Ann Thorac Surg 2002;74:767-770
© 2002 The Society of Thoracic Surgeons
Original article: cardiovascular
Overexpression of heat shock protein 60/10 in myocardium of patients with chronic atrial fibrillation
Alfons E. Schäfler, MD*a,b,
Kiriakos Kirmanogloub,
Peter Pecher, MDa,b,
Andreas Hannekum, MDa,
Bernd Schumacher, MDa,b
a Department of Cardiac Surgery, University of Ulm, Ulm, Germany
b Cardiovascular Research Center, University of Ulm, Ulm, Germany
Accepted for publication May 29, 2002.
* Address reprint requests to Dr Schäfler, Department of Cardiac Surgery and Cardiovascular Research Center, University of Ulm, Steinhövelstr. 9, 89075 Ulm, Germany
e-mail: aschaefler{at}gmx.de
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Abstract
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Background. Cardiomyocytes respond to chronic atrial fibrillation with increased expression of heat shock protein 60 (HSP60). The aim of this study was to investigate whether expression of the coprotein HSP10 is also increased.
Methods. Right atrial samples from 16 patients undergoing elective cardiac operation were excised and immediately frozen in liquid nitrogen. Eight patients had chronic atrial fibrillation and 8 patients were in sinus rhythm. The HSP60 and HSP10 protein levels were determined by SDS-PAGE, Western blot, and quantified by optical densitometry according to the immunoreactive bands of actin.
Results. In myocardial samples from patients with chronic atrial fibrillation we found simultaneous upregulation of both stress proteins. HSP60 expression was more than 2.3-fold and HSP10 expression was more than 2.4-fold increased in atrial myocardium of patients with chronic atrial fibrillation.
Conclusions. These results indicate functional upregulation of mitochondrial HSP60 and HSP10 in response to chronic atrial fibrillation.
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Introduction
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Atrial fibrillation (AF) is the most common cause of arrhythmias, occurring in 0.4% of the general population, in 4% of the hospital population, and in 40% of patients with congestive heart failure. The mortality rate in patients with atrial fibrillation is twice as high as that of patients in sinus rhythm [1].
Studies have shown that AF can be caused by a rapid firing focus [2] or by multiple independent reentrant wavelets [3] without prolonging the refractory period when the heart rate slowed [4]. Microscopically massive interstitial fibrosis, cellular hypertrophy, and a loss of myocytes were found [5]. On a cellular level, the stress of chronic AF leads to a loss of myofibrils, accumulation of glycogen, and fragmentation of sarcoplasmic reticulum [6].
The increased atrial activity also leads to an increase in heat shock protein (HSP) production. In myocardium from patients with chronic atrial fibrillation (CAF) we found more than a twofold increase of HSP60 [7]. Because HSP60 works in conjunction with HSP10 we measured the HSP10 level in myocardium of patients with CAF in this study.
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Material and methods
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Patients
The present study was reviewed and approved on 10/6/2000 by the ethical committee of the University of Ulm. Analyses of protein levels of HSP60 and HSP10 were performed on myocardium from 16 human hearts. Atrial myocardium was obtained after extracorporeal circulation from 8 patients in sinus rhythm and 8 patients with AF undergoing elective cardiac operation for coronary revascularization, aortic or mitral valve replacement, and combined coronary revascularization with mitral valve replacement. The specimens of the right atria were obtained after decannulation of the venous line. Chronic AF was considered permanent AF if it lasted for more than 3 months [8]. All patients except one, had documented CAF on serial electrocardiograms, ultrasound, and a history of more than 3 month’s duration. The clinical characteristics of the patients are given in Table 1.
Their mean age was 69 ± 9 years. Anesthesia was performed for each patient as total intravenous anesthesia (TIVA). Cardiopulmonary bypass was established with a priming solution (1,000 mL of Ringer’s solution, 400 mL of human albumin, 200 mL of Trasylol, 5,000 IU of heparin) at a flow rate of 2.4 L · min-1 · m-2 body surface area. All patients were cooled to 32°C (esophageal). In addition, there was aortic cross-clamping, with a myocardial arrest induced by antegrade infusion of cold Brettschneider cardioplegic solution at a myocardial temperature of approximately 10°C.
