Caveolin-1/3 Double-Knockout Mice Are Viable, but Lack Both Muscle and Non-Muscle Caveolae, and Develop a Severe Cardiomyopathic Phenotype
2002; Elsevier BV; Volume: 160; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61168-6
ISSN1525-2191
AutoresDavid Park, Scott E. Woodman, William Schubert, Alex W. Cohen, Philippe G. Frank, Madhulika Chandra, Jamshid Shirani, Babak Razani, Baiyu Tang, Linda A. Jelicks, Stephen M. Factor, Louis M. Weiss, Herbert B. Tanowitz, Michael P. Lisanti,
Tópico(s)RNA Research and Splicing
ResumoThe caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy. The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy. Caveolae are 50- to 100-nm omega-shaped invaginations of the plasma membrane found in various tissue types. The principal structural proteins of caveolar membranes are encoded by the caveolin gene family (caveolin-1, -2, and -3). Caveolin-1 and -2 are co-expressed in numerous tissue types, with particularly high expression in adipocytes, endothelial cells, fibroblasts, and epithelial cells.1Razani B Lisanti MP Caveolin-deficient mice: insights into caveolar function and human disease.J Clin Invest. 2001; 108: 1553-1561Crossref PubMed Scopus (204) Google Scholar, 2Scherer PE Okamoto T Chun M Nishimoto I Lodish HF Lisanti MP Identification, sequence, and expression of caveolin-2 defines a caveolin gene family.Proc Natl Acad Sci USA. 1996; 93: 131-135Crossref PubMed Scopus (495) Google Scholar Caveolin-3, on the other hand, is muscle-specific, being highly expressed in all muscle tissues, such as skeletal muscle, diaphragm, and heart.3Song KS Scherer PE Tang Z Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells: caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (614) Google Scholar, 4Galbiati F Razani B Lisanti MP Role of caveolae and caveolin-3 in muscular dystrophy.Trends in Molecular Medicine. 2001; 7: 435-441Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Caveolin-1 and -3 form ∼350-kd homo-oligomers made up of ∼14–16 caveolin monomers. These homo-oligomers serve as the basic structural units that drive the formation of caveolae membranes. In contrast, Cav-2 either homodimerizes or forms high molecular mass hetero-oligomers with Cav-1.5Galbiati F Razani B Lisanti MP Emerging themes in lipid rafts and caveolae.Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 6Sargiacomo M Scherer PE Tang Z-L Kubler E Song KS Sanders MC Lisanti MP Oligomeric structure of caveolin: implications for caveolae membrane organization.Proc Natl Acad Sci USA. 1995; 92: 9407-9411Crossref PubMed Scopus (480) Google Scholar, 7Tang Z-L Scherer PE Okamoto T Song K Chu C Kohtz DS Nishimoto I Lodish HF Lisanti MP Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle.J Biol Chem. 1996; 271: 2255-2261Crossref PubMed Scopus (613) Google Scholar Cav-1 and Cav-3 are both independently necessary and sufficient to drive caveolae formation in heterologous expression systems, while Cav-2 requires the presence of Cav-1 for proper membrane targeting and stabilization. In the absence of Cav-1, Cav-2 localizes to the Golgi complex where it is degraded by the proteasome.8Parolini I Sargiacomo M Galbiati F Rizzo G Grignani F Engelman JA Okamoto T Ikezu T Scherer PE Mora R Rodriguez-Boulan E Peschle C Lisanti MP Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane: retention of caveolin-2 at the level of the Golgi complex.J Biol Chem. 1999; 274: 25718-25725Crossref PubMed Scopus (192) Google Scholar, 9Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou Jr, H Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar Initially considered to be mere conduits for endocytosis, caveolae are now recognized to have pleiotropic effects on numerous cellular events. Caveolin family members have been proposed to participate in vesicular trafficking, 10Schubert W Frank PG Razani B Park DS Chow CW Lisanti MP Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.J Biol Chem. 2001; 276: 48619-48622Crossref PubMed Scopus (279) Google Scholar lipid metabolism, 11Smart E Ying Y-S Conrad P Anderson RGW Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation.J Cell Biol. 1994; 127: 1185-1197Crossref PubMed Scopus (378) Google Scholar, 12van Meer G Caveolin, cholesterol, and lipid droplets?.J Cell Biol. 2001; 152: F29-F34Crossref PubMed Scopus (100) Google Scholar and various signal transduction processes. The “caveolae