Secondary Coronary Artery Vasospasm Promotes Cardiomyopathy Progression
2004; Elsevier BV; Volume: 164; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63193-8
ISSN1525-2191
AutoresMatthew T. Wheeler, Claudia E. Korcarz, Keith A. Collins, Karen A. Lapidos, Andrew A. Hack, Matthew R. Lyons, Sara Zarnegar, Judy U. Earley, Roberto M. Lang, Elizabeth M. McNally,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoGenetic defects in the plasma membrane-associated sarcoglycan complex produce cardiomyopathy characterized by focal degeneration. The infarct-like pattern of cardiac degeneration has led to the hypothesis that coronary artery vasospasm underlies cardiomyopathy in this disorder. We evaluated the coronary vasculature of γ-sarcoglycan mutant mice and found microvascular filling defects consistent with arterial vasospasm. However, the vascular smooth muscle sarcoglycan complex was intact in the coronary arteries of γ-sarcoglycan hearts with perturbation of the sarcoglycan complex only within the adjacent myocytes. Thus, in this model, coronary artery vasospasm derives from a vascular smooth muscle-cell extrinsic process. To reduce this secondary vasospasm, we treated γ-sarcoglycan-deficient mice with the calcium channel antagonist verapamil. Verapamil treatment eliminated evidence of vasospasm and ameliorated histological and functional evidence of cardiomyopathic progression. Echocardiography of verapamil-treated, γ-sarcoglycan-null mice showed an improvement in left ventricular fractional shortening (44.3 ± 13.3% treated versus 37.4 ± 15.3% untreated), maximal velocity at the aortic outflow tract (114.9 ± 27.9 cm/second versus 92.8 ± 22.7 cm/second), and cardiac index (1.06 ± 0.30 ml/minute/g versus 0.67 ± 0.16 ml/minute/g, P < 0.05). These data indicate that secondary vasospasm contributes to the development of cardiomyopathy and is an important therapeutic target to limit cardiomyopathy progression. Genetic defects in the plasma membrane-associated sarcoglycan complex produce cardiomyopathy characterized by focal degeneration. The infarct-like pattern of cardiac degeneration has led to the hypothesis that coronary artery vasospasm underlies cardiomyopathy in this disorder. We evaluated the coronary vasculature of γ-sarcoglycan mutant mice and found microvascular filling defects consistent with arterial vasospasm. However, the vascular smooth muscle sarcoglycan complex was intact in the coronary arteries of γ-sarcoglycan hearts with perturbation of the sarcoglycan complex only within the adjacent myocytes. Thus, in this model, coronary artery vasospasm derives from a vascular smooth muscle-cell extrinsic process. To reduce this secondary vasospasm, we treated γ-sarcoglycan-deficient mice with the calcium channel antagonist verapamil. Verapamil treatment eliminated evidence of vasospasm and ameliorated histological and functional evidence of cardiomyopathic progression. Echocardiography of verapamil-treated, γ-sarcoglycan-null mice showed an improvement in left ventricular fractional shortening (44.3 ± 13.3% treated versus 37.4 ± 15.3% untreated), maximal velocity at the aortic outflow tract (114.9 ± 27.9 cm/second versus 92.8 ± 22.7 cm/second), and cardiac index (1.06 ± 0.30 ml/minute/g versus 0.67 ± 0.16 ml/minute/g, P < 0.05). These data indicate that secondary vasospasm contributes to the development of cardiomyopathy and is an important therapeutic target to limit cardiomyopathy progression. Cardiomyopathy occurs from mutations in genes encoding the dystrophin glycoprotein complex in both humans and mice.1Heydemann A Wheeler MT McNally EM Cardiomyopathy in animal models of muscular dystrophy.Curr Opin Cardiol. 2001; 16: 211-217Crossref PubMed Scopus (19) Google Scholar, 2Cox GF Kunkel LM Dystrophies and heart disease.Curr Opin Cardiol. 