α-Linolenic Acid-Enriched Diet Prevents Myocardial Damage and Expands Longevity in Cardiomyopathic Hamsters
2006; Elsevier BV; Volume: 169; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.051320
ISSN1525-2191
AutoresRoberta Fiaccavento, Felicia Carotenuto, Marilena Minieri, Laura Masuelli, Alba Vecchini, Roberto Bei, Andrea Modesti, Luciano Binaglia, Angelo Fusco, A. Bertoli, Giancarlo Forte, Luciana Carosella, Paolo Di Nardo,
Tópico(s)Cardiac Valve Diseases and Treatments
ResumoRandomized clinical trials have demonstrated that the increased intake of ω-3 polyunsaturated fatty acids significantly reduces the risk of ischemic cardiovascular disease, but no investigations have been performed in hereditary cardiomyopathies with diffusely damaged myocardium. In the present study, δ-sarcoglycan-null cardiomyopathic hamsters were fed from weaning to death with an α-linolenic acid (ALA)-enriched versus standard diet. Results demonstrated a great accumulation of ALA and eicosapentaenoic acid and an increased eicosapentaenoic/arachidonic acid ratio in cardiomyopathic hamster hearts, correlating with the preservation of myocardial structure and function. In fact, ALA administration preserved plasmalemma and mitochondrial membrane integrity, thus maintaining proper cell/extracellular matrix contacts and signaling, as well as a normal gene expression profile (myosin heavy chain isoforms, atrial natriuretic peptide, transforming growth factor-β1) and a limited extension of fibrotic areas within ALA-fed cardiomyopathic hearts. Consequently, hemodynamic indexes were safeguarded, and more than 60% of ALA-fed animals were still alive (mean survival time, 293 ± 141.8 days) when all those fed with standard diet were deceased (mean survival time, 175.9 ± 56 days). Therefore, the clinically evident beneficial effects of ω-3 polyunsaturated fatty acids are mainly related to preservation of myocardium structure and function and the attenuation of myocardial fibrosis. Randomized clinical trials have demonstrated that the increased intake of ω-3 polyunsaturated fatty acids significantly reduces the risk of ischemic cardiovascular disease, but no investigations have been performed in hereditary cardiomyopathies with diffusely damaged myocardium. In the present study, δ-sarcoglycan-null cardiomyopathic hamsters were fed from weaning to death with an α-linolenic acid (ALA)-enriched versus standard diet. Results demonstrated a great accumulation of ALA and eicosapentaenoic acid and an increased eicosapentaenoic/arachidonic acid ratio in cardiomyopathic hamster hearts, correlating with the preservation of myocardial structure and function. In fact, ALA administration preserved plasmalemma and mitochondrial membrane integrity, thus maintaining proper cell/extracellular matrix contacts and signaling, as well as a normal gene expression profile (myosin heavy chain isoforms, atrial natriuretic peptide, transforming growth factor-β1) and a limited extension of fibrotic areas within ALA-fed cardiomyopathic hearts. Consequently, hemodynamic indexes were safeguarded, and more than 60% of ALA-fed animals were still alive (mean survival time, 293 ± 141.8 days) when all those fed with standard diet were deceased (mean survival time, 175.9 ± 56 days). Therefore, the clinically evident beneficial effects of ω-3 polyunsaturated fatty acids are mainly related to preservation of myocardium structure and function and the attenuation of myocardial fibrosis. A direct relationship between increased intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), either from dietary sources or as pharmacological supplementation, and beneficial effects on the cardiovascular system has become evident throughout the years. Dietary sources