Skip Navigation

Cardiovascular Research 2000 46(2):346-355; doi:10.1016/S0008-6363(00)00034-1
© 2000 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Oliviéro, P.
Right arrow Articles by Samuel, J.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oliviéro, P.
Right arrow Articles by Samuel, J.-L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Expression of laminin {alpha}2 chain during normal and pathological growth of myocardium in rat and human

Patricia Oliviéroa, Catherine Chassagnea, Nathalie Salichona, Alain Corbierb, Gilles Hamonb, Françoise Marottea, Danièle Charlemagnea, Lydie Rappaporta and Jane-Lise Samuela,*

aU127 INSERM, IFR—Circulation Paris VII, Hopital Lariboisière, 41 Bd Chapelle, Université D. Diderot, 75475 Paris, France
bHoechst Marion Roussel, 111 route de Noisy, Romainville, France

* Corresponding author. Tel.: +33-144-631-741; fax: +33-148-742-315

Received 5 October 1999; accepted 28 January 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Fibrosis is a classical feature of cardiac hypertrophy. To date changes within the basal lamina during normal and pathological cardiac growth have been poorly investigated. The goal of the present study was to determine if the expression of the muscle specific subunit of merosin (laminin {alpha}2 chain) together with that of fibronectin (FN) is modified in the diseased human heart. Laminin {alpha}2 chain expression was also investigated during physiological and pathological cardiac growth in the rat. Methods: In ten normal human hearts and ten hearts with idiopathic dilated cardiomyopathy (IDCM), the laminin-{alpha}2 and FN mRNA levels were quantified by slot-blot using total RNA and the protein distribution was analysed using an immunofluorescence approach. In Wistar rats, laminin {alpha}2 and FN mRNA expression was analyzed using RNase protection assay (RPA) and slot-blot assays. Results: The amount of laminin {alpha}2 mRNA did not vary in normal and pathological human hearts whereas it was significantly decreased in renovascular hypertensive rats (–20%) P<0.05 versus normal tissue). The amount of fibronectin mRNA increased in IDMC patients (x2, P<0.05 versus normal tissue), but was unchanged in hypertensive rats. A negative correlation was found between the cardiac laminin-{alpha}2 level and the age of the patients whatever the cardiac status. During postnatal development in the rat, a similar decrease in cardiac laminin-{alpha}2 level was observed between 3 and 30 weeks of age. Finally, the immunofluorescent approach failed to detect any alteration in laminin {alpha}2 distribution within the human myocardium. Conclusion: These data indicate that an imbalance between myocyte hypertrophy and the level of laminin-{alpha}2 might contribute to alterations in sarcolemmal properties, which occur during the development of cardiac hypertrophy and its transition to cardiac failure.

KEYWORDS Extracellular matrix; Hypertrophy


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Laminin and fibronectin are two glycoproteins which together with type IV collagen, and heparan sulfate proteoglycan, constitute a network in the basal lamina (reviewed in Ref. [1]). Fibronectin is localized predominantly in the vicinity of smooth muscle cells and fibroblasts whereas laminin is also found in the basal lamina of cardiomyocytes. Both proteins modulate cell adhesion by interacting with cellular receptors, such as integrins [1,2], but laminin also attaches to {alpha}-dystroglycan, forming a transmembrane link to the intracellular cytoskeleton via dystrophin [3]. Laminin is composed of three subunits ({alpha}, β, {gamma}) which are organized in a three-dimensional intertwinned structure. Among the numerous {alpha}-chain isoforms that have been described, the {alpha}1 chain is found in the basement membrane of epithelial, endothelial and smooth muscle cells, while the {alpha}2 chain isoform has only been identified in striated muscles and peripheral nerves [4,5]. Thus, the laminin {alpha}2 chain appears to be expressed specifically in the basal lamina of striated muscles, and a deficiency in its expression results in a muscular dystrophy at birth or in early childhood which is accompanied by a dilated cardiomyopathy [6–9].

Fibronectin is a dimeric glycoprotein. The protein is expressed by all the cell types within the heart and binds to integrins through the specific amino acid sequence RGD. Multiple forms of FN arise by the alternative splicing of a primary transcript. Alternatively spliced elements, (FN-EIIIA or FN ED-A and FN-EIIIB or FN ED-B) are specifically expressed at the embryonic and fetal stages of development and are reexpressed in pathological conditions [10].

Fibronectin and laminin mediate cell migration, growth and differentiation but with different characteristics and specificity [11,12]: fibronectin promotes the modulation of cultured smooth muscle cells from a contractile to a synthetic phenotype, while laminin maintains the cells in a contractile phenotype during the first few days in primary culture [11]. Fibronectin and laminin also have opposite effects in the control of adhesiveness of cardiac myocytes since only laminin is essential for cell survival in culture conditions [12]. The maturation of arterial human smooth muscle is accompanied by a switch in the expression from {alpha}1β1 {gamma}1 to {alpha}1β2 {gamma}1 laminin, the only protein variant associated with the differentiated phenotype [13]. However it is not known whether the cardiac expression of the {alpha}2 isoform in the heart is developmentally regulated.

