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J Thorac Cardiovasc Surg 1995;110:89-98
© 1995 Mosby, Inc.

CARDIOPULMONARY BYPASS, MYOCARDIAL MANAGEMENT, AND SUPPORT TECHNIQUES

Ischemia induces early changes to cytoskeletal and contractile proteins in diseased human myocardium

Bad Nauheim, Germany

Supported by a grant from the Minna-James-Heineman Foundation, München, Germany.

Received for publication May 26, 1994. Accepted for publication Nov. 14, 1994. Address for reprints: Jutta Schaper, MD, PhD, Max-Planck-Institute, Department of Experimental Cardiology, Benekestrasse 2, D-61231 Bad Nauheim, Germany.

Abstract

Ischemia is known to produce damage to subcellular organelles, such as nuclei and mitochondria, in myocardial tissue. We tested the hypothesis that during myocardial ischemia various cytoskeletal and contractile proteins also undergo changes. We induced total global ischemia by incubation in buffer of tissue samples from six human left ventricles that were obtained from heart transplant recipients. Samples were removed from the incubation medium at different time intervals and investigated by immunohistochemistry using monoclonal antibodies against myosin, actin, tropomyosin, troponin T, myomesin, desmin, tubulin, and vinculin. The degree of ischemic injury was determined by electron microscopy. Ischemic cardiomyopathic human tissue showed disturbances of the localization pattern of myosin, actin, tropomyosin, and troponin T as early as 10 minutes after the onset of ischemia; this disruption was complete at 20 minutes. Tubulin also started changing at 10 minutes, but complete disruption was only evident after 120 minutes. Desmin and myomesin showed an intermediate response; changes began at 30 to 40 minutes, and disruption was complete at 90 to 120 minutes. Vinculin was most resistant to ischemia. Ultrastructurally, the tissue showed moderate reversible ischemic injury during the entire period of 180 minutes. Measuring the exposure time in seconds allowed quantitation of the intensity of the fluorescence. We reached the following conclusions: (1) Ischemia causes damage to the contractile proteins sooner than to the cytoskeleton and subcellular organelles. (2) Diseased human hearts are extremely susceptible to the effects of ischemia. These findings are important for the situation of induced cardiac arrest in heart operations and for preservation of donor hearts for transplantation. (J THORACCARDIOVASCSURG1995;110:89-98)

The effects of ischemia on animal and human myocardium have been extensively studied during the last three decades by means of functional methods, biochemical methods and electron microscopy. ^1-6 Our own previous studies were mainly concerned with the ultrastructural changes in reversible and irreversible types of injury. ¹ Our findings provided criteria for the evaluation of the quality of cardioprotection during heart operations.

Electron microscopy can estimate structural changes occurring in mitochondria, nuclei, or the T-tubular system. It can also estimate the degree of disorganization of the myofilaments. Finer defects in molecular structure of the contractile apparatus, however, cannot be detected in this way. Furthermore, many components of the contractile machinery and the cytoskeleton cannot be seen at all by electron microscopy because they lack contrast and specific morphologic definition. Other microscopic methods are therefore needed to study morphologic signs of injury of the various cardiac muscle proteins. Immunohistochemistry allows the microscopic visualization of proteins with the aid of specific antibodies. This visualization helps to detect early changes in localization and integrity of these molecules.

Our aim in this study was to investigate ischemic human myocardium for possible early changes in the contractile and cytoskeletal proteins. It will be shown that these proteins undergo degenerative alterations sooner than do subcellular organelles. Myosin and the thin filament complex seem to be extremely susceptible to the effects of ischemia, whereas the components of the cytoskeleton have a higher ischemic tolerance.

METHODS AND MATERIAL

Human myocardium
Cardiac tissues from explanted hearts from six patients who were undergoing cardiac transplantation, four of whom had dilated cardiomyopathy and two of whom had coronary artery disease, were investigated in this study. All patients had New York Heart Association stage IV conditions and an ejection fraction of less than 20%. Several left ventricular myocardial samples from each heart were immediately frozen in liquid nitrogen. These were used as "zero time" control tissues. The remaining tissues were used for experiments.

