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European Journal of Heart Failure 2006 8(3):284-289; doi:10.1016/j.ejheart.2005.09.004
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© 2005 European Society of Cardiology

Enhanced myocardial cathepsin B expression in patients with dilated cardiomyopathy

Junbo Gea,*,1, Gang Zhaoa,1, Ruizhen Chena,1, Shuangjie Lib, Shijun Wanga, Xingang Zhanga, Yamin Zhuanga, Jiuzhong Dub, Xiaohua Yub, Gaoping Lia and Yingzhen Yanga

a Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University Fenglin Road 180, Shanghai 200032, P.R. China
b The First Affiliated Hospital, Nanhua University Hengyang of Hunan Province, China

* Corresponding author. Tel.: +86 21 64041990 2745; fax: +86 21 64223006. E-mail address: gejunbo{at}zshospital.net (J. Ge).


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Cathepsin B is a prominent lysosomal protease and is involved in apoptosis as well as degradation of myofibrillar proteins in myocardial infarction. The aim of this study was to investigate myocardial cathepsin B expression in failing and non-failing human hearts.

Methods: Tissue samples were taken from transplanted left ventricles from 20 patients with dilated cardiomyopathy and 5 non-failing donor hearts that could not be transplanted for technical reasons. Myocardial cathepsin B expression was determined by immunohistochemistry, the reverse transcription–polymerase chain reaction (RT–PCR) and Western blotting. Apoptosis was assessed by TUNEL staining.

Results: Positive cathepsin B staining was found in failing and non-failing hearts. The expression of cathepsin B at mRNA and protein levels was significantly higher in failing hearts compared with non-failing hearts. Correlation analysis revealed that cathepsin B at mRNA and protein levels negatively correlated with EF (r=0.66, p=0.002 and r=0.492, p=0.028, respectively) in patients with heart failure. The apoptotic index was 0.015±0.006 in failing hearts and 0.002±0.001 in non-failing hearts (p<0.01).

Conclusion: Increased myocardial expression of cathepsin B was found in patients with heart failure suggesting that cathepsin B might play a role in the genesis and development of heart failure.

Key Words: Cathepsin B • Dilated cardiomyopathy • Heart failure • Apoptosis

Received November 12, 2004; Revised July 3, 2005; Accepted September 8, 2005


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cathepsin B is a prominent lysosomal protease and plays an important role in the apoptosis process [1,2]. Hill [3] found cathepsin B expression was increased during neuronal death in rats following global ischemia or decapitation necrosis. Tsuchida et al. demonstrated that cysteine proteinases, particularly cathepsin B, are involved in degradation of myofibrillar proteins in myocardial infarction [4]. Canbayl et al. [5] showed that hepatocyte apoptosis and liver injury are cathepsin B dependent and cathepsin B inactivation (both by genetic and pharmacologic inhibition) could attenuate liver injury, inflammation and fibrogenesis in the bile duct ligated mice model. Korolenko [6] reported that cathepsin B content and activity were reduced in tumour tissues and antitumour therapy increased activity and content of cathepsin B in tumour tissues suggesting that cathepsin B could promote apoptosis in tumour tissues. The pathogenesis of dilated cardiomyopathy (DCM) has not been completely defined, recent studies suggest that the apoptosis of abundant myocardial cells is a key factor in the pathogenesis of DCM [7,8]. Myocardial cathepsin B expression from patients with DCM and its relationship to cardiac function have not been studied previously. The aim of this present study was, therefore, to compare cathepsin B expression and apoptosis in myocardial samples from DCM patients to normal control hearts. The correlation between myocardial cathepsin B expression and cardiac function as well as apoptosis in DCM patients was also studied.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Myocardial tissue sampling
Hearts were obtained from 20 end-stage DCM patients (NYHA III-IV) who were admitted to Zhongshan hospital from May 2000 to December 2003 for heart transplantation. There were 13 male and 7 female patients. The average age was 38 years (12-64). Left ventricular end-diastolic diameter (LVEDD) was 77.5±9.6 mm, left ventricular end-systolic diameter (LVESD) was 66.9±9.8 mm and the ejection fraction (EF) was 28.9±6.8% (Table 1). Five donor hearts that could not be transplanted for technical reasons were used as controls. Immediately after explantation, the hearts were put directly into heart protection solution (University of Wisconsin solution: Osmotic pressure, 325 mOsm/L; pH, 7.4; Mg+, 5 mmol/L; Na+, 30 mmol/L; K+, 120 mmol/L; Lactose solvate, 100 mmol/L; Phosphate 25 mmol/L; Hetastarch, 50 g/L; Melitriose, 17.8g/L; Adenosine, 5 mmol/L; Allopurinol, 1 mmol/L; Glutathione, 3 mmol/L; Insulin, 100 U/L; Trimethoprim, 8 mg/L; Hexadecadrol, 8 mg/L). Transmyocardial samples were taken from the left ventricular apex, fixed in 4% formaldehyde for 24 h and embedded in paraffin for histology, immunohistochemistry and Tunnel assay or frozen and stored under –70 °C for cathepsin B mRNA and protein measurements. The study conforms with the principles outlined in the Declaration of Helsinki and was reviewed and approved by the local Ethics Committee.


