Author + information
- Received November 5, 2010
- Revision received December 22, 2010
- Accepted December 23, 2010
- Published online May 31, 2011.
- Yousuke T. Horikawa, PhD⁎,
- Mathivadhani Panneerselvam, PhD⁎,
- Yoshitaka Kawaraguchi, MD⁎,
- Yasuo M. Tsutsumi, MD, PhD†,
- Sameh S. Ali, PhD⁎,
- Ravi C. Balijepalli, PhD¶,
- Fiona Murray, PhD‡,
- Brian P. Head, PhD⁎,
- Ingrid R. Niesman, MS⁎,
- Timo Rieg, MD§∥,
- Volker Vallon, MD‡,§∥,
- Paul A. Insel, MD‡∥,
- Hemal H. Patel, PhD⁎ and
- David M. Roth, PhD, MD⁎,§,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. David M. Roth, VA San Diego Healthcare System, 9125, 3350 La Jolla Village Drive, San Diego, California 92161–9125
Objectives We hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy.
Background Natriuretic peptides modulate cardiac hypertrophy and are potential therapeutic options for patients with heart failure. Caveolae, microdomains in the plasma membrane that contain caveolin proteins and natriuretic peptide receptors, have been implicated in cardiac hypertrophy and natriuretic peptide localization.
Methods We generated transgenic mice with cardiac myocyte-specific overexpression of caveolin-3 (Cav-3 OE) and also used an adenoviral construct to increase Cav-3 in cardiac myocytes.
Results The Cav-3 OE mice subjected to transverse aortic constriction had increased survival, reduced cardiac hypertrophy, and maintenance of cardiac function compared with control mice. In left ventricle at baseline, messenger ribonucleic acid for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were increased 7- and 3-fold, respectively, in Cav-3 OE mice compared with control subjects and were accompanied by increased protein expression for ANP and BNP. In addition, ventricles from Cav-3 OE mice had greater cyclic guanosine monophosphate levels, less nuclear factor of activated T-cell nuclear translocation, and more nuclear Akt phosphorylation than ventricles from control subjects. Cardiac myocytes incubated with Cav-3 adenovirus showed increased expression of Cav-3, ANP, and Akt phosphorylation. Incubation with methyl-β-cyclodextrin, which disrupts caveolae, or with wortmannin, a PI3K inhibitor, blocked the increase in ANP expression.
Conclusions These results imply that cardiac myocyte-specific Cav-3 OE is a novel strategy to enhance natriuretic peptide expression, attenuate hypertrophy, and possibly exploit the therapeutic benefits of natriuretic peptides in cardiac hypertrophy and heart failure.
- atrial natriuretic peptide (ANP)
- brain natriuretic peptide (BNP)
In response to chronic stress, the heart undergoes hypertrophy that can progress to adverse remodeling and heart failure. The signaling events in this progression are complex and involve cell surface receptors, signal transduction pathways, and transcriptional and post-transcriptional events (1). The subcellular organization of signaling components in highly ordered lipid microdomains within the plasma membrane helps provide spatial and temporal regulation of signaling (2). Caveolae are specialized cholesterol- and sphingolipid-enriched membrane microdomains that contain the structural proteins caveolins (3). Caveolins organize and regulate receptors and signaling molecules that are involved in a wide array of cell functions, including cell growth and hypertrophy (4). These molecules include: G-protein coupled receptors, natriuretic peptide receptors, heterotrimeric G-protein subunits, G-protein-regulated effectors, and PI3 kinase (PI3K) (5,6).
There are 3 caveolin isoforms: caveolin 1, 2, and 3 (7). Caveolin-3 (Cav-3) is an isoform predominantly found in skeletal and cardiac muscle. Caveolin-3 knockout mice develop peripheral muscle myopathic changes, cardiac hypertrophy, and cardiomyopathy (8). By contrast, overexpression of caveolin-3 (Cav-3 OE) in neonatal cardiac myocytes (CMs) results in the inability of an adrenergic agonist (i.e., phenylephrine) and endothelin-1 to induce myocyte hypertrophy (9). Thus, loss of Cav-3 induces cardiac hypertrophy and cardiomyopathy, and increased Cav-3 in CMs attenuates hypertrophy.
