Vol. 284, Issue 5, H1872-H1878, May 2003
SPECIAL COMMUNICATIONS
Multitrack system for superfusing isolated cardiac
myocytes
Lois Jane
Heller1,
David
E.
Mohrman1,
Juline A.
Smith1, and
Kendall B.
Wallace2
1 Department of Physiology and
2 Department of Biochemistry and Molecular
Biology, School of Medicine, University of Minnesota, Duluth,
Minnesota 55812
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ABSTRACT |
A new
system for studying mechanical activity of freshly isolated cardiac
myocytes from up to four experimental groups simultaneously is
described. Suspensions of cardiac myocytes isolated from adult rat
hearts were drawn into microhematocrit capillary tubes, which were then
mounted in parallel fashion between two four-channel tubing manifolds
placed on the movable stage of an inverted microscope. Within a few
minutes, cells settled and attached to the bottom of the tubes and then
could be superfused with various test solutions. The system allowed for
electrical field stimulation, rapid changes in bathing solutions,
control of temperature, and simulation of ischemia and
reperfusion with measurements of the effects of such interventions on
both populations of cells (low power survey) and individual myocytes
(high power). Myocyte responses to these various interventions are
described. The primary advantage of this system is the ability to
conduct experiments on cardiac myocytes isolated concurrently from
multiple experimental groups at the same time and under identical conditions.
simulated ischemia and reperfusion; caffeine contracture; temperature effects; electrical field stimulation; myocyte shortening; test chambers
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INTRODUCTION |
THE ABILITY to study
the contractile activity of isolated cardiac myocytes under a variety
of experimental conditions has led to significant understanding of
myocyte function. However, with the use of the standard approaches, it
has not been possible to conduct simultaneous experiments on more than
one preparation of isolated cardiac myocytes at a time. In addition, it
has been necessary to use separate specialized apparati for studying
different aspects of myocyte behavior such as responses to electrical
field stimulation, rapid changes in bathing solution composition,
simulated ischemia, and reperfusion.
We have developed a simple experimental system for subjecting isolated
cardiac myocytes from up to four experimental groups simultaneously to
identical treatments. This method allows for electrical field
stimulation, rapid changes in bathing solutions, control of
temperature, and simulation of ischemia and reperfusion with
measurements of the effects of such interventions on both populations
of cells and individual myocytes.
This report describes the experimental system and characterizes
contractile responses of cardiac myocytes isolated from adult rat
hearts to differences in temperature, changes in stimulus parameters,
rapid exposure to caffeine, and simulated ischemia and reperfusion.
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METHODS |
Cardiac myocyte preparations.
Adult male Harlan Sprague-Dawley rats (body weights = 373 ± 5 g) were housed in the University's climate-controlled American Association for Accreditation of Laboratory Animal Care-accredited animal care facility on a 12-h:12-h light-dark cycle with free access
to food and water. On the day of the experiment, rats were injected
with heparin (1.0 IU/g body wt ip) 30 min before being euthanized with
CO2. Hearts were rapidly removed from the thorax, placed in
a preweighed beaker containing iced perfusate (Joklik's calcium-free
modified minimal essential media containing 0.1% BSA), and then
weighed with aortic stump attached (weights = 1.55 ± 0.03 g). Cardiac myocytes were isolated from the rat hearts using
standard enzymatic dispersal techniques (3-5, 17).
Briefly, hearts were cannulated via the aorta for Langendorff-type
constant pressure (40 mmHg) perfusion at 37°C with the perfusate
passing through a gas-exchange column to equilibrate with 100%
O2. After 5-10 min of nonrecirculating perfusion to
wash blood from the coronary vasculature, the perfusate was switched to
one containing 0.7% (200 U/ml) collagenase (Worthington), and
perfusion was continued in a recirculating mode (with ~60 ml of
solution) until the vascular bed deteriorated as indicated by a
doubling of coronary flow (usually achieved after 20-30 min).
