Disruption of Collagen Homeostasis Can Reverse Established Age-Related Myocardial Fibrosis
2015; Elsevier BV; Volume: 185; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2014.11.009
ISSN1525-2191
AutoresNicole L. Rosin, Mryanda Sopel, Alec Falkenham, Timothy D.G. Lee, Jean‐François Légaré,
Tópico(s)Cardiovascular Function and Risk Factors
ResumoHeart failure, the leading cause of hospitalization of elderly patients, is correlated with myocardial fibrosis (ie, deposition of excess extracellular matrix proteins such as collagen). A key regulator of collagen homeostasis is lysyl oxidase (LOX), an enzyme responsible for cross-linking collagen fibers. Our objective was to ameliorate age-related myocardial fibrosis by disrupting collagen cross-linking through inhibition of LOX. The nonreversible LOX inhibitor β-aminopropionitrile (BAPN) was administered by osmotic minipump to 38-week-old C57BL/6J male mice for 2 weeks. Sirius Red staining of myocardial cross sections revealed a reduction in fibrosis, compared with age-matched controls (5.84 ± 0.30% versus 10.17 ± 1.34%) (P < 0.05), to a level similar to that of young mice at 8 weeks (4.9 ± 1.2%). BAPN significantly reduced COL1A1 mRNA, compared with age-matched mice (3.5 ± 0.3-fold versus 15.2 ± 4.9-fold) (P < 0.05), suggesting that LOX is involved in regulation of collagen synthesis. In accord, fibrotic factor mRNA expression was reduced after BAPN. There was also a novel increase in Ly6C expression by resident macrophages. By interrupting collagen cross-linking by LOX, the BAPN treatment reduced myocardial fibrosis. A novel observation is that BAPN treatment modulated the transforming growth factor-β pathway, collagen synthesis, and the resident macrophage population. This is especially valuable in terms of potential therapeutic targeting of collagen regulation and thereby age-related myocardial fibrosis. Heart failure, the leading cause of hospitalization of elderly patients, is correlated with myocardial fibrosis (ie, deposition of excess extracellular matrix proteins such as collagen). A key regulator of collagen homeostasis is lysyl oxidase (LOX), an enzyme responsible for cross-linking collagen fibers. Our objective was to ameliorate age-related myocardial fibrosis by disrupting collagen cross-linking through inhibition of LOX. The nonreversible LOX inhibitor β-aminopropionitrile (BAPN) was administered by osmotic minipump to 38-week-old C57BL/6J male mice for 2 weeks. Sirius Red staining of myocardial cross sections revealed a reduction in fibrosis, compared with age-matched controls (5.84 ± 0.30% versus 10.17 ± 1.34%) (P < 0.05), to a level similar to that of young mice at 8 weeks (4.9 ± 1.2%). BAPN significantly reduced COL1A1 mRNA, compared with age-matched mice (3.5 ± 0.3-fold versus 15.2 ± 4.9-fold) (P < 0.05), suggesting that LOX is involved in regulation of collagen synthesis. In accord, fibrotic factor mRNA expression was reduced after BAPN. There was also a novel increase in Ly6C expression by resident macrophages. By interrupting collagen cross-linking by LOX, the BAPN treatment reduced myocardial fibrosis. A novel observation is that BAPN treatment modulated the transforming growth factor-β pathway, collagen synthesis, and the resident macrophage population. This is especially valuable in terms of potential therapeutic targeting of collagen regulation and thereby age-related myocardial fibrosis. Myocardial fibrosis, a common pathological feature of many cardiovascular disorders, is characterized by an overabundant deposition of extracellular matrix (ECM) molecules and/or a deficit in their degradation.1Berk B.C. Fujiwara K. Lehoux S. ECM remodeling in hypertensive heart disease.J Clin Invest. 2007; 117: 568-575Crossref PubMed Scopus (685) Google Scholar The excess ECM protein results in loss of myocardial contractility and increased stiffness.1Berk B.C. Fujiwara K. Lehoux S. ECM remodeling in hypertensive heart disease.J Clin Invest. 