The remaining explanted heart tissues were incubated in a humid chamber at 20º C for 30, 60, 90, 120, and 180 minutes. Four to six samples per heart and per time interval were removed from the container and frozen in liquid nitrogen. A total of about 20 to 25 tissue pieces from each heart were investigated. In addition, at each time interval small samples (about 1 mm long and 0.5 to 1.0 mm thick) from each heart were fixed for electron microscopy in cold 3% glutaraldehyde buffered with 1 mol/L sodium cacodylate.

Immunohistochemistry
Cryostat sections 4 µm in thickness were fixed with 4% paraformaldehyde at room temperature. They were first stained with hematoxylin and eosin for evaluation of the state of tissue preservation and selection of longitudinal sections. Sections cut transversally or diagonally were reembedded to a longitudinal orientation. Antibodies against myosin, actin, tropomyosin, troponin T, and myomesin were used to detect the contractile proteins. Antibodies to desmin, tubulin, and vinculin were used to detect the proteins associated with the cytoskeleton. The first antibodies are listed in Table I. The detection system was biotinylated donkey antimouse immunoglobulin 1:50 (Amersham International Ltd., Little Chalfont, U.K.), followed by fluorescein isothiocyanate-labeled streptavidin at a dilution of 1:50. Nuclei were stained with 0.0001% propidium iodide according to the modified protocol from Jones and Kniss ⁷ . The sections were viewed in a Leitz Aristoplan (Leitz, Wetzlar, Germany) or Olympus Vanox T (Olympus Optical Co. Ltd., London, U.K.) light microscope equipped with fluorescence filters. Micrographs were taken on Kodak professional 200 ASA color slide film (Eastman Kodak Company, Rochester, N.Y.). All micrographs are reproductions from color slides that are representative of all fields examined. Immunohistochemical results were assessed in several ways: (1) qualitative estimation of the localization of different antibodies, (2) determination of the time at which the first changes in localization and pattern were observed and the time when the changes were maximal, and (3) measurement of the intensity of fluorescent labeling according to the photographic exposure time (in seconds) in the microscope. For these measurements, glass slides were prepared with control (nonischemic) and ischemic tissue to standardize the exposure times measured. All sections from the entire experiment were stained for a given antibody at the same time. Care was also taken to avoid photobleaching and quenching. Ten randomly selected areas of each section were measured, for a total of at least 100 areas per animal. Spot measurements were used.

Electron microscopy
All samples were immersion fixed in 3% buffered glutaraldehyde, postfixed in osmium tetroxide, rinsed in buffer, dehydrated in a graded series of alcohol and propylen oxide, and embedded in Epon (Ladd Research Inc., Burlington, Vt.). Semithin sections were stained with toluidin blue, viewed in the light microscope for artefact free areas and ultrathin 50 nm sections were prepared. These were stained with uranyl acetate and lead citrate and viewed in a Philips EM 201 electron microscope (Philips, Eindhoven, The Netherlands). All micrographs were evaluated for the degree of ischemic injury according to a semiquantitative scoring system, ¹ and these data were compared with the results from immunohistochemical studies.

Western blotting and immunoblots
The frozen tissue samples were homogenized in extraction buffer (0.1 mol/L tromethamine-hydrochloride pH 8.0, 10% sodium dodecyl sulfate, 10 mmol/L ethylenediamine tetraacetic acid, 40 mmol/L dithiothreitol), heated and centrifuged. The supernatant was diluted with the extraction buffer to 10 mg/ml and 60% glycerin and 0.05% bromophenol blue were added. From each sample, a 5 µl portion was analyzed in a 12.5% sodium dodecyl sulfate polyacrylamide gel.

