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Human Molecular Genetics, 2004, Vol. 13, No. 2 151-157
DOI: 10.1093/hmg/ddh014

Overexpression of P104L mutant caveolin-3 in mice develops hypertrophic cardiomyopathy with enhanced contractility in association with increased endothelial nitric oxide synthase activity

Yutaka Ohsawa1, Haruhiro Toko2, Masashi Katsura3, Kazue Morimoto1, Haruki Yamada1, Yaeko Ichikawa1, Tatsufumi Murakami1, Seitaro Ohkuma3, Issei Komuro2 and Yoshihide Sunada1,*

1Division of Neurology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki-City, Okayama 701-0192, Japan, 2Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan and 3Department of Pharmacology, Kawasaki Medical School, 577 Matsushima, Kurashiki-City, Okayama 701-0192, Japan

Received July 8, 2003; Accepted November 11, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effect of endogenous nitric oxide synthase (NOS) on cardiac contractility and architecture has been a matter of debate. A role for NOS in cardiac hypertrophy has recently been demonstrated by studies which have shown hypertrophic cardiomyopathy (HCM) with altered contractility in constitutive NOS (cNOS) knockout mice. Caveolin-3, a strong inhibitor of all NOS isoforms, is expressed in sarcolemmal caveolae microdomains and binds to cNOS in vivo: endothelial nitric oxide synthase (eNOS) in cardiac myocytes and neuronal nitric oxide synthase (nNOS) in skeletal myocytes. The current study characterized the biochemical and cardiac parameters of P104L mutant caveolin-3 transgenic mice, a model of an autosomal dominant limb-girdle muscular dystrophy (LGMD1C). Transgenic mouse hearts demonstrated HCM, enhanced basal contractility, decreased left ventricular end diastolic diameter, and loss and cytoplasmic mislocalization of caveolin-3 protein. Surprisingly, cardiac muscle showed activation of eNOS catalytic activity without increased expression of all NOS isoforms. These data suggest that a moderate increase in eNOS activity associated with loss of caveolin-3 results in HCM.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Caveolae are 50–100 nm flask-shaped invaginations of the plasma membrane that are primarily composed of the 21–24 kDa integral membrane proteins, caveolin-1, -2 and -3 (1). These structures participate in vesicular trafficking events and signal transduction by acting as scaffold proteins for specific lipids and lipid-modified signaling molecules (e.g. cholesterol, G-proteins, G-protein coupled receptors, receptor tyrosine kinases and nitric oxide synthase) (2–4).

Caveolin-3 is a myocyte-specific isoform that assembles to ~350 kDa homo-oligomers in the sarcoplasmic reticulum (SR) (2). Caveolin-3 homo-oligomers are translocated to the plasma membrane via the trans-Golgi network (2,4) and inhibit all NOS isoforms in vitro and bind to constitutive NOS (cNOS) in vivo: endothelial nitric oxide synthase (eNOS) in cardiac myocytes and neuronal nitric oxide synthase (nNOS) in skeletal myocytes (5–8).

Autosomal dominant limb-girdle muscular dystrophy 1C (LGMD1C) and autosomal dominant rippling muscle disease (AD-RMD) result from heterozygous mutations of the skeletal muscle caveolin-3 gene (CAV3) (9,10). We previously generated transgenic (Tg) mice (TgCAV3M1 mice) with severe myopathy secondary to overexpression of P104L mutant caveolin-3 as a model of LGMD1C (11). Further study of this transgenic animal model revealed increased sarcolemmal nNOS activity without alternations of nNOS expression at both mRNA and protein levels. Other reports demonstrated that caveolin-3 is down-regulated in the hypertrophic hearts of spontaneously hypertensive rats (12) and that circulatory NO (13) or eNOS mRNA in cardiac muscle (14) increases in human heart failure. These observations suggest that loss of caveolin-3 may modulate cardiac architecture and function via disinhibition of NOS activity. The present study investigated this hypothesis by characterizing the biochemical and cardiac parameters of TgCAV3M1 mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TgCAV3M1 mice show hypertrophic cardiomyopathy with enhanced contractility
TgCAV3M1 mice were generated as described previously (11). Tg mice showed poor growth and were significantly smaller than their wild-type (Wt) littermates at 4, 12, 24 and 36 weeks of age (Fig. 1A) and demonstrated kyphosis of the spine and paralysis of the hindlimbs from 12 weeks of age. The Tg mice exhibited increased cardiac weight and statistically significant increases in the cardiac weight-to-body weight ratio at 4, 12, 24 and 36 weeks of age when compared with their Wt littermates (Fig. 1B and C). However, from the clinical standpoint, the Tg mice have so far not developed any significant symptoms of heart failure, nor have any significant differences in life span been observed between the Wt and Tg mice.



