confirming
that the internal body temperature in the region of the heart
closely approximates external water temperature.
Data analysis
For both in vivo and in vitro experiments, the frequency of
the heart beat was measured over 60·s at each temperature,
Pericardial sinus
External water
20
Temperature (°C)
returned to 2°C and serotonin (1·␮mol·l–1) was added to the
saline perfusing the heart through the sternal artery cannula.
After waiting for the serotonin effects to reach steady state
(defined as a potentiation of contraction amplitude that varied
by no more than 5% over a period of 5·min), temperature was
again warmed from 2 to 22°C in the presence of serotonin.
15
10
5
0
0
10
20
30
Time (min)
40
50
Fig.·2. Temperature of the pericardial sinus increases as external water
temperature warms. The water temperature begins warming from 2°C
at time zero. In this experiment internal and external temperature
differed by 1.03±0.14° (mean ± s.e.m.) over the temperature range
2–22°C.
with at least five heartbeats recorded in each trial. In some in
vivo experiments the heart occasionally stopped temporarily.
If the heart failed to beat for 15·s or longer, the frequency data
were analyzed by excluding the period of heart failure. The
strength of the heartbeat was measured as the peak amplitude
of the contraction with respect to baseline, at least five
heartbeats were measured at each temperature. Acute values of
Q10 were calculated as Q10=(fH2/fH1)exp[10/(t2–t1)] where fH2
is the heart rate at temperature t2 and fH1 is the heart rate at
temperature t1 (DeWachter and Wilkens, 1996). Stroke volume
was calculated by dividing cardiac output by the heart rate.
Statistical methods
Repeated measures models (Crowder and Hand, 1990) were
used for the analyses of experiments involving multiple
measurements on the same lobster as well as to investigate
specific hypotheses concerning comparisons between groups
or to specific temperatures. F tests were used to compare the
values for cardiac parameters at different temperatures.
Analyses were done using SAS software (The SAS Institute,
Cary, NC, USA) version 9.1 module ‘PROC MIXED’.
Results
To examine the temperature dependence of cardiac
performance in the lobster, spontaneously beating isolated
hearts were exposed in vitro to a steady temperature increase
throughout the physiological range 2–22°C. As shown in the
tension recordings in Fig.·3, temperature alters both the
strength and frequency of the heartbeat. At the coldest
temperatures the contractions of the heart are large in
amplitude and heart rate is relatively low. Both parameters
change as temperature warms, the heartbeats become smaller
in amplitude and faster in frequency. In addition, the shape of
the heartbeat changes, contraction and relaxation during each
heartbeat proceed relatively slowly at cold temperatures but
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Lobster heart and temperature 1027
0.5 g
10 s
2
4
6
8
10
12
14
16
18
20
22
Temperature (°C)
Fig.·3. Temperature affects the amplitude and frequency of heartbeats
recorded from isolated lobster hearts. Tension recordings of
heartbeats at different temperatures are shown; all are from the same
in vitro preparation and are displayed on the same scale.
become faster at warmer temperatures. Overall, therefore,
heartbeats are long in duration and large in amplitude at the
cold extreme of the temperature range where heart rate is slow,
and they become faster and weaker as temperature warms and
heart rate increases. Finally, in this preparation the baseline
tonus of the heart also changed, increasing with temperature
up to 16°C and decreasing at higher temperatures. Similar
changes in tonus were observed in three other experiments, but
were variable and not analyzed further.
Mean contraction (normalized)
Temperature affects the amplitude of the heartbeat as well as
the rates of contraction and relaxation
To describe these phenomena quantitatively, data from nine
isolated hearts were pooled. Contraction amplitude decreases
significantly as the temperature warms (Fig.·4). At
1.2
1.0
0.8
0.6
0.4
0.2
0
2
6
10
14
18
Temperature (°C)
22
Fig.·4. The strength of the heartbeat depends on temperature. Symbols
represent the estimated mean ratios (±95% confidence intervals) of
contraction amplitudes measured at each temperature relative to those
measured at 2°C. Statistical significance was determined from
repeated-measures models using log scale. Compared to the values at
2°C, contraction amplitudes are significantly different at temperatures
from 12 to 22°C (P<0.05). For temperatures of 2–16°C, N=9; 18 and
20°C, N=6; 22°C N=5.