In Western blotting experiments, the proteins were transferred from the gel to a nitrocellulose membrane (02 µm; Schleicher & Schuell GmbH, Dassel, Germany) with 0.8 mA/cm² for 60 minutes. The membrane was treated with 5% nonfat milk powder to block nonspecific binding. The antibodies for vinculin, desmin, myosin, troponin T, actin, tropomyosin, and tubulin were the same as those used in immunohistochemical tests (Table I). They were used in dilutions ranging from 1:50 to 1:500 in 2% phosphate-buffered saline solution. Goat-antimouse immunoglobulin labeled with peroxidase was applied as secondary antibody in a dilution of 1:50 (Boehringer Mannheim GMBH, Mannheim, Germany). Multiple washing steps after each incubation were included in the protocol. Binding of the antibody was detected by means of 4-chloro-1-naphthol and water in triethanolamine buffer. A specific-molecular weight marker (broad range; BioRad, München, Germany) was stained with colloidal gold (BioRad).

RESULTS

Electron microscopy
For the evaluation of the degree of ischemic injury, we used our semiquantitative evaluation system as previously described. ¹ Reversible injury is characterized by an increasing clearing of the mitochondrial matrix and fragmentation of the cristae. Nuclei show clearing and clumping of the chromatin. Reversible injury can be light, moderate, or severe, depending on the degree of progression of the mitochondrial and nuclear changes. Irreversible injury is characterized by the occurrence in the mitochondria of amorphous, dense areas consisting of calcium and lipoprotein deposits. All control tissues had a normal ultrastructural appearance. In human ischemic myocardium, there was light but reversible ischemic injury at 20 minutes. At the end of the experiment, we found moderate but still reversible injury. All ultrastructural data are listed in Table II, which also describes the immunohistochemical results.

Myosin.
Myosin was located exclusively in the A-band in nonischemic human myocardium. This location gave the appearance of small quadrangles of constant length, independent of the state of contraction (Fig. 1, a). Ten minutes after the onset of ischemia, the cross striation pattern in the human hearts was not homogeneous and had partially disappeared (Fig. 1, b). At 20 minutes and subsequent time intervals, the localization of the myosin antibody was completely diffuse and cross striations were absent (Fig. 1, c). In Fig. 1 (d) the negative control preparation obtained by omission of the first antibody is shown. Specific staining is absent.

View larger version (669K): [in this window] [in a new window]   Fig. 1. Staining for myosin (green fluorescence). a, Nonischemic human myocardium shows a regular cross striation for myosin. b, Partial loss of the cross striation at 10 minutes of ischemia. c, Total loss of the specific labeling at 20 minutes of ischemia. d, Negative control with omission of the first antibody shows absence of specific green fluoresence. Yellow dots are lipofuscin granules. Nuclei are stained red.

Tropomyosin.
Staining for tropomyosin in nonischemic cardiomyocytes was seen as fine cross striation corresponding to that of actin. Both antibodies stained both isoforms, and this resulted in a homogeneous labeling pattern. Ischemia produced early changes at 10 minutes, and the fully developed changes were observed at 20 minutes and all subsequent time intervals.

Troponin T.
Troponin T showed a labeling pattern similar to that of actin (Fig. 2, a). The early changes occurred at 10 minutes after ischemia, and the fully developed changes were seen at 20 minutes (Fig. 2, b).

View larger version (288K): [in this window] [in a new window]   Fig. 2. Staining for troponin T (green fluorescence). a, In nonischemic myocardium, the cross striation pattern is regular and intercalated disks remain unstained (arrows). b, Complete absence of a regular cross striation at 20 minutes of ischemia. Nuclei are stained red.

View larger version (391K): [in this window] [in a new window]   Fig. 3. Tubulin staining (green fluorescence). a, In nonischemic myocardium, the staining is intense but not homogeneous, mostly in the longitudinal direction of the cells. b, At 10 minutes of ischemia, the staining is less intense and extremely irregular. c, Complete disturbance of the labeling pattern at 120 minutes. Nuclei are stained red.

View larger version (413K): [in this window] [in a new window]   Fig. 4. Desmin staining (green fluorescence) in human myocardium. a, Nonischemic myocardium shows a regular cross striation but also lack of desmin (arrows) and accumulation in other areas (arrowheads). b, At 30 minutes of ischemia, the cross striation pattern is disturbed. Intercalated disks are clearly labeled (arrow). c, Complete disappearance of the cross striation pattern at 90 minutes of ischemia. Nuclei are stained red.