Hearts were then removed from the perfusion apparatus, and the tissue
was minced and suspended in a beaker containing ~20 ml of fresh
collagenase-containing solution. Digestion was continued in the beaker
placed in a gyrating water bath at 37°C for an additional ~20 min
with CaCl2 added after 10 min to a concentration of 50 µM. The tissue digestate was then slowly triturated through a wide-tipped pipette several times to help separate the cells and filtered through cheese cloth to remove nondigested material, and the
cells were allowed to settle out of the filtrate. After removal of the
supernatant, the cell preparation was resuspended at room temperature
and resettled in collagenase-free, calcium-free Joklik's solution
containing 1.5% BSA. This step was repeated twice. Initial yield of
myocytes was assessed at this point by determining the number of
rod-shaped cells obtained per heart (average = 5.5 × 106). Initial viability of myocytes was determined as the
percentage of rod-shaped cells in the total sample of cells that
retained the ability to exclude the vital dye trypan blue (average = 82 ± 1%). The cell suspension was then returned to the 37°C
water bath for calcium addition. This was achieved in five successive steps over a 30-min period to a final concentration of 1.0 mM CaCl2. Viability was reassessed at the end of this
procedure (average = 61 ± 1%).
After isolation, cardiac myocytes were settled and resuspended at room
temperature two times in a normal Tyrode solution containing (in mM)
140 NaCl, 5.0 KCl, 1.0 MgCl2, 5.0 HEPES, 2.0 CaCl2, and 10.0 glucose, and pH was adjusted to 7.4. The
solution was pregassed with 100% oxygen. All preparations were stored
at room temperature for at least 30 min before being loaded into the
perfusion chambers. All measurements were taken within 6 h of
myocyte isolation.
Multitrack suffusion system for studies of myocyte function.
The new system for studying mechanical properties of isolated cardiac
myocytes used in this study is shown in Fig.
1. Myocytes suspended in normal Tyrode
solution were drawn into microhematocrit capillary tubes, which served
as "test chambers" for evaluating contractile behavior. The ends of
each capillary tube were attached via Silastic tubing between two
four-channel tubing manifolds. Specific bathing solutions were gassed
in elevated reservoirs and could be selected for perfusion through the
four parallel test chambers either by manually adjusting stopcocks or
electronically by activating solenoid switches in the inflow tubing
upstream of the test chambers. The outflow of the downstream tubing
manifold passed through a 22-gauge needle that provided a resistance
sufficient to maintain overall flow rate at ~6 ml/min. The capillary
tubes were viewed on the movable stage of an inverted video microscope on either low (×10) or high power (×40) to visualize either
populations of cells or single cells adhered to the bottom of the
tubes.
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Fig. 1.
Apparatus for monitoring contractile characteristics of
isolated cardiac myocytes. Myocytes are loaded into the capillary tube
test chambers, perfused with selected test solutions, field stimulated,
and observed with an inverted microscope.
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When the temperature was to be regulated above room temperature (37°C
or 27°C), the system was enclosed in a heatable, thermostated Plexiglas and styrofoam case, which fit over the reservoirs, the connecting tubing, and the myocyte-containing capillary tubes above the
microscope stage. The temperature was then adjusted to the chosen level.
The heated enclosure was actually a circular ductwork system that was
mounted on the microscope stage and moved with it. Air flowed in a
closed circuit formed by 1) a horizontal duct across the top
of the microscope stage, 2) a vertical duct leading upward past one side of the microscope lamp, 3) a horizontal duct
passing above the lamp, and 4) a vertical duct leading
downward on the other side of the lamp. A fan (Comair-Rotron standard
electronic chassis model FN12B3) located in the upward vertical duct
produced continuous and rapid air circulation around the circuit. Heat was supplied electrically by a 3" × 5" self-adhesive electrical heating pad (model HR5490R57.9L12B, Minco Products) attached to a
radiator (Wakefield model 394-2AB standard finned aluminum
semiconductor heat sink) located within the upper horizontal duct. The
downward vertical duct housed the perfusion reservoirs, associated
tubing, and solenoid-operated perfusion control valves. The horizontal duct across the stage contained the microcapillary tube test chamber apparatus and had a clear Plexiglas cover (to allow illumination from
the lamp above) and a plastic film window immediately beneath the test
chambers (to allow viewing from below with the microscope objectives).
