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(Circulation Research. 1995;76:1071-1078.)
© 1995 American Heart Association, Inc.

Articles

Role of Calcium-Activated Neutral Protease (Calpain) in Cell Death in Cultured Neonatal Rat Cardiomyocytes During Metabolic Inhibition

Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993.

Douwe E. Atsma, E.M. Lars Bastiaanse, Anastazia Jerzewski, Lizet J.M. Van der Valk, Arnoud Van der Laarse

From the Department of Cardiology, University Hospital, Leiden, Netherlands.

Correspondence to D.E. Atsma, MD, Department of Cardiology, C5-P, University Hospital, PO Box 9600, 2300 RC Leiden, Netherlands.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Calcium-activated neutral protease (CANP), also known as calpain, has been implicated in the development of cell death in ischemic hearts. CANP is thought to be activated by the calcium overload that develops during ischemia. We studied the involvement of CANP in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition (5 mmol/L NaCN+10 mmol/L 2-deoxyglucose). First, we isolated CANP using ion exchange and affinity chromatography. Then the efficacy of the CANP inhibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CANP activity was tested with the use of fluorescently labeled ß-casein as a substrate. The IC₅₀ for the inhibitors was between 2.1 and 56 µmol/L. Uptake of the inhibitors by intact cells was assessed with the use of ^99mTc-radiolabeled inhibitors. The calculated intracellular inhibitor concentrations were sufficiently high to yield substantial inhibition of intracellular CANP activity. Intracellular CANP activity was measured directly with the use of the cell-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin. During metabolic inhibition, intracellular CANP activity was increased compared with control incubation. The time course of CANP activation was compatible with that of the rise in [Ca²⁺]_i, as measured by fura 2 and digital imaging fluorescence microscopy. Calpain I inhibitor and leupeptin inhibited intracellular CANP activity both during metabolic inhibition and control incubation, whereas E64 did not. Despite their substantial inhibition of intracellular CANP activity, calpain I inhibitor and leupeptin did not attenuate cell death during metabolic inhibition. We therefore conclude that intracellular CANP in cardiomyocytes is activated during metabolic inhibition, but it does not play a major role in the development of cell death.

Key Words: myocytes • cell death • metabolic inhibition • calcium-activated neutral protease • protease inhibitors

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanism of myocardial cell death during ischemia, anoxia, and metabolic inhibition is still unclear. The rise in [Ca²⁺]_i in cardiomyocytes observed under these conditions is considered to be a pivotal event in the process of cell death.^{1 2} The resulting calcium overload is thought to massively activate dormant [Ca²⁺]_i-activated enzymes, such as calcium-activated neutral protease (CANP), leading to structural damage of the sarcolemmal membrane and eventually cell death.^{3 4}

The ubiquitously distributed thiol protease CANP (Enzyme Commission 3.4.22.17) exists in two forms, CANP I and CANP II, which are activated at micromolar and millimolar concentrations of Ca²⁺, respectively. The physiological significance of the two isoforms of CANP is not clear.

CANP is thought to play a harmful role in a variety of pathological states, such as in Duchenne's muscular dystrophy⁵ ; neurodegenerative conditions including Alzheimer's disease,⁶ ischemia,⁷ and multiple sclerosis⁸ ; toxic⁹ and anoxic^{10 11} injury in hepatocytes; oxidative stress in endothelial cells¹² ; and the development of cataract.¹³

Several studies have addressed the involvement of CANP in anoxic or ischemic cell death in heart cells. In these studies conflicting results have been reported as to the activation of CANP during anoxia or ischemia and the effects of inhibitors of CANP on cell injury. Increased CANP activity was found in regionally ischemic rat hearts in vivo,¹⁴ in Langendorff-perfused rat hearts after ischemia/reperfusion,¹⁵ and in anoxic cultured rat cardiomyocytes.¹⁶ In contrast, decreased CANP activity was found in ischemic dog heart.¹⁷ In addition, a CANP inhibitor temporarily protected the ischemic heart in one study,¹⁷ whereas in another study no effect of inhibitors of CANP on infarct size in ischemic rat hearts was found.¹⁸

In these studies the activity of myocardial CANP was measured post hoc after the isolation of the enzyme, which procedure could potentially influence the measured enzyme activity¹⁹ and therefore may not accurately reflect the intracellular CANP activity.

