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Molecular and Functional Alterations in a Mouse Cardiac Model of Friedreich Ataxia

2013; Elsevier BV; Volume: 183; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2013.05.032

ISSN

1525-2191

Autores

Michael Huang, Sutharshani Sivagurunathan, Samantha Ting, Patric J. Jansson, Christopher Austin, Matt Kelly, Christopher Semsarian, Daohai Zhang, Des R. Richardson,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

Friedreich ataxia (FA) is a neurodegenerative and cardiodegenerative disease resulting from marked frataxin deficiency. The condition is characterized by ataxia with fatal cardiomyopathy, but the pathogenic mechanisms are unclear. We investigated the association between gene expression and progressive histopathological and functional changes using the muscle creatine kinase conditional frataxin knockout (KO) mouse; this mouse develops a severe cardiac phenotype that resembles that of FA patients. We examined KO mice from 3 weeks of age, when they are asymptomatic, to 10 weeks of age, when they die of the disease. Positive iron staining was identified in KO mice from 5 weeks of age, with markedly reduced cardiac function from 6 weeks. We identified an early and marked up-regulation of a gene cohort responsible for stress-induced amino acid biosynthesis and observed markedly increased phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), an activator of the integrated stress response, in KO mice at 3 weeks of age, relative to wild-type mice. Importantly, the eIF2α-mediated integrated stress response has been previously implicated in heart failure via downstream processes such as autophagy and apoptosis. Indeed, expression of a panel of autophagy and apoptosis markers was enhanced in KO mice. Thus, the pathogenesis of cardiomyopathy in FA correlates with the early and persistent eIF2α phosphorylation, which precedes activation of autophagy and apoptosis. Friedreich ataxia (FA) is a neurodegenerative and cardiodegenerative disease resulting from marked frataxin deficiency. The condition is characterized by ataxia with fatal cardiomyopathy, but the pathogenic mechanisms are unclear. We investigated the association between gene expression and progressive histopathological and functional changes using the muscle creatine kinase conditional frataxin knockout (KO) mouse; this mouse develops a severe cardiac phenotype that resembles that of FA patients. We examined KO mice from 3 weeks of age, when they are asymptomatic, to 10 weeks of age, when they die of the disease. Positive iron staining was identified in KO mice from 5 weeks of age, with markedly reduced cardiac function from 6 weeks. We identified an early and marked up-regulation of a gene cohort responsible for stress-induced amino acid biosynthesis and observed markedly increased phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), an activator of the integrated stress response, in KO mice at 3 weeks of age, relative to wild-type mice. Importantly, the eIF2α-mediated integrated stress response has been previously implicated in heart failure via downstream processes such as autophagy and apoptosis. Indeed, expression of a panel of autophagy and apoptosis markers was enhanced in KO mice. Thus, the pathogenesis of cardiomyopathy in FA correlates with the early and persistent eIF2α phosphorylation, which precedes activation of autophagy and apoptosis. Friedreich ataxia (FA), the most prevalent hereditary neurodegenerative and cardiodegenerative ataxia, is caused by a reduction in frataxin expression.1Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.L. Vescovi A. Pandolfo M. Koenig M. Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes.Hum Mol Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (613) Google Scholar A wide variety of models have been assessed to examine the pathological processes in FA with respect to the cardiomyopathy and neurological effects observed in the disease.2Puccio H. Simon D. Cossée M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.Nat Genet. 