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  		Figure 1. Comparison of body weight (A), cardiac weight (B) and the cardiac weight-to-body weight ratio (C) between Wt (open circle, n=5) and Tg (solid circle, n=5) mice at 4, 12, 24 and 36 weeks of age. Note that the smaller body weight and larger cardiac weight in the Tg mice results in statistically significant increases in the mean cardiac weight-to-body weight ratios. Error bars are ±SD. *Statistical significance was determined by Welch's t-test (P<0.05).

 
Gross appearance of the hearts from 6-week-old Wt and Tg mice at the lower ventricular level showed thickening of the interventricular septum and the posterior wall of the left ventricle, resulting in a smaller left ventricular chamber in the Tg mice (Fig. 2A). Hematoxylin and eosin staining of cardiac muscle sections showed hypertrophy of cardiac myofibers in the Tg mice (Fig. 2B). The diameters of 1000 cardiac muscle fibers from five Tg mice and five Wt mice, respectively, were measured. The frequency distribution (percentage) of the cardiac myofiber diameter in Tg mice showed a distribution skew to the right and marked size variability on both longitudinal and transverse sections when compared to Wt mice (Fig. 2C). The mean cardiac muscle fiber diameter and standard deviation were significantly larger (P<0.05) in Tg mice (8.53±1.43 µm on longitudinal section and 9.35±1.17 µm on transverse sections) than in Wt mice (6.55±0.91 and 7.32±0.83 µm, respectively; Fig. 2C). Gene expression of cardiac myocyte hypertrophic markers, atrial natriuretic peptide (ANP) and brain-derived natriuretic peptide (BNP) was analyzed by northern blotting. Densitometry using BAS2000 Image Analyzer demonstrated that both the ANP and BNP transcripts in the Tg mouse hearts were up-regulated by 1.69- and 1.54-fold, respectively (Fig. 2D). Transthoracic echocardiogram performed in 24-week-old Tg mice (n=7) revealed unique pathophysiological characteristics for HCM; increased thickness of the interventricular septum and left ventricular posterior wall, hypercontractility (increased left ventricular fractional shortening) and diastolic dysfunction (decreased left ventricular end diastolic diameter; Table 1).



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  		Figure 2. Histology of hearts from 6-week-old Wt and Tg mice. (A) Gross appearance of transverse lower ventricular sections from Wt and Tg mouse hearts. Note that apparent thickening of the interventricular septum and posterior wall of the left ventricle results in a tiny ventricular chamber in the Tg mouse heart (right). LV, left ventricle; IVS, interventricular septum; LVPW, left ventricular posterior wall; RV, right ventricle. Bar, 1 mm. (B) H&E staining of cardiac muscle sections from 6-week-old Wt or Tg mice. Note the myofiber hypertrophy in the Tg mouse hearts in both longitudinal (Upper panels) and transverse (lower panels) sections. Bar, 10 µm. (C) Histogram of cardiac myofiber diameter from 6-week-old Wt or Tg mice on longitudinal sections (left) and on transverse sections (right). The diameters of 1000 cardiac muscle fibers from five Tg mice and five Wt mice were measured. Note that the frequency distribution (%) of the cardiac muscle fiber diameter in the Tg mice compared with that in the Wt mice shows a skewed distribution to the right and marked size variability, thus indicating cardiac myocyte hypertrophy. The diameter of each cardiac muscle fiber was measured by the IBAS 2000 image analysis system (Zeiss). (D) Northern blot analysis of cardiac myocyte hypertrophic markers; ANP and BNP. Radiointensities of ANP and BNP transcripts in the Tg mouse hearts were up-regulated by 1.69- and 1.54-fold, respectively.