2007; 117: 568-575Crossref PubMed Scopus (685) Google Scholar The functional result of myocardial fibrosis is diastolic dysfunction and ultimately diastolic heart failure; affected patients have preserved systolic function but impaired relaxation associated with significant adverse health outcomes.2Biernacka A. Frangogiannis N.G. Aging and cardiac fibrosis.Aging Dis. 2011; 2: 158-173PubMed Google Scholar Of particular clinical importance is the relationship of age with the development of myocardial fibrosis. The aging myocardium is associated with progressive and significant ECM deposition, which is termed age-related fibrosis.3Chen W. Frangogiannis N.G. The role of inflammatory and fibrogenic pathways in heart failure associated with aging.Heart Fail Rev. 2010; 15: 415-422Crossref PubMed Scopus (119) Google Scholar The degree of fibrosis, as measured by the amount of interstitial collagen, correlates with diastolic dysfunction during aging.4Reed A.L. Tanaka A. Sorescu D. Liu H. Jeong E.M. Sturdy M. Walp E.R. Dudley Jr., S.C. Sutliff R.L. Diastolic dysfunction is associated with cardiac fibrosis in the senescence-accelerated mouse.Am J Physiol Heart Circ Physiol. 2011; 301: H824-H831Crossref PubMed Scopus (77) Google Scholar, 5Besse S. Delcayre C. Chevalier B. Hardouin S. Heymes C. Bourgeois F. Moalic J.M. Swynghedauw B. Is the senescent heart overloaded and already failing?.Cardiovasc Drugs Ther. 1994; 8: 581-587Crossref PubMed Scopus (25) Google Scholar Diastolic heart failure is reported to be the highest cause of hospitalization in elderly patients.6Barasch E. Gottdiener J.S. Aurigemma G. Kitzman D.W. Han J. Kop W.J. Tracy R.P. Association between elevated fibrosis markers and heart failure in the elderly: the Cardiovascular Health Study.Circ Heart Fail. 2009; 2 ([Erratum appeared in Circ Heart Fail 2009, 2(6):e4]): 303-310Crossref PubMed Scopus (102) Google Scholar The mechanisms responsible for the development of age-related myocardial fibrosis have yet to be fully characterized. Nonetheless, there is convincing evidence that the increased collagen in the myocardium of aging mice represents an imbalance involving one or more of the following: i) collagen biosynthesis by fibroblasts, ii) postsynthetic collagen processing (cross-linking), which makes collagen less prone to degradation, and iii) collagen degradation. To date, few studies have addressed the fundamental cause of such imbalance. The mechanical strength of fibril-forming collagens is dependent on cross-links. Cross-linking can involve various processes, including enzymatically driven lysyl oxidation and nonenzymatic glycation.2Biernacka A. Frangogiannis N.G. Aging and cardiac fibrosis.Aging Dis. 2011; 2: 158-173PubMed Google Scholar, 7Ricard-Blum S. The collagen family.Cold Spring Harb Perspect Biol. 2011; 3: a004978Crossref Scopus (1036) Google Scholar Nonenzymatic cross-linking and the resulting advanced glycation end products increase during aging, in part because of the accumulation over time of reactions between collagen and carbohydrates.8Baynes J.W. The role of AGEs in aging: causation or correlation.Exp Gerontol. 2001; 36: 1527-1537Crossref PubMed Scopus (285) Google Scholar, 9Jensen L.J. Østergaard J. Flyvbjerg A. 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Influence of age and long-term dietary restriction on enzymatically mediated crosslinks and nonenzymatic glycation of collagen in mice.J Gerontol. 1994; 49: B71-B79Crossref PubMed Scopus (80) Google Scholar It remains unclear, however, whether the cause of functional improvement is a reduction in cross-links or a decrease in the number of advanced glycation end products. Enzymatic cross-linking is initiated by lysyl oxidase (LOX), which is an extracellular copper-dependent enzyme. LOX oxidizes the amino group on lysine or hydroxylysine, leading to spontaneous nonreducible bonds forming with native lysine or hydroxyproline residues on other collagen molecules.12López B. González A. Hermida N. Valencia F. de Teresa E. Díez J. Role of lysyl oxidase in myocardial fibrosis: from basic science to clinical aspects.Am J Physiol Heart Circ Physiol. 