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1Centre de Recherche Cardiovasculaire Inserm Lariboisière U689, Institut Fédératif de Recherche 139, Hôpital Lariboisière, Paris, France; and 2Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska, Czech Republic
Submitted 17 November 2004 ; accepted in final form 15 February 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its 
-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1 labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1 expression, suggesting that caveolin-1 and -1 are coregulated; the caveolin-1 levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1 and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1 expression. membrane microdomain; cardiomyocyte; vessel; coronary artery
s, and Gi proteins, etc.]. The importance of membrane microdomains such as caveolae in the control of these signaling pathways has been demonstrated in vessels and myocardium (23, 27). Caveolin is a family of constituent proteins of caveolae. Currently, three caveolin genes are described. Caveolin-1 and -2 are relatively ubiquitously expressed (27), whereas caveolin-3 is specifically present in muscles (9, 27, 28).
Caveolin-1 is expressed in two isoforms derived from a single gene: caveolin-1 and caveolin-1 (23); the - and -isoforms start from methionine at positions 1 and 32 of the protein sequence, respectively (25). Within caveolae, caveolin-1 or -3 interact with a cohort of proteins and control the activity of the signaling pathways involved in cardiac development (27). For example, caveolin-1 has been involved in the formation of endothelial capillary tubule (16) and the control of cell proliferation (23). On the other hand, caveolin-3 is involved in the mechanism of T-tubule biogenesis (12). In utero, the expression of caveolin-1 and -3 appears to be related to cell differentiation. Although caveolin-1 and -3 emerged as key elements implicated in growth signal transduction, only a few studies (15, 24) have reported some expressional changes in caveolin abundance.
However, the precise caveolin distribution and expression during normal and pathological cardiac postnatal development have not been defined. Here, we have analyzed patterns of caveolin-1 and -3 distributions and expressions during postnatal rat heart development. We have also examined the effect of thyroid hormone on caveolin expressions and caveolin-1 phosphorylation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The following materials were used in this study: nitrocellulose transfer membrane (0.45-µm pore) (Schleicher & Schuell; Dassel, Germany); antibodies, mouse anti-caveolin-1 (clone 2297), mouse anti-phospho-caveolin (P-Tyr 14) (clone 56), and mouse anti-caveolin-3 (clone 26) (Transduction Laboratories-Interchim; Asnières sur Seine, France); rabbit anti-caveolin-1 (sc-894) and goat anti-caveolin-3 (sc7665) (Santa Cruz-Tébu; Le-Perray-en-Yvelines, France); rabbit anti-laminin (Chemicon-Euromedex; Souffelweyersheim, France); mouse anti-vinculin (clone hVin-1) (Sigma; St.-Quentin-Fallavier, France); mouse anti-smooth muscle actin (clone 1A4) (Dako; Trappes, France); FITC-conjugated sheep anti-mouse IgG and Texas red-conjugated donkey anti-rabbit polyclonal IgG (Amersham Pharmacia; Orsay, France); Cy3-conjugated anti-goat IgG (Sigma); Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (Vector-ABCys; Paris, France); anti-mouse IgG- and anti-rabbit IgG-conjugated to horseradish peroxidase (Amersham Pharmacia); enhanced chemiluminescence immunoblotting reagents (Amersham); BCA protein assay kit (Pierce-Perbio; Brebières, France); and 4-methyl-2-thiouracil (Fluka; St.-Quentin-Fallavier, France).
Experimental Model
The investigation was done in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health (NIH publication no. 85-23, revised 1996). Wistar rats were from Iffa-Credo (Lyon, France). Care of animals conformed to the Helsinki declaration, and the study was approved by our institution (INSERM).
Ontogenic development. Hearts from 1-, 3-, 5-, 10-, 14-, 16-, and 21-day-old and 3-mo-old rats (n = 4–6 per groups) were included in the study. Until 2 wk of age, both male and female rats were used; afterward, only male rats were included in the study.
Animal treatment. Postnatal hypothyroidism was induced by treatment of pregnant Wistar rats from day 16 of gestation (embryonic day 16) with 0.1% 4-methyl-2-thiouracil in the drinking water and a low-iodine food regimen ad libitum. The treatment was continued throughout lactation until the animals were killed. Classic signs of hypothyroidism, such as reduced weight and delayed eye opening in the pups, were observed. The hearts were removed and weighed. The free wall of left ventricle was rapidly frozen in isopentane precooled in liquid nitrogen, then stored at –80°C.