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  		Table 1 Patient characteristics

 
2.2. Immunohistochemistry
The slides (5 µm thick) were deparaffinized through xylenol, and dehydrated with alcohol series, the endogenous peroxidase activity was blocked by incubating slides for 30 min with 0.3% H2O2 in methanol, treated with 10% normal horse serum in Tris-buffered saline (TBS) for 30 min at room temperature and then incubated with anti-cathepsin B polyclonal antibody (dilution 1:100, Santa Cruz Biotechnology Inc.) overnight at 4 °C. After washing with TBS, the slides were incubated with avidine-biotin-conjugated horse antigoat IgG (dilution 1:200, Pufei Biotechnology Inc. Shanghai) for 1 h at room temperature. The avidine-biotin complex activity was then revealed with 0.1% 3, 3'-diaminobenzidine tetrahydrochloride (DAB) and 0.3% H2O2 in 50 mM TBS for 5 min. Immunostained sections were counterstained with haematoxylin.

2.3. TUNEL assay
Apoptotic cardiomyocytes were detected in tissue sections by terminal transferase mediated DNA nick end-labelling assay (TUNEL, Oncogene Inc.). The number of apoptotic cardiomyocytes was counted in 10 randomly selected light microscopy fields (200x magnification, 0.20 mm2 field area) and the data shown are the mean values from the 10 examined fields of each heart sample. The apoptotic index (AI), as described by Narula et al. [8], is expressed by the formula: number of apoptotic cardiomyocytes/total number of cardiomyocytes. The apoptotic nucleus was also observed under an electron microscope.

2.4. RT-PCR
Total mRNA of 100 mg myocardial tissue was isolated using Trizol (Gibco Inc.) reagent according to the instructions of the supplier, and cDNA was generated using Superscript First-Strand Synthesis System for RT-linked PCR (Invitrogen). cDNA (1 µl) was removed for amplification by PCR in a final volume of 25 µl. The 5'primer (5'-ACAGTGTCCCACCATCAAAG) and the 3'primer (5'-CACCATTACAGCCGTCCC) generated a 185-bp product specific for the cathepsin B mRNA. At the same time, 303-bp β-actin was amplified by PCR with 5'primer (5'-GTGGACATCCGCAAAGAC) and the 3'primer (5'-GAAAGGGTGTAACGCAACT). All these primers were synthesized by Shanghai Genebase Gene-Tech Co., Ltd. The amplified transcripts were visualized on 1.5% agarose gels with the use of ethidium bromide. Specific amplification products of the expected size were observed. Cathepsin B mRNA was normalized to housekeeping gene β-actin mRNR concentration.

2.5. Western blot analyses
Total protein was extracted from 100 mg of myocardial tissue using lysis buffer (containing 10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Triton X-100, 10 mM EDTA [ethylenediaminetetraacetic acid], 10 µg/mL leupeptin, 1 mM phenylmethylsufonyl fluoride [PMSF]). Proteins were separated on 12% SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane and probed with goat polyclonal anti-Cathepsin B antibody(dilution 1:200, Santa Cruz Biotechnology Inc.), avidine-biotin-conjugated horse antigoat IgG(dilution 1:200, Pufei Biotechnology Inc. Shanghai), avidine-biotin complex activity was then revealed with 0.1% 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0.3% H2O2 in 50 mM TBS for about 5 min. The molecular weight of Cathepsin B was 27 kd. β-actin protein (43 kd, dilution 1:10000, Promega Inc.) was also detected. Cathepsin B protein was normalized to housekeeping gene β-actin protein concentration.

2.6. Statistical analysis
Data are expressed as mean±SD. Unpaired Student's T test was used for the comparison between the two groups. P<0.05 was considered as significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. HE staining
HE stained myocardial tissues obtained from the 20 DCM patients showed typical pathological changes (Fig. 1): huge myocardial cell nucleus, irregular and diffuse fibrosis.