Natriuretic peptides have numerous actions that inhibit hypertrophy; these include diuretic, natriuretic, and vasodilatory properties as well as autocrine/paracrine actions on CMs (10–12). In atrial myocytes, atrial natriuretic peptide (ANP) is secreted from caveolae and found closely associated with Cav-3 (5,13). Little is known about the role of caveolae and caveolins in natriuretic peptide biology in ventricular CMs. Although prior studies have implicated a role for Cav-3 and caveolae in cardiac hypertrophy (9), relationships among caveolin expression, natriuretic peptide signaling, and cardiac hypertrophy have not been described. In the current study, we show that CM-specific Cav-3 OE attenuates cardiac hypertrophy and increases natriuretic peptide expression and signaling.
Animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science), and protocols were approved by the Veterans Affairs San Diego Healthcare System Institutional Animal Care and Use Committee. Animals were kept on a 12-h light/dark cycle in a temperature-controlled room with ad libitum access to food and water. A subset of Cav-3 OE mice and control littermates were individually housed so that food and water intake could be measured daily. Transgenic mice were generated in our laboratory with the alpha-myosin heavy chain promoter to produce CM-specific Cav-3 OE (14). In some experiments, we isolated CMs from 200- to 300-g male Sprague-Dawley rats.
Concentrations of sodium ion and potassium ion in plasma and urine were determined in a subset of animals with a flame photometer (Cole-Parmer Instrument Company, Vernon Hills, Illinois) as described previously (15).
A subset of Cav-3 OE and control mice were trained to allow conscious blood pressure measurements without anesthesia with the CODA noninvasive tail blood pressure system (Kent Scientific, Torrington, Connecticut). Data were analyzed with the CODA version 2.5 software.
Transverse aortic constriction
Eight- to sixteen-week-old transgenic Cav-3 OE mice and transgene negative littermate mice (control) underwent transverse aortic constriction (TAC), as previously described (16). In brief, mice were anesthetized with isoflurane, intubated, and mechanically ventilated. An incision was made in the second intercostal space, a 7-0 silk suture was placed around the aorta, and a 27-g needle was placed between the innominate and left carotid artery. A double surgeon's knot was tied down to the aorta and needle, and the needle was removed, resulting in a 0.41-mm stenosis. Mice were allowed to recover with 100% oxygen for 1 h. Mice were killed after 4 weeks of TAC. Sham animals underwent all aspects of the surgery except placement of the stenosis.
Echocardiography was performed in a subset of Cav-3 OE and control mice before TAC surgery and before killing. Mice were anesthetized with isoflurane, and echocardiography was performed with a L15/6-MHz transducer (Sonos 5500, Philips Medical Systems, Andover, Massachusetts). Mice were evaluated for presence of transverse aortic stenosis with pulse wave Doppler; all animals had >3.5 m/s gradient across the stenosis. Bilateral carotid catheterization performed in pentobarbital-anesthetized mice by the use of 2 high-fidelity 1.4-F microtip pressure transducers 48 h after TAC showed mean pressure gradients between 40 and 50 mm Hg for both experimental groups (p = NS, control vs. Cav-3 OE, n = 5/group).
A subset of Cav-3 OE and control mice were anesthetized with pentobarbital, and cardiac catheterization was performed with a high-fidelity 1.4-F microtip pressure transducer (SPR-671, Millar Instruments, Inc., Houston, Texas). The catheter was advanced via the right carotid artery into the left ventricle (LV) after measuring systolic, diastolic, and mean arterial pressure. Parameters were determined by an algorithm from EMKA Technologies (Falls Church, Virginia).
