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Am J Physiol Heart Circ Physiol 285: H1521-H1527, 2003. First published June 12, 2003; doi:10.1152/ajpheart.00890.2002
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Cyclical mechanical stretch-induced apoptosis in myocytes from young rats but necrosis in myocytes from old rats

B. Husse, A. Sopart, and G. Isenberg

Department of Physiology, Martin-Luther-University Halle, D-06097 Halle, Germany

Submitted 11 October 2002 ; accepted in final form 10 June 2003


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanical load as stimulus for apoptosis and necrosis could be responsible for the loss of cardiomyocytes. Ventricular myocytes from young (3 mo) and old (14–24 mo) rats underwent cyclical mechanical stretch (CMS; 5% elongation, 1 Hz) for 24 h. Spontaneous apoptosis was in myocytes from young rats 0.33 ± 0.12% and from old rats 1.05 ± 0.35% [Tdt-mediated dUTP nick-end labeling (TUNEL) assay]; associated with a decrease of Bcl-2. CMS increased the apoptosis to 0.58 ± 0.18% in myocytes from young rats. Western blot analysis showed that CMS reduced Bcl-2 and increased p53 (young rats). Bax was not changed by CMS. These were confirmed by cytochrome c release (31 ± 13%) and by the enrichment of cytosolic nucleosomes (11 ± 8%). CMS did not influence the apoptosis in myocytes from old rats (TUNEL assay, Bcl-2, Bax, or p53). CMS did not cause necrosis in myocytes from young rats. CMS increased the number of necrotic cells by showing the cell membrane rupture in myocytes from old rats (50 ± 13% 5-hexadecanoylaminofluorescein-positive and 38 ± 6% propidium iodide-positive cells) as well as by measuring the lactate dehydrogenase release. The results suggest that CMS-induced apoptosis in myocytes of young rats but necrosis in myocytes from old rats, which could be attributed to more stress sensitivity of cells from old rats.

ventricular myocytes; age

SEVERAL STUDIES HAVE INDICATED that the aging process of the human and the animal hearts is characterized by a significant loss of myocytes and a hypertrophy of the remaining cells (21). The cell loss could be responsible for the diminished ability of the aged heart to sustain increases in pressure and volume loads (19) and for the development of ventricular dysfunction and failure (7). Identification of the molecular mechanisms implicated in the limitation of stress adaptation of the aged heart was attempted (39). As age-dependent changes of the intracellular processes, a decrease in calcium transients and contractility (40, 42) as well as many other changes of the mitochondria (23) and protein kinases (22) were reported.

The two forms of cell death involved in the reduction of the total number of myocytes are the programmed cell death (apoptosis) and the nonapoptotic cell death (necrosis) (1). Apoptosis is determined by morphological and biochemical criteria that are the condensation of chromatin leading to the development of apoptotic bodies or membrane-enclosed vesicles containing oligonucleosomal DNA fragments. This is an energy-dependent process, which leads to cell death. The analytic tools of apoptosis detect the DNA strand breaks or the proteins of the apoptotic pathways (11, 17). Necrosis is characterized by the swelling of cells and their organelles, thereby leading to the disruption of the cell membrane (29, 35).

In ventricular papillary muscles, an increased mechanical loading and passive tensions were reported to induce apoptosis coupled with oxidative stress and Fas expression (6). Experimental infarction or pressure overload in hearts of young rats induced apoptosis (5, 41). The myocyte loss analyzed in the human failing heart was attributed to apoptosis and necrosis (27, 33).

The myocyte loss is documented for cardiomyocytes from young and old hearts. Stretch was reported to increase the apoptotic cell death only in myocytes from young hearts; however, correspondent data on myocytes from old animals are missing. The aim of this study was to analyze the influence of stretch on ventricular myocytes from young and old rats. To exclude paracrine effects on the ventricular myocytes during the strain, which would have been possible in a multicellular experiment, we used isolated ventricular myocytes from young and old rats under culture conditions with the Flexercell Strain Unit for the application of stretch. We investigated which stretch-induced form of cell death, apoptosis or necrosis, is dominant in myocytes from young and old rats. We found that cyclical mechanical stretch (CMS) applied on single ventricular myocytes induced apoptosis only in myocytes from young not from old rats. In contrast, CMS did not cause necrosis in myocytes from young rats but only in myocytes from old rats.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Myocyte Isolation, Culture, and Stretch

