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First published online June 11, 2007
Journal of Experimental Biology 210, 2192-2198 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02784
Assessment of the pressure–volume relationship of the single ventricle of the grass shrimp, Palaemonetes pugio
1 College of Osteopathic Medicine, Touro University – Nevada,
Henderson, NV 89014, USA
2 Department of Biological Sciences, University of Nevada, Las Vegas, NV,
USA
3 Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC,
Pittsburgh, PA, USA
* Author for correspondence (e-mail: jguadagnoli{at}touro.edu)
Accepted 26 March 2007
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Summary |
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Key words: invertebrate, cardiac function, stroke work, Palaemonetes pugio
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). More
recently, pressure–area (PA) loops have been used to define cardiac
dynamics in mammalian (Senzaki et al.,
2001) and avian systems
(Tobita and Keller, 2000).
Both PV and PA loops have been used extensively to study the energetics of
cardiac contraction and dynamics of cardiac function in the ventricles of
mammalian and avian systems, yet this tool has found limited use in the study
of cardiac dynamics in the single ventricle of animals possessing an open
circulatory system (Wilkens,
1999). We believe that PV and PA loops provide a valuable tool in
the assessment of the cardiovascular dynamics of the single ventricle.
The cardiovascular system can be loosely defined as a three-part system
with (1) a pump for generating force to move (2) blood or hemolymph through a
(3) distribution pathway or arterial system. Alterations in any of the three
components can alter the amount of work required of the pump. In decapod
crustaceans the pump consists of a single ventricle suspended within a
pericardial sinus by a three-dimensional array of suspensory ligaments located
anteriorly, laterally and posteriorly on the dorsal and ventral surface of the
heart (Maynard, 1960;
Blatchford, 1971). All
ligaments are paired, with the exception of dorsal posterior suspensory
ligament (Blatchford, 1971).
Unlike the typical vertebrate four-chambered heart, with only one entrance and
one exit from the ventricle, crustaceans must coordinate the opening and
closing of multiple ostial and aortic valves. Pre-branchial hemolymph from
active tissues is collected in large, paired infrabranchial sinuses that guide
the hemolymph back to the gills to become reoxygenated. Post-branchial
hemolymph enters defined branchio-cardiac veins that deliver the oxygenated
hemolymph to the pericardial sinus surrounding the heart. This defined path
serves to minimize admixture of oxygenated and deoxygenated hemolymph
(McLaughlin, 1983). Hemolymph
from the pericardial sinus enters the heart passively through three pairs of
muscular ostia (Fig. 1B,C), and
leaves the heart via six aortic valves that lead to five arterial
systems (Fig. 1A). Contraction
of the ventricle then distributes the oxygenated hemolymph to the arterial
systems that branch repeatedly to terminate as open tubes through which
hemolymph flows to directly bathe the tissues
(McLaughlin, 1983).
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Physiologically, the initiation of cardiac contraction in the single
ventricle of a decapod is neurogenic, as compared with the myogenic properties
of vertebrate cardiac myocytes, and is driven by a burst of action potentials
from the cardiac ganglion located on the inner dorsal surface of the heart
(Florey, 1960;
Sullivan and Miller, 1984).
Overall cardiac function depends on ganglionic burst frequency and duration
(for a review, see Cooke,
2002), which is further altered by both cardio-excitatory and
cardio-inhibitory nerves originating from the central nervous system. Beyond
the difference in the initiation of contraction, the microscopic architecture
of cardiac myocytes of crustaceans is similar to that of typical mammalian
myocytes, with each sarcomere spanning the area between two Z-lines and
surrounded by the sarcoplasmic reticulum (SR) and T-tubule system
(Nylund et al., 1987). The SR
membrane system is involved in excitation–contraction (EC) coupling
(Yazawa et al., 1999;
Shinozaki et al., 2002) with
activation of voltage-dependent sarcolemmal Ca2+-release channels
that allow the entry of Ca2+ required to initiate contraction. As
in all muscle cells, relaxation occurs when released Ca2+ is
sequestered back into the SR or pumped out to the extracellular fluid, an
energetically demanding process. From the available literature, the
microscopic architecture of the contractile apparatus of crustacean myocardium
shares many similarities with that of mammalian myocardium
(Shinozaki et al., 2002;
Yazawa et al., 1999).
The PV loop (Fig. 2)
provides a tool for the estimation of myocardial O2 consumption.
