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DOI: 10.1369/jhc.4C6527.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (7): 803-807, 2005
Copyright ©The Histochemical Society, Inc.

RAPID COMMUNICATION

Immunohistochemical Determination of Cytosolic Cytochrome c Concentration in Cardiomyocytes

Brechje J. van Beek-Harmsen and 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

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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Cytochrome c release from the intermembrane space of mitochondria is one of the triggers of apoptosis. There is no histochemical method available to demonstrate cytochrome c in cryostat sections, possibly because small cytosolic proteins diffuse readily into aqueous fixation media. This report shows that it is possible to demonstrate cytochrome c release in cardiomyocytes in failing myocardium using vapor fixation of cryostat sections and immunohistochemistry. The method is calibrated using sections from gelatin blocks containing known concentrations of cytochrome c. The method is applied to the hypertrophied right ventricular wall of rats in which pulmonary hypertension was induced by monocrotaline. Cytochrome c release is found in a fraction of the cardiomyocytes, leading to a mosaic-staining pattern. Cytochrome c release was found in myocytes over the full range of cross-sectional area (from 1 to 3.9 times control) in the hypertrophied myocardium. Cytosolic cytochrome c concentrations up to 0.4–0.5 mM occur frequently.

(J Histochem Cytochem 53:803–807, 2005)

Key Words: pulmonary hypertension • heart • hypertrophy • heart failure • cytochrome c release • monocrotaline • immunohistochemistry


   Introduction
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CYTOCHROME C IS A 12.4-kDa electron carrier localized in the intermembrane space of mitochondria. Reduced cytochrome c contains an Fe2+ atom and is the substrate of the last reaction in the electron transport chain: the reduction of oxygen by cytochrome c oxidase. Cytochrome c can be released from the mitochondria into the cytosol, for example, during ischemia when the free energy of ATP hydrolysis in the cell is diminished and the cytosolic calcium concentration increases. The released cytochrome c impairs mitochondrial function and can activate caspases, which in turn induce apoptosis.

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 2003Go; Saikumar and Venkatachalam 2003Go; Weiss et al. 2003Go; Duchen 2004Go; Halestrap et al. 2004Go).

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. 2002Go; Waterhouse and Trapani 2003Go). Although other stages of the apoptotic process can be demonstrated using histochemistry and/or fluorescent dyes (Otsuki et al. 2003Go), 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 1999Go).

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. 1991Go; Jockusch et al. 2003Go). 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. 2004Go). 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.


   Materials and Methods
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Pulmonary hypertension in two male Wistar rats (body weights 163 and 200 g) was induced by a subcutaneous injection of 40 mg monocrotaline/kg in saline (Okumura et al. 1992Go). An untreated rat served as control. The institutional ethics committee approved the experiments. Myocardial changes related to oxygen supply, and demand of cardiomyocytes induced by this treatment have been described in some detail elsewhere (Des Tombe et al. 2002Go; van Beek-Harmsen et al. 2003Go,2004Go). Right ventricular heart failure, indicated by a reduction of body weight, occurs 3 to 4 weeks after the injection. After 22 and 24 days, when body weights were 238 and 271 g and decreased by 2.4% per day and 3% per day, respectively, the rats were anesthetized with ether, and the hearts were excised and perfused with cold (10C) Tyrode's solution (in mM: NaCl 120, KCl 5, MgSO4 1.2, Na2HPO4 2, NaHCO3 27, CaCl2 1, 2,3-butanedione monoxime 20, equilibrated with 95% O2/5% CO2, pH 7.6), to remove the blood and to prevent contraction. The right ventricular wall was dissected and frozen in liquid nitrogen. Sections were cut 5 µm thick in a cryostat at –20C, collected on slides coated with Vectabond (Vector Laboratories Inc.; Burlingame, CA), air-dried for 30 min, and stored at –80C until used.

Fixation of Cytochrome c
Cytochrome c was fixed using the procedure developed for the demonstration of myoglobin described previously (van Beek-Harmsen et al. 2004Go). 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 delays—but does not prevent—diffusion 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 (22C–25C), 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.ml–1 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 0–0.8 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 c–positive cardiomyocytes.

Statistics
Values are given as the mean ± SD. ANOVA was used to determine differences between means.