The animal data of the young and old rats are presented in the Table 1. The ratio of the heart weight to body weight remained age-independently constant. Ventricular myocytes were isolated from the hearts of both groups by means of retrograde perfusion of collagenase-containing solutions. Details have been reported previously (18). After the perfusion, the cells were incubated in Kraftbruhe medium composed of (in mM) 70 KOH, 40 KCl, 50 L-glutamic acid, 20 taurine, 20 KH2PO4, 3 MgCl2, 10 glucose, 5 Na-pyruvate, and 0.5 EGTA, pH titrated with KOH to 7.4 for 2–3 h. The yield of cell isolations was 67.4 ± 10.2% myocytes with intact plasma membrane from young rats and 61.2 ± 7.3% myocytes with intact plasma membrane from old rats measured with vital dye. The myocytes from both ventricles were cultured for 24 h with medium 199 (Earle's salts) containing 1% penicillin/streptomyocin (Sigma) and 10 µg/ml amikacin (Sigma) on the Bioflex culture plates coated with 20 µg/ml laminin. The number of attached cells was 40 ± 12 cells/mm2 (young rats) and 23 ± 6% cells/mm2 (old rats). After the medium was changed, the culture plates were put in the gasket of the Flexercell Strain Unit (Flexercell; McKeesport, PA) of a tissue incubator (5% CO2, 37°C). The Flexercell computer system connected the unit with a vacuum pump and controlled the stretch parameters (2). The unit cyclically stretched the foil with the adherent myocytes at a frequency of 1 Hz and an amplitude of 5% for 24 h. The control groups were handled the same way but without cyclic deformation.


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  		Table 1. Animal data of young and old male rats

 

Apoptosis Assay

Tdt-mediated dUTP nick-end labeling assay. For in situ detection of apoptosis at the level of single cells, we used terminal deoxynucleotidyl transferase (TdT) to incorporate fluorescein-labeled dUTP into DNA strand breaks (In Situ Cell Death Detection Kit; Boehringer-Mannheim). Tdt-mediated dUTP nick-end labeling (TUNEL) assay was performed with cardiomyocytes, which were fixed with 4% paraformaldehyde in PBS. The apoptotic nuclei were detected as red nuclei by using Fast Red Tablets (Boehringer-Mannheim) a substrate for alkaline phosphatase. The apoptotic cells were counted (magnification x100) and related to the total number of myocytes in 20 different areas of each experiment.

ELISA for histone-associated DNA fragments. Apoptosis was quantified in both groups of rats with the use of a commercially available ELISA for in vitro determination of cytoplasmic histone-associated DNA fragments (Boehringer-Mannheim). One well of the Bioflex culture plate after stretch as well as one well of the control culture plate were washed with PBS and lysed according to the business direction. Samples were run in triplicate, with optical density (OD) measured at 405 nm. The relative enrichment of cytosolic nucleosomes was calculated for each group of the experiments as (OD405 stretch/OD405 control) x 100, after subtraction of background OD490.

Western blot analysis. For the analysis of Bcl-2, Bax, and p53, myocytes of control and stretched groups were washed with ice-cold PBS and solubilized with a sample buffer consisting of 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 4 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1 mM DTT, and 0.001% protease inhibitor cocktail (P8340; Sigma). In 10-µl solubilized samples, the protein content was analyzed with the Bio-Rad protein assay. The protein concentration was 0.50–2.50 mg/ml or 0.1–0.5 mg per well. Equal amounts of protein (50 µg) were loaded and run together on the same 12% SDS-polyacrylamide gel. After being transferred to nitrocellulose, the membranes were strained with Ponceau red to control for equal transfer of protein. Membranes were treated with 5% Blotto, composed of 5% wt/vol nonfat dry milk in TBS (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, with 0.1% Tween 20) for 2 h at room temperature. After being washed, the blots were incubated for 1 h with antibody against Bcl-2 (1 µg/ml monoclonal mouse, BioMol) or Bax (3.6 mg/ml polyclonal rabbit, BioMol) or p53 (1.4 µg/ml monoclonal mouse, BioMol). Horseradish peroxidase (HRP) conjugated anti-mouse IgG (Bcl-2 and p53) and HRP-conjugated anti-rabbit IgG (Bax) were used as secondary antibody in a dilution of 1:1,000. The blots were visualized with the use of an enhanced chemiluminescence (ECL) detection system (Amersham).