of ω-3 PUFAs include mainly fish oils rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid and vegetable (eg, soybean, canola, walnut, and flaxseed) oils rich in α-linolenic acid (ALA).1Collomb M Sollberger H Butikofer U Sieber R Stoll W Schaeren W Impact of a basal diet of hay and fodder beet supplemented with rapeseed, linseed and sunflowerseed on the fatty acid composition of milk fat.Int Dairy J. 2004; 14: 549-559Crossref Scopus (97) Google Scholar Randomized secondary prevention clinical trials with either EPA and docosahexaenoic acid2Trichopoulou A Orfanos P Norat T Bueno-de-Mesquita B Ocke MC Peeters PH van der Schouw YT Boeing H Hoffmann K Boffetta P Nagel G Masala G Krogh V Panico S Tumino R Vineis P Bamia C Naska A Benetou V Ferrari P Slimani N Pera G Martinez-Garcia C Navarro C Rodriguez-Barranco M Dorronsoro M Spencer EA Key TJ Bingham S Khaw KT Kesse E Clavel-Chapelon F Boutron-Ruault MC Berglund G Wirfalt E Hallmans G Johansson I Tjonneland A Olsen A Overvad K Hundborg HH Riboli E Trichopoulos D Modified Mediterranean diet and survival: EPIC-elderly prospective cohort study.BMJ. 2005; 330: 991-998Crossref PubMed Scopus (626) Google Scholar, 3Harper CR Jacobson TA Beyond the Mediterranean diet: the role of omega-3 fatty acids in the prevention of coronary heart disease.Prev Cardiol. 2003; 6: 136-146Crossref PubMed Scopus (32) Google Scholar or ALA4de Lorgeril M Salen P Alpha-linolenic acid and coronary heart disease.Nutr Metab Cardiovasc Dis. 2004; 14: 162-169Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 5Harper CR Jacobson TA Usefulness of omega-3 fatty acids and the prevention of coronary heart disease.Am J Cardiol. 2005; 96: 1521-1529Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar demonstrated a strong association between the intake of these ω-3 PUFAs and significant reductions in cardiovascular risk and compared favorably with landmark secondary prevention trials with lipid-lowering drugs.6Klungel OH Heckbert SR de Boer A Leufkens HG Sullivan SD Fishman PA Veenstra DL Psaty BM Lipid-lowering drug use and cardiovascular events after myocardial infarction.Ann Pharmacother. 2002; 36: 751-757Crossref PubMed Scopus (21) Google Scholar ω-3 PUFAs exhibit positive effects on hemostatic factors, thrombogenesis, blood pressure, plasma lipids, and heart susceptibility to ventricular arrhythmias.7Kris-Etherton PM Hecker KD Binkoski AE Polyunsaturated fatty acids and cardiovascular health.Nutr Rev. 2004; 62: 414-426Crossref PubMed Google Scholar, 8Albert CM Oh K Whang W Manson JE Chae CU Stampfer MJ Willett WC Hu FB Dietary α-linolenic acid intake and risk of sudden cardiac death and coronary heart disease.Circulation. 2005; 112: 3232-3238Crossref PubMed Scopus (200) Google Scholar Their administration in the form of dietary fish or fish oil capsules has been shown to cause a 30% reduction in the mortality of infarcted patients compared with untreated controls9Calder PC N-3 fatty acids and cardiovascular disease: evidence explained and mechanisms explored.Clin Sci. 2004; 107: 1-11Crossref PubMed Scopus (463) Google Scholar and to induce relevant protective effects in primary prevention of cardiovascular disease in animal models including monkeys.7Kris-Etherton PM Hecker KD Binkoski AE Polyunsaturated fatty acids and cardiovascular health.Nutr Rev. 2004; 62: 414-426Crossref PubMed Google Scholar Accordingly, long-chain ω-3 fatty acid consumption has been promoted for all individuals, especially those at risk of developing cardiovascular diseases.7Kris-Etherton PM Hecker KD Binkoski AE Polyunsaturated fatty acids and cardiovascular health.Nutr Rev. 2004; 62: 414-426Crossref PubMed Google Scholar, 9Calder PC N-3 fatty acids and cardiovascular disease: evidence