signaling hypothesis” states that caveolae serve as an integrated platform to concentrate and modulate the activity of specific lipid-modified signaling molecules, including Src family tyrosine kinases, H-Ras, eNOS, and heterotrimeric G proteins.13Engelman JA Zhang XL Razani B Pestell RG Lisanti MP p42/44 MAP kinase-dependent and -independent signaling pathways regulate caveolin-1 gene expression.J Biol Chem. 1999; 274: 32333-32341Crossref PubMed Scopus (149) Google Scholar, 14Dessy C Kelly RA Balligand JL Feron O Dynamin mediates caveolar sequestration of muscarinic cholinergic receptors and alteration in NO signaling.EMBO J. 2000; 19: 4272-4280Crossref PubMed Scopus (91) Google Scholar, 15Razani B Zhang XL Bitzer M von Gersdorff G Bottinger EP Lisanti MP Caveolin-1 regulates transforming growth factor (TGF)-β/SMAD signaling through an interaction with the TGF-β type I receptor.J Biol Chem. 2001; 276: 6727-6738Crossref PubMed Scopus (290) Google Scholar, 16Engelman JA Lee RJ Karnezis A Bearss DJ Webster M Siegel P Muller WJ Windle JJ Pestell RG Lisanti MP Reciprocal regulation of Neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Implications for mammary tumorigenesis.J Biol Chem. 1998; 273: 20448-20455Crossref PubMed Scopus (191) Google Scholar With the centralized role that caveolins assume in multiple cellular processes, it is not surprising that mutations within the CAV1 and CAV3 loci have been identified in human breast cancers17Hayashi K Matsuda S Machida K Yamamoto T Fukuda Y Nimura Y Hayakawa T Hamaguchi M Invasion activating caveolin-1 mutation in human scirrhous breast cancers.Cancer Res. 2001; 61: 2361-2364PubMed Google Scholar and muscular dystrophy (limb girdle muscular dystrophy, type 1C; LGMD-1C), 18Minetti C Sotogia F Bruno C Scartezzini P Broda P Bado M Masetti E Mazzocco P Egeo A Donati MA Volonte' D Galbiati F Cordone G Bricarelli FD Lisanti MP Zara F Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.Nat Genet. 1998; 18: 365-368Crossref PubMed Scopus (497) Google Scholar respectively. Gene deletion studies have confirmed and challenged our views of these crowded little caves. Targeted gene disruption of the Cav-1 locus in mice leads to a loss of caveolae in caveolin-1-expressing tissues, but a retention of caveolae in striated muscle tissues. The major tissue-specific defects include abnormalities in pulmonary structure and function, as characterized by hypercellularity, thickened alveolar septa, and exercise intolerance; decreased vascular tone, as assessed by aortic ring studies and determined to be secondary to eNOS activation; resistance to diet-induced obesity and fibrosis of fat pads with increasing age; and defects in caveolar endocytosis and marked hyperproliferation in mouse embryonic fibroblasts.9Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou Jr, H Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 19Drab M Verkade P Elger M Kasper M Lohn M Lauterbach B Menne J Lindschau C Mende F Luft FC Schedl A Haller H Kurzchalia TV Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1318) Google Scholar, 20Razani B Combs TP Wang XB Frank PG Park DS Russell RG Li M Tang B Jelicks LA Scherer PE Lisanti MP Caveolin-1 deficient mice are lean, resistant to diet-induced obesity, and show hyper-triglyceridemia with adipocyte abnormalities.J Biol Chem. 2002; 277: 8635-8647Crossref PubMed Scopus (472) Google Scholar Interestingly, Cav-3 null mice show a loss of caveolae specifically within muscle tissues. Myopathic changes ranging from mild to moderate were noted in skeletal muscle and characterized by variability in muscle fiber size and the presence of necrotic fibers. Although no changes were noted in the expression levels of the members of the dystrophin-glycoprotein (DG) complex, the DG complex was no longer properly targeted to cholesterol-rich lipid rafts/caveolae. In addition, the T-tubule system appeared immature and was longitudinally oriented; diffuse mislocalization of T-tubule marker proteins was also noted.21Galbiati F Engelman JA Volonte D Zhang XL Minetti C Li M Hou Jr, H Kneitz B Edelmann W Lisanti MP Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities.J Biol Chem. 2001; 276: 21425-21433Crossref PubMed Scopus (366) Google Scholar, 22Hagiwara Y Sasaoka T Araishi K Imamura M Yorifuji H Nonaka I Ozawa E Kikuchi T Caveolin-3 deficiency causes muscle degeneration in mice.Hum Mol Genet. 