temperatures of 16°C and higher the mean amplitude of the
heartbeat is less than 35% of its initial value at 2°C. The
temperature dependence of the shape of the heartbeat is
described in Fig.·5. Contractions develop and relax relatively
slowly at the coldest temperatures but become faster as
temperature warms (Fig.·5A). This phenomenon is illustrated
quantitatively by the phase plots (Fig.·5B,E) describing the
relation of the rate of change in force to the contractile force
recorded during the heartbeat. These plots show pronounced
differences in shape as temperature changes because at warm
temperatures the amplitude of the heartbeat is comparatively
small while the rate of change in force is comparatively fast
for the contraction and the relaxation phase of the heartbeat.
From 2 to 10°C the rates of contraction and relaxation of each
heartbeat more than double, before slowing as temperature
becomes warmer (Fig.·5C). To examine the temperature
dependence of contraction and relaxation without the
confounding effect of temperature on heartbeat strength, data
from Fig.·5C were replotted after normalizing for contraction
amplitude (Fig.·5F). Normalization changes the shape of these
plots without changing the conclusion of the analysis.
Calculation of acute Q10 values for changes in rates of
contraction and relaxation demonstrate these parameters are
strongly temperature dependent in the 2–4°C range (acute Q10
values >20 and >7 for raw and normalized data, respectively;
Fig.·5D,G). At warmer temperatures rates of contraction and
relaxation vary from weakly temperature dependent to
temperature independent.
Temperature affects heart rate both in vitro and in vivo
As shown in the raw data of Fig.·3, the frequency of the
heartbeat tends to increase as a function of temperature.
Responses of ten individual isolated hearts to temperature
change are shown in Fig.·6A. Heart rates increased in all hearts
between 2 and 10°C, but there was considerable variability at
warmer temperatures with some of the isolated hearts slowing
or failing. Fig.·6B compares the mean heart rates recorded as
a function of temperature from isolated hearts in vitro and from
hearts in intact animals in vivo. In both isolated hearts and
intact animals, increases in heart rate occurred gradually as
temperature warmed with no observed periods of bradycardia
or tachycardia. Both sets of data show significant increases in
heart rate as temperature warms (see Fig.·6 legend). Over the
temperature range from 2 to 22°C, the heart rates of isolated
hearts increased approximately fourfold in vitro (closed
squares) whereas heart rates measured in vivo increased
approximately 2.5-fold (closed circles). Isolated hearts tend to
beat more slowly than hearts in intact animals, these difference
are statistically significant at 2°C and in the temperature range
from 12 to 22°C. In isolated hearts, the maximum heart rate
observed was 1.53·Hz (99·beats·min–1) at a temperature of
20°C. In intact animals the maximum heart rate observed was
1.85·Hz (111·beats·min–1) at 22°C, slightly faster than the
previously reported upper limit of 100·beats·min–1 for lobsters
walking on treadmills (O’Grady et al., 2001). Finally, to test
whether heart rates in intact animals might thermoadapt over
THE JOURNAL OF EXPERIMENTAL BIOLOGY
22°C
5
B
2°C
Normalized
dF/dt (g s–1)
dF/dt (g s–1)
1.5 s
0
–5
5
22°C
2°C
E
0
0
2
Rate of rise
Rate of decay
0
2
30
20
6
D
14 18
6
6
F
1
Rate of rise
Rate of decay
4
50
0.5
0
0
2
6
2.0
1.5
*
1.0
0.5
8
G
10 14 18 22
Rise
Decay
4
6
long periods at thermal equilibrium, lobsters were warmed
from 2°C to a new steady state level of 12°C. Fig.·6C shows
that heart rate increases as temperature warms from 2 to 12°C
and remains constant for 45·min under steady state temperature
conditions. Thus, we find no evidence that thermoadaptation
alters heart rate. This observation is consistent with the fact
that our data, recorded from lobsters under laboratory
conditions, is in good agreement with heart rates measured in
lobsters in the wild (Fig.·6B, open symbols).