For the cellular fractionation, myocytes from young or old rats of control and stretched groups were washed in ice-cold PBS and scraped in 200 µl buffer consisting of 20 mM HEPES/KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM DTT, 20 µl/ml protease inhibitor cocktail (P8340; Sigma) per well. The cells were homogenized with the use of a glass Dounce homogenizer and then centrifuged at 1,000 g for 10 min at 4°C to sediment the nuclear fraction. The supernatant was centrifuged at 100,000 g for 1 h at 4°C. The resulting supernatant was used as the cytosolic fraction. The pellet was resuspended in 300 µlofthe above-described buffer and used as membrane fraction. After the protein concentraction was determined, the samples (20 µg of young rats and 10 µg of old rats) were electrophoresed on a 15% SDS-polyacrylamide gel and subjected to the above described immunoblotting with antibody against cytochrome c (1 µl/ml monoclonal mouse, Zymed) and HRP-conjugate anti-mouse IgG as secondary antibody, which was detected by ECL (Amersham).

Necrosis Assay

Fluorescence labeling with 5-hexadecanoylaminofluorescein. Plasma membrane damage was analyzed in myocytes of three different groups from young and old rats. The first group was measured after 24 h of culture time. At this point, the myocytes that did not attach on the culture surface were already removed from the measured area. The second group was measured after 48 h of culture time and the third group was measured after 24 h of culture and 24 h of stretch. The myocytes were washed twice with PBS and incubated with 5 µM and 5-hexadecanoylaminofluorescein (HEDAF) for 10 min at room temperature. HEDAF integrated in the plasma membrane of the intact cells and into all membranes of damaged cells. The number of damaged cells relative to the total number of cells was measured by x200 magnification over an area of 200 mm2 with the use of a fluorescence microscope.

Fluorescence labeling with propidium iodide. The myocytes of the above-described groups were washed twice with PBS and incubated with 1.5 mM propidium iodide (PI) for 20 min at room temperature to quantify the nuclear stain. PI is an exclusion fluorescent dye that binds to chromatin only on loss of membrane integrity. The number of damaged cells relative to the total number of cells was measured by a x200 magnification over an area of 200 mm2 with the fluorescence microscope.

Assay of lactate dehydrogenase activity. Lactate dehydrogenase (LDH) activity released from myocytes of young and old rats was determined with the use of an LDH assay kit (Sigma). LDH catalyzes the reduction of pyruvate to lactate, resulting in an equimolar amount of NADH being oxidized to NAD. The rate of increase in absorbance at 495 nm is directly proportional to LDH activity in the sample. LDH activity was measured in the cell medium, and the cells were then lysed with a 1% Triton X-100 solution to measure the LDH activity in the myocytes. The LDH activity was expressed as relative LDH activity, meaning that it was in relationship to the LDH activity in the cell medium plus LDH activity in the cells.

Statistical Analysis

Data are expressed as means ± SD. The differences of Figs. 1 and 5 were assessed by one-way analysis of variance combined with Bonferroni test. A statistically significant difference in Figs. 2, 3, 4 was analyzed with the use of unpaired Student's t-test. A value of P < 0.05 was considered to be statistically significant.



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  		Fig. 1. Quantitative analysis of the number of apoptotic cells in ventricular myocytes from young (A; n = 7) and old (B; n = 11) rats by Tdt-mediated dUTP nick-end labeling (TUNEL) assay (means ± SD).

 


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  		Fig. 5. Quantitative analysis of the number of necrotic cells in ventricular myocytes from young (n = 8, open bars) and old (n = 9, hatched bars) rats by measurement of 5-hexadecanoylaminofluorescein (HEDAF)-positive cells (A), propidium iodide (PI)-labeled cells (B), and analysis of the relative lactate dehydrogenase activity (C) in ventricular myocytes from young (n = 6) and old (n = 9) rats after 24 h, 48 h, and 24 h of culture plus 24 h of stretch (means ± SD).