1997; 12: 329-343Crossref PubMed Scopus (225) Google Scholar, 3Tsubata S Bowles KR Vatta M Zintz C Titus J Muhonen L Bowles NE Towbin JA Mutations in the human delta-sarcoglycan gene in familial and sporadic dilated cardiomyopathy.J Clin Invest. 2000; 106: 655-662Crossref PubMed Scopus (306) Google Scholar In heart and skeletal muscle, genetic mutations destabilize components of the dystrophin glycoprotein complex leading to membrane defects and myocyte loss. Dystrophin is a large cytoskeletal protein that binds to the transmembrane and cytoskeletal elements of the dystrophin glycoprotein complex.4Rafael JA Brown SC Dystrophin and utrophin: genetic analyses of their role in skeletal muscle.Microsc Res Tech. 2000; 48: 155-166Crossref PubMed Scopus (26) Google Scholar Dystroglycan and sarcoglycan comprise the transmembrane dystrophin glycoprotein complex elements.5Bonnemann CG McNally EM Kunkel LM Beyond dystrophin: current progress in the muscular dystrophies.Curr Opin Pediatr. 1996; 8: 569-582Crossref PubMed Scopus (68) Google Scholar, 6Hack AA Groh ME McNally EM Sarcoglycans in muscular dystrophy.Microsc Res Tech. 2000; 48: 167-180Crossref PubMed Scopus (119) Google Scholar Dystroglycan directly binds the extracellular matrix protein laminin completing the link from the internal cytoskeleton through the membrane to the extracellular matrix.7Ibraghimov-Beskrovnaya O Ervasti JM Leveille CJ Slaughter CA Sernett SW Campbell KP Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1175) Google Scholar The sarcoglycan complex consists of single transmembrane domain-containing proteins that form a multimeric complex.6Hack AA Groh ME McNally EM Sarcoglycans in muscular dystrophy.Microsc Res Tech. 2000; 48: 167-180Crossref PubMed Scopus (119) Google Scholar The sarcoglycan complex serves a mechanosignalling role through its interactions with dystroglycan and filamin-2.8Thompson TG Chan YM Hack AA Brosius M Rajala M Lidov HG McNally EM Watkins S Kunkel LM Filamin 2 (FLN2): a muscle-specific sarcoglycan interacting protein.J Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (233) Google Scholar There are six sarcoglycan family members: α-, β-, γ-, δ-, ε-, and ζ-sarcoglycan.6Hack AA Groh ME McNally EM Sarcoglycans in muscular dystrophy.Microsc Res Tech. 2000; 48: 167-180Crossref PubMed Scopus (119) Google Scholar, 9Wheeler MT Zarnegar S McNally EM Zeta-sarcoglycan, a novel component of the sarcoglycan complex, is reduced in muscular dystrophy.Hum Mol Genet. 2002; 11: 2147-2154Crossref PubMed Google Scholar Mutations in the β-, γ-, and δ-sarcoglycan genes produce muscular dystrophy and dilated cardiomyopathy, and mouse models of sarcoglycan mutations recapitulate the human phenotype.10Durbeej M Cohn RD Hrstka RF Moore SA Allamand V Davidson BL Williamson RA Campbell KP Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell. 2000; 5: 141-151Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 12Hack AA Lam MY 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 Sci. 2000; 113: 2535-2544PubMed Google Scholar, 13Hack AA Ly CT Jiang F Clendenin CJ Sigrist KS Wollmann RL McNally EM Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol. 1998; 142: 1279-1287Crossref PubMed Scopus (248) Google Scholar Cardiomyopathy in the murine models is progressive. By 8 to 10 weeks of age, small foci of degeneration are present and regional uptake of the vital tracer Evans blue dye into cardiomyocytes is distributed randomly and is typically present in 1 to 2% of cardiomyocytes. With time regional fibrosis becomes more evident. Sarcoglycan mutant mice have an increased mortality without evidence of severe edema, consistent with the human clinical course in sarcoglycan deficiency.14Politano L Nigro V Passamano L Petretta V Comi LI Papparella S Nigro G Rambaldi PF Raia P Pini A Mora M Giugliano MA Esposito MG Evaluation of cardiac and respiratory involvement in sarcoglycanopathies.Neuromuscul Disord. 