2010; 299: H1-H9Crossref PubMed Scopus (191) Google Scholar In addition to adding to physical strength, collagen cross-linking is believed to be responsible for making collagen more resistant to degradation by collagenases.12López B. González A. Hermida N. Valencia F. de Teresa E. Díez J. Role of lysyl oxidase in myocardial fibrosis: from basic science to clinical aspects.Am J Physiol Heart Circ Physiol. 2010; 299: H1-H9Crossref PubMed Scopus (191) Google Scholar, 13Chang K. Uitto J. Rowold E.A. Grant G.A. Kilo C. Williamson J.R. Increased collagen cross-linkages in experimental diabetes: reversal by beta-aminopropionitrile and D-penicillamine.Diabetes. 1980; 29: 778-781Crossref PubMed Scopus (121) Google Scholar LOX expression and activity are reported to increase with age.12López B. González A. Hermida N. Valencia F. de Teresa E. Díez J. Role of lysyl oxidase in myocardial fibrosis: from basic science to clinical aspects.Am J Physiol Heart Circ Physiol. 2010; 299: H1-H9Crossref PubMed Scopus (191) Google Scholar It has been postulated that an increase in collagen cross-linking by LOX directly contributes to the increase in tissue stiffness within the myocardium that occurs during aging, but to date clear evidence regarding this process is lacking.14Bradshaw A.D. Baicu C.F. Rentz T.J. Van Laer A.O. Bonnema D.D. Zile M.R. Age-dependent alterations in fibrillar collagen content and myocardial diastolic function: role of SPARC in post-synthetic procollagen processing.Am J Physiol Heart Circ Physiol. 2009; 298: H614-H622Crossref PubMed Scopus (100) Google Scholar Transforming growth factor-β (TGF-β) has been suggested as the main cytokine involved in regulating age-related myocardial fibrosis.2Biernacka A. Frangogiannis N.G. Aging and cardiac fibrosis.Aging Dis. 2011; 2: 158-173PubMed Google Scholar This notion is supported by work demonstrating that TGF-β is chemotactic for fibroblasts, stimulates fibroblast proliferation, and increases the synthesis of a number of ECM proteins.15Khan R. Sheppard R. Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia.Immunology. 2006; 118: 10-24Crossref PubMed Scopus (417) Google Scholar, 16Wynn T.A. Cellular and molecular mechanisms of fibrosis.J Pathol. 2008; 214: 199-210Crossref PubMed Scopus (3060) Google Scholar, 17Ikedo H. Tamaki K. Ueda S. Kato S. Fujii M. Ten Dijke P. Okuda S. Smad protein and TGF-beta signaling in vascular smooth muscle cells.Int J Mol Med. 2003; 11: 645-650PubMed Google Scholar Mice that have diminished levels of TGF-β (Tgfb1+/−) exhibit decreased age-related fibrosis and preserved diastolic function.18Brooks W.W. Conrad C.H. Myocardial fibrosis in transforming growth factor beta(1) heterozygous mice.J Mol Cell Cardiol. 2000; 32: 187-195Abstract Full Text PDF PubMed Scopus (145) Google Scholar Given the complexity of TGF-β regulation, however, many investigators have advocated looking at downstream mediators of TGF-β, especially for identification and development of antifibrotic therapies. TGF-β exposure increases expression of connective tissue growth factor (CTGF).19Rosin N.L. Falkenham A. Sopel M.J. Lee T.D. Légaré J.F. Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.Am J Pathol. 2013; 182: 714-726Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar CTGF has been shown to be involved in ECM deposition, wound repair, angiogenesis, migration, differentiation, and cell survival and/or proliferation in animal models of hypertension-related myocardial fibrosis or myocardial infarction.19Rosin N.L. Falkenham A. Sopel M.J. Lee T.D. Légaré J.F. Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.Am J Pathol. 2013; 182: 714-726Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Rupérez M. Lorenzo O. Blanco-Colio L.M. Esteban V. Egido J. Ruiz-Ortega M. Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis.Circulation. 2003; 108: 1499-1505Crossref PubMed Scopus (225) Google Scholar, 21Wang X. McLennan S.V. Allen T.J. Twigg S.M. Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF in H9c2 cardiomyocytes.J Cell Commun Signal. 