Several groups, including ours, have described an increased expression of basal lamina associated proteins, such as fibronectin [14–17], collagen III [16–19] and IV [16] and laminin β chain [20] in the hearts of hypertensive rats. However the increase in fibronectin or collagen I—III reflected the development of fibrosis whereas changes in collagen type IV and laminin β reflected changes in basal lamina independently of their location in the endothelium or around smooth and striated muscle cells. Therefore in the heart, the laminin-{alpha}2 chain is a unique marker for the cardiomyocyte basal lamina. We have analyzed the accumulation of laminin-{alpha}2 chain during normal and pathological cardiac growth (i) in human (normal versus dilated idiopathic cardiomyopathy), (ii) in Wistar rats (normal versus renovascular hypertension model), and have compared the laminin pattern to that of fibronectin.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Human samples
The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovasc Res. 1997;35:2–3). The study was approved by the consultative Committee for the protection of Human Subjects in Biomedical Research at the Pitié–Salpétrière in Paris. The ten non failing hearts (8 males, 2 females) were obtained from adult patients who died from head trauma (n=9) or smoke inhalation (n=1). They were ultimately rejected for transplantation for reasons which were not related to their cardiac function. Ten failing hearts from patients with IDCM (9 males, 1 female) were obtained at the time of transplantation. The left ventricular ejection fraction was less than 20%, the heart weight was 547±79 g. Transmural samples (0.5–2 g) were quickly excised from the anterior free wall of the left ventricle and immersed in liquid nitrogen. Besides, the study included four dilated cardiomyopathies which exhibited signs of ischemic disease (mixed cardiomyopathies: MCM) [21]. Tissues were kept frozen (–80°C) until use, some fragments were embedded in OCT (RUA, Torcy France) prior to immunohistochemical studies (n=4–5 per group).

2.2 Experimental models
This study was conducted in a accordance with the institutional guidelines and those formulate by the European Community for the use of experimental animals. Wistar rats were from Iffa-Credo (Lyon, France).

2.2 Ontogenic development
Hearts from 19-day post-coitus fetuses, 1-day and 2, 3, 8 and 30-week old rats (n=6–8 per groups) were included in the study. Until 2 weeks of age both male and female rats were used, afterwards only males were included in the study.

2.2 Renovascular hypertension
Twelve male Wistar rats (2 month-old), weighing 200±20 g were used in this study. The animals were randomly distributed in two groups of six experimental and six control animals. The same surgery was performed on all animals excepted that a clip was positioned around the isolated right renal artery of the experimental animals (Goldblatt: one clip, two kidneys). The systolic blood pressure (SBP) was measured by the tail-cuff method before and then weekly after surgery. Four weeks after surgery, the rats were weighted and then sacrificed under anesthesia. The hearts were rapidly removed, trimmed free of large vessels and weighed. The cardiac ventricles were divided in half. The lower halves were immediately frozen in liquid nitrogen and stored at –80°C prior to RNA isolation.

2.3 Immunolabelling
Serial ventricular cryosections (5 µm) were labeled using a double immunolabelling technique previously described in detail [22]. Briefly, cardiac sections were incubated overnight at 4°C with monoclonal antibodies directed against either merosin (laminin {alpha}2) or FN-EDA diluted (1/10 or 1/3, respectively) in phosphate-buffered saline (PBS) (mmol/l: NaCl, 150; KCl, 2.5; phosphate buffer, 10; pH 7.2) containing 2% bovine serum albumin (BSA). Sections were rinsed twice in PBS at room temperature, then incubated for 30 min at 37°C with rabbit polyclonal IgGs directed against the plasma form of FN (total-FN, 1/50) or total laminin (1/100), in PBS/BSA. After two washings in PBS, sections were incubated for 30 min at room temperature with biotinylated anti-mouse IgGs (1/30 dilution in PBS containing 2% rat serum, Vector Laboratories, Biosys, Compiègne, France). Sections were washed again in PBS and incubated with anti-rabbit IgGs conjugated to FITC (1/40 Amersham, Les Ulis, France), and then with a streptavidin Texas Red complex (1/40 dilution in PBS, Amersham). Sections were mounted in aqueous medium (Fluoprep, Biomerieux, Marcy l’Etoile, France). Fluorescence was observed using a Leica microscope equipped with epifluorescence optics (Leica, Reuil-Malmaison, France).

2.4 Analysis and quantification of mRNAs
2.4 RNA extraction
Total RNA was extracted from human and rat tissue by the RNA-quick procedure (Trizol, Bioprobe), according to Chomczynski and Sacchi [23]. RNA concentrations were measured by their absorbance at 260 nm, assuming that 40 µg gives one absorbance unit. The RNA were resuspended in Tris—HCl—EDTA (TE pH 7.4) and aliquots were stored at –80°C.

2.4 Probes
The human laminin {alpha}2 cDNAs were obtained by RT-PCR using human placenta total RNA and primers specific for the coding region of human laminin {alpha}2 DNA [oligonucleotide antisense 5'-GCA GCC AGT GAA TGT AAT CAC ACG TAC-3' and oligonucleotide sense 5'-AAA GTA TCT GTG TCT TCA GGA GGT GAC-3'] which corresponds to a sequence of 474 bp. The RT-PCR product was subcloned into the EcoRI site of pCR.2.1TM vector (Kit TA cloning, Invitrogen) and its sequence was confirmed.

