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European Journal of Heart Failure 2006 8(4):343-346; doi:10.1016/j.ejheart.2005.10.006
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© 2006 European Society of Cardiology

MLP accumulation and remodelling in the infarcted rat heart

James R. Wildinga, Craig A. Lygateb, Kay E. Daviesc, Stefan Neubauerb and Kieran Clarkea,*

a Departments of Physiology, University of Oxford Oxford, England, UK
b Cardiovascular Medicine, University of Oxford Oxford, England, UK
c Human Anatomy and Genetics, University of Oxford Oxford, England, UK

* Corresponding author. University Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK. Tel.: +44 1865 282248; fax: +44 1865 282272. E-mail address: james.wilding{at}cep.u-psud.fr, Kieran.Clarke{at}physiol.ox.ac.uk


   Abstract
Top
 Abstract
1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
 Acknowledgments
 References
 
Mutation of cytoskeletal protein genes results in abnormal protein function and causes cardiomyopathy. We hypothesised that cardiac levels of cytoskeletal proteins, such as dystrophin, desmin and muscle LIM protein (MLP), would be altered during remodelling caused by myocardial infarction (MI). We measured left-ventricular morphology, function and cytoskeletal protein levels 10 weeks after coronary artery ligation or sham operation in male Wistar rats. Two-dimensional echocardiography revealed significant impairment of systolic function and decreased ejection fraction in infarcted hearts compared with sham (47±5% versus 73±4%), commensurate with the development of heart failure. Western blotting was used to measure levels of β-myosin heavy chain (β-MyHC), a marker of hypertrophy, and levels of dystrophin, desmin, MLP, β-tubulin, utrophin and syncoilin, using GAPDH for normalization. Relative to shams, β-MyHC and MLP levels were increased 1.9-fold and 1.7-fold, respectively, in infarcted rat hearts, whereas the levels of other cytoskeletal proteins were unchanged. Both MLP and desmin protein levels correlated negatively with ejection fraction, with the strongest relation between MLP and ejection fraction (r=–0.95, n=13, p<0.0001). This work suggests that MLP may play an important compensatory role in cardiac remodelling following MI.

Key Words: Myocardial infarction (MI) • Cytoskeleton • Muscle LIM protein (MLP) • Echocardiography

Received November 29, 2004; Revised October 5, 2005; Accepted October 10, 2005


   1. Background
Top
 Abstract
 1. Background
2. Aims
 3. Methods
 4. Results
 5. Conclusions
 Acknowledgments
 References
 
Mutation or ablation of the genes encoding dystrophin, desmin and muscle LIM protein (MLP) causes cardiomyopathy [1,2]. It is unclear whether changes in the function of these cytoskeletal proteins also contribute to non-genetic forms of heart disease, but alterations in their expression, localization, organization and structure post-translation have been reported in various cardiac pathologies [3-8]. In addition, increased β-tubulin levels with microtubule accumulation caused contractile dysfunction in pressure-overload hypertrophy [9,10]. Here, we studied the relationship between left-ventricular remodelling and proteins of the dystrophin/utrophin-protein complex, intermediate filaments (desmin, syncoilin), microtubules (β-tubulin) and the interface between the Z-disk and the costamere/intercalated disk (MLP).


   2. Aims
Top
 Abstract
 1. Background
 2. Aims
3. Methods
 4. Results
 5. Conclusions
 Acknowledgments
 References
 
To determine whether levels of dystrophin, desmin, MLP, β-tubulin, utrophin and syncoilin were altered in the rat left ventricle (LV) following myocardial infarction (MI), and to define the relationship between changes in cardiac cytoskeletal protein levels and in vivo contractile function.


   3. Methods
Top
 Abstract
 1. Background
 2. Aims
 3. Methods
4. Results
 5. Conclusions
 Acknowledgments
 References
 
3.1. Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Male Wistar rats were obtained from a commercial breeder (Harlan, UK) and kept under controlled conditions for temperature, humidity and light, with rat chow and water available ad libitum.

3.2. Coronary artery ligation and echocardiography
The left-anterior descending coronary artery of 200-250 g male Wistar rats was surgically ligated (n=9) or sham-operated (n=4). Ten weeks later, 2D echocardiography was performed, as described previously [11]. Images were stored digitally and analyzed off-line to measure end-diastolic and end-systolic cross-sectional area (EDA and ESA). From these, LV ejection fraction (EF) was estimated (EDA-ESA/EDAx100).

3.3. Protein extraction and immunoblotting
Rats were heparinised (5000 U/kg body weight) and anaesthetised with pentobarbitone (140 mg/kg body weight), administered intraperitoneally. Hearts were removed, weighed and scar tissue was excised. The remaining LV tissue was homogenized in lysis buffer, electrophoretically separated and immunoblotted with antibodies specific for β-myosin heavy chain (β-MyHC), dystrophin, utrophin, syncoilin, β-tubulin, desmin, MLP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described previously [12]. GAPDH was used for normalization because we found unaltered levels of this protein in infarcted hearts, as described previously for GAPDH gene expression [13].

3.4. Statistics
All data are expressed as mean±S.E.M. Differences between two groups were assessed using analysis of variance followed by a Student's t-test, and the relations between cytoskeletal protein levels and ejection fraction by Pearson correlation analysis, using the software program GraphPad Instat v.3.05 (GraphPad Sotware, Inc., San Diego). Significance was defined using a p-value of less than 0.05 (95% confidence).


