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European Journal of Heart Failure 2008 10(2):129-132; doi:10.1016/j.ejheart.2007.12.013
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© 2008 European Society of Cardiology

Quantitative analysis of apoptotic markers in human end-stage heart failure

Lorenz Bott-Flügela,*,1, Hans-Jörg Weiga,1,2, Heiko Ühleina, Michael Nabauerb, Karl-Ludwig Laugwitza and Melchior Seyfartha

a Medizinische Klinik und Deutsches Herzzentrum München, Technische Universität Munich, Germany
b Medizinische Klinik, Klinikum Großhadern der Ludwigs-Maximilians-Universität München Munich, Germany

* Corresponding author. Deutsches Herzzentrum München, Lazarettstr. 36, 80636 Munich, Germany. Tel.: +49 89 1218 4566; fax: +49 89 1218 4593. E-mail address: bott-fluegel{at}med1.med.tu-muenchen.de (L. Bott-Flügel).


   Abstract
Top
 Notes
 Abstract
1. Background
 2. Aims
 3. Materials and methods
 4. Results
 5. Conclusions
 References
 
Apoptosis – programmed cell death – has been implicated in a variety of cardiac diseases, including myocardial infarction and chronic heart failure. This study was conducted to quantify the amount of apoptotic markers in human end-stage heart failure and to correlate the results to clinical parameters of heart failure.

Myocardial samples from 44 patients with end-stage heart failure and 5 controls were collected at the time of heart transplantation. Lysates of tissue samples were analysed for cleavage of alpha actin, alpha actinin, troponin T, tropomyosin, essential myosin light chain-1 (MLC-1v), and gelsolin. We observed cleavage of alpha actin, and alpha actinin. Troponin I, tropomyosin, and MLC-1v were not detectably cleaved. The amount of active caspase-3 was low in all samples (1.10±0.1 ng/ml). The same applied for DNA histone fragments (0.61±0.04). In patients with acutely decompensated heart failure we observed a striking increase in caspase-3 activity, but not DNA fragmentation. When calculated for the entire group there was no correlation between caspase-3 activity, DNA fragmentation and haemodynamic or echocardiographic variables. Relevant increases in apoptosis were only observed in patients with acute decompensated heart failure.

Key Words: Apoptosis • Heart failure • Caspase-3 • Contractile proteins

Received August 11, 2007; Revised October 29, 2007; Accepted December 19, 2007


   1. Background
Top
 Notes
 Abstract
 1. Background
2. Aims
 3. Materials and methods
 4. Results
 5. Conclusions
 References
 
The process of programmed cell death has been implicated in a wide variety of myocardial diseases including myocardial ischaemia and reperfusion [1,2], ischaemic cardiomyopathy [3,4], or myocarditis [5]. The role of apoptosis in human heart failure is still under discussion. Recent evidence suggests that cardiomyocytes can start apoptotic self-destruction, but do not complete it, thus rendering the cell functionally compromised, but still viable. After removal of the noxious stimulus which activated the apoptotic process, cells could completely recover [6]. These principles have been demonstrated in animal models of heart failure, but there are still conflicting results from human studies.


   2. Aims
Top
 Notes
 Abstract
 1. Background
 2. Aims
3. Materials and methods
 4. Results
 5. Conclusions
 References
 
The aim of the present study was to address the role of apoptosis in human heart failure by detecting the amount of apoptotic protein and DNA cleavage, and to correlate it with clinical variables in patients with end-stage heart failure at the time of heart transplantation.


   3. Materials and methods
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Materials and methods
4. Results
 5. Conclusions
 References
 
This study complies with the principles outlined in the Declaration of Helsinki. All experiments and the collection of human myocardial tissue were conducted following approval of the local ethics review board. Written informed consent was obtained from each patient. Myocardial samples were collected from 44 patients immediately after explantation of the heart during a heart transplantation procedure. Samples from 5 donors with no apparent cardiac disease served as controls (Table 1). All samples were lysed, homogenized, and prepared for analysis via immunoblotting or ELISA.


