1 Department of Molecular Pharmacology, Albert Einstein College of Medicine; 2 Division of Hormone-Dependent Tumor Biology, The Albert Einstein Cancer Center; 3 Divisions of Cardiology and Infectious Disease, Department of Medicine, Albert Einstein College of Medicine and The Montefiore Medical Center; and 4 Department of Pathology and 5 Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Recently, development of a caveolin-1-deficient (Cav-1 null) mouse model has allowed the detailed analysis of caveolin-1's function in the context of a whole animal. Interestingly, we now report that the hearts of Cav-1 null mice are markedly abnormal, despite the fact that caveolin-1 is not expressed in cardiac myocytes. However, caveolin-1 is abundantly expressed in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelia. Quantitative imaging studies of Cav-1 null hearts demonstrate a significantly enlarged right ventricular cavity and a thickened left ventricular wall with decreased systolic function. Histological analysis reveals myocyte hypertrophy with interstitial/perivascular fibrosis. Because caveolin-1 is thought to act as a negative regulator of the p42/44 MAP kinase cascade, we performed Western blot analysis with phospho-specific antibodies that only recognize activated ERK1/2. As predicted, the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (i.e., interstitial fibrotic lesions) and isolated cardiac fibroblasts. In addition, endothelial and inducible nitric oxide synthase levels are dramatically upregulated. Thus loss of caveolin-1 expression drives p42/44 MAP kinase activation and cardiac hypertrophy.
caveolae; cardiomyopathy; signal transduction; cardiac fibroblasts
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INTRODUCTION |
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FIRST IDENTIFIED IN THE 1950S by electron microscopists, caveolae membrane domains are 50- to 100-nm flask-shaped organelles located at or near the plasma membranes of numerous cell types (47, 78). These organelles are particularly abundant in fibroblasts and endothelial cells, where they were originally thought to function as mere conduits for cellular uptake and transport.
Over the past decade, the study of caveolae has blossomed into a rapidly expanding field due to the identification of caveolin-1, the principal structural component of caveolae organelles in nonmuscle cells (27, 35). The molecular cloning of this marker protein allowed for the biochemical purification of caveolae microdomains, which are characterized by a high content of caveolin-1, cholesterol, sphingolipids, and cytoplasmic signaling molecules. This unique structure is now known to constitute a regulatory platform from which numerous signaling cascades are held in check by direct molecular interactions with the caveolin-1 scaffolding domain (residues 82-101). Some of these molecules include receptor tyrosine kinases (EGF-R and PDGF-R), Src family tyrosine kinases, endothelial nitric oxide synthase (eNOS), and members of the p42/44 MAP kinase cascade (MEK1/2 and ERK1/2) (9, 21, 45). The majority of these interactions are those of negative regulation, i.e., where caveolin-1 binding serves to hold a given signaling molecule in an inactive or repressed state.
Caveolin-1 is the first of a member of a gene family consisting of three structurally related proteins. Shortly after the identification of caveolin-1, two other caveolin-related genes were identified and cloned, i.e., caveolin-2 and caveolin-3 (63, 74). Caveolin-1 and -2 are coexpressed in a variety of cells and tissue types but are abundantly expressed in fibroblasts and endothelial cells, whereas the expression of caveolin-3 is muscle specific (cardiac myocytes and skeletal muscle fibers) (62, 74).
Caveolin-1 and -3 are capable of forming stable homooligomers containing ~14-16 monomers per oligomer (59, 74), whereas caveolin-2 requires the presence of caveolin-1 for its stability and proper targeting to the plasma membrane (49). In the absence of caveolin-1, caveolin-2 remains trapped within the Golgi apparatus, where it is rapidly degraded by the proteasome (54).
What is known about the human caveolin genes? Human Cav-1 and Cav-2 are colocalized to the D7S522 locus on chromosome 7 (7q31.1) at a known fragile site that is frequently deleted in a variety of cancers, including breast cancers (15, 16). In addition, the Cav-1 gene is sporadically mutated (P132L) in up to 16% of human breast cancer samples (31), consistent with the notion that the Cav-1 gene normally functions as a transformation suppressor or negatively regulates cell growth (55). The human Cav-3 gene is localized on chromosome 3 (3p25) and is mutated in a novel form of muscular dystrophy, termed LGMD-1C (limb-girdle muscular dystrophy type 1C) (24, 43).
The recent generation of caveolin-deficient mice by targeted gene deletion has now allowed for the exploration of the role of each caveolin gene in the context of a whole organismal model. The first reports on Cav-1 null mice describe the hyperproliferation of mouse embryo fibroblasts in culture and lung hypercellularity due to the presence of an increased number of endothelial cells (10, 54), consistent with the idea that caveolin-1 normally functions as a negative regulator of cell growth (23). Furthermore, we have recently demonstrated that Cav-1 null mice develop mammary epithelial cell hyperplasia, even in 6-wk-old virgin mice (36). Thus Cav-1 null mice may possess other hyperproliferative phenotypes.
In a further search for the functional significance of the caveolin-1 protein in vivo, we chose to more closely examine a tissue that contains an abundance of caveolin-1-expressing cell types in relation to its overall makeup, the heart. The murine heart, like that of humans, is composed of ~60% cardiac myocytes that express caveolin-3 and ~40% other cell types (most notably fibroblasts and endothelial cells) that express caveolin-1 (40). As such, the heart represents an interesting area of investigation for the role of caveolin-1 in an intact organ system.
