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1 Cardiac Muscle Research
Laboratory, To test whether
contractile function in "hypoxic" myocytes treated with high
glucose (19.5 mM) can be improved by increasing intracellular
Ca2+ without accelerating cell
contracture or death, we challenged metabolically inhibited, paced
myocytes with high extracellular Ca2+ concentration
([Ca2+]o)
and measured simultaneously cell shortening and intracellular Ca2+ concentration
([Ca2+]i).
NaCN exposure at a physiological
[Ca2+]o
level (1.2 mM) caused a decline of contractile function to 58 ± 8%
of the pre-NaCN value (P < 0.001)
but increased systolic and diastolic
[Ca2+]i
by 104 ± 17 and 37 ± 9% above baseline
(P < 0.01), respectively. Consequent
doubling of
[Ca2+]o
to 2.4 mM, in the presence of NaCN, immediately restored contractile function, and twitch amplitude after 18 min was 123 ± 14%
(P < 0.001) of baseline pre-NaCN
values, whereas systolic
[Ca2+]i
increased further to 225 ± 63% (P < 0.05) and diastolic
[Ca2+]i
to 73 ± 16% above baseline (P < 0.01). This marked increase in
[Ca2+]i
had no deleterious effect on myocyte diastolic function or survival.
These results suggest that if adequate metabolic substrate is provided,
contractile function in metabolically inhibited, hypoxic
myocytes can be restored by increasing
[Ca2+]i
without causing short-term cell injury.
anoxia; fura 2; glucose; hypoxia; heart failure; metabolic
inhibition
MYOCARDIAL ISCHEMIA initially causes contractile
dysfunction and, when prolonged, cell death and necrosis (38).
Cytosolic Ca2+ overload and severe
depletion of ATP have been associated, along with other factors, with
the transition from reversible to irreversible myocardial injury (5,
30, 37). The reversal of ischemic contractile dysfunction by positive
inotropic interventions is of considerable clinical interest. However,
most positive inotropic agents increase intracellular
Ca2+, and potentially decrease ATP
content, and may therefore accelerate ischemic cell injury and death.
Hypoxia itself causes an increase in intracellular
Ca2+ concentration
([Ca2+]i)
(5, 11, 14, 20). Because
[Ca2+]i
overload following high extracellular
Ca2+ intoxication (31, 34),
ouabain (17), strophanthidin, or veratrine intoxication (33) or
exposure to free radicals (23) causes cellular contracture, it may
cause hypoxic or ischemic contracture as well (11). Blocking or
reducing the increase in intracellular
Ca2+ with lanthanum or verapamil
(18, 26) delayed contracture, and depletion of
Ca2+ in the sarcoplasmic reticulum
(SR) prolongs myocyte survival during complete metabolic inhibition
(29). These results support the notion that
Ca2+ is a mediator of cell death.
Reversible contractile dysfunction occurs in acutely ischemic
myocardium. A partial decrease in coronary flow causes a proportional decrease in contractile function (40, 42), from which recovery can be
complete on restoration of normal blood flow. This decreased level of
function can be overcome by inotropic agents, but at the expense of
accelerated depletion of metabolic reserves (42). During such moderate
ischemic or hypoxic failure, depletion of energy reserves rather than
intracellular Ca2+ overload may be
more important for the development of contracture. For example, during
sustained low-flow ischemia, in which contractile function was
depressed but sustained, increasing the perfusate Ca2+ level improved systolic
function but did not worsen the ischemic diastolic dysfunction. This
result seems inconsistent with intracellular Ca2+ overload as the direct
mediator of contracture (9). Thus, during moderate hypoxic or ischemic
failure, the relationships among intracellular
Ca2+, systolic and diastolic
function, and cell energetics are not completely defined.
The purpose of this study was to investigate the effect of increased
intracellular Ca2+ on contractile
function and the development of contracture in a model of prolonged,
moderate metabolic dysfunction in cardiac myocytes. Our goals were
1) to induce a stable level of
contractile dysfunction in isolated adult cardiomyocytes,
2) to monitor continuously intracellular Ca2+ levels,
3) to determine whether and how
intracellular Ca2+ levels changed
throughout a sustained period during which metabolic inhibition reduced
systolic function by ~50%, and then
4) to increase substantially the
intracellular Ca2+ level of the
metabolically inhibited myocyte to determine whether systolic function
can be increased without accelerating diastolic dysfunction,
contracture (rigor), or cell death.