Therefore, in our study we used the membrane-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin (Suc-Leu-Leu-Val-Tyr-AMC)²⁰ to investigate the role of CANP in the development of cell injury in intact cardiomyocytes during metabolic inhibition. This approach allows the serial monitoring of CANP activity during the course of the experiment. In addition, we assessed the efficacy of inhibitors of CANP, namely, calpain I inhibitor, leupeptin, and E64, to inhibit CANP activity both in vitro and in intact cells and determined their ability to reduce cell death in cardiomyocytes during metabolic inhibition. It was found that CANP activity is increased during metabolic inhibition and that it can be inhibited in intact cells by calpain I inhibitor and leupeptin. Since this inhibition of CANP does not lead to improved viability of the cardiomyocytes during metabolic inhibition, we conclude that CANP does not play a major role in the development of cell death in cardiomyocytes during metabolic inhibition.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Calpain I inhibitor, leupeptin, and E64 were purchased from Boehringer Mannheim. DEAE cellulose and phenyl-sepharose were obtained from Whatman. ß-Casein, fluorescamine, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), fluorescein isothiocyanate (FITC), Suc-Leu-Leu-Val-Tyr-AMC, and 7-amido-4-methyl-coumarin (AMC) were obtained from Sigma Chemical Co. Fura 2-AM was purchased from Molecular Probes. All other reagents used were of analytical grade.

Cell Culture
The myocyte cultures were prepared with the use of the method described by Van der Laarse et al.²¹ Briefly, hearts of 2-day-old Wistar rats were dissected and transferred to a solution containing the following (mmol/L): NaCl 137, KCl 5, Na₂HPO₄ 0.4, KH₂PO₄ 0.4, glucose 5.5, HEPES 20, and phenol red 0.5 mg/L (pH 7.4). The ventricles were minced into small fragments and dissociated with the use of collagenase (type I CLS, Worthington) during two periods of 20 minutes in a shaking water bath at 37°C. After centrifugation at 50g for 15 minutes, the cells were suspended in a culture medium consisting of Ham's F-10 medium (Flow Laboratories) supplemented with 10% fetal bovine serum (Flow Laboratories) and 10% horse serum (Flow Laboratories). The cells were plated in 60-mm culture dishes (Becton-Dickinson) in a density of 4.2x10⁶ cells per dish. Myocytes were separated from nonmuscle cells with the use of a selective adhesion technique.²² After 45 minutes the medium, enriched in myocytes, was transferred to either 24-well plates or 35-mm culture dishes, some of which contained 25-mm round glass coverslips. The myocyte cultures were kept in a humidified incubator (37°C) with an atmosphere of 95% air and 5% CO₂. Culture medium was changed after 3 hours and after 48 hours. After 3 days the monolayers of spontaneously beating myocytes were used for the experiment.

CANP Isolation
To test whether CANP was present in our model, we isolated the enzyme using the method described by Spalla et al.²³ Briefly, ventricular myocytes obtained from 5-day-old Wistar rats were homogenized in a basic buffer containing (mmol/L) MOPS 20, EGTA 1, EDTA 1, and 2-mercaptoethanol 3 (pH 7.2) supplemented with 10 mmol/L NaCl. After centrifugation of the homogenate at 12 000g for 30 minutes, the supernatant was collected and the pellet resuspended and centrifuged as before. After the supernatants were pooled, the mixture was loaded on a 2.5x10-cm DEAE cellulose column. After elution with a NaCl gradient from 0.01 to 0.5 mol/L in basic buffer, the fractions containing CANP I and CANP II were identified on the basis of 280 nm absorbance.²³ To separate CANP I from its endogenous inhibitor calpastatin, the CANP I pool was adjusted to 0.25 mol/L NaCl and loaded on a 1x10-cm phenyl-sepharose column. After the column was washed with a gradient of basic buffer supplemented with 0.25 mol/L NaCl and basic buffer supplemented with 20% ethylene glycol, CANP I was eluted from the column with basic buffer containing 20% ethylene glycol alone. The CANP I and CANP II fractions were then concentrated with the use of membrane filters (Amicon), and protein concentration in the fractions was determined by means of the method of Lowry et al.²⁴

In Vitro CANP Activity
The proteolytic activity of the isolated CANP was studied with the use of a sensitive kinetic in vitro protease assay described by Farmer and Yuan, with ß-casein as a substrate.²⁵ The ß-casein was double labeled with DACM and FITC, yielding a fluorescence donor and acceptor pair. In the intact molecule, the DACM fluorescence at 470 nm, upon excitation at 385 nm, is quenched by the adjacent FITC and is therefore very low. However, degradation of the FITC/DACM/ß-casein complex by CANP separates the two moieties and releases the quenching of DACM fluorescence by FITC. This results in an increase of DACM fluorescence in proportion to CANP proteolytic activity.

To a mixture of 980 µL of a 20 mmol/L Tris-HCl buffer (pH 7.0) and 20 µL casein substrate solution (1.34 mg protein per milliliter), either 400 µL CANP I solution (0.87 mg protein per milliliter) or 400 µL CANP II solution (0.79 mg protein per milliliter) was added. The increase in DACM fluorescence was measured in a spectrofluorometer (Perkin-Elmer LS-3) for at least 5 minutes under constant stirring at a temperature of 37°C. CANP proteolytic activity was expressed as the increase of fluorescence in arbitrary units per minute. The calcium dependency of the CANP proteolytic activity was demonstrated by the use of the Tris-HCl buffer supplemented either with 5 mmol/L CaCl₂ to measure calcium-dependent proteolysis or with 1 mmol/L EGTA to measure calcium-independent proteolysis.