2001; 27: 181-186Crossref PubMed Scopus (600) Google Scholar, 3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar, 4Seznec H. Simon D. Bouton C. Reutenauer L. Hertzog A. Golik P. Procaccio V. Patel M. Drapier J.C. Koenig M. Puccio H. Friedreich ataxia: the oxidative stress paradox.Hum Mol Genet. 2005; 14: 463-474Crossref PubMed Scopus (190) Google Scholar, 5Miranda C.J. Santos M.M. Ohshima K. Smith J. Li L. Bunting M. Cossée M. Koenig M. Sequeiros J. Kaplan J. Pandolfo M. Frataxin knockin mouse.FEBS Lett. 2002; 512: 291-297Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 6Coppola G. Choi S.H. Santos M.M. Miranda C.J. Tentler D. Wexler E.M. Pandolfo M. Geschwind D.H. Gene expression profiling in frataxin deficient mice: microarray evidence for significant expression changes without detectable neurodegeneration.Neurobiol Dis. 2006; 22: 302-311Crossref PubMed Scopus (50) Google Scholar, 7Ventura N. Rea S.L. Caenorhabditis elegans mitochondrial mutants as an investigative tool to study human neurodegenerative diseases associated with mitochondrial dysfunction.Biotechnol J. 2007; 2: 584-595Crossref PubMed Scopus (40) Google Scholar The cardiomyopathy is of significant concern, because it leads to premature death in FA patients.8Weidemann F. Rummey C. Bijnens B. Störk S. Jasaityte R. Dhooge J. Baltabaeva A. Sutherland G. Schulz J.B. Meier T. Mitochondrial Protection with Idebenone in Cardiac or Neurological Outcome (MICONOS) study groupThe heart in Friedreich ataxia: definition of cardiomyopathy, disease severity, and correlation with neurological symptoms.Circulation. 2012; 125: 1626-1634Crossref PubMed Scopus (99) Google Scholar A prominent cardiac pathology in FA is left ventricular (LV) hypertrophy, which develops into a hypokinetic dilated cardiomyopathy characterized by enlarged end-diastolic and end-systolic diameters coupled with reduced cardiac function.8Weidemann F. Rummey C. Bijnens B. Störk S. Jasaityte R. Dhooge J. Baltabaeva A. Sutherland G. Schulz J.B. Meier T. Mitochondrial Protection with Idebenone in Cardiac or Neurological Outcome (MICONOS) study groupThe heart in Friedreich ataxia: definition of cardiomyopathy, disease severity, and correlation with neurological symptoms.Circulation. 2012; 125: 1626-1634Crossref PubMed Scopus (99) Google Scholar, 9Casazza F. Morpurgo M. The varying evolution of Friedreich’s ataxia cardiomyopathy.Am J Cardiol. 1996; 77: 895-898Abstract Full Text PDF PubMed Scopus (57) Google Scholar Frataxin is a nuclear-encoded mitochondrial protein.1Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.L. Vescovi A. Pandolfo M. Koenig M. Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes.Hum Mol Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (613) Google Scholar Its down-regulation leads to mitochondrial iron loading and perturbed cellular iron homeostasis.3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar, 10Whitnall M. Suryo Rahmanto Y. Sutak R. Xu X. Becker E.M. Mikhael M.R. Ponka P. Richardson D.R. The MCK mouse heart model of Friedreich’s ataxia: alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation.Proc Natl Acad Sci USA. 2008; 105: 9757-9762Crossref PubMed Scopus (101) Google Scholar, 11Whitnall M. Suryo Rahmanto Y. Huang M.L. Saletta F. Lok H.C. Gutiérrez L. Lázaro F.J. Fleming A.J. St Pierre T.G. Mikhael M.R. Ponka P. Richardson D.R. Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.Proc Natl Acad Sci USA. 2012; 109: 20590-20595Crossref PubMed Scopus (77) Google Scholar This mitochondrial iron accumulation can potentiate further mitochondrial defects from oxidative stress.12Huang M.L. Lane D.J. Richardson D.R. Mitochondrial mayhem: the mitochondrion as a modulator of iron metabolism and its role in disease.Antioxid Redox Signal. 2011; 15: 3003-3019Crossref PubMed Scopus (77) Google Scholar Studies using models of FA4Seznec H. Simon D. Bouton C. Reutenauer L. Hertzog A. Golik P. Procaccio V. Patel M. Drapier J.C. Koenig M. Puccio H. Friedreich ataxia: the oxidative stress paradox.Hum Mol Genet. 2005; 14: 463-474Crossref PubMed Scopus (190) Google Scholar, 13Cortopassi G. Danielson S. Alemi M. Zhan S.S. Tong W. Carelli V. Martinuzzi A. Marzuki S. Majamaa K. Wong A. 