We recognized that unloaded isolated cells perform less work than
myocytes of intact myocardium; nonetheless, we wished to simulate the
in vivo
[Ca2+]i
and metabolic demand as closely as possible. Therefore, the cells were
maintained at 37°C and paced at 5 Hz. These parameters differ
significantly from most of the prior studies of metabolically inhibited
myocytes, which were done under hypothermia, and with slow stimulation
rates, both of which decrease intracellular
Ca2+ levels and metabolic demand.
In prior studies we determined that a stable reduction of contractile
function could be achieved by inhibiting cellular respiration with 2 mM
NaCN and providing a relatively high glucose concentration (19.5 mM) as
substrate; a normal (5.5 mM) glucose level did not sustain stable
contractile function in the majority of NaCN-treated myocytes (45).
Therefore, in the current study a state of sustained "partial
hypoxic failure" was achieved by the combination of 2 mM NaCN and
19.5 mM glucose.
Cell Isolation
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Measurement of Cell Shortening
Aliquots of cell suspension solution were placed on coverslips of a heated (37 ± 1°C), flow-through (1 ml/min) perfusion chamber on the stage of a Nikon Diaphot inverted microscope (Nikon, Melville, NY). The myocytes were continuously superfused with Tyrode solution (in mM: 137 NaCl, 5.4 KCl, 1.2 or 2.4 CaCl2, 0.5 MgCl2, 10 HEPES, and 19.5 D-glucose, pH 7.4) and electrical-field stimulated at 5 Hz by 2 platinum electrodes (1 mm apart) connected to a Grass stimulator (Grass Instruments, Quincy, MA) with 2-ms square-wave bipolar pulses. Cells were illuminated by the microscope light, filtered by a long-pass red filter (
> 650 nm; Omega Optical, Brattleboro, VT). The cell image, collected by a ×40 ultraviolet (UV) epifluorescence objective, was diverted to the microscope's side
port, where the cell image was recorded by a video camera (Pulnix,
Sunnyvale, CA). The video camera was specially adapted to acquire
images at 240 Hz. Contractile amplitude and velocity of shortening of
the myocytes were analyzed in real time by a video edge detector
(Crescent Electronics, Salt Lake City, UT) and a personal
computer-based data acquisition system as well as recorded on a Gould
chart recorder. Calibration of the system was accomplished with a
micrometer, which was visualized by the objective lens.
Reporting of Cell Shortening
The end-diastolic and peak systolic cell lengths are expressed as a percentage of the baseline (pre-NaCN) end-diastolic cell length. Twitch amplitude is expressed as a percentage of (end-diastolic cell length
peak systolic cell length)/end-diastolic cell length.
Measurement of [Ca2+]i
Cytosolic Ca2+ was measured by the fluorescent Ca2+ indicator fura 2 (Molecular Probes, Eugene, OR), using a dual-excitation fluorescence, Ca2+ ion-sensing system (Ionoptix, Milton, MA). The fura-loaded myocytes were excited at 360 ± 6.5 and 380 ± 6.5 nm, and the resulting fluorescence at 510 ± 15 nm was measured. The ratio of the fluorescence excited by 360 nm to that excited by 380 nm was independent of the intracellular fura 2 concentration, cell geometry, and excitation light intensity and reflected [Ca2+]i (16).Light from a UV xenon lamp (Photon Technology International, Monmouth
Junction, NJ) passed through one of two filters, centered at 360 and
380 nm (Chroma Technology, Brattleboro, VT) and placed on a rotary
wheel whose position was controlled by the data acquisition system. An
externally driven shutter was provided, to minimize the presentation of
light to the cell. A dichroic mirror (cutoff = 430 nm, Omega
Optical) directed the UV light to the objective lens. The excited
fluorescence was collected by the epifluorescence lens, transmitted
through the previously described dichroic mirror, and reflected to the
side port of the microscope, where another dichroic mirror (cutoff = 550 nm, Omega Optical) reflected the light ( < 550 nm) to a
photomultiplier tube (Hamamatsu, Bridgewater, NJ). An adjustable
aperture stop restricted the collected light area to the observed cell,
and a barrier filter (Omega Optical), centered at 510 nm and with a
bandwidth of 30 nm, restricted the detected photons to those in the
emission spectra of fura 2. These photons were counted by the
photomultiplier detector and analyzed by the data acquisition system
provided by Ionoptix, consisting of an 80486 processor-based personal
computer with an analog-to-digital board and a photon-counting
(ThornEMI, Rockaway, NJ) board. The system included software to control
the presentation of UV light to the cell and the simultaneous
acquisition of the fluorescence signal and the length signal.