60
0
0
2.0
C
1.5
6
10
14
18
Temperature (°C)
22
90
2
12°C
12°C
60
1.0
30
0.5
0
0
10 14 18 22
Fig.·5. The rates of contraction and relaxation of the heartbeat depend on
temperature. (A) Tension recordings averaged over 12 successive heartbeats
show the shape of the heartbeat at the indicated temperatures. The inset shows
heartbeats at 2°C and 22°C normalized to the maximal amplitude of the
heartbeat. (B) Phase plots of the rate of change of force (y-axis) as a function
of the force of contraction (x-axis) of the heartbeat. Plots represent all
heartbeats recorded during 1·min at each indicated temperature. (C) Rates of
contraction and of relaxation measured as the mean (±s.d.) of the maximum
rising and falling slopes (dF/dt) of the heartbeat at indicated temperatures.
(D) Acute Q10 values for the contraction and relaxation of the heartbeat
calculated from the change in dF/dt as a function of temperature (see
Materials and methods). (E) Phase plots of B normalized for contraction
amplitude at each temperature. (F) Rates of contraction and relaxation from
C normalized for contraction amplitude at each temperature. (G) Acute Q10
values calculated from normalized data in F. All data are from a single
isolated heart.
******
* **
*
0
0
10 14 18 22
2
Temperature (°C)
22
120
2
6
10
14
18
Temperature (°C)
B
2
22
2
Temperature (°C)
Rise
Decay
3
2
1
0
2
10
Normalized
maximum dF/dt (g s–1)
4
Acute Q10
Maximum dF/dt (g s–1)
Acute Q10
C
100
2°C
0
Force (g)
8
A
1.0
22°C
–5
22°C
1.5
Heart rate (beats min–1)
4°C
Mean heart rate (Hz)
1.2 g
2°C
Mean heart rate (Hz)
A
Heart rate (Hz)
1028 M. K. Worden and others
20
40
60
Time (min)
80
Fig.·6. Heart rate depends on temperature. (A) Scatter plots
show the frequency of the heartbeat in isolated hearts (N=10)
in vitro as a function of temperature. Each symbol represents
a different heart. The number of hearts failing at temperatures
of 18, 20 and 22°C, was 2, 3 and 5, respectively. (B) Heart
rates from isolated hearts (filled squares) plotted for
comparison with heart rates recorded in intact lobsters in vivo
(filled circles) as a function of temperature. In both data sets
there are significant differences in mean heart rate across
temperature (P<0.0001). Assuming a linear trend, the
estimated slopes (means ± s.e.m.) of the lines relating heart
rate to temperature in intact animals and in isolated hearts are
0.040±0.004 and 0.026±0.006, respectively. *Data are
significantly different at P<0.05; **P<0.001; P-value based
on contrast in repeated-measures model. The P-values are not
adjusted for the multiple comparisons. Even with adjusted
differences the data from 12 to 22°C are still significant.)
Only data from beating hearts were included for calculation
of the mean, therefore, for isolated hearts values of N are as
follows: from 2–16°C, N=10; at 18°C, N=8; at 20°C, N=7; at
22°C, N=5. In intact animals only one heart failed as
temperature increased (at 20°C). Heart rates (means ± s.e.m.)
measured in intact animals in the wild [open symbols; data
reproduced from (Mercaldoallen and Thurberg, 1987)]. (C)
Heart rates in intact animals (N=4) measured over time as
temperature increased from 2°C to a steady state level of
12°C. Horizontal bar indicates time period of temperature
change (2 to 12°C) and time period of steady state
temperature at 12°C.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Lobster heart and temperature 1029
*
2
2
1
1
3
Intact animals (N=21)
Isolated hearts (N=10)
2
5
1
4
0
0
0 0.5 1.0 1.5
3
*
2
1
0
2
6
10
14
18
Temperature (°C)
22
Fig.·7. Values for acute Q10 (means ± s.e.m.) calculated for heart rates
in intact animals and isolated hearts. Values are calculated from data
in Fig.·6B. *Data are significantly different at P<0.05.