 

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  		Table 1. Transthoracic echocardiographic analysis of hearts from 24-week-old Wt and Tg mice
 
Tg CAV3M1 mouse hearts show loss and cytoplasmic mislocalization of caveolin-3
Northern blot analysis of Tg mouse cardiac muscle showed overexpression of smaller-sized (~1.1 kb) mutant caveolin-3 mRNA (Fig. 3A). Detection of endogenous caveolin-3 mRNA (~1.4 kb) in the Tg mice was interfered with by an excessive amount of the mutant caveolin-3 mRNA. Then, the differential expression of endogenous and mutant caveolin-3 mRNA was analyzed by RT–PCR using two distinct reverse primers as described previously (11). RT–PCR confirmed the expression of endogenous caveolin-3 mRNA in Tg mouse hearts as well as in Wt mouse hearts, as shown in Figure 3B.



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  		Figure 3. (A) Northern blot analysis of caveolin-3 in cardiac muscle from 6-week-old Wt and Tg mice. The smaller-sized (~1.1 kb) mutant caveolin-3 mRNA was expressed excessively in the Tg mice compared with the Wt mice. (B) RT–PCR analysis of endogeneous or mutant caveolin-3 in cardiac muscle from 6-week-old Wt and Tg mice. The endogenous caveolin-3 was detectable in Tg mice as well as Wt mice using primers of F1/R1 (upper panel, left). The mutant caveolin-3 mRNA was detectable only in Tg mice using primers of F1/R2 (upper panel, right). R1 corresponds to the 5'-untranslated region (5'-UTR) of the caveolin-3 transcript, whereas R2 corresponds to the SV40 polyadenylation site (pA) of the transgene transcript (lower panel). (C) Immunoblot analysis of caveolin-3 in total protein extracts from 6-week-old Wt and Tg mouse cardiac muscles. Caveolin-3 expression was markedly reduced in the Tg mice. In contrast, ß-DG expression was similar in the Wt and Tg mice. (D) Immunohistochemical analysis of cardiac muscle from 6-week-old Wt and Tg mice. Lower ventricular cryosections from the Wt and Tg mice were stained with antibodies against caveolin-3 (Cav-3), dystrophin (Dys) and caveolin-1 (Cav-1). Caveolin-3 localized to the sarcolemma in the Wt mice, but the Tg mice generally lacked caveolin-3 except for a few fibers that showed weak cytoplasmic localization of caveolin-3. The expression of dystrophin and caveolin-1 was similar in the Wt and Tg mice. Bar, 10 µm.

 
In sharp contrast to overexpression of mutant caveolin-3 mRNA, immunoblot analysis showed a marked reduction (~95%) of caveolin-3 protein in Tg mouse cardiac muscle (Fig. 3C). Immunohistochemical analysis demonstrated sarcolemmal localization of caveolin-3 protein in the Wt mice, while the majority of cardiac muscle fibers of the Tg mice showed only weak sarcolemmal immunoreactivity with small, cytoplasmic, dot-like immunoreactivity (Fig. 3D). There was no change in sarcolemmal dystrophin expression in the Tg mice (Fig. 3D). Caveolin-1 was expressed in endothelial cells and showed no compensatory overexpression in Tg mouse cardiac myocytes (Fig. 3D). ß-Dystroglycan expression was similar in the Wt and Tg mice (Fig. 3C).