Figure 1
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  		Fig. 1 HE staining of control and DCM hearts. Hearts from DCM patients show uneven myocardial hypertrophy, huge myocardial cell nucleus, irregular and diffuse fibrosis.

 
3.2. Immunohistochemistry
Fig. 2 shows immunohistochemistry myocardial cathepsin B expression. Brown yellow positive pellets were visualized both in the cytoplasm of DCM and control hearts. Cathepsin B positive cell counts were similar between normal and DCM hearts.


Figure 2
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  		Fig. 2 Immunohistochemistry cathepsin B staining (avidine-biotin complex method) of control (A) and DCM (B) hearts. Brown yellow positive pellets indicate cathepsin B inside the cardiomyocytes in both the DCM and control hearts. Negative control (C) was stained without the 1st antibody and for positive control (D) the cardiac muscle sample with definite cathepsin B expression was stained as cathepsin B is commonly expressed in mammalian cells.

 
3.3. TUNEL assay
The apoptotic myocyte count was 0.015±0.006 in DCM hearts and 0.002±0.001 in control hearts (P<0.01, Fig. 3A,B). Electron microscopy showed typical apoptotic nucleus in DCM heart (Fig. 3D) compared to a normal nucleus in control heart (Fig. 3C).


Figure 3
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  		Fig. 3 TUNEL assay. (A) Brown yellow positive signals (arrows) represent the apoptotic cells. (B) Apoptotic cell counts were significantly higher in DCM hearts (0.015±0.006) compared to control hearts (0.002±0.001). (C) Electron microscopy of a control heart showing normal nucleus. (D) Electron microscopy of a DCM heart showing apoptotic nucleus.

 
3.4. Myocardial Cathepsin B mRNA expression
Cathepsin B mRNA (Fig. 4) was significantly increased in the DCM hearts compared to the control hearts (0.93±0.30 vs. 0.44±0.25, P<0.01).


Figure 4
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  		Fig. 4 Cathepsin B mRNA detected by RT-PCR. M, marker; N: control heart; 1~3: DCM hearts; NC: cDNA of cathepsin B was omitted; PC: cDNA of cathepsin B synthesized by gene transfection was added. Cathepsin B mRNA was significantly increased in DCM hearts compared to control hearts.

 
3.5. Myocardial Cathepsin B protein expression
Cathepsin B at protein level (Fig. 5) was also significantly increased in the DCM hearts compared to the control hearts (0.45±0.08 vs. 0.13±0.03, P<0.01).


Figure 5
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  		Fig. 5 Western blot analysis of cathepsin B. N1~N2: control hearts; 1~3: DCM hearts. NC: negative control, the 1st antibody was omitted; PC: positive control, recombinant human cathepsin B protein purchased from RnD Co. (No. 953-CY-010) was added. Cathepsin B expression at protein level was significantly increased in DCM hearts compared to control hearts.

 
3.6. Correlation between cathepsin B expression, apoptosis and EF in failing human hearts
There was a trend towards a positive correlation between cathepsin B mRNA and apoptosis index (r=0.405, p=0.077) but cathepsin B at protein level did not correlate with apoptosis index in DCM hearts (r=0.177, p=0.455), however, cathepsin B at both mRNA and protein level was negatively correlated with EF in patients with DCM (Fig. 6A,B).


Figure 6
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  		Fig. 6 Correlation between cathepsin B at mRNA (A) and protein levels (B) to ejection fraction (EF) in patients with DCM.

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we found that the expression of cathepsin B at protein and mRNA levels, and the apoptotic index were significantly increased in failing hearts compared with non-failing hearts. Cathepsin B at mRNA and protein levels was also negatively correlated with EF in patients with DCM. To our knowledge, this is the first study showing increased cathepsin B expression in human failing hearts compared to non-failing hearts.