Contraction amplitude (g)
Acute Q10
100
75
50
3
3
2
2
1
1
The strength and the frequency of the heartbeat are
independent parameters
Because the spontaneously beating heart is driven by
rhythmic output of the cardiac ganglion, the relationship
between the frequency of the heartbeat and the strength of the
heartbeat is of particular interest. The neurocardiac synapses
on the heart muscle gate calcium influx into heart muscle
through voltage-dependent channels, and these synapses
facilitate strongly as a function of frequency of motoneuron
firing (Anderson and Cooke, 1969; Mahadevan et al., 2004).
Previously, Mahadevan et al. have observed a positive
correlation between frequency and strength of the lobster heart
beat that they attributed to enhanced synaptic depolarization at
high burst frequencies (Mahadevan et al., 2004). However, in
our experiments we observed that these parameters behave
differently in response to temperature change: the strength of
contractions decreases (Fig.·4) whereas the frequency of
contractions increases (Fig.·6; see also raw data in Fig.·3).
To examine the potential coupling between strength and
frequency of the heartbeat we plotted the relation between
these parameters for each of seven isolated hearts that were
exposed to temperatures warming from 2 to 22°C (Fig.·8).
None of these plots shows a positive relationship between the
amplitude and the frequency of contraction. In general, the
contraction amplitude of the heartbeat appears to decrease as
0 0.5 1.0 1.5
0
0
0
0.5
1.0
0
0.5
1.0
0.5
1.0
2
5
4
3
Values for acute Q10 calculated for heart rates in vivo and in
vitro are shown in Fig.·7. For isolated hearts, the acute Q10
value associated with the increase in heart rate between 2 to
4°C is exceptionally high (acute Q10=60), owing to the steep
increase in beat frequency as the heart speeds up from an
exceptionally low heart rate at 2°C. Otherwise acute Q10 values
up to 20°C ranged from 3.5 to 1.0 and were similar in both
datasets, suggesting the temperature dependence of the beating
frequency in isolated hearts is similar to that in intact animals
over most of the temperature range.
0
0 0.5 1.0 1.5
1
2
1
0
0
0
0.5 1.0 1.5
0
Frequency (Hz)
Fig.·8. Plots showing the relationship between the amplitude of the
heartbeat contraction and the frequency of the heartbeat for seven
isolated hearts. Data were collected as each preparation was warmed
from 2 to 22°C. Arrows indicate direction of warming temperature.
frequency increases. Almost all of the plots showed regions
where similar amplitudes were recorded at different
frequencies, or temperature ranges where contraction
amplitude changed while heartbeat frequency remained
constant. Therefore, measured as a function of changing
temperature, the parameters of heart rate and strength of the
heartbeat do not positively correlate.
Cardiac output is relatively constant as a function of
temperature
The cardiac output depends both on the stroke volume and
the heart rate. To test the possibility that the temperaturedependent decrease in heartbeat strength (Fig.·4) might be
offset by the concurrent temperature-dependent increase in
heart rate (Fig.·6) we measured cardiac output directly. Fig.·9A
shows examples of cardiac flow recorded from the same
isolated heart at two different temperatures. Measurements of
this type in four isolated hearts were analyzed over the
temperature range from 2 to 22°C. In agreement with earlier
results (Fig.·6B), heart rate increases with temperature from
2°C up to 12°C to 14°C (Fig.·9B). In contrast, stroke volume
is maximal at the coldest temperatures and decreases as
temperature warms (Fig.·9C). Interestingly, cardiac output
shows a temperature-dependent trend that is well described by
a quadratic equation with a maximum at approximately 10°C
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1030 M. K. Worden and others
0.5 ml min–1
A
2°C
A
18°C
5s
10 min
Serotonin (1 μmol l–1)
B
Heart rate
(beats min–1)
75
2°C
50
25
3
Stroke volume
(ml beat–1)
Serotonin
B
10°C
0
Cardiac output
(ml min–1)
Control
C
2
* * *
* * * * * *
1
18°C
0.5 g
0
60
10 s
D
40
*
20
0
2
6
10
14
Temperature (°C)
18
Fig.·10. Serotonin increases the frequency and strength of the
heartbeat. (A) Serotonin increases contraction amplitude to a level that
remains at steady state for more than 10·min at 2°C. (B) Traces show
tension recordings at indicated temperatures under control conditions
and after application of serotonin. All traces in B are from the same
isolated heart.