Fluorescence Ratio F360/F380 and Time Resolution of the System
The fluorescence excited by 360 nm was independent of [Ca2+]i changes (16) (Fig. 1). During a typical data collection run, the 360-nm light was presented to the myocytes for 0.5 s at the beginning and end of the run, which lasted 250 ms. Between these two points, only the 380-nm light was presented, and the fluorescence was acquired by the system at 500 samples/s. At each of these sampling points, a calculated 360-nm excited fluorescence was determined, using an interpolation of the 360-nm excited fluorescence collected at the beginning and end of the data collection run. The fluorescence ratio was formed by dividing the calculated 360-nm excited fluorescence by the measured 380-nm excited fluorescence. The data from 16 sequential runs were averaged to reduce photon noise. This technique had a sampling period of 2 ms.
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Fura 2 Loading
The cells were loaded with the membrane-permeant ester form of fura 2 by incubating them in the presence of 5 µM fura 2-AM for 20 min at room temperature. After we washed out the fura 2 in the loading solution, 60 min were allowed for the deesterification of the fura 2 ester in the cells. Fifty micromolars probenecid was included throughout this procedure to help inhibit leakage of fura 2 before experimentation (8).Distribution of Fura 2
The extent of loading of fura 2 into noncytosolic compartments (predominantly the mitochondria) was tested by quenching the fluorescence of Ca2+-sensitive fura 2 in the cytosol with 100 µM MnCl2 (36). The residual fluorescence after Mn2+ was 10 ± 7% (n = 5), indicating that 90% of the recorded fura 2 fluorescence was sensitive to Ca2+ and emanated from the cytosolic space.Field Study
Morphology of fields of cells were imaged via the Pulnix video camera and recorded on a videocassette recorder. A ×20 bright-field objective lens (Nikon) was used, allowing up to 15 cells to be observed simultaneously.Cell Inclusion Criteria
The myocytes were paced at 5 Hz and 37°C for 5-15 min. Cells, which showed no damage after this warm-up period, were chosen according to following criteria: 1) rod shape, 2) clear striations, 3) no sarcolemmal blebs, 4) square edges, and 5) stable mechanical behavior at 5 Hz and 37°C for a further 5 min. Additionally, the fura 2-loaded cardiomyocytes were required to have sufficient fura 2-dependent fluorescence [360-nm-excited fura 2 fluorescence/360-nm-excited cell autofluorescence (AF)
3].
Adjustment for Autofluorescence Changes
Noncellular background (BG) and cellular AF were subtracted from the measured fluorescence from fura 2-loaded cardiomyocytes before calculation of the fura 2 ratio. Because AF varied in different preparations, it was determined daily, by averaging the fluorescence in 6-8 cardiomyocytes (from the same heart) not loaded with fura 2. Furthermore, inhibition of oxidative metabolism by NaCN caused a rapid, sustained increase in cellular AF and could potentially affect the measurement of the Ca2+-dependent change in fluorescence from fura 2-loaded cardiomyocytes. To account for this effect, cellular fluorescence (excited by 360 and 380 nm) in paced myocytes not loaded with fura 2 was measured in 2-min intervals before and after exposure to NaCN over a 30-min period (n = 8 cells, 4 hearts). The results from these experiments were then used to compute a scaling factor [a360(t) and a380(t)] to adjust for NaCN-induced changes in AF at a given time point. The scaling factors were calculated as follows
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(1) |
The daily determined AF was then adjusted as follows
![AF′<SUB>360</SUB>(<IT>t</IT>) = AF<SUB>360</SUB> [1 + a<SUB>360</SUB>(<IT>t</IT>)]](/content/vol275/issue6/fulltext/H2272/img002.gif)
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![AF′<SUB>380</SUB>(<IT>t</IT>) = AF<SUB>380</SUB> [1 + a<SUB>380</SUB>(<IT>t</IT>)]](/content/vol275/issue6/fulltext/H2272/img003.gif)
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(2) |
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(3) |
Normalization and Reporting of Fura 2 Fluorescence Changes
The end-diastolic and peak systolic Ca2+ levels are expressed as the fura 2 fluorescence ratio values relative to the magnitude of the baseline (pre-NaCN) fura 2 transient amplitude in each cell. The fura 2 ratios were normalized such that the baseline peak systolic value was considered to be 100%, the end-diastolic value was set at 0%, and the baseline Ca2+ transient amplitude height was therefore 100%.Time Constant of the Ca2+ Transient
The time constant (
) was calculated for certain fura 2 ratio
transients by fitting the decay of the transient to an exponential curve. The curve was fitted from the fura 2 ratio starting from 10%
below the peak value until the next transient as determined by the
positive upstroke of the derivative of the fura 2 transient.