 


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  		Fig. 2. Effect of stretch on the released nucleosomes in the cytoplasm of ventricular myocytes of young (n = 12, open bars) and old (n = 10, hatched bars) rats; analysis of the percentage of enrichment of cytosolic nucleosomes by ELISA (means ± SD; *P < 0.002).

 


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  		Fig. 3. Bcl-2 (A), Bax (B), and p53 (C) expression of ventricular myocytes from young (n = 6, open bars) and old (n = 6, hatched bars) rats after 24 h of culture plus 24 h of stretch relative to control without stretch (48 h of culture); means ± SD. *P < 0.001; **P < 0.0001.

 


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  		Fig. 4. Detection of cytochrome c in the cytosolic and membrane fraction by Western blots. Comparison between cytosolic fractions of ventricular myocytes from young (n = 4) and old (n = 3) rats after 48 h of culture (A). Effect of stretch on the cytochrome c level in the cytosolic and membrane fraction of ventricular myocytes from young (B; n = 4) and old (C; n = 3) rats; comparison of 48 h of culture with 24 h of culture plus 24 h of stretch (means ± SD).

 


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Apoptosis

The spontaneous apoptosis was measured after a culture time of 24 and 48 h. In this time, the spontaneous apoptosis did not vary significantly. The percentage of TUNEL-positive cells was 0.38 ± 0.24% (n = 6) after 24 h and 0.33 ± 0.12% (n = 7) after 48 h in ventricular myocytes from young rats. In myocytes from old rats, 1.24 ± 0.44% (n = 6; after 24 h) and 1.05 ± 0.35% (n = 11; after 48 h) TUNEL-positive myocytes were measured. The influence of CMS on the number of apoptotic cells was determined in the ventricular myocytes of both age groups, which were cultured for 24 h and followed by CMS for 24 h. CMS increased the number of TUNEL-positive cells to 0.58 ± 0.18% in myocytes from young rats (Fig. 1A), whereas the number of TUNEL-positive cells with 0.98 ± 0.22% in ventricular myocytes from old rats was not changed compared with spontaneous apoptosis (Fig. 1B).

The in vitro determination of cytosolic histone-associated DNA fragments showed that CMS increased the cytosolic nucleosomes by 11 ± 8% (n = 12) in ventricular myocytes from young rats but not in ventricular myocytes from old rats (–1 ± 8%, n = 10; Fig. 2).

The expression of Bcl-2, Bax, and p53 was measured by densitometric analysis of Western blots in ventricular myocytes from young and old rats after 48 h of culture. The expression of Bcl-2 protein in myocytes from old rats was by 24 ± 16% (n = 6; P < 0.004) lower than in myocytes from young rats. The Bax and p53 proteins did not show differences between ventricular myocytes from young and old rats (n = 6).

CMS decreased the Bcl-2 protein by 28 ± 18% (n = 6) in ventricular myocytes from young rats but not in those from old rats (n = 6; Fig. 3A). The effect of CMS on the quantity of Bax protein was not significant but with big variations of the values in ventricular myocytes from young (n = 6) and old (n = 6) rats (Fig. 3B).

CMS increased the p53 protein by 20 ± 7% (n = 6, P < 0.001) in ventricular myocytes from young rats but did not significantly increase in ventricular myocytes from old rats (n = 6; Fig. 3C).

The cytochrome c level was analyzed by Western blots in the cytosolic and membrane fractions of ventricular myocytes from young and old rats after 48 h of culture. Under these conditions, the cytochrome c level was neither increased in the cytosol nor decreased in the fractions of membrane and mitochondria of myocytes from old rats (n = 3) compared with young rats (n = 4; Fig. 4A).

Because CMS increased apoptosis only in ventricular myocytes from young rats, the mitochondrial cytochrome c release can attributed to CMS as an apoptotic marker only in ventricular myocytes from young rats. As expected, CMS increased the amount of cytochrome c by 31 ± 13% (n = 4, P < 0.003) in the cytosol and decreased it by 48 ± 16% (n = 4, P < 0.001) in the fractions of membrane and mitochondria (Fig. 4B).