2001; 11: 178-185Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar The infarct-like pattern of necrosis in β-sarcoglycan or δ-sarcoglycan-null mice and in another model of sarcoglycan deficiency, the δ-sarcoglycan mutant BIO14.6 cardiomyopathic hamster, suggested that coronary vascular spasm leads to cardiomyopathy progression.10Durbeej M Cohn RD Hrstka RF Moore SA Allamand V Davidson BL Williamson RA Campbell KP Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell. 2000; 5: 141-151Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 15Factor SM Minase T Cho S Dominitz R Sonnenblick EH Microvascular spasm in the cardiomyopathic Syrian hamster: a preventable cause of focal myocardial necrosis.Circulation. 1982; 66: 342-354Crossref PubMed Scopus (253) Google Scholar, 16Sonnenblick EH Fein F Capasso JM Factor SM Microvascular spasm as a cause of cardiomyopathies and the calcium-blocking agent verapamil as potential primary therapy.Am J Cardiol. 1985; 55: 179B-184BAbstract Full Text PDF PubMed Scopus (78) Google Scholar, 17Factor SM Sonnenblick EH Hypothesis: is congestive cardiomyopathy caused by a hyperreactive myocardial microcirculation (microvascular spasm)?.Am J Cardiol. 1982; 50: 1149-1152Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 18Cohn RD Durbeej M Moore SA Coral-Vazquez R Prouty S Campbell KP Prevention of cardiomyopathy in mouse models lacking the smooth muscle sarcoglycan-sarcospan complex.J Clin Invest. 2001; 107: R1-R7Crossref PubMed Scopus (100) Google Scholar Disruption of the smooth muscle sarcoglycan complex was noted in both δ-sarcoglycan and β-sarcoglycan mutant mice10Durbeej M Cohn RD Hrstka RF Moore SA Allamand V Davidson BL Williamson RA Campbell KP Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell. 2000; 5: 141-151Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 18Cohn RD Durbeej M Moore SA Coral-Vazquez R Prouty S Campbell KP Prevention of cardiomyopathy in mouse models lacking the smooth muscle sarcoglycan-sarcospan complex.J Clin Invest. 2001; 107: R1-R7Crossref PubMed Scopus (100) Google Scholar and proposed as a mechanism underlying vasospasm and responsible as a primary etiology of cardiomyopathy. Supporting this, microvascular-filling experiments of coronary arteries demonstrated focal stenoses in β-sarcoglycan and δ-sarcoglycan mice consistent with vasospasm. To reduce vasospasm, δ-sarcoglycan mutant mice or the BIO14.6 hamster model were treated with the calcium channel antagonist verapamil with reduction of the pathological features of cardiomyopathy and microvascular filling defects.18Cohn RD Durbeej M Moore SA Coral-Vazquez R Prouty S Campbell KP Prevention of cardiomyopathy in mouse models lacking the smooth muscle sarcoglycan-sarcospan complex.J Clin Invest. 2001; 107: R1-R7Crossref PubMed Scopus (100) Google Scholar, 19Factor SM Cho SH Scheuer J Sonnenblick EH Malhotra A Prevention of hereditary cardiomyopathy in the Syrian hamster with chronic verapamil therapy.J Am Coll Cardiol. 1988; 12: 1599-1604Abstract Full Text PDF PubMed Scopus (47) Google Scholar γ-Sarcoglycan mutant mice lack exon 2 and represent a null allele.13Hack AA Ly CT Jiang F Clendenin CJ Sigrist KS Wollmann RL McNally EM Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol. 1998; 142: 1279-1287Crossref PubMed Scopus (248) Google Scholar Like other sarcoglycan gene mutations, disruption of γ-sarcoglycan destabilizes the entire sarcoglycan complex at the plasma membrane with secondary reduction of the remaining sarcoglycan subunits.20Hack AA Cordier L Shoturma DI Lam MY Sweeney HL McNally EM Muscle degeneration without mechanical injury in sarcoglycan deficiency.Proc Natl Acad Sci USA. 1999; 96: 10723-10728Crossref PubMed Scopus (108) Google Scholar Focal degeneration develops in the heart and skeletal muscle of γ-sarcoglycan mutants similar to δ- or β-sarcoglycan mutants. We now evaluated the role of smooth muscle sarcoglycan disruption and vasospasm in