Simulated Ischemia Increases the Susceptibility of Rat Cardiomyocytes to Hypercontracture
The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca2+ levels than did normoxic cells (Camax, 0.80±0.05 μmol/L versus 1.27±0.05 μmol/L; P<.01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 μmol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardiomyocytes to hypercontracture. This seems to be due to protein dephosphorylation.
Reperfusion of the heart after prolonged periods of ischemia can aggravate the ischemic injury of the myocardium by eliciting hypercontracture of the myofibrils.1 In myocardial tissue where adjacent cells can mutually exchange mechanical forces, hypercontracture causes membrane destruction and cell death. The underlying pathomechanism of reoxygenation-induced hypercontracture was characterized in previous studies using the model of anoxic/reoxygenated isolated cardiomyocytes.2 3 4 5 In prolonged energy depletion, cardiomyocytes develop cytosolic Ca2+ overload. Hypercontracture is, therefore, elicited when cardiomyocytes are reenergized at elevated cytosolic Ca2+ levels. Strong contractile activation, based on high cytosolic Ca2+ concentrations and a sufficient energy supply, causes irreversible distortion of the cytoskeleton and thus of cell shape. Irreversible structural distortions seem to occur when the contractile force exceeds the elastic deformability of the cytoskeleton. In several studies, it has been shown that the cytoskeleton becomes injured when cardiomyocytes are subjected to prolonged energy depletion (for review, see Reference 6). This gives rise to the question, central to the present study, whether the propensity of anoxic/reoxygenated cardiomyocytes to develop hypercontracture when reenergized in presence of a cytosolic Ca2+ overload is increased. If the answer is affirmative, reoxygenated cardiomyocytes are in jeopardy not only because they are Ca2+-overloaded but also because this Ca2+ overload is more likely to produce structural destruction.
In the present study, isolated cardiomyocytes from adult rat were used as a model. The susceptibility to hypercontracture (SH) of the cells was defined as the minimum cytosolic Ca2+ rise producing manifest hypercontracture. A test protocol was applied to the cells in which gradual Ca2+ overload was intentionally produced, and the corresponding stepwise reduction of cell length was monitored until cells developed maximal hypercontracture. This protocol was carried out in intact cardiomyocytes under normoxic control conditions as well as after simulated ischemia/reoxygenation. We found that SH is indeed increased in reoxygenated cardiomyocytes.
When energy depletion reaches a critical degree in oxygen-deprived cardiomyocytes, their myofibrils pass into the rigor state. In isolated cells, this event is indicated by a reduction of length by approximately one third (rigor shortening). Rigor shortening is reversible if the cells are immediately reenergized but becomes irreversible if the cells are left in this distorted shape for ≥5 minutes.2 7 8 Rigor shortening is followed by a progressive rise of cytosolic Ca2+, because Ca2+ overload is another result of critical loss of energy. In the present study, we attempted to determine the following: (1) We investigated whether extensive loss of energy, characterized by rigor shortening, is a sufficient cause for increased SH in the reoxygenated cell. (2) We also studied whether the development of Ca2+ overload is necessary for the increase of SH. (3) Being part of the conditions of simulated ischemia, the importance of acidosis as another possible causal factor was analyzed. (4) Finally, experiments were carried out in the presence of cantharidin, an inhibitor of protein phosphatase 1 and 2A. In these experiments, the question was addressed whether phosphatase activity inhibitable by this agent contributes to the increase in SH.
Materials and Methods
Preparation of Isolated Cardiomyocytes
Ventricular heart muscle cells were isolated from 200- to 250-g adult male Wistar rats and plated in medium 199 with 4% fetal calf serum on glass coverslips that had been preincubated overnight with 4% fetal calf serum.9 Four hours after plating, the coverslips were washed with medium 199. As a result of the wash, broken cells were removed, leaving a homogeneous population (>95%) of rod-shaped quiescent cardiomyocytes attached to the coverslip. From each isolation, two coverslips were used. On each coverslip, one cell was investigated. Only cells exhibiting a rod-shaped morphology and no signs of sarcolemmal blebbing were used for the experiments. These cells were always found to have a low resting cytosolic Ca2+ concentration (see below).