explained and mechanisms explored.Clin Sci. 2004; 107: 1-11Crossref PubMed Scopus (463) Google Scholar However, the major studies on ω-3 PUFAs' beneficial effects have been performed in patients or experimental models suffering from cardiac ischemic disease, whereas no epidemiological or experimental studies have investigated their effects in hereditary cardiomyopathies. In addition, most investigations have been performed using marine-derived ω-3 PUFAs,9Calder PC N-3 fatty acids and cardiovascular disease: evidence explained and mechanisms explored.Clin Sci. 2004; 107: 1-11Crossref PubMed Scopus (463) Google Scholar and only a few epidemiological studies have evaluated ALA's potential against cardiovascular diseases. Furthermore, although the cardioprotective ability of ω-3 PUFAs has been mainly related to anti-arrhythmic and anti-fibrillatory effects,10McLennan PL Abeywardena MY Membrane basis for fish oil effects on the heart: linking natural hibernators to prevention of human sudden cardiac death.J Membr Biol. 2005; 206: 85-102Crossref PubMed Scopus (53) Google Scholar and among others, recent studies indicate that they could act by altering lipid composition11Ma DWL Seo J Davidson LA Callaway ES Fan YY Lupton JR Chapkin RS N-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon.FASEB J. 2004; 1: 1040-1042Google Scholar and plasma membrane structure to regulate intracellular signaling12Serhan CN Clish CB Brannon J Colgan SP Chiang N Gronert K Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.J Exp Med. 2000; 192: 1197-1204Crossref PubMed Scopus (963) Google Scholar and metabolism,13Pepe S McLennan PL Cardiac membrane fatty acid composition modulates myocardial oxygen consumption and postischemic recovery of contractile function.Circulation. 2002; 105: 2303-2308Crossref PubMed Scopus (181) Google Scholar further in-depth research is needed to establish the amount of dietary ω-3 PUFAs that maximally affects the greatest number of cardiovascular risk factors and to determine the exact cellular and molecular mechanisms through which ω-3 PUFAs elicit their beneficial effects on the cardiovascular system. The present study was designed to test the hypothesis that ω-3 PUFAs could beneficially affect the pathophysiological mechanisms of hereditary cardiac hypertrophy. The UM-X7.1 hamster strain was used as the experimental model because this strain displays several pathological characteristics, among which are an abnormal accumulation of ω-6 fatty acids in the heart14Vecchini A Binaglia L Di Nardo P Minieri M Panagia V Dhalla NS Altered lipid metabolism in the failing heart of cardiomyopathic hamsters (UM-X7.1).Prostaglandins Leukot Essent Fatty Acids. 1995; 52: 199-203Abstract Full Text PDF PubMed Scopus (7) Google Scholar, 15Vecchini A Binaglia L Bibeau M Minieri M Carotenuto F Di Nardo P Insulin deficiency and reduced expression of lipogenic enzymes in cardiomyopathic hamster.J Lipid Res. 2001; 42: 96-105PubMed Google Scholar and severely damaged cardiac mitochondrial16Minieri M Zingarelli M Shubeita H Vecchini A Binaglia L Carotenuto F Fantini C Fiaccavento R Masuelli L Coletti A Simonelli L Modesti A Di Nardo P Identification of a new missense mutation in the mtDNA of hereditary hypertrophic, but not dilated cardiomyopathic hamsters.Mol Cell Biochem. 