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Ann Thorac Surg 2005;79:1333-1337
© 2005 The Society of Thoracic Surgeons

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Original articles: Cardiovascular

Is the Myofibrillarlytic Myocyte a Forme Fruste Apoptotic Myocyte?

^a Departments of Pathology and Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania
^b Division of Cardiology, University of California, Irvine, California

Accepted for publication September 7, 2004.

^_* Address reprint requests to Dr J. Narula, UCI Medical Center, 101 The City Dr, Bldg 53, Route 81, Orange, CA 92868-4080 (E-mail: narula{at}uci.edu).

	Abstract

Top Abstract Introduction Material and Methods Results Comment References

 
BACKGROUND: Myofibrillarlytic (MFL) cells are commonly observed in subendocardial myocardium in myocardial infarction. Because ischemic damage to myocytes is also known to induce apoptosis, we evaluated the prevalence of apoptosis in MFL cells in nine ischemic cardiomyopathic hearts explanted during transplantation.

METHODS: Myocytes with partial or complete clearing of cytoplasm, observed commonly in the subendocardium, were recognized as MFL cells. Prevalence of apoptosis was defined by TUNEL and ISOL staining and further characterized by immunohistochemical staining for caspase-3, Bcl2, BCL-X_L, Bax, proliferating cell nuclear antigen (PCNA), and Ki67.

RESULTS: Of 4131 MFL cells examined, 1305 (32%) possessed nuclei in a given histologic section; 1140 (88%) of the nucleated myocardial cells were TUNEL positive. Of 842 cells with normal appearance, 257 (31%) cells demonstrated nuclei in the given histologic section. TUNEL staining was observed in 5 (1.9%) in these control areas. All MFL cells stained positive for caspase 3. The antiapoptotic proteins, Bcl2 and BCL-X_L, demonstrated intense upregulation within and surrounding MFL cells, whereas pro-apoptotic protein Bax expression was only seen at control level. The MFL cells had Ki67 negative and PCNA positive nuclei.

CONCLUSIONS: The present study demonstrates that the majority of MFL cells are apoptotic and are associated with upregulation of caspase 3. Simultaneous upregulation of Bcl2 represents a survival effort in these myocytes. This is consistent with the review of the literature that MFL cells are viable, persist in myocardium for long time and may be functionally reversible. Evidence for concurrent apoptosis and survival instinct represent a conceptual paradox and suggests that myocytes undergoing apoptosis should be amenable to reconstitution of function.

	Introduction

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Myofibrillarlytic (MFL) cells are commonly observed in subendocardial myocardium and at the periphery of large myocardial infarct [1]. Scattered MFL cells are also observed frequently in various cardiomyopathic states [2, 3] including adriamycin toxicity [4]. Such cells are known to exist in myocardium for a prolonged period of time and are considered to be viable [2]. Morphologically, MFL cells are characterized by a variable degree of cytoplasmic clearing with or without dilatation of sarcoplasmic reticulum [4]. The mechanism for development of myofibrillarlysis is not known. The morphologic and topographic characteristics suggest that MFL cells may constitute a part of spectrum of apoptosis, because MFL cells are distributed at the border of infarct or in the subendocardium where the ischemic injury is likely to be milder. Myocardial ischemic injury is known to be associated with apoptosis [5–8]. In addition, the contractile proteins are selectively cleared in MFL cells, which need upregulation of specific proteolytic enzymes. Caspases, which get induced and activated in apoptosis, have been shown to result in contractile protein damage [9, 10].

We undertook this study to evaluate the prevalence of apoptosis in MFL cells in ischemic cardiomyopathic (ISCM) hearts explanted from patients undergoing orthotopic heart transplantation. Apoptosis in myofibrillarlytic cells was evaluated by demonstration of DNA fragmentation by the TUNEL staining, and further characterized by immunohistochemical staining for proteolytic enzyme (caspase 3), antiapoptotic proteins (Bcl2 and BCL-X_L), and the pro-apoptotic protein Bax.

	Material and Methods

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Histologic sections from nine ISCM hearts obtained during transplantation over the past 2 years were reviewed. Section stained by hematoxylin-eosin, which showed large populations of MFL cells in subendocardial layers, were identified. A myofibrillarlytic cell was defined as a myocyte wherein a part or all of the sarcoplasm had been replaced by a clear, intracellular vacuole. The myocytes with only perinuclear clearing were rejected as sectioning artifact. Contiguous sections of the selected paraffin blocks with myofibrillarlytic cells were evaluated for the presence of apoptosis by TUNEL staining, and immunohistochemical staining for Bcl2, BCL-X_L, Bax, caspase 3 [11, 12], Ki67, and PCNA was performed.

TUNEL staining
The histologic sections were deparafinized by washing in xylene, and absolute ethanol followed by a wash in phosphate buffered saline (PBS). The sections were then treated with freshly diluted protein digesting enzyme for 15 minutes and washed in two changes of distilled water [11]. Next, the sections were treated with 3% hydrogen peroxide in PBS for 5 minutes at room temperature to quench the endogenous peroxidase. Equilibration buffer was applied for 10 seconds at room temperature. Sections were then incubated with the digoxigenin-labeled TdT enzyme at 37°C in a humidified chamber. After washing, antidigoxigenin antibody-peroxidase conjugate was applied to the tissue sections and incubated for another 30 minutes. Following a PBS wash, color reaction was developed with DAB peroxidase. Glass slides were then washed, counterstained lightly with hematoxylin, and mounted.