Because caveolin-1 is expressed in cardiac fibroblasts, where it is thought to negatively regulate the p42/44 MAP kinase cascade (22), one possible outcome of loss of caveolin-1 protein expression in cardiac fibroblasts is the development of cardiac myocyte hypertrophy. The idea that changes in cardiac fibroblasts can lead to secondary alterations in cardiac myocyte structure and function, i.e., cardiac hypertrophy and cardiomyopathy, is not new. A plethora of studies have established a direct causal relationship between alterations in cardiac fibroblast functioning/extracellular matrix composition and the development of cardiac myocyte hypertrophy (5, 6, 29, 50, 76, 79). A singular theme resonates in all of these findings: the cardiac fibroblast and the matrix it secretes are active participants in, if not the main cause of, the hypertrophic changes found in multiple forms of cardiac disease. Reasons for this include the following: 1) the increased deposition of extracellular matrix results in disruption of the myocardium, alterations in the electrical conduction system, and impairment of myocyte contraction (6, 76); and 2) fibroblast-secreted factors activate hypertrophic signals in the cardiac myocytes (5). Within the context of this knowledge base, we sought to examine the potential effects of loss of caveolin-1 in cardiac fibroblasts on cardiac myocyte structure and function in the intact murine heart.
Here, we demonstrate that caveolin-1-deficient mice develop cardiac hypertrophy and show hyperactivation of the p42/44 MAP kinase cascade in interstitial fibrotic lesions in vivo and in isolated cardiac fibroblasts in culture. As such, this is the first in vivo demonstration that loss of caveolin-1 expression leads to hyperactivation of the Ras-p42/44 MAP kinase cascade in a mammalian organism, directly supporting the conclusions of earlier in vitro studies (12, 21, 31, 38). In addition, RNA-interference (RNAi)-based ablation of Cav-1 gene expression in Caenorhabditis elegans leads to progression of the meiotic cell cycle, a phenotype that mirrors Ras activation (61).
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MATERIALS AND METHODS |
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Materials
Anti-caveolin-1, -2, and -3 IgG (mouse MAbs) (62, 64, 70) were the generous gift of Dr. Roberto Campos-Gonzalez (BD Transduction Laboratories). Other antibodies and their sources were as follows: anti-GLUT-4 [monoclonal antibody (MAb); Genzyme], anti-ERK1/2 and anti-phospho-ERK1/2 [polyclonal antibodies (PAb); Cell Signaling Technology], anti-PKCAnimal Studies
All animals were housed and maintained in a barrier facility at the Institute for Animal Studies, Albert Einstein College of Medicine. Cav-1 and Cav-2 null mice were generated, as we previously described (54, 56).Immunoblot Analysis
Wild-type and Cav-1 null mice were killed by CO2 asphyxiation, and their hearts were immediately harvested. Heart tissue samples were then homogenized in lysis buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 2 nM sodium orthovanadate, 0.1 µg/ml okadaic acid, 40 nM bpVphen, 0.1% SDS, 0.5% deoxycholic acid, and 1% Igepal (formerly NP-40)] containing protease inhibitors (Boehringer Mannheim). Tissue lysates were then centrifuged at 12,000 g for 10 min to remove insoluble debris. Protein concentrations were analyzed by using the bicinchoninic acid reagent (BCA; Pierce), and the volume required for 10-40 µg of protein was determined. Samples were then separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein bands), followed by immunoblot analysis. All subsequent wash buffers contained 10 mM Tris, pH 8.0-150 mM NaCl-0.05% Tween 20, which was supplemented with 1% bovine serum albumin (BSA) and 2% nonfat dry milk (Carnation) for the blocking solution and 1% BSA for the antibody diluent. Primary antibodies were used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; Pierce) were used to visualize bound primary antibodies with the Supersignal chemiluminescence substrate (Pierce).Preparation of Heart and Lung Paraffin Sections
Mice were killed, and their hearts and lungs were removed and placed in buffered formalin (10%). The tissue was fixed for ~24 h, washed in PBS for 20 min, dehydrated through a graded series of ethanol washes, treated with xylene for 40 min, and then embedded in paraffin for 1 h at 55°C. Paraffin-embedded 5-µm-thick sections were then prepared using a Microm (Baxter Scientific) microtome and placed on Superfrost Plus slides (Fisher). Slides were then stained with hematoxylin and eosin (H&E), according to standard laboratory protocols. Areas of the myocardium (left ventricle, right ventricle, and intraventricular septum) were selected for imaging. Samples were examined in a blinded fashion by S. M. Factor.Preparation of Heart Frozen Sections and Immunohistochemistry
Mice were killed, and their hearts were immediately removed, embedded in optimal cutting temperature (OCT) compound, and flash-frozen in liquid nitrogen. Five-micrometer-thick sections were then cut with a cryostat (Leica) maintained atNorthern Blot Analysis
Mice were killed, and the cardiac ventricular tissue was carefully separated from atrial tissue by dissection. Total RNA was extracted from 100 mg of cardiac ventricular tissue for each sample using the Trizol reagent protocol (GIBCO). Twenty micrograms of total RNA for each sample were separated by using a 1.2% agarose gel under RNase-free conditions and transferred to a Hybond-XL nylon membrane (Amersham Pharmacia Biotech). The filters were hybridized using ExpressHyb solution (Clontech). The blots were probed with radiolabeled ANF cDNA.Noninvasive Cardiac Imaging
Gated cardiac magnetic resonance imaging. Magnetic resonance imaging (MRI) experiments were performed by using a GE Omega 9.4T vertical-bore MR system equipped with a microimaging accessory and custom-built coils designed specifically for mice. Just before each image acquisition, the heart rate was determined from the electrocardiogram, and the spectrometer gating delay was set to acquire data in diastole and systole. Multislice spin-echo imaging with an echo time of 18 ms and a repetition time of ~100-200 ms was performed. A 35-mm field of view (with a 256 × 256-pixel image matrix) was used. Short- and long-axis images of the heart were acquired, and MRI data were processed off-line with MATLAB-based custom-designed software.