CMS did not stimulate apoptosis in ventricular myocytes from old rats. An enhanced level of cytochrome c in the cytosol by 72 ± 19% (n = 3; P < 0.004) and a decreased amount of cytochrome c in the membrane fraction by 29 ± 8% (n = 3; P < 0.04) can only be attributed to necrosis connected with the damage of mitochondria (Fig. 4C).

Necrosis

The total number of analyzed myocytes from young (4,320 ± 118 cells in each group) and old (2,387 ± 81 cells in each group) rats remained constant over an area of 200 mm2 during the analyzed time. The myocytes with a damaged plasma membrane were recognized and counted with the membrane fluorescence dye HEDAF. After 24 h, the number of necrotic cells was 31.7 ± 8.7% (n = 9) in myocytes from young rats. In the following 24 h, the number of necrotic cells did not change in the myocytes from young rats. After a culture time of 48 h, the percentage was 35.7 ± 6.5% (n = 9) and under the influence of 24 h of CMS, 35.7 ± 7.7% (n = 9) necrotic cells were measured (Fig. 5A).

In contrast, the myocytes from old rats showed an increase of the number of necrotic cells from 27.3 ± 6.9% (n = 9) after 24 h of culture to 34.6 ± 6.5% (n = 9) after 48 h of culture. After 24 h of culture, 24-h CMS enhanced the number of necrotic cells further to 50.0 ± 13.3% (n = 9), as illustrated in Fig. 5A. Myocytes from young rats which had lost their membrane integrity and were labeled with PI did not show significant changes in the number of necrotic cells. The percentage was 24.7 ± 5.5% (n = 9) after the 24-h culture, 30.7 ± 10.6% (n = 9) after the 48-h culture time and 33.8 ± 12.8% (n = 9) after the 24-h culture plus 24-h CMS (Fig. 5B).

In the myocytes from old rats, however, an increase in the number of necrotic cells from 19.4 ± 5.5% (n = 9) after 24 h of culture to 28.2 ± 5.9% (n = 9) after 48 h culture and with 37.9 ± 6.4% (n = 9) after 24 h of culture and 24 h of CMS was measured (Fig. 5B).

The LDH release was analyzed as relative LDH activity of 16.01 ± 3.59% (n = 6) in the medium of myocytes from young rats and of 17.65 ± 5.11% (n = 8) in the medium of old rats after 24 h. These relative high values were attributed to the fact that the cell medium also contained not attached and dead myocytes. In myocytes from young rats, the relative LDH activity was 5.59 ± 1.43% (n = 6) after 48 h of culture, which was not different from 5.58 ± 1.89% (n = 6) measured after 24 h of culture plus 24 h of CMS (Fig. 5C). In contrast, CMS increased the relative LDH activity from 5.59 ± 1.15% (n = 8) after 48 h of culture to 8.40 ± 3.33% (n = 8) after 24 h of culture plus 24 h of CMS in myocytes from old rats (Fig. 5C).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study attempted to investigate the possible effect of CMS on the cell death of ventricular myocytes from young compared with old rats. The isolated ventricular myocytes were chosen to exclude the interaction of other cell types over paracrine mechanisms with cardiomyocytes. The stimulus for the cell death should be only CMS. The time course of the apoptotic cascade in adult ventricular cardiomyocytes is represented (38). The DNA fragmentation is completed 14 h after stimulation. On the basis of this information, the apoptotic markers were selected in these experiments where CMS was the main stimulus.

We found that the number of apoptotic cells is high in ventricular myocytes from old rats relative to young rats if we compare the spontaneous apoptosis in the cell culture. A potential reason might be in the age-dependent decrease of the Bcl-2 (the antiapoptotic protein) whereas Bax (the proapoptotic protein) and p53 (the transcriptional regulator) did not change with the age. The mechanism for the regulation of Bcl-2 and Bax are incompletely investigated. Both proteins are located in the outer mitochondrial membrane and are responsible for the regulation of the permeability of cytochrome c (9, 34). In this connection a decrease of Bcl-2 could promote to form lower Bcl-2/Bax heterodimers and more Bax homodimers lead to an enhanced level of cytochrome c release. But we did not find an increased release of cytochrome c in cultured myocytes from aging rats. The cytochrome c release is also discussed to be independent on the changes of Bcl-2 family protein (32). A comparable change of Bcl-2 and Bax with the age was found in isolated mitochondria from different aging rats (30). Other studies (20, 26) of the apoptotic and necrotic myocyte cell death in aging rats showed that both forms of cell death are presented. Another report (4) hypothesized that the aging heart becomes more sensitive to damaging factors due to an exploited molecular control of cell death.