NOS activity is increased in TgCAV3M1 mouse hearts
Expression of all NOS isoforms was similar when Wt and Tg mice cardiac muscles were compared by northern blot and immunoblot analyses (Fig. 4A and B). In addition, subsarcolemmal and vascular endothelial localization of eNOS was similar in Tg and Wt mouse hearts (Fig. 4C). NOS activity was quantified in crude extracts from freshly isolated hearts from six-week-old Wt and Tg mice (n=8). Total NOS and eNOS activities in the Tg mouse hearts were significantly higher (P<0.05) than those in the Wt mouse hearts: total NOS activity (pmol/mg protein/min)—Tg mouse, 5.10±0.32, Wt mouse, 3.38±0.32; eNOS activity (pmol/mg protein/min)—Tg mouse, 4.03±0.32, Wt mouse, 2.31±0.24. No significant differences were observed in either nNOS activity (pmol/mg protein/min)—Tg mouse, 0.77±0.07, Wt mouse, 0.75±0.06—or iNOS activity (pmol/mg protein/min)—Tg mouse, 0.30±0.02, Wt mouse, 0.31±0.03 (Fig. 4D).



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  		Figure 4. Northern blot analysis (A) and immunoblot analysis (B) of NOS isoforms in cardiac muscle from 6-week-old Wt and Tg mice. Note the comparable expression of all NOS isoforms in the Wt and Tg mice. (C) Immunohistochemical analysis of eNOS in cardiac muscle from 6-week-old Wt and Tg mice. The expression pattern of eNOS was similar in the Wt and Tg mice. Bar, 10 µm. (D) NOS activities in crude extracts from cardiac muscle from 6-week-old Wt and Tg mice. Note that significant increases in total NOS and eNOS activity were observed in the Tg mice, compared to the Wt mice (n=8). In contrast no significant difference was observed in either nNOS or iNOS activity between the Tg and Wt mice. Data are expressed as mean±SD. *Statistical significance was determined using Welch's t-test (P<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Caveolin-3 is a strong physiological inhibitor of NOS and expressed specifically in cardiac and skeletal myocytes (5–8). The present study demonstrated the occurrence of hypertrophic cardiomyopathy with enhanced contractility in association with increased eNOS activity in caveolin-3-deficient transgenic mice without changes in NOS expression. Therefore, loss of NOS inhibition secondary to deficient caveolin-3 may contribute to the pathogenesis of hypertrophic cardiomyopathy.

NOS activity may modulate cardiac contractility and architecture (15–18). eNOS is localized in caveolae (5,8) and plays a role in inhibition of ß-adrenergic-induced contractility (19,20). NO generated by eNOS may also interact with the SR ryanodine receptor (21). In contrast, nNOS, located in the SR (15,22), increases SR Ca2+ release and enhances cardiac contractility (15,23). eNOS knockout mice show enhanced contractility (15), while overexpression of eNOS results in reduced cardiac size and contractility (24). Unexpectedly, our Tg mouse hearts with moderately increased eNOS activity showed enhanced contractility and hypertrophic cardiomyopathy. It is noteworthy that exogenous NO, derived from pharmacological NO donors, produces a biphasic contractile response in cardiac tissue with augmentation at low NO levels and depression at high NO levels (25–27). Based on these exogenous NO donor experiments, we postulate that a moderate activation of eNOS activity, as seen in the mutant caveolin-3 Tg mouse hearts enhances contractility and leads to hypertrophic cardiomyopathy, whereas an extremely high activation of eNOS activity, as seen in mouse hearts in which eNOS is overexpressed, reduces contractility and cardiac size.

Woodman et al. (28) reported that caveolin-3 null mice showed cardiomyopathy and suppressed contractility, possibly secondary to alterations in the p42/44 MAPK pathway, but they did not characterize changes in NOS isoform profiles. Based on previous studies and the present data, we would predict greater NOS activity in caveolin-3 null mouse hearts than in the hearts of mutant caveolin-3 transgenic mice. This higher NOS activity in caveolin-3 null mouse hearts may result in suppression of cardiac contractility.