   4. Results
Top
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
5. Conclusions
 Acknowledgments
 References
 
4.1. Cardiac morphology and function
Ten weeks after surgery, rat heart morphology and function were characterized using echocardiography. Rats with infarcted hearts weighed about 20% less than sham-operated animals, suggesting cachexia (Table 1). Heart and LV (minus scar) weights were therefore normalized to tibia length, a more reliable measure of animal size, but no significant differences were observed, suggesting that hypertrophy occurred in infarcted hearts such that LV weight was maintained. Echocardiography revealed 3.2-fold higher end-systolic areas (p<0.05) and lower ejection fractions (p<0.005) in infarcted hearts than in shams. Expression of β-MyHC, a hypertrophy marker, was 1.9-fold higher in infarcted hearts than in shams (Fig. 1).


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  		Table 1 Cardiac morphology and function in sham and infarcted rat hearts

 


Figure 1
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  		Fig. 1 Levels of β-myosin heavy chain were higher in infarcted rat hearts (n=9) than shams (n=4). Protein levels were measured by Western blot and normalized to GAPDH. *p<0.05 vs. shams.

 
4.2. Cytoskeletal protein levels
Representative immunoblots of cytoskeletal proteins from sham and infarcted rat hearts, plus protein levels, are shown in Fig. 2. Levels of MLP were 1.7-fold higher in infarcted hearts than shams (p<0.005), whereas levels of dystrophin, utrophin, syncoilin, desmin and total β-tubulin were unchanged. Cytoskeletal protein levels were directly related to cardiac function as negative correlations between MLP or desmin levels and ejection fractions were found, such that higher levels of these proteins occurred in hearts with lower ejection fractions (Fig. 3). The relationship between MLP levels and EF was especially strong (r=–0.95, n=13, p<0.0001). MLP and desmin also both correlated negatively with EDA and ESA (data not shown). No other correlations were found between cytoskeletal proteins and LV parameters or β-MyHC levels.


Figure 2
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  		Fig. 2 Representative immunoblots of cytoskeletal proteins in sham-operated (sham, n=4) and infarcted (n=9) rat hearts (upper panel), with quantification of immunoblot band intensities (lower panel). *p<0.005 vs. shams.

 


Figure 3
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  		Fig. 3 Correlation between MLP (upper graph) and desmin (lower graph) levels with LV ejection fraction in sham and infarcted rat hearts (n=13). Statistical significance of correlations: MLP-ejection fraction, p<0.0001; desmin-ejection fraction, p<0.05.

 

   5. Conclusions
Top
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
Acknowledgments
 References
 
Elevated β-MyHC levels after MI indicated foetal gene program activation, characteristic of rodent cardiac hypertrophy and failure [14,15]. Consistent with our data, others reported MLP accumulation in infarcted mouse heart [16] and in hypertrophied myocytes, where it promoted sarcomeric organization [17]. By contrast, MLP loss caused dilated cardiomyopathy and augmented chamber dilation post-MI, suggesting that MLP is important for the hypertrophic response to mechanical stress [2,12,16]. Myocyte hypertrophy is a well-known characteristic of post-infarction remodelling [16,18]; therefore, MLP accumulation here was perhaps related to the extent of myocyte hypertrophy, which is itself related to infarct size [18], and thus functional deterioration. One infarcted heart had 2.1-fold higher MLP levels than the sham average, with EDA, EF and lung weight/tibia length of 0.9 cm2, 29% and 56 mg/mm, respectively, indicating elevated MLP levels in heart failure. This contrasts with a human study reporting lower MLP levels in end-stage heart failure with ischaemic cardiomyopathy [8]. There, patients were treated with nitrates to increase levels of nitric oxide, which causes MLP down-regulation [17], perhaps explaining the discrepancy.

Human studies have reported increased or decreased cardiac desmin levels post-MI [5,19]. Here, desmin accumulation was perhaps associated with myocyte hypertrophy, as in aortic-banded hearts [20]. Dystrophin levels were unchanged in infarcted hearts, in contrast to another study that reported lower levels [3]. However, the previous study found only a small (15%) decrease in dystrophin content, without normalizing to a housekeeping protein. Normal dystrophin, syncoilin and β-tubulin expression in MI suggested that altered levels of these proteins and microtubules did not contribute to post-infarction remodelling. Accumulation of MLP, and possibly desmin, may contribute to compensatory remodelling after MI, with progressive cytoskeletal changes occurring during the transition to heart failure. Cytoskeletal proteins also accumulated in heart failure caused by idiopathic dilated cardiomyopathy and MLP gene deletion [6,12], suggesting the process may be common to heart failure of different aetiologies.


   Acknowledgments
Top
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
 Acknowledgments
References
 
This work was supported by the Wellcome Trust, Worcester College Oxford (JRW) and the British Heart Foundation (CAL, SN and KC). We are grateful to Dr. L. Anderson (University of Newcastle, UK) for the anti-dystrophin antibody, Dr. G.E. Morris (NEWI, UK) for the anti-utrophin antibody and Dr. P. Caroni (FMI, Basel, Switzerland) for the anti-MLP antibody.


   References
Top
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
 Acknowledgments
 References
 

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