Immunolabeling
Cryostat sections (7 µm thick) of heart tissue were postfixed in methanol at –20°C. To prevent nonspecific binding after rinsing in PBS (pH 7.4), sections were preincubated in PBS containing 5% bovine serum albumin (BSA) for 30 min at room temperature (RT). Monoclonal antibodies (MAb) to caveolin-1 (5 µg/ml) and caveolin-3 (2.5 µg/ml), or polyclonal antibody (PAb) to caveolin-1 (0.2 µg/ml) and caveolin-3 (0.2 µg/ml) were applied to sections for 1 h at RT. After three washings in PBS, slides treated with MAb were incubated with PAb anti-laminin (0.2 µg/ml), or slides treated with PAb were treated with either MAb anti--smooth muscle actin (2 µg/ml) or MAb anti-vinculin (10 µg/ml) for 1 h at RT. Binding of primary antibodies was detected by incubating the sections for 1 h at RT with fluorescein-conjugated sheep anti-mouse IgG (1:40), fluorescein-conjugated donkey anti-rabbit IgG (1:40), or Cy3-conjugated anti-goat IgG (1:40). Laminin, -smooth muscle actin antibodies, and vinculin were detected by incubating the sections for 1 h at RT with Texas red-conjugated donkey anti-rabbit IgG (1:40), Texas red-conjugated sheep anti-mouse IgG (1:40), and fluorescein-conjugated donkey anti-mouse IgG (1:40), respectively. Sections were finally washed as above and mounted in aqueous medium Vectashield (Vector) containing 4,6-diamidino-2-phenylindole. Fluorescence was observed using DMRB Leica microscope equipped with epifluorescence optics.
Protein Extractions
Ten left ventricular cryostat sections (15 µm width) were lysed in boiling SDS buffer (1% SDS, 10 mM Tris·HCl, pH 7.4, 1 mM orthovanadate, 0.3 mM aprotinin, 4 mM leupeptin, 60 mM phenylmethanesulfonyl fluoride). Lysates were warmed in a microwave oven for 15 s (900 W) and were clarified by centrifugation (11,000 g for 15 min at 15°C). Lysate protein concentrations were measured using the BCA protein assay kit with BSA as a standard. Supernatants were stored at –20°C.
Western Blot Analysis
The cellular contents in caveolin-1, caveolin-1, caveolin-3, and caveolin-1 phosphorylation on tyrosine 14 were determined by Western blot analysis. Proteins [20 µg to detect caveolin-1, caveolin-3, and caveolin-1 phosphorylation profile of tyrosine14-caveolin-1 (PY14); 5 µg to detect caveolin-1] were resolved by electrophoresis on 12% SDS-acrylamide gel and electrophoretically transferred to nitrocellulose transfer in 25 mM Tris, 192 mM glycin, 0.01% SDS, and 15% methanol. Ponceau staining of each membrane confirmed that equal amounts of protein were loaded onto each lane. The membranes were blocked with TBS (pH 7.4) containing 0.1% Tween-20, 5% nonfat dry milk, and 1% BSA for 1 h at RT before incubation for 1 h at RT with either MAb anti-caveolin-1 (50 ng/ml), MAb caveolin-3 (25 ng/ml), or PAb caveolin-1 (20 ng/ml) in TBS-Tween-20. For incubation with MAb anti-caveolin-1 PY14 (25 ng/ml), the membranes were blocked prior with TBS (pH 7.4) containing 0.1% Tween-20 and 2.5% BSA for 1 h at RT. After being washed, membranes were incubated for 1 h at RT with either anti-mouse IgG- or anti-rabbit IgG-conjugated to horseradish peroxidase (1:5,000). After being washed, immunoreactive bands were visualized by enhanced chemiluminescence, quantified by densitometry using a computer-based imaging system (Gel Doc 1000; Bio-Rad), and normalized to actin amount present on the gel. To allow normalization between experiments, a same sample (link) was loaded on each gel.
Statistical Analysis
ANOVA tests were used in statistical evaluation of the data, which are given as means ± SE. A Scheffé test, except when indicated, was used; a value of P < 0.05 was considered as significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Postnatal heart growth is characterized by a rapid increase in heart weight (Table 1), which reflects both hyperplasia and hypertrophy of the cardiomyocytes (19).