Loading of Fura 2 and BCECF
To measure cytosolic Ca2+ or H+ concentrations, cardiomyocytes were loaded at 35°C with fura 2 or BCECF, respectively. For loading, cells attached to glass coverslips were incubated for 30 minutes in medium 199 with the acetoxymethyl ester of fura 2 (2.5 μmol/L) or BCECF (1.5 μmol/L). After loading, the cells were washed twice with medium 199 containing 1% bovine serum albumin. This washing step was followed by a 30-minute postincubation period in medium 199 to allow hydrolysis of the acetoxymethyl esters within the cell. The fluorescence from dye-loaded cells was 20 to 30 times higher than background fluorescence from unloaded cells.
Ca2+, pH, and Cell-Length Measurements
The coverslip with the loaded cells was introduced into a gastight, temperature-controlled (37°C), transparent perfusion chamber positioned in the light path of an inverted microscope (Diaphot TMD, Nikon). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura 2 or 450 and 490 nm for BCECF was performed with an AR-Cation measurement system adapted to the microscope (Spex Industries). Emitted light (490 to 510 nm for fura 2 and 520 to 560 nm for BCECF) from a 10×10-μm area within a single fluorescent cell was collected by the photomultiplier of the Spex system. The light signal was recorded and analyzed by an IBM PC/AT–based data analysis system (model DM3000CM, Spex Industries).
Simultaneous to the measurement of the fluorescence, the cell's microscopic image was recorded with a video camera and stored on tape. From these recordings, changes of the cell length were determined later. In the case of hypercontracted cells, the cell dimension along its previous longitudinal axis was determined.
The loading protocols used were selected from a number of variations because they provided the highest yield in fluorescence and minimal dye compartmentation. To assess the extent of intracellular dye compartmentation, cells were chemically “skinned” with digitonin as described previously.10 Briefly, cardiomyocytes were metabolically inhibited with 1 mmol/L KCN to prevent hypercontracture and superfused for 5 minutes with EGTA buffer containing (mmol/L) KCl 135, NaCl 5, HEPES 5, EGTA 1, and KCN 1, pH 7.2 at 37°C. After this procedure, 2.5 μmol/L digitonin was added. Digitonin permeabilizes the sarcolemmal membrane but leaves membranes of the organelles intact.11 After release of the dyes from the cytosol, the residual fluorescence was measured, which was a sum of fluorescence from compartment and background fluorescence. To separate them, 1 mmol/L MnCl2 and 5 μmol/L ionomycin were added to the buffer. This quenched the fluorescence of the dyes within organelles leaving background fluorescence.10 The background fluorescence was subtracted from the initial fluorescence. Excitation of the fluorescent dyes was performed at wavelengths of 360 nm for fura 2 and 450 nm for BCECF. This test showed that the fluorescent signal from intracellular stores did not exceed 10% for fura 2 and 12% for BCECF compared with the signal from whole cells. Furthermore, extent of dye compartmentation did not differ significantly between control cells and cells after anoxia and reoxygenation. For the purpose of the present study, therefore, correction of the data for this small extent of dye compartmentation seemed unnecessary.