The rat laminin {alpha}2 cDNAs were obtained by RT-PCR using rat heart RNA and primers specific for human laminin {alpha}2 DNA [oligonucleotide sense: 5'-AAA GTA TCT GTG TCT TCA GGA-3', antisense 5'-AGT GAA TGT AAT CAC ACG TAC AGC-3'] (Eurogentec, Seraing, Belgium) were subcloned into pBS plasmid according to the manufacturers instructions (R&D Systems, London). The rat {alpha}2 laminin DNA sequence analysis indicated a homology >90% with the corresponding human sequence.

For RNase protection assays (RPA), Sp6 and T7 polymerase (Boehringer) generated transcripts derived from vectors containing the laminin {alpha}2 and GAPDH or MyHC cDNA, were used. The rat laminin {alpha}2 plasmid was linearized with EcoRV and transcribed in vitro in the presence of SP6 RNA polymerase, generating a 563-bp probe. A rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA subcloned into the ApaI/XbaI site of pBluescript IISK±, was linearized with StyI and transcribed in vitro using T7 RNA polymerase generating a 185-bp probe. For MyHC cRNA probe, the {alpha},β MyHC clone [24] was linearized with HindIII and transcribed in vitro with T7 polymerase generating a 220 nt cRNA identical for both {alpha}-and β-MyHC sequence except for four substitutions in the last 14 nucleotides allowing the identification of the {alpha}- and β-MyHC transcripts. All the cRNA probes were labeled using [{alpha}-32P]UTP (3000 Ci/mmol, Amersham), and the full-length fragments were purified on acrylamide—urea denaturing gels,

For slot-blot hybridization, the EcoRI/BamHI (247 nt) insert from FN212 clone [14], the EcoRI insert of both rat (466 nt) and human (583 nt) laminin-{alpha}2 clones, the XbaI/ApaI insert from GAPDH (860 nt) clone and the PST1 insert from {alpha},β MyHC clone (158 nt) were used. The fragments were purified and radiolabeled with 32P to obtain a specific activity of 1x106 cpm/ml using random primers, DNA polymerase I (Klenow, Boehringer Mannheim), [{alpha}-32P]dCTP (3000 Ci/mmol) and the Multiprime DNA labelling system (Amersham). A 24 oligonucleotide (5'-ACGGTATCTGATCGTCTTCGA-ACC-3') complementary to part of the rat 18S RNA (Institut Pasteur, Paris, France) was 5'-end-labelled in the presence of [{gamma}32P]ATP and polynucleotide kinase. The labelled probes were separated from unincorporated 32P-dCTP or 32P-ATP by gel filtration (BioSpin-30 or BioSpin-6, BioRad) and the specific activity of each probe was measured before utilization.

2.4 RNase protection analysis
The general procedure followed was that outlined in the instructions supplied with RPA IITM kit (Ambion). The assays were performed on 10 µg of total RNA using laminin {alpha}2 and GAPDH or MyHC cRNA, respectively. The full-length cRNA labelled probes were hybridized to total RNA, in 80% formamide, 100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), 1 mM EDTA at 45°C overnight, then digested with 0.5 µg of RNaseA and 10 Units of RNaseT1 (Ambion, Clinisciences, France), at 37°C for 30 min. Each experiment included a control reaction in which total RNA was replaced by 10 µg of yeast tRNA. After digestion, the protected probes were separated by electrophoresis on 6% polyacrylamide gels. The gels were dried and exposed to the screen of a phospho-imager (Fuggi). The images were analysed using Mac-Bas software to quantify the protected labeled RNAs.

2.4 Slot-blot and Northern blot analysis
For slot blots, serial dilutions from 0.25 to 7.5 µg of total RNA were denatured in 15x SSC, 3% formaldehyde at 65°C for 15 min, rapidly cooled on ice and spotted onto the Hybond N membrane (Amersham) using a minifold apparatus (Schleicher & Schuell, Inc., Keene, NH). For Northern-blots, 10 µg of total RNA were fractionated by electrophoresis on 1% agarose gels containing 3% formaldehyde and transferred to nylon membranes (Hybond N, Amersham). After crosslinking of RNA to the membrane by UV, the membranes were prehybridized at 42°C for at least 4 h in a buffer containing [50% formamide, 5x Denhart’s solution; 5x SSC; 0.5% SDS and 100 µg/ml sonicated denatured herring sperm DNA]. The membranes were hybridized with 32P-labeled laminin {alpha}2, FN, GAPDH, MyHC or 18S probes. Hybridization of the probes was performed overnight at 42°C in the prehybridization medium. Excess of probes was removed at 24°C in 0.2x SSC, 0.1% SDS. The membranes were then exposed to the screen of a phosphor-imager (Fuggi). The images were analyzed using MacBas software to quantify the signals.

Values were normalized to the amount of GAPDH mRNA, MyHC mRNA (RPA assays) or total RNA (slot-blot). The equal amounts of total RNA loaded onto the slot-blot were checked by hybridization with 18S probe. Because in human tissue the amounts of GAPDH mRNAs vary with cardiac status we preferred to refer to the µg of total RNA since the yield of RNA per gram of tissue is constant [21]

2.5 Statistical analysis
Results for the various groups are expressed as means±S.E.M. Differences across groups were evaluated using one-way analysis of variance (ANOVA), and group-to-group comparisons were done using Student’s t-test and Bonferroni analysis. In all experiments, the accepted level of significance was P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Distribution of laminin {alpha}2 chain and fibronectin in human cardiomyopathies and quantification of the corresponding mRNAs.
The distribution of the laminin {alpha}2 chain subunit was analyzed and compared to that of total laminin and fibronectin isoforms, using an immunofluorescence approach with either polyclonal antibodies that recognize all isoforms of either laminin or fibronectin or monoclonal antibodies specific for the laminin {alpha}2 chain or FN-EDA isoform.