Inhibition of CANP In Vitro
The effect of the protease inhibitors calpain I inhibitor, leupeptin, and E64 on the proteolytic activity of CANP was tested in vitro by means of the assay described above. To this end, the inhibitors were added to the reaction mixture of the proteolytic assay in a final concentration between 0.1 and 200 µmol/L, and the reduction in proteolytic activity was expressed as the percent inhibition compared with the proteolytic activity in the absence of the inhibitors. Calpain I inhibitor was dissolved in ethanol, E64 in 50% ethanol, and leupeptin in water. Control experiments showed that the final concentrations of ethanol used (<0.25%) did not influence the assay.

Cellular Protease Inhibitor Uptake
To study whether the protease inhibitors calpain I inhibitor, leupeptin, and E64 were taken up by cultured myocytes in sufficiently high quantities to yield inhibitory effects, the inhibitors were labeled with ^99mTc using the method previously described by Pauwels et al.²⁶ Purity of the labeled compounds was verified by high-performance liquid chromatography before further use, which procedure also allowed the calculation of the specific radioactivity of the radiolabeled inhibitors. The myocardial cells were incubated in a cell incubation solution containing (mmol/L) NaCl 140, KCl 4, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 0.44, Na₂HPO₄ 0.34, NaHCO₃ 21, and sodium pyruvate 5 (pH 7.4) in an atmosphere of 95% air and 5% CO₂ at 37°C. Then 10 µmol/L ^99mTc-leupeptin, 10 µmol/L ^99mTc–calpain I inhibitor, or 10 µmol/L ^99mTc-E64 was added, and the cultures were incubated for 2 hours at 37°C. After this period the medium was separated from the cells, and the radioactivity in the cells and in the incubation medium was counted in a well-type counter (Scalar Ratemeter R4). After we determined the uptake of radioactivity by the cells, the intracellular protease inhibitor concentration was calculated with the use of the specific radioactivity values determined after high-performance liquid chromatography.

Metabolic Inhibition of Cardiomyocytes
Metabolic inhibition of cardiomyocyte cultures was imposed by incubating the cells with 5 mmol/L NaCN and 10 mmol/L 2-deoxyglucose in a HEPES balanced salt solution (HBSS) containing (mmol/L) NaCl 125, KCl 5, MgSO₄ 1, KH₂PO₄ 1, CaCl₂ 2.5, NaHCO₃ 10, HEPES 20, and sodium pyruvate 5 (pH 7.4) for 5 hours at 37°C. Before the start of metabolic inhibition, each culture was allowed to equilibrate in HBSS for 1 hour at 37°C.

[Ca²⁺]_i
To record the changes in [Ca²⁺]_i in the myocytes during metabolic inhibition and control incubation, [Ca²⁺]_i was measured with the use of the fluorescent calcium indicator fura 2.²⁷ The cardiomyocytes, cultured on round glass coverslips, were loaded with fura 2 by incubation with 2 µmol/L fura 2-AM in 1 mL HBSS for 30 minutes at 37°C. After the glass coverslip was rinsed three times, it was mounted in a culture dish containing two compartments, previously described by us.²⁸ With the two-compartment culture dish, the two halves of a single cell culture grown on a standard coverslip can be exposed to different treatments simultaneously, allowing the effect of one treatment to be compared with that of the other treatment in the same culture. This arrangement circumvents the natural variability that might exist between different individual cultures. In addition, by simultaneously conducting two experiments per dish, the time and the number of cultures needed for the experiment are essentially halved. After the coverslip was mounted with the culture in the two-compartment culture dish, each compartment of the dish was incubated with 500 µL HBSS at 37°C.

The cells were studied by means of digital imaging fluorescence microscopy, as described in detail previously.²⁸ The fluorescence microscope consists of an inverted microscope body (Leitz Diavert) equipped with a x20 fluorite objective (Nikon) and a mercury light source (HBO-100, Osram). A filter wheel (Sutter Instruments) allows the selection of excitation filters of 340 nm or 380 nm. Emission fluorescence is led through a 490-nm high-pass filter and is imaged by a high-sensitivity SIT camera (Hamamatsu C2400-08). The resulting video signal is digitized by a frame-grabber board (PCVISIONplus, Imaging Technologies Inc) in a PC-AT 486 computer. Spatial resolution of the images is 256x256 pixels, with an 8-bit intensity resolution. Every 5 minutes, 16 images of each wavelength were averaged to improve the signal-to-noise ratio. This was achieved within 3 seconds, minimizing photo bleaching of the fura 2. The images were processed and analyzed with the use of dedicated image processing software (TIM, Difa). After the subtraction of the background fluorescence, the 340-nm image was divided by the 380-nm image on a pixel-by-pixel basis to yield a ratio image. The software allows the analysis of individual cells. Statistical parameters (mean, median, SD) were calculated by the software and used to calculate [Ca²⁺]_i.