In Vivo Calibration of Fura 2 and BCECF
The fura 2 signal was calibrated according to the method described by Li et al.12 For this purpose, the cells were exposed to 5 μmol/L ionomycin in modified Tyrode's solution (pH 7.4) containing either 3 mmol/L Ca2+ or 5 mmol/L EGTA to obtain, respectively, the maximum (Rmax) and the minimum (Rmin) ratio of fluorescence. To prevent morphological alterations during calibration, cells were ATP-depleted with 1 mmol/L KCN. The free cytosolic Ca2+ concentration ([Ca2+]i) was calculated according to the equation [Ca 2+]i=Kd×β×(R−Rmin)/(Rmax−R), where β is the ratio of the 380-nm excitation signals of ionomycin-treated cells at 5 mmol/L EGTA and at 3 mmol/L Ca2+, and Kd is the dissociation constant of fura 2. For fura 2 dissolved in buffer, Grynkiewicz et al13 determined a Kd of 224 nmol/L. The affinity of fura 2 to Ca2+ inside a cell may differ from the affinity in solution, however. We found that the Kd in vivo was higher than the Kd in vitro (312±9 nmol/L [n=8] versus 200±11 nmol/L [n=7]). The conversion of fura 2 ratios into absolute values of intracellular Ca2+ was performed, therefore, with an in vivo Kd of 312 nmol/L. Calibration of the BCECF ratio signal was performed, as previously described by Koop and Piper,14 with 10 μg/mL nigericin, a K+-H+ ionophore, and incubation media with various pH values.
ATP and CrP Measurements
For metabolic analysis, experiments were terminated by adding 0.5 mL 1.2 mol/L HClO4 to the culture dishes free from medium. Protein was determined according to Bradford15 using bovine serum albumin as a standard. After neutralization, perchloric acid extracts of cultures were analyzed for ATP16 and creatine phosphate (CrP).17
The perfusion chamber (1-mL filling volume) placed on the microscope stage was perfused at a flow rate of 0.5 mL/min with modified glucose-free Tyrode's solution containing (mmol/L) NaCl 125.0, KCl 2.6, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.0, and HEPES 25.0; pH was 7.4 at 37°C. Medium was made anoxic by autoclaving as described previously18 and was equilibrated before and during use with 100% N2. Normoxic medium was equilibrated with air.
In standard anoxic experiments, cardiomyocytes were superfused with anoxic (pH 6.4) medium at 37°C until rigor shortening occurred (≈30 minutes). After an additional 30 minutes, they were reoxygenated. Reoxygenation was performed by using medium of pH 6.4 for the first 10 minutes to protect cardiomyocytes against reoxygenation-induced hypercontracture.19 Then the pH of the extracellular medium was switched to 7.4, which allows recovery of cytosolic pH to control value within 10 minutes.19 After 20 minutes of reoxygenation, the Ca2+ overload protocol was applied, and susceptibility of cardiomyocytes to hypercontracture was determined.
Determination of SH
The graded cytosolic Ca2+ overload was produced under normoxic conditions. Cells were treated with 3 μmol/L ryanodine to inhibit cytosolic Ca2+ oscillations and superfused with 37°C modified Tyrode's solution (pH 7.4) containing 10 mmol/L Ca2+. The osmolality was corrected by appropriate reduction of NaCl concentration. After 10 minutes, cells were electrically stimulated with two silver chloride electrodes immersed into the medium to accelerate influx of Ca2+ into the cytosol. Biphasic electrical stimuli composed of two equal but opposite rectangular 40-V stimuli each of 2-millisecond duration were applied. Variation of the stimulation frequencies between 2 and 10 Hz allowed us to raise cytosolic Ca2+ concentration in a stepwise fashion. With the increase of cytosolic Ca2+, the cell length was gradually reduced, as shown in Fig 1⇓. Shortening of the cells exceeding 40% of resting length was not reversed upon Ca2+ removal. The resulting relationship between cell length and sustained rise of cytosolic Ca2+ must, therefore, not be mistaken for the relationship between reversible phasic cell shortening (not exceeding 30% of resting cell length) and transient elevation of cytosolic Ca2+ as found in normal excitation-contraction cycles. In the present study, cytosolic Ca2+ was allowed to rise until the maximal hypercontracture was developed, ie, until further elevation of cytosolic Ca2+ did not lead to further reduction of cell length. Therefore, the Ca2+ concentration at which cardiomyocytes reached the maximal hypercontracture was used as an index for SH.