Firstly, we verified that in control hearts, total laminin was detected at the level of the basal laminae of the cardiomyocytes, capillary endothelial cells and smooth muscle cells from the arteries (Fig. 1a), whereas the laminin {alpha}2 chain subunit was present only in the basal lamina of the cardiomyocytes (Fig. 1b). In the pathological tissue sections, the laminin staining of the basal lamina revealed the hypertrophy of the myocytes but no significant alteration in the distribution of either total laminin or laminin {alpha}2 was observed (Fig. 1c,d). Total FN was detected in the interstitial space and the basal laminae of the cardiomyocytes as well as in the wall of coronary arteries, whereas the cellular isoform, FN-EDA, was present mainly in the endothelium of the vessels in normal heart (Fig. 2a,b). In the pathological tissues, both forms accumulated in the fibrotic areas (Fig. 2c,d).


Figure 1
View larger version (159K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Immunofluorescent localization of total laminin and laminin {alpha} 2 chains in the human heart. Cardiac sections from normal (a,b) and pathological (c,d) myocardium were incubated with antibodies directed against total laminin (a,c) or the {alpha}2 chain of laminin (b,d). Noted that total laminin staining is detected in all basal lamina whereas {alpha} 2 chain staining is observed only around the striated myocytes (magnification: x200).

 

Figure 2
View larger version (149K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Expression of c-FN-EDA and total-FN in the human normal (a,b) and pathological myocardium (c,d). Sections were incubated with antibodies directed against total-FN (a,c) and FN-EDA (b,d). The arrows indicate the FN-EDA accumulation in fibrotic area. Note the poor abundance of FN-EDA in normal heart (magnification: x200).

 
The amounts of laminin {alpha}2 chain and fibronectin mRNAs in tissue samples were analyzed on Northern Blots (Fig. 3A) and then quantified on slot-blots. The relative concentrations of specific mRNAs were evaluated per µg of total RNA loaded. Indeed the level of the classical house keeping gene GAPDH transcripts decreased in the failing human hearts (9.1±4.6 arbitrary units (AU)) when compared to normal tissue (30.5±6.3 AU, P<0.05). The relative amount of the laminin {alpha}2 chain mRNA did not vary significantly in the pathological group (Fig. 3B). In fact, when all samples including controls and pathological tissues were considered, a negative linear correlation (P<0.005) was found between the relative amount of the laminin {alpha}2 chain mRNA and the age of the patients (ranging from 9 to 58 years old), independent of the cardiac status (Fig. 3C). In contrast, the relative amount of FN mRNAs increased in the pathological hearts (+100%, P<0.05) (Fig. 2D) but the level of FN transcript did not vary according to patient’s age (Fig. 3E).


Figure 3
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Expression of laminin {alpha}2 chain and fibronectin (FN) mRNAs in human heart. (A) Northern blots indicated that in the human heart, the two mRNA species are present. In B, the relative level of laminin {alpha}2 chain mRNAs, obtained from slot-blot as described in Methods did not vary significantly among groups whereas a linear negative correlation is observed as a function of the patient’s age (n=22, r2=0.3547, P<0.005). The quantification of FN mRNAs indicated that the level is significantly increased in IDCM groups (D) but the expression of this transcript is not related to the age (E). *P<0.05 versus controls. U.A. are arbitrary units.

 
In order to obtain a more precise characterization of the modulation in the cardiac patterns of FN and laminin expression in normal and pathological situations, we have investigated the relative level of these two transcripts in the growing rat heart, during post-natal development and in a model of experimental hypertension.

3.2 Accumulation of the laminin {alpha}2 chain and fibronectin mRNAs during physiological and pathological cardiac growth in the rat
The relative level of mRNAs encoding the laminin {alpha}2 chain was analyzed during the period of perinatal cardiac growth using RNase protection assay (Figs. 4A and 5AGo). The relative amount of the laminin {alpha}2 chain isoform was rather low at the end of the intra-uterine period, but clearly increased after birth to reach a maximum at 3 weeks of age and thereafter progressively decreased. These results were confirmed using slot-blot analysis (not shown) and RNase protection assay with MyHC probes (Fig. 4B). The increase in laminin {alpha}2 mRNA during the first 3 weeks of age paralleled that of total MyHC mRNA (Fig. 5B). Thus the increase in the level of laminin {alpha}2 chain mRNA observed during the perinatal period (Fig. 5A) is related to the rapid enlargement of the cardiomyocytes [15]. In contrast, during the same period, the amount of FN mRNAs which was abundant in utero, progressively decreased after birth to reach a basal level at 8 weeks of life (Fig. 5C).