The temperature of the heating radiator was sensed by a self-adhesive
thermocouple sensor (Minco Products no. 5665) and actively controlled
by a temperature controller (Minco Products CT 15). The temperature in
the outflow manifold of the test chamber apparatus was monitored with a
small thermocouple (BAT-12, Bailey Instruments) and recorded as the
experimental temperature in the test chambers.
The arrangement allowed free external access to the microscope and
stage controls. Moreover, the rapid internal air circulation scheme
greatly assisted in solving the difficult problem of maintaining a
single constant temperature throughout any heated enclosure. Myocytes
were allowed to settle in the capillary tubes for about 5 min before
perfusion of the tubes was begun. During this time some of the myocytes
attached quite firmly to the glass and remained attached when perfusion
with oxygenated normal Tyrode solution was initiated. After 30 min of
perfusion, each capillary tube contained between 50 and 150 viable
contracting, rod-shaped myocytes. Care was taken throughout all steps
in each protocol to prevent gas bubbles from passing through the tubes
because these were immediately toxic to the cells (as made evident by
rapid implosion, rounding up and sometimes detachment from the glass).
Bubbles formed in the inflow tubing were trapped and intermittently
"bled out" through a bubble-trap sidearm of the fluid line upstream to the test chambers.
The effects of several experimental interventions are included in this
report to characterize the behavior of normal rat isolated cardiac
myocytes in this system.
Population responses to electrical field stimulation.
Electrical field stimulation of the cells was achieved by applying
voltage from a Grass S44 stimulator to stimulating electrodes attached
to stainless steel connectors in the inflow and outflow lines at each
end of the capillary tubes. Stimulus parameters in most of these
experiments were set at 70 V and 7 ms. Measurement of the population
response to a given set of stimulus parameters was achieved by
monitoring the percentage of rod-shaped myocytes that contracted in
response to the stimulus. The effect of changes in voltage intensity on
the percentage of cells responding to the stimulus is reported in this study.
Mechanical responses of single myocytes to field stimulation.
Vigorously contracting, rod-shaped myocytes (arbitrarily limited to
those with lengths greater than 3 times their diameters) were chosen
for studies of single-cell mechanical shortening capabilities. Myocyte
lengths were determined by standard video edge-detection methods
(Crescent Electronics) and captured on-line using PowerLab/410 (AD
Instruments). Although only one cell could be monitored at a time, it
took only a few seconds to switch the view between cells in a given
chamber and only 1-2 min to switch the view between chambers. Thus
it was possible to measure mechanical activity of separate experimental
groups of cells within a very short time period. In the initial
experiments reported here, myocytes were stimulated at 1.0 Hz, and
steady-state twitch characteristics were determined after 30-min
equilibration at 37°C, 27°C, or 22°C.
Caffeine contractures.
Caffeine contractures have been used to estimate characteristics of
some calcium-dependent processes involved in cardiac muscle contraction
and relaxation (1, 2, 9, 15, 16, 21, 22). In the
experiments reported here, such contractures were induced by adapting
the method of McCall et al. (13) to our perfusion system.
(In these studies, cells were loaded into only one of the test
chambers, and the other 3 were plugged.) Once a cell was selected for
study, responses to 1.0-Hz field stimuli were recorded, and then the
stimulus was abruptly stopped. Within 2-5 s, the perfusate
solution was abruptly switched to one containing 10 mM caffeine. (Given
that the length and diameter of a capillary tube is 75 mm and 1.2 mm
respectively, a flow rate of 6 ml/min is estimated to change the entire
chamber volume within 850 ms and that the passage of the wavefront of
caffeine-containing solution over a given myocyte occurs within ~1.6
ms). Application of caffeine in this fashion evokes an immediate
release of calcium from sarcoplasmic reticular stores and prevents its
reuptake (2, 13, 22). Thus the magnitude of the
contracture has been taken to reflect the amount of calcium stored in
the sarcoplasmic reticulum, and the time course of subsequent
relaxation has been taken to reflect the combined effect of all
nonsarcoplasmic reticulum calcium removal processes in the cell (i.e.,
the Na+/Ca2+ exchanger, the mitochondrial
uniporter, and the plasma membrane Ca-ATPase).