   Results
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Discussion
 Literature Cited
 
Figure 1 shows sections of the right ventricular wall of a pulmonary hypertensive rat and a control rat. In the right ventricular wall of the pulmonary hypertensive rat, a mosaic of positive and negative cardiomyocytes is observed, while the cardiomyocytes in the control myocardium are hardly distinguishable. There is some staining in control myocardium (A436 = 0.032 ± 0.017; n=15), which can be due to mitochondrial cytochrome c exposed by sectioning of intact mitochondria (see below). A comparison of Figure 1B and 1D demonstrates that nuclei in cytochrome c–positive cells are present and are positive for cytochrome c. [Only a fraction of the cardiomyocytes show a nucleus—this is because cell length (~200 µm) is more than 10 times longer than the length of the nucleus.]



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  		Figure 1

Sections from the right ventricular walls of rat hearts. Left: a control rat, incubated with anti–cytochrome c and photographed using a 436-nm light (A), and the same cells after counterstaining with hematoxylin photographed with white light (B). Right: a failing heart incubated with anti–cytochrome c at 436 nm (C) and after counterstaining in white light (D). The arrows indicate cardiomyocyte nuclei. Section thickness = 5 µm. Bar = 50 µm.

 
The absorbance of sections that have been preincubated in PBS before vapor fixation is very low (A436 = 0.009 ± 0.003; n=10), and the differentiation between cytochrome c–positive and –negative cells is lost after preincubation. The differentiation is also lost by addition of cytochrome c to the incubation medium containing the antibodies. The absorbance in cardiomyocytes in these sections (A436 = 0.037 ± 0.007; n=10) is slightly higher (p<0.001) than the absorbance in the preincubated sections. The latter value is similar to the absorbance measured in cardiomyocytes in control myocardium given in Figure 2 (0.032 ± 0.013; n=15).



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  		Figure 2

Cytosolic cytochrome c (arbitrary units) in individual cardiomyocytes related to the cross-sectional area of the cell. The open circles and squares indicate different hearts. The filled square indicates the mean ± SD of control cardiomyocytes [the cross-sectional areas were normalized to a sarcomere length of 2 µm (see text)].

 
Figure 2 shows the relationship between the absorbance due to cytosolic cytochrome c and the cross-sectional area of the cardiomyocytes. Little variation in cardiomyocyte cross-sectional area (207 ± 36 µm2; n=15) is found in the control rat, whereas a wide range of values is found in the monocrotaline-treated rats. The mean cross-sectional area of the cardiomyocytes in the failing hearts is 428 µm2 at a sarcomere length of 2 µm, which is similar to the value reported before (Des Tombe et al. 2002Go). Cytochrome c–positive cells are found over the whole range of cross-sectional areas, from 160 to 780 µm2. There were no cytochrome c–negative cardiomyocytes with cross-sectional areas larger than 600 µm2.

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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  		Figure 3

The relationship between the absorbance measured in sections cut from gelatin blocks containing cytochrome c. The different symbols (square, upward triangle, downward triangle) indicate three separate calibration experiments. The dashed line is the regression line for all data points [A436 = 0.006 + 0.128·(cyt c) (in mM)]. The uninterrupted line is the best fit to values between 0 and 0.5 mM [A436 = 0.004 + 0.149·(cyt c)] and covers the range of absorbance values measured in the sections.

 
After subtraction of the mean absorbance measured in control cardiomyocytes, the absorbance measured in cardiomyocytes in the failing hearts ranges from A436 = 0 to 0.1. Using the calibration in Figure 3, this corresponds to a range of cytoplasmic cytochrome c concentrations of 0 to 0.8 mM. Absorbances of ~0.1 at 436 nm occur frequently. After subtraction of the absorbance in control cardiomyocytes, this value corresponds to a cytosolic cytochrome c concentration of {approx}0.46 mM.


   Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
The results show that the cytosolic cytochrome c concentration can be determined via immunohistochemistry in vapor-fixed sections, and that it is possible to investigate cytochrome c release together with other cellular changes in cells in situ, using double staining procedures or serial sections.

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 ({Delta}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. 1998Go). 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)Go, 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 c–positive cardiomyocytes. The value derived from the results of Dallman and Schwartz (1964)Go 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. 1982Go) and the mitochondrial cytochrome c concentration of 0.4 mM (Radi et al. 1991Go; Phaneuf and Leeuwenburgh 2002Go). 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 (0.5–5 mM) (Forman and Azzi 1997Go). 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 1969Go). 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. 2002Go, van Beek-Harmsen et al. 2003Go). 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.