2003; 252: 73-81Crossref PubMed Scopus (12) Google Scholar and cellular membranes.17Masuelli L Bei R Sacchetti P Scappaticci I Francalanci P Albonici L Coletti A Palumbo C Minieri M Fiaccavento R Carotenuto F Fantini C Carosella L Modesti A Di Nardo P Beta-catenin accumulates in intercalated disks of hypertrophic cardiomyopathic hearts.Cardiovasc Res. 2003; 60: 376-387Crossref PubMed Scopus (69) Google Scholar These features are part of a more complex pathophysiological pattern. In fact, UM-X7.1 hamsters exhibit a cardiomyopathic phenotype associated with the deletion of the δ-sarcoglycan (δ-SG) gene,18Nigro V Okazaki Y Belsito A Piluso G Matsuda Y Politano L Nigro G Ventura C Abbondanza C Molinari AM Acampora D Nishimura M Hayashizaki Y Puca GA Identification of the Syrian hamster cardiomyopathy gene.Hum Mol Gene. 1997; 6: 601-607Crossref PubMed Scopus (249) Google Scholar representing a unique model for investigating well-defined patterns of myocardial degeneration that ultimately result in heart failure.19Di Nardo P Fiaccavento R Natali A Minieri M Sampaolesi M Fusco A Janmot C Cuda G Carbone A Rogliani P Peruzzi G Embryonic gene expression in nonoverloaded ventricles of hereditary hypertrophic cardiomyopathic hamsters.Lab Invest. 1997; 77: 489-502PubMed Google Scholar, 20Nakamura T Matsumoto K Mizuno S Sawa Y Matsuda H Nakamura T Hepatocyte growth factor prevents tissue fibrosis, remodeling, and dysfunction in cardiomyopathic hamster hearts.Am J Physiol. 2005; 288: H2131-H2139Google Scholar, 21Fiaccavento R Carotenuto F Minieri M Fantini C Forte G Carbone A Carosella L Bei R Masuelli L Palumbo C Modesti A Prat M Di Nardo P Stem cell activation sustains hereditary hypertrophy in hamster cardiomyopathy.J Pathol. 2005; 205: 397-407Crossref PubMed Scopus (21) Google Scholar The ablation of δ-SG, a structural glycoprotein of skeletal and cardiac muscle cell membranes,22Hack AA Lam MJ Cordier L Shoturma DI Ly CT Hadhazy MA Hadhazy MR Sweeney HL McNally EM Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin-glycoprotein complex.J Cell Science. 2000; 113: 2535-2544PubMed Google Scholar causes diffuse alterations of cell/cell and cell/extracellular matrix (ECM) contacts, detachment of the basal membrane, and an aberrant intracellular signaling pattern.17Masuelli L Bei R Sacchetti P Scappaticci I Francalanci P Albonici L Coletti A Palumbo C Minieri M Fiaccavento R Carotenuto F Fantini C Carosella L Modesti A Di Nardo P Beta-catenin accumulates in intercalated disks of hypertrophic cardiomyopathic hearts.Cardiovasc Res. 2003; 60: 376-387Crossref PubMed Scopus (69) Google Scholar, 23Ambra R Di Nardo P Fantini C Minieri M Canali R Natella F Virgili F Selective changes in DNA binding activity of transcription factors in UM-X7.1 cardiomyopathic hamsters.Life Sci. 2002; 71: 2369-2381Crossref PubMed Scopus (9) Google Scholar Therefore, the aim of this study was to modulate the lipid composition of the hearts of cardiomyopathic hamsters (CMPHs) by administering an ALA-enriched diet, in an attempt to attenuate cardiomyopathic structural and functional damages. ALA was chosen because the hamster's capability to uptake, transport, and store this specific PUFA has been extensively investigated.24Morise A Combe N Boue C Legrand P Catheline D Delplanque B Fenart E Weill P Hermier D Dose effect of alpha-linolenic acid on PUFA conversion, bioavailability, and storage in the hamster.Lipids. 2004; 39: 325-334Crossref PubMed Scopus (41) Google Scholar In the present study, CMPHs (strain UM-X7.1), affected by δ-SG gene deletion, were used as the experimental model and were compared with healthy Golden Syrian hamsters (GSHs) bred under the same conditions. Three different groups of hamsters were considered: CMPHs and GSHs fed with a standard pellet chow diet (PT) (Rieper, Bolzano, Italy) and CMPHs fed with an ALA-enriched diet (flaxseeds, apples, and carrots) (FS). A fourth group of FS-fed GSHs was initially considered; however, because after 250 days their survival curve was overlapping with that of the GSH-PT, and no significant signs were detectable when myocardial samples were analyzed with light microscopy, only the GSH-PT group