There are four distinct phases that include (1) isovolumic contraction as
pressure is generated by the ventricle, (2) ventricular emptying as the
pressure in the ventricle overcomes peripheral pressure, (3) isovolumic
relaxation as the ventricle relaxes and (4) rapid ventricular filling at low
pressure (Berne and Levy,
1986). The x-axis provides an estimate of stroke volume
and the y-axis represents changes in pressure during the cardiac
cycle. Although ventricular area and pressure can be determined independently
for the determination of stroke volume, or total pressure change, the
integration of this data is the basis for determining myocardial O2
consumption. Once integrated, the area enclosed by a PV loop is an index of
kinetic energy or ventricular stroke work
(Sagawa et al., 1988).
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Materials and methods |
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Grass shrimp were attached to the flattened end of a wooden applicator
stick at the lateral cephalothorax with cyanoacrylate glue. The animal was
held in place and positioned within the experimental chamber with a
micromanipulator (World Precision Instruments, Sarasota, FL, USA). The video
camera was placed over the chamber so that video images of the heart could be
captured through the transparent exoskeleton [see methods from Harper and
Reiber (Harper and Reiber,
1999)]. The transparent exoskeleton allows for the measurements of
area and pressure in vivo.
Experimental design
Seawater (30±2 ppt) within a flow-through experimental chamber was
maintained at 20°C and the partial pressure of oxygen
(PO2) in the water was maintained at normoxic
levels by bubbling room air into the flow-through chamber. All animals were
placed in the experimental chamber in normoxic water
(PO2=20.5 kPa) and acclimated for 1 h.
Thereafter a minimum of three recordings of pressure and volume were made for
each animal.
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Video image processing
Video images were acquired in vivo through the transparent
exoskeleton at a rate of 60 Hz using a stereo-microscope (Leica MZ12.5; McBain
Instruments, Chatsworth, CA, USA) equipped with a video camera (World
Precision Instruments), frame-grabber board (LG-3; Scion, Frederick, MD, USA)
and programmed frame-grabbing software (Scion Image; Scion). Each video image
was analyzed using custom-programmed image analysis software (LabView;
National Instruments) commonly used in the study of chick embryos
(Tobita and Keller, 2000).
First, maximum and minimum ventricular borders were traced from recorded
sequences to determine ventricular cross-sectional area. The number of pixels
and individual pixel values in the area contained between the maximum and
minimum borders was stored in memory as a region of interest (ROI)
(Fig. 3). Assuming that
movement of the ventricular border would be associated with changes in the
pixel values within the image of the heart, changes in ventricular area from
the minimum area during the cardiac cycle were identified automatically by
detecting the pixels that changed value in the ROI for sequential video
fields. Total ventricular cross-sectional area in each video field was then
calculated as the sum of the changes in area within the ROI defined by the
maximum (Fig. 3B) and minimum
(Fig. 3A) ventricular areas.
The pressure signal (600 Hz) and video images (60 Hz) were acquired
simultaneously for 4 s by an output trigger to the AD board and the
frame-capturing board. Using a custom computer program (K. Tobita using
LabView; National Instruments) the pressure waveform was interpolated with the
image data to yield a series of x, y coordinates required for the PA
loop.
Area was converted to volume in a method used in previous studies
(Harper and Reiber, 1999;
Guadagnoli and Reiber, 2005).
The use of dimensional analysis, with the heart modeled as a trapezoid
{cardiac volume=w [0.5 h(b + a)], where
w is width, h is height, a is base length and b is
top length; the width (w) of the heart was determined to be 0.64 h
during systole and 0.67 h during diastole}, differed only 13% from dye
dilution techniques (Harper and Reiber,
1999). We therefore used the same model and by incorporating the
known changes in the depth of the heart from a lateral view, converted area to
volume (Harper and Reiber,
1999; Guadagnoli and Reiber,
2005). The volume data was then used to generate PV loops.
Heart rate (fH), maximum pressure
(Pmax), minimum pressure (Pmin),
change in pressure (
P), maximum area
(Amax), minimum area (Amin) and change
in area (A) were determined by independently analyzing the
pressure and video output from LabView (National Instruments) using a
customized computer program, MATLAB (The Mathworks, Inc., Natwick, MA, USA).