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Shirasawa T. Oxidative Stress Causes Heart Failure with Impaired Mitochondrial Respiration.J Biol Chem. 2006; 281: 33789-33801Crossref PubMed Scopus (183) Google Scholar, 16Fujita Y. Ito M. Nozawa Y. Yoneda M. Oshida Y. Tanaka M. CHOP (C/EBP homologous protein) and ASNS (asparagine synthetase) induction in cybrid cells harboring MELAS and NARP mitochondrial DNA mutations.Mitochondrion. 2007; 7: 80-88Crossref PubMed Scopus (27) Google Scholar have identified up-regulation of several stress-inducible transcripts, which can be activated by the integrated stress response (ISR).17Harding H.P. Zhang Y.H. Zeng H.Q. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2376) Google Scholar, 18Kilberg M.S. Shan J.X. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar The ISR is a highly conserved eukaryotic adaptation to various cellular stresses that results in phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α).17Harding H.P. Zhang Y.H. Zeng H.Q. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2376) Google Scholar, 18Kilberg M.S. Shan J.X. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar The phosphorylated form of eIF2α (p-eIF2α) inhibits general protein synthesis, but selectively activates the translation of stress-inducible transcripts, particularly the activating transcription factor 4 (Atf4).17Harding H.P. Zhang Y.H. Zeng H.Q. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2376) Google Scholar, 18Kilberg M.S. Shan J.X. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar Four mammalian kinases are known to phosphorylate eIF2α in response to stress stimuli: i) heme-regulated inhibitor (Hri), which is stimulated by heme deficiency; ii) interferon-induced, double-stranded RNA-activated protein kinase (Pkr), which is activated by viral infection; iii) Pkr-like endoplasmic reticulum kinase (Perk), which is triggered by endoplasmic reticulum (ER) stress; and iv) general control nonrepressed 2 protein kinase (Gcn2), which is activated by amino acid deficiency.17Harding H.P. Zhang Y.H. Zeng H.Q. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2376) Google Scholar, 18Kilberg M.S. Shan J.X. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar Significantly, ISR activation plays a role in the development of cardiac dysfunction via its downstream adaptive processes, namely, autophagy19Kouroku Y. Fujita E. Tanida I. Ueno T. Isoai A. Kumagai H. Ogawa S. Kaufman R.J. Kominami E. Momoi T. ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation.Cell Death Differ. 2006; 14: 230-239Crossref PubMed Scopus (768) Google Scholar, 20Gustafsson Å B. Gottlieb R.A. Autophagy in ischemic heart disease.Circ Res. 2009; 104: 150-158Crossref PubMed Scopus (311) Google Scholar and apoptosis.21Fu H.Y. Okada K. Liao Y. Tsukamoto O. Isomura T. Asai M. Sawada T. Okuda K. Asano Y. Sanada S. Asanuma H. Asakura M. Takashima S. Komuro I. Kitakaze M. Minamino T. Ablation of C/EBP homologous protein attenuates endoplasmic reticulum-mediated apoptosis and cardiac dysfunction induced by pressure overload.Circulation. 2010; 122: 361-369Crossref PubMed Scopus (213) Google Scholar, 22Okada K. Minamino T. Tsukamoto Y. Liao Y. Tsukamoto O. Takashima S. Hirata A. Fujita M. Nagamachi Y. Nakatani T. Yutani C. Ozawa K. Ogawa S. Tomoike H. Hori M. Kitakaze M. Prolonged endoplasmic reticulum stress in hypertrophic and failing heart after aortic constriction: possible contribution of endoplasmic reticulum stress to cardiac myocyte apoptosis.Circulation. 2004; 110: 705-712Crossref PubMed Scopus (443) Google Scholar Autophagy is responsible for the recycling of long-lived and damaged organelles by lysosomal degradation.20Gustafsson Å B. Gottlieb R.A. Autophagy in ischemic heart disease.Circ Res. 2009; 104: 150-158Crossref PubMed Scopus (311) Google Scholar Although it is often considered to be a protective mechanism, emerging evidence suggests that excessive stimulation of the autophagic pathway can potentiate cardiomyocyte death.20Gustafsson Å B. Gottlieb R.A. Autophagy in ischemic heart disease.Circ Res. 2009; 104: 150-158Crossref PubMed Scopus (311) Google Scholar, 23Knaapen M.W.M. Davies M.J. De Bie M. Haven A.J. Martinet W. Kockx M.M. Apoptotic versus autophagic cell death in heart failure.Cardiovasc Res. 2001; 51: 304-312Crossref PubMed Scopus (223) Google Scholar, 24Shimomura H. Terasaki F. Hayashi T. Kitaura Y. Isomura T. Suma H. Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy.Jpn Circ J. 2001; 65: 965-968Crossref PubMed Scopus (159) Google Scholar Apoptosis also plays a significant role in mediating heart failure from cardiomyocyte death.21Fu H.Y. Okada K. Liao Y. Tsukamoto O. Isomura T. Asai M. Sawada T. Okuda K. Asano Y. Sanada S. Asanuma H. Asakura M. Takashima S. Komuro I. Kitakaze M. Minamino T. Ablation of C/EBP homologous protein attenuates endoplasmic reticulum-mediated apoptosis and cardiac dysfunction induced by pressure overload.Circulation. 2010; 122: 361-369Crossref PubMed Scopus (213) Google Scholar, 22Okada K. Minamino T. Tsukamoto Y. Liao Y. Tsukamoto O. Takashima S. Hirata A. Fujita M. Nagamachi Y. Nakatani T. Yutani C. Ozawa K. Ogawa S. Tomoike H. Hori M. Kitakaze M. Prolonged endoplasmic reticulum stress in hypertrophic and failing heart after aortic constriction: possible contribution of endoplasmic reticulum stress to cardiac myocyte apoptosis.Circulation. 2004; 110: 705-712Crossref PubMed Scopus (443) Google Scholar, 25Olivetti G. Abbi R. Quaini F. Kajstura J. Cheng W. Nitahara J.A. Quaini E. DiLoreto C. Beltrami C.A. Krajewski S. Reed J.C. Anversa P. Apoptosis in the failing human heart.N Engl J Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1482) Google Scholar Importantly, both processes are associated with FA pathogenesis3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar, 26Simon D. Seznec H. Gansmuller A. Carelle N. Weber P. Metzger D. Rustin P. Koenig M. Puccio H. Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia.J Neurosci. 2004; 24: 1987-1995Crossref PubMed Scopus (159) Google Scholar and both can be induced by ISR activation.19Kouroku Y. Fujita E. Tanida I. Ueno T. Isoai A. Kumagai H. Ogawa S. Kaufman R.J. Kominami E. Momoi T. ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation.Cell Death Differ. 2006; 14: 230-239Crossref PubMed Scopus (768) Google Scholar, 22Okada K. Minamino T. Tsukamoto Y. Liao Y. Tsukamoto O. Takashima S. Hirata A. Fujita M. Nagamachi Y. Nakatani T. Yutani C. Ozawa K. Ogawa S. Tomoike H. Hori M. Kitakaze M. Prolonged endoplasmic reticulum stress in hypertrophic and failing heart after aortic constriction: possible contribution of endoplasmic reticulum stress to cardiac myocyte apoptosis.Circulation. 2004; 110: 705-712Crossref PubMed Scopus (443) Google Scholar The ISR may therefore potentiate the cardiomyopathy of FA. We investigated the progression of FA cardiomyopathy using the muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse, which harbors a frataxin deletion in striated muscle (skeletal and heart muscle) and develops a severe cardiomyopathy that closely mimics that of FA patients.2Puccio H. Simon D. Cossée M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.Nat Genet. 2001; 27: 181-186Crossref PubMed Scopus (600) Google Scholar The histopathological, functional, and molecular changes in these mice were characterized and compared with those observed in FA patients. Previous characterization studies of this KO model were focused on the identification of the endpoint phenotype, with very limited assessment of chronological disease development and its molecular consequences.2Puccio H. Simon D. Cossée M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.Nat Genet. 2001; 27: 181-186Crossref PubMed Scopus (600) Google Scholar In the present study, for the first time in a mammalian FA model and using molecular analyses, we identified early ISR activation via eIF2α phosphorylation, which was followed chronologically by the activation of autophagic and apoptotic markers in KO mice. The findings highlight the association of the ISR with frataxin deficiency and its potential in mediating the cardiac phenotype. The MCK mouse strain was bred from animals provided by Hélène Puccio and Michel Koenig (Louis Pasteur University, Strasbourg, France), who developed the model.2Puccio H. Simon D. Cossée M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.Nat Genet. 