22
Fig.·9. Cardiac performance in isolated hearts (N=4) in vitro as a
function of temperature. (A) Examples of blood flow in sternal artery
measured at the indicated temperatures. (B) Heart rate (mean ± s.d.)
measured as a function of temperature. (C) Stroke volume (mean ±
s.d.) measured as a function of temperature. The data show a linear
trend with an estimated slope of –0.070 (s.e.m.=0.017, P<0.001). (D)
Cardiac output (mean ± s.d.), measured as a function of temperature,
is represented by the symbols and solid lines. The broken line
represents the estimated means based on the quadratic equation as
follows:
mean
cardiac
output=19.18(±5.4)+5.01(±0.91)⫻
(temperature)–0.25(±0.04)⫻(temperature)2,
where
values
in
parentheses are standard errors. The estimated maximum for the fitted
curve is at temperature=9.90, 95% confidence interval (3.6, 16.2).
*Data are significantly different from values at 2°C (P<0.05).
(Fig.·9D). Between 2 and 20°C there are no statistically
significant differences in cardiac output, thus cardiac output
appears relatively constant over this temperature range. At
22°C cardiac output decreases significantly compared to the
data at 2°C.
Modulatory effects of serotonin are temperature independent
In agreement with earlier reports, serotonin increases both
the strength and the frequency of the heartbeat (Fig.·10). At
2°C the effects of serotonin on seven isolated hearts reached
steady state within 12.2±8.5·min (mean ± s.d.; range
2.3–21·min). In these experiments serotonin increased
contraction amplitude of the heart by an average of 128%
(range 13–497%) and increased the frequency of the heartbeat
by an average of 64% (range 18–171%). Fig.·11 shows the
effects of serotonin on contraction amplitude and frequency
over the entire temperature range, expressed as a ratio between
the value measured in the presence of serotonin as compared
to the control value at that temperature. In some experiments
performed in summer months serotonin was particularly potent
in increasing contraction amplitude at the warm temperatures
of 16–18°C (raw data not shown). However, results averaged
from all seven experiments showed no statistically significant
temperature dependence of the effect of serotonin on
contraction amplitude (Fig.·11A). Serotonin’s effect in
increasing the heart rate was also temperature independent,
serotonin increased the frequency of the heartbeat
approximately two fold at all temperatures (Fig.·11B). The
shapes of the phase plots for heartbeats in the presence and
absence of serotonin are similar (Fig.·11C), indicating that the
effects of serotonin on contraction and relaxation rates are
proportional to its effect in increasing contraction amplitude.
Discussion
Temperature is a critically important parameter both for
lobsters in the wild and for physiologically excitable cell
membranes. Here we focus on the temperature dependence of
a crustacean heart, a complex physiological system paced by
the rhythmic output of the cardiac ganglion motoneurons and
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Lobster heart and temperature 1031
modulated by neural inputs and circulating hormones.
However, unlike neurohormones and transmitters, which are
targeted to cells expressing the appropriate receptors,
temperature will affect all cells by altering the activity of ion
channels and membrane pumps both in the plasma membranes
and in the intracellular membranes. Temperature, therefore, is
a ubiquitous modulator, potentially affecting cardiac
physiology at multiple levels. In this study we describe the
temperature dependencies of several parameters of cardiac
performance in the lobster Homarus americanus. Interestingly,
although contraction amplitude, heart rate, stroke volume and
heartbeat kinetics have different patterns of temperature
45
40
Ratio 5-HT/Control
6
A
B
Frequency
Amplitude
5
35
30
4
25
20
3
15
10
2
5
0
2
7
12
17
1
22
2
7
Temperature (°C)
12
17
22
C
4
dF/dt (g s–1)
Serotonin
2
Control
0
–2
–4
0
1
2
3
Force (g)
Fig.·11. Serotonin increases the strength and frequency of the
heartbeat in isolated hearts (N=7). (A) Means of ratios of the
amplitude of the contraction in the presence and absence of serotonin
(5-HT) for each preparation are shown as a function of temperature.