Area was converted to volume to obtain end-diastolic volume (EDV),
end-systolic volume (ESV) and stroke volume (Vs). After
interpolation of the PV data to generate multiple loops in LabView, the data
were analyzed using MATLAB to obtain a mean PV loop as well as the area
enclosed by the loop. The area of the PV loop is an estimate of stroke work
(SW). The PV loop does not account for heart rate; therefore, the product of
area and fH yields an estimate of minute cardiac work
(CW). However, either a PV or PA loop can be used to elucidate the phases of
the cardiac cycle and cardiac dynamics in general. All values are means
± s.e.m. (N=12).
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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P, an
increase in Vs and a decrease in PV area (SW) from 377.3
to 343.2 mmHg mm3 (Fig.
7).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Although all four phases are present in the PA loop, the timing of the
phases is considerably different than in the mammalian ventricle. In the
mammalian four-chambered heart, approximately 33% of the cardiac cycle
accounts for time in systole, with the remaining 67% in diastole. In this
study we find that a much greater portion of the cardiac cycle is spent in
systole (53%). In crayfish with heart rates in the 160–200 beats
min–1 range, systole accounts for more than 60% of the
cardiac cycle (Reiber, 1995).
The negative filling pressure or diastolic sucking observed in this study may
account for the reduction in diastolic filling time when compared with the
filling times observed in the mammalian ventricles.
In a closed vertebrate circulatory system EDV is affected by venous filling
pressure, distensability of the ventricular wall and time available for
filling (Sagawa et al., 1988).
When comparing ventricular EDV in an open circulatory system to that of a
ventricular EDV in a closed circulatory system the most obvious difference is
the effect of venous filling pressure. In the closed vertebrate system blood
flows into the ventricle via a discrete pathway supplied by the vena
cava, with the remainder of ventricular filling accomplished by contraction of
the atria. In the open circulatory system of crustaceans there is no direct
venous return path; nor as in multi-chambered hearts is there atrial
contraction to enhance filling of the ventricle. Instead, filling occurs as a
result of the pressure difference between the pericardial sinus and the
expanding ventricle through the open ostia. During the cardiac cycle the
ventral pericardial membrane is depressed during diastole and relaxes during
systole, enhancing hemolymph flow from the branchio-cardial veins into the
pericardial sinus (Belman,
1975; Reiber,
1994). The hemolymph in the pericardial sinus bathes the ventricle
and then passively enters the relaxed ventricle through the open ostial
valves.
At the onset of systole in the grass shrimp, the six ostial valves close,
as evidenced by the rapid rise in pressure, with no change in volume. The
ostial valves have an inward-pointing arrangement that prevents backflow
during systole (Yazawa et al.,
1999). In decapod crustaceans, there are seven arteries leaving
the ventricle with outlets that are regulated by muscular bicuspid valves. The
valves prevent passive reflux of hemolymph during diastole, but actively
control outflow during systole via neural innervation
(Alexandrowicz, 1932). Both
excitatory and inhibitory neurons are present in the valves
(Kuromoto et al., 1992), with
excitation causing valve muscle contraction that impedes flow and inhibition,
causing relaxation that facilitates flow
(Wilkens, 1997). Given that
each of the valves is innervated, the ventricle must not only generate
sufficient pressure to overcome resistance in the vasculature to open the
valves (afterload), but the amount of resistance is also altered depending on
the contractile state of the valves. The isovolumic contraction phase
therefore requires the overcoming of peripheral resistance along with the
nervous coordination of the timing and tension in the individual valves.
The emptying phase of the cycle is characterized by an initial drop in
pressure, followed by a more stable pressure during the remainder of emptying
in all PV loops that were analyzed. In macruran decapod crustaceans, between
50–60% of cardiac output is delivered to the large sternal artery, which
travels ventrally and then branches in the anterior and posterior direction to
supply the ventral nerve cord as well as other tissues
(Fig. 1)
(Reiber, 1994;
Guadagnoli and Reiber, 2005).
The sternal artery is the primary vessel responsible for the delivery of
hemolymph to nervous tissue. Although we do not have specific data on the
sequential opening of the arterial valves, the nature of the pressure tracings
may be because of the independent neural innervation and timing of the valves.
If the sternal artery were to open first, this could account for the drop in
pressure associated with the first portion of the emptying phase. Thereafter,
emptying of the ventricle occurs at a steady rate until the closing of the
valves at the end of systole.
