stress signal to survival and apoptosis

427

Stress Signal to Survival and Apoptosisa

ATSUSHI TAKEDAb AND NOBUAKIRA TAKEDA

Department of Internal Medicine, Aoto Hospital, Jikei University School of Medicine,6-41-2 Aoto, Katsushika-ku, 125 Tokyo, Japan

ABSTRACT: This investigation focused on whether apoptosis can be observedin some heart diseases. Apoptosis was examined immunochemically using mon-oclonal antibodies such as p53, Bcl-2 and cyclin E, A, and B1 in parallel withflow cytometry. Left ventricular myocardium was obtained at autopsy from 40patients with acute myocarditis (AM; N � 10, 6 males, 4 females, mean age 56� 13 years), chronic myocarditis (CM; N � 10, 5 males, 5 females, mean age 48� 16 years), dilated cardiomyopathy (DCM; N � 10, 7 males, 3 females, meanage 60 � 11 years), and no heart disease (Cont; N � 10, 5 males, 5 females, meanage 63 � 14 years). Cell cycle analysis of myocytes by flow cytometry revealedthat the relative content of G2M phase in acute myocarditis was far higher thanthose in other heart diseases (AM, 12.3 � 3.7%; CM, 5.2 � 4.5%; DCM, 6.3 �4.0%; Cont, 3.4 � 1.8%; Mean � SD). Expression of p53 was observed mainlyin myocytes from chronic myocarditis. Expression of Bcl-2, on the other hand,was detected in myocytes from acute myocarditis. Results suggest that apoptosismay play some role in the repairing process of myocardial inflammation.

INTRODUCTION

Inflammation is a dynamic process by which living tissues react to injury. It isdefined as a focal defensive response with circulation disturbance, exudation, and in-flammatory cell infiltration and proliferation in the tissue. It is still unknown wheth-er regressive and regenerative changes occur at the same time in this inflammatorylesion. Apoptosis, in contrast to inflammation, is a natural phenomenon and has beenthought to be an active as well as reactive process.

Using immunohistochemical and flow cytometrical analysis, we examined whetherapoptosis and cell cycle disturbances can be observed in some heart diseases.

MATERIALS AND METHODS

Materials

At autopsies performed in our hospital, hearts were sampled for this study. Leftventricular myocardium was obtained at autopsy from 40 patients: 10 with acute my-

aThis study was partly supported by the Research Committee for Epidemiology and Etiologyof Idiopathic Cardiomyopathy of the Ministry of Health and Welfare of Japan, and a researchgrant from the Vehicle Racing Commemorative Foundation.

bAddress correspondence to: Atsushi Takeda, M.D., Ph.D., Department of Internal Medicine,Aoto Hospital, Jikei University, 6-41-2 Aoto, Katsushika-ku, 125 Tokyo, Japan; Telephone: 81-3-3603-2111, Fax: 81-3-3602-2839; E-mail: [email protected].

428 ANNALS NEW YORK ACADEMY OF SCIENCES

ocarditis (AM; 6 males, 4 females, mean age 56 ± 13 years), 10 with chronic (per-sistent) myocarditis (CM; 5 males, 5 females, mean age 48 ± 16 years), 10 withdilated cardiomyopathy (DCM; 7 males, 3 females, mean age 60 ± 11 years), and 10with no heart disease (Cont; 5 males, 5 females, mean age 63 ± 14 years).

Methods

Pathological Study

Myocardium was stained with hematoxylin-eosin stain, Masson-trichrome stainperiodic acid-Schiff stain, Elastica von Gieson stain, and phosphotungstic acid he-matoxylin stain after routine 10% formalin fixation, paraffin embedding, andsectioning.

Immunohistochemical Study

These sections were also investigated immunohistochemically with anti-cyclin E,A, and B1 (Pharmingen Co.) and anti-p53, Bcl-2 monoclonal antibodies (DAKOCo.) by the streptavidin-biotin complex (SAB) method (FIG. 1).10

FIGURE 1. Pathological and immunohistological methods.

429TAKEDA & TAKEDA: APOPTOSIS IN DCM AND MYOCARDITIS

Cell Cycle Analysis with Flow Cytometry

The percent ratios of each phase were calculated automatically with the integralcalculus by the FACS flow cytometry (Becton Dickinson Co.)1−3 The cell cycleswere measured by flow cytometry and statistical analysis were perfomed in compar-ison with normal control hearts. Several 60-µm sections were cut from each paraffinblock of tissue.4 These sections were placed in tubes with xylene for 20 min and thengradually rehydrated with distilled water. Then the sample tubes were incubated for1 h at 37°C in a water bath after adding 1 ml of 0.5% pepsin solution (pH 1.5) in0.9% NaCl. After pipetting off this solution, target nuclei were purified by filtering

FIGURE 2. Fluorescein DNA histogram of a myocyte. The first high peak is the G0-1phase and the second one is the G2M phase.


through a 30−60 µm Teflon mesh to remove debris (modified Hedley method).5,6

Then the nuclei were stained with 50 µg/ml propidium iodide (PI) and 0.1% TritonX-100 in 4 mM sodium citrate (Vindelov method). This solution was centrifuged(4°C, 400×g, 10 min), the supernatant was discarded, 0.5 ml PI solution was discard-ed, 0.5 ml PI solution was added, and then a FACScan (Becton Dickinson Co.) wasused for flow cytometry.1,2,7−9

In the fluorescein DNA histogram, the first peak indicates the G0-1 phase of cellcycle and the second peak is the G2M phase of cell cycle. The valley between G0-1and G2M shows the S phase (FIG. 2).1,2

The DNA frequency histogram was deconvoluted and results were computer an-alyzed using the Multicycle software (Phoenix Flow System, San Diego, CA).1,2 Re-sults are presented as means ± SD. Comparisons between values were performedusing a Student’s t test. A value of p < 0.05 was considered statistically significant.