In some of the experiments after the first caffeine contracture of a
given cell is measured, the chamber was perfused again with normal
Tyrode solution and the cell stimulated as before for 2 min (during
which time the contractile response returned to its previous
steady-state characteristics). The bathing solution was then switched
to one containing no sodium and no calcium, and the field stimulation
was stopped for 2 min. This "modified" solution contained (in mM)
140 LiCl, 5.0 KCl, 1.0 MgCl2, 5.0 HEPES, 1.0 EGTA, and 10.0 glucose, and pH was adjusted to 7.4. At the end of this period, a
second caffeine contracture was evoked by abruptly switching to
caffeine-containing (10 mM) "modified" solution. The absence of
sodium prevents normal operation of the
Na+/Ca2+ exchanger and eliminates that route of
calcium removal from the cell following caffeine-induced contracture
(2, 13). Thus the magnitude of the second caffeine
contracture has been taken to reflect the amount of calcium stored in
the sarcoplasmic reticulum, and the time course of the subsequent
relaxation has been taken to reflect the effects of the remaining
calcium removal processes in the cell (i.e., the mitochondrial
uniporter and the plasma membrane Ca- ATPase).
After these caffeine contractures of a given cell were recorded, the
solution was switched back to normal Tyrode, and after a brief
reequilibration period (2-5 min), the procedure was repeated on
other cells. Experiments reported here were conducted at 22°C.
Myocyte responses under conditions simulating ischemia
and reperfusion.
This protocol was adapted from one described by Maddiford et al.
(12) in which isolated cardiac myocytes were exposed to low-flow, ischemia-mimetic conditions for various intervals
with subsequent reperfusion. Our closed-system capillary tube
arrangement allowed us to impose sustained periods of hypoxia and mimic
true ischemia by totally stopping the flow while we continued
electrical field stimulation of the cells. For these studies, capillary
tubes containing isolated cardiac myocytes were initially perfused with well-oxygenated normal Tyrode solution, and myocytes were continually stimulated at 1.0 Hz at either 22°C, 27°C, or 37°C. At the end of
a 15-min equilibration period, the capillary tubes were observed at low
power (×10) for determination of the percentage of rod-shaped cells
that responded to the stimulus. Specific cells in each capillary tube
were identified and visualized at high power (×40) for analysis of
contractile activity using video edge-detection methods.
After the steady-state responses of the myocytes under these control
conditions were recorded, the solution perfusing the capillary tubes
was switched to one that simulated ischemic conditions. This
solution contained all the components of the normal Tyrode solution
except it had no glucose and no insulin, pH was 7.0, and argon was
bubbled through the solution (for 45 min) to reduce the oxygen content
to negligible levels. Tubes were perfused with this ischemo-mimetic
solution for 2 min, and then the flow through the tubes was stopped for
a prolonged period of time (90 min at 22°C and 27°C and 45 min at
37°C). Stimulation of myocytes at 1.0 Hz continued throughout the
solution changes and the entire ischemic period. This protocol
mimics an in vivo ischemic situation in that with continual
stimulation and cessation of flow, the microenvironment surrounding
individual cells will progressively deteriorate. Myocytes were
periodically monitored during the ischemic period for overall
viability, responsiveness to the stimulus, changes in rest lengths, and
twitch characteristics.
At the end of the ischemic period, flow of normal,
well-oxygenated solution was reinstituted, and myocytes continued to be stimulated for another 30 min. All variables were measured again at the
end of this reperfusion period.
Data analysis.
Data are reported throughout as means ± SE. Statistical
comparisons of data obtained under different testing conditions was achieved by applying two-tailed Student's unpaired or paired
t-tests. Significant differences were declared at
P < 0.05.
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RESULTS |
Steady-state twitch characteristics of individual myocytes at
37°C, 27°C, and 22°C.
Characteristics of the steady-state twitch response to 1.0-Hz
electrical field stimulation at the end of an initial 30-min equilibration period at three different temperatures are indicated in
Table 1. The average of responses from
six to nine myocytes was determined for each separate preparation of
myocytes. The average initial resting length of myocytes studied was
126 ± 2 µm with no significant differences between groups. Not
unexpectedly, as the temperature decreased, the extent of myocyte
shortening in response to the field stimuli increased, the time course
of shortening was prolonged, and the maximum rates of shortening and
relaxation slowed.