Transthoracic echocardiography.
Transthoracic echocardiography was performed on 2- and 4-mo-old mice,
as we described previously (8). Echocardiography was
performed with mice in supine position on a heating pad set at 38°C.
Light anesthesia was achieved by using an intraperitoneal injection of
chloral hydrate (300 mg/kg). Continuous, standard electrocardiograms
were taken from electrodes placed on the extremities. Echocardiographic
images were obtained by using an annular array, broadband, 10/5-MHz
transducer attached to an HDI 5000 CV ultrasound system (Advanced
Technology Laboratories, Bothel, WA). A small gel standoff was placed
between the probe and chest. Two-dimensional and M-mode images of the
heart were obtained from the basal short-axis view of the heart and
stored on 3/4-in. SVHS video tapes for off-line measurements
using the Nova-Microsonic (Kodak) Imagevue DCR workstation (Indianapolis, IN). All measurements were made in three to six consecutive cardiac cycles, and the averaged values were used for
analysis. Left ventricular end-diastolic and end-systolic diameters as
well as diastolic ventricular septal and posterior wall thickness were
measured from M-mode tracings. Diastolic measurements were performed at
the point of greatest cavity dimension, and systolic measurements were
made at the point of minimal cavity dimension, using the leading-edge
method of the American Society of Echocardiography (65).
In addition, the following parameters were calculated by using the
above-mentioned measurements: left ventricular diastolic wall thickness
as the average of ventricular septal and left ventricular posterior
wall thickness; left ventricular percent fractional shortening as
100 × [(end-diastolic diameter end-systolic
diameter)/end-diastolic diameter]; and relative wall thickness as
(2 × left ventricular diastolic wall thickness)/end-diastolic diameter.
Statistical Analysis
All results were analyzed using a one-sided unpaired t-test. A P value of <0.05 was deemed significant.Isolation and Culture of Primary Cardiac Fibroblasts
Under sterile conditions, hearts from 4-mo-old wild-type (n = 5) and Cav-1 null (n = 5) mice were removed and carefully cleaned of all surrounding tissue. Each heart was then washed in sterile PBS, minced with a razor blade, and placed in a 10-ml solution of warm Hanks' balanced salt solution (HBSS; Sigma) containing 10 mM HEPES (Sigma) and 2% collagenase type I (Worthington Biochemical). Each sample was then agitated for 90 min at ~175 rpm at 37°C. At the end of this period, samples were spun at 1,000 rpm for 5 min, the HBSS solution was removed, and the pellets were washed twice in DMEM containing 10% FBS. Cells were then plated in 10-cm dishes and allowed to attach for ~2 days, at which time the medium was replaced. Fibroblasts were purified by selective attachment to plastic tissue culture dishes. Populations were allowed to grow to ~60% confluence and then expanded at a dilution of 1:3. With the use of this approach, in passage 3 cultures >98% of the cells showed a fibroblastic morphology. Western blot analysis was performed only on cells that were early passage ( ![]() |
RESULTS |
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Cav-1 Null Mice Develop Cardiac Hypertrophy With Right Ventricular Dilation as Shown by Gated Cardiac MRI and Gross Morphology
We first investigated hearts of Cav-1 null mice using a noninvasive technique, gated MRI. Current MRI technology provides the most accurate and reliable noninvasive method for determining several parameters in the murine heart, especially right ventricular (RV) chamber size (77). Cardiac gating allows for the timing of images that correspond to systolic and diastolic portions of the cardiac cycle, thus allowing for measurements of the fully dilated and fully contracted ventricular chamber.Using this technique, we examined the hearts of 2- and 4-mo-old Cav-1
null mice and age-matched controls for several parameters (Fig.
1). We found that at 2 mo of age, Cav-1
null mice demonstrate significant left ventricular (LV) wall thickening
(~19% thicker) compared with wild-type control mice (Table
1). Most notable are changes observed in
the RV of the Cav-1 null mice; this chamber is ~62% larger than in
wild-type mice of the same age (Table 1). Virtually identical results
were also obtained at 4 mo of age, when Cav-1 null mice demonstrate an
~18% increase in LV wall thickness and an ~70% larger RV chamber
compared with age-matched wild-type mice.
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To further evaluate this phenotype, we killed the mice and removed
their hearts by gross dissection. Each heart was then carefully freed
of surrounding tissue, washed in PBS, and weighed on a standard laboratory balance. Because of variations in body weight between Cav-1
null and wild-type mice, we chose to express the heart wet weight as a
percentage of total body weight. This normalizes differences in heart
size that can be attributed to the overall size of the mouse.
Consistent with our findings by MRI, we show in Table
2 that the hearts of 2- and 4-mo-old
Cav-1 null mice are ~25-40% heavier than those of age-matched
wild-type mice.
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Cav-1 Null Mice Show Concentric LV Hypertrophy as Revealed by Transthoracic Echocardiography
Heart rate. Transthoracic echocardiography was performed on wild-type and Cav-1 null mice at 2 and 4 mo of age. In all groups tested, there was no significant difference in heart rate (data not shown). No cardiac arrhythmias or conduction defects were noted during continuous electrocardiographic monitoring. However, several parameters were significantly altered in Cav-1 null mice.