We investigated SH in six sets of experiments. In the first set of experiments, the influence of the duration of anoxic incubation on SH was investigated. For this, cardiomyocytes were superfused with anoxic medium until rigor shortening occurred (≈30 minutes). Anoxic superfusion was then continued for 5, 15, or 30 minutes.
In the second set of experiments, the role of anoxic Ca2+ overload for SH was estimated. Cardiomyocytes were exposed to anoxia with or without cytosolic Ca2+ overload. Anoxic Ca2+ overload was prevented by incubation of cardiomyocytes in nominally Ca2+-free anoxic medium.
In the third set of experiments, the role of anoxic acidosis for SH was tested. Anoxic acidosis was imitated in normoxic cells incubated for 50 minutes in medium with pH 6.2. This incubation was followed by another one for 30 minutes in medium with pH 7.4.
In the fourth set of experiments, cardiomyocytes were exposed to anoxia in the presence of 10 μmol/L cantharidin, an inhibitor of protein phosphatases 1 and 2A.20 Cantharidin was omitted during the subsequent 20-minute period of reoxygenation.
In the fifth set of experiments, normoxic cardiomyocytes were treated with 10 μmol/L cantharidin for 50 minutes. This was followed by 20 minutes of incubation without cantharidin.
In the sixth set of experiments, normoxic cardiomyocytes were treated for 50 minutes with 20 mmol/L 2,3-butanedione monoxime (BDM), a chemical phosphatase,21 with a following incubation without BDM for 20 minutes. The osmolality was corrected by appropriate reduction of NaCl. All procedures were performed at 37°C.
Medium 199 was purchased from Boehringer-Mannheim; fetal calf serum, from GIBCO; acetoxymethyl esters of fura 2 and BCECF, from Paesel and Lorey; and cantharidin and BDM, from Sigma-Chemie. All other chemicals were from Merck and were of the highest purity available.
Data are given as mean±SE. For each experimental protocol, individual cells (n=6 to 12) were used, with not more than two cells from the same cell isolates. Metabolic data were obtained from four coverslips from four cell isolates. Statistical comparisons were performed by one-way ANOVA and use of the Bonferroni test for post hoc analysis.22 Differences at a value of P<.01 were regarded as significant.
Influence of Time of Anoxic Incubation on SH
In normoxic quiescent cardiomyocytes, cytosolic free Ca2+ was 78±8 nmol/L (n=14 cells). Incubation of the cells in anoxic glucose-free medium at pH 6.4 caused a cell-length reduction of one third, as a result of rigor shortening. The time until onset of rigor was 23±4 minutes (n=14 cells). Within 30 minutes after the onset of rigor shortening, the fura 2 fluorescence ratio rose to its saturation level, indicating a severe cytosolic Ca2+ overload4 (Fig 2A⇓). When the cells were then reoxygenated, cytosolic Ca2+ recovered to its initial level. Reoxygenation was performed by using medium at pH 6.4 for the first 10 minutes to protect cardiomyocytes against reoxygenation-induced hypercontracture and was switched then to medium at pH 7.4, which allows recovery of cytosolic pH to the control value within 10 minutes.19 After 20 minutes of reoxygenation, the susceptibility of cardiomyocytes to hypercontracture was tested.
The occurrence of rigor shortening indicates that ATP depletion has reached a critical degree.23 When anoxia is continued for ≥5 minutes, rigor shortening is not reversible. We tested how the duration of anoxic incubation after rigor shortening (5, 15, and 30 minutes) influences the susceptibility of cardiomyocytes to hypercontracture.