2000; 9: 3047-3054Crossref PubMed Scopus (145) Google Scholar Although much insight into caveolar function has been gained from the individual Cav-1 and Cav-3 knockouts, several questions remain unanswered. For example, (i) Does the persistence of tissue-specific caveolae in the single knock-outs (Cav-1 and Cav-3 null mice) allow for a functional compensation in certain tissues (ie, the heart, where ∼40% of the cells are non-muscle and therefore normally express Cav-1)?23MacKenna D Summerour SR Villarreal FJ Role of mechanical factors in modulating cardiac fibroblast function and extracellular matrix synthesis.Cardiovasc Res. 2000; 46: 257-263Crossref PubMed Scopus (320) Google Scholar and (ii) What are the developmental and physiological consequences of a caveolae-deficient animal? These remaining questions prompted us to generate a mouse model that lacks caveolae in both muscle and non-muscle cells; this was achieved by interbreeding Cav-1 null and Cav-3 null mice, to yield Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Surprisingly, we show here that Cav-1/3 dKO mice are viable and fertile. Loss of Cav-1 and Cav-3 protein expression was verified by Western blot analysis and the pleiotropic ablation of caveolae formation was demonstrated by transmission electron microscopy. Routine histopathological examination revealed that Cav-1/3 dKO mice exhibited lung, fat, and skeletal muscle defects of comparable severity to their single-knockout counterparts. However, Cav-1/3 dKO mice displayed grossly enlarged hearts, ie, cardiomegaly, greatly exceeding those of Cav-1 KO, Cav-3 KO, and wild-type mice. Gated MRI analysis of the Cav-1/3 dKO hearts revealed a significant increase of ∼40% in left ventricular wall thickness. Further functional analysis using transthoracic echocardiography demonstrated an ∼35% increase in left ventricular wall thickness, with left ventricular dilation. Moreover, systolic function, as determined by left ventricular fractional shortening, was significantly compromised in Cav-1/3 dKO mice. Histopathological examination of sections of cardiac tissue from Cav-1/3 dKO mice demonstrated cardiac myocyte hypertrophy, generalized myocyte disorganization and myocytolysis, accompanied by chronic inflammation and fibrosis. Finally, Northern blot analysis indicated that an embryonic marker, atrial natriuretic factor (ANF), was up-regulated in ventricular samples prepared from Cav-1/3 dKO mice. The cardiac defects observed in Cav-1/3 dKO mice emphasize the importance of both caveolin isoforms for normal cardiac structure and function. Future studies will be needed to define the diastolic and systolic phenotype and address the development of contractile failure in Cav-1/3 dKO mice, such as monitoring the generation of calcium sparks24Hoit BD Walsh RA Cardiovascular Physiology in the Genetically Engineered Mouse. Kluwer Academic Publishers, Dordrecht, The Netherlands2002Crossref Google Scholar and their exercise responses.24Hoit BD Walsh RA Cardiovascular Physiology in the Genetically Engineered Mouse. Kluwer Academic Publishers, Dordrecht, The Netherlands2002Crossref Google Scholar Caveolin-1, -2, and -3 mouse mAbs (used for immunoblotting;3Song KS Scherer PE Tang Z Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells: caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (614) Google Scholar, 25Scherer PE Tang Z-L Chun MC Sargiacomo M Lodish HF Lisanti MP Caveolin isoforms differ in their N-terminal protein sequence and subcellular distribution: identification and epitope mapping of an isoform-specific monoclonal antibody probe.J Biol Chem. 1995; 270: 16395-16401Crossref PubMed Scopus (323) Google Scholar, 26Scherer PE Lewis RY Volonte D Engelman JA Galbiati F Couet J Kohtz DS van Donselaar E Peters P Lisanti MP Cell-type and tissue-specific expression of caveolin-2: caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo.J Biol Chem. 1997; 272: 29337-29346Crossref PubMed Scopus (474) Google Scholar) were the generous gift of Dr. Roberto Campos-Gonzalez, BD-Transduction Laboratories, Inc. The ANF cDNA was the generous gift of Dr. Jil Tardiff, Albert Einstein College of Medicine. A variety of other reagents were of the highest purity grade and were obtained from the usual commercial sources. All animals were housed and maintained in a barrier facility at the Institute for Animal