To date hypertrophic cardiomyopathy has not been reported in LGMD1C patients. However, in only one German pedigree of autosomal dominant rippling muscle disease (AD-RMD) (29), which was proven to carry a caveolin-3 missense mutation (A45V) (10), two patients died suddenly of possible cardiac arrhythmia. An autopsy study of one patient in this pedigree disclosed non-obstructive cardiomyopathy, which was poorly described (29). Cardiac involvement, cardiomyopathy and/or arrhythmia may be a rare but possible feature of CAV-3 missense mutations. Further extensive clinical examination of cardiac function in LGMD1C patients is necessary to test this possibility. Furthermore, it is plausible that the higher copy number of the mutant caveolin-3 gene in TgCAV3M1 mice promotes a disease process leading to HCM.

In conclusion, we propose that moderate eNOS activation caused by loss of caveolin-3 may be involved in a caveolin-3-mediated hypertrophic signal pathway of cardiac myocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
RT–PCR
cDNA templates were reverse-transcribed from 1 µg of total RNA from mouse cardiac muscle, primed with an oligo(dT)12–18 primer and then subjected to PCR. For amplification of the endogenous caveolin-3 gene, we used F1 (5'-CCCAGCCTCACAATGATGACCGAAG-3') and R1 (5'-CATGTGAACGCAAAGCCTTGC-3'). For amplification of the transgene, we used F1 and R2 (5'-GCATTCTAGTTGTGGTTT-3'). As illustrated in Figure 3B, R1 and R2 correspond to the 5'-untranslated region of caveolin-3 cDNA and the SV40 polyadenylation site, respectively, as described previously (11).

Northern blot analysis
RT–PCR product of endogenous caveolin-3 was subcloned into TA vector pCR2.1 (Invitrogen) and then digested by EcoRI. The digested insert was extracted from agarose gel using Concert Kit (GibcoBRL). This cDNA was then labeled as a probe with [{alpha}-32P]dCTP using MegaPrime DNA labeling system (Amersham Pharmacia Biotech). Ten micrograms of total RNA from 6-week-old Wt and Tg cardiac muscle were separated on 0.7% agarose gels containing 7% formaldehyde and blotted onto Hybond-N+ (Amersham Pharmacia Biotech). Hybridization was performed at 42°C for 12–24 h and autoradiography was performed on a Fuji imaging plate (Fuji Film). Signal intensity of transcripts was measured by BAS2000 Image Analyzer (Fuji Film). Using the same method, we hybridized northern blots with RT–PCR generated and labeled fragments of ANP cDNA, BNP cDNA, eNOS cDNA, nNOS cDNA, iNOS cDNA and internal control GAPDH cDNA.

Immunoblot analysis
Six-week-old Wt or Tg mouse cardiac muscle was homogenized in 10 vols (w/v) of a buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ß-mercaptoethanol, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 1 mM benzamidine. These crude extracts were denatured and size fractionated by SDS–PAGE (3–12%) and then transferred to a polyvinylidine difluoride (PVDF) membrane. The membrane was blocked using 5% milk in phosphate-buffered saline (PBS) and incubated with rabbit polyclonal antibody against caveolin-3 (Transduction Laboratories, C38330), raised against synthetic peptide corresponding to the amino terminus of caveolin-3 of rat and mouse origin, a monoclonal antibody against ß-dystoglycan (Novo Castra), a rabbit polyclonal antibody against eNOS (Santa Cruz Biotechnology), a rabbit polyclonal antibody against nNOS (Santa Cruz Biotechnology) and a rabbit polyclonal antibody against iNOS (Transduction Laboratories) overnight at room temperature. After washing with 5% milk in PBS, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (Amersham Pharmacia Biotech). Immunoreactive bands were visualized with ECL (Amersham Pharmacia Biotech).

