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Cardiovascular Research 2002 54(2):380-389; doi:10.1016/S0008-6363(02)00289-4
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Copyright © 2002, European Society of Cardiology

Activation of proteolysis by calpains and structural changes in human paroxysmal and persistent atrial fibrillation

Bianca J.J.M Brundela,b,*, Jannie Ausmad, Isabelle C van Gelderb, Johan J.L Van Der Wantc, Wiek H van Gilsta, Harry J.G.M Crijnse and Robert H Henninga

aDepartment of Clinical Pharmacology, Groningen University Institute for Drug Exploration (GUIDE), Groningen, The Netherlands
bDepartment of Cardiology, Thoraxcenter University Hospital, University of Groningen, Groningen, The Netherlands
cDepartment of Cell Biology and Electron Microscopy, University of Groningen, Groningen, The Netherlands
dDepartment of Physiology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands
eDepartment of Cardiology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands

* Corresponding author. Department of Radiation and Stress Cell Biology, Deusinglaan 1, 9713 AV Groningen, The Netherlands. Tel.: +31-50-363-2911; fax: +31-50-363-2913 b.j.j.m.brundel{at}med.rug.nl

Received 30 November 2001; accepted 6 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Atrial fibrillation (AF) is accompanied by electrical, structural and ion-channel protein remodeling. We tested if proteolysis by calpain and proteasome is activated during AF, and studied the relation with the remodeling processes. Methods: Right atrial appendages were obtained from patients with paroxysmal (n=7) or persistent (n=10) lone AF and compared to controls (n=10) in sinus rhythm undergoing coronary artery bypass grafting (CABG). Proteolysis was measured using Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin. Protein expression of calpain I and II was assessed by Western-blot and calpain I localization by immunohistochemistry. Structural changes were quantified by counting atrial myocytes with contraction bands or hibernation. Results: Calpain activity was significantly increased in paroxysmal AF (2-fold, P<0.001) and persistent AF (3-fold, P<0.001), mainly due to calpain I activation. Increased calpain I protein expression was found in AF with Western blot and immunohistochemistry. Myocytes from all AF groups showed increased contraction bands, whereas hibernation was only found in persistent AF. Calpain activity correlated with L-type Ca2+ channel and Kv1.5 protein amounts (r=–0.80, P<0.001 and r=–0.72, P<0.001, respectively), degree of structural changes (r=0.90, P<0.001), shortening of atrial effective refractory period (AERP) (basic cycle length 500 ms, r=–0.60, P<0.001) and AERP rate adaptation (r=–0.80, P<0.001). Conclusions: Calpain activity is induced during AF and correlates with parameters of ion-channel protein, structural and electrical remodeling. The results suggest that calpain activation represents an important mechanism linking calcium overload to cellular adaptation mechanisms in human AF.

KEYWORDS Apoptosis; Arrhythmia (mechanisms); Calcium (cellular); Hibernation; Remodeling; Supraventr. arrhythmia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Atrial fibrillation (AF) is characterized by heterogeneity in the electrical activation pattern [1,2] and loss of contractility [3,4]. Recent experimental research has demonstrated that AF induces electrophysiological changes, influences protein expression and leads to morphological alterations which in turn increase the vulnerability to AF [5–7]. To explain the electrical remodeling during AF, the expression level of ion-channels have been investigated [8,9]. A reduction in protein levels of a variety of calcium and potassium ion-channels was found [8], of which the L-type Ca2+ channel is believed to play a key role [6]. A relationship between down-regulation of the L-type Ca2+ channel and changes in AERP was recently reported in human AF [8].

The molecular mechanism underlying changes in expression of these proteins is poorly understood. An important clue may be obtained from the finding that reduction in protein expression occurs in absence of changes in mRNA in patients with paroxysmal lone AF [8,9]. Such a discrepancy was also observed by others [10,11] and suggests activation of a protein degradation mechanism. An obvious candidate is proteolysis invoked by calcium dependent neutral proteases, calpain I and calpain II, as the cytosolic calcium increases during AF [12,13]. An increased cardiac calpain activity in cardiac myocytes has been demonstrated following metabolic inhibition, cardiac stunning and calcium overload [14,15]. Alternatively, activation of the proteasome may also underlie increased proteolysis in cardiac cells, as shown for degradation of myosin heavy chain and connexin43 in rat [16,17].