Ca2+-Cell Length Phase Loops
Ca2+-cell length phase loops, as first introduced by Spurgeon et al. (44), were plotted to assess changes in Ca2+ sensitivity of myofilaments. Fura 2 ratio is plotted versus the simultaneous cell length. The time resolution of these loops was 2 ms.Data and Statistical Analysis
Data are reported as means ± SE. Data acquired by repeated sequential measurements in individual myocytes were tested by analysis of variance for repeated measures with individual differences tested using the least-squares differences comparison. Analysis was performed on the raw data, although percentage data are presented in Figs. 3, 6, and 7. The field study results were tested using Fisher's exact test. A P value <0.05 was considered significant.Experimental Protocols
In all protocols, the cells were continuously superfused with Tyrode solution containing 19.5 mM D-glucose. Cells were paced at 5 Hz.Protocol 1. Cell shortening and [Ca2+]i changes during sustained NaCN-induced contractile dysfunction. After the baseline period, fura 2-loaded cardiomyocytes were exposed to 2 mM NaCN for 20 min. Six of seven myocytes, from seven hearts, exhibited sustained, albeit reduced, function during the 20-min NaCN exposure; one developed contracture and was excluded for not representing sustained stable contractile dysfunction. Cell shortening and fura 2 fluorescence were recorded simultaneously at 2-min intervals.
Protocol 2. Effect of increasing extracellular Ca2+ on cell shortening and [Ca2+]i changes in metabolically inhibited cardiomyocytes. After the baseline period, 15 fura 2-loaded cardiomyocytes, from seven hearts, were superfused with Tyrode solution containing 2 mM NaCN and 1.2 mM Ca2+ for 20 min. Thirteen of the 15 cardiomyocytes had sustained reduced function during this 20-min NaCN exposure; two developed contracture and were excluded. In these 13 myocytes, after 20 min of sustained contractile dysfunction, the extracellular solution was either 1) unchanged at 1.2 mM Ca2+ (n = 6) or 2) replaced with the same solution, but with twice the Ca2+ concentration, i.e., 2.4 mM Ca2+ (n = 7). Cell shortening and fura 2 fluorescence were recorded at 5-min intervals. To minimize heart-to-heart variability, pairs of cells from the same heart were used for pairs of experiments, one myocyte for a 1.2-mM Ca2+ run and one for a 2.4-mM Ca2+ run. The two cells were studied sequentially within 2 h of each other. To control for the age of the cells after isolation, the order of the protocols was alternated on successive runs.
Protocol 3. Effect of increasing extracellular Ca2+ on myocyte survival (field study). To test whether increasing [Ca2+]i could have a deleterious effect on survival in metabolically inhibited myocytes, the response of groups of myocytes (field study) was observed. After the baseline period, groups of cardiomyocytes were exposed to 1) 40 min of the normal Tyrode solution (control), 2) 40 min of 2 mM NaCN at 1.2 mM CaCl2, or 3) 20 min of 2 mM NaCN at 1.2 mM CaCl2 and 20 min of 2 mM NaCN at 2.4 mM CaCl2. The time from the start of NaCN exposure until the onset of rigor was recorded. All three of these field study protocols were applied to cells from the same cell preparation from the same six hearts, and the order in which the last two protocols were performed was alternated for successive studies.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Protocol 1. Cell Shortening and [Ca2+]i Changes During Sustained NaCN-Induced Contractile Dysfunction
Cell stability in the absence of NaCN. Fura 2 fluorescence, cell length, and cell shortening were recorded in eight cardiomyocytes (from 6 hearts) paced at 5 Hz at 37°C in Tyrode solution, containing 19.5 mM glucose, without NaCN. All parameters were stable over a 30-min time period.
Effects of NaCN.