View larger version (388K): [in this window] [in a new window]   Fig. 5. Myomesin staining (green fluorescence). a, In nonischemic human myocardium, a regular cross striation pattern can be observed. The dark spaces between cells correspond with extracellular fibrotic material. b, Disturbance of the cross striation at 40 minutes of ischemia. c, At 120 minutes of ischemia, the cross striation pattern is totally disturbed. Nuclei are stained red.

View larger version (253K): [in this window] [in a new window]   Fig. 6. Vinculin staining (green fluorescence). a, In nonischemic myocardium, vinculin is localized at the intercalated disks and the lateral sarcolemma. b, Slight reduction of the staining intensity and the intracellular localization of vinculin at 60 minutes, with no changes thereafter until 180 minutes. Nuclei are stained red.

View larger version (50K): [in this window] [in a new window]   Fig. 7. Measurements of the intensity of the fluorescent light in the microscope. The exposure time (in seconds) increases with ischemia; that is, the fluorescence intensity decreases. A, Actin; M, myosin; TT, troponin T; TM, tropomyosin; Tb, tublin; D, desmin; Mm, myomesin; Vc, vinculin.

View larger version (66K): [in this window] [in a new window]   Fig. 8. Western blotting of ischemic human myocardium with the different antibodies used in this study in control and at 30 and 120 minutes of ischemia. Protein fragments cannot be detected, but the bands become thinner with increasing time of ischemia. A, Actin; M, myosin; TT, troponin T; TM, tropomyosin; Tb, tublin; D, desmin; Mm, myomesin; Vc, vinculin.

In this study, we showed that the contractile proteins are more sensitive to the effects of ischemia than are the components of the cytoskeleton. Vinculin is the most resistant of all the proteins investigated. Damage to the proteins occurred during the reversible phase of injury, as determined by electron microscopy.

Methodology
The model of in vitro ischemia has been shown to be suitable for the investigation of the in vivo ^{8, 9} effects of ischemia. Herdson, Kaltenbach, and Jennings ¹⁰ compared myocardial ischemia in vitro with that observed in vivo and concluded that the results obtained with the two models are comparable. Grochowski and colleagues ¹¹ and Armiger and coworkers ¹² confirmed this observation. The results presented here therefore apply to the in vivo situation of global ischemia as used in heart operations.

Measurements of fluorescence
An excellent agreement was found between the qualitative evaluation and the intensity of the fluorescent light. Direct measurements of antibody fluorescence intensity offer significant advantages compared with quantitative determinations of antibody concentration in tissue extracts by means of enzyme-linked immunoassay or Western blot techniques. In results obtained with both techniques, the composition of the tissue (the content of myocytes versus fibrous tissue) is unknown and a reference value for the measurements is therefore lacking. This problem is avoided with direct measurements in the microscope because the areas of interest can be identified. It is, however, necessary to stain and measure control and ischemic tissue simultaneously and to express all values with respect to control values.

Effects of ischemia on contractile proteins
Actin, myosin, tropomyosin, and troponin T showed early alterations in antibody binding and significant decreases in the intensity of fluorescence. To exclude the possibility that this rather unexpected finding was caused by the binding properties of one particular antibody, several antibodies against different epitopes of the same molecule were tested. These tests provided identical results. We therefore concluded that depolymerization of filaments was occurring during ischemia. This would explain the disturbed cross striation pattern in the presence of a persistent, if reduced, fluorescence intensity.

The findings for actin presented here are in agreement with data published by Nishida, Hiruma, and Hashimoto, ¹³ who found an early disappearance of labeling for actin in regionally ischemic rat myocardium. Iwai and coworkers, ¹⁴ on the other hand, noted a late distortion of actin labeling in the ischemic dog heart; this result corresponds with our pig and rabbit data (unpublished).

