CARS2 Hypermethylation Is a Risk Factor for Stage B Heart Failure and Hospitalizations in the Project Baseline Health Study

CARS2 Hypermethylation Is a Risk Factor for Stage B Heart Failure and Hospitalizations in the Project Baseline Health Study | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search CARS 2 Hypermethylation Is a Risk Factor for Stage B Heart Failure and Hospitalizations in the Project Baseline Health Study View ORCID Profile Jessica A. Regan , Jordan Franklin , Kalyani Kottilil , Nicholas Cauwenberghs , Kenneth W. Mahaffey , Pamela S. Douglas , Fatima Rodriguez , Francois Haddad , Adrian F. Hernandez , Svati H. Shah , Lydia Coulter Kwee , the Project Baseline Health Study Group doi: https://doi.org/10.1101/2024.12.20.24319449 Jessica A. Regan 1 Duke Molecular Physiology Institute, Duke University School of Medicine , Durham, NC, USA 2 Department of Medicine, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jessica A. Regan Jordan Franklin 2 Department of Medicine, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kalyani Kottilil 1 Duke Molecular Physiology Institute, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicholas Cauwenberghs 3 Research Unit Hypertension and Cardiovascular Epidemiology, KU Leuven Department of Cardiovascular Sciences, University of Leuven , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kenneth W. Mahaffey 4 Stanford Center for Clinical Research, Department of Medicine, Stanford University School of Medicine , Stanford, CA, USA 5 Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine , Stanford, CA, USA 6 Stanford Cardiovascular Institute, Stanford University School of Medicine , Stanford, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pamela S. Douglas 2 Department of Medicine, Duke University School of Medicine , Durham, NC, USA 7 Duke Clinical Research Institute, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fatima Rodriguez 5 Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine , Stanford, CA, USA 6 Stanford Cardiovascular Institute, Stanford University School of Medicine , Stanford, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Francois Haddad 5 Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine , Stanford, CA, USA 6 Stanford Cardiovascular Institute, Stanford University School of Medicine , Stanford, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adrian F. Hernandez 2 Department of Medicine, Duke University School of Medicine , Durham, NC, USA 7 Duke Clinical Research Institute, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Svati H. Shah 1 Duke Molecular Physiology Institute, Duke University School of Medicine , Durham, NC, USA 2 Department of Medicine, Duke University School of Medicine , Durham, NC, USA 7 Duke