Test results were as follows: (1) In control cells not exposed to anoxia/reoxygenation, a maximal shortening to 32±2% of control length corresponded to a cytosolic Ca2+ concentration of 1.27±0.05 μmol/L (n=12 cells; Fig 3⇓, open circles). (2) Cells exposed to anoxia for only 5 additional minutes after rigor shortening developed maximal hypercontracture (31±2%) at the same level of cytosolic Ca2+ (1.21±0.07 μmol/L; n=8 cells; P=NS versus control; Fig 3⇓, open triangles). (3) Continuation of anoxic incubation for another 15 minutes after rigor led to a significant shift of the length-Ca2+ curve to lower Ca2+ concentrations. In this case, the maximal hypercontracture of 31±1% was observed already at 0.80±0.05 μmol/L of cytosolic Ca2+ (n=6 cells; P<.01 versus control; Fig 3⇓, closed circles). (4) Prolongation of anoxic incubation to 30 minutes after rigor shortening led only to a little additional shift of the length-Ca2+ relationship. These cardiomyocytes developed maximal hypercontracture (31±2%) at 0.74±0.05 μmol/L of cytosolic Ca2+ (n=7 cells; P<.01 versus control; Fig 3⇓, closed squares). In summary, susceptibility of cardiomyocytes to hypercontracture was increased with the time of anoxic incubation after rigor shortening.
Role of Ca2+ Overload for Anoxia-Induced Increase in SH
In this set of experiments, the question was addressed whether increased SH was caused by Ca2+ overload observed during prolonged anoxic incubation. For this purpose, anoxic superfusion was performed with nominally Ca2+-free medium. With this protocol, cardiomyocytes underwent rigor shortening, but cytosolic Ca2+ did not increase during anoxia and reoxygenation (Fig 2B⇑). In spite of the absence of Ca2+ overload during anoxia, the susceptibility of these cardiomyocytes to hypercontracture did not differ from that in cells exposed to Ca2+ overload (Fig 4⇓).
Role of Acidosis for Anoxia-Induced Increase in SH
When cardiomyocytes were exposed to simulated ischemia (incubation in anoxic medium at pH 6.4), the cytosolic pH decreased from 7.2±0.1 to 6.5±0.2 (n=12 cells) during the first 30 minutes. To investigate whether cytosolic acidosis represents a sufficient cause for the change in susceptibility of cardiomyocytes to hypercontracture, normoxic cells were superfused with a medium of pH 6.2. This protocol produced the same degree of cytosolic acidosis as in simulated ischemia (Fig 5⇓). After normoxic acidosis, the pHi was allowed to recover, and SH was then investigated. As Fig 6⇓ shows, there was no difference between the length-Ca2+ relationships of control cells and cells subjected to cytosolic acidosis.
Effect of Protein Phosphatase Inhibition on SH
We hypothesized that dephosphorylation of proteins during anoxia can be an important factor causing the increase in SH. To investigate this hypothesis, 10 μmol/L cantharidin, an inhibitor of protein phosphatases 1 and 2A,20 was added to the anoxic medium. Presence of cantharidin did not alter rigor shortening or the development of anoxic Ca2+ overload. Determination of SH showed, however, that anoxic/reoxygenated cardiomyocytes treated with cantharidin during anoxia developed maximal hypercontracture at significantly higher cytosolic Ca2+ concentrations (1.00±0.06 μmol/L; n=8 cells; Fig 7⇓, open triangles) than did untreated cells (0.72±0.04 μmol/L; n=8 cells; Fig 7⇓, closed squares). This Ca2+ concentration was of the same magnitude as in the control group (1.09±0.08 μmol/L; n=12 cells; Fig 7⇓, open circles). These results suggest that protein dephosphorylation is responsible for the increase in SH. With another set of experiments, we tested whether the effect of cantharidin on SH is specific for the changes occurring in the energy-depleted cells. For this purpose, normoxic cardiomyocytes were treated with cantharidin for 50 minutes at normoxia followed by a 20-minute period without cantharidin. The susceptibility to hypercontracture of cells treated in this manner was not different from that of untreated cells (Fig 8⇓).