There are no statistically significant differences in amplitude across
temperature (P=0.40). (B) Means of ratios of the frequency of the
heartbeat in the presence and absence of serotonin for each
preparation are shown as a function of temperature. There are no
significant differences in heart rate across temperature (P=0.69). (C)
Phase plots for heartbeats recorded in the absence (control) and
presence of serotonin show similar shapes. Each trace shows the rate
of change of force (y-axis) as a function of the force of the contraction
(x-axis) for heartbeats recorded over 1·min at 2°C. In this experiment
serotonin increased contraction amplitude at 2°C by 39.6%.
dependence, cardiac output remains relatively constant over a
wide temperature range.
A major finding in this study is that heart rates increase with
temperature both in vitro and in vivo. These results are in
agreement with several previous reports on Homarus
(Hokkanen and DeMont, 1997; McMahon, 1999; Schreiber et
al., 2000; Schreiber et al., 1998) as well as other species of
lobster (Nakamura et al., 1994; Zainal et al., 1992). We observe
faster heart rates in intact lobsters compared to isolated hearts,
especially in the range 12–20°C. Similar observations have
been made previously, both in lobsters (Schreiber et al., 1998)
and crabs (Wilkens, 1994), and attributed to the overall
cardioexcitatory effects of the cardioregulatory nerves and
endogenous hormones in intact animals. It is also possible that
the different heart rates observed in isolated and intact hearts
reflect differences in filling pressure, vascular resistance and
stretch of the heart muscle.
Our demonstration that the acute Q10 values for heart rate
are similar in isolated heart and in intact animals (except at the
extreme of 2°C) suggests that the temperature dependence of
the lobster heart rate arises mainly from the intrinsic properties
of the cardiac ganglion, rather than from activity in the
cardioregulatory nerves or from hormonal signals arising from
other regions of the CNS. Interestingly, Jury and Watson (Jury
and Watson, 2000) reached a different conclusion. Based on
their observations that severing the cardioregulatory nerves in
intact animals abolished the response of the heart to changes
in temperature, they suggested that the cardioregulatory nerves
mediate temperature dependence of the heart rate (Jury and
Watson, 2000). It is not entirely clear how to reconcile their
results with ours. However, we note that their experiments
were performed in a relatively narrow temperature range
(starting at 15°C and warming or cooling by 1.5°C) and are in
agreement with our data showing a small but significant
difference in temperature dependence of intact and isolated
hearts at 14°C (see Fig.·7). Nevertheless, over the relatively
large temperature range of 4–22°C our observation that the
temperature dependence of heart rate is similar in vitro and in
vivo supports the hypothesis that temperature dependence of
the heartbeat arises mainly from temperature effects on the
cardiac ganglion neurons. A minor contribution by
cardioregulatory nerves and/or hormones to the temperature
dependence of the heart rate is suggested by the difference in
the slopes of the plots describing the temperature dependence
of the heart rate in vivo and in vitro, as shown in Fig.·6B.
McMahon has emphasized the importance of not relying
solely on the heart rate as an indicator of cardiac performance
(McMahon, 1999; McMahon, 2001) and our observations
strongly support this view. Whereas the lobster heart rate
increases linearly as a function of temperature over most of the
range from 2 to 22°C two other parameters of cardiac
performance, the strength of the heartbeat and the stroke
volume, both decrease. Similar observations have been
reported for crab (Cancer magister) hearts (DeWachter and
McMahon, 1996; DeWachter and Wilkens, 1996). Moreover,
cardiac output in the lobster displays a pattern of temperature
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1032 M. K. Worden and others
dependence that probably results from the opposing
temperature-dependent effects on heart rate and stroke volume.