2010; 4: 15-23Crossref PubMed Scopus (54) Google Scholar A similar link between CTGF expression and age-related fibrosis in the myocardium has not been addressed previously, nor the relationship between expression of other highly active profibrotic factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), with myocardial fibrosis in the aging heart. In the present study, we characterized the expression of several profibrotic factors early in the trajectory of aging and examined how they relate to collagen homeostasis. We also demonstrated that disruption of collagen cross-linking with LOX inhibition can have profound effects on overall collagen homeostasis, beyond structural effects on collagen, which might contribute to novel insights into the development of age-related myocardial fibrosis and thus could lead to potential new therapies. All work was approved by the Dalhousie University Committee on Laboratory Animals (reference number: 12-028) and was in accordance with Canadian Council on Animal Care guidelines. Male C57BL/6J mice at approximately 8, 12, or 38 weeks of age (young to middle-aged) were purchased from the Jackson Laboratory (Bar Harbor, ME) and were housed within the Carleton Animal Care Centre at Dalhousie University. Mice were provided food and water ad libitum for 1 week before experimentation. In a randomly selected group of 38-week-old mice, LOX activity was inhibited using the compound β-aminopropionitrile (BAPN). Mice under general anesthesia were implanted either with an Alzet osmotic minipump (Durect, Cupertino, CA) to deliver BAPN (150 mg/kg per day BAPN) (Sigma-Aldrich, St. Louis, MO) or with subcutaneous saline, as described previously.22Sopel M.J. Rosin N.L. Lee T.D. Légaré J.F. Myocardial fibrosis in response to angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2010; 91: 565-578Crossref PubMed Scopus (54) Google Scholar Animals were anesthetized using inhaled isoflurane (Baxter International, Deerfield, IL) in oxygen and were provided the analgesic buprenorphine subcutaneously. The pumps remained in place for 7 days, with fresh pumps for a further 7 days, during which time the mice were provided food and water ad libitum and were monitored for signs of morbidity. A sonographer, masked to experimental group, performed transthoracic echocardiography using a Vivid 7 ultrasound device (GE Healthcare Canada, Mississauga, ON, Canada; GE Healthcare, Little Chalfont, UK). Mice were anesthetized with isoflurane, body temperature was maintained on a heating pad, and electrocardiograms were monitored during the procedure. Two-dimensional M-mode measurements in short-axis view at the mid-papillary included left ventricular interior diameter in diastole (LVIDd) and systole (LVIDs), septal wall thickness in diastole (ISd) and systole (ISs), and left ventricular posterior wall thickness in diastole (LVPWd) and systole (LVPWs). The percentage fractional shortening (FS) was calculated, and the Teicholz method was used to calculate ejection fraction (EF). Pulsed wave tissue Doppler was used to determine the early and late mitral inflow velocities (E and A, respectively), and the E/A ratio was calculated. Animals were anesthetized with isoflurane and blood was collected using cardiac puncture, followed by sacrifice via exsanguination. Hearts were harvested, weighed, and divided into three sections along the vertical axis. The base section was processed for histological examination; the other two portions were snap-frozen immediately for molecular analysis. The blood collected was allowed to coagulate at 4°C, and then was centrifuged at 1200 × g for 10 minutes for serum isolation. Serum was aliquoted and stored at −80°C. Formalin-fixed tissues were paraffin-embedded and serially sectioned at 5 μm on a microtome. Basic myocardial histology and cellular infiltration were examined using heart cross sections stained with hematoxylin and eosin. Immunohistochemistry for α-smooth muscle actin (SMA) (Sigma-Aldrich) was performed on sectioned paraffin-embedded tissue that was deparaffinized and heat-treated for antigen retrieval before staining. In brief, endogenous peroxidases were quenched with 3% hydrogen peroxide, endogenous biotin was blocked (Dako biotin blocking system; Dako, Carpinteria, CA), and nonspecific staining was blocked with normal goat serum. Sections were incubated