mice lacking γ-sarcoglycan. Despite an identical pattern of cardiomyopathy to γ-sarcoglycan-null mice, we found no disruption of the vascular smooth muscle sarcoglycan complex in γ-sarcoglycan mutant mice. However, microvascular-filling defects were present in γ-sarcoglycan-null coronary arteries. As there is no intrinsic defect in γ-sarcoglycan-mutant vascular smooth muscle, these findings support a paracrine mechanism whereby degenerating cardiomyocytes produce vascular spasm. Vascular spasm, in this model, may further contribute to cardiomyopathy progression and the development of congestive heart failure. To test whether inhibition of secondary vasospasm limits cardiomyopathy progression, we treated γ-sarcoglycan mutant mice with verapamil and found that verapamil-treated mice had improved cardiac function. These data support that secondary vasospasm arises from primary cardiomyocyte degeneration and identify secondary vasospasm as an important target for therapeutic intervention. Mice lacking γ-sarcoglycan (gsg−/−) or δ-sarcoglycan (dsg−/−) were previously reported.12Hack AA Lam MY 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 Sci. 2000; 113: 2535-2544PubMed Google Scholar, 13Hack AA Ly CT Jiang F Clendenin CJ Sigrist KS Wollmann RL McNally EM Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol. 1998; 142: 1279-1287Crossref PubMed Scopus (248) Google Scholar Mice were maintained by backcrossing heterozygous null animals into the 129T2/SvEmsJ strain. Animals used in the treatment study were products of a gsg−/− × gsg−/− mating after one gsg+/−/gsg+/− cross and seven generations of crosses between gsg+/− and normal 129T2/SvEmsJ mice (Jackson Laboratories, Bar Harbor, ME). Animals were housed, treated, and handled in accordance with the guidelines set forth by the University of Chicago's IACUC, the Animal Welfare Act regulations, and the NIH Guide for the Care and Use of Laboratory Animals. Heart and quadriceps skeletal muscle were dissected and processed as described.12Hack AA Lam MY 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 Sci. 2000; 113: 2535-2544PubMed Google Scholar Cryosections of 7 to 10 μm thickness were incubated with polyclonal anti-γ-sarcoglycan (1:1000),21McNally EM Duggan D Gorospe JR Bonnemann CG Fanin M Pegoraro E Lidov HG Noguchi S Ozawa E Finkel RS Cruse RP Angelini C Kunkel LM Hoffman EP Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy.Hum Mol Genet. 1996; 5: 1841-1847Crossref PubMed Scopus (135) Google Scholar polyclonal anti-δ-sarcoglycan (1:500),12Hack AA Lam MY 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 Sci. 2000; 113: 2535-2544PubMed Google Scholar polyclonal anti-β-sarcoglycan (1:150),22Bonnemann CG Passos-Bueno MR McNally EM Vainzof M de Sa Moreira E Marie SK Pavanello RC Noguchi S Ozawa E Zatz M Kunkel LM Genomic screening for beta-sarcoglycan gene mutations: missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E).Hum Mol Genet. 1996; 5: 1953-1961Crossref PubMed Google Scholar affinity-purified polyclonal anti-ε-sarcoglycan (1:50),23McNally EM Ly CT Kunkel LM Human epsilon-sarcoglycan is highly related to alpha-sarcoglycan (adhalin), the limb girdle muscular dystrophy 2D gene.FEBS Lett. 1998; 422: 27-32Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar affinity-purified polyclonal anti-ζ-sarcoglycan (1:500),9Wheeler MT Zarnegar S McNally EM Zeta-sarcoglycan, a novel component of the sarcoglycan complex, is reduced in muscular dystrophy.Hum Mol Genet. 2002; 11: 2147-2154Crossref PubMed Google Scholar polyclonal anti-dystrophin (1:5000),24Lidov HG Byers TJ Watkins SC Kunkel LM Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons.Nature. 