was considered as healthy control. Animals were allowed to consume each diet component ad libitum from weaning to death. In the FS diet, fresh fruit and vegetables supplied carbohydrates and vitamins, whereas flaxseeds were the only source of fats, with ALA representing 52% of total lipids. Flaxseeds were selected assuming that they would be more palatable for hamsters. Every 24 hours, at the end of the dark period, food remnants were carefully harvested and weighed. The total amount of food consumed by each animal was not significantly different between healthy and cardiomyopathic individuals (CMPH/FS, 6.9 ± 0.7 g/day/100 g body weight; CMPH/PT, 6.5 ± 1.1 g/day/100 g body weight; GSH/PT, 7.1 ± 1.3 g/day/100 g body weight; n = 15 per group). In addition, the amount of each component ingested by every animal per day was estimated to be 50% apple, 20% carrot, and 30% flaxseeds. Assuming this composition as the closest to the diet actually consumed, two independent nutritional analyses of the PT and FS diets were performed on the basis of international standard procedures; one was performed by the Laboratory for Food Control of the Italian National Institute of Health (ISS) and another by a private organization (Biodigit srl, Campobasso, Italy) involved in nutritional studies. The analyses indicated that all macro- and micronutrients needed for animal health maintenance were in due proportion in both dietary regimens. In 100 g of fresh PT or FS diet, the caloric power was 222.5 and 202.8 kcal, respectively, whereas major components were differently represented, as shown in Table 1. However, every 7 days, animal weights were recorded to exclude possible decrements attributable to calorie restriction.Table 1Major Components of Estimated Experimental DietsPTFSKcal/100 g222.5 ± 48.1202.8 ± 44.7*P < 0.01 andMoisture (%)37.4 ± 5.253.8 ± 4.1Ash (%)4.6 ± 0.71.7 ± 0.6*P < 0.01 andCrude carbohydrate (%)37.9 ± 4.29.9 ± 3.6*P < 0.01 andCrude fat (%)2.9 ± 0.214.4 ± 0.5*P < 0.01 andTotal ω-3 fatty acids (%)0.13 ± 0.032.58 ± 0.05†P < 0.005 versus PT.Total ω-6 fatty acids (%)1.11 ± 0.080.64 ± 0.05†P < 0.005 versus PT.Values are means ± SD of eight different diet lots shared at random between laboratories in charge for nutritional analyses. PT, standard diet; FS, flaxseed diet (apple 50%, carrot 20%, flaxseeds 30%).* P < 0.01 and† P < 0.005 versus PT. Open table in a new tab Values are means ± SD of eight different diet lots shared at random between laboratories in charge for nutritional analyses. PT, standard diet; FS, flaxseed diet (apple 50%, carrot 20%, flaxseeds 30%). Animals were anesthetized with urethane (400 mg/kg i.p.) and sacrificed. Ventricles were rapidly excised, washed in ice-cold 1× phosphate-buffered saline (PBS), pH 7.4, weighed, frozen in liquid nitrogen, and stored at −80°C until use. Alternatively, ventricles were fixed with 4% formaldehyde and embedded in paraffin for light microscopy or with 2.5% glutaraldehyde in PBS for electron microscopy. All animal handling procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the institutional Animal Care and Use Committee of the University of Rome Tor Vergata. To analyze fatty acid composition, plasma and heart samples were obtained from seven animals per group. Heart tissues were homogenized in H2O, at 48°C, using an Ultra-Turrax T25 homogenizer (6 × 15 seconds, setting 5; Janke & Kunkel, Staufen, Germany). Lipids were extracted from aliquots of plasma or tissue homogenates according to Folch and colleagues.25Folch J Lees M Sloane-Stanley GM A simplified method for the isolation and purification of total lipids from animal tissues.J Biol Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar Fatty acid methyl esters were obtained by