In Situ Oligo Ligation
The ApopTag in situ oligo ligation (ISOL) kit (Intergen #7200; Serological Corp, Norcross, GA) was used to detect apoptotic DNA fragmentation. This kit utilizes T4 DNA ligase to bind synthetic biotinylated oligonucleotides to 3'-dT overhangs. Tissue sections were first deparaffinized in three changes of xylene, followed by three changes of absolute ethanol. Subsequently, endogenous peroxidase was quenched in 3% hydrogen peroxide in PBS. After washing the tissue sections, they were treated with 20 µg/mL of proteinase K in PBS, washed again, and placed in equilibration buffer. A solution of T4 DNA ligase and oligonucleotides was then applied to the slides and incubated overnight at 16 to 22°C. Detection of ligated oligonucleotides was accomplished by first applying a streptavidin-peroxidase conjugate that was developed with diaminobenzidine (DAB). Finally, tissue sections were counterstained in hematoxylin.

Immunohistochemical Staining for Caspase-3, BCL-X_L, and BAX
Deparaffinize slides with three changes of xylene for 5 minutes each, then rehydrate in three changes of 100% alcohol for 2 minutes each. After washing with denatured H₂O for 5 minutes, antigen retrieval is done by boiling in citrate buffer for 10 minutes. This is followed by blocking for endogenous biotin with Dako's avidin and biotin system (Dako, Hamburg, Germany), and subsequent blocking of endogenous peroxidase. Wash with 1x automation buffer with 1% BSA 2 times for 5 minutes each and incubate slides with 2% normal horse serum for 20 minutes at room temperature. After rinsing, the sections were layered with monoclonal caspase-3 antibody (MS-1123-PO; NeoMarkers, Fremont, CA), BCL-X_L (1:200; SC-8392 [Santa Cruz Biotechnology, Inc., Santa Cruz, CA]) and Bax antibody (MS-711-P; NeoMarkers), dilution 1:100, for 60 minutes at room temperature. This is followed by wash and incubation with secondary (biotinylated antimouse, 1:250; Vector Labs, Burlingame, LA) and biotin-HRP complex (Dako) 1 hour at room temperature and DAB plus for 5 minutes. The slides are counterstained with hematoxylin, mounted, and coverslipped.

Immunohistochemical Staining for Bcl2, Ki-67, and PCNA
Sections (5-µm thick) of formalin fixed paraffin embedded tissue were immunostained for monoclonal Ki67 (1:25; M7187 [Dako]), monoclonal PCNA (1:2000; M0879 [Dako]), and monoclonal BCL-2 (1:50; M0887 [Dako]). Tissue sections were first deparaffinized in three changes of xylene and rehydrated in three changes of absolute ethanol followed by one change of distilled water. Ki67 and BCL-2 tissue sections were then treated in boiling 1x target retrieval solution (S1699; Dako) for 20 minutes and allowed to cool for 20 minutes. The PCNA tissue sections were heat treated in a Panasonic microwave (model NN-5602A; Panasonic, Secaucus, NJ) on medium-high in 1x citrate buffer (Lab Vision, AP-9003-500), followed by 15 minutes of cooling. All sections were then immunostained on the Dako autostainer (Dako Corporation) using Dako Envision+System, HRP (DAB; K4007 [Dako]).

Counting of MFL Cells
Three-to-nine MFL cell rich areas in the hematoxylin-eosin stained tissue sections were identified in all specimens. The number of total cells and MFL myocytes were determined. Because nuclei may not be observed in all myocytes, the number of myocardial cells with nuclei was determined. After TUNEL staining, MFL cells with positive TUNEL-stained nuclei were counted and the proportion of TUNEL-positive cells was calculated. The results were expressed as percent nucleated myocytes. Non-MFL myocyte areas were counted for control.

	Results

Top Abstract Introduction Material and Methods Results Comment References

 
A total of 4131 MFL cells (Fig 1A) were examined; 1305 of these cells possessed nuclei (32%) in the examined sections. Of 1305 MFL cells, 1146 nucleated cells (88%) revealed TUNEL-positive nuclei (Fig 2A). For comparison, 842 normal appearing myocytes were evaluated. Of these, 257 cells (31%) revealed nuclei in the section, merely 5 (1.9%) nuclei were TUNEL positive. No caspase 3 staining was observed in the cytoplasm or nuclei in normal appearing myocytes. On the other hand, all MFL cells stained positive for caspase 3 with variable intensity (Fig 1B). The caspase 3 staining was more prominent in MFL cells with relatively milder vacuolization and lesser degrees of cytoplasmic clearing. Mild upregulation of caspase 3 was also observed in the adjacent non-MFL cells. However, areas of the myocardium that appeared normal ie, had no evidence of myocyte hypertrophy in terms of nuclear enlargement and had no clearing of the cytoplasm were negative for caspase 3 staining. All cells within and surrounding MFL cellular area demonstrated marked upregulation of antiapoptotic proteins Bcl2 (Fig 1C) and BCL-X_L (Fig 1D) compared with the control myocardial areas. On the other hand, no difference was observed in the staining for pro-apoptotic protein Bax in MFL or non-MFL cells. Immunostaining with TUNEL, ISOL, Ki67, and PCNA was performed in serial sections. As illustrated in Figure 2, the MFL cells are TUNEL positive (A), ISOL negative (B), PCNA positive (C), and Ki67 negative (D).