1990; 348: 725-728Crossref PubMed Scopus (360) Google Scholar monoclonal anti-dystrophin (NCL-Dys2, 1:200; Novocastra Laboratories, Newcastle on Tyne, UK), monoclonal anti-α-sarcoglycan (NCL-asarc, 1:100; Novocastra), or monoclonal anti-smooth muscle α-actin (clone 1A4, 1:1000; Sigma, St. Louis, MO). Secondary goat anti-rabbit or goat anti-mouse Cy3 or fluorescein isothiocyanate-conjugated antibodies (Jackson Immunoresearch, West Grove, PA) were used. Images were acquired using an Axioscope (Carl Zeiss, Oberkochen, Germany), Axiocam, and Axiovision. Whole aorta, bladder, and uterus were dissected from strain-matched control mice, dsg−/− or gsg−/− and solubilized in lysis buffer [50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 2 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L NaF, 10 mmol/L Na pyrophosphate, 1 mmol/L Na orthovanadate, 10% glycerol, 1% Triton X-100, 50 μmol/L phenylmethyl sulfonyl fluoride, plus Complete protease inhibitor (Roche, Mannheim, Germany)]. Heavy microsomes were prepared from normal control mouse skeletal muscle as described.12Hack AA Lam MY 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 Sci. 2000; 113: 2535-2544PubMed Google Scholar, 25Ohlendieck K Ervasti JM Snook JB Campbell KP Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma.J Cell Biol. 1991; 112: 135-148Crossref PubMed Scopus (240) Google Scholar Five μg of skeletal muscle microsomes and 50 μg of smooth muscle lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Equal loading was verified by Coomassie staining of duplicate gels. Protein was transferred to Immobilon-P membranes (Millipore, Bedford, MA) and incubated with polyclonal anti-δ-sarcoglycan (1:5000), polyclonal anti-γ-sarcoglycan (1:1000), or anti-smooth muscle actin (clone 1A4, 1:200; Sigma). Goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated antibodies were used (1:10,000). Blots were imaged using ECLPLUS (Amersham Pharmacia, Piscataway, NJ) and Biomax MS film (Eastman Kodak, Rochester, NY). Evans blue dye (20 mg/ml) (Sigma) was dissolved in sterile phosphate-buffered saline. Mice were given intraperitoneal injections at 5 μl/g body weight 18 hours before sacrifice.13Hack AA Ly CT Jiang F Clendenin CJ Sigrist KS Wollmann RL McNally EM Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol. 1998; 142: 1279-1287Crossref PubMed Scopus (248) Google Scholar Hearts were excised and frozen for immunofluorescence analysis. Verapamil was administered to mice through dissolution in their drinking water at a concentration of 1 mg/ml.18Cohn RD Durbeej M Moore SA Coral-Vazquez R Prouty S Campbell KP Prevention of cardiomyopathy in mouse models lacking the smooth muscle sarcoglycan-sarcospan complex.J Clin Invest. 2001; 107: R1-R7Crossref PubMed Scopus (100) Google Scholar, 26Morris SA Weiss LM Factor S Bilezikian JP Tanowitz H Wittner M Verapamil ameliorates clinical, pathologic and biochemical manifestations of experimental chagasic cardiomyopathy in mice.J Am Coll Cardiol. 1989; 14: 782-789Abstract Full Text PDF PubMed Scopus (73) Google Scholar Average daily water consumption of 3.5 ml per day provides an average daily dose of 3.5 mg/mouse/day or ∼100 mg/kg/day. Steady-state serum concentration using this protocol was approximated at 1 μg/ml.26Morris SA Weiss LM Factor S Bilezikian JP Tanowitz H Wittner M Verapamil ameliorates clinical, pathologic and biochemical manifestations of experimental chagasic cardiomyopathy in mice.J Am Coll Cardiol. 1989; 14: 782-789Abstract Full Text PDF PubMed Scopus (73) Google Scholar gsg−/− mice (n = 10) were treated with oral verapamil starting at 4 weeks of age and treated for 22 weeks. gsg−/− control mice (n = 11) were housed under identical conditions without verapamil. All animals were monitored daily. Coronary microvascular perfusion was performed as described.11Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 27Chutkow WA Pu J Wheeler MT Wada T Makielski JC Burant CF McNally EM Episodic coronary artery vasospasm and hypertension develop in the absence of Sur2 K(ATP) channels.J Clin Invest. 