Figure 4
View larger version (51K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Ribonuclease protection assay of laminin-{alpha}2 chain mRNAs during cardiac development of the rat. (A) Ten µg of total RNA from rat ventricles at different stages: fetal (F), newborn (Nb), 1 week (8d) and 10 weeks old animals (Ad), and yeast tRNA (Y) were hybridized with the rat 32P-laminin-{alpha}2 cRNA and GAPDH probes. The partially protected fragments of 550 and 164 bp correspond to the laminin-{alpha}2 and GAPDH mRNA, respectively. P: undigested probe. (B) Ten µg of total RNA from rat ventricles at different stages: fetal (f), 1, 4, 8 days and 1, 2 and 3 weeks after birth were hybridized with the rat 32P-laminin-{alpha}2 cRNA and MyHC probes. The partially protected fragments of 90 and 75 bp correspond to the β-MyHC and {alpha}-MyHC, respectively. Note that only {alpha}-MyHC transcripts are detected in the adult rat. p: undigested probe.

 

Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Quantification of laminin {alpha}2 chain and fibronectin mRNAs at different stages of development. (A) The relative amount of laminin transcript was normalized to GAPDH level evaluated on the same RPA assay (see Fig. 4A). Note that the maximal amount of laminin {alpha}2 chain is reached in 3 week old rats. *P<0.05 and *P<0.01 versus 3-week-old rats. (B) The relative amount of laminin {alpha}2 chain mRNAs was normalized to MyHC mRNA accumulation ({alpha}-MyHC+β-MyHC) evaluated on the same RPA assay (see Fig. 4B). Note that this ratio remained constant between 1 and 3 weeks of age. (C) The relative amount of fibronectin mRNA decreased progressively during post-natal development.

 
In addition we verified that in cultured neonatal cardiomyocytes, laminin {alpha}2 chain mRNA level was not dependent on the level of thyroxin (not shown), a growth hormone whose concentration in the plasma increases particularly during this period of ontogenic development [25].

Goldblatt rats were hypertensive 4 weeks after surgery (Fig. 6A) and showed a 40% hypertrophy of the left ventricle (Fig. 6B) without any clinical sign of cardiac failure. The amount of laminin {alpha}2 chain mRNAs was 20% lower than in non hypertensive control rats of the same age (Fig. 6C), whereas FN mRNAs did not vary significantly in the same groups of animals (Fig. 6D).


Figure 6
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Expression of laminin {alpha}2 chain mRNA and fibronectin mRNA in Goldblatt rat heart. (A) Systolic arterial pressure measured in sham-operated and Goldblatt rats 4 weeks after surgery indicated a significant hypertension in Goldblatt rats. (B) The left ventricle weights indicate a significant cardiac hypertrophy in the Goldblatt group. (C) The relative amount of laminin {alpha}2 chain mRNA expressed per µg total RNA. Note the significant decrease of this transcript in Goldblatt rat left ventricle. (D) The relative amount of fibronectin mRNA expressed per µg total RNA. *P<0.05 versus control rats.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study used human and rat cardiac tissues to investigate the expression of laminin-{alpha}2 chain and fibronectin during postnatal development and in pathological situations.

Recent data have suggested that, in human, laminin expression, at the protein level, is constant throughout life [26]. The present results indicate clearly that the cardiac expression of laminin {alpha}2 chain mRNA is developmentally regulated. At the mRNA levels, we observed a progressive decrease in the level of laminin-{alpha}2 chain mRNAs both from 8 to 60 years of age in human and from weaning to adulthood in rat. Such a decrease in laminin {alpha}2 transcript may reflect the low turn-over of the protein surrounding cardiomyocytes, the minor changes in both density and size of myocytes in adult (reviewed in Refs. [27,28]). Moreover, we found that the relative level of laminin {alpha}2 transcript increased markedly during the phase of rapid perinatal growth of the rat heart (3 first weeks of age). In fact the pattern of laminin isoform expression varies during development since in mice, whereas the β chain is expressed at all developmental stages, the expression of the {alpha}1 and {alpha}4 chain isoforms is relatively high in the embryo but weak in neonates and adults, and the {alpha}2 chain isoform is virtually absent in the embryo, weakly expressed in the neonatal and strongly expressed in the adult [29]. Differential expression of the integrin subunits that bind laminin has also been described during the development of the murine heart [30]. In two mouse models, both characterized by a muscular degeneration typical of congenital muscle dystrophy (CMD), the complete absence of laminin {alpha}2 chain did not alter (i) the heart development, (ii) differentiation or maturation of muscle cells but did cause a fatal muscle dysfunction in the adult [31–34], probably as a result of a disruption of the linkage between the sarcolemma membrane and the extracellular matrix [10]. The relatively late accumulation of laminin {alpha}2 chain mRNAs during ontogenic development ([29], and this issue) could be related to the contribution of this protein in the maintenance of the tissue architecture [1,8]. This hypothesis is supported by the fact that during the early postnatal phase of cardiac growth in rat, the level of the mRNAs encoding laminin {alpha}2 chain increased in parallel with that of myosin heavy chain (Fig. 5). The factors involved in the postnatal regulation of laminin {alpha}2 in the heart are not known. In vivo growth hormone appears to favor laminin accumulation but not that of fibronectin and collagen [35]. In the rat cerebellum, the expression of laminin during post-natal development is dependent upon the presence of thyroid hormone [36]. We show herein that this hormone is not the primary determinant of the laminin {alpha}2 mRNA increase in the cardiac tissue.