Studies, Albert Einstein College of Medicine. Cav-1(−/−)/Cav-3 (−/−)-deficient mice were generated by interbreeding Cav-1 (−/−)9Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou Jr, H Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar and Cav-3 (−/−) mice,21Galbiati F Engelman JA Volonte D Zhang XL Minetti C Li M Hou Jr, H Kneitz B Edelmann W Lisanti MP Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities.J Biol Chem. 2001; 276: 21425-21433Crossref PubMed Scopus (366) Google Scholar that were in a C57Bl/6 background. Mice of various genotypes were sacrificed and fat, lung, heart, muscle, and aortic tissue samples were harvested. Approximately 100 mg of a given tissue sample was then homogenized in lysis buffer (10 mmol/L Tris, pH 7.5, 50 mmol/L NaCl, 1% Triton X-100, 60 mmol/L octyl glucoside), containing protease inhibitors (Boehringer Mannheim). Tissue lysates were then centrifuged at 12,000 × g for 10 minutes to remove insoluble debris. Protein concentrations were analyzed using the BCA reagent (Pierce) and the volume required for 10 μg of protein was determined. Samples were then separated by SDS-PAGE (12.5% 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 mmol/L Tris pH 8.0, 150 mmol/L 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 (1:5000 dilution, Pierce) were used to visualize bound primary antibodies with the Supersignal chemiluminescence substrate (Pierce). Fat, lung, heart, skeletal muscle, and aortic tissue samples were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, postfixed with OsO4, and stained with uranyl acetate and lead citrate. Microtome sections were examined under a JEOL 1200 EX transmission electron microscope and photographed at a magnification of ×16,000.9Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou Jr, H Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar Caveolae were identified by their characteristic flask shape, size (50–100 nm), and location at or near the plasma membrane.27Yamada E The fine structure of the gall bladder epithelium of the mouse.J Biophys Biochem Cytol. 1955; 1: 445-458Crossref PubMed Scopus (527) Google Scholar, 28Farquhar M Palade G Junctional complexes in various epithelia.J Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2144) Google Scholar Mice were sacrificed and their hearts were removed and placed in buffered formalin (10%). The tissue was fixed for ∼24 hours, washed in PBS for 20 minutes, dehydrated through a graded series of ethanol washes, treated with xylene for 40 minutes, and then embedded in paraffin for 1 hour 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 and intraventricular septum) were selected for imaging. Samples were examined in a blinded fashion by one of us (S.M.F.). Mice were sacrificed and the ventricular tissue was carefully separated from atrial tissue by dissection. Total RNA was extracted from 100 mg of left ventricular tissue from each sample using Trizol reagent protocol (Gibco). Twenty micrograms of total RNA for each sample was separated using a 1.2% agarose gel under RNase-free conditions and transferred to nitrocellulose. The filters were hybridized using the ExpressHyb solution (Clontech). The blots were probed with the radiolabeled ANF cDNA. Magnetic resonance imaging (MRI) experiments were performed using a GE Omega 9.4T vertical bore magnetic resonance 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 ECG, 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 approx. 100 to 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 was performed on 2- and 4-month-old mice, as we described previously.29Chandra M Shirani J Shtutin V Weiss LM Factor SM Petkova SB Rojkind M Dominguez-Rosales JA Jelicks LA Morris SA Wittner M Tanowitz HB Cardioprotective effects of verapamil on myocardial structure and function in a murine model of chronic Trypanosoma cruzi infection (Brazil Strain): an echocardiographic study.Int J Parasitol. 2002; 32: 207-215Crossref PubMed Scopus (48) Google Scholar Echocardiography was performed with mice in supine position on a heating pad set at 38°C. Light anesthesia was achieved using isoflurane inhalation.29Chandra M Shirani J Shtutin V Weiss LM Factor SM Petkova SB Rojkind M Dominguez-Rosales JA Jelicks LA Morris SA Wittner M Tanowitz HB Cardioprotective effects of verapamil on myocardial structure and function in a murine model of chronic Trypanosoma cruzi infection (Brazil Strain): an echocardiographic study.Int J Parasitol. 