transesterification with 3% sulfuric acid in methanol under nitrogen (1 hour, 75°C). Gas chromatography of fatty acid methyl esters was performed with a HRGC 5300 gas chromatograph (Carlo Erba Instruments, Milan, Italy) equipped with a SP-2330 capillary column (30 cm × 0.25 mm, Supelco; Sigma-Aldrich srl, Milan, Italy) and flame ionization detector. Serial paraffin-embedded ventricular sections (4 μm) were stained with hematoxylin and eosin (H&E). Morphometric analysis was performed using the DS Software (Delta Sistemi, Rome, Italy), as previously described.19Di Nardo P Fiaccavento R Natali A Minieri M Sampaolesi M Fusco A Janmot C Cuda G Carbone A Rogliani P Peruzzi G Embryonic gene expression in nonoverloaded ventricles of hereditary hypertrophic cardiomyopathic hamsters.Lab Invest. 1997; 77: 489-502PubMed Google Scholar The extent of myocardial fibrosis was quantified after Masson trichrome staining (Bio-Optica, Milan, Italy); the green area corresponding to fibrosis was quantified (DS Software) from eight visual fields selected at random from 11 animals per group (objective, ×40; Leica DMRB microscope; Wetzlar, Germany). Ultrastructural analysis was performed on myocardial fragments processed for transmission electron microscopy (Philips CM10; Milan, Italy), as previously described.26Modesti A Masuelli L Modica A D'Orazi G Scarpa S Bosco MC Forni G Ultrastructural evidence of the mechanisms responsible for interleukin-4-activated rejection of a spontaneous murine adenocarcinoma.Int J Cancer. 1993; 53: 988-993Crossref PubMed Scopus (38) Google Scholar Two independent observers evaluated at least five different hearts per each hamster group (CMPH/FS, CMPH/PT, and GSH/PT). Heart serial sections (4 μm) of at least eight animals per group were analyzed by immunofluorescence (Leica DMRB microscope; objective, ×20): sections were incubated with a primary anti-α-dystroglycan antibody (1:200) (Novocastra, Newcastle, UK) and a secondary fluorescein isothiocyanate-labeled antibody (1:300) (Vector Laboratories Inc., Burlingame, CA). Anti-collagen type I (1:200; Santa Cruz Biotechnologies, Santa Cruz, CA) and anti-laminin α-2 (1:200; Santa Cruz Biotechnologies) were used to evaluate the expression of the corresponding genes, which was assessed by peroxidase immunostaining (Vector Laboratories Inc.). Myofibrillar extracts were prepared using the Caforio's procedure, as previously described.27Caforio ALP Grazzini M Mann JM Keeling PJ Bottazzo GF McKenna WJ Schiaffino S Identification of alpha- and beta-cardiac myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy.Circulation. 1992; 85: 1734-1742Crossref PubMed Scopus (259) Google Scholar Briefly, tissue samples were homogenized in a low-salt buffer solution (20 mmol/L KCl, 2 mmol/L K2HPO4, and 1 mmol/L EGTA, pH 6.8); after centrifugation at 2000 × g, pellets were resuspended in high-salt solution (40 mmol/L Na4P2O7, 1 mmol/L MgCl2, and 1 mmol/L EGTA, pH 9.5) and centrifuged at 10,000 × g. The protein content of the clarified supernatant samples was evaluated by the Bradford method (Amresco, Solon, OH), and equivalent protein amounts were separated onto 8 to 22% gradient polyacrylamide gels and blotted to polyvinylidene difluoride membranes. Specific antibodies were against α and β myosin heavy chain (α-MHC and β-MHC) (Novocastra) and myosin light chain 1 (MLC1) (BiosPacific, Emeryville, CA) and 2 (MLC2) (Santa Cruz Biotechnologies). Secondary antibodies were peroxidase-linked (Vector Laboratories Inc.) and revealed with enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Western blot bands were quantified by the DS Software. Band intensities were expressed as fold increase versus band of the same protein from age-matched GSH/PT. Protein loading was checked by probing for β-actin expression. This assay measures the sliding rate of rhodamine-phalloidin-labeled actin filaments translocated by myosin monomers bound to a nitrocellulose-coated surface. The assay was performed by essentially following the Cuda et al procedure, as previously described.28Cuda G Fananapazir L Zhu WS Sellers JR Epstein ND Skeletal muscle expression and abnormal function of beta-myosin in hypertrophic cardiomyopathy.J Clin Invest. 