Ventricular myocytes from young rats responded to stretch with an increase in the apoptotic cell death. A similar increase in apoptosis due to stretch was reported (6) for papillary muscles caused by the formation of reactive oxygen species and for adult ventricular myocytes by using sustained equibiaxial stretch attributed to release of angiotensin II (24). The present experiments are based on the role of mitochondria for the apoptosis, which is already known (3, 14, 28). In the ventricular myocytes from young rats, CMS induced an increase of the cytochrome c release in the cytosol, a decrease of the antiapoptotic protein Bcl-2 and an increase of the tumor suppressor protein p53. The function of p53 as transcription regulator protein was reported in context with the regulation of the angiotensinogen gene or angiotensin type 1 receptor gene (25) as well as the cell death-regulating genes Bax and Bcl-2 (10). Thus our results are in line with the previous studies on the stretch-mediated apoptosis. CMS did not cause necrotic cell death in ventricular myocytes from young rats. The total number of myocytes remained constant and the high percentage of myocytes with damaged membranes can be attributed to the isolation of the myocytes and the characteristic of the cell culture that dead cells also could attach and did not remove.

This is the first study that used ventricular myocytes from old rats to investigate the influence of stretch on the cell death. Studies (21, 36) that used samples of the failing human heart have indicated relatively big differences in the level of the cell death in different forms of cardiomyopathy. The reasons for these results could not be explained, unless the differences of the myocyte death were considered dependent on the gender (12). In this study, we used myocytes only from healthy male rats. We found a relatively high apoptotic level in the control group that was not increased by stretch. The measured markers of apoptosis Bcl-2, Bax, and p53 remained constant after CMS in ventricular myocytes from old rats. The ventricular myocytes from old rats responded to CMS exclusively with an increase of the necrotic cell death while it was evaluated from the number of cells with damaged membranes and the release of LDH. We speculatively attribute the prevalence of necrosis to the mitochondria. The mitochondria in old cells, compared with those in young cells, have a higher proton leakage and a lower ATP turnover (15). The lower membrane fluidity and the shift toward more long-chain polyunsaturated fatty acids in the mitochondrial membranes of old rats are associated with an increase of lipid peroxidation (23). The age-related changes of the mitochondria can be contributed to the cardiac stiffness, myocardial oxidative stress and apoptosis (13), which could be the cause for the response of ventricular myocytes of old rats to mechanical stress.

As a stimulus for the induction of apoptosis in the heart, hypoxia (8), palmitate as metabolic component (37), or pacing of the heart (16) as well as mechanical load (24) were reported. In our study, CMS was used as a stimulus to investigate the induction of cell death. Such experiments were used before with cell cultures of neonatal rat cardiomyocytes, which responded with an increase of reactive oxygen species associated with differential activation of kinases and induction of apoptosis (31). Our results relating to CMS showed that only ventricular myocytes from young rats responded with an enhanced level of apoptotic cell death, whereas ventricular myocytes from old rats responded with necrotic cell death. In conclusion, the loss of cardiomyocytes with age can be attributed to more stress sensitivity of cells from old rats.


   ACKNOWLEDGMENTS
 
We thank Prof. Dr. M. F. Gallitelli and Frank Rudolf for providing the ventricular myocytes from young and old rats for our experiments. We also thank Nicole Duerrschmidt and colleagues of the Pathophysiological Institute for expert technical assistance with the Western blot analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Husse, Dept. of Physiology, Martin-Luther-Univ. Halle/Wittenberg, 6 Magdeburger St., D-06097 Halle, Germany (E-mail: britta.husse{at}medizin.uni-halle.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
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A. M. Samarel
Costameres, focal adhesions, and cardiomyocyte mechanotransduction
Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2291 - H2301.
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