Dissected mouse hearts were placed in ice-cold saline to evacuate remaining blood before dissecting atria and ventricles. Dissected tissue was dried on gauze, immediately weighed, and frozen. Heart weight was defined as that of the left and right ventricles. Lungs and tibias were also dissected. Lungs were dried on gauze to remove excess blood and then weighed. Tibias were stored frozen in eppendorf tubes and measured with calipers.
Animals were transcardially perfused with ice-cold cardioplegic solution (20 mmol/l potassium chloride) and then with either 10% formalin or 4% paraformaldehyde in phosphate buffer (pH = 7.4) and removed. Tissues were fixed overnight at 4°C and embedded in paraffin. Fibrosis was assessed with Masson's Trichrome staining (Sigma, St. Louis, Missouri) and hematoxylin-eosin staining of 5-μm sections.
For wheat germ agglutinin (WGA) immunofluorescence, 5-μm sections of hearts were prepared, washed in PBS, incubated for 2 h in WGA-Alexa 488 lectin (Invitrogen, Carlsbad, California), and washed and mounted in anti-fade reagent. Five images (40×) from each heart were taken, and the diameter and areas from 70 to 100 of cross-sectionally arranged myocytes were measured individually and analyzed with National Institutes of Health ImageJ software.
Electron microscopy was performed in a subset of Cav-3 OE and control whole hearts as previously described (14).
Plasma peptide extraction and enzyme immunoassay
Plasma ANP and brain natriuretic peptide (BNP) concentrations were determined by fluorescent immunoassay kits (Phoenix Pharmaceuticals, Inc., Burlingame, California). Plasma was isolated from mouse blood obtained by cardiac puncture from a subset of Cav-3 OE and control littermate animals. Peptides were extracted from the plasma with an established protocol (Phoenix Pharmaceuticals) that involved addition of an acidifying buffer and centrifugation of samples at 17,000× g for 20 min at 4°C. The acidified plasma was loaded on a pre-equilibrated C-18 SEP-column (Phoenix Pharmaceuticals) and then eluted for analysis of eluted proteins by use of enzyme immunoassay kit protocols.
Cyclic guanosine monophosphate assay in mouse heart
For cyclic guanosine monophosphate (cGMP) measurements, hearts from control and Cav-3 OE mice were weighed (n = 5 to 6 hearts) and homogenized in 10 volumes of cold 5% trichloroacetic acid with a polytron homogenizer. Samples were centrifuged at 1,500× g for 10 min, and the supernatants were then extracted with water-saturated ether. The cGMP was measured in acetylated supernatants with an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, Michigan). The cGMP content is expressed as fmol/mg wet tissue.
Isolation of CMs
Cardiac myocytes were isolated from adult male Sprague-Dawley rats (250 to 300 g) as previously described (17). The CMs were incubated with 1.0 × 108 plaque-forming units Cav-3 adenovirus or green fluorescent protein adenovirus (Control) for 24 h.
Whole tissue homogenates and cell lysates (prepared as previously described ), nuclear and cytoplasmic fractions (18), or blood samples were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis with 10% polyacrylamide precast gels (Invitrogen, Carlsbad, California) and transferred to polyvinylidene difluoride membranes via electroelution. Membranes were blocked in 20 mmol/l tris buffered saline Tween (1%) containing 3% bovine serum albumin and incubated with primary antibodies (Cav-3 and ANP, Santa Cruz Biotechnology, Santa Cruz, California; PErk, PAKT, and TAKT, Cell Signaling, Danvers, Massachusetts; glyceraldehyde-3-phosphate dehydrogenase, Imgenex, San Diego, California) overnight at 4°C. Blots were visualized with secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) and enhanced chemiluminescence reagent (GE Healthcare, Waukesha, Wisconsin).
