RAPID COMMUNICATION |
Immunohistochemical Determination of Cytosolic Cytochrome c Concentration in Cardiomyocytes
Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
Correspondence to: Dr. Willem J. van der Laarse, Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail: wj.vanderlaarse{at}vumc.nl
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Summary |
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(J Histochem Cytochem 53:803807, 2005)
Key Words: pulmonary hypertension heart hypertrophy heart failure cytochrome c release monocrotaline immunohistochemistry
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Introduction |
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Cytochrome c release plays an important role in disease and in reperfusion injury. It may also play a role in the transition from myocardial hypertrophy to chronic heart failure. Exactly how apoptosis is induced and whether it causes the transition from myocardial hypertrophy to heart failure or is a consequence of failure is not known (for recent reviews, see Borutaite and Brown 2003; Saikumar and Venkatachalam 2003
; Weiss et al. 2003
; Duchen 2004
; Halestrap et al. 2004
).
The release of cytochrome c from mitochondria can be studied in various ways: in homogenates via Western blotting, ELISA, or HPLC, on fixed cultured cells via immunocytochemistry, and in live cells by using green fluorescent protein-labeled cytochrome c (Lim et al. 2002; Waterhouse and Trapani 2003
). Although other stages of the apoptotic process can be demonstrated using histochemistry and/or fluorescent dyes (Otsuki et al. 2003
), none of the methods allows the determination of the cytochrome c concentration in the cytoplasm of individual cells. This is important because pharmaceutical intervention of cell death pathways requires an understanding of the mechanisms and consequences of cytochrome c release in individual, identifiable cells in situ. Determination of the cytosolic cytochrome c concentration in cryostat sections is important, because the concentration at which apoptosis is induced is not known (Willingham 1999
).
The demonstration of cytochrome c in cryostat sections is complicated by the fact that small proteins diffuse into the incubation medium or into the fixative. For other small proteins, parvalbumin, and green fluorescent protein, this can be prevented through the use of vapor fixation (Füchtbauer et al. 1991; Jockusch et al. 2003
). We have recently developed a vapor fixation technique to determine the myoglobin concentration in individual muscle cells in cryostat sections (van Beek-Harmsen et al. 2004
). In this report, we show that vapor fixation in combination with immunohistochemistry can be used to determine the cytosolic cytochrome c concentration in individual cardiomyocytes.
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Materials and Methods |
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Fixation of Cytochrome c
Cytochrome c was fixed using the procedure developed for the demonstration of myoglobin described previously (van Beek-Harmsen et al. 2004). Sections were transferred from the freezer to a freeze dryer and dried and warmed to room temperature overnight. This was essential, because condensation of water on cold slides causes a redistribution of soluble molecules in the section. The sections were vapor-fixed for 1 hr in a stainless steel box at 70C. The box contained a beaker with paraformaldehyde, of which 0.3 g evaporated during 1 hr. This fixation delaysbut does not preventdiffusion of small proteins into aqueous media. Therefore, after vapor fixation the sections were fixed in 2.5% glutaraldehyde in 70 mM sodium phosphate buffer, pH 7.4 for 10 min at room temperature (22C25C), and washed with water. The solution was prepared immediately before use from 25% glutaraldehyde in water (G-5882; Sigma, St. Louis, MO) stored at 20C.
Immunohistochemistry of Cytochrome c
A rabbit polyclonal antibody, cytochrome c (H-104), raised against a recombinant protein corresponding to amino acids 1-104 representing full-length cytochrome c of horse, was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used in a 1:100 dilution in PBS (150 mM NaCl, 10 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.6), for 17 hr at 7C. A biotinylated goat-anti rabbit IgG in a dilution of 1:100 in PBS for 30 min at room temperature was used as secondary antibody (BA-1000, Vector Laboratories Inc.). Endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide in PBS for 30 min at room temperature. The biotin was visualized using peroxidase (Vectastain ABC kit), following the instructions of the supplier (Vector Laboratories Inc.). The peroxidase activity was demonstrated with 0.3 mg diaminobenzidine/ml buffer, which consisted of 50 mM TRIS, 10 mM imidazole, and 10 mM sodium azide, adjusted to pH 7.6 with HCl, and 0.003% H2O2. The diaminobenzidine was dissolved in dimethyl sulphoxide (3 mg in 3 µl).
Two control incubations were performed. First, sections were vapor-fixed as described above and were incubated with the incubation medium supplemented with 0.2 mg.ml1 cytochrome c to bind the specific antibodies. Second, sections were preincubated in PBS for 10 min, followed by 10 min in distilled water to remove the salts; they were then dried and vapor-fixed. This treatment is expected to remove all soluble proteins from the cytoplasm, including cytochrome c released from mitochondria.
For calibration cytochrome c from bovine heart (C-2037, Sigma; final concentration 00.8 mM) was dissolved in 15% gelatin in PBS. Blocks were cast in a mold, cooled on ice, and frozen in liquid nitrogen. Sections were cut, stored, and fixed as described above.