It is known that apoptosis induced cardiomyocyte loss plays a key role in DCM pathogenesis [7], that increased apoptotic cells are found in failing hearts [7-9] and the quantity of apoptotic cells is related to pathological progression of DCM [10]. However, the exact mechanisms by which cardiomyocyte apoptosis occurs in DCM patients remain largely unknown. Although our study could not show the underlying mechanisms by which cathepsin B mediated the onset of apoptosis in failing human myocardium, the increased expression of cathepsin B at protein and mRNA levels in failing hearts and the negative correlation between cathepsin B expression and EF in patients with DCM shown in this study, suggest that cathepsin B might be involved in the pathogenesis of DCM. In the present study, we only found a weak positive correlation between cathepsin B mRNA and apoptosis index and cathepsin B at protein level did not correlate with apoptosis index in DCM hearts, this may be due to the limited number of cases studied. Cardiomyocyte apoptosis has been clearly demonstrated in end-stage failing hearts and is triggered by a variety of factors, including reactive oxygen species, cardiomyocyte over-stretch, hypoxia, Ca2+-overload, cytokines, and neurohormones. These different factors activate a precisely orchestrated genetic program culminating in the activation of executioner caspases, the final common pathway of different proapoptotic stimuli. Caspases promote cell death by degrading critical target proteins in the nucleus, cytosol and mitochondria (reviewed in [13]). It is known that cathepsin B is released from lysosomes after caspase-8 activation by various stimuli and activated cathepsin B in turn promotes the release of cytochrome c from mitochondria by cleaving one or more cytosolic substrates. Release of cytochrome c results in cleavage of caspase-9 and caspase-3 followed by further apoptotic changes [1,2]. Previous studies have shown that TNF-P readily induced apoptosis in isolated hepatocytes in wild type mice, however, deletion of the cathepsin B gene resulted in diminished liver injury and enhanced survival after treatment in vivo with TNF-P suggesting that cathepsin B played a key role in the apoptotic process [2]. Studies have also shown that specific cathepsin B inhibitors, such as CA-074 [11] and Spi2A [12] could attenuate cell apoptosis death and reduce ischaemic hippocampal neuronal death. It has also been shown that degradation of myocardial structural proteins in myocardial infarcted dogs is reduced by Ep459, a cysteine proteinase inhibitor [4]. Future studies are warranted to investigate the possible mechanisms underlying how cathepsin B might mediate the onset of apoptosis in the failing human myocardium and to show if cathepsin B inhibitors could modulate myocardial apoptosis and affect the disease progress in patients with DCM.


   Acknowledgements
 
This study was supported by the Major State Basic Research Development Program of People's Republic of China (G2000056903).


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
1 Junbo Ge, Gang Zhao and Rhuizen Chen contributed equally to this work. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Stoka V., Turk B., Schendel S.L., et al. Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route. J Biol Chem (2001) 276:3149–3157.[Abstract/Free Full Text]
  2. Guicciardi M.E., Deussing J., Miyoshi H., et al. Cathepsin B contributes to TNF-P-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest (2000) 106:1127–1137.[Web of Science][Medline]
  3. Hill I.E., Preston E., Monette R., et al. A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis. Brain Res (1997) 751:206–216.[CrossRef][Web of Science][Medline]
  4. Tsuchida K., Aihara H., Isogai K., Hanada K., Shibata N. Degradation of myocardial structural proteins in myocardial infarcted dogs is reduced by Ep459, a cysteine proteinase inhibitor. Biol Chem Hoppe Seyler (1986) 367:39–45.[Web of Science][Medline]
  5. Canbay A., Guicciardi M.E., Higuchi H., et al. Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis. J Clin Invest (2003) 112:152–159.[CrossRef][Web of Science][Medline]
  6. Korolenko T.A., Zhyanaeva S.Y., Poteryaeva O.N., et al. Activity and concentration of cathepsin B as prognostic criteria for the development of mouse LS lymphosarcoma and Lewis lung adenocarcinoma. Bull Exp Biol Med (2002) 133:392–395.[CrossRef][Web of Science][Medline]
  7. Olivetti G., Abbi R., Quaini F., et al. Apoptosis in the failing human heart. N Engl J Med (1997) 336(16):1131–1141.[Abstract/Free Full Text]
  8. Narula J., Haider N., Virmani R., et al. Apoptosis in myocytes in end-stage heart failure. N Engl J Med (1996) 335(16):1182–1189.[Abstract/Free Full Text]
  9. Kostin S., Pool L., Elsasser A., et al. Myocytes die by multiple mechanisms in failing human hearts. Circ Res (2003) 92:715–724.[Abstract/Free Full Text]
  10. Saraste A., Pulkki K., Kallajoki M., et al. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Invest (1999) 29:380–386.[CrossRef][Web of Science][Medline]
  11. Yamashima T., Kohda Y., Tsuchiya K., et al. Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on ‘calpain-cathepsin hypothesis’. Eur J Neurosci (1998) 10(5):1723–1733.[CrossRef][Web of Science][Medline]
  12. Liu N., Raja S.M., Zazzeroni F., et al. NF-kappa B protects from the lysosomal pathway of cell death. EMBO J (2003) 22(19):5313–5322.[CrossRef][Web of Science][Medline]
  13. Wollert K.C., Drexler H. Regulation of cardiac remodeling by nitric oxide: focus on cardiac myocyte hypertrophy and apoptosis. Heart Fail Rev (2002) 7:317–325.[CrossRef][Medline]

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