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Using double fluorescent immunolabeling, caveolin-3 distribution was analyzed in the different animal groups and compared with laminin, a major component of basal lamina, or vinculin, a costamere element. Whatever the developmental stage, caveolin-3 appeared codistributed with either laminin or vinculin, clearly indicating that caveolin-3 was present at sarcolemma and intercalated disk levels of cardiomyocytes (Figs. 1 and 2). However, at the level of cardiomyocytes, caveolin-3 staining appeared as a dotted line in 1- and 5-day-old rats, whereas after 14 days of age caveolin-3 antibodies labeled continuously the sarcolemma (Figs. 1 and 2). Higher magnification of caveolin-3 staining evidenced the slightly heterogeneous pattern in 3-day-old rat (Fig. 3A) that became homogeneously distributed with age, as shown in 90-day-old rat (Fig. 3C).
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The pattern of expression of isoform- and - was investigated by using antibodies that recognized either the two isoforms or specifically caveolin-1 only.
In accordance with the literature, caveolin-1, as well as the isoform caveolin-1, was detected in endothelial cells of both large vessels and capillaries (Fig. 5) throughout the postnatal development stage studies. Around birth, at the level of large vessels, caveolin-1 and its -isoform were detected in smooth muscle cells (revealed by -smooth muscle actin antibodies). With increasing age, the expression of caveolin-1 tends to decrease in the smooth muscle cells, with the level of isoform- being very weak in adults (Fig. 5H).
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expression were similar, suggesting that the two isoforms were coregulated (Fig. 6B). The time course analysis showed that the caveolin-1 level already increased fourfold by day 5, reached a maximum value by day 14 after birth, and remained at this high level afterward. Because the caveolin-1 isoform is characterized by a tyrosine in position 14, which is phosphorylated by the src kinase family, we analyzed PY14 to get insight into the maturation process of caveolin within endothelium (Fig. 6C). The signal for PY14 that was low in the early postnatal period (days 1–10) dramatically increased by day 14 (8-fold vs. day 1; P < 0.05), with this increase being very transient, as shown by the weak signal found by day 16.
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expression and its phosphorylation are controlled by the thyroid hormone. The hypothyroid rat displayed delayed growth compared with euthyroid animals, as indicated by both body and heart weights (Table 1). The level of expression of caveolin-1, which was significantly lower (–50%) than that of age-matched animals at 14 days of age, was similar at 21 days after birth. In addition, the level of PY14 was dramatically altered in 14-day-old hypothyroid rats compared with controls (3.91 ± 0.12 vs. 6.87 ± 0.92, respectively), but the PY14-to-caveolin-1 ratio did not change significantly (Fig. 7A). In contrast, the variation in caveolin-3 expression did not reach statistical significance (Fig. 7, A and C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-isoform, and caveolin-3 during postnatal development in normal and hypothyroid rat hearts. We demonstrate first that the postnatal cardiac expression of caveolin-3 is developmentally regulated in rat. Caveolin-3 is specifically expressed in muscle tissue, including cardiomyocytes (23), and transcripts of caveolin-3 mRNA are first detectable in vivo at day 10 of gestation in developing heart, well before T-tubule maturation (3). The protein is expressed at birth, and its expression increases to reach a maximum by day 5 (Fig. 4). Such a developmental pattern of caveolin-3 expression appeared to be parallel with the time course of cardiac growth. Indeed, the rate of cardiac growth increases just after birth, attaining a maximum value on postnatal day 4 (21). This phase of rapid growth is predominantly due to the hyperplastic growth of rat cardiac cells that markedly decreases after postnatal day 4 (1). Therefore, caveolin-3 level reached its maximum at the onset of the hypertrophic phase of growth that follows the hyperplastic one. Analogously, caveolin-3 is not expressed in proliferating skeletal myoblasts but increases as skeletal muscle differentiation ensues (13). The caveolin-3 expression profile is thus in accordance with caveolin-3 function as a negative regulator of muscle cell proliferation. In skeletal muscle, it was shown that caveolin-3 plays a critical role in T-tubule biogenesis (5). In ventricular cardiomyocyte, the repetitive generation of caveolae has been involved in the formation of the transverse-axial tubular system, which began to develop by the time of birth and is achieved after day 14 (10), and when caveolae and T tubules proliferated, the dyadic couplings become more abundant (8). In caveolin-3-null mice, the abnormalities in the organization of the T-tubule system (12) lead to pathophysiological changes in Ca2+ signaling (17). By day 5, we observed a transition from a discontinuous to continuous distribution of caveolin-3 at the sarcolemma level. It emerged that both the qualitative and quantitative changes observed for caveolin-3 during postnatal growth correlate with the increasing contractile performance that reflects substantial changes in myocardial Ca2+ handling (6).