with primary antibody overnight at 4°C, followed by host-specific biotin-conjugated secondary antibody. Antibody complexes were then conjugated to avidin–biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and developed using 3,3′-diaminobenzidine as the chromogen (Dako). Images were captured with an AxioCam HRC color camera (Carl Zeiss Microscopy, Jena, Germany). Collagen content was analyzed using two methods. Histological analysis using Sirius Red and fast green stains and semiquantification using a technique modified from Underwood et al23Underwood R.A. Gibran N.S. Muffley L.A. Usui M.L. Olerud J.E. Color subtractive-computer-assisted image analysis for quantification of cutaneous nerves in a diabetic mouse model.J Histochem Cytochem. 2001; 49: 1285-1291Crossref PubMed Scopus (55) Google Scholar were performed by a masked observer (N.L.R.). Image analysis software (Photoshop CS5; Adobe Systems, San Jose, CA) was used to quantify the area of tissue positive for Sirius Red relative to the total cross-sectional area, as described previously.19Rosin N.L. Falkenham A. Sopel M.J. Lee T.D. Légaré J.F. Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.Am J Pathol. 2013; 182: 714-726Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Biochemical analysis using a hydroxyproline assay was used to quantify collagen content in myocardial tissue and in serum. Approximately 10 mg of frozen tissue was homogenized in 100 μL of 1 mol/L NaCl and incubated overnight at 4°C. Concentrated hydrochloric acid was added to separated supernatant and pellet and incubated at 120°C for 3 hours. The hydroxyproline assay was performed according to the manufacturer's protocol (Sigma-Aldrich). The assay was read on an Infinite 200 Pro plate reader (Tecan, Männedorf, Switzerland) at 560 nm. To quantify degradation products in the serum, equal volumes of serum and concentrated hydrochloric acid were incubated at 120°C for 3 hours. Activated charcoal (5 mg) was added to each sample, the sample was centrifuged at 13,000 × g for 2 minutes, and hydroxyproline assay was performed according to the manufacturer's instructions. Total RNA was isolated from snap-frozen heart sections using TRIzol reagent (Life Technologies, Burlington, ON, Canada; Carlsbad, CA) according to the manufacturer's protocol. First-strand cDNA was synthesized from RNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). Real-time quantitative RT-PCR (RT-qPCR) was completed using iQ SYBR Green Supermix (Bio-Rad Laboratories); an iCycler iQ multicolor real-time PCR detection system thermocycler (Bio-Rad Laboratories) was used for detection. Primers were designed against mRNA sequences of the genes of interest using PrimerBlast24Ye J. Coulouris G. Zaretskaya I. Cutcutache I. Rozen S. Madden T.L. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.BMC Bioinformatics. 2012; 13: 134Crossref PubMed Scopus (3385) Google Scholar (Table 1). Expression was normalized to the 18S rRNA gene using the Pfaffl method.Table 1qPCR PrimersPrimerForwardReverseCTGF5′-TCAACCTCAGACACTGGTTTCG-3′5′-TAGAGCAGGTCTGTCTGCAAGC-3′TGFB15′-GGTCTCCCAAGGAAAGGTAGG-3′5′-CTCTTGAGTCCCTCGCATCC-3′PDGFB5′-GCATCTGCCTGAAGTGTGTACC-3′5′-TTAAGGACTTGACCCTGCTTCC-3′FGF5′-CCAGCGGCATCACCTCGCTT-3′5′-GGGTCGCTCTTCTCGCGGAC-3′COL1A15′-CAACAGTCGCTTCACCTACAGC-3′5′-GTGGAGGGAGTTTACACGAAGC-3′LOX5′-TACTCCAGACTCTGTGCGCT-3′5′-GGACTCAGATCCCACGAAGG-3′18S5′-TCAACTTTCGATGGTAGTCGCCGT-3′5′-TCCTTGGATGTGGTAGCCGTTTCT-3′ Open table in a new tab Frozen tissue was homogenized in radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl base, 0.1% SDS, 0.1% Triton X-100, 0.5% deoxycholic acid) with the addition of protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Samples were denatured, and the protein was separated on a 12% SDS-PAGE gel and transferred to Immobilon polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA). Membranes were blocked using 5% nonfat milk before incubation with Smad2/3 (Cell Signaling Technology, Danvers, MA), phospho-Smad2 (Cell Signaling Technology), LOX (EMD Millipore), or β-tubulin (loading control) (Sigma-Aldrich) antibody overnight at 4°C. Blots were developed