To examine whether proteolytic activation plays a role in human AF, we determined the activity of calpains and the proteasome in atrial tissue of patients with paroxysmal and persistent lone AF and of controls in sinus rhythm. An increased calpain-mediated proteolysis was found in AF. Therefore, the protein levels of calpain I and II were determined and localization of calpain I was performed by immunohistochemistry. Since calpains degrade structural proteins [18], the structural changes of atrial tissue were investigated by light and electron microscopy. Finally, we examined the relationship of calpain activation with structural changes found in this study and with changes in the expression of various ion-channels and electrophysiology as reported for the same patient population previously [8].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Patients
Right atrial appendices (RAAs) were obtained as described before [8] from patients with normal left ventricular function and paroxysmal (n=7) or persistent (n=10) lone AF undergoing MAZE surgery and controls in sinus rhythm undergoing coronary bypass grafting (CABG, n=10, Table 1). The Institutional Review Board approved the study and patients gave written informed consent. Changes in electrophysiology were reported previously [8] by obtaining intra-operative AERP at five basic cycle lengths (BCL: 250–600 ms). The adjustment of AERP to BCL was quantified by calculating the rate adaptation coefficient as the slope of the linear regression after logarithmic transformation of BCL [8]. The mRNA and protein levels of ion-channels in this population were also reported previously [8].


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  		Table 1 Baseline characteristics of patients with lone paroxysmal AF (PAF), lone persistent AF (CAF) and control patients in sinus rhythm (SR)

 
2.2 Protein extraction
For analysis of proteolysis, frozen RAAs were homogenized in buffer (100 mM Tris–HCl, 145 mM NaCl, pH=7.3) and centrifuged at 26 000xg (30 min, 4 °C). For Western-blot analysis, parts of the same RAAs were homogenized in Radio-Immuno-Precipitation-Assay (RIPA) buffer [8]. Protein concentration was determined using the DC assay (Bio-Rad) with a bovine albumin standard.

2.3 Proteolytic assay
Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (AMC, Sigma) was used as substrate. A 25 µg protein extract was added to 20 µM AMC in 300 µl Tris-buffered saline. AMC release was measured by fluorometry (360-nm excitation; 430-nm emission, Spectrometer LS50B, Perkin Elmer) after incubation for 30 min at 25 °C. Standard curves were generated using known concentrations of 7-amino-4 methyl-coumarin (Sigma) and 25 µg heat denatured protein. Maximal calpain activation was assessed after reconstitution of calcium at 1 mM. E-64 (10–4 M, Roche), calpain I inhibitor (N-acetyl-Leu-Leu-norleucinal, 10–4 M, Sigma) and calpain II inhibitor (N-acetyl-Leu-Leu-methioninal, 10–4 M, Sigma) were used to assess calpain activation. Lactacystin (10–4 M, Calbiochem) was used to investigate proteasome activity. All inhibitors were added 30 min before measuring AMC release. The amount of native protein chosen was in the linear AMC release signal area. Protease inhibitors were used at concentrations rendering maximal inhibition. Assays were performed in triplicate. Tissue calpain activity was calculated as the amount of protease activity specifically blocked by E-64.

2.4 Western blot
Protein levels of calpains were determined by Western blot and expressed as ratio to levels of GAPDH. Denatured protein (10 µg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes (Stratagene) followed by staining with Ponceau S solution (Sigma). Membranes were incubated with primary antibody against GAPDH (Affinity Reagents, USA), large subunit of calpain I or calpain II (both Research Diagnostics, USA). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa-Cruz Biotechnology) was used as secondary antibody. Signals were detected by the ECL-detection method (Amersham) and quantified by densitometry. The amount of protein chosen was in the linear immunoreactive signal range. The specificity of the antibodies was checked by pre-incubation with control peptide antigen.