NaCN exposure caused characteristic changes in cell length, twitching
amplitude, and cytosolic Ca2+
transients. Typical tracings of simultaneously measured cell length and
[Ca2+]i
in two myocytes are shown in Fig. 2,
A and
B, and group values are reported in
Fig. 3. During 18 min of NaCN exposure,
end-diastolic cell length became longer by 2.3 ± 0.8%
(P < 0.001), twitch
amplitude decreased to 65 ± 9% of the pre-NaCN value
(P < 0.005), end-diastolic [Ca2+]i
increased by 33 ± 16% (P < 0.05), peak systolic
[Ca2+]i
increased to 191 ± 34% of baseline
(P < 0.01), and the amplitude of the
Ca2+ transient increased to 157 ± 17% of baseline (P < 0.005)
(Figs. 3 and 4). There was no significant difference between the time constant of Ca2+ transient decay
() at baseline and after 18 min of exposure to NaCN (52 ± 5 vs.
46 ± 3 ms), indicating that there was no impairment of
Ca2+ removal when diastolic
[Ca2+]i
was significantly elevated.
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Protocol 2. Effects of Increasing Ca2+ on Cell Shortening and [Ca2+]i in Metabolically Inhibited Myocytes
The effect of increasing extracellular Ca2+ concentration on myocyte function during NaCN-induced hypoxia was studied in 13 myocytes. Figure 2C shows representative cell length and fura 2 ratio transient tracings from a single cardiomyocyte subjected to this protocol, and Fig. 6 reports group results. Before the high-Ca2+ intervention, NaCN caused the same degree of failure in both groups, reducing the twitch amplitude of the high (2.4 mM) Ca2+ group (n = 7) and the normal (1.2 mM) Ca2+ control group (n = 6) to similar levels (58 ± 8 and 61 ± 13% of baseline, respectively). In the high Ca2+ group, 5 min after the high-Ca2+ intervention, twitch amplitude increased to 156 ± 16% of the pre-NaCN level and after 20 min was 123 ± 14% of the pre-NaCN value (P < 0.025 vs. pre-NaCN, P < 0.001 vs. before high-Ca2+ intervention), indicating a small but significant decline during the high-Ca2+ intervention (P < 0.01, Fig. 6C). During the high-Ca2+ period, end-diastolic cell length became shorter (1.3 ± 0.3%, P < 0.02) but remained significantly
longer than the pre-NaCN length (101.5 ± 1.0% of pre-NaCN,
P < 0.025). The restoration of
extracellular Ca2+ to 1.2 mM (at
42 min in Fig. 6) removed the inotropic effect, and the twitch
amplitude was reduced to a level not different from that before the
elevation of extracellular Ca2+.
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Eighteen minutes of exposure to 2 mM NaCN increased the Ca2+ transient amplitude and systolic and diastolic [Ca2+]i equally in both the high Ca2+ and normal Ca2+ (control) groups (Fig. 6, D-F). After the high-Ca2+ intervention, the Ca2+ transient amplitude further rose to 232 ± 45% of the pre-NaCN value (P < 0.001 vs. pre-NaCN, P < 0.05 vs. before high-Ca2+ intervention). Similarly, systolic [Ca2+]i rose to 279 ± 65% of the pre-NaCN baseline (P < 0.001 vs. pre-NaCN, P < 0.05 vs. before high-Ca2+ intervention), which is consistent with the high-Ca2+ intervention achieving its impact via an increase of cytosolic [Ca2+]i. End-diastolic [Ca2+]i rose by 66 ± 13% at 5 min after the increase of superfusate Ca2+ (P < 0.001 vs. pre-NaCN, P < 0.02 vs. before high-Ca2+ intervention). Peak systolic and end-diastolic [Ca2+]i did not change significantly subsequently during the high-Ca2+ intervention.
Figure 5B shows the shifts of the Ca2+-cell length phase loops of a representative myocyte subjected to the NaCN-high Ca2+ protocol. The rightward and downward shift of the loop after 20 min of NaCN is consistent with the observations in myocytes exposed to NaCN bathed in 1.2 mM Ca2+ as shown in Fig. 5A. After 5 min of the high-Ca2+ intervention, the loop shifted upward and slightly rightward such that the relaxation phases of the pre- and post-Ca2+ intervention loops inscribed a common trajectory. Such a shift is consistent with no change in the myofilament responsiveness to Ca2+ as a result of the Ca2+ intervention and suggests that the functional changes were due to increases in intracellular Ca2+ and not altered Ca2+ sensitivity.