Calibration of Fura 2 Fluorescence
At the end of the experiment, 10 µmol/L of the Ca²⁺ ionophore ionomycin (Boehringer Mannheim) was added to obtain the highest obtainable ratio value, R_max. Then 20 mmol/L EGTA was added to obtain the lowest obtainable ratio value, R_min. Calculation of [Ca²⁺]_i was then performed by means of the formula described by Grynkiewicz et al.²⁷

Lactate Dehydrogenase Release
In all experiments, cell death was quantified by measuring the lactate dehydrogenase (LDH) activity released from the cardiomyocytes into the medium. To this end, 15-µL samples of the medium were taken at the indicated time points, and the LDH activity in the samples was measured with the use of a photometric assay (1442597, Boehringer Mannheim). At the end of the experiment the cells were lysed with the use of 0.1% Triton X-100, and total LDH activity in the cell culture, ie, in cells plus medium, was determined. After correction for the changes in medium volume during the course of the experiment, LDH activity in the medium samples was expressed as percentage of total LDH activity.

Effect of CANP Inhibitors on Cell Death During Metabolic Inhibition
To study the effect of calpain I inhibitor, leupeptin, and E64 on cell death during metabolic inhibition, cardiomyocyte cultures in 24-well plates were preincubated with HBSS containing either 0 (control), 2.5, 5, or 10 µmol/L of the inhibitors for 2 hours at 37°C. Then the medium was changed and metabolic inhibition was initiated. The CANP inhibitors were present during the entire experiment. In separate control experiments, the effect of the inhibitors on cell viability during incubation without metabolic inhibition was determined. In the absence of metabolic inhibition, none of the inhibitors induced LDH release in concentrations 10 µmol/L. However, E64 caused LDH release at concentrations >15 µmol/L, whereas calpain I inhibitor and leupeptin failed to induce LDH release in concentrations up to 80 µmol/L (data not shown).

Intracellular CANP Activity
To study the involvement of CANP in cell death in more detail, the activity of CANP in intact cells was assessed with the use of the cell-permeable synthetic fluorogenic substrate for CANP, Suc-Leu-Leu-Val-Tyr-AMC,²⁰ previously used to measure CANP activity in intact liver cells.^{10 11} The intact substrate exhibits little fluorescence at 430 nm upon excitation at 360 nm. However, specific proteolysis of the substrate by CANP liberates the fluorescent AMC group, leading to an increase of its fluorescence that is proportional to the proteolytic activity of CANP.

Cardiomyocyte cultures in 35-mm culture dishes were incubated with 3 mL HBSS containing either 5 mmol/L NaCN and 10 mmol/L 2-deoxyglucose (metabolic inhibition) or no addition (control). Immediately after the start of the metabolic inhibition or control incubation, Suc-Leu-Leu-Val-Tyr-AMC (20 µmol/L) was added to each culture dish, and after an equilibration period of 3 minutes, a sample of 400 µL was taken (time [t]=0). Subsequent samples were taken after 30, 60, 90, and 120 minutes, and AMC fluorescence was measured in a spectrofluorometer (Hitachi F4500). Control experiments showed that the concentration of Suc-Leu-Leu-Val-Tyr-AMC used was adequate and was not a limiting factor in the determination of CANP activity. In addition, autofluorescence of the medium was negligible during the entire experiment.

It would be expected that CANP, if it plays a causal role in cell death during metabolic inhibition, is activated before the actual onset of cell death. To draw a firm conclusion concerning the time course of CANP activation during metabolic inhibition, we found that it was unavoidable to select a subpopulation from the cultures used. Preliminary experiments showed that after the onset of cell death an additional steep increase in CANP activity takes place, possibly due to the interaction of cellular CANP with the Ca²⁺-rich medium. This phenomenon would make it difficult to discern increased CANP activity in still-intact cells from the increased CANP activity in already irreversibly injured cells, hampering the correct identification of the time course of CANP activation.

To resolve this problem, we also measured in each culture cell death by LDH release (see above) in addition to CANP activity to ensure that the CANP activity was from viable cardiomyocytes. When LDH release in a culture during metabolic inhibition exceeded the LDH release in control cultures at the corresponding time point by >2 SDs, the CANP activity data were discarded. At t120 minutes all cultures in the metabolic inhibition group exceeded this limit. After correction for the changes in medium volume during the course of the experiment, the AMC concentration in the sample was calculated by comparing the measured fluorescence with a calibration curve made with various known concentrations of AMC.