2.5 Immunohistochemistry
Immunohistochemistry was performed in six patients with paroxysmal AF, eight with persistent AF and eight control patients. Two groups of 5 µm thick frozen RAA sections were made. Sections were air dried before use or immediately fixed for 10 min in 4% paraformaldehyde in phosphate buffered saline (PBS). After three washes (PBS, 10 min) and 30 min blocking with 1% BSA in PBS, sections were incubated with anti-calpain I antibodies (1:100) (Research Diagnostics, USA) overnight at room temperature. After three washes with PBS (5 min), sections were incubated with peroxidase conjugated rabbit-anti-mouse IgG (DAKO A/S, Glostrup, Denmark, 1 h). Three washes with PBS were followed by one with aqua dest (5 min), peroxidase activity was detected using a buffer of 40 mg 3-amino-9-ethylcarbazole (Sigma), 10 ml N,N dimethylformamide (Merck), hydrogen peroxide 0.01% (v/v) and 190 ml 0.05 M sodium acetate (pH 4.95). After staining for 10 min, sections were rinsed with water, counterstained with haematoxylin (Sigma) and mounted with Kaiser's glycerol gelatin (Merck).

2.6 Morphological evaluation
Biopsies from RAAs were immediately fixed for 2 h in 2% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.4) with post-fixation for 2 h in 1% osmium tetroxide (supplemented with 1.5% K4Fe(CN)6 in cacodylate buffer, pH 7.4) at 4 °C and embedded in Epon. Light microscopy was performed using semi-thin sections (1 µm) stained with 1% toluidine blue. Changes were evaluated in at least 300 cells from six randomly chosen regions with the nucleus in the plane of the section by an investigator blinded for patient groups, who scored: (1) hibernating cells, showing areas of loss of sarcomeres (<10% of the cell surface) and pale nuclei [7]; and (2) cells displaying intensely stained contraction bands (<10% of sarcomere surface) [19]. Ultrastructural changes were studied by electron microscopy (Philips 201, 60 kV) on ultrathin sectons (60 nm) stained with uranylacetate and lead citrate.

2.7 Statistical analysis
Results are expressed as mean±S.D. One-way ANOVA was used for multiple group comparisons. Correlation was determined with Spearman correlation test (SPSS 8.0). P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Proteolytic activity
Proteolytic activity was significantly increased in tissue from patients with paroxysmal and persistent AF compared to patients in sinus rhythm (Fig. 1A, Table 2).


Figure 1
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  		Fig. 1 Significant increase of tissue calpain activity (A) in RAAs of patients with sinus rhythm (SR; {circ}), paroxysmal AF (PAF; {odot}) and persistent AF (CAF; bullet). (B) Significant correlation for maximal and tissue calpain activity. Calpain activity was expressed as nM AMC/mg protein/30 min. (C) Typical Western-blot of calpain I expressed as ratio to GAPDH and group protein ratios for calpain I. (D) Significant correlation of calpain activity and calpain I levels. * P<0.01 compared to SR.

 

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  		Table 2 Proteolytic activity (nM AMC/mg protein/30 min) in atrial tissue of patients with lone paroxysmal AF (PAF), lone persistent AF (CAF) and control patients in sinus rhythm (SR)

 
Proteolytic activity was measured in the presence of inhibitors to assess the involvement of different pathways (Table 2). The non-selective calpain inhibitor E-64 (10–4 M) and calpain I inhibitor (10–4 M) significantly reduced proteolytic activity to a similar level in all groups. Calpain II inhibitor (10–4 M) only partially attenuated the increased proteolytic activity observed in AF, while the proteasome inhibitor lactacystin (10–4 M) did not reduce proteolytic activity in any group. To test whether increased calpain activity was due to calcium in the protein extract, experiments were conducted in the presence of 10 mM EDTA. No differences were found in proteolysis in the presence or absence of EDTA (sinus rhythm: 96%±8, paroxysmal AF: 93%±7; persistent AF:101%±10).

Maximal calpain activity was determined by addition of 1 mM calcium, resulting in an extra activation of proteolysis across all patient groups (Table 2). The extra activation due to addition of calcium was abolished by E-64, calpain I inhibitor and calpain II inhibitor, but unaffected by lactacystin. Furthermore, tissue calpain activity across all groups correlated significantly with the maximal calpain activity (r=0.89, P<0.001; Fig. 1B).