Quantitative real-time polymerase chain reaction analysis
Total ribonucleic acid (RNA) was isolated from CM with a RNeasy Mini Kit (Qiagen, Valencia, California). First strand complementary deoxyribonucleic acid (cDNA) synthesis (iScript cDNA synthesis kit, Bio-Rad, Hercules, California) was performed with random hexamers on 1 to 2 μg of total RNA. The concentration of cDNA was determined and adjusted to 50 ng/μl for real-time polymerase chain reaction analysis, which was performed on a MJ Research Opticon 2 (Bio-Rad) in triplicate with the IQ SYBR Green Supermix (Bio-Rad) with 100 ng cDNA and 0.5 μmol/l forward/reverse primer mix in 20 μl final reaction volume. The ANP, BNP, and alpha-myosin heavy chain primers were QuantiTect Primers (Qiagen) (primer sequences for alpha-sk-actin forward: GTGTCACCCACAACGTGC, reverse: AGGGCCACATAGCACAGC; β-myosin heavy chain forward: GCTGAAAGCAGAAAGAGATTATC, reverse: TGGAGTTCTTCTCTTCTGGAG). Thermal cycler conditions were as follows: 94°C-10 min (1 cycle); 94°C-20 s, 55°C-20 s, and 72°C-30 s (40 cycles). Resulting polymerase chain reaction products were confirmed by melt curve analysis. Analysis of cycle threshold was performed with Opticon 2 analysis software (Bio-Rad); normalized values were obtained for each group by subtracting matched glyceraldehyde-3-phosphate dehydrogenase cycle threshold values.
Statistical analysis was performed with GraphPad Prism Software (version 4.0, GraphPad, La Jolla, California). All values are presented as mean ± SEM. One-way analysis of variance, followed by post-hoc Bonferroni correction for multiple comparisons (Figs. 1C,2A to 2F, and 5C) (all comparisons were performed, and all p values were corrected) and unpaired and paired Student t test (Figs. 1D, 3C to 3F, 4A to 4F, and 5D) were used. A Kaplan-Meier analysis was performed on mouse survival after TAC banding (Fig. 1A), and significance was calculated with a log-rank test. A p value < 0.05 was considered statistically significant.
Cardiac myocyte-specific Cav-3 OE increases survival, attenuates cardiac hypertrophy, and maintains cardiac function in vivo
The Cav-3 OE and control mice underwent 4 weeks of TAC to produce pressure-induced cardiac hypertrophy. A log-rank test on a Kaplan-Meier survival curve revealed that Cav-3 OE mice have increased survival after TAC (Fig. 1A). Control mice showed an increase in concentric hypertrophy, nearly doubling heart size in response to TAC, but TAC produced less hypertrophy in Cav-3 OE mice (Fig. 1B). Transverse aortic constriction nearly doubled the ratio of LV to body weight and tibia length in control mice, but these changes were blunted in Cav-3 OE mice with TAC (Fig. 1C). Cardiac hypertrophy, assessed by WGA staining of LV cross-sections, was reduced in Cav-3 OE mice. Surface area of CMs from control TAC-treated mice was nearly 60% greater than that of myocytes from Cav-3 OE mice subjected to TAC (Fig. 1D). Echocardiography confirmed that Cav-3 OE mice had a blunted hypertrophic response to TAC compared with control subjects. Although, Cav-3 OE mice had a hypertrophic response in interventricular septal wall thickness at end diastole and LV posterior wall thickness at end diastole after TAC banding, hypertrophy was attenuated compared with control mice; no changes were observed in interventricular septal wall thickness at end systole or LV posterior wall thickness at end systole (Table 1). The Cav-3 OE mice also had less perivascular and interstitial cardiac fibrosis associated with TAC (Fig. 1E).
Serial echocardiography revealed that control mice had decreased ejection fraction and percentage fractional shortening after 4 weeks of TAC (Figs. 2A and 2B), whereas Cav-3 OE mice subjected to TAC had no change in either measure of cardiac function. Carotid catheterization showed that control mice but not Cav-3 OE mice had reduced LV systolic function (dP/dtmax) after 4 weeks of TAC (Fig. 2C). Thus, Cav-3 OE mice maintained LV function in the presence of pressure overload. Furthermore, LV relaxation (dP/dtmin) was decreased in control mice but not Cav-3 OE mice after 4 weeks of TAC (Fig. 2D), a result consistent with the increased fibrosis observed in the control mice. Wet lung weight to body weight and tibia length ratios were increased in control but not Cav-3 OE mice after TAC, suggesting that control mice had increased fluid accumulation consistent with heart failure (Fig. 2E).