Protocol 3. Effect of Increasing Extracellular Ca2+ on Myocyte Survival (Field Study)
The effect of increasing extracellular Ca2+ concentration on myocyte survival during metabolic inhibition was investigated in field studies, and the result is shown in Fig. 7. Forty minutes of beating in control solution had no effect on myocyte survival (n = 49 cells from 6 hearts), but 20 min of 2 mM NaCN (n = 73 cells from 9 hearts) caused a significant number of cells to go into contracture. However, in the group in which extracellular Ca2+ was raised to 2.4 mM (n = 88 cells from 9 hearts), the incidence of contracture was not different from that of the 1.2-mM Ca2+ group. As shown in Fig. 7, at the end of the protocol, the fraction of myocytes that had survived 40 min of NaCN was practically identical in the two Ca2+ groups. Thus, during metabolic inhibition, increasing cell Ca2+ to a level that more than completely reversed contractile failure (see Fig. 6) did not increase injury in these myocytes provided with supraphysiological levels of glucose.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Mechanism of Contractile Dysfunction in This Model
Our results indicate that during moderate metabolic inhibition, both systolic and diastolic levels of intracellular Ca2+ increased. However, diastolic length increased, and twitch amplitude decreased, consistent with the previous report of Ikenouchi et al. (20), indicating a decrease in myofilament sensitivity to the increased levels of Ca2+. The rightward and downward shift of the cell length-Ca2+ loops (Fig. 5) is also consistent with a decrease in myofilament Ca2+ sensitivity (44). Doubling the extracellular Ca2+ level, which increased intracellular Ca2+, more than completely reversed the 50% depression of systolic function caused by the NaCN.These observations suggest that decreased myofilament sensitivity to Ca2+ was the cause of the moderate degree of contractile failure in this model. The failure was not due to inadequate Ca2+ availability because systolic Ca2+ was elevated during metabolic inhibition.
The mechanisms responsible for the decreased Ca2+ sensitivity were not studied but were most likely changes in metabolic milieu, to which an increase in Pi and decrease in pH both potentially contributed (11, 24, 28, 30). The increase in Pi, which results from the partial depletion of high energy reserves, most likely interfered with cross-bridge cycling, whereas the decrease in pH affected Ca2+ activation of the myofilaments. Studies show that Pi is likely the more important factor, as has been demonstrated by NMR studies of buffer-perfused ferret hearts exposed to hypoxia (27) and rat hearts exposed to mild coronary flow reduction (12). The continuous superfusion of the cardiomyocytes prevented the extracellular accumulation of acidotic products (e.g., lactic acidosis) and thus allowed uninhibited H+ efflux. Two previous studies showed a variable development of modest acidosis caused by metabolic inhibition (11, 20), and therefore acidosis may have contributed to the reduced Ca2+ responsiveness of the myofilaments.
A lack of energy supply does not explain the contractile dysfunction in this model. We did not measure energetics and cannot say with absolute certainty whether decreased energy production contributed to the contractile dysfunction. However, during continued NaCN exposure, sufficiently high energy reserves existed to support systolic function at a greater level than pre-NaCN baseline levels. After extracellular Ca2+ was doubled, the existing energy supply was adequate to provide sustained support for greater contraction and for increased energy-consuming cycling of Ca2+ (Fig. 6). We had expected that the increased work and energy requirement imposed by the high-Ca2+ intervention would decrease energy reserves over time, as has been observed in inotropic stimulation of hypoperfused myocardium in intact animals (42). Surprisingly, this was not the case. Increased extracellular Ca2+ resulted in increased systolic and diastolic levels of intracellular Ca2+ but did not cause contracture or accelerate failure in this model.
Tolerance to Increased Ca2+
The surprising tolerance of the hypoxic myocytes to the increased levels of intracellular Ca2+ may have been due to one or more of the following factors.Relatively brief exposure to metabolic inhibition and the high level of Ca2+. Although "sustained" relative to current isolated myocyte protocols, the 40-min period of metabolic inhibition, 20 min of which comprised the exposure to high Ca2+, is relatively brief in comparison with clinical periods of hypoxia. It is possible that with longer periods of metabolic inhibition or exposure to Ca2+, more cell injury would have occurred. Nonetheless, this degree and duration of metabolic inhibition was lethal to a majority of myocytes when a normal (5.5 mM) or subnormal glucose level was provided as substrate (45), so the degree of inhibition of oxidative phosphorylation per se must be considered significant. Furthermore, during the 20-min exposure to the high extracellular Ca2+, intracellular Ca2+ levels did not progressively rise but rather reached a level plateau (Fig. 6); therefore, there is no a priori basis to assume that a longer exposure would have resulted in progressive Ca2+ overload.