Treatment of Normoxic Cardiomyocytes With BDM Increases SH
In this set of experiments, protein dephosphorylation was induced in normoxic cells. Cardiomyocytes were treated with 20 mmol/L BDM, a chemical phosphatase,21 for 50 minutes. Thereafter, BDM was removed, and superfusion was continued for 20 minutes. When the Ca2+-overload protocol was then applied, cardiomyocytes developed maximal hypercontracture at significantly lower Ca2+ concentrations (0.61±0.04 μmol/L; n=9 cells; P<.01 versus control; Fig 9⇓, closed triangles) than did control cells (1.06±0.11 μmol/L; n=10 cells; Fig 9⇓, open circles).
State of Energy of Cardiomyocytes
The cellular state of energy was determined for conditions under which the cells were submitted to the SH test protocol (Table⇓).
In anoxic/reoxygenated cells, ATP contents recovered to 36% of the control level within 20 minutes of reoxygenation; CrP recovered fully to the control level. Treatment of the cells with cantharidin or BDM did not affect ATP and CrP contents.
In the present study, we investigated the hypothesis that cardiomyocytes reoxygenated after simulated ischemia exhibit an increased SH. The main findings are as follows: (1) After prolonged simulated ischemia, reoxygenated cardiomyocytes indeed exhibit increased SH. (2) Increased SH was observed only in cells reoxygenated at times ≥15 minutes after the initiation of ischemic rigor shortening. (3) Acidosis and cytosolic Ca2+ overload developing in simulated ischemia are not causes of increased SH. (4) Increased SH can be prevented by protein phosphatase inhibition applied during simulated ischemia and provoked in normoxic cells by treatment with the chemical phosphatase BDM.
It has been investigated in previous studies why cardiomyocytes develop hypercontracture upon reoxygenation.7 24 25 Briefly, reoxygenation-induced hypercontracture is elicited by the coincidence of two factors: (1) resupply of energy to the cells, caused by reactivation of mitochondrial ATP production, and (2) an excessive rise in the cytosolic Ca2+ concentration, accumulated during anaerobic conditions. In the present study, spontaneous development of hypercontracture upon reoxygenation was prevented by prolongation of acidosis for the time needed by the cells to establish a normal cytosolic Ca2+ control.19 After overcoming this initial period of reoxygenation, susceptibility of the cells to hypercontracture was tested. The result is that reoxygenated cardiomyocytes respond to Ca2+ overload more strongly than do normal cells. They develop hypercontracture at lower cytosolic Ca2+ concentrations than normally needed; ie, their SH is increased.
Under oxygen and substrate depletion, cardiomyocytes develop a partial shortening after an extensive loss of their energy reserves due to the onset of rigor. To achieve the aim of the present study, it was necessary to determine whether this partial cell shortening is responsible for the increase in SH found upon reoxygenation, because the distortion of cytoskeletal structures produced by partial cell shortening could already weaken the cytoskeletal anchoring of the myofibrils and thereby favor hypercontracture in the reoxygenated cells. It seemed also conceivable that the extensive loss of ATP, responsible for rigor shortening, represents itself as the immediate cause for structural changes underlying increased SH. Both these hypotheses were invalidated by the finding that in cells reoxygenated 5 minutes after rigor shortening, the susceptibility to hypercontracture was not increased. An increase in SH in reoxygenated cells was observed only if simulated ischemia had been extended to times ≥15 minutes after rigor shortening.