The pattern of fibronectin expression during development clearly differed from that of laminin. During in utero life, fibronectin mRNAs and their corresponding proteins are normally abundant in both humans [26] and rodents [15], the inactivation of the fibronectin gene induces major lethal cardiac and nervous system abnormalities in the embryo [37]. After birth in humans as in rats, the expression of fibronectin mRNA is very low in the heart (Figs. 3 and 5Go). These quantitative results are in agreement with immunofluorescence and in situ hybridization studies which indicated that c-FN is poorly expressed in adult heart [14,38]. From the developmental studies it can be concluded that FN is highly expressed during the embryonic and fetal periods [15,26,39,40] and then decreases as the cardiomyocytes mature, whereas laminin-{alpha}2 expression increases during the phase of rapid enlargement of cardiomyocytes, both transcripts are then only weakly expressed in the adult. The differences in the time course evolution of laminin {alpha}2 chain and fibronectin mRNA accumulation during early postnatal myocardial growth have to be related to the specific functions of fibronectin and laminin during cell growth and differentiation [4,5].

During development of cardiac hypertrophy and failure, the expression of fibronectin has been extensively explored [14,15,17,20,22,38–42] while that of laminin and particularly laminin {alpha}2 chain expression has been poorly investigated. We observed that in both the failing human myocardium and the hypertrophied rat heart the relative level of laminin {alpha}2 chain mRNAs did not increase. A thickening of the laminin layer has been described in the rat heart in isoproterenol-induced cardiac failure, and during hypertrophy induced by myocardial infarction [43], whereas the pattern of laminin remained normal in the pressure overloaded rat heart [22]. Using specific laminin {alpha}2 chain (merosin) antibodies we were unable to detect any significant alteration in the laminin {alpha}2 chain distribution, even in the failing human myocardium exhibiting signs of fibrosis. In adult human failing heart a thickening of the laminin pattern has also been described by Schaper and Hein [45]. Human dilated cardiomyopathy is accompanied by alterations in the staining pattern of proteins of the extracellular matrix and cytoskeleton, that appear prominent in some areas of the heart muscle and are associated with disorganized myofibrils. The pattern is however completely normal in other areas of the tissue sections and thus discrepancies in the results may depend on tissue heterogeneity, nature of explants, stage of the disease [45]. It is possible that in the failing heart, the non stimulation of the laminin-{alpha}2 chain gene could lead to further alterations in the basal laminae network and as a consequence in membrane fragility, which in turn could participate to myocyte necrosis and development of fibrosis, as shown with fibronectin labeling in IDCM patients (Fig. 2). This hypothesis is supported by the fact that congenital defect in laminin-{alpha}2 chain leads to skeletal muscle disorders which can be partially restored when cells expressing laminin-{alpha}2 chain are grafted within the muscle [46].

In the present study, FN quantification and immunolabelling indicated an active fibrotic process in human dilated cardiomyopathies at the terminal stage whereas the amount of fibronectin mRNAs was unchanged in the Goldblatt rat hearts suggesting the completion of fibrosis in these cardiac tissues. Indeed, in the pressure-overloaded heart [14], and during ischemia [41], the increase in fibronectin mRNA is an early and transient event associated with the development of fibrosis [41,42] which is no longer observed once the hypertrophy has developed and reached a compensated stage. It is well known that the active components of the renin—angiotensin—aldosterone system induce an increase in the mRNAs and corresponding extracellular matrix proteins such as fibronectin and collagen in the cardiovascular system [18,19,40–44]. These effects are limited by ACE inhibitors, despite an ongoing cardiac pressure overload [20]. However, it should be noted that laminin {alpha}2 chain synthesis does not seem to be associated with the angiotensin II dependent growth of myocardium in adult rat, since, according to our results, the level of accumulation of laminin {alpha}2 chain mRNA was decreased by 20–30% in the hypertrophied heart of Goldblatt rats.

Taken together our results demonstrate that the expression of laminin {alpha}2 chain gene is developmentally regulated in both human and rodents. Its relative level increases rapidly after birth to follow normal cardiomyocyte growth and then decreases progressively to adulthood. During the development of cardiac hypertrophy and failure, the imbalance between the increased size of myocyte and the steady state or decreased level of laminin-{alpha}2 chain might contribute to the altered sarcolemma properties in the failing heart. In contrast cellular fibronectin expression decreases slowly from birth to aging but is stimulated when fibrosis occurs. The differences in laminin and fibronectin expression could be associated to the different properties of the 2 proteins. Fibronectin is mostly associated together with collagen I/III to fibrosis while laminin is mostly considered as a structural component of the basal membrane forming one of the links between extracellular domains and the cytoskeleton and/or the myofibrils.

Time for primary review 37 days.


   Acknowledgements
 
The work was supported by AFM, INSERM, CNRS. The authors thanks Dr Gillian Butler-Brown for fruitful discussions and careful editing of the manuscript.