2002; 110: 203-208Crossref PubMed Scopus (199) Google Scholar Microfil red (1 to 1.5 ml; FlowTech, Carver, MA) was injected directly into the left ventricular apex. Heart contractions perfused the coronary microvasculature, after which the heart was rapidly excised; fixed in 10% formalin for 24 hours; and cleared by sequential incubations in 25%, 50%, 75%, 95%, and 100% ethanol followed by storage in methyl salicylate. Hearts were scored for the presence or absence of stenosis in coronary vessels, and filled major coronary vessel trees and branches were scored for presence or absence of stenosis. Data collection was performed blinded to treatment group and genotype. All animals in the verapamil treatment study, plus normal, age-matched 129T2/SvEmsJ controls (n = 11), underwent echocardiographic study at age 6 months as previously described28Weiss RE Korcarz C Chassande O Cua K Sadow P Koo E Samarut JRL Thyroid hormone and cardiac function in mice deficient in thyroid hormone receptor-alpha or -beta: an echocardiograph study.Am J Physiol. 2002; 283: E428-E435Google Scholar, 29Collins KA Korcarz CE Shroff SG Bednarz JE Fentzke RC Lin H Leiden JM Lang RM Accuracy of echocardiographic estimates of left ventricular mass in mice.Am J Physiol. 2001; 280: H1954-H1962Google Scholar blinded to treatment group and genotype. Anesthesia was induced by administering isoflurane in a closed chamber at 3 to 5% (Ohmeda Fluotec 3; Matrix Medical, Orchard Park, NY) in 80% room air/20% O2, followed by 0.5 to 2.0% isoflurane through a nose cone throughout the experiment. A high-frequency 15-MHz linear transducer was used (Sonos 5500; Agilent, Andover, MA) at a frame rate of 120 frames/second. Measurements were performed as described previously.28Weiss RE Korcarz C Chassande O Cua K Sadow P Koo E Samarut JRL Thyroid hormone and cardiac function in mice deficient in thyroid hormone receptor-alpha or -beta: an echocardiograph study.Am J Physiol. 2002; 283: E428-E435Google Scholar, 29Collins KA Korcarz CE Shroff SG Bednarz JE Fentzke RC Lin H Leiden JM Lang RM Accuracy of echocardiographic estimates of left ventricular mass in mice.Am J Physiol. 2001; 280: H1954-H1962Google Scholar Stroke volume was determined from aortic Doppler recordings and two-dimensionally targeted M-mode echocardiographic measurements of the diameter of the proximal ascending aorta during systole from the parasternal long axis view. Stroke volume was calculated from the flow velocity integral of each beat multiplied by the aortic cross-sectional area. Cardiac output was calculated as the product of stroke volume and heart rate. Cardiac index was calculated from cardiac output correcting for animal weight. Six animals from each treatment group were monitored for 24 to 72 hours by radio frequency telemetry after implantation of an electrocardiographic radio transmitter (TA10EA-F20; Data Sciences International, St. Paul, MN).27Chutkow WA Pu J Wheeler MT Wada T Makielski JC Burant CF McNally EM Episodic coronary artery vasospasm and hypertension develop in the absence of Sur2 K(ATP) channels.J Clin Invest. 2002; 110: 203-208Crossref PubMed Scopus (199) Google Scholar Additionally, more than 40 untreated gsg−/− mice ranging in age from 6 weeks to 1 year of age have similarly been examined with a minimum of 72 hours monitoring. gsg−/− mice (ages 3 to 4 months) underwent 10 minutes of exercise by running downhill 10 minutes per day for 3 days at 9 m/minute on a 14 degree incline. After the exercise session on day 3, mice were immediately sacrificed for study. Data are presented as means ± SD. Groups were tested for statistical significance using one-way analysis of variance followed by Student-Newman-Keuls post test (echocardiography); the chi-square test for independence followed by Fisher's exact test (microvascular filling); and Student's unpaired t-test (heart rate variance). All values given are two-tailed P values. Heart sections were taken through