3.2 Calpain protein levels
To examine the relation between calpain activation and expression, proteins levels of calpains were determined by Western-blotting (Fig. 1C,D). Protein levels of calpain I were significantly increased by 35% (P<0.01) in patients with persistent AF compared to controls (Fig. 1C), whereas calpain II levels were similar in all groups (sinus rhythm: 0.75±0.05; paroxysmal AF: 0.68±0.06; persistent AF: 0.7±0.06). A positive correlation was found between tissue calpain activity and protein expression of calpain I (r=0.61, P<0.001, Fig. 1D), whereas no correlation was found with calpain II expression (r=–0.20, P=0.33). GAPDH levels did not differ between the groups (SR: 102±10, PAF: 109±7, CAF: 98±11, arbitrary units).

3.3 Localization of calpain I
Calpain I localization was studied by immunohistochemistry to determine the cell type responsible for its increase in activity and expression. Calpain I was predominantly localized in the nucleus, intercalated discs and in the cytoplasm of atrial myocytes (Fig. 2A,B). To obtain an indication about the amount of calpain I protein in the myocytes, the staining intensity was scored semi-quantitatively (Fig. 2C–E). Staining intensity of the nucleus and the intercalated discs increased significantly from sinus rhythm, via paroxysmal AF to reach a maximum in persistent AF (Fig. 2C,D). In contrast, staining intensity in the cytoplasm did not differ between the groups (Fig. 2E).


Figure 2
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  		Fig. 2 (A) Immunohistochemical localization of calpain I in myocytes in the nucleus (arrow) and (B) at the intercalated discs (arrow) (magnification (A) x400, (B) x300). Intensity of the calpain I staining in patients with PAF (n=6; {odot}), CAF (n=8; bullet) and SR (n=8; {circ}) in the nucleus (C), at the intercalated discs (D) and in the cytosol (E). ± low staining – +++ intense staining; * P<0.05, ** P<0.01, compared to SR.

 
3.4 Structural changes
Structural and ultra-structural changes in atrial tissue were examined by light and electron microscopy. Two types of structural changes were found in the atrial myocytes, i.e. contraction bands (Fig. 3C) and with hibernation, characterized mainly by a reduced number of sarcomeres (myolysis) (Fig. 3E). Atrial myocardium of sinus rhythm patients showed normally structured myocytes mainly without myolysis (5%) and contraction bands (12%) (Figs. 3A and 4AGo). At the ultrastructural level, a highly organized sarcomeric structure with mitochondria in between, and a typical clustering of heterochromatin at the nuclear membrane were found (Fig. 3B). In contrast, in patients with paroxysmal AF, contraction bands were abundantly present (Fig. 3C) and these myocytes showed pyknotic nuclei. Also secondary lysosome like bodies were present at the ultrastructural level (Fig. 3D). Patients with persistent AF revealed both contraction bands and hibernation (Fig. 3E,F). The number of myocytes with contraction bands was increased 3-fold in both AF groups compared to controls (Fig. 4A), while the amount of hibernating cells was only significantly increased in persistent AF (Fig. 4A). Furthermore, a correlation was found between the calpain activity and the total number of affected cells (either contraction bands or hibernation; r=0.71, P<0.001; Fig. 4B).


Figure 3
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  		Fig. 3 Typical examples of structural and ultrastructural changes in atrial myocytes. Control patient examined by light (A) and electron microscopy (B), showing normal myocytes without myolysis, contraction bands and a normal nucleus. Paroxysmal AF patient show contraction bands (C, arrow) and degenerative features as clumping of nuclear chromatin and secondary lysosomelike bodies (D, arrow). Persistent AF patient show extensive perinuclear myolysis and pale nuclei (E,F). Further, only a rim of sarcomeres is present at the border of the cell, glycogen (g) is visible and the nucleus has a homogenously dispersed chromatin (F). (Magnification A, C and E x250, B x4250, D x3250 and F x3400).

 

Figure 4
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  		Fig. 4 (A) Percentage structural changes of atrial myocytes affected by contaction bands or hibernation in patients with sinus rhythm (SR), paroysmal AF (PAF) and chronic, persistent AF (CAF). (B) Significant correlation between the total amount of affected cells and calpain activity. (C) Significant correlation between the type of structural change and the duration of persistent AF. (bullet) contraction bands and ({blacksquare}) hibernation.