Only a moderate increase in intracellular Ca2+ level was imposed. Our studies do not contest the well-established role of Ca2+ overload in causing cell injury. A loss of Ca2+ regulation is a feature of irreversibly injured ischemic and hypoxic myocardium. Ca2+-loaded, disrupted mitochondria have been observed in reperfused and severely ischemic dog hearts (43). High levels of mitochondrial Ca2+ have been associated with the transition between reversibly injured and irreversibly injured hypoxic, isolated cardiomyocytes (35). High levels of cytosolic Ca2+ may impair myocardial relaxation (25, 31), may activate Ca2+-dependent proteolytic enzymes (3, 13, 15), and may promote the ability of oxidant stress to cause injury (4).
Our goal was to attempt to reverse contractile dysfunction by increasing Ca2+ availability without causing lethal Ca2+ overload, and a doubling of the extracellular Ca2+ achieved this end. Greater increases of extracellular and intracellular [Ca2+] may accelerate the development of irreversible injury. For example, in a small series of preliminary experiments we increased the extracellular Ca2+ threefold, to 3.6 mM, and observed that two of four metabolically inhibited myocytes underwent immediate contracture. Thus there is clearly a limit to the amount of Ca2+ load a hypoxic myocyte can tolerate.The decreased myofilament sensitivity to Ca2+ reduced the deleterious effects of the Ca2+ increase. The increased level of Ca2+ may have been tolerated in part because of the decrease in myofilament Ca2+ sensitivity. For example, Barry et al. (5) have proposed that hypercontracture itself can damage the sarcolemma and accelerate Ca2+ entry and lethal Ca2+ overload (29, 39). Thus the decreased Ca2+ sensitivity may have conferred protection against this mechanism of cell injury.
Buffering effect of fura 2. Fura 2 is a Ca2+ chelator and could have protected the fura 2-loaded cardiomyocytes from a Ca2+ overload via its buffering capacity. However, the field study experiment (Fig. 7) was conducted with cells not loaded with fura 2, and the failure of the high-Ca2+ intervention to accelerate contracture in these cells could not be the consequence of a fura 2 protective effect. Thus, in the absence of fura 2, the degree of Ca2+ loading we employed was not deleterious. Therefore, a protective effect of fura 2 is not a satisfactory explanation for the tolerance to the increased Ca2+ level.
A high level of glycolytic activity resulting from the high substrate glucose concentration. Previous work from this laboratory and others has shown an important protective effect of an active glycolytic pathway during hypoxia and low-flow ischemia (1, 2, 7, 10, 19, 41, 45). In studies of isolated cardiac myocytes, the fraction of myocytes that suffered contracture in the presence of 2 mM NaCN was reduced in a dose-dependent fashion by increasing the concentration of exogenous glucose (45). Therefore, it is likely that the relatively high level of exogenous glucose that we used in the current study protected the metabolically inhibited myocytes from injury during the increase in superfusate Ca2+ and resultant inotropic stimulation. How an active glycolytic pathway might protect metabolically inhibited myocytes was not investigated in this study. Possible mechanisms include the preservation of glycolytic ATP preferentially used for the SR Ca2+ pump and Na+ pump (47), attenuation of an increase of ADP with a consecutive preservation of free-energy availability and SR Ca2+ pump function (46), activation of second messengers that could affect the excitation-contraction coupling (21), and activation of nonmetabolic pathways by glucose. Continued functioning of the Na+ pump could aid Ca2+ homeostasis by mitigating an intracellular Na+ overload with its attendant adverse effect on Ca2+ efflux via the Na+/Ca2+ exchanger. Activation of protein kinase C by glucose (4) could lead to phosphorylation of phospholamban and to higher rates of SR Ca2+ pump activity.