LV hypertrophy.
As shown in Table 3, 2-mo-old Cav-1 null
mice exhibit significant hypertrophy with increases of ~26% in
intraventricular septal thickness, posterior wall thickness, and LV
wall thickness during diastole compared with age-matched wild-type
control mice. At 4 mo of age, the Cav-1 null mice show an even greater
degree of hypertrophy, with increases of ~50% in the same
parameters.
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Decreased systolic function. Cav-1 null mice at 2 and 4 mo of age demonstrate a significant reduction in LV systolic function, as evidenced by decreased fractional shortening (Table 3). In both age groups, Cav-1 null mice show a ~20% decrease in fractional shortening compared with age-matched wild-type controls. With significant LV hypertrophy, there is first a loss of diastolic function (which cannot be quantified with our equipment), and eventually there is a loss of systolic function as well. This is a common response, regardless of the molecular mechanism that has resulted in cardiac hypertrophy.
Histopathological Analysis of Cav-1 Null Hearts Reveals Cardiac Myocyte Hypertrophy and Degeneration With Interstitial Fibrosis And Inflammation
We next subjected Cav-1 null hearts to routine histological analysis using H&E and Tri-chrome-stained paraffin sections derived from wild-type and Cav-1 null animals at 2 and 4 mo of age. Figures 2, 3, and 4 illustrate some of the pertinent pathological differences between wild-type and Cav-1 null mouse hearts.
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Right ventricle. At 2 mo of age, Cav-1 null mice display interstitial hypercellularity, enlarged nuclei (arrows) and focal double nuclei (double arrows), and cellular hypertrophy (Fig. 2, C and D). Comparative wild-type images demonstrate normal tissue architecture (Fig. 2, A and B). At 4 mo of age, similar pathology is found in the RV of Cav-1 null mice, with the most prominent features being interstitial hypercellularity and hypertrophic myocytes (Fig. 3, C and D).
Left ventricle. Figure 2, E and F, shows representative images of a normal 2-mo-old wild-type LV. In comparison, the LV of Cav-1 null mice at 2 mo of age shows significantly enlarged myocellular nuclei (arrows), focal perivascular fibrosis with interstitial inflammatory cells, and myocyte hypertrophy (Fig. 2, G and H). At 4 mo of age, Cav-1 null mice show cellular vacuolization (Fig. 3, G, H, and J, arrowheads), interstitial inflammation (Fig. 3I), fibrosis and necrosis (Fig. 3J, box), and lipofuscin deposits (Fig. 3J, arrows).
Atria. The atria of Cav-1 null mice also demonstrate some unique pathological findings. Both the right and left atrial chambers are dilated and thin walled, and show increased interstitial cellularity and abnormally small cells (data not shown).
Fibrosis. Paraffin-embedded wild-type and Cav-1 null mouse hearts were sectioned and subjected to Tri-chrome staining to identify possible areas of fibrosis that contain collagen (Fig. 4). Interestingly, the hearts of Cav-1 null mice show a significant increase in Tri-chrome staining throughout the myocardium and particularly around large vessels (Fig. 4, B-D, arrows point at the areas of blue-green staining/fibrosis). In contrast, the hearts of wild-type mice show very little fibrous tissue (Fig. 4A).
Upregulation of ANF mRNA in Cav-1 Null Hearts Indicates a Switch to Fetal Programming
ANF is normally produced by the cardiac atria and plays a crucial role in the regulation of blood pressure and fluid volume. During embryogenesis and fetal development, the atria and ventricles both produce ANF; however, after birth, ANF production ceases in the ventricles and is restricted to the atria.Most forms of cardiac hypertrophy result in a reversion to a fetal
program of gene expression in ventricular tissue (42). Therefore, we used Northern blot analysis to examine the levels of ANF
mRNA in ventricular tissue from Cav-1 null hearts. Figure 5 shows that there is a dramatic increase
in the ANF transcript in ventricular tissue isolated from Cav-1 null
hearts compared with those of age-matched wild-type control mice. These
findings are consistent with the notion that Cav-1 null mice develop
significant cardiac hypertrophy.
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Hyperactivation of p42/44 MAP Kinase Cascade in Cav-1 Null Heart Tissue
Multiple lines of experimental evidence now indicate that caveolin-1 functions as an endogenous inhibitor of the Ras-p42/44 MAP kinase cascade (12-14, 22, 81). Thus a negative reciprocal relationship exists between caveolin-1 and the p42/44 MAP kinase cascade, because 1) caveolin-1 expression is downregulated by sustained activation of the Ras-p42/44 MAP kinase cascade at the level of transcriptional control (i.e., caveolin-1 promoter studies; Refs. 17, 48); and 2) the caveolin-1 scaffolding domain (residues 82-101) directly interacts with both MEK and ERK and inhibits their kinase activity (12). Similarly, antisense-mediated ablation of caveolin-1 expression in NIH/3T3 cells causes sustained hyperactivation of the Ras-p42/44 MAP kinase cascade (22). Finally, RNAi-based ablation of caveolin-1 in C. elegans leads to progression of the meiotic cell cycle, a phenotype that mirrors that of Ras activation (61).Because the Ras-p42/44 MAP kinase cascade is known to be hyperactivated
in in vitro and in vivo models of cardiac hypertrophy, we next
investigated the status of this cascade in the hearts of Cav-1 null
mice (46, 66, 79). Wild-type and Cav-1 null hearts were
carefully harvested, homogenized in boiling sample buffer, and
subjected to Western blot analysis with phospho-specific antibodies
that selectively recognize only the activated/phosphorylated form of
ERK1/2. Figure 6 shows that in the Cav-1
null heart tissue samples, ERK1/2 is dramatically hyperactivated.