1993; 91: 2861-2865Crossref PubMed Scopus (202) Google Scholar Northern blot analysis of atrial natriuretic peptide (ANP) and transforming growth factor (TGF)-β1 was performed essentially as previously described.19Di Nardo P Fiaccavento R Natali A Minieri M Sampaolesi M Fusco A Janmot C Cuda G Carbone A Rogliani P Peruzzi G Embryonic gene expression in nonoverloaded ventricles of hereditary hypertrophic cardiomyopathic hamsters.Lab Invest. 1997; 77: 489-502PubMed Google Scholar In brief, total RNA, extracted from hamster hearts, was electrophoretically separated under denaturing conditions and transferred to nylon membranes. After hybridization with 32P-labeled cDNA probes, nylon membranes were subjected to autoradiography. cDNA of the GAPDH housekeeping gene was used as probe to check the amount of loaded RNAs; bands were quantified by DS Software. Aortic and intraventricular pressures were measured in 150-day-old CMPH/PT, CMPH/FS, and GSH/PT anesthetized with urethane (400 mg/kg i.p.), as previously described.19Di Nardo P Fiaccavento R Natali A Minieri M Sampaolesi M Fusco A Janmot C Cuda G Carbone A Rogliani P Peruzzi G Embryonic gene expression in nonoverloaded ventricles of hereditary hypertrophic cardiomyopathic hamsters.Lab Invest. 1997; 77: 489-502PubMed Google Scholar In brief, a polyethylene catheter (PE50), connected to a Statham P23Gb transducer (Gould Instruments, Cleveland, OH), was introduced into the left ventricular cavity via the right carotid artery; intra-arterial and intraventricular pressures were recorded by a multichannel polygraph (Gould Instruments) after a 20-minute stabilization. Three hamster groups were considered: GSH/PT (n = 46), as healthy controls, and two CMPH groups, CMPH/PT (n = 47) and CMPH/FS (n = 47), of which CMPH was randomly divided at the beginning of the study. The survival study was performed from weaning (30 days of age) up to 450 days. Data analysis was performed with SPSS for Windows (version 11.5; SPSS Inc., Chicago, IL). Hamster survival curves were built by the Kaplan-Meier analysis. Differences between survival mean ages were analyzed by one-way analysis of variance and were considered statistically significant when P was < 0.05. The significance of differences between curves was verified by log-rank test; differences was considered statistically significant when P < 0.05. Results are presented as mean ± SD. Unless stated otherwise, the comparison between FS and PT data was performed by the unpaired t-test. To evaluate the capability of an ALA-enriched diet to target the myocardium, the molar percent presence of each fatty acid species, obtained by trans-esterification of total lipids from hearts and plasma of 150- and 90-day-old CMPH/PT, CMPH/FS, and GSH/PT, was assessed (Table 2). At 150 days of age, CMPH/FS exhibited up to 20- and 7-fold increases in the molar percentage of ALA (18:3 ω-3) in all of the ventricular regions (left ventricular free wall, right ventricular free wall, interventricular septum) and plasma, respectively, compared with CMPH/PT. Moreover, in these animals, EPA (20:5 ω-3), which was not detectable in either plasma or myocardial tissue or plasma of GSH/PT or CMPH/PT, was present (up to 1.7 molar percent) in the different heart regions of CMPH/FS