using horseradish peroxidase–conjugated goat anti-rabbit IgG (Vector Laboratories) and an Amersham ECL enhanced chemiluminescence kit (GE Healthcare). Aliquoted serum samples were thawed and the manufacturer's protocol for CTGF enzyme-linked immunosorbent assay was performed [Uscn Life Science (USCNK), Wuhan, China]. Samples were run in duplicate at a 1:5 dilution with diluent buffer and undiluted. Hearts from mice at 8 weeks, 40 weeks, and 40 weeks + BAPN were harvested under sterile conditions and used for cell isolation in a modification of methods described previously.22Sopel M.J. Rosin N.L. Lee T.D. Légaré J.F. Myocardial fibrosis in response to angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2010; 91: 565-578Crossref PubMed Scopus (54) Google Scholar In brief, hearts were mechanically and enzymatically digested in 1 mg/mL collagenase II solution (Cedarlane Laboratories, Burlington, ON, Canada; Burlington, NC) in Dulbecco's modified Eagle's medium (Life Technologies) at 37°C with agitation for 20 minutes. After two washes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies), cells were strained over a 70 μm cell strainer (Thermo Fisher Scientific, Waltham, MA) and resuspended in 37% isotonic Percoll medium (GE Healthcare) in Dulbecco's phosphate-buffered saline containing 5% fetal bovine serum. The 37% cell–Percoll mixture was layered over 70% isotonic Percoll medium (GE Healthcare) and was centrifuged for 25 minutes at 850 × g with the brake off. The cell layer containing mononuclear cells was isolated and washed before being resuspended in fluorescence-activated cell sorting buffer (Dulbecco's phosphate-buffered saline, 1% bovine serum albumin, and 0.1% sodium azide). Cell surface expression of F4/80, CD11b, and Ly6C was assessed to determine the relative proportion of resident macrophages. In brief, isolated cells were incubated with fc receptor blocker (TrueStain fcX anti-mouse CD16/32; BioLegend, San Diego, CA) for 10 minutes, followed by incubation with fluorescently conjugated anti–F4/80-PerCP-Cy5.5, anti–CD11b-APC, and anti–Ly6C-PE antibodies (BioLegend) for 1 hour at 4°C. After antibody incubations, cells were washed and fixed in 1% formalin solution for storage before analysis. Cells were analyzed with a BD FACSCalibur flow cytometer (BD Biosciences, Mississauga, ON, Canada; San Jose, CA) within 5 days of labeling. Findings were confirmed using isotype controls (rat IgG2a-PerCP-Cy5.5, rat IgG2b-APC, and rat IgG2c-PE; BioLegend). Results are expressed as mean fluorescence index of Ly6C gated on the F4/80+ and then the CD11b+ population. One-way analysis of variance tests were completed using Dunnett's post test to compare experimental groups with saline controls or the Bonferroni post test for comparing multiple groups. All qPCR results and direct comparisons between two groups were evaluated using a one-tailed t-test to compare changes in relative expression. All statistical calculations were performed using GraphPad Prism software version 4 (GraphPad Software, La Jolla, CA), and significance was determined if P < 0.05. A minimum of six mice were evaluated per group. The group allocations were based on age: 8, 12, and 40 weeks (±1 week). Body weight and heart weight did not significantly change between groups (Table 2). Hypertrophy, defined in terms of the heart weight/body weight ratio, did not differ significantly between groups.Table 2Two-Dimensional M-Mode and Tissue Doppler Echocardiography in Young, Aged, and BAPN-Treated Aged MiceMeasure8 weeks40 weeks40 weeks + BAPNMeanSEMnMeanSEMnMeanSEMnM-mode LVIDd (cm)0.390.0250.380.0250.350.034 LVIDs (cm)0.280.0150.270.0250.240.034 EF58.762.32559.331.17565.444.514 FS (%)27.481.50527.650.97532.852.994 IVSd (cm)0.100.0150.110.0150.090.014 IVSs (cm)0.120.0150.140.0150.140.014 LVPWd (cm)0.110.0150.110.0150.13∗P < 0.05 versus 8 weeks; †P < 0.05 and ††P < 0.01 versus 40 weeks.0.014 LVPWs (cm)0.130.0150.130.0150.160.024Doppler E (cm/sec)59.563.67453.273.89550.366.314 A (cm/sec)27.252.85430.551.44523.34P < 0.05 versus 8 weeks; †P < 0.05 and ††P < 0.01 versus 40 weeks.1.964 E/A2.250.2441.760.1652.140.144Morphometry