In normal human myocardium, Hayakawa and associates ¹⁵ used a polyclonal antibody against tropomyosin and observed slow disappearance of staining for tropomyosin until 24 hours after death. This finding may not conflict with our results, because Hayakawa and associates ¹⁵ studied normal hearts. Normal hearts may behave similarly to that of normal animal myocardium. With the Western blot technique, however, Westfall and Solaro ¹⁶ showed a decrease in tropomyosin in rat myocardium kept regionally ischemic for 60 minutes and reported the appearance of a band of lower molecular weight, suggestive of proteolysis.

Ischemia of cytoskeletal proteins
Desmin, tubulin, and myomesin showed similar time courses of qualitative changes. Alterations of the intensity of fluorescence were minimal in the case of desmin and myomesin but were significant for tubulin. Ganote and Vander Heide ¹⁷ described desmin as more resistant to ischemia than vinculin, which is in contrast to our findings. Differences in tissue preservation and the use of different antibodies, however, may have influenced the results.

Studies describing the effects of ischemia on myomesin were not found in the literature, and there was only one report on tubulin organization in ischemia Iwai and coworkers ¹⁴ found a time course of the disappearance of tubulin in canine myocardium similar to that in our study. Rupture and depolymerization of the microtubules were considered to indicate irreversible cell injury. Our study does not confirm this view, however, because disappearance of tubulin was already observed during the reversible stage of ischemic injury, as determined by electron microscopy.

Vinculin in normal myocardium is found in the intercalated disks and at the costameres, confirming the localization pattern described earlier by Pardo, Siliciano, and Craig ¹⁸ and by Shear and Bloch ¹⁹ Steenbergen, Hill, and Jennings ²⁰ used polyclonal antibodies on totally ischemic dog myocardium and found an early disturbance of vinculin localization. This difference from the results presented here may have been caused by the use of different antibodies in the two studies. Ganote and Vander Heide ¹⁷ also found a drastic reduction of specific fluorescence for vinculin after 90 minutes of total ischemia, whereas Iwai and coworkers ¹⁴ showed an increased resistance of vinculin to ischemic damage. The increased tubulin fragility observed by this group is also in agreement with our findings. On the basis of our results, we believe that vinculin is more resistant to ischemic injury than are many other cardiac proteins and that this resistance is partially responsible for the maintenance of cellular structure during ischemia.

CONCLUSION

Depolymerization of filaments may be the first biochemical lesion of proteins in ischemia. This is indicated by the fact that the bands in the Western blot became thinner with progressing ischemia. We did not detect bands of lower molecular weight, which suggests either that smaller fragments were not abundant enough to be detected, or that the fragments did not carry the epitope and were therefore nonreactive with the antibody. In addition, it is possible that in diseased human hearts depolymerization has already started and that proteolytic enzymes are more abundant and possibly more active than in normal hearts. In ultrastructural studies of human hearts with dilated cardiomyopathy or ischemic heart disease, a significant increase of lysosomes has been shown. ^{21, 22} Cytosolic proteases may also increase. In ischemia, a drop in tissue pH may then activate these enzymes to produce protein degradation. Because the antigenic epitopes may remain intact, the degradation fragments are still able to bind antibody and produce a certain fluorescence intensity.

We conclude that ischemia in diseased human hearts causes important alterations of the cardiac contractile and cytoskeletal proteins. These alterations occur simultaneously with or frequently even precede ultrastructural ischemic injury. This severe damage may significantly influence the ability of the myocytes to recover structural integrity and contractile function during postischemic reperfusion. We therefore concluded that these alterations of contractile and cytoskeletal proteins in addition to the damage to subcellular organelles play a decisive role in impeding the recovery of cardiac function after heart operations.

The findings presented here were obtained in human hearts with either dilated cardiomyopathy or ischemic heart disease. In an earlier study with electron microscopy, we found that hypertrophic human hearts possess a lower threshold for ischemic injury than do hearts with ischemic heart disease. ²³ We therefore assume that these results apply to all types of patients undergoing heart operations and that care should be taken to avoid damage to structural proteins during induced cardiac arrest.

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