   Acknowledgments
 
This study was supported by the Netherlands Heart Foundation Grant 00.191.


   Footnotes
 
Received for publication September 21, 2004; accepted February 24, 2005


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Anversa P, Beghi C, Levicky V, McDonald SL, Kikkawa Y (1982) Morphometry of right ventricular hypertrophy induced by strenuous exercise in rat. Am J Physiol 243:H856–861

Borutaite V, Brown GC (2003) Mitochondria in apoptosis of ischemic heart. FEBS Lett 541:1–5[CrossRef][Medline]

Dallman PR, Schwartz HC (1964) Cytochrome c concentrations during rat and guinea pig development. Pediatrics 33:106–110[Abstract/Free Full Text]

Des Tombe AL, van Beek-Harmsen BJ, Lee-de Groot MBE, van der Laarse WJ (2002) Calibrated histochemistry applied to oxygen supply and demand in hypertrophied rat myocardium. Microsc Res Tech 58:412–420[CrossRef][Medline]

Duchen MR (2004) Roles of mitochondria in health and disease. Diabetes 53(suppl 1):S96–S102[Abstract/Free Full Text]

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Füchtbauer E-M, Rowlerson AM, Götz K, Friedrich G, Mabuchi K, Gergely J, Jockusch H (1991) Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogenase activity on frozen sections of rodent muscle. J Histochem Cytochem 39:355–361[Abstract]

Halestrap AP, Clarcke SJ, Javadov SA (2004) Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc Res 61:372–385[Abstract/Free Full Text]

Jockusch H, Voigt S, Eberhard D (2003) Localization of GFP in frozen sections from unfixed mouse tissues: immobilization of a highly soluble marker protein by formaldehyde vapor. J Histochem Cytochem 51:401–404[Abstract/Free Full Text]

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Lee-de Groot MBE, des Tombe AL, van der Laarse WJ (1998) Calibrated histochemistry of myoglobin concentration in cardiomyocytes. J Histochem Cytochem 46:1077–1084[Abstract/Free Full Text]

Lim MLR, Lum M-G, Hansen TM, Roucou X, Nagley P (2002) On the release of cytochrome c from mitochondria during cell death signaling. J Biomed Sci 9:488–506[Medline]

Munn EA (1969) Ultrastructure of mitochondria. In Lima-de-Faria A, ed. Handbook of Molecular Cytology. Amsterdam, North-Holland, 875–913

Okumura K, Kondo J, Shimuzu K, Yoshino M, Toki Y, Hashimoto H, Ito T (1992) Changes in ventricular 1,2-diacylglycerol content in rats following monocrotaline treatment. Cardiovasc Res 26:626–630[Abstract/Free Full Text]

Otsuki Y, Li Z, Shibata M-A (2003) Apoptotic detection methods—from morphology to gene. Progr Histochem Cytochem 38:273–340

Phaneuf S, Leeuwenburgh C (2002) Cytochrome c release from mitochondria in the aging heart: a possible mechanism for apoptosis with age. Am J Physiol Regul Integr Comp Physiol 282:R423–430[Abstract/Free Full Text]

Radi R, Turrens JF, Freeman BA (1991) Cytochrome c–catalyzed membrane lipid peroxidation by hydrogen peroxide. Arch Biochem Biophys 288:118–125[CrossRef][Medline]

Saikumar P, Venkatachalam MA (2003) Role of apoptosis in hypoxic/ischemic damage in the kidney. Semin Nephrol 23:511–521[CrossRef][Medline]

van Beek-Harmsen BJ, Feenstra HM, van der Laarse WJ (2003) Body weight changes explained by lung weight and myocardial HIF 1 alpha expression in monocrotaline-treated rats. Europ J Heart Fail 2(suppl):30–31

van Beek-Harmsen BJ, Bekedam MA, Feenstra HM, Visser FC, van der Laarse WJ (2004) Determination of myoglobin concentration and oxidative capacity in cryostat sections of human and rat skeletal muscle fibres and rat cardiomyocytes. Histochem Cell Biol 121:335–342[CrossRef][Medline]

Waterhouse NJ, Trapani JA (2003) A new quantitative assay for cytochrome c release in apoptotic cells. Cell Death Differ 10:853–855[CrossRef][Medline]

Weiss JN, Korge P, Honda HM, Ping P (2003) Role of the mitochondrial permeability transition in myocardial disease. Circ Res 93:292–301[Abstract/Free Full Text]

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