It must be considered whether the differences in susceptibility to hypercontracture observed in the present study can be attributed to differences in ATP contents of cardiomyocytes under the various experimental conditions. Altschuld et al11 observed that in permeabilized cardiomyocytes incubated with Mg2+-ATP solutions in the absence of CrP, the apparent Ca2+ sensitivity of contractile activation may be increased when the ATP concentration is gradually lowered from a physiological level to one causing Ca2+-independent rigor shortening. Since CrP was absent in their study and permeabilized cells were used, a direct extrapolation of their observations to the state of energy in intact cells with high CrP contents is not possible. In the present study, anoxia/reoxygenation experiments were carried out with or without cantharidin. This led to different susceptibilities for hypercontracture, but ATP and CrP contents were the same under both experimental conditions. In normoxic experiments with and without the presence of BDM, susceptibility to hypercontracture was also different, and again ATP and CrP contents were identical. These findings render it unlikely that the variations in susceptibility to hypercontracture are due to variations of the cellular state of energy.
Under the chosen conditions of simulated ischemia, cells developed intracellular cytosolic Ca2+ overload and acidosis. We tested whether either of these factors was causally related to the development of increased susceptibility to hypercontracture. First, the role of cytosolic Ca2+ overload was investigated using a protocol of simulated ischemia with a Ca2+-depleted medium. Under this protocol, the cells developed rigor shortening but did not thereafter accumulate Ca2+ within their cytosol. When the cells were reoxygenated 30 minutes after rigor shortening (as in simulated ischemia with extracellular Ca2+ present), their susceptibility to hypercontracture was found to be increased to the same degree as in cells that had accumulated Ca2+ during simulated ischemia. Thus, in spite of the potentially deleterious effects of high cytosolic Ca2+ on some aspects of cell integrity, Ca2+ overload is not the cause for the changes underlying increased susceptibility to hypercontracture in the reoxygenated cardiomyocyte. Second, the role of acidosis was investigated by exposing cells to a time course and an extent of intracellular acidification (pHi 6.5) comparable to those observed after simulated ischemia. These experiments showed that acidosis alone is not a sufficient cause to provoke increased susceptibility to hypercontracture.
Furthermore, the role of protein dephosphorylation was tested with the use of cantharidin, an inhibitor of protein phosphatases 1 and 2A.20 It was found that the susceptibility of cardiomyocytes to hypercontracture was significantly reduced by treatment with cantharidin during simulated ischemia. Reoxygenated cantharidin-treated cells developed maximal hypercontracture at the same Ca2+ concentration as found in control cells. Treatment of normoxic cardiomyocytes with cantharidin had no effect on their susceptibility to hypercontracture. Therefore, the protective effect of cantharidin on SH must be due specifically to its action during simulated ischemia. Since cantharidin acts as an inhibitor of protein phosphatases, it seems to be the dephosphorylation of proteins in the ischemic cardiomyocytes that elicits an increase in SH. That an agent with an opposite mode of action (ie, the chemical phosphatase BDM) is able to induce an increase in SH in cells not exposed to simulated ischemia is in agreement with this conclusion. It was beyond the scope of the present study to identify the dephosphorylated proteins responsible for the changes in the cells' susceptibility to hypercontracture.
Susceptibility to hypercontracture is a complex phenomenon that must not be mistaken for Ca2+ sensitivity of the myofibrils. Cardiomyocytes develop hypercontracture if (1) the supply of energy is sufficient for contractile activation, (2) a large contractile force is generated because the cytosolic Ca2+ concentration is high and/or the Ca2+ sensitivity of the myofibrils is increased, and (3) this force overrides the reversible deformability of the cytoskeleton. In the protocol applied in the present study, cardiomyocytes were forced into hypercontracture under well-energized conditions by defined steps of cytosolic Ca2+ overload. The increase in SH found in cardiomyocytes reoxygenated after simulated ischemia may thus be due to either an increase in myofibrillar Ca2+ sensitivity or a reduction in structural stiffness. According to the data of the present study, these possible causes cannot be differentiated. Data from the literature indicate that myofibrillar Ca2+ sensitivity is reduced in postischemic myocardium.26 27 This is supposed to be one of the causes of the phenomenon called “stunned myocardium.”28 Structural fragility is likely to increase under ischemic conditions.29 Data obtained by Armstrong and Ganote30 also indicate that protein dephosphorylations can cause structural fragility. However, they tested a fragility phenomenon different from hypercontracture, namely, the propensity of cells to lyse upon osmotic shock. It is an open question whether the protein dephosphorylations responsible for that phenomenon are the same as those favoring hypercontracture.