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  		Table 1 Baseline characteristics of the patient cohort according to the aetiology of heart failure

 
To detect cleavage fragments of myocardial proteins, samples were immunoblotted and stained with primary antibodies against monoclonal anti-alpha sarcomeric actin (mouse clone 5C5, Sigma Aldrich, Germany), monoclonal anti-alpha actinin (mouse clone EA-53, Sigma Aldrich), monoclonal anti-essential myosin light chain-1 (MLC-1v, BiosPacific, Emeryville, USA), monoclonal anti-troponin T (Sigma Aldrich), and monoclonal anti-tropomyosin (sarcomeric, mouse clone CH1, Sigma Aldrich). MLC-1v was detected after immunoprecipitation with purified 2C8 Myosin LC-1 antibody (BiosPacific), and subsequent blotting as described above. DNA fragmentation was detected with the Cell Death Detection ELISA (Roche Diagnostics, Mannheim, Germany), and caspase-3 activity was analysed with the human active caspase-3 immunoassay (R&D Systems, Wiesbaden, Germany). TUNEL staining was performed on 10 µm cryosections, as well as staining with phalloidin to verify the myocytic nature of the cells. Caspase-3 was stained with primary antibody against the active fragments of caspase-3 (Sigma Aldrich). All data were analysed using SPSS v. 12.0.


   4. Results
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Materials and methods
 4. Results
5. Conclusions
 References
 
We observed an elevation of caspase-3 activity in samples from the eight patients with acutely decompensated heart failure, 3 in the ICM group and 5 in the DCM group (Figs. 1 and 2, Table 2). In this subgroup of patients with acute heart failure, two patients with ICM and 3 with DCM were on intravenous catecholamines, 2 had received a ventricular assist device (Novacor) due to cardiogenic shock, and one had an intraaortic balloon pump. The causes of acute heart failure were known in 4 patients, 1 had an acute viral myocarditis, 1 an acute myocardial infarction due to occlusion of the left main coronary artery, 1 had decompensated heart failure due to mitral regurgitation, and 1 had decompensated ischaemic cardiomyopathy. Caspase-3 activity was significantly increased in these acute heart failure patients compared to patients with chronic heart failure (1.6±0.38 ng/ml vs 1.1±0.16 ng/ml in DCM, 0.8±0.08 ng/ml in ICM, p=0.004, see Fig. 3). The amount of DNA fragmentation was not increased in the acute heart failure patients (0.6±0.1 vs 0.6±0.1 in chronic DCM, and 0.6±0.1 in chronic ICM).


Figure 01
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  		Fig. 1 Markers of apoptotic activity. Caspase-3 activity (A) and DNA fragmentation (B) in patients with acute or chronic heart failure and controls. Individual values and means are shown. The p-value shown is for analysis of caspase-3 activity. DCM dilated cardiomyopathy, ICM ischaemic cardiomyopathy.

 


Figure 02
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  		Fig. 2 Cryosections (10 mm) of human heart tissue from a patient with acute heart failure. Panel A depicts an overlay image of the following stainings: Panel B shows blue fluorescence after staining with antibody against active caspase-3, panel C shows single green fluorescent nuclei after TUNEL staining, which marks cells with DNA single strand breaks, and panel D shows cardiomyocytes in red after phalloidin staining marking cardiomyocytes.

 


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  		Table 2 Outcome variables of patients with acute heart failure vs chronic heart failure

 


Figure 03
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  		Fig. 3 Representative immunoblots from tissue lysates of patients with end-stage heart failure. Shown are cleavage fragments of cardiac contractile proteins: (from above) alpha actin, alpha actinin, troponin T, MLC-1v, and uncleaved alpha actin as loading control. On the right the respective purified protein with and without addition of caspase-3. MLC-1v myosin light chain ventricular isoform-1.

 
We did not detect any relevant correlation between caspase-3 activity and fractional shortening or ejection fraction or any of the clinical characteristics in the whole population (data not shown). The same applied for DNA fragmentation.

We observed cleavage fragments of alpha actin (25 kDa-fragment) in 77% of samples from DCM patients and 60% from ICM patients, and cleavage of alpha actinin (30 kDa-fragment) in 27% and 35%, respectively (Fig. 3). Other contractile proteins showed no cleavage.