Other factors unique to metabolic inhibition and/or unloaded cells. The relative tolerance to inotropic stimulation of our isolated myocytes, in contrast to the depletion of energy reserves that occurred when hypoperfused myocardium was inotropically stimulated in intact hearts (42), may also be due to important differences between experimental conditions. In the working ischemic heart other factors may contribute to cell injury and the deleterious effects of Ca2+. In ischemic myocardium, other agents, such as oxygen-derived free radicals, inflammatory cytokines, and amphiphiles, may have a greater deleterious impact on the Ca2+ regulation system, or these factors may be additive to an increase in cell Ca2+ in causing injury. Also, in unloaded cells the workload is relatively low compared with the intact heart, and this may ameliorate the effect of an inotropic stimulation to decrease energy reserves.
Ca2+ Overload and Diastolic Dysfunction
Our observations may be pertinent to considerations of the mechanisms responsible for hypoxic and ischemic diastolic dysfunction. ATP depletion and/or cytosolic Ca2+ overload have been proposed as mechanisms for impaired myocyte relaxation during hypoxia and/or ischemia, with resultant diastolic dysfunction. However, our observations indicate a remarkable tolerance of myocyte relaxation to an elevated diastolic [Ca2+]i level. For example, in Fig. 6, after the high-Ca2+ intervention, the diastolic Ca2+ level was markedly elevated to a level ~60% of the height of the Ca2+ transient at baseline (Fig. 6D). The diastolic cell length decreased during exposure to high Ca2+ but remained longer than diastolic cell length at baseline (Fig. 6A). The increased Ca2+ levels did not harm the Ca2+ removal mechanisms, as is reflected by the lack of any change in, the time constant for
Ca2+ decay. The observed increase
in diastolic cell length at a time of marked diastolic
Ca2+ overload is consistent with a
decrease in myofilament sensitivity to
Ca2+, but it also demonstrates
that hypoxic impairment of myocyte relaxation does not always occur
during elevated diastolic cell Ca2+ levels.
The source of the increase in cell Ca2+ after NaCN exposure is not certain. The Mn2+ quenching experiments indicated that 90% of the fluorescent signal emanated from the cytosolic space, thereby making it likely that changes in fura 2 fluorescence reflected an increase in cytosolic Ca2+. During normoxic as well as hypoxic conditions the mitochondrial Ca2+ concentration is lower than cytosolic Ca2+ concentration, and therefore it is unlikely that an increase in mitochondrial Ca2+ contributed significantly to the change in fura 2 fluorescence (35). Because the time constant of Ca2+ decay was not prolonged and because the height of the Ca2+ transient amplitude was able to increase after the high-Ca2+ intervention, it seems unlikely that impaired SR Ca2+ reuptake can significantly account for the increase in cytosolic Ca2+ levels. A leak from the SR or the NaCN-induced activation of the ryanodine receptor remain other possibilities. However, leakage from the SR would eventually lead to SR Ca2+ depletion and to a decrease rather than an increase in the Ca2+ transient amplitude. Furthermore, the decrease in ATP, increase in cytosolic Mg2+, and intracellular acidosis accompanying metabolic inhibition should inhibit rather than activate the ryanodine receptor activity (6). An alternative explanation is that sarcolemmal flux of Ca2+ was altered by NaCN in a manner to favor net cytosolic Ca2+ accumulation.
In conclusion, during metabolic inhibition intracellular Ca2+ increased and contractile function decreased secondary to decreased myofilament Ca2+ sensitivity. Normal contractile function could be restored by an increase in intracellular Ca2+. This increase had no adverse effect on diastolic cell length or onset of contracture, when cells were provided with increased glycolytic substrate. These observations suggest that, during hypoxic and ischemic pump failure, contractile function might be effectively improved by interventions that increase intracellular Ca2+, and the deleterious effect of increased intracellular Ca2+ can be prevented by simultaneously increased glucose availability.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-48715 (to C. S. Apstein). T. M. Suter was the recipient of a grant for young investigators from the Swiss National Foundation of Science.
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FOOTNOTES |
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Address for reprint requests: T. M. Suter, Boston Univ. School of Medicine, Cardiac Muscle Research Laboratory, 80 East Concord St., W611, Boston, MA 02118.
Received 25 November 1997; accepted in final form 2 September 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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) and peak
systolic (
) cell length (normalized such that pre-NaCN diastolic
length = 100%) both increased after NaCN exposure. Onset of transient
decrease of end-diastolic cell length observed in Fig. 
). Increase
in superfusate Ca2+ level had no
effect on myocyte survival.