2002; 32: 207-215Crossref PubMed Scopus (48) Google Scholar Continuous, standard electrocardiograms were taken from electrodes placed on the extremities. Echocardiographic images were obtained 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-inch 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.30Schiller NB Two-dimensional echocardiographic determination of left ventricular volume, systolic function, and mass: summary and discussion of the 1989 recommendations of the American Society of Echocardiography.Circulation. 1991; 84: I280-I287PubMed Google Scholar Additionally, the following parameters were calculated 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 minus end-systolic diameter)/end-diastolic diameter]}; and relative wall thickness as (2 × left ventricular diastolic wall thickness)/end-diastolic diameter. Also, see this more recent review on the echocardiographic examination of the mouse.31Hoit BD New approaches to phenotypic analysis in adult mice.J Mol Cell Cardiol. 2001; 33: 27-35Abstract Full Text PDF PubMed Scopus (72) Google Scholar Note that differences between the “absolute” wall thicknesses measured using MRI and echocardiography are commonly observed and are likely due to technical factors, such as differences in the time of gating; echocardiography may underestimate these values, while MRI may overestimate these values. Most importantly, however, the relative changes measured in left ventricular wall thickness using MRI and echocardiography are in agreement, ie, an increase of ∼34–41% for Cav-1/3 dKO mice (see below). We have previously shown the Cav-1 null mice lack caveolae in non-muscle cells (fibroblasts, adipocytes, endothelial cells), but continue to form caveolae in skeletal muscle fibers9Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou Jr, H Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 20Razani B Combs TP Wang XB Frank PG Park DS Russell RG Li M Tang B Jelicks LA Scherer PE Lisanti MP Caveolin-1 deficient mice are lean, resistant to diet-induced obesity, and show hyper-triglyceridemia with adipocyte abnormalities.J Biol Chem. 2002; 277: 8635-8647Crossref PubMed Scopus (472) Google Scholar (and data not shown). Conversely, Cav-3 null mice fail to form caveolae in skeletal muscle, but show abundant caveolae in non-muscle cells, such as endothelia.21Galbiati F Engelman JA Volonte D Zhang XL Minetti C Li M Hou Jr, H Kneitz B Edelmann W Lisanti MP Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities.J Biol Chem. 2001; 276: 21425-21433Crossref PubMed Scopus (366) Google Scholar These findings are consistent with the relatively ubiquitous expression of caveolin-1 in non-muscle cell types and the restricted tissue-specific expression of caveolin-3 in muscle cells. Thus, whole-body ablation of caveolae would require a complete loss of both caveolin-1 and caveolin-3 protein expression. Here, we generated caveolin-1/caveolin-3 double-knockout mice by interbreeding Cav-1-null and Cav-3-null mice. Surprisingly, Cav-1/3 dKO mice are viable and fertile and exhibit no obvious external gross defects. As predicted based on their genotype, we show that Cav-1/3 dKO mice fail to express both the caveolin-1 and caveolin-3 protein products (Figure 1). Note that in wild-type mice, fat and lung tissues abundantly co-express the Cav-1 and Cav-2 proteins, while the Cav-3 protein is highly expressed in striated muscle tissues (cardiac and skeletal); finally, all three caveolin family members are co-expressed in smooth muscle cells (such as in the aorta).2Scherer PE Okamoto T Chun M Nishimoto I Lodish HF Lisanti MP Identification, sequence, and expression of caveolin-2 defines a caveolin gene family.Proc Natl Acad Sci USA. 1996; 93: 131-135Crossref PubMed Scopus (495) Google Scholar, 3Song KS Scherer PE Tang Z Okamoto T Li S Chafel M Chu C Kohtz DS Lisanti MP Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells: caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.J Biol Chem. 1996; 271: 15160-15165Crossref PubMed Scopus (614) Google Scholar, 32Rothberg KG Heuser JE Donzell WC Ying Y Glenney JR Anderson RGW Caveolin, a protein component of caveolae membrane coats.Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1873) Google Scholar Western blot analysis using caveolin isoform-specific mAb p
Referência(s)