 
When the type of structural change, i.e. contraction bands or hibernation, of patients with persistent AF was plotted against the duration of AF an intriguing relationship was observed (Fig. 4C). Atrial myocytes of patients with the shortest duration of AF (<10 months) showed high amounts of contraction bands and low amounts of hibernating cells, whereas the opposite pattern was found in patients with the longest duration of AF (<10 months).

3.5 Relation between calpain activation, ion-channel expression and AERP
Previously we reported changes in ion-channel mRNA and protein levels in atrial tissue of this patient population [8]. In brief, the reduction in protein expression of the L-type Ca2+ channel, Kv1.5 and HERG was accompanied with little or minor changes in mRNA levels. In contrast, a reduction in both mRNA and protein levels, showing a significant correlation, was observed for Kv4.3. To investigate a potential role of calpain activation in reduction of these ion-channels, calpain activity was correlated with ion-channel protein levels. A highly significant correlation was found between calpain activity and reduction in L-type Ca2+-channel and Kv1.5 protein, whereas correlation with HERG was less prominent and with Kv4.3 was absent (Fig. 5).


Figure 5
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  		Fig. 5 Correlation between tissue calpain activity and expression of various plasma membrane ion-channels. (A) anti-L-type calcium channel, (B) anti-Kv1.5, (C) anti-Kv4.3 and (D) HERG. ({odot}) indicates patients with PAF, (bullet) CAF and ({circ}) SR.

 
Patients with AF demonstrated significantly shorter AERPs and poorer rate adaptation than control patients in sinus rhythm, as reported previously [8]. To investigate whether calpain activation relates to electrical remodeling, a correlation between calpain activity, AERPs and rate adaptation coefficient was made. Calpain activity showed a significant negative correlation with AERP at different BCLs and with the rate adaptation coefficient (Table 3).


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  		Table 3 Correlation of calpain activity with AERP and rate adaptation coefficient

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The main finding of this study is the activation of calpain-mediated proteolysis in atrial tissue of patients with paroxysmal and persistent lone AF. Increased calpain activity was accompanied by increased expression of calpain I and intensified staining of calpain I in atrial myocytes. Furthermore, calpain activity correlated well with several parameters of electrical, structural and protein remodeling. Activation of proteolysis by calpain explains the reduction of ion-channel proteins found in the presence of unchanged mRNA levels in patients with lone, paroxysmal AF [8], and may represent one important molecular event in various remodeling processes observed in AF.

4.1 Calpain activation
This study demonstrates increased proteolytic activity in atrial myocytes of patients with AF due to activation of calpains. This is evidenced by the reduction of proteolysis to similar levels in all groups by the non-selective calpain inhibitor E-64, even under high calcium conditions known to activate calpain II. In contrast, the selective inhibitor of the proteasome, lactacystin, did not influence proteolysis in any group. In addition, Western-blot revealed increased protein levels of calpain I in persistent AF, but unchanged levels of calpain II. Finally, intensity of calpain I staining was increased in AF patients. Taken together, these findings represent a report of activation of the calpain pathway in human cardiac disease.

The relative contribution of calpain I and II is difficult to establish by using inhibitors, due to their partial selectivity. However, calpain I inhibitor completely attenuated the increased proteolysis in AF both under basal and high calcium conditions, whereas calpain II inhibitor did not. Moreover, calpain I protein level was increased and correlated with increased calpain activity, in contrast to that of calpain II. Therefore, our results suggest that the increased calpain activity in AF is mainly due to activation and up-regulation of calpain I, rather than calpain II.

The staining intensity of calpain I was increased at the intercalated discs and nucleus in patients with AF, in accord with the observed increase in activity and protein expression. Increased calpain I expression in AF seems confined to cellular areas that are vital for action potential conduction and structural integrity of the atrial myocytes. Under normal conditions, calpain is localized diffusely in the cytosol. After an increase in intracellular calcium, calpain rapidly translocates to the inner surface of the plasma membrane, aggregates at the intercalated discs, which is followed by its activation [20]. At the intercalated discs, activated calpain I may degrade important ion-channels, like the Na+-channel [21] and Kv1.5 [22], but also proteins involved in excitation-contraction coupling [23] and conduction [10]. The increased expression of calpain I at the nucleus might be suggestive for a role in promoting cell death [24], indicating that calpain I may play a role in the degeneration observed in AF [25].