Natriuretic peptide expression in Cav-3 OE mice at baseline
The LVs from Cav-3 OE mice showed an increased number of caveolae in the sarcolemmal membrane (Fig. 3A), a result consistent with previous observations (14). To define a role for natriuretic peptides as a mechanism for the attenuation of cardiac hypertrophy in Cav-3 OE mice, we examined fetal gene expression. The RNA isolated from LV homogenates showed a nearly 7- and 3-fold increase in ANP and BNP expression, respectively, when compared with transgene negative littermates (control) with little to no difference in other fetal genes (Fig. 3B). Consistent with the change in RNA expression, ANP and BNP protein expression in LV (Figs. 3C and 3D) were also increased in Cav-3 OE mice. However, plasma ANP and BNP levels were not significantly different between control and Cav-3 OE mice (Figs. 3E and 3F). We also observed no significant differences in conscious blood pressures (136 ± 6 mm Hg for Cav-3 OE, and 141 ± 3 mm Hg for control subjects, p = NS, n = 11/group), water or food intake, natriuresis, or diuresis between control and Cav-3 OE mice (Table 2).
Cav-3 OE mice have increased natriuretic peptide receptor A expression and cGMP levels, reduced nuclear factor of activated T-cells nuclear translocation and increased nuclear pAkt
Left ventricular homogenates from Cav-3 OE and control littermates were immunoblotted for natriuretic peptide receptor A (NPR-A), the dominant receptor involved in ANP and BNP signaling. We found that NPR-A expression was increased in Cav-3 OE mice (Fig. 4A) and that, consistent with the signaling by NPR-A, cGMP levels were also increased in those mice (Fig. 4B). To investigate downstream signaling molecules involved in hypertrophy, we isolated nuclear and cytoplasmic fractions from Cav-3 OE and control LV homogenates and assessed the expression and localization of nuclear factor of activated T-cells (NFATc3) and Akt. The LV from Cav-3 OE mice had a greater cytoplasmic/nuclear ratio of NFATc3 (Figs. 4C and 4D) and decreased cytoplasmic/nuclear expression of pAkt due to increased nuclear expression than did LV from control littermates (Figs. 4E and 4F).
ANP expression is caveolae-dependent and dependent on Akt phosphorylation
To assess whether increased expression of caveolins or caveolae were alone sufficient to observe the increase in ANP expression, we isolated ventricular CM from adult Sprague-Dawley rats and treated them with increasing Cav-3 adenovirus (from 0 to 30 × 107 plaque-forming units) for 24 h (Fig. 5A). We found that Cav-3 expression, ANP expression, and Akt phosphorylation all increased in CM incubated with the Cav-3 adenovirus but that adenovirus for enhanced green fluorescent protein (control) produced no increase in Cav-3, ANP, or pAkt expression (Fig. 5A).
To determine whether Cav-3 expression or the formation of caveolae was involved in the increase in ANP expression, we incubated CM for 24 h with methyl-β-cyclodextrin (MβCD) (500 nmol/l), an agent that disrupts caveolae, during incubation with the Cav-3 adenovirus. MβCD disrupted caveolae (Fig. 5B), and in the presence of MβCD, neither ANP nor pAkt expression increased even though Cav-3 was substantially increased (Fig. 5C). Thus, the presence of caveolae seems to be necessary for the increase in ANP expression in CM engineered to overexpress Cav-3.
To assess the role of Akt in ANP expression, we incubated CM with Cav-3 adenovirus for 24 h in the presence of wortmannin, a PI3K inhibitor, or dimethyl sulfoxide (vehicle). Wortmannin decreased Akt phosphorylation and ANP expression in myocytes incubated with Cav-3 adenovirus (Fig. 5D), thus implying that the increased ANP expression in response to Cav-3 OE is dependent on Akt phosphorylation.