2001; 27: 181-186Crossref PubMed Scopus (600) Google Scholar These animals were bred and handled according to a protocol approved by the University of Sydney Animal Ethics Committee. Genotyping was performed using tail DNA according to standard techniques.2Puccio H. Simon D. Cossée M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.Nat Genet. 2001; 27: 181-186Crossref PubMed Scopus (600) Google Scholar Heart autopsy specimens from two male and two female FA patients (Supplemental Table S1) were kindly provided by Dr. Arnulf Koeppen (Albany Medical College, Albany, NY). Heart specimens (pre-embedded in paraffin blocks) from four age- and sex-matched control subjects who died from causes unrelated to FA (Supplemental Table S1) were purchased from the National Disease Research Interchange (Philadelphia, PA). For histological analysis, heart specimens from FA patients were embedded in paraffin blocks. Both FA and control hearts were then sectioned and stained with Perls’ Prussian blue, Gömöri trichrome, or H&E for microscopic assessment of iron deposits, fibrosis, and general tissue architecture, respectively. Human tissues were handled according to a protocol approved by the University of Sydney Human Ethics Committee. KO and WT mouse littermates were euthanized at different time points, from 3 to 10 weeks of age. The hearts were excised, washed in PBS, blotted dry on tissue paper, and fixed in 10% formalin. For histological analysis, mouse hearts were cut and embedded in paraffin blocks and then sectioned and stained with Perls’ Prussian blue, Gömöri trichrome, or H&E. Echocardiography was performed on eight littermates (four WT and four KO mice) at 5, 6, 7, 9, and 10 weeks of age. Echocardiography of these mice was performed implementing two-dimensional and M-mode studies, as described previously.27Tsoutsman T. Kelly M. Ng D.C. Tan J.E. Tu E. Lam L. Bogoyevitch M.A. Seidman C.E. Seidman J.G. Semsarian C. Severe heart failure and early mortality in a double-mutation mouse model of familial hypertrophic cardiomyopathy.Circulation. 2008; 117: 1820-1831Crossref PubMed Scopus (60) Google Scholar Left ventricular end-diastolic (LVEDD) and end-systolic (LVESD) chamber dimensions and wall thickness were obtained from M-mode tracings based on measurements averaged from three separate cardiac cycles. Left ventricular fractional shortening (FS) was calculated as FS = (LVEDD − LVESD)/LVEDD. Heart rate was recorded throughout the study. Total RNA was isolated from hearts of eight female littermates (two 4-week-old WT, two 4-week-old KO, two 10-week-old WT, and two 10-week-old KO mice). Total RNA was isolated using Invitrogen TRIzol reagent (Life Technologies, Carlsbad, CA). First-strand cDNA and biotin-labeled cRNA synthesis was performed and hybridized to the mouse Affymetrix GeneChip 430 version 2.0 (Affymetrix, Santa Clara, CA).3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar A two-phase strategy was used to identify differentially expressed genes.3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar Genome-wide screening was performed using Affymetrix GeneChip microarrays, and then low-level analysis was performed with Affymetrix GeneChip operating software version 1.3.0, followed by the GC robust multiarray average (GCRMA) method for background correction and quantile–quantile normalization of expression. Tukey’s method for multiple pairwise comparisons was applied to acquire fold-change estimations. Tests for significance were calculated and adjusted for multiple comparisons by controlling the false discovery rate at 5%.28Benjamini Y. Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing.J R Stat Soc B Methodol. 1995; 57: 289-300Google Scholar Definitive evidence of differential expression was obtained from RT-PCR and Western blot assessment of samples used for the microarray analysis and at least three other independent samples. Functional annotation of genes was assigned via Gene Ontology29Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. the Gene Ontology ConsortiumGene Ontology: tool for the unification of biology.Nat Genet. 