HW (mg)138.57.610162.98.66154.76.38 BW (g)27.21.01033.8‡P < 0.05 versus 8 weeks; †P < 0.05 and ††P < 0.01 versus 40 weeks.0.7632.6†P < 0.05 versus 8 weeks; †P < 0.05 and ††P < 0.01 versus 40 weeks.1.08 HW/BW (mg/g)5.10.2104.80.264.80.28A, late mitral inflow velocity; BAPN, β-aminopropionitrile; BW, body weight; E, early mitral inflow velocity; EF, ejection fraction; FS, fractional shortening; HW, heart weight; HW/BW, hypertrophic index; IVSd, interventricular septal wall thickness in diastole; IVSs, interventricular septal wall thickness in systole; LVIDd, left ventricular interior diameter in diastole; LVIDs, left ventricular interior diameter in systole; LVPWd, left ventricular posterior wall thickness in diastole; LVPWs, left ventricular posterior wall thickness in systole.∗ P < 0.05 versus 8 weeks; †P < 0.05 and ††P < 0.01 versus 40 weeks. Open table in a new tab A, late mitral inflow velocity; BAPN, β-aminopropionitrile; BW, body weight; E, early mitral inflow velocity; EF, ejection fraction; FS, fractional shortening; HW, heart weight; HW/BW, hypertrophic index; IVSd, interventricular septal wall thickness in diastole; IVSs, interventricular septal wall thickness in systole; LVIDd, left ventricular interior diameter in diastole; LVIDs, left ventricular interior diameter in systole; LVPWd, left ventricular posterior wall thickness in diastole; LVPWs, left ventricular posterior wall thickness in systole. Myocardial fibrosis was assessed using Sirius Red for collagen visualization. Interstitial and perivascular collagen increased in older mice, with darker red staining indicating the presence of denser collagen fibers in the myocardium of 40-week-old mice, compared with 8-week-old or 12-week-old mice (Figure 1, A–C). The amount of collagen was semiquantified and expressed as a percentage of total cross-sectional area affected. The myocardium of mice at 40 weeks had significantly more collagen deposition (10.2 ± 1.3%), compared with mice at either 8 weeks (5.7 ± 1.8%) or 12 weeks (5.5 ± 1.2%) (P < 0.05) (Figure 1D). Collagen type I is the most abundant ECM protein within the myocardium.25Weber K.T. Sun Y. Tyagi S.C. Cleutjens J.P. Collagen network of the myocardium: function, structural remodeling and regulatory mechanisms.J Mol Cell Cardiol. 1994; 26: 279-292Abstract Full Text PDF PubMed Scopus (433) Google Scholar It is actively and continually synthesized, processed (including cross-linking), and degraded. We therefore assessed collagen homeostasis within the myocardium and its relationship to aging. The relative expression of collagen type I α1 (COL1A1) mRNA in myocardium, as assessed by qPCR, did not change significantly between 8 and 12 weeks, but was significantly elevated by 40 weeks (15.2 ± 4.9-fold) (P < 0.05) (Figure 1E). This suggests that the increase in collagen deposition observed during aging is due in part to an increase in new collagen mRNA production. Having shown that collagen increased in both mRNA and protein as early as 40 weeks of age, we stained sections of myocardium for SMA to assess the presence of myofibroblasts, a differentiated form of fibroblasts responsible for ECM production. We were able to identify myofibroblasts in the interstitium of 40-week-old mice (Figure 1F), something that was not possible in the myocardium of younger mice (Figure 1G). Hydroxyproline is present largely in collagen, making it an ideal biochemical marker for the measurement of total collagen content per microgram of tissue.14Bradshaw A.D. Baicu C.F. Rentz T.J. Van Laer A.O. Bonnema D.D. Zile M.R. Age-dependent alterations in fibrillar collagen content and myocardial diastolic function: role of SPARC in post-synthetic procollagen processing.Am J Physiol Heart Circ Physiol. 2009; 298: H614-H622Crossref PubMed Scopus (100) Google Scholar The proportion of cross-linked collagen was assessed by comparing the amount of hydroxyproline in non–NaCl-solubilized (cross-linked) to that in NaCl-solubilized (non–cross-linked) fractions of homogenized myocardium. There was a significant increase in total
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