but remained absent from the FS-fed animal plasma, suggesting that its synthesis was myocardium-specific. By contrast, there was a 30% decrease in arachidonic acid (20:4 ω-6, AA) molar percent, and a consequent higher EPA/AA ratio, in all ventricular regions considered and in plasma of 150-day-old CMPH/FS compared with age-matched CMPH/PT. Furthermore, concerning saturated fatty acids, plasma and cardiac lipids from CPMH/FS hearts were enriched in stearic acid (18:0) and depleted in palmitic acid (16:0), compared with those from CMPH/PT. Comparable effects of the FS diet were attained for all ventricular regions and plasma of 90-day-old CMPH groups (data not shown). The effects of the FS diet on the levels of docosahexaenoic acid (22:6 ω-3) in all heart and plasma samples were not uniform regardless of age.Table 2Molar Percent Composition of Individual Fatty Acids from 150-Day-Old CMPH and GSH Ventricles and PlasmaLVRVGSH/PTCMPH/PTCMPH/FSGSH/PTCMPH/PTCMPH/FS16:016.64 ± 0.8916.72 ± 1.4010.34 ± 1.22†P < 0.01 versus age-matched CMPH/PT. (table continues)16.91 ± 0.5821.30 ± 1.8611.95 ± 1.22†P < 0.01 versus age-matched CMPH/PT. (table continues)18:012.69 ± 0.4215.72 ± 0.7219.70 ± 0.94†P < 0.01 versus age-matched CMPH/PT. (table continues)11.76 ± 0.2716.61 ± 1.3519.77 ± 0.75†P < 0.01 versus age-matched CMPH/PT. (table continues)18:120.31 ± 3.2212.72 ± 0.8514.24 ± 1.1118.91 ± 2.8614.44 ± 1.1312.37 ± 0.4418:232.29 ± 2.5131.93 ± 0.4532.59 ± 0.6734.91 ± 2.3526.35 ± 2.2830.62 ± 0.91†P < 0.01 versus age-matched CMPH/PT. (table continues)18:3 ω-60.14 ± 0.110.43 ± 0.12ND0.28 ± 0.110.23 ± 0.15ND18:3 ω-30.59 ± 0.120.37 ± 0.117.3 ± 1.23*P < 0.005 and0.65 ± 0.150.45 ± 0.117.56 ± 0.89*P < 0.005 and20:4 ω-66.63 ± 0.6810.12 ± 1.347.6 ± 0.72*P < 0.005 and6.88 ± 0.359.35 ± 0.427.18 ± 0.48*P < 0.005 and20:5 ω-3NDND1.04 ± 0.28*P < 0.005 andNDND1.64 ± 0.12*P < 0.005 and22:5 ω-60.38 ± 0.130.53 ± 0.11ND0.43 ± 0.110.73 ± 0.12ND22:5 ω-30.59 ± 0.090.92 ± 0.240.93 ± 0.150.62 ± 0.120.85 ± 0.151.13 ± 0.0922:6 ω-36.35 ± 0.539.31 ± 0.615.77 ± 0.49†P < 0.01 versus age-matched CMPH/PT. (table continues)6.19 ± 0.496.92 ± 0.447.6 ± 0.17SPPlasmaGSH/PTCMPH/PTCMPH/FSGSH/PTCMPH/PTCMPH/FS17.11 ± 1.1618.57 ± 1.3511.78 ± 0.89†P < 0.01 versus age-matched CMPH/PT. (table continues)25.15 ± 1.1223.85 ± 1.1319.01 ± 0.93†P < 0.01 versus age-matched CMPH/PT. (table continues)12.62 ± 1.7217.61 ± 1.7719.49 ± 1.01†P < 0.01 versus age-matched CMPH/PT. (table continues)8.8 ± 0.7210.56 ± 1.115.12 ± 2.97†P < 0.01 versus age-matched CMPH/PT. (table continues)20.36 ± 1.4213.25 ± 3.7712.61 ± 1.1517.59 ± 1.5316.77 ± 2.1615.07 ± 2.8432.84 ± 4.8229.46 ± 5.2930.36 ± 1.2434.84 ± 1.7533.07 ± 2.5233.35 ± 2.910.16 ± 0.060.18 ± 0.15NDNDNDND0.57 ± 0.090.38 ± 0.187.36 ± 1.57*1.29 ± 0.362.07 ± 0.8415.03 ± 3.91*6.17 ± 0.379.53 ± 1.17.17 ± 0.55*4.57 ± 0.237.21 ± 2.464.37 ± 0.94*NDND1.72 ± 0.43*NDNDND0.31 ± 0.050.65 ± 0.13ND0.17 ± 0.030.18 ± 0.05ND0.77 ± 0.200.81 ± 0.071.45 ± 0.250.36 ± 0.070.53 ± 0.211.56 ± 0.196.94 ± 0.457.05 ± 0.768.05 ± 0.234.72 ± 0.193.16 ± 0.572.77 ± 0.91Values are mean ± SD of seven animals per group. LV, left ventricle; RV, right ventricle; SP, interventricular septum; PT, standard diet; FS, ω-3 PUFA-enriched diet; ND, not detectable.* P < 0.005 and† P < 0.01 versus age-matched CMPH/PT. (table continues) Open table in a new tab Values are mean ± SD of seven animals per group. LV, left ventricle; RV, right ventricle; SP, interventricular septum; PT, standard diet; FS, ω-3 PUFA-enriched diet; ND, not detectable. The hamster ventricular weight to body weight ratio was assessed throughout the entire study period. This ratio was slightly, but not significantly smaller in CMPH fed with FS (
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