Population responses to field stimulation.
The intensity of the field stimulation directly influenced the
percentage of rod-shaped myocytes that responded. These studies were
conducted at 22°C with stimulus duration at 7 ms and included seven
experiments with 137 ± 22 total myocytes monitored per
experiment. Not surprisingly, decreasing the stimulus voltage from 70 to 60, 50, and 40 V resulted in a decrease in the percentage of rods responding from 72 ± 3% to 60 ± 4%, 50 ± 4%, and
28 ± 5%, respectively. Increases in voltage above 70 V had
little effect on the percentage of cells responding but tended to
promote electrolysis at the points of electrode attachment and bubble
formation. Large bubbles could either occlude the fluid pathway between
electrodes and block transmission of the field stimulus or could break
loose and pass through the tube, causing myocyte death.
In experiments done at 37°C and 27°C, the percentage of myocytes
responding to stimulus parameters of 70 V and 7 ms was 77 ± 3%
and 75 ± 2%, respectively. In another set of experiments assessing the long-term stability of the preparations under
steady-state conditions, the percentage response to constant stimulus
of 70 V and 7 ms at 1.0 Hz did not change significantly over 2 h
(from 72 ± 8% to 70 ± 3%). Thus the percentage of
myocytes responding to a given field stimulation was not significantly
influenced by either temperature or perfusion time.
Caffeine contractures of individual myocytes.
Records of caffeine contractures obtained from a single myocyte in
the presence and absence of sodium in the bathing media are shown in
Fig. 2 in comparison with the normal
twitch response of that myocyte. Characteristics of these contractions
are summarized in Table 2. Note that the
caffeine contractures are larger and longer than the twitch responses
and that the absence of sodium and calcium in the bathing solution
significantly increases the contractile amplitude and prolongs the
contraction and relaxation times.
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Fig. 2.
Records of shortening of a single cardiac myocyte with
resting length of 110 µm. A: steady-state twitch responses
in normal Tyrode solution (22°C) to 1.0-Hz field stimulation.
B: response to rapid superfusion with normal Tyrode solution
containing 10 mM caffeine. C: response to rapid superfusion
with modified Tyrode solution (0 Na+ and 0 Ca2+) containing 10 mM caffeine.
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Ischemia-reperfusion simulations.
Responses of isolated cardiac myocytes to simulated ischemia
and reperfusion were characterized at 37°C, 27°C, and 22°C. In preliminary experiments conducted at 37°C, it was apparent that the
myocytes rapidly deteriorated when exposed to more than 45 min of
simulated ischemia with the number of rod-shaped cells decreasing to very low numbers during ischemia and the
remainder of the myocytes washing away upon reperfusion. Thus the
duration of simulated ischemia was limited to 45 min in
experiments conducted at 37°C. When experiments were conducted at
27°C and 22°C, the myocytes could easily withstand 90 min of
simulated ischemia. Data in Table
3 show that, at all three temperatures,
the total number of rod-shaped cells decreased during ischemia
(because of cell death) and during reperfusion (because of washout).
The percentage of rod-shaped cells continually responding to the
stimulus (70 V, 7 ms, and 1.0 Hz) decreased during ischemia and
recovered during reperfusion at 27°C and 22°C. This recovery was
not complete, however, when experiments were conducted at 37°C.
Characteristics of the contractile responses of individual isolated
cardiac myocytes during simulated ischemia and reperfusion are
shown in Table 4. As can be seen by
comparing the responses recorded before, during, and after the
simulated ischemia at any one of the three temperatures, the
amplitude of myocyte shortening decreased during ischemia as
did the rates of contraction and relaxation. Furthermore, on
reperfusion, the rates of contraction and relaxation decreased even
further. Some recovery of contractile amplitude was apparent in
experiments conducted at 22°C, but none was apparent at 27°C, and
there was actually a further decline in contractile amplitude in
experiments conducted at 37°C. Under the conditions used, there were
no observed changes in myocyte resting length during ischemia
or reperfusion.
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Table 4.