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

  1. Hay E.D., ed. Cell biology of extracellular matrix. (1991) 2nd ed. New York: Plenum Press.
  2. Engvall E. Laminin variants. Why, where and when? Kidney Int (1993) 43(1):2–6.[Web of Science][Medline]
  3. Henry M.D., Campbell K.P. Dystroglycan: an extracellular matrix receptor linked to the cytoskeleton. Curr Opin Cell Biol (1996) 8(5):625–631.[CrossRef][Web of Science][Medline]
  4. Ehrig K., Leivo I., Argraves W.S., Ruoslahti E., Engvall E. Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc Natl Acad Sci USA (1990) 87(9):3264–3268.[Abstract/Free Full Text]
  5. Leivo I., Engvall E. Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development. Proc Natl Acad Sci USA (1988) 85(5):1544–1548.[Abstract/Free Full Text]
  6. Cox G.F., Kunkel L.M. Dystrophies and heart diseases. Curr Opin Cardiol (1997) 12(3):329–343.[Web of Science][Medline]
  7. Campbell K.P. Three muscular dystrophies: loss of cytoskeleton—extracellular matrix linkage. Cell (1995) 80:675–679.[CrossRef][Web of Science][Medline]
  8. Tome F.M., Evangelista T., Leclerc A., et al. Congenital muscular dystrophy with merosin deficiency. C R Acad Sci Ser III (1994) 317(4):351–357.[Medline]
  9. Helbling-Leclerc A., Zhang X., Topaloglu H., et al. Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat Genet (1995) 11:216–218.[CrossRef][Web of Science][Medline]
  10. Farhadian F., Contard F., Sabri A., Samuel J.L., Rappaport L. Fibronectin and basement membrane in cardiovascular organogenesis and disease pathogenesis. Cardiovasc Res (1996) 32:433–442.[Free Full Text]
  11. Hedin U., Bottger B.A., Forsberg E., Johansson S., Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol (1988) 107(1):307–319.[Abstract/Free Full Text]
  12. Lundgren E., Gullberg D., Rubin K., Borg T.K., Terracio M.J., Terracio L. In vitro studies on adult cardiac myocytes. attachment and biosynthesis of collagen type IV and laminin. J Cell Physiol (1988) 136(1):43–53.[CrossRef][Web of Science][Medline]
  13. Glukhova M., Koteliansky V., Fondacci C., Marotte F., Rappaport L. Laminin variants and integrin laminin receptors in developing and adult human smooth muscle. Dev Biol (1993) 157(2):437–447.[CrossRef][Web of Science][Medline]
  14. Samuel J.L., Barrieux A., Dufour S., et al. Accumulation of fetal fibronectin mRNAs during the development of rat cardiac hypertrophy induced by pressure overload. J Clin Invest (1991) 88:1737–1746.[Web of Science][Medline]
  15. Farhadian F., Barrieux A., Lortet S., et al. Differential splicing of fibronectin pre-messenger ribonucleic acid during cardiac ontogeny and development of hypertrophy in the rat. Lab Invest (1994) 71(4):552–559.[Web of Science][Medline]
  16. Chapman D., Weber K.T., Eghbali M. Regulation of fibrillar collagen types I and III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium. Circ Res (1990) 67:787–794.[Abstract/Free Full Text]
  17. Villareal F.J., Dillmann W.H. Cardiac hypertrophy-induced changes in mRNA levels for TGF-beta 1, fibronectin, and collagen. Am J Physiol (1992) 262(6Pt2):H1861–1866.[Web of Science][Medline]
  18. Robert V., Van Thiem N., Cheav S.L., Mouas C., Swynghedauw B., Delcayre C. Increased cardiac types I and III collagen mRNAs in aldosterone—salt hypertension. Hypertension (1994) 24:30–36.[Abstract/Free Full Text]
  19. Robert V., Silvestre J.S., Charlemagne D., et al. Biological determinants of aldosterone-induced cardiac fibrosis in rats. Hypertension (1995) 26:971–978.[Abstract/Free Full Text]
  20. Grimm D., Kromer E.P., Bocker W., et al. Regulation of extracellular matrix proteins in pressure-overload cardiac hypertrophy: effects of angiotensin converting enzyme inhibition. J Hypertens (1998) 16:1345–1355.[CrossRef][Web of Science][Medline]
  21. Sainte Beuve C., Allen P.D., Dambrin G., et al. Cardiac calcium release channel (ryanodine receptor) in control and cardiomyopathic human hearts: mRNA and protein contents are differentially regulated. J Mol Cell Cardiol (1997) 29:1237–1246.[CrossRef][Web of Science][Medline]
  22. Contard F., Koteliansky V., Marotte F., Dubus I., Rappaport L., Samuel J.L. Specific alterations in the the distribution of extracellular matrix components within rat myocardium during the development of pressure overload. Lab Invest (1991) 64:65–67.[Web of Science][Medline]
  23. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate—phenol—chloroform extraction. Anal Biochem (1987) 162:156–159.[Web of Science][Medline]
  24. Schiaffino S., Samuel J.L., Sassoon D., et al. Nonsynchronous accumulation of {alpha}-skeletal actin and β-myosin heavy chain mRNAs during early stages of pressure overloaded-induced cardiac hypertrophy demonstrated by in situ hybridization. Circ Res (1989) 64:937–948.[Abstract/Free Full Text]
  25. Dussault J.H., Labrie F. Development of the hypothalamic—pituitary—thyroid axis in the neonatal rat. Endocrinology (1975) 97(5):1321–1324.[Abstract/Free Full Text]