Effect of CANP Inhibitors on Intracellular CANP Activity
To test whether calpain I inhibitor, leupeptin, and E64 are able to inhibit CANP proteolytic activity in intact heart cells during control incubation and during metabolic inhibition, the cultures were preincubated in HBSS with 10 µmol/L of each inhibitor for 2 hours at 37°C. Then the medium was changed to HBSS with (metabolic inhibition) or without (control) 5 mmol/L NaCN and 10 mmol/L 2-deoxyglucose, and again 10 µmol/L of each inhibitor was added. After the addition of 20 µmol/L Suc-Leu-Leu-Val-Tyr-AMC, CANP activity was measured for 2 hours as described above.

Statistical Analysis
All results are expressed as mean±SD. Statistical analysis was performed with the use of ANOVA, followed by the Bonferroni t test when appropriate. Differences were considered significant at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Vitro Protease Assay
Proteolytic activity of CANP I and CANP II isolated from neonatal rat heart, as measured by means of the protease assay with FITC/DACM/ß-casein as a substrate, is shown in Fig 1. In the presence of 5 mmol/L Ca²⁺, a rapid and linear increase in DACM fluorescence was recorded for both isoforms for at least 20 minutes, whereas in the absence of Ca²⁺ no degradation of the casein substrate occurred. These results show that calcium-activated proteolytic activity is present in neonatal rat heart.

View larger version (20K): [in this window] [in a new window]   Figure 1. Graph showing proteolytic activity of calcium-activated neutral protease I (CANP I; squares) and calcium-activated neutral protease II (CANP II; circles), isolated from neonatal rat myocardium, in the presence (filled symbols) or absence (open symbols) of 5 mmol/L Ca2+, measured in vitro with the use of fluorescently labeled ß-casein as a substrate. Proteolytic degradation of the substrate led to an increase in fluorescence, expressed in arbitrary fluorescence units (A.F.U.).

In Vitro Efficacy of CANP Inhibitors
The potency of calpain I inhibitor, leupeptin, and E64 to inhibit CANP proteolytic activity in vitro was also tested with the FITC/DACM/ß-casein substrate. All three inhibitors were able to inhibit CANP I and CANP II (Fig 2). CANP I activity was half-maximally inhibited at 2.1, 5.9, and 8.8 µmol/L of calpain I inhibitor, leupeptin, and E64, respectively. Complete inhibition of CANP I was achieved with 45, 65, and 168 µmol/L, respectively. For CANP II proteolytic activity, half-maximal inhibition was observed at 56 µmol/L calpain I inhibitor, 4.6 µmol/L leupeptin, and 6.2 µmol/L E64, whereas complete inhibition was obtained at 240, 103, and 101 µmol/L of the inhibitors, respectively.

View larger version (23K): [in this window] [in a new window]   Figure 2. Graphs showing inhibition in vitro of the proteolytic activity of isolated calcium-activated neutral protease I (CANP I; top) and calcium-activated neutral protease II (CANP II; bottom) by various concentrations of the protease inhibitors calpain I inhibitor (), leupeptin (), and E64 ().

Cellular Uptake of Radiolabeled Protease Inhibitors
To test the ability of calpain I inhibitor, leupeptin, and E64 to enter the cells, we measured the uptake of the radiolabeled inhibitors. After incubation with 10 µmol/L inhibitor in 500 µL medium for 2 hours, 13±3% of calpain I inhibitor, 15±3% of leupeptin, and 5±2% of E64 were incorporated in the cells (Fig 3). Given a cell volume of 3.3 pL²⁹ and a cell number of 410 000 cells per well,³⁰ the calculated intracellular concentrations of calpain I inhibitor, leupeptin, and E64 were 470, 520, and 150 µmol/L, respectively. Based on the results shown in Fig 2, these concentrations are expected to result in nearly complete inhibition of intracellular CANP activity.

View larger version (42K): [in this window] [in a new window]   Figure 3. Bar graph showing uptake of 99mTc-labeled calpain I inhibitor (inhib.), leupeptin, or E64 by cultured neonatal rat cardiomyocytes after incubation with 10 µmol/L of each inhibitor for 2 hours at 37°C. Values are mean±SD. *P<.05 compared with E64; n=6 to 8.

[Ca²⁺]_i Values
[Ca²⁺]_i in the cardiomyocytes during metabolic inhibition and control incubation is shown in Fig 4. The number of cells analyzed was 53 cells in the control incubation group and 42 cells in the metabolic inhibition group. Cells were from at least five different cultures. During control incubation, [Ca²⁺]_i did not change significantly from the basal value of 82±30 nmol/L during the 120-minute incubation. In contrast, upon metabolic inhibition [Ca²⁺]_i started to rise after 25 minutes from a basal value of 86±24 nmol/L (n=42 cells) and reached the micromolar range after 45 minutes. At 120 minutes, [Ca²⁺]_i had risen to a value of 3278±636 nmol/L.