In a closed system, ventricular relaxation begins with an isovolumic phase
with all valves closed and a rapid drop in pressure toward zero. This
isovolumic relaxation phase in the ventricle of the grass shrimp continues
until pressure falls below zero. As pressures drop, there is clear evidence of
a `diastolic sucking' phase as the ventricle begins to fill during negative
pressure (Kraner, 1959) and
completes its filling at low, but positive pressures. Negative pressures are
not usually observed in crustaceans, with passive ventricular filling
resulting from the pressure difference between the pericardial sinus and the
ventricle (Belman, 1975;
Reiber, 1994). Active
diastolic sucking has been documented in mammalian ventricles and in chick
hearts during development (Keller et al.,
1990; Keller,
1994). In mature hearts negative pressures during ventricular
filling are thought to result from restoring forces generated from the recoil
of titin molecules within myocytes. A restoring force stores potential energy
that can be converted to suction during the succeeding systole. In rat
myocytes titin is responsible for 
90% of passive force during stretch and
60% of the restoring force (Helmes et al.,
2003). Titin has been described in striated muscles of
invertebrate species including crayfish
(Fukuzawa et al., 2002), but
its functional significance remains unclear.
The heart of decapod crustaceans also has an external mechanism for
generating restoring forces. The decapod crustacean heart is held within the
pericardial sinus via suspensory ligaments that stretch during
systole and recoil during diastole. As suspensory ligament tension is
increased, diastolic expansion enlarges because of greater elastic recoil
(Volk, 1988). Although the
ventricle begins to fill under negative pressure, the remainder of filling is
accomplished via the pressure difference between the ventricle and
the pericardial sinus. The ventricle of the grass shrimp may have both active
(recoil) and passive (P) properties available during the
filling phase.
The area enclosed by the PV loop is an indicator of SW. Analysis of SW is useful in determining the efficiency of cardiac contraction and how this may change under various conditions. Based on the hypoxic PV loop, the pressure difference is decreased, stroke volume is increased and, overall, total SW is reduced (Fig. 7). During hypoxia, heart rate decreases, contributing to a decline in total CW. The fall in pressure may be the result of a decreased resistance in the branchial vasculature and a reduction in valve tension by the nerves regulating the arterial valves. Stroke volume may be increased simply because of the increased amount of time available for filling or enhanced tension across the suspensory ligaments via the muscles attached to the epimeral wall. The future use of the PV loops in evaluating the cardiac response to stress, neurohormones and toxins will allow for a more detailed understanding of cardiac function than can be provided by independent measures of volume or pressure.
In multi-chambered hearts of closed systems, pressure and volume data have been used extensively to understand the mechanics and energetics of ventricular functions. Investigation of the decapod crustacean heart continues in an effort to obtain a clearer understanding of its filling and contractile properties. These investigations may be further enhanced using PV loops. Given the ongoing study of physiological stressors and interactions in this model, the use of PV loops provides a new tool for researchers to evaluate multiple levels of ventricular function in the open-circulatory system of decapod crustaceans.
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Acknowledgments |
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|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alexandrowicz, J. S. (1932). The innervation of the heart of the Crustacea. I. Decapoda. Q. J. Microsc. Sci. 75,181 -249.
Belman, B. W. (1975). Some aspects of the circulatory physiology of the spiny lobster Panulirus interruptus.Mar. Biol. 29,295 -305.[CrossRef]
Berne, R. M. and Levy, M. N. (1986). Cardiovascular Physiology (5th edn). St Louis: Mosby.
Blatchford, J. G. (1971). Haemodynamics of Carcinus maenas (L.). Comp. Biochem. Physiol. 39A,193 -202.
Cooke, I. M. (2002). Reliable, responsive
pacemaking and pattern generation with minimal cell numbers: the crustacean
cardiac ganglion. Biol. Bull.
202,108
-136.
Florey, E. (1960). Studies on the nervous
regulation of the heart beat in decapod Crustacea. J. Gen.
Physiol. 43,1061
-1081.