Microdensitometry
The sections were studied with a Leica DMRB microscope (Wetzlar, Germany) fitted with calibrated gray filters. Images were obtained using an interference filter at 436 nm with a 40x objective and a monochrome charge-coupled devices camera (Sony XC-77CE; Towada, Japan) connected to an LG-3 framegrabber (Scion; Frederic, MD) in an Apple Macintosh G4 computer, and analyzed using NIH Image (http://rsb.info.nih.gov/nih-image/). Gray values were converted to absorbance values using the gray filters and a third order polynomial fit in the calibrate option of NIH Image.
Areas in the preparation where cardiomyocytes were cut perpendicular to the longitudinal axis were selected for analysis. The boundaries of the cardiomyocytes, which are barely visible in the image files, were checked under the microscope using a 40x phase contrast objective. The sarcomere length was determined using a 100x phase contrast objective in areas of the section where the cardiomyocytes were cut along their longitudinal axis. This value was used to normalize the cross-sectional area to a sarcomere length of 2 µm, assuming that the volume of cardiomyocytes does not change when the cells contract. This normalization allows for comparison of results from different hearts, which may contract to different degrees before freezing. Sections were counterstained with hematoxylin after the absorbance measurements, for an impression of nuclear morphology in cytochrome cpositive cardiomyocytes.
Statistics
Values are given as the mean ± SD. ANOVA was used to determine differences between means.
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Results |
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Figure 3 shows the relationship between the absorbance measured in sections cut from gelatin blocks containing known amounts of cytochrome c. A proportional relationship is found between the absorbance and the cytochrome c concentration. The calibration is reproducible for the lower cytochrome c concentrations (correlation coefficient = 0.94 for cytochrome c concentrations up to 0.5 mM; p<0.001). Inclusion of the higher cytochrome c concentrations does not significantly change the relationship, but results in a smaller correlation coefficient (r = 0.92; p<0.001), reflecting reduced reproducibility at higher concentrations.
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Discussion |
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The method is specific judging from the preincubated sections and incubation in media to which cytochrome c is added. The slightly increased background staining compared with sections that have been preincubated in PBS (A436 = 0.032 0.009 = 0.023) may be explained by endogenous peroxidase activity, for example, of myoglobin, that is not completely quenched, or due to the absorbance of myoglobin itself (Lee-de Groot et al. 1998
). The very weak staining observed in control cardiomyocytes when cytochrome c release is not expected (e.g., in the right ventricular wall of untreated rats) indicates that cytochrome c cannot be demonstrated when it is present only in the intermembrane space of mitochondria. The reason for this is not clear. It may be that the intact outer membrane of the mitochondria shields the binding sites on cytochrome c for the antibodies, allowing binding only to cytochrome c in the mitochondria that have been sectioned, or that the high cytochrome c concentration in the intermembrane space of the mitochondria interferes with proportional antibody binding (e.g., due to steric hindrance).
The expected maximum cytochrome c concentration in the cytoplasm of rat heart after complete release from the mitochondria can be calculated from Dallman and Schwartz (1964), and corresponds to 0.025 mM. Judging from Figure 3, it is impossible to detect this cytochrome c concentration in sections without amplification. Their value is 18 times smaller than the 0.46 mM found in a substantial fraction of cytochrome cpositive cardiomyocytes. The value derived from the results of Dallman and Schwartz (1964)
is also small compared with the estimate of 0.13 mM based on the volume density of mitochondria, 32% of the cardiomyocyte volume (Anversa et al. 1982
) and the mitochondrial cytochrome c concentration of 0.4 mM (Radi et al. 1991
; Phaneuf and Leeuwenburgh 2002
). This estimate is still 3.5 times smaller than the maximum value that we observe. However, estimates of the cytochrome c concentration in the intermembrane space vary considerably (0.55 mM) (Forman and Azzi 1997
). Assuming a cytochrome c concentration of 5 mM in the intermembrane space in heart mitochondria, and that all cytochrome c is released when the cytoplasmic concentration is 0.46 mM, the calculated total intermembrane space in cardiomyocytes is 34% of the volume of mitochondria. This value is not unrealistic (Munn 1969
). Paired biochemical and histochemical determinations of cytochrome c release are required to solve the discrepancy, but are complicated by the fact that the release occurs in a fraction of the cardiomyocytes only.
Based on the results in Figure 2, it can be speculated that mitochondria in hypertrophying rat cardiomyocytes release cytochrome c when the cross-sectional area of the cell increases beyond 600 µm2. This value will then be the maximum cross-sectional area of metabolically functional rat cardiomyocytes. Cytochrome c release in relatively small cardiomyocytes and the large variability of the cross-sectional area of cardiomyocytes in failing hearts may be due to various degrees of cell shrinkage after cytochrome c release. This is a serious possibility, because in rats that do not lose weight after administration of 40 mg/kg monocrotaline, cytochrome c release and the relatively small cardiomyocytes are absent (results not shown).
It has been shown recently that the number of cardiomyocytes in the right ventricular free wall of monocrotaline-treated rats does not change during the development of heart failure (Korstjens et al. 2002, van Beek-Harmsen et al. 2003
). Therefore, it is unlikely that apoptosis is the cause of the transition from myocardial hypertrophy to heart failure in this model of pulmonary hypertension. It remains a possibility that monocrotaline-treated rats die from heart failure as a consequence of cytochrome c release in cardiomyocytes.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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