View larger version (21K): [in this window] [in a new window]   Figure 4. Graph showing [Ca2+]i in cultured neonatal cardiomyocytes during control incubation (; n=53 cells from six different cultures) and during metabolic inhibition (; n=42 cells from five different cultures), measured with the use of fura 2 and digital imaging fluorescence microscopy (see "Materials and Methods"). Values are mean±SD. The increase in [Ca2+]i during metabolic inhibition was significantly higher than during control incubation from t=25 minutes onward (P<.05).

Effect of CANP Inhibitors on Cell Death During Metabolic Inhibition
The effects of calpain I inhibitor, leupeptin, and E64 on cell death during metabolic inhibition are shown in Fig 5. After preloading the cardiomyocytes with 0 (control), 2.5, 5, or 10 µmol/L of each inhibitor and in the presence of the inhibitors in these concentrations during the experiment, the time course of cell death during metabolic inhibition in the treated groups was not different from the untreated group for all three CANP inhibitors. Extension of the preloading period of the inhibitors from 2 hours to 18 hours did not lead to protection of the cardiomyocytes during metabolic inhibition.

View larger version (20K): [in this window] [in a new window]   Figure 5. Graphs showing cumulative release of lactate dehydrogenase (LDH) activity from cultured neonatal rat cardiomyocytes during metabolic inhibition, in the absence (No Addition, ) or presence of 2.5 µmol/L (), 5 µmol/L (), or 10 µmol/L () of the calcium-activated neutral protease inhibitors calpain I inhibitor (Inh) (top), leupeptin (middle), or E64 (bottom). No statistical differences were observed between treated and untreated groups for all three inhibitors (n=4 to 6 triplicate experiments).

Higher concentrations of calpain I inhibitor (40 µmol/L) or leupeptin (80 µmol/L) did not protect the cells from cell death during metabolic inhibition, whereas higher concentrations of E64 (15 µmol/L) accelerated rather than delayed cell death during metabolic inhibition (data not shown).

Intracellular CANP Activity
Invariably, when CANP activity data from all cultures exposed to metabolic inhibition were combined, an increased CANP activity compared with control incubation was observed at all time points from t=30 minutes onward (data not shown). However, as explained in "Materials and Methods," part of this increase in CANP activity was caused by CANP activity from cardiomyocytes that were already grossly damaged. Because this phenomenon would make it impossible to establish the exact time course of CANP activation in intact cells, we measured in each culture LDH release in addition to CANP activity and selected the still viable cardiomyocyte cultures (see "Materials and Methods").

During control incubation, a gradual increase in AMC fluorescence was observed, reflecting basal CANP proteolytic activity (Fig 6A). In contrast, upon metabolic inhibition CANP activity was significantly increased at 60 and 90 minutes after onset of metabolic inhibition, as indicated by a higher rate of fluorescence increase (P<.05) (Fig 6A). Because in the cultures exposed to metabolic inhibition no significant cell death took place at t90 minutes (Fig 6B), the values measured in these cultures at these time points reflect CANP activity in still-intact cells. However, at t=120 minutes, cell death in all metabolically inhibited cultures was higher than in control cultures, and therefore the values measured at t=120 minutes also represent CANP activity in fatally damaged cells.

View larger version (21K): [in this window] [in a new window]   Figure 6. A, Graph showing intracellular proteolytic activity of calcium-activated neutral protease (CANP) in viable cultured neonatal rat cardiomyocytes during control incubation () and during metabolic inhibition (), measured with the use of the membrane-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin (see "Materials and Methods"). Proteolysis of the substrate by CANP led to the liberation of the fluorescent 7-amido-4-methyl-coumarin (AMC) moiety, leading to an increase of its fluorescence that was proportional to the proteolytic activity of CANP. B, Graph showing cumulative release of lactate dehydrogenase (LDH) activity from cultured neonatal rat cardiomyocytes in the same cultures shown in panel A, during control incubation () and during metabolic inhibition (). Values are mean±SD. *P<.05, **P<.01, control incubation vs metabolic inhibition; n13 cultures.

Effect of CANP Inhibitors on Intracellular CANP Activity
Calpain I inhibitor (10 µmol/L) and leupeptin (10 µmol/L) inhibited intracellular CANP activity almost completely during control incubation (Fig 7A). In addition, during metabolic inhibition calpain I inhibitor and leupeptin inhibited intracellular CANP activity to values comparable to basal CANP activity (Fig 7B). The inhibition of the intracellular CANP activity by calpain I inhibitor or leupeptin was significant at all time points compared with control incubation or metabolic inhibition without inhibitors (P<.05). In contrast, E64 (10 µmol/L) did not decrease intracellular CANP activity during either control incubation or metabolic inhibition (Fig 7A and 7B). In addition, at higher concentrations of E64 (40 µmol/L) no inhibition of CANP activity was observed. The increase of the concentrations of calpain I inhibitor and leupeptin up to 40 µmol/L did not cause a further inhibition of the intracellular CANP activity.