   5. Conclusions
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Materials and methods
 4. Results
 5. Conclusions
References
 
In the present study we investigated the occurrence of apoptotic markers in myocardial tissue from patients with end-stage heart failure undergoing heart transplantation. In previous studies, activation of caspases in terminal heart failure, like caspase-3, 8 or -9, [6-8], and subsequent cleavage of contractile proteins has been demonstrated [9] but no direct measurement of enzyme activity in tissue lysates had been done so far. It has been demonstrated that the number of apoptotic nuclei correlates with clinical deterioration [10].

We demonstrated a significant increase in caspase-3 activity in our 8 patients with acute decompensated heart failure, whereas the levels in patients with chronic heart failure or controls were low. Corroborating our finding of activated caspase-activity, we also demonstrated apoptotic cleavage of structural proteins like alpha actin and actinin. However, we could not demonstrate a clear correlation between clinical and haemodynamic characteristics and caspase-activity in the overall population. This lack of correlation could be due to the small number of cases.

In conclusion, our study demonstrates the activation of apoptotic proteins in tissue samples of patients with acutely decompensated heart failure, indicating an important role of apoptosis in exacerbations of chronic heart failure. Based on animal studies in which blockade of caspase-3 stopped the progression of heart failure [9], future studies should be designed to investigate the beneficial role of blocking apoptosis in human heart failure.


   Notes
Top
 Notes
Abstract
 1. Background
 2. Aims
 3. Materials and methods
 4. Results
 5. Conclusions
 References
 
1 L. Bott-Flügel and H.J. Weig contributed equally to this manuscript. Back

2 H.J. Weig moved since conclusion of the project: III. Medizinische Klinik und Poliklinik, Eberhard-Karls-Universität, Tübingen, Germany. Back


   References
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Materials and methods
 4. Results
 5. Conclusions
 References
 

  1. Gottlieb R.A., Engler R.L. Apoptosis in myocardial ischemia-reperfusion. Ann N Y Acad Sci (1999) 874:412–426.[CrossRef][Web of Science][Medline]
  2. Nakamura M., Wang N.P., Zhao Z.Q., et al. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res (2000) 45:661–670.[Abstract/Free Full Text]
  3. Saraste A., Pulkki K., Kallajoki M., Henriksen K., Parvinen M., Voipio-Pulkki L.M. Apoptosis in human acute myocardial infarction. Circulation (1997) 95:320–323.[Abstract/Free Full Text]
  4. Yue P., Long C.S., Austin R., Chang K.C., Simpson P.C., Massie B.M. Post-infarction heart failure in the rat is associated with distinct alterations in cardiac myocyte molecular phenotype. J Mol Cell Cardiol (1998) 30:1615–1630.[CrossRef][Web of Science][Medline]
  5. Kyto V., Saraste A., Saukko P., et al. Apoptotic cardiomyocyte death in fatal myocarditis. Am J Cardiol (2004) 94:746–750.[CrossRef][Web of Science][Medline]
  6. Narula J., Arbustini E., Chandrashekhar Y., Schwaiger M. Apoptosis and the systolic dysfunction in congestive heart failure. Story of apoptosis interruptus and zombie myocytes. Cardiol Clin (2001) 19:113–126.[CrossRef][Medline]
  7. Scheubel R.J., Bartling B., Simm A., et al. Apoptotic pathway activation from mitochondria and death receptors without caspase-3 cleavage in failing human myocardium: fragile balance of myocyte survival? J Am Coll Cardiol (2002) 39:481–488.[Abstract/Free Full Text]
  8. Narula J., Pandey P., Arbustini E., et al. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci U S A (1999) 96:8144–8149.[Abstract/Free Full Text]
  9. Laugwitz K.L., Moretti A., Weig H.J., et al. Blocking caspase-activated apoptosis improves contractility in failing myocardium. Hum Gene Ther (2001) 12:2051–2063.[CrossRef][Web of Science][Medline]
  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]

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This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
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