Our results show that CM-specific Cav-3 OE blunts pressure-induced cardiac hypertrophy and increases natriuretic peptide expression and downstream signaling. This increase in natriuretic peptide expression depended on the presence of caveolae and was associated with increased phosphorylation of Akt. Thus, increasing cardiac Cav-3 expression might provide a novel therapeutic approach to enhance natriuretic peptide expression.
The localization of natriuretic peptides with caveolae and caveolins was suggested 20 years ago, but little further work has investigated these relationships (5,13,19). Previously we have shown that CM-specific Cav-3 OE mimics ischemia-induced preconditioning and protects the heart from ischemic injury by increasing pAkt signaling, effects that are blocked with 5-hydroxydecanoate, a mitochondrial KATP channel inhibitor, and wortmannin, a PI3K inhibitor (14). Treatment of rat hearts with ANP also has cardioprotective effects that are blocked with 5-hydroxydecanoate (20). The Cav-3 knockout mice display enhanced hypertrophic signaling (8), an effect similar to that observed in mice with a knockout of NPR-A, the major natriuretic signaling receptor in CMs (21). The current results show that Cav-3 OE mice are resistant to pressure-induced hypertrophy, potentially as a consequence of increased natriuretic peptide and NPR-A expression accompanied by increased cGMP and pAkt signaling. Our conclusion is consistent with previous work that has documented the antihypertrophic (22), antifibrotic (23), and inotropic (24) actions of natriuretic peptides.
Overexpression of NPR-A has been shown to reduce TAC-induced cardiac hypertrophy via a nonmitogen-activated protein kinase signaling pathway (25). The NPR-A has an intracellular guanylyl cylase domain, which upon activation increases cGMP levels and can inhibit hypertrophic signaling. The Cav-3 OE mice have increased levels not only of NP but also of NPR-A and cGMP, suggesting that NPR-A activity is increased. Although we have shown that phosphorylated endothelial nitric oxide synthase and nitric oxide synthase activity are not significantly elevated in cardiac homogenates of Cav-3 OE mice (14), recent studies suggest an interplay between ANP and nitric oxide (26) and implicate a synergistic augmentation of NPR-A signaling via nitric oxide-activated guanylyl cyclases (27). The mechanism for this crosstalk will require further investigation.
Akt localization in the nucleus can prevent cardiac hypertrophy, and nuclear Akt can induce ANP expression, prevent pathological hypertrophy, and maintain cardiac function after TAC (28). The increase in natriuretic peptides that occurs with cardiac hypertrophy might help compensate for the increase in afterload via its diuretic, natriuretic, and vasodilating actions as well as by its ability to inhibit aldosterone synthesis and renin secretion (10–12). Natriuretic peptides have been used to reduce post-myocardial infarction remodeling (29) and to improve stroke volume index in patients with heart failure (24). However, the use of natriuretic peptides as heart failure therapeutics has been hampered by the relatively short half-life and instability of ANPs and hypotension-dependent reductions in renal perfusion that occur with BNP (30). The current data show that Cav-3 OE creates a cellular pool of natriuretic peptides that can lead to long-term positive cardiovascular effects in a setting of cardiac hypertrophy.
We find that increased Cav-3 expression in CMs is associated with increases in pAkt and ANP expression and that the latter increases depend on caveolae expression and localization of Cav-3 in caveolae, because MβCD treatment decreases caveolae but not Cav-3 expression and significantly lowers pAkt and ANP expression (Fig. 5C). On the basis of results obtained with wortmannin, which inhibits expression of pAkt and ANP, pAkt potentially contributes to the enhanced expression of ANP (28). Thus, our results imply that caveolae not only localize ANP but might also decrease its degradation and facilitate the ability of PI3K and pAkt to up-regulate ANP protein expression. We speculate that the increase in natriuretic peptides in Cav-3 OE mice might prevent the dephosphorylation and thus decrease nuclear translocation of NFATc3, thereby inhibiting cardiac hypertrophy (31). The Cav-3 OE mice also have greater nuclear pAkt expression, which perhaps contributes to their increase in natriuretic peptide expression.