Importantly, immunoblotting with phospho-independent ERK1/2 antibodies
revealed that equivalent amounts of total ERK1/2 are present in both
wild-type and Cav-1 null samples. Thus hyperactivation of the
Ras-p42/44 MAP kinase cascade may provide an underlying mechanism to
explain the cardiac hypertrophy observed in Cav-1 null mice.
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Hyperactivation of p42/44 MAP Kinase Cascade is Localized to Areas of Interstitial Fibrosis Within Cav-1 Null Cardiac Tissue
Because caveolin-1 is normally expressed only in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelial cells, we next used immunocytochemistry to localize the hyperactivation of ERK1/2 to a particular cell type. The hearts of wild-type and Cav-1 null mice were quickly harvested by dissection, embedded in OCT, and frozen in liquid nitrogen. Frozen sections of cardiac tissue were then prepared, fixed in acetone, and immunostained with anti-phospho-ERK1/2 IgG.Figure 7 shows representative
phase-contrast images of wild-type and Cav-1 null heart sections.
Cardiac myocytes appear striated under phase contrast and are easily
recognized (Fig. 7, A, B, E, and
F, arrows). However, areas of interstitial/perivascular fibrosis in Cav-1 null hearts do not show a typical myocytic appearance (Fig. 7, B and F, asterisks). Interestingly,
immunostaining with anti-phospho-ERK1/2 IgG stains only the areas of
interstitial/perivascular fibrosis, not the cardiac myocytes themselves
(Fig. 7, D and F). An overlay of Fig. 7,
B and D, shows that the phospho-ERK1/2 staining (red) is indeed confined to the area of interstitial fibrosis (Fig.
7F). These findings are consistent with the previous
observation that caveolin-1 is not normally expressed in cardiac
myocytes in vivo. In contrast, little or no immunostaining with
anti-phospho-ERK1/2 IgG is observed in wild-type heart tissue sections
(Fig. 7, C and E).
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We next performed double-labeling with anti-phospho-ERK1/2 IgG (rabbit
PAb) and anti-caveolin-3 IgG (mouse MAb) because caveolin-3 is
selectively expressed in the cardiac myocytes but not in
cardiac fibroblasts or endothelial cells. Interestingly,
Fig. 8 shows that caveolin-3 strongly
labels the adjacent cardiac myocytes but not the area of
interstitial fibrosis. Thus immunostaining with anti-phospho-ERK1/2 IgG
is selectively localized to the areas of interstitial fibrosis and
clearly excluded from the Cav-1 null cardiac myocytes.
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Cardiac Fibroblasts Derived From Cav-1 Null Mice Also Show Hyperactivation of ERK1/2
We have presented in vivo evidence the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (Fig. 6). Immunostaining of frozen sections reveals that activated ERK1/2 is confined to areas of interstitial fibrosis, suggesting that cardiac fibroblasts are the cell type that is affected (Figs. 7 and 8).To test this hypothesis directly, we next isolated cardiac fibroblasts
from wild-type and Cav-1 null hearts and examined the activation state
of ERK1/2 by Western blotting. Cells were grown to confluence in
six-well plates and lysed in boiling sample buffer. Figure
9 shows that ERK1/2 is clearly
hyperactivated in Cav-1 null cardiac fibroblasts compared with
wild-type control cardiac fibroblasts. Importantly, immunoblotting with
phospho-independent ERK1/2 antibodies revealed that equivalent amounts
of total ERK1/2 are present in both wild-type and Cav-1 null samples.
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PKC Levels Remain Unchanged in Hearts of Cav-1 Null Mice
Recently, Takeishi et al. (73) showed that transgenic
overexpression of the -isoform of PKC leads to concentric cardiac hypertrophy. The signaling pathway by which this isoform of PKC leads
to cardiac hypertrophy was shown to be unique from the
- and
-isoforms in that the
-isoform involves the activation of ERK1/2
(73). Because ERK 1/2 is hyperactivated in Cav-1 null mouse hearts, we sought to determine whether PKC
is upregulated under these conditions. Figure 10 shows
that PKC
expression remains unchanged in lysates derived from the
hearts of Cav-1 null mice. Immunoblotting with
-tubulin is shown as
a control for equal protein loading. Therefore, hyperactivation of
ERK1/2 in the hearts of Cav-1 null mice occurs independently of PKC
protein levels.
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Upregulation of Endothelial and Inducible Nitric Oxide Synthase Protein Levels in Hearts of Cav-1 Null Mice
We next examined the expression levels of several signaling molecules that are known to associate with caveolae and have been implicated in hypertrophic cardiac states. Several reports have indicated a role for nitric oxide synthase (NOS) isoforms in the development of cardiac hypertrophy. Caveolin-1 is known to be a negative regulator of the endothelial form of nitric oxide synthase (eNOS), which has been shown to play a role in hypertrophic cardiomyopathy (4, 28). The inducible form of NOS (iNOS) has been shown to be upregulated in the hearts of mice with Chagas' disease (an infectious disease that results in similar functional changes and RV pathology to those described here) as well as in other models of cardiomyopathy (11, 26, 32, 60). Cyclin D1 plays a key role in proliferative and mitogenic signaling and has been shown be transcriptionally regulated by caveolin-1 (33). Levels of caveolin-3 have been shown to be decreased in the hearts of dogs with cardiomyopathy (51). GLUT-4 null mice develop a hypertrophic cardiomyopathy (1), and protein levels of GLUT-4 are perturbed in adipocytes derived from Cav-1 null mice (A. W. Cohen and M. P. Lisanti, unpublished observations).Therefore, we examined the hearts of Cav-1 null mice for changes in the
expression levels of eNOS, iNOS, cyclin D1, GLUT-4, and caveolin-3.