the short axis from apex to base from the verapamil-treated and untreated groups (n = 4 in each group). Masson Trichrome staining was used to evaluate fibrosis. Sections were scored blinded to genotype and drug treatment status using a scale as follows: 0, no fibrosis; 1, fewer than three areas of fibrosis; 2, greater than three areas of fibrosis; 3, greater than three areas of fibrosis and at least one area greater than 10 μm in diameter. Sections were each assigned a score, and the average score from each animal was determined. The mean score from each treatment group was then determined and analyzed using a Student's t-test. Ten to 65 sections were examined from each animal. To assess inflammation, three different biotinylated antibodies were used: CD45.2, Ly-6G/Ly-6C, and CD11b/Mac-1. Sections were taken from five different animals in each treatment group. Fifteen fields per animal were examined for the number of immunoreactive cells in each field at ×20 magnification. The number of cells in each treatment group was averaged and compared. γ-Sarcoglycan mice develop cardiomyopathy between 12 and 24 weeks of age.13Hack AA Ly CT Jiang F Clendenin CJ Sigrist KS Wollmann RL McNally EM Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol. 1998; 142: 1279-1287Crossref PubMed Scopus (248) Google Scholar To determine how the γ-sarcoglycan-null allele affects expression of the vascular smooth muscle complex, we used antibodies specific to each of the sarcoglycan subunits on sections from cardiac tissue that contained arterial structures. When the vascular smooth muscle layer of coronary arteries from normal mice was examined, we found that β-, δ-, ε-, and ζ-sarcoglycan were expressed (Figure 1A). In the adjacent normal cardiomyocytes, γ-sarcoglycan in addition to β-, δ-, ε-, and ζ-sarcoglycan were expressed (Figure 1A). An anti-smooth muscle actin antibody was used to confirm the integrity and identity of vascular smooth muscle and to demonstrate that γ-sarcoglycan was not expressed in the arterial smooth muscle (data not shown). Mutations in a single sarcoglycan subunit produce disruption of the remainder of the sarcoglycan subunits.12Hack AA Lam MY 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 Sci. 2000; 113: 2535-2544PubMed Google Scholar Consistent with this, δ-, β-, and, to a lesser extent, ε- and ζ-sarcoglycan, were reduced in gsg−/− cardiomyocytes (Figure 1A, adjacent cardiomyocytes). In contrast, the vascular smooth muscle expression pattern did not differ between normal coronary artery vascular smooth muscle and γ-sarcoglycan mutant (gsg−/−) coronary vascular smooth muscle. β-Sarcoglycan, δ-sarcoglycan, ε-sarcoglycan, and ζ-sarcoglycan were all found at levels comparable to that seen in normal coronary smooth muscle. α-Sarcoglycan was not found in coronary vessels (data not shown). Using antibodies specific to γ- or δ-sarcoglycan, we tested aorta, bladder, and uterus isolated from gsg−/−, dsg−/−, and normal mice. γ-Sarcoglycan could not be detected in any of the smooth muscle-containing tissues studied (Figure 1B). Furthermore, γ-sarcoglycan was readily detected in skeletal muscle from control animals. δ-Sarcoglycan was detected in gsg−/− smooth muscle tissues comparable to levels seen in wild-type tissues. δ-Sarcoglycan mutants (dsg−/−), by contrast, show a complete absence of δ-sarcoglycan in the smooth muscle tissues, and loss of other smooth muscle sarcoglycans.11Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar The smooth muscle sarcoglycan complex is composed of δ-, β-, ε-, and ζ-sarcoglycan. In mice mutant for δ-sarcoglycan, there is disruption of the vascular smooth muscle sarcoglycan complex whereas in γ-sarcoglycan mutant mice, the vascular smooth muscle sarcoglycan complex remains intact. Microfil microvascular filling was used to examine coronary ves
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