Increased natriuretic peptide levels are generally thought to reflect cardiac dysfunction and have been used as a “biomarker” of cardiac hypertrophy (32). However, unlike in other hypertrophic models, Cav-3 OE mice have increased ANP and BNP RNA levels but not those of other hypertrophic markers. Thus, Cav-3 OE seems to have the unique ability to alter natriuretic peptide levels without inducing the complete fetal gene response, a result akin to the ability of nuclear localization of pAkt to prevent hypertrophy (28). Treatment with hypertrophic agonists can increase Cav-3 protein and caveolae formation, but it is not clear whether this effect is harmful or helpful in the setting of cardiac hypertrophy (9,33). The current results show that overexpressing caveolae and Cav-3 does not exacerbate but instead prevents cardiac hypertrophy, thus implicating a protective role for increased Cav-3 expression.
The current findings in mice with myocyte-specific Cav-3 OE contrast with data for mice with non–CM-specific Cav-3 OE, which develop a cardiomyopathy-like phenotype (34). Cav-3 expression is normally limited to skeletal and cardiac muscle, and as a result, other effects of Cav-3 OE—such as induction of vasculopathies—cannot be ruled out as the cause of cardiac dysfunction in such mice.
A limitation of the current study was that we did not directly investigate the role of NPR-A antagonism on downstream hypertrophic signaling. We were unable to obtain an inhibitor of NPR-A (e.g., HS-142-1, which was used in some prior studies but is no longer manufactured).
The data shown here indicate that a plasma membrane structural protein, Cav-3, not only increases caveolae formation but also induces a “protective phenotype” that is characterized by increased natriuretic peptide expression and decreased cardiac hypertrophy. The present data thus define a physiologically important relationship between Cav-3 and natriuretic peptide levels in the LV and identify a novel mechanism and therapeutic rationale for the potential of increasing Cav-3 expression to enhance the expression of natriuretic peptides and their beneficial effects against heart failure.
The authors thank Ana Moreno and Stephanie Cipta for quantification of WGA immunofluorescence.
This work was supported by the American Heart Association: Predoctoral Fellowship06150217Y (to Dr. Horikawa), Beginning Grant in Aid 0765076Y (to Dr. Tsutsumi), Grant in Aid 3440038 (to Dr. Vallon), and Scientist Development Grant 0630039N (to Dr. Patel), 0730010N (Dr. Balijepalli), and 2610034 (to Dr. Rieg); National Institutes of Health: HL081400 (to Dr. Roth), HL66941 (to Drs. Insel and Roth), HL091071 (to Dr. Patel), GM66232 (to Dr. Insel), HL007261 (to Dr. Horikawa), GM007198 (to Dr. Horikawa), HL094728 (to Dr. Vallon), and DK079337 (to Dr. Vallon); American Society of Nephrology Carl W. Gottschalk Research Grant (to Dr. Rieg); and a Merit Award from the Department of Veterans Affairs (to Dr. Roth). The authors have reported that they have no relationships to disclose. W. H. Wilson Tang, MD, served as Guest Editor for this paper.
- Abbreviations and Acronyms
- atrial natriuretic peptide
- brain natriuretic peptide
- Cav-3 OE
- overexpression of caveolin-3
- complementary deoxyribonucleic acid
- cyclic guanosine monophosphate
- cardiac myocyte
- left ventricle/ventricular
- nuclear factor of activated T-cell
- ribonucleic acid
- transverse aortic constriction
- wheat germ agglutinin
- Received November 5, 2010.
- Revision received December 22, 2010.
- Accepted December 23, 2010.
- American College of Cardiology Foundation
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