Effect of simulated ischemia and reperfusion on contractile
characteristics of isolated cardiac myocytes
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To determine whether the decrease in percentage of myocytes responding
during ischemia was a result of altered sensitivity to the
stimulus, experiments were conducted in which responses to altered
stimulus intensity were investigated at specific time points during
ischemia and reperfusion at 22°C. In these experiments, the
steady-state stimulus intensity was set at 50 V and, at the intervals
shown in Fig. 3, was briefly altered to
40, 60, and 70 V to assess the percentage of myocyte response at each
intensity. The results shown in Fig. 3 indicate that at each stimulus
intensity, simulated ischemia resulted in a late suppression in
the percentage of cells responding to a given stimulus intensity and
then a restoration of percentage of cells responding on reperfusion.
There was no stimulus-dependent change in the pattern of myocyte
response to ischemia or reperfusion. These results suggest that
the ischemia-induced decrease in the percentage of myocytes
responding might be due to an ischemia-induced increase in
myocyte electrical threshold. For example, the suppression in myocyte
response to 50-V stimulus observed at 90 min of ischemia could
be completely negated by increasing the voltage intensity to 70 V.
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Fig. 3.
Effect of changes in stimulus intensity on percentage of
rod-shaped myocytes responding before, during, and after a 90-min
period of simulated ischemia. Experiment number, 7;
temperature, 22°C; stimulus duration, 7 ms. *P < 0.05 compared with preceding value (paired t-test).
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DISCUSSION |
This method of suffusing isolated cardiac myocytes offers several
advantages over other techniques used to evaluate cardiac myocyte
function. First, the parallel arrangement of four separate test
chambers allows for assessment of function of cardiac myocytes isolated
from hearts of up to four separate experimental groups during a single
experimental protocol and permits immediate comparisons of myocyte
responses under identical testing conditions. Second, it is possible to
tightly control the extracellular environment of the myocytes by
selecting the suffusion solution, its flow rate, the experimental
temperature, and the external stimulus conditions. Third, the suffusion
solution can be rapidly changed so that all cells in the test chambers
are exposed to altered conditions within 1 s. The transit time of
the wavefront generated by a solution change over a given cell is
estimated to be <2 ms. Fourth, responses of entire populations of
cells as well as of individual cells can be monitored and compared by
switching from low- to high-power microscope objectives. Fifth, with
the exception of the inverted microscope, a stimulator, and the video
edge-detection system, the rest of the apparatus is easily and
inexpensively built with readily available laboratory supplies.
The data obtained using this system are similar to those reported by
others. The amplitude and rates of myocyte shortening and relaxation
obtained in this study under control conditions (Table 1) are within
the same range as those obtained by others using open chambers with
myocytes attached to the bottom or to coverslips placed in the chambers
(1, 7, 8, 11, 20, 23). Rapid application of caffeine in
this study evoked alterations in magnitude and rates of
contraction/relaxation of myocytes (Table 2) that were similar to those
obtained by others using a micropipette placed near the myocyte being
evaluated (1, 13, 14, 20). Simulation of ischemia
in this study suppressed myocyte activity, and reperfusion resulted in
incomplete recovery of responsiveness and contractile activity (Tables
3 and 4 and Fig. 3). Although the magnitude of the responses to
simulated ischemia and reperfusion depend greatly on the
species, the duration of the ischemia, and the specific testing
conditions used, the results of the present studies show similar
patterns to those reported by others who used low-flow hypoxia (with
solutions that mimicked ischemia) followed by reperfusion
with well-oxygenated physiological salt solutions (6, 7, 11,
12).
These studies provide baseline information about the effect of changes
in temperature on basic contractile properties of isolated rat cardiac
myocytes (Table 1). These data are similar to those reported for rabbit
and ferret cardiac myocytes (14), in which under control
conditions, the amplitude of shortening increased and the rates
decreased as temperature decreased. In addition, the present study
indicated that changes in temperature did not influence the
ischemia-induced decrease in the percentage of myocytes responding to field stimulation (Table 3) or the proportional alterations in the ischemia-induced decreases in contractile
amplitude and rates of shortening and relaxation (Table 4). Recovery of myocyte responsiveness to stimulation and amplitude of myocyte shortening during reperfusion were better at 22°C and 27°C than at
37°C (Tables 3 and 4).