  26. Kim H., Yoon C.S., Kim H., Rah B. Expression of extracellular matrix components fibronectin and laminin in the human fetal heart. Cell Struct Funct (1999) 24:19–26.[CrossRef][Web of Science][Medline]
  27. Anversa P., Kajstura J. Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res (1998) 83:1–14.[Free Full Text]
  28. Soonpaa M.H., Field L.J. Survey of studies examining mammalian cardiomyocyte DNA synthesis. Circ Res (1998) 83:15–26.[Free Full Text]
  29. Miner J.H., Patton B.L., Lentz S.I., et al. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8–11, and cloning of a novel alpha3 isoform. J Cell Biol (1997) 137:685–701.[Abstract/Free Full Text]
  30. Hierck B.P., Poelmann R.E., van Iperen L., Brouwer A., Gittenberger-de-Groot A.C. Differential expression of alpha-6 and other subunits of laminin binding integrins during development of the murine heart. Dev Dyn (1996) 206:100–111.[CrossRef][Web of Science][Medline]
  31. Sunada Y., Bernier S.M., Kozak C.A., Yamada Y., Campbell K.P. Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin M chain gene to dy locus. J Biol Chem (1994) 269(19):13729–13732.[Abstract/Free Full Text]
  32. Xu H., Christmas P., Wu X.R., Wewer U.M., Engvall E. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc Natl Acad Sci USA (1994) 91(12):5572–5576.[Abstract/Free Full Text]
  33. Kuang W., Xu H., Vachon P.H., et al. Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models. J Clin Invest (1998) 102:844–852.[Web of Science][Medline]
  34. Kuang W., Xu H., Vachon P.H., Engvall E. Disruption of the lama2 gene in embryonic stem cells. laminin alpha 2 is necessary for sustenance of mature muscle cells. Exp Cell Res (1998) 25:117–125.
  35. Grimm D., Cameron D., Griese D.P., Riegger G.A., Kromer E.P. Differential effects of growth hormone on cardiomyocyte and extracellular matrix protein remodeling following experimental myocardial infarction. Cardiovasc Res (1998) 40:297–306.[Abstract/Free Full Text]
  36. Farwell A.P., Dubord-Tomasetti S.A. Thyroid hormone regulates the expression of laminin in the developing rat cerebellum. Endocrinology (1999) 140:4221–4227.[Abstract/Free Full Text]
  37. George E.L., Baldwin H.S., Hynes R.O. Fibronectins are essential for heart and blood vessel morphogenesis but are dispensable for initial specification of precursor cells. Blood (1997) 90:3073–3081.[Abstract/Free Full Text]
  38. Crawford D.C., Chobanian A.V., Brecher P. Angiotensin II induces fibronectin expression associated with cardiac fibrosis in the rat. Circ Res (1994) 74:727–739.[Abstract/Free Full Text]
  39. Farhadian F., Contard F., Corbier A., Barrieux A., Rappaport L., Samuel J.L. Fibronectin expression during physiological and pathological cardiac growth. J Mol Cell Cardiol (1995) 27:981–990.[CrossRef][Web of Science][Medline]
  40. Samuel J.L., Farhadian F., Sabri A., et al. Expression of fibronectin during fetal and post natal development: An in situ hybridisation and immunohistochemical study. Cardiovasc Res (1994) 28:1653–1661.[Abstract/Free Full Text]
  41. Knowlton A.A., Connelly C.M., Romo G.M., Mamuya W., Apstein C.S., Brecher P. Rapid expression of fibronectin in the rabbit heart after myocardial infarction with and without reperfusion. J Clin Invest (1992) 89:1060–1068.[Web of Science][Medline]
  42. Ulrich M.M., Janssen A.M., Daemen M.J., et al. Increased expression of fibronectin isoforms after myocardial infarction in rats. J Mol Cell Cardiol (1997) 29:2533–2543.[CrossRef][Web of Science][Medline]
  43. Grimm D., Elsner D., Schunkert H., et al. Development of heart failure following isoproterenol administration in the rat: role of the renin—angiotensin system. Cardiovasc Res (1998) 37:91–100.[Abstract/Free Full Text]
  44. Iwami K., Ashizawa N., Do Y.S., Graf K., Hsueh W.A. Comparison of ANG II with other growth factors on Egr-1 and matrix gene expression in cardiac fibroblasts. Am J Physiol (1996) 270:H2100–H2107.[Medline]
  45. Schaper J., Hein S. The structural correlate of reduced cardiac function in human dilated cardiomyopathy. Heart Failure (1993) 1:95–111.
  46. Vilquin J.T., Kinoshita I., Roy B., et al. Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation. J Cell Biol (1996) 133:185–197.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
N. S. Dhalla, H. K. Saini-Chohan, D. Rodriguez-Leyva, V. Elimban, M. R. Dent, and P. S. Tappia
Subcellular remodelling may induce cardiac dysfunction in congestive heart failure
Cardiovasc Res, February 15, 2009; 81(3): 429 - 438.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. Lal, J. P. Veinot, and F. H.H. Leenen
Critical role of CNS effects of aldosterone in cardiac remodeling post-myocardial infarction in rats
Cardiovasc Res, December 1, 2004; 64(3): 437 - 447.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Oliviéro, P.
Right arrow Articles by Samuel, J.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oliviéro, P.
Right arrow Articles by Samuel, J.-L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?