Fukuzawa, A., Hiroshima, M., Maruyama, K., Yonezawa, N., Tokunaga, M. and Kimura, S. (2002). Single-molecule measurement of elasticity of serine-, glutamate-, lysine-rich repeats of invertebrate connectin reveals that its elasticity is caused entropically by random coil structure. J. Muscle Res. Cell Motil. 23,449 -453.[CrossRef][Medline]
Guadagnoli, J. A. and Reiber, C. L. (2005). Changes in cardiac output and hemolymph flow during hypoxic exposure in the gravid grass shrimp, Palaemonetes pugio. J. Comp. Physiol. B 175,313 -322.[CrossRef][Medline]
Harper, S. L. and Reiber, C. L. (1999). Influence of hypoxia on cardiac functions in the grass shrimp (Palaemonetes pugio Holthuis). Comp. Biochem. Physiol. 124A,569 -573.
Helmes, M., Lim, C. C., Liao, R. L., Bharti, A., Cui, L. and
Sawyer, D. B. (2003). Titin determines the Frank-Starling
relation in early diastole. J. Gen. Physiol.
121,97
-110.
Keller, B. B. (1994). Embryonic ventricular diastolic and systolic pressure-volume relations. Cardiol. Young 4,19 -27.
Keller, B. B., Hu, N., Serrino, P. J. and Clark, E. B. (1990). Ventricular pressure-area loop characteristics in the stage 16-24 chick embryo. Circ. Res. 68,226 -231.
Kraner, J. C. (1959). Effects of increased
residual volume, increased cardiac output resistance and autonomic drugs on
ventricular suction in the turtle. Circ. Res.
7, 101-106.
Kuromoto, T., Hirose, E. and Tani, M. (1992). Neuromuscular transmission and hormonal modulation in the cardioarterial valve of the lobster, Homarus americanus. Comp. Physiol. 11, 62-69.
Maynard, D. M. (1960). Circulation and heart function. In Metabolism and Growth: The Physiology of Crustacea. Vol. 1 (ed. T. H. Waterman), pp. 161-226. New York: Academic Press.
McLaughlin, P. A. (1983). Internal anatomy. In The Biology of Crustacea. Vol.5 (ed. L. Mantel), pp. 1-53. New York: Academic Press.
Nylund, A., Okland, S. and Tjonneland, A. (1987). The crustacean heart ultrastructure and its bearing upon the position of the isopods in eumalacostracan phylogeny. Zool. Scr. 16,235 -241.[CrossRef]
Reiber, C. L. (1994). Hemodynamics of the crayfish Procambarus clarkii. Physiol. Zool. 67,449 -467.
Reiber, C. L. (1995). Physiological adaptations of crayfish to the hypoxic environment. Am. Zool. 35, 1-11.
Sagawa, K., Maughan, L., Suga, H. and Sunagawa, K. (1988). Cardiac Contraction and the Pressure-volume Relationship. New York: Oxford University Press.
Senzaki, H., Chen, C., Masutani, S., Taketazu, M., Kobayashi,
J., Kobyashi, T., Sasaki, N., Asano, H., Kyo, S. and Yokote, Y.
(2001). Assessment of cardiovascular dynamics by pressure-area
relations in pediatric patients with congenital heart disease. J.
Thorac. Cardiovasc. Surg. 122,535
-547.
Shinozaki, T., Wilkens, J. L., Yazawa, T., Miura, M. and ter Keurs, H. E. D. J. (2002). Excitation-contraction coupling in cardiac muscle of lobster (Homarus americanus): the role of the sarcolemma and sarcoplasmic reticulum. J. Comp. Physiol. B 172,125 -136.[CrossRef][Medline]
Sullivan, R. E. and Miller, M. W. (1984). Cholinergic activation of the lobster cardiac ganglion. J. Neurobiol. 21,639 -650.[CrossRef]
Tobita, K. and Keller, B. (2000). Maturation of end-systolic stress-strain relations in chick embryonic myocardium. Am. J. Physiol. 279,H216 -H224.
Volk, E. L. (1988). The role of suspensory ligaments in modifying cardiac output in crustaceans. MSc Thesis, University of Calgary, Alberta, Canada.
Wilkens, J. L. (1997). Possible mechanisms of control of vascular resistance in the lobster Homarus americanus.J. Exp. Biol. 200,487 -493.[Abstract]
Wilkens, J. L. (1999). The control of cardiac rhythmicity and of blood distribution in crustaceans. Comp. Biochem. Physiol. 124A,531 -538.
Yazawa, T., Wilkens, J. L., ter Keurs, H. E. D. J. and Cavey, M. J. (1999). Structure and contractile properties of the ostial muscle (musculus orbicularis ostii) in the heart of the American lobster. J. Comp. Physiol. B 169,529 -537.[CrossRef]
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