Lysates were prepared from freshly isolated hearts and were
then subjected to Western blot analysis. Figure
11 shows that both eNOS and iNOS
protein levels are dramatically upregulated in Cav-1 null hearts.
However, the levels of cyclin D1, GLUT-4, and caveolin-3 remain
unchanged compared with wild-type control hearts. Immunoblotting with
-tubulin is shown as a control for equal protein loading. Thus
upregulation of eNOS and iNOS expression may contribute to the
cardiomyopathic phenotype of Cav-1 null mice.
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Analysis of Cav-2 Null Mouse Hearts Reveals Normal Structure and Function
As previously reported by our group and others (10, 54), Cav-1 null mice exhibit a hyperproliferative lung phenotype that is characterized by hypercellularity, with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (VEGF-R/Flk-1)-positive endothelial cells. In a subsequent study, we demonstrated that 1) Cav-2 null mice develop the same hyperproliferative lung phenotype and 2) this lung phenotype in Cav-1 null mice is due to a secondary deficiency in caveolin-2, because Cav-1 null mice also lack caveolin-2 protein expression (56). Downregulation of caveolin-2 expression occurs in Cav-1 null mice, because caveolin-1 is normally required to stabilize caveolin-2 and to allow caveolin-2 to be transported from the Golgi complex to the plasma membrane. In the absence of caveolin-1, caveolin-2 is misfolded, retained at the level of the Golgi, and undergoes rapid proteasomal degradation (56).Because this type of hyperproliferative lung disease might be expected
to produce increased pulmonary vascular resistance and a consequential
dilation of the RV, we examined the hearts of Cav-2 null mice for
evidence of the cardiac changes that we observed in Cav-1 null mice.
Importantly, using gated cardiac MRI, we have shown that Cav-2 null
mice do not show the RV dilation seen in Cav-1 null mice (Table
4). Whereas Cav-2 null mice appear to
have a slightly larger RV than wild-type mice, this difference is not
statistically significant (P 0.05). These data argue that the cardiac phenotype observed in Cav-1 null mice is due to loss
of caveolin-1 expression and is not secondary to the development of
pulmonary hypertension.
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We next examined the hearts of Cav-2 null mice for histopathological
abnormalities as well as possible changes in activation state of the
Ras-p42/44 MAP kinase cascade. Figure
12A shows representative H&E-stained images of wild-type and Cav-2 null RV and LV tissue. Importantly, these sections do not show any pathological alterations, further demonstrating that the hearts of Cav-2 null mice are normal. In
addition, Western blot analysis of Cav-2 null hearts did not reveal any
detectable increases in phospho-ERK1/2 levels (Fig. 12B) in
contrast to our results with Cav-1 null hearts (Fig. 6). Thus
lung hyperplasia and loss of caveolin-2 does not appear to play a role
in the cardiac phenotype of Cav-1 null mice.
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DISCUSSION |
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In this study, we have defined a new role for caveolin-1 in the
development of cardiac hypertrophy and dilation. Using noninvasive measurements, we have shown that Cav-1 null mice develop a severely dilated RV and a concentrically hypertrophied LV. Histopathological examination of Cav-1 null cardiac tissue directly supports these findings and shows interstitial hypercellularity with fibrosis, inflammation, and necrosis. The ANF mRNA, a well-known marker for
hypertrophy, is upregulated in the ventricular tissue of Cav-1 null
mice. Using Western blot analysis, we have found that the Ras-p42/44
MAP kinase pathway is hyperactivated and that this hyperactivation is
confined to areas of interstitial fibrosis but does not occur within
the cardiac myocytes themselves. Consistent with these findings,
isolated cardiac fibroblasts show hyperactivation of ERK1/2. We also
have shown that eNOS and iNOS protein levels are upregulated in Cav-1
null cardiac tissue but that the levels of PKC, GLUT-4, caveolin-3,
and cyclin D1 remain unchanged. Taken together, our results demonstrate
that loss of caveolin-1 leads to dilation and hypertrophy of the RV and
concentric hypertrophy of the LV. Mechanistically, this cardiac
phenotype appears to be due to the hyperactivation of ERK1/2 signaling
in the Cav-1 null cardiac fibroblasts.
Numerous studies have suggested a role for caveolin-1 in the
development of cardiac hypertrophy. In dogs with experimental hypertension, caveolin-1 was shown to be downregulated in the heart,
primarily in the subendocardium (51). Expression of
caveolin-1 is also decreased in the hearts of mice subjected to chronic
-adrenergic stimulation, a treatment known to cause cardiac
hypertrophy and remodeling (44). However, the exact role
of caveolin-1 in the development of cardiac hypertrophy, whether
passive or active, remained unclear. In this study, we have shown that
loss of caveolin-1 is clearly sufficient to drive the development of a
primary cardiomyopathic phenotype.