View larger version (26K): [in this window] [in a new window]   Figure 7. Graphs showing intracellular proteolytic activity of calcium-activated neutral protease (CANP) in intact cultured neonatal rat cardiomyocytes, measured with the use of the membrane-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin (see "Materials and Methods"). A, Intracellular CANP activity during control incubation in the absence (Control Incubation, ) or the presence of 10 µmol/L of the CANP inhibitors calpain I inhibitor (), leupeptin (), or E64 (). The lower CANP activity in the presence of calpain I inhibitor and leupeptin compared with CANP activity during control incubation was significant at all time points (n=9 to 11 cultures). B, Intracellular CANP activity during metabolic inhibition in the absence (Metabolic Inhibition, ) or the presence of 10 µmol/L of the CANP inhibitors calpain I inhibitor (), leupeptin (), or E64 (). The lower CANP activity in the presence of calpain I inhibitor and leupeptin compared with CANP activity during metabolic inhibition was significant at all time points (n=7 to 9 cultures). AMC indicates 7-amido-4-methyl-coumarin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The role of CANP in ischemic cell death in cardiomyocytes remains unclear. The involvement of CANP in cell death as an effector of irreversible cell injury constitutes an attractive hypothesis because the proteolytic action of CANP is Ca²⁺ dependent, and among its purported substrates are several key cytoskeletal proteins.¹⁹ Thus, massive supraphysiological activation of CANP during ischemia would link the developing calcium overload with the subsequent degradation of the sarcolemmal membrane, thereby causing myocardial cell death.

In our study we used cultured neonatal rat cardiomyocytes as a model. Although developmental differences may exist compared with adult heart cells, the neonatal cardiomyocytes constitute an attractive model in that several key events during metabolic inhibition or ischemia mimic the events occurring in adult heart cells. For example, during energy depletion neonatal and adult cells have comparable time courses of (1) the rise in [Ca²⁺]_i,^{31 32} (2) development of sarcolemmal blebs,^{33 34} and (3) cumulative enzyme release.^{33 35} These findings indicate that the differences in development between neonatal and adult cells have only a slight influence with respect to the aforementioned cellular dysfunction and cell death.

We isolated the two isoforms of CANP, namely, CANP I and CANP II, from neonatal rat heart and demonstrated the Ca²⁺ dependency of the proteolytic activity of both isoforms, using a sensitive kinetic proteolytic assay with a double-labeled fluorescent ß-casein substrate. With the use of this assay, it was established that the CANP inhibitors calpain I inhibitor, leupeptin, and E64 were able to effectively inhibit the proteolytic activity of both CANP isoforms in vitro. Until now, CANP activity has not been directly assessed in intact myocardial cells but instead was measured post hoc after isolation of the enzyme from ischemic or anoxic tissue. Because the isolation procedure itself may induce artifacts leading to a less accurate estimation of intracellular CANP activity,¹⁹ we used the cell-permeant fluorogenic CANP-specific substrate Suc-Leu-Leu-Val-Tyr-AMC to serially measure CANP activity in intact cells during the course of the experiment.

Although with the use of this method it was found that CANP activity was significantly increased as soon as 30 minutes after addition of the metabolic inhibitors when compared with the CANP activity in control cultures, this result proved deceptive. Control experiments showed that after the onset of cell death, CANP activity in cardiomyocyte cultures was greatly increased, most likely because of massive activation of the intracellular CANP by the millimolar concentration Ca²⁺ present in the incubation medium. Because cardiomyocyte cultures contain a heterogeneous population of heart cells, it is to be expected that fairly soon after the start of metabolic inhibition, cell death occurs in a number of cells, which leads to an early rise in measured CANP activity. Because these values of CANP activity do not provide insight into the role of CANP in the development of sarcolemmal damage but rather reflect sarcolemmal damage that has already been inflicted, cell death was assessed concurrently with the determination of CANP activity. In this manner a distinction could be made between CANP activity data obtained from viable cells and those obtained from cells with already gross irreversible sarcolemmal damage. Data from the latter group were discarded. Only through this correction method could a firm conclusion be drawn concerning the time course of CANP activation in cardiomyocytes during metabolic inhibition.

With the use of this correction method, it was found that during metabolic inhibition the intracellular CANP activity was significantly increased 60 minutes and 90 minutes after addition of the metabolic inhibitors when compared with intracellular CANP activity during control incubation.