A major finding of this study is that 1) the expression of caveolin-1 and its phosphorylation level are highly regulated during postnatal growth and 2) caveolin-1 expression is influenced by thyroid hormone level. The similar temporal pattern of caveolin-1 ( and ) and caveolin-1 expression during cardiac postnatal development suggested that the expression of the two caveolin-1 isoforms- and - are coregulated, despite the fact that caveolin-1 and -1 have a different potential for forming caveolae structure (11). The precise time course analysis also showed that caveolin-1 levels increased rapidly during the hyperplastic growth of the hearts (day 4), reached its maximum by day 14, and remained at this high level afterward (Fig. 6). It is well assessed that increased expression of caveolin-1 results on one hand in cell growth inhibition (23) and on the other hand in an enhancement of endothelial tubule capillary formation (16). Morphometric analysis has clearly demonstrated that the quantitative development of microcirculation in the heart occurs after birth and the ultrastructural differentiation of capillary endothelium in rats is completed by the end of the second postnatal week (20). Consequently, the developmental pattern of caveolin-1 expression observed herein correlates with the angiogenesis process in the heart.
In the normal rat endothelium, tyrosine phosphorylation of caveolin-1, which occurs at tyrosine-14 in vivo, may induce caveolar vesiculation and/or fusion (2).
Using caveolin-1-null mice, Schubert et al. (26) demonstrated that caveolae/caveolin-1 are involved in the efficient uptake and transport of albumin. Within caveolar endothelial membrane, the activation of the cell surface 60-kDa glycoprotein gp60 by albumin stimulates the Src protein tyrosine kinase signaling pathway (29), and the activation of Src leads to the phosphorylation of caveolin-1 on tyrosine 14 (4). Caveolin phosphorylation, in turn, might induce the formation of preassembled signaling complexes containing inactive Src family kinases at specific sites at the cell membrane that are primed for transient activation in response to extracellular stimuli. Together with the maximum increase in tyrosine-14 phosphorylation around day 14, this would indicate that this period is of primary importance for changes in signaling pathway at the endothelial level (e.g., vascular endothelial growth factor, AT1, endothelin, mechanical forces) and could correspond to a maturation process. The present study is, to our knowledge, the first demonstration of a transient phosphorylation level in caveolin-1 during normal development in vivo.
Our comparison of expressional changes of caveolin in heart between euthyroid and hypothyroid rats indicated that thyroid hormone could play a role in the regulation of caveolin-1 expression during the postnatal development of the heart. Among numerous factors, hormones such as T3 and T4 modulate the capillary growth during postnatal development and participate to establish the normal hierarchy of the arteriolar tree (14). Neonatal hypothyroidism slowed the formation of new capillary branches in proportion to the reduction of cardiac growth (14). The low levels of caveolin-1 observed in 4-methyl-2-thiouracil-treated rat heart by day 14 correlated well with the delay in capillary maturation.
Previously, it has been reported that hypothyroidism in adult rat is associated with blunted endothelium-dependent relaxation of arterial vessels (22), whereas endothelium-dependent vascular smooth muscle contraction is not altered (7). However, in adult rat heart, nitric oxide synthase (NOS) activity increased during hypothyroidism (22). Because caveolin-1 is a major component involved in NOS activity, the low level of caveolin-1 in 14-day-old hypothyroid rat heart could result in a dysinhibition of endothelial NOS. Therefore, we might propose that changes in caveolin-1 expression might contribute to alterations in both nitric oxide bioavailability and vasculogenesis process during neonatal hypothyroidism.
In summary, the data presented herein demonstrated that during heart development the caveolin protein exhibits a temporal and cell-specific pattern of expression, with thyroid hormone being one of the factors involved in the control of caveolin-1 expression.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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, caveolin-1
, total caveolin-1). *P < 0.05 vs. d1. C: quantification of caveolin-1