Cardiac output was maximal at 10°C, but became
compromised as the temperature warmed above 20°C, with a
significant decrease in cardiac output in hearts that continued
to beat and an increase in the number of hearts failing. It is
possible that cardiac performance in intact animals might be
protected somewhat from thermal stress by endogenous neural
and hormonal signals, since nearly all the hearts in intact
animals (20 out of 21) continued to beat at 22°C. Although we
did not make measurements at temperatures warmer than 22°C,
it is likely that cardiac performance will degrade as animals
approach their lethal temperatures of 25 to 30°C (McLeese,
1956).
It is important to note that the rate of temperature change
in our experiments (approximately 1°C·min–1) is considerably
faster than temperature changes occurring in the wild during
a tide or a season. (For example, the fastest rate of temperature
change during the course of tide is approximately 3°C·h–1 in
the record shown in Fig.·1.) In the ocean, it is possible that
the animals might be able to acclimate to warmer temperatures
if temperature changes sufficiently slowly, thereby improving
cardiac performance at warm temperatures. However, it is also
likely that animals moving through thermoclines on the ocean
floor will experience relatively rapid and extreme changes in
temperature. It is interesting to note that our measurements of
heart rate in lobsters exposed to acute changes in temperature
in the lab are in good agreement with those obtained from
ocean animals experiencing seasonal temperature changes
over the course of a year (see Fig.·6B). These results suggest
that our relatively fast thermal challenges in the laboratory
stimulate cardiac responses very similar to those in lobsters
experiencing seasonal thermal shifts in their natural habitat.
Future experiments will be directed toward examining
whether the temperature dependence of cardiac performance
differs for animals acclimated to different seasonal
temperatures or animals exposed to temperature change at
different rates.
Finally, the temperature dependence of cardiac performance
in the lobster differs in several respects from that reported for
crab C. magister (DeWachter and Wilkens, 1996), a Pacific
coast species that inhabits a similar thermal range. In isolated
crab hearts the temperature dependence of the heart rate is only
one third that measured in intact animals and cardiac output is
maximal at 2°C and decreases as a function of temperature
until heart failure occurs at 20°C. In contrast, isolated lobster
hearts appear to be more robust in the face of warming
temperature. Most are viable up to 22°C, show temperaturedependent increases in heart rate that are similar to those
observed in hearts in intact animals, and exhibit an increase in
cardiac output as temperature warms above 2°C that reaches a
maximum at 10°C.
Frequency and amplitude of the heartbeat
A previous study of isolated lobster hearts concluded that
the strength of the heartbeat and the frequency of the heartbeat
are coupled (Mahadevan et al., 2004), because these
parameters show a positive correlation in spontaneously
beating hearts and in hearts paced by external stimuli. This
interpretation seems reasonable, given that the same study
showed that the magnitude of synaptic depolarization at
neurocardiac synapses is linearly related to burst frequency.
However, the experiments were performed at constant
temperature (fixed between 10 and 14°C), thereby constraining
the range of possible physiological responses. Our results,
collected over a broad temperature range, show no evidence
for coupling between the strength and frequency of the
heartbeat. We favor the viewpoint that strength and frequency
of the heartbeat are independent parameters and independently
regulated. At constant temperatures heartbeat frequency will
be a critical determinant of contraction amplitude, but changes
in temperature will affect both parameters independently, and
alter any relationship between them. The ‘uncoupling’ of
heartbeat frequency and amplitude in the lobster has been
observed previously in response to hypoxia (McMahon, 1999)
and during adaptation to stimulation by nitrous oxide
(Mahadevan et al., 2004).
Although we have not investigated the physiological
mechanisms underlying temperature dependence in this
system, our results suggest several possible mechanisms. As
heart rate is determined by the rhythmic neural output of the
cardiac ganglion, it is possible that temperature regulates the
frequency and patterns of bursting of the Homarus cardiac
ganglion motoneurons and pacemaker cells. Temperaturedependent decreases in the strength of the heartbeat might arise
either from the properties of the Homarus cardiac muscle
fibers, or of the cardiac ganglion motoneuron synapses that
innervate them. For example, temperature may affect both the
size of the neurocardiac synaptic potentials and the extent to
which they facilitate, as shown in the myocardium of another
lobster species Panulirus japonicus (Kuramoto, 1994). In
addition, it is also probable that the calcium dynamics within
the cardiac muscle fibers might be temperature dependent,
thereby regulating the availability of calcium in the cytoplasm
and the rate at which it activates the contractile machinery.