4.2 Structural changes in myocytes
This study in human AF investigates myocyte degeneration and hibernation in atrial tissue. Contraction bands were increased in equal amounts both in patients with persistent and paroxysmal AF. Contraction bands were previously observed in degenerative myocardium [25,26]. In contrast, hibernating cells were only increased in patients with persistent AF. The latter is in agreement with the experimental goat model for AF, in which hibernation develops only at a relatively late stage of the arrhythmia [7]. Thus, these findings suggest that contraction bands represent an early structural abnormality in human lone AF, whereas hibernation develops only after a protracted period of sustained AF. Similar alterations in myocardial structure were described in patients with atrial arrhythmias of various aetiology [27]. Moreover, in persistent AF, the number of hibernating cells increased with the duration of AF, while contraction bands decreased. Hibernating myocardium is suggested to be protected against degeneration [28]. Taken together, the observed structural changes are indicative of a substantial deterioration of normal tissue architecture, likely to promote AF through heterogeneity of atrial refractoriness [1,2] and slowed atrial conduction [29–31].

4.3 Calpain activation and remodeling
Calpain activity correlated well with the observed structural changes and previously measured AERP adjustments. Furthermore, a correlation with protein levels was present for three ion-channels, in which mRNA levels did not account for reduction in channel protein [8]. The significant correlation with protein levels of L-type Ca2+ channel and Kv1.5 may be explained by the fact that these undergo the most profound protein reduction and that both have been described as targets for calpain degradation [22,32].

As a consequence, most parameters show a correlation with each other. Therefore, it remains unclear whether calpain activation causes the molecular changes associated with remodeling, or merely reflects gross cellular damage. However, the up-regulation of calpain I protein seems incompatible with general cellular damage. Also on theoretical grounds, calpain activation represents a likely candidate to convey important cellular changes in AF. Calpains have been demonstrated to degrade cytoskeletal [18], contractile [15] and L-type Ca2+ channel [32] proteins that are involved in atrial remodeling in AF. The reduction in expression of L-type Ca2+ channel is thought to play a major role in electrical remodeling in AF [6]. Calpains are readily activated by elevated cellular calcium levels [33,34], while calcium overload plays a key role in the pathogenesis of AF [8,12,13,35]. Of note is that calcium overload is also responsible for the initiation of additional short-term adaptation mechanism through inactivation of functional Ca2+ current [36].

Irrespective of whether calpain activation is an initiator or a consequence of molecular remodeling, its activation is likely to contribute to further deterioration of atrial tissue. Therefore, interference with the calpain pathway may represent an important tool to unravel the sequence of molecular events in AF, and perhaps a future strategy for pharmacological intervention.

4.4 Possible clinical relevance
The success rate of chemical and electrical cardioversion and the time needed to recover atrial contractile function depends on the AF duration [4]. In addition to electrical remodeling, the structural remodeling of atrial myocytes may contribute to the intractability of long-term AF. After restoration of normal sinus rhythm, it may take the cardiomyocyte a certain period to normalize levels of sarcomeres, if possible at all (J. Ausma et al. unpublished results). The early structural changes observed in paroxysmal AF underscores the importance to restore sinus rhythm as soon as possible, thereby preventing the continuation of the atrial structural, ion-channel protein and electrical remodeling. Whether calpain inhibitors may serve to protect atria in this respect, remains to be investigated.

4.5 Limitations of the study
Ideally, the quantification of calpain I staining intensity would need staining of a reference protein to correct for potential differences during development. However, it is hard to define a suitable reference protein in AF, in view of the notable structural changes of myocytes. To avoid this experimental bias, samples were cut, fixed, incubated and stained simultaneously. The good agreement between upregulation of calpain I protein found in Western Blot and in the staining, suggests—but does not prove—that the method was adequate. Therefore staining quantification data should be interpreted with care.

Time for primary review 17 days.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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