Western blot analysis
Protein preparation
The myocardium for Western blot analyses was dissected, immediately frozen in liquid nitrogen, and stored at –80°C until use. Care was taken not to use fibrotic or adipose tissue. For protein preparation, 
40 mg of frozen atrial tissue was homogenized by use of a Ultra-Turrax T8 micro dismembrator (IKA Labortechnik, Staufen, Germany) for 60 seconds in a ninefold lysis buffer (Na4P2O7 100 mmol/L, EGTA 5 mmol/L, MgCl2 5 mmol/L, KCl 300 mmol/L, DTT 1 mmol/L at pH 8.5) along with a protease inhibitor cocktail (0.1 mmol/L AEBSF, containing E-64, estatin, leupeptin, and aprotinin [product no. P2714, Sigma Ltd]) at 4°C. The homogenate was centifuged at 4,000 rpm for 10 minutes at 4°C in a Labofuge GL (Heraeus Sepatech, Hanau, Germany). After centrifugation, the supernatant was carefully removed.
HSP60
HSP60 separation and detection was carried out as previously described [7].
HSP10
HSP10 separation and immunodetection was accomplished as with HSP60. Differences of the procedure are the following: SDS-PAGE was performed under reducing conditions on a 17% separation gel with 4% stacking gel. Running time was 45 minutes. The gel was equilibrated for 10 minutes in blotting buffer and proteins were transferred at 3 mA/cm2 for 20 minutes. The blot was blocked for 1 hour in 10% nonfat milk solution, incubated with a 1:2,000 diluted rabbit antihuman HSP10 polyclonal antibody solution (product no. SPA-110, StressGen, Hamburg, Germany) for 1 hour, and washed twice for 1 minute and three times for 5 minutes in 10 mL of tween tris buffered saline (TTBS) solution. Immunodetection of the primary antibody against the HSP10 protein was carried out with a 1:5,000 diluted peroxidase conjugated antirabbit secondary antibody (product no. NA 934, Amersham Ltd, Freiburg) for 45 minutes and washed again twice for 1 minute and four times for 15 minutes in TTBS. After incubation with 0.125 mL/cm2 enhanced chemiluminescense-detection reagent (Amersham buchler Ltd) for 1 minute, blots were exposed to Hyperfilm ECL (Amersham Ltd) for 5 minutes.
Quantification of immunoreactive bands
After development, the blots were scanned with a Umax Mirage II densitometer (Umax, Freemont, CA) and a Epson Perfection 1240 Photo (Epson, Düsseldorf, Germany). Bands were quantified with an analysis software (mars 98, version 1.0.1, Karl Dubies, Ulm) according to the densitometric integral derived from each sample band. The integral of the density over the measured area was taken to calculate the amount in each sample according to the known standard values. A linear relationship was found between the known HSP60 and HSP10 amount and the densitometric integrals. On the basis of this linear relationship, HSP60 and HSP10 of each atrial sample was calculated (r = 0.98 and r = 0.96, respectively). The values for HSP60/HSP10 are presented as a ratio of the levels of HSP60/HSP10 compared to the level of actin found in the human right atria.
Statistical analysis
All data are presented as mean ± standard deviation. To detect differences between groups, the Mann-Whitney rank sum test was performed. A p value less than 0.05 was considered statistically significant. The correlation between standard HSP60/HSP10 and the densitometric integral was examined by linear regression analysis.
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Results
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The surgical outcome was uneventful in all patients, and there was no complication from right atrial dissection. The bypass time was approximately 120.4 ± 32 minutes and the ischemic time 70.4 minutes ± 22 minutes.