Further experiments will be required to investigate these
possibilities.
Serotonergic modulation of heart rate and contraction
amplitude is temperature independent
In agreement with earlier reports, we find that serotonin
increases both the rate and the strength of the heartbeat. The
onset of serotonin effects on the heart at 2°C is comparatively
slow, reaching steady state in approximately 12·min, on
average. By comparison, other studies performed at 12–13°C
have demonstrated that potentiation of lobster heart rate by
serotonin reaches steady state within 5·min (Guirguis and
Wilkens, 1995; Kuramoto et al., 1995). In our experiments
modulation of the heart by serotonin was effective over the
whole temperature range tested. Although there was some
variability between preparations, we did not observe a
statistically significant temperature dependence of the
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Lobster heart and temperature 1033
modulatory effects overall. In contrast, serotonin elicits several
temperature-dependent effects on a lobster skeletal muscle
neuromuscular system. Serotonin increases muscle tonus most
strongly at the coldest temperatures (2°C), increases nerveevoked contractions most strongly at warm temperatures
(16–18°C) and enlarges the temperature range over which the
inhibitory motoneuron is effective in relaxing the muscle
(M.K.W., unpublished observations).
There are likely to be multiple sites of action for serotonin
in the lobster cardiac system. Serotonin increases the
heartbeat of intact animals even after the cardioregulatory
nerves are severed (Guirguis and Wilkens, 1995), suggesting
that serotonin acts directly on the cardiac ganglion. This was
subsequently verified by Berlind’s observation that serotonin
increases and prolongs the bursting output of the cardiac
ganglion in vitro (Berlind, 1998). Such effects would be
consistent with serotonin increasing heartbeat frequency and
contraction amplitude. In Homarus serotonin also decreases
output through the sternal arterial valve, thereby redirecting
arterial flow (Wilkens et al., 1996). However, it is also
probable that serotonin acts on the neurocardiac synapses as
well as the muscle fibers to increase contractility. Cooke
reported preliminary evidence that serotonin potentiates
synaptic transmission at synapses on the lobster heart
(Cooke, 1966). Serotonin potentiates both synaptic
transmission and muscle contractility in lobster dactyl opener
neuromuscular systems (Kravitz et al., 1980) as well as other
invertebrate neuromuscular systems (Worden, 1998).
Finally, it is important to note that the temperature can be a
confounding variable in physiology experiments, because
multiple physiological processes underlie cellular function and
each can depend differently on temperature. For example, a
recent study of excitation–contraction coupling in lobster cardiac
muscle at constant temperature concluded with the prediction
that an increase in heart rate will enhance calcium loading of the
sarcoplasmic reticulum, thereby accelerating relaxation of the
heartbeat, increasing diastolic filling and contraction amplitude
(Shinozaki et al., 2002). Further, these authors predicted that
heartbeat frequency-dependent increases in contraction
amplitude will cause stroke volume and cardiac output to
increase. However, using an experimental protocol that changes
temperature we observe different relationships between the
parameters underlying cardiac performance. As the lobster heart
rate increases both contraction amplitude and stroke volume
decrease, while cardiac output itself remains relatively constant
with a maximum at 10°C and a significant decrease at
temperatures warmer than 20°C. Changes in temperature can
therefore alter the relationships between parameters of cardiac
function to produce physiological effects that cannot be
predicted from observations at a constant fixed temperature.
The authors are grateful to Claire Edwards and Joseph
Camacho for help with data collection, Deforest Mellon and
Lynne Fieber for comments on the manuscript, and Brian
Duling for helpful discussions. This work was supported by
funds from the University of Virginia School of Medicine.
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