Because caveolin-1 has emerged as a regulatory element of multiple
cellular signaling cascades, there are several mechanisms by which a
loss of caveolin-1 could lead to the development of cardiac
hypertrophy. For example, it has been well documented that components
of the Ras-p42/44 MAP kinase cascade reside within caveolae membrane
domains and that signaling via this cascade is negatively regulated by
caveolin-1 (12, 38). Furthermore, the activation of this
cascade generally results in the downregulation of caveolin-1 protein
levels via transcriptional control; caveolin-1 promoter activity is
sharply diminished by activation of the p42/44 MAP kinase cascade
(14, 17). Also, a variety of independent investigators
have shown that the p42/44 MAP kinase cascade is activated in model
systems of cardiac hypertrophy (46, 57, 66, 79). Moreover,
PKC is a known mediator of ERK1/2 activation, and mice
transgenically overexpressing this PKC isoform develop concentric
cardiac hypertrophy (73). Here, we have shown that the
p42/44 MAP kinase cascade is hyperactivated and ERK 1/2 is hyperphosphorylated in the hearts of Cav-1 null mice. Because of the
known relationships between caveolin-1, p42/44 MAP kinase, and cardiac
hypertrophy, it is perhaps not unexpected that loss of caveolin-1
expression leads to hyperactivation of ERK 1/2 and development of
concentric cardiac hypertrophy. However, PKC
levels remain unchanged
in Cav-1 null hearts.
The cardiac fibroblast has traditionally been considered a passive
player in the development of cardiac disease. Its role was often
thought to be limited to reactive fibrosis, exemplified by the
remodeling that occurs post-myocardial infarction (post-MI). Only
within the past 10 years has the true nature of the cardiac fibroblast's contribution to disease pathogenesis been appreciated. A
multitude of studies have led to the understanding that cardiac fibroblasts can indeed be the main cause of hypertrophic changes in the
cardiac myocyte (5, 6, 29, 50, 76, 79). One group even
stated that the cardiac fibroblast "plays a dominant role in
governing the structure, architecture, and mechanical behavior of the
myocardium" (6). The mechanisms by which the cardiac
fibroblast exercises its control have only begun to be elucidated.
Angiotensin II (ANG II) has long been known to contribute to the
development of cardiac hypertrophy and heart failure. Until recently,
it was assumed that ANG II exerted its effect by direct binding to ANG
II type 1 (AT1) receptors on the cardiac myocyte. However,
with the use of radioligand binding, immunohistochemistry, and other
methodologies, it was recently demonstrated that ANG II binding occurs
primarily on cardiac fibroblasts, not on the cardiac myocytes as
previously thought (29). In this context, ANG II binding
has been shown to activate the Ras-p42/44 MAP kinase pathway in
cultured cardiac fibroblasts (57, 66). Furthermore, it has
been demonstrated that another consequence of ANG II-mediated activation of cardiac fibroblasts is the secretion of peptide growth
factors/hormones, such as TGF-1 and endothelin-1, from the
fibroblast (29). It has been shown that, when added to
cardiac myocytes in culture, conditioned media taken from primary
cardiac fibroblast cultures can activate the hypertrophic program in
cultured cardiac myocytes (5, 29). Ultimately, a model has
been developed whereby ANG II binds to the AT1 receptor on
the cardiac fibroblast, causing activation of p42/44 MAP kinase cascade
and the release of the above factors, which, via a paracrine mechanism,
leads to hypertrophic responses in neighboring cardiac myocytes
(5, 6, 29, 41, 46, 50, 57, 66). In the present study, we
have presented dramatic in vivo support for these ideas, showing that
activation of the p42/44 MAP kinase pathway specifically in cardiac
fibroblasts/fibrotic lesions is associated with myocyte hypertrophy and
the development of cardiomyopathy in the intact murine heart.
We also have shown here that two NOS isoforms are overexpressed in
cardiac tissue derived from Cav-1 null mice. Recently, the role of the
various NOS isoforms in the development of cardiac pathology has been
the subject of intense study. Transgenic mice that overexpress TNF-
develop a dilated cardiomyopathy. In studying these mice, it was noted
that iNOS expression was significantly increased in the hearts of the
transgenic animals, leading to inotropic hyporesponsiveness
(20). Rats with experimental volume-overload heart failure
are also hyporesponsive to
-stimulation, resulting from increased
iNOS activity (58). In the human population, it has been
shown that patients with MIs and those with end-stage heart failure
have increased cardiac expression of iNOS (2, 19). This
increase in iNOS levels contributes to the development of myocardial
dysfunction and the progression of an MI to heart failure
(58). A recent study demonstrated that a direct
interaction exists between caveolin-1 and iNOS. Using a human colon
carcinoma cell line that normally expresses very low levels of
caveolin-1, Felley-Bosco et al. (18) showed that ectopic
expression of caveolin-1 results in diminished iNOS activity and
protein levels. Furthermore, these authors demonstrated that
caveolin-1-induced degradation of iNOS occurs via a proteasomal pathway
(18). Therefore, caveolin-1 acts to reduce both the amount
of iNOS protein and its enzymatic activity. Consistent with these
findings, we have shown that in cardiac tissue derived from the Cav-1
null mice, the amount of iNOS protein is greatly upregulated.
The role of the p42/44 MAP kinase cascade in the induction of iNOS has
also been recently explored. In both cardiac endothelial cells and
myocytes, IFN- causes an increase in iNOS transcription that is
blocked by inhibitors of the p44/42 MAPK pathway. These results suggest
that the activation of MAP kinase is necessary for the induction of
iNOS in both of these cells types (68). In light of our
findings regarding the phosphorylation state of ERK1/2 and in
accordance with the idea that caveolin-1 downregulates levels of iNOS,
the observed increases in iNOS protein may be due to hyperactivation of
the p42/44 MAP kinase cascade caused by loss of caveolin-1.