View larger version (148K): [in this window] [in a new window]   Fig 1. Immunohistochemical characterization of myofibrillarlytic cells. (A) Myofibrillarlytic cells are observed in an ischemic cardiomyopathic myocardial sample with vacuolated cytoplasm (hematoxylin & eosin, original magnification x20). (B) Although these cells stain modestly for the upregulation caspase-3 (Anticaspase-3 antibody, immunoperoxidase, original magnification x20), these cells stain intensely for the (C) antiapoptotic protein, Bcl2 (anti-Bcl-2 antibody; immunoperoxidase, original magnification x20) and (D) BCL-XL (anti-BCL-XL antibody; immunoperoxidase, original magnification x20).

View larger version (147K): [in this window] [in a new window]   Fig 2. Immunohistochemical characterization of myofibrillarlytic cells. Staining of the myofibrillarlytic cells with (A) TUNEL, (B) ISOL, (C) PCNA, and (D) Ki67 suggests lack of completion of apoptotic process. TUNEL-positivity probably represents myocyte repair or hypertrophy. The staining has been performed in serial sections. As shown, the myofibrillarlytic cell nuclei are positive for TUNEL and PCNA, and negative for ISOL and Ki67 (immunoperoxidase, original magnification x20).

	Comment

Top Abstract Introduction Material and Methods Results Comment References

Although, a large number of MFL cells were TUNEL positive, the importance of TUNEL staining in cardiomyopathic hearts has recently been questioned [17]. Kanoh and colleagues' study [17] of ultrastructural examination of TUNEL-positive cells failed to demonstrate morphologic characteristics of apoptosis in the cytoplasm or nuclei. They further characterized that nuclei of the TUNEL-positive cells also stained for PCNA but not for cyclin-dependent Ki67. These results suggested that TUNEL-positive cells comprise nuclei under repair. Although significance of TUNEL results is suspect, caspase 3 upregulation in MFL cells suggests that apoptotic cascade has been initiated in these cells. However, this assumption creates a conceptual paradox for the following reasons. The MFL cells exist in myocardium for longer periods, sometimes as long as years and are lost rather slowly unlike the usual rapidity of apoptotic process [18]. In addition, these cells are probably reversibly damaged. A coronary bypass surgery study has demonstrated that postoperative wall motion improvement only occurs if the transmural biopsy from the myocardial segments predominantly contained MFL cells [19]. Myocardial segments with fibrosis did not improve. Similarly, dilated cardiomyopathy patients presenting with acute onset congestive heart failure demonstrate higher likelihood of functional improvement over time if their endomyocardial biopsy specimens contained significant MFL cells [3]. It is intriguing to conceive of apoptotic cells as reversibly damaged. Some recent findings of apoptotic myocytes may explain this paradoxical phenomenon. It has been found that apoptosis in terminally differentiated heart muscle cells is initiated and results in extensive loss of cytochrome c from mitochondria [20, 21]. Caspase 3 is upregulated downstream to cytochrome c [20, 22], and while some cytoplasmic apoptosis may continue, the nuclear apoptosis does not necessarily complete [20, 21]. Therefore the cell may continue to live. Upregulation of a major antiapoptotic protein, Bcl2, and no change in its counterpart, Bax, confirms the survival instinct of these cells. Because Bcl2 also may act as a paradoxical proapoptotic protein depending upon the levels of expression and the target organ [23], we also performed BCL-X_L characterization [24]. BCL-X_L staining paralleled that of Bcl2 stain confirming the upregulation of antiapoptotic factors. This phenomenon of a balance sheet in favor of survival has been reported earlier in end-stage cardiomyopathy [25, 26]. The cytoplasmic clearance, to variable degrees, results from partial inactivation of active caspases and nuclei escape damage probably due to loss of DNAses [26]. Although the cytochrome c release and cytoplasmic proteolysis may contribute to systolic dysfunction, preservation of the genetic blueprint may allow revitalization of the cell upon heart muscle unloading [21]. This may partially explain the reversibility of MFL containing myocardial walls after coronary bypass surgery in ischemic cardiomyopathy.

Conclusions
The present study suggests that MFL cells may constitute forme fruste apoptotic cells. A majority of these cells are associated with caspase 3 upregulation. A review of the literature suggests that MFL cells are viable, persist in myocardium for a long time, and demonstrate functional reversibility. Upregulation of antiapoptotic proteins may form the basis of survival instinct in these cells.

	References

Top Abstract Introduction Material and Methods Results Comment References

 

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