This time course of CANP activation during metabolic inhibition is consistent with that of the rise in [Ca²⁺]_i under these conditions. Measurement of [Ca²⁺]_i with the use of fura 2 and digital imaging fluorescence microscopy showed that [Ca²⁺]_i starts to rise 25 minutes after onset of metabolic inhibition from the basal value of approximately 80 nmol/L and reaches the micromolar range after 45 minutes. This finding is in agreement with results obtained in other models of metabolic inhibition.^{36 37} Thus, the time course of the rise in [Ca²⁺]_i in our model is compatible with the finding of an increased CANP activity at t=60 minutes and t=90 minutes. The increased CANP activity during metabolic inhibition reported in the present study is in agreement with the rise in CANP activity found by Tolnai and Korecky¹⁴ and Yoshida et al¹⁵ in ischemic rat hearts and Iizuka et al¹⁶ in hypoxic cultured rat cardiomyocytes. The reason Toyo-oka et al¹⁷ found a decreased CANP activity in ischemic dog heart is not clear. It is possible that in this study the previously activated CANP had already been inactivated, and therefore its increased proteolytic activity could no longer be measured.

In addition to an increased CANP activity in ischemic hearts, Tolnai and Korecky¹⁴ also found a concomitant decrease in the activity of the endogenous CANP inhibitor calpastatin, which may well contribute to the observed rise in CANP activity.

In our experiments the protease inhibitors calpain I inhibitor and leupeptin were able to decrease intracellular CANP activity in the cardiomyocytes during both control conditions and metabolic inhibition. Treatment with E64 did not lead to inhibition of CANP activity in control cells or in metabolically inhibited cells. Since E64 does inhibit CANP in vitro, lack of CANP inhibition in intact cells could, at least in part, be due to the inability of the charged E64 molecule to readily cross the sarcolemmal membrane.³⁸ This explanation is corroborated by our finding that radiolabeled calpain I inhibitor and leupeptin were taken up by the cells in quantities three to four times higher than E64. However, even this lower uptake of E64 is expected to result in an intracellular concentration sufficiently high (150 µmol/L) to inhibit CANP activity considerably. An explanation for the failure of E64 to exert its inhibitory action despite its substantial intracellular concentration could be that on entering the cells, E64 is bound by intracellular components that prevent the charged molecule from reaching the CANP molecule.

Having established that CANP activity could be inhibited intracellularly by calpain I inhibitor and leupeptin, we studied the effect of these CANP inhibitors and of E64 on cell death during metabolic inhibition. We found that none of the three inhibitors protected the cardiomyocytes from cell death under these conditions. Even with concentrations of calpain I inhibitor up to 40 µmol/L or of leupeptin up to 80 µmol/L, no protective effect was observed. Higher concentrations of E64 (15 µmol/L) caused an accelerated rather than a delayed cell death. Also, prolongation of the period of loading the cells with the inhibitors up to 18 hours did not alter the outcome of the experiment. These results are in agreement with the observations of Bolli et al,¹⁸ who also did not find a protective effect of the protease inhibitors leupeptin, antipain, or pepstatin on infarct size in ischemic rat hearts. In contrast, Toyo-oka et al¹⁷ did find a beneficial effect of the protease inhibitor NCO-700, previously shown to inhibit CANP,³⁹ on infarct size in ischemic dog heart. The protection provided by NCO-700 in this model (a decrease in infarct size from 14.4% to 10.2%) was only observed at 3 hours after onset of ischemia and was no longer present at 6 hours after onset of ischemia. A possible explanation for the protective effect of NCO-700 could lie in its ability to attenuate myocardial acidosis during ischemia rather than in its CANP-inhibiting properties.⁴⁰

Iizuka et al¹⁶ found a protective effect of the CANP inhibitors calpain I inhibitor and E64-c, a more membrane-permeant derivative of E64, on cell death in hypoxic cultured rat cardiomyocytes. The reason for the discrepancy with our results is not clear, although it could be due to differences in model used, ie, metabolic inhibition versus hypoxia.

In summary, we isolated CANP from neonatal rat heart and demonstrated the efficacy of the protease inhibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CANP activity in vitro. We then showed that CANP in intact cardiomyocytes is activated during metabolic inhibition as measured using the fluorogenic CANP-specific substrate Suc-Leu-Leu-Val-Tyr-AMC. Using fura 2 and digital imaging fluorescence microscopy, we showed that the time course of the rise in [Ca²⁺]_i in the cardiomyocytes during metabolic inhibition is compatible with the observed intracellular CANP activation. We showed that calpain I inhibitor and leupeptin were taken up by the cardiomyocytes, as assessed by the accumulation of radiolabeled inhibitors and by the inhibition of CANP activity in intact cardiomyocytes. Finally, despite substantial inhibition of intracellular CANP activity during metabolic inhibition by calpain I inhibitor and leupeptin, no effect of these inhibitors was observed on cell death during metabolic inhibition. Therefore, we conclude that CANP in cardiomyocytes is activated during metabolic inhibition but that it does not play a major role in the development of cell death.

	Acknowledgments

 
This study was supported by grant 90.089 from the Netherlands Heart Foundation.

Received September 19, 1994; accepted February 28, 1995.

	References

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