Upregulation of iNOS could also explain some of the histopathological findings that we observed. We have found that in the hearts of Cav-1 null mice, there is evidence of myocyte degeneration and myocyte dropout. Several reports suggest that an overproduction of NO from iNOS can have deleterious effects on cardiac myocytes, leading to cell death presumably via apoptosis (37, 52, 53, 71, 72, 80). Therefore, hyperactivation of ERK1/2 and upregulation of iNOS may both play a significant role in the development of the cardiac phenotype of Cav-1 null mice.
The endothelial form of NOS is constitutively expressed in the endocardium and the endothelial cells of arteries, veins, and capillaries, as well as in the ventricular cardiac myocytes of rodents. However, the literature contains many conflicting reports regarding the relationship between eNOS and the development of cardiac hypertrophy (3, 51). Several studies have shown that there is no change in eNOS protein levels in models of experimental hypertrophy, whereas others have shown a decrease in eNOS protein content (4, 39, 51). An increase in eNOS mRNA has also been demonstrated (4). Here, we show a dramatic upregulation of eNOS protein content in the hearts of Cav-1 null mice. NO derived from eNOS has been shown to accelerate ventricular relaxation during diastole, without affecting systolic function (30, 69). Furthermore, studies in isolated hearts have shown that NO is released only during diastole and that its release is augmented by increased preload or increased end-diastolic volume (53); the source of this NO was ascribed to intramyocardial endothelial cells expressing eNOS. Interestingly, eNOS is known to be localized to caveolae membrane domains, where its activity is negatively regulated by either caveolin-1 or caveolin-3, depending on cell type (7, 25, 67). Therefore, loss of caveolin-1 in Cav-1 null mice could drive the upregulation of the eNOS protein and its subsequent effects on the myocardium.
It is important to consider the topic of primary vs. secondary development of the cardiac phenotype observed in Cav-1 null mice. As reported previously, Cav-1 null mice have a pulmonary phenotype characterized by hypercellularity and alveolar wall thickening (10, 54). This phenotype could possibly lead to pulmonary hypertension and a consequential dilation of the RV and is, therefore, a potential confounding variable in this study.
However, we directly addressed this issue by studying the hearts of Cav-2 null mice. Because caveolin-2 is unstable in the absence of caveolin-1, the Cav-1 null mouse is also functionally deficient in caveolin-2. In studying Cav-2 null mice, our group previously showed that the pulmonary pathology identified in Cav-1 null mice is actually due to loss of caveolin-2, and not caveolin-1 (56). Because Cav-2 null mice have the same pulmonary defect as Cav-1 null mice, we used Cav-2 null mice to determine whether the pulmonary pathology plays any role in the cardiac dilation observed in Cav-1 null mice. Importantly, using MRI analysis, we have shown that Cav-2 null mice do not exhibit significant RV dilation. Thus we can conclude that the pulmonary phenotype does not contribute to the RV dilation observed in Cav-1 null mice.
In summary, here we have clearly defined a new role for caveolin-1 in the development of concentric LV hypertrophy and RV dilation. We have shown that loss of caveolin-1 in the hearts of these mice leads to hyperactivation of p42/44 MAP kinase cascade selectively in areas of interstitial fibrosis and in isolated cardiac fibroblasts. In addition, we have observed that Cav-1 null hearts also show overexpression of eNOS and iNOS. Finally, we have demonstrated that the pathological changes observed in Cav-1 null hearts are not secondary to lung defects and/or pulmonary hypertension.
Recently, a report by Zhao et al. (82) appeared describing
similar findings in a caveolin-1-deficient mouse model independently generated by this group. Importantly, their results are consistent with
our current findings, including dilation of the RV, decreased systolic
function, an increased heart weight-to-body weight ratio, and
upregulation of ventricular ANF mRNA in Cav-1 null mice. In contrast to
our current results, they observed thinning and dilation of the LV,
whereas we observed thickening of the LV. However, the ventricular
dilation that they noted is based solely on echocardiographic measurements, whereas our data were obtained from both echocardiography and MRI analysis. Thus one likely explanation for this discrepancy may
be the use of different anesthetics during the echocardiographic measurements. For example, in performing our experiments, we found that
Cav-1 null mice are hypersensitive to inhaled drugs, such as isoflurane
and metofane (not shown). In Cav-1 null mice, use of these sedatives
caused an unnatural dilation of the LV during echocardiography.
Unfortunately, it is unclear which anesthetic Zhao et al. used during
echocardiography. Finally, these authors did not examine the activation
state of the Ras-p42/44 MAP kinase cascade in Cav-1 null hearts or the
behavior of caveolin-1 (/
) cardiac fibroblasts in culture, and they
did not perform any other mechanistic studies. Nevertheless, both
reports (our paper and Ref. 82) conclude that caveolin-1
expression in the heart is important for maintaining normal cardiac functioning.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Roberto Campos-Gonzalez for generously donating antibodies directed against Cav-1, Cav-2, and Cav-3.
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FOOTNOTES |
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This work was supported by grants from the National Institutes of Health (NIH), Muscular Distrophy Association, and American Heart Association, as well as a Hirschl/Weil-Caulier Career Scientist Award (all to M. P. Lisanti). A. W. Cohen, S. E. Woodman, and T. M. Williams were supported by NIH Medical Scientist Training Grant T32-GM-07288. D. S. Park is supported by NIH Graduate Training Program Grant TG-CA-09475. H. B. Tanowitz was supported by NIH Grant AI-12770.
Address for reprint requests and other correspondence: M. P. Lisanti, Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: lisanti{at}aecom.yu.edu).
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.
First published October 9, 2002;10.1152/ajpcell.00380.2002
Received 22 August 2002; accepted in final form 3 October 2002.
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