A number of techniques have been devised for rapidly changing the
bathing solution of isolated cardiac myocytes (1, 10, 18,
19). These techniques have all involved placing a single- or
double-barreled micropipette within a few micrometers from a single
myocyte and observing various physiological responses (e.g., myocyte
shortening, intracellular ion transients, and membrane potential
alterations) to either squirting substances directly onto the cell or
to changes between two solutions flowing over the cell. The technique
described here is not useful for studies of membrane potential with
micropipettes because the perfusion chamber is glass enclosed. However,
it is useful for applying rapid changes to a single cell within
milliseconds without disturbing flow patterns across the cell as well
as achieving rapid changes to an entire population of cells within a second.
Limitations of this system are similar to those of other systems used
to monitor cardiac myocyte behavior. First, the firmness of the
adherence of the myocyte to the underlying material (glass, in this
case) will influence the magnitude of shortening of the cell such that
the actual contractile ability is difficult to discern. Second,
selection of which particular cells to study among the wide variety of
sizes, shapes, and contractile responses that are present in any given
experiment is a concern for any experimental protocol using isolated
myocytes. In this system, it is possible to mark a position of the test
chamber and return to a given cell to compare its responses under a
variety of extracellular conditions. With each cell as its own control,
variability that might arise from comparing effects of a given
intervention using populations of cells can be minimized. Third, the
estimation of the time to change the suffusate solution over a given
cell ignores the parabolic shape of the interface between the two
solutions as the new solution moves through the capillary tube. Thus
the actual time for the microenvironment surrounding the cell attached to the capillary wall to equilibrate with the new solution will be
slightly longer than predicted. Such situations exist whenever solution
changes are made by directing a stream of solution over a cell attached
to a wall or bottom of a chamber.
There are some special methodological cautions that need to be taken
when using this system. First, the cells chosen for a study need to be
attached on or near the bottom of the tube. The optical distortion due
by the tube curvature can introduce significant measurement errors.
(Capillary tubes with square lumens are available and would overcome
this difficulty.) Second, sheer stresses produced by high flow rates
can overcome attachment forces and wash the cells away. Third, the
passage of gas bubbles through the test chambers can have catastrophic
effects on the myocytes. High stimulus intensity promotes electrolysis
and bubble formation at the site of electrode attachment. These bubbles
can be isolated and "bled off" by placing the upstream
electrode on a stainless steel connector in the bubble-trap sidearm of
the fluid line. During the ischemic episodes, cessation of flow
through the test chambers can be accomplished by blocking the outflow
tube, and a low flow of fluid out of the upstream sidearm can wash out
any upstream bubbles that formed. Another cause of bubble formation in
the line arises from heating the perfusion solutions in the reservoirs.
This often occurs in lines from reservoirs of perfusate that are used
only intermittently in the experiment. Caution must be taken to
"bleed off" the bubbles from these lines via the upstream
sidearm before allowing the fluid from these reservoirs to flow through
the test chambers.
We have successfully used this technique to assess contractile
properties of cardiac myocytes isolated from hearts of rats chronically
treated with the antineoplastic agent adriamycin and to compare these
properties with those of sham-treated rats (4) The ability
to do side-by-side myocyte isolations and contractile evaluations has
greatly simplified the conduct of these experiments.
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ACKNOWLEDGEMENTS |
The authors acknowledge the expert technical contributions of Jamie
Denninger in isolating the cardiac myocytes and conducting many of the
experimental protocols described in the study.
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FOOTNOTES |
The work was supported by grants from the Minnesota Medical Foundation
and National Heart, Lung, and Blood Institute Grant HL-58016.
Address for reprint requests and other correspondence:
L. J. Heller, Dept. of Physiology, Univ. of Minnesota,
Duluth, School of Medicine, 1035 University Dr., Duluth, MN
55812 (E-mail: lheller{at}d.umn.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 16, 2003;10.1152/ajpheart.00914.2002
Received 22 October 2002; accepted in final form 8 January 2003.
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ABSTRACT
INTRODUCTION