In conclusion, the present study has shown that simulated ischemia increases the susceptibility of cardiomyocytes to hypercontracture. Activation of protein phosphatases seems to be part of the pathomechanisms. Therefore, reoxygenated cardiomyocytes are jeopardized not only by Ca2+ overload but also by an enhanced responsiveness to Ca2+ overload.
This work was supported by the BIOMED-2 program of the European Union and grant Si 618/1-1 of Deutsche Forschungsgemeinschaft. The technical help of H. Holzträger and D. Schreiber is gratefully acknowledged.
- Received July 22, 1996.
- Accepted October 18, 1996.
Hohl C, Ansel A, Altschuld R, Brierley GP. Contracture of isolated rat heart cells on anaerobic to aerobic transition. Am J Physiol. 1982;242:H1022-H1030.
Stern MD, Chien AM, Capogrossi MC, Pelto DJ, Lakatta EG. Direct observation of the ‘oxygen paradox’ in single rat ventricular myocytes. Circ Res. 1985;56:899-903.
Siegmund B, Zude R, Piper HM. Recovery of anoxic-reoxygenated cardiomyocytes from severe Ca2+ overload. Am J Physiol. 1992;263:H1262-H1269.
Siegmund B, Ladilov YV, Piper HM. Importance of sodium for recovery of calcium control in reoxygenated cardiomyocytes. Am J Physiol. 1994;267:H506-H513.
Ganote C, Armstrong S. Ischaemia and the myocyte cytoskeleton: review and speculation. Cardiovasc Res. 1993;27:1387-1403.
Siegmund B, Koop A, Klietz T, Schwartz P, Piper HM. Sarcolemmal integrity and metabolic competence of cardiomyocytes under anoxia-reoxygenation. Am J Physiol. 1990;258:H285-H291.
Borzak S, Kelly RA, Krämer BK, Motoba Y, Marsh JD, Reers M. In situ calibration of fura-2 and BCECF fluorescence in adult rat ventricular myocytes. Am J Physiol. 1990;259:H973-H981.
Altschuld RA, Wenger WC, Lamka KG, Kindig OR, Capen CC, Mizuhira V, Vander Heide RS, Brierley GP. Structural and functional properties of adult rat heart myocytes lysed with digitonin. J Biol Chem. 1985;260:14325-14334.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.
Lamprecht W, Stein P, Heinz F, Weisser H. Creatin-phosphat. In: Bergmeyer HU, ed. Methoden der enzymatischen Analyse. Weinheim, FRG: Verlag Chemie; 1974:1825-1829.
Allshire A, Piper HM, Cuthbertson KS, Cobbold PH. Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation. Biochem J. 1987;244:381-385.
Ladilov YV, Siegmund B, Piper HM. Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange. Am J Physiol. 1995;268:H1531-H1539.
Neumann J, Herzig S, Boknik P, Apel M, Kaspareit G, Schmitz W, Scholz H, Tepel M, Zimmermann N. On the cardiac contractile, biochemical and electrophysiological effects of cantharidin, a phosphatase inhibitor. J Pharmacol Exp Ther. 1995;274:530-539.
Holmstedt B. Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol Rev. 1959;11:567-688.
Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1-9.
Hajjar RJ, Gwathmey JK. Direct evidence of changes in myofilament responsiveness to Ca2+ during hypoxia and reoxygenation in myocardium. Am J Physiol. 1990;259:H784-H795.
Kusuoka H, Kortsune Y, Chacko VP, Weisfeldt ML, Marban E. Excitation-contraction coupling in postischemic myocardium: does failure of activator Ca2+ transients underlie stunning? Circ Res. 1990;66:1268-1276.