RESULTS

Histopathological and Immunohistochemical Study

There was extensive lymphocyte infiltrate in the acute myocarditis samples (FIG.3) and focal lymphocyte infiltrate in the interstitium in chronic (persistent) myo-carditis samples (FIG. 4). Interstitial fibrosis is perinuclear in distribution and the in-terstitium is involved by patchy fibrosis in dilated cardiomyopathy (FIG. 5).

FIGURE 3. Myocardial tissue (left ventricle) of acute myocarditis (Masson-trichromestain, original magnification ×400).


FIGURE 4. Myocardial tissue (left ventricle) of persistent (chronic) myocarditis (Mas-son-trichrome stain, original magnification ×400).

FIGURE 5. Myocardial tissue (left ventricle) of dilated cardiomyopathy (Masson-trichrome stain, original magnification ×200).


In case of acute myocarditis, scattered Bcl-2–positive myocytes were detected inthe myocardial tissues of patients (5/10 cases) (FIG. 6), but no (0/10 cases) p53-pos-itive myocytes were found immunohistochemically in the tissue. In case of chronicmyocarditis on the other hand, no Bcl-2–positive myocytes were detected in the tis-sue, but p53-positive myocytes were found in the tissue (4/10 cases) (FIG. 7).

Cell Cycle Analysis with Flow Cytometry

According to the cell cycle analysis, the G2M phase was more prominent in my-ocytes of acute myocarditis than in control (acute myocarditis, 12.3 ± 3.7% versusControl, 3.4 ± 1.8%; p < 0.01) or chronic myocarditis (acute myocarditis, 12.3 ±3.7% versus chronic myocarditis, 5.2 ± 4.5%; p < 0.05). However, there was no sig-nificant increase of the G2M phase in chronic myocarditic and dilated cardiomyo-pathic myocytes compared to Control.

In the same manner, the extent of the G0-1 phase was significantly less in acutemyocarditis and dilated cardiomyopathy than in controls (acute myocarditis, 77.4 ±4.6% versus Control, 90.1 ± 1.6%; p < 0.01. Dilated cardiomyopathy, 86.0 ± 2.8%versus Control, 90.1 ± 1.6%; p < 0.05). However, there was no significant differencein the G0-1 phase of chronic myocarditis than that of control. The S phase was alsomore prominent in myocytes of acute myocarditis than in Control (acute myocardi-tis, 12.3 ± 3.7% versus Control, 5.2 ± 4.5%; p < 0.05) (TABLE 1).

With regard to the selection of nuclei, the average size of nuclei in the myocar-dium was measured in hematoxylin-eosin–stained tissue using the light microscope.Only myocardial nuclei were filtrated through the optimum Teflon mesh, whose holesize was a little smaller than size of the nuclear myocytes, and almost all the nucleiof the interstitial cells were taken out (FIG. 8). However, the mixing ratio (percent-age) of interstitial cell nuclei was technically between about 2−8% (somewherearound 6%) after the measurement.

TABLE 1. Cell cycle of myocytes in myocarditis, dilated cardiomyopathy, and controla

AcuteMyocarditis

Chronic Myocarditis

Dilated Cardiomyopathy Control

G0-1 phase (%) 77.4 ± 4.6 87.1 ± 5.5 86.0 ± 2.8 90.1 ± 1.6

S phase (%) 10.4 ± 2.5 7.7 ± 2.1 8.1 ± 2.9 6.5 ± 2.0

G2M phase (%) 12.3 ± 3.7 5.2 ± 4.5 6.2 ± 4.0 3.4 ± 1.8

aN = 10, mean ± S.D., *p < 0.05, **p < 0.01.

* *

* * *

* *

*

* *

*


FIGURE 6. Bcl-2–positive myocyte in myocardial tissue (left ventricle) of acute myo-carditis (Masson-trichrome stain, original magnification ×200).

FIGURE 7. p53-positive myocytes in myocardial tissue (left ventricle) of chronic my-ocarditis (Masson-trichrome stain, original magnification ×400).


DISCUSSION

In the case of acute myocarditis, a scattering of Bcl-2–positive myocytes couldbe detected in myocardial tissues of patients (5/10 cases) (FIG. 6), but no p53-posi-tive myocytes could be found immunohistochemically in the tissue. This suggeststhat the Bcl-2 expression in myocytes could protect the myocardial damage from theseveral kind of cytokines induced by inflammatory cell infiltration against the viralinfection. In the case of chronic myocarditis on the other hand, p53 expression inmyocytes could be thought of as a repairing process for remodeling damaged tissue.In DCM cases, there were few p53 and Bcl-2–positive cells in the myocardial tissue.According to the cell cycle analysis, the G2M and S phases were more prominent inmyocytes of acute myocarditis than other cases. It could be that the myocytes ofacute myocarditis were strongly influenced on the cell cycle compared to the stablephase of control myocytes. And also, there was no correlation in S and G2M phasebetween DCM and Control. It shows that the myocytes of DCM are little influencedand some myocytes are stable in a polyploidy state. These results suggest that apo-ptosis may play some role in the repairing process of myocardial inflammation.

REFERENCES

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FIGURE 8. The naked nucleus after nuclear DNA analysis (hematoxylin-eosin stain,original magnification ×600).


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stress signal to survival and apoptosis

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