To assess whether there were any changes in HSP60 and HSP10 expression in chronic atrial fibrillation, Western blot analysis was carried out on SDS-PAGE separations of total protein isolated from eight hearts in sinus rhythm (Fig 1)
and eight hearts in AF (Fig 2).
Visual inspection of the actin bands, middle lane in Figures 1 and 2, demonstrates that wells in each group were loaded with equal amounts of protein.
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Fig 1. Western blot of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separated HSP60 (top lane), actin (middle lane), and HSP10 (lower lane) from human right atrial specimen in sinus rhythm.
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Fig 2. Western blot of immunochemical detection of HSP60 (top lane), actin (middle lane), and HSP10 (bottom lane) from human right atrial specimen with chronic atrial fibrillation.
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When expressed according to actin level, a significant difference was noted in the mean HSP60 and HSP10 content (± standard deviation). There was more than a 2.3-fold increase in HSP60 content (291 ± 91 ng) in myocardium of patients with AF compared with the mean HSP60 level in myocardium of patient with sinus rhythm (124 ng ± 18 ng; p < 0.001). There was more than a 2.4-fold increase in HSP10 content (34 ± 18 ng) in myocardium of patients with AF compared with the mean HSP10 level in myocardium of patient with sinus rhythm (14 ± 15 ng; p < 0.01).
As shown in Figure 3,
we measured 570 ± 83 ng of HSP60 versus 64 ± 69 ng of HSP10 (per gram wet heart) in the right atrial myocardium from patients in sinus rhythm and 1,119 ± 350 ng of HSP60 versus 130 ± 69 of HSP10 in the myocardium of patients in AF.
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Fig 3. Computerized densitometric HSP60 and HSP10 levels of right atrial myocardium from patients in sinus rhythm (SR) and patients with chronic atrial fibrillation (AF). Values are given as nanograms per gram wet heart. *p < 0.001, **p < 0.01.
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CMMENT
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The majority of proteins for ATP production (eg, F1-ATPase) are synthesized in the cytoplasm and transported to the mitochondrium [9]. Once they are imported in an unfolded state HSP60 and HSP10 are responsible for correct folding and translocation of the mitochondrial proteins [10].
HSP60 is a large donut-shaped protein with a central cavity that is essential for the folding of a huge spectrum of proteins in the mitochondrial matrix. It functions in conjunction with a coprotein, HSP10, which enhances its ability to eject proteins during the ATPase cycle [11].
In CAF, a state of increased atrial activity, there is a need for increased ATP requirements and subsequently higher protein turnover. High levels of unfolded protein are the primary initiator of the HSP synthesis [12]. The presence of high HSP60/HSP10 levels in mitochondria seems to reflect high activity (eg, ATP synthesis) [13].
Inducible HSP60 and HSP10 when coexpressed have a protective effect against ischemic injury as determined by the release of enzymes like creatine kinase and LDH [14]. Cells overexpressing HSP60 and HSP10, forming a chaperon-in-complex, were found to be protected against simulated ischemia [15].
The chronic fibrillating stress leads to an adaptive response of the heart cells. HSP60 mRNA and protein are increased during stimulation [16]. One mechanism to cope with the high energy and protein metabolism might be the upregulation of HSP60 [17]. This could also be a mechanism that the failing myocardium uses to adapt to increased hemodynamic stress [18].
The novel aspect of this study is the detection of an increased HSP60 and HSP10 content in myocardium of patients with CAF. The common and proportional increase in protein expression in CAF might indicate a functional relationship.
Differences in the HSP60 and HSP10 levels could be due to different duration and severity of CAF. Further studies elucidating the role of HSP60 and the coprotein HSP10 in AF need to be undertaken.
In summary, HSP60 and HSP10 contents were elevated in atrial specimens from patients with CAF. Although the exact reason for the hearts of these patients expressing HSP60/HSP10 remains unknown, it may reflect a higher energy metabolism.
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Acknowledgments
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We thank Susanne Heiß for excellent technical support.
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References
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Abstract
Introduction