2000; 25: 25-29Crossref PubMed Scopus (27147) Google Scholar (http://www.geneontology.org, last accessed March 8, 2012), and classifications were obtained through the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 (http://david.abcc.ncifcrf.gov, last accessed March 8, 2012). The complete data set has been deposited with the Gene Expression Omnibus (http://ncbi.nlm.nih.gov/geo; accession number GSE31208). RNA was isolated and RT-PCR was performed by established methods using the primers listed in Table 1.3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar, 10Whitnall M. Suryo Rahmanto Y. Sutak R. Xu X. Becker E.M. Mikhael M.R. Ponka P. Richardson D.R. The MCK mouse heart model of Friedreich’s ataxia: alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation.Proc Natl Acad Sci USA. 2008; 105: 9757-9762Crossref PubMed Scopus (101) Google Scholar RT-PCR was shown to be semiquantitative by an optimization protocol demonstrating that it was in the log phase of amplification. Densitometry was performed using Quantity One software version 4.6 (Bio-Rad Laboratories, Hercules, CA) and was normalized using the relative Gapdh loading control.Table 1Primers for Amplification of Mouse mRNAPair no.Target geneGenBank accession no.Oligonucleotide sequenceProduct size (bp)ForwardReverse1AsnsNM_012055.25′-ACAACGCTATCAAGAAACGC-3′5′-CGGGCAGAAGGGTCAGT-3′9312Ces3NM_053200.25′-CTTATGGGCTATCCACTCGC-3′5′-CTCCTTTGACTCCAGGTTCTT-3′6603Cmtm8NM_027294.25′-AGGGCACAACTTCAACAGC-3′5′-GGAGACTTCAGGGTTCATTAGA-3′3774Dusp4NM_176933.45′-AGCATCATCTCGCCCAACT-3′5′-TTTCTCCTCTTTCCTTTCCCT-3′6415Efnb3NM_007911.45′-CCAGGAATACAGCCCTAACCT-3′5′-TCACCGCTCACCTTCTCGT-3′6006Gpr22NM_175191.35′-TGTTTCGACAGCAATCAACG-3′5′-TTATTAGGCATGGGATCAGC-3′8137Ift81NM_009879.25′-GGAGATGAGTTCAAGCGATAC-3′5′-GCTCCTCTTGGGTGTAA-3′2148Itgb1bp3NM_027120.25′-AAACTCATCATAGGCATTGGAG-3′5′-GCACGGTCAGGAAGTAGCG-3′3649LoxNM_010728.15′-GCCAGACAATCCAATG-3′5′-TGCGGAAATCGTAGC-3′67310Lrtm1NM_176920.35′-GTGCCACCCATCATCAAACT-3′5′-ACATACGATCCCACATACTACCC-3′82011Mthfd2NM_008638.15′-ACAATCCCGCCAGTC-3′5′-CAGCCACCCGTTTA-3′94212Myh7NM_080728.25′-TGCCAATGACGACC-3′5′-AGCCGCAGTAGGTT-3′60613Pla2g5NM_011110.35′-TGATTGGTGCTGTCAGATGC-3′5′-GAGGGTTGTAAGTCCAGAGGTT-3′20314Psat1NM_177420.15′-TTTCTTGACAAGGCGGTAGA-3′5′-TGCCCTGATGTTTAGTCGTTATTC-3′31315Rgs2NM_009061.35′-CTACAAGTGGCTGCTTCACC-3′5′-CATTGCATCGGATCTTTCAT-3′44516Rpl3lNM_025425.25′-CAAGGAAGACCCACAAAGG-3′5′-GACCGGACAGATAGCAAGAGT-3′55717Serpina3nNM_009252.25′-TGAAACCCAGGATGATAGATGA-3′5′-GTGCCAGATGTGGACAAAGTG-3′88918Tbl1xr1NM_030732.35′-GGGACCCAACTGGCAATC-3′5′-TGTGAACTAGAGCACCTGTCTGTG-3′37419Timp1NM_011593.25′-CCCCAGAAATCAACGAGACC-3′5′-ACGCCAGGGAACCAAGAA-3′24220Wisp2NM_016873.15′-TTCTGCCCTTGTCACTC-3′5′-ACCTATTACTGTCACTATCCC-3′55221Trib3NM_175093.25′-TGGGCACGTTTCCTACCG-3′5′-CCAGAACAGGGCCTGAGATT-3′40322ChopNM_007837.35′-GAGTCCCTGCCTTTCACCTT-3′5′-TTCTCCTTCATGCGTTGCTT-3′35623BnpNM_008726.45′-CGAGACAAGGGAGAACACGG-3′5′-GCCCAAACGACTGACGGA-3′39224GapdhNM_008084.25′-ATTCAACGGCACAGTCAA-3′5′-CTTCTGGGTGGCAGTGAT-3′394 Open table in a new tab Protein isolation and Western blot analysis were performed using established techniques.3Huang M.L. Becker E.M. Whitnall M. Suryo Rahmanto Y. Ponka P. Richardson D.R. Elucidation of the mechanism of mitochondrial iron loading in Friedreich’s ataxia by analysis of a mouse mutant.Proc Natl Acad Sci USA. 2009; 106: 16381-16386Crossref PubMed Scopus (173) Google Scholar The primary antibodies used were against TfR1 (13-6890; Life Technologies), Atf4 (sc-200; Santa Cruz Biotechnology, Santa Cruz, CA), Mthfd2 (ab37840; Abcam, Cambridge, UK), Asns (1732-1; Epitomics, Burlingame, CA), Chop (SC-575; Santa Cruz), p-eIF2α (Ser51; 3398; Cell Signaling Technology, Danvers, MA), eIF2α (2103; Cell Signaling Technology), Atg3 (3415; Cell Signaling Technology), LC3 (PD014; MBL International, Woburn, MA), p62 (GP62-C; Progen Biotechnik, Heidelber

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