Immunohistochemical analysis
Unfixed cardiac muscle samples were snap frozen in liquid nitrogen-cooled isopentane, sectioned on a cryostat (10 µm), and melted directly onto glass slides. Sections were then postfixed in 4% freshly depolymerized paraformaldehyde in PBS for 15–30 min at 4°C. After blocking with 3% BSA in PBS, sections were immunostained with a goat polyclonal antibody against caveolin-3 (Santa Cruz Biotechnology, sc-7665), recognizing the epitope mapped at the amino terminus of caveolin-3 of mouse origin, a rabbit polyclonal antibody against caveolin-1 (Santa Cruz Biotechnology), a goat polyclonal antibody against dystrophin (Santa Cruz Biotechnology), and a rabbit polyclonal antibody against eNOS (Santa Cruz Biotechnology) for 1 h at room temperature. After extensive washing with PBS, sections were incubated with FITC-conjugated or Cy3-conjugated secondary antibody. Rabbit and goat polyclonal antibodies against caveolin-3 used in immunoblot and immunohistochemical analyses can recognize both mutant and wild type forms of caveolin-3.

Transthoracic echocardiogram
Transthoracic echocardiography was performed on 24-week-old Tg mice and Wt littermates (n=7) under light anesthesia using intraperitoneal pentobarbital as described previously (30).

NOS assay
Total NOS activity was measured by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline, as previously described (18,31–33). Briefly, freshly prepared cardiac muscle from 6-week-old Tg mice and Wt littermates (n=8) was homogenized in 10 vols (w/v) of a buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 1 µM leupeptin. Aliquots from crude homogenates were quickly assayed in 100 µl reactions containing 100 000 cpm (40 Ci/mmol) of L-[3H]arginine, 1 mM NADPH, 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1.2 mM CaCl2, 10 µg/ml calmodulin, 1 mM DTT and 10 µM each of tetrahydrobiopterin, FAD and FMN. After an incubation of 10 min at 37°C, assays were terminated with 900 µl of ice cold H2O. After brief sonication, samples were applied to 2 ml of Dowex AG 50W-X8 (Na+ form) column. L-[3H]citrulline was quantified using 1 ml flow-through by liquid scintillation spectroscopy. The combined activity of eNOS plus iNOS was also measured in the same reaction buffer containing specific inhibitor of nNOS (0.1 µM N{omega}-propyl-L-arginine) (TOCRIS) (34,35). iNOS activity was also measured in the presence of 1.5 mM EDTA and 1.5 mM EGTA, which replaced Ca2+ ion in the reaction buffer. The above three assays were performed simultaneously. Then, nNOS activity was calculated by subtraction of the combined activity of eNOS and iNOS from total NOS activity. eNOS activity was calculated by subtraction of iNOS activity from the combined activity of eNOS and iNOS. Data are expressed as mean±SD. Statistical significance was determined using Welch's t-test (P<0.05).


   ACKNOWLEDGEMENTS
 
We thank Drs Tsutomu Ogura, Yukiko Kurashima (Investigative Treatment Division, National Cancer Center Research Institute East) and Hiroshi Shima (Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University) for their appropriate advice about the NOS assay method. We also thank Kenzo Uehira (Electron Microscopy Center, Kawasaki Medical School) and Megumu Kita (Laboratory Animal Center, Kawasaki Medical School) for their technical assistance. This work was supported by Research Grant (14B-4) for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Research Grant (15130301) for Research on Psychiatric and Neurological Diseases and Mental Health from the Ministry of Health, Labour and Welfare, Research Grant (14370212) from the Ministry of Education, Culture, Sports, Science and Technology, and Research Project Grants (no. 13-105, 14-117, 14-505, 14604 and 14-208) from Kawasaki Medical School.


   FOOTNOTES
 
* To whom correspondence should be addressed: Tel: +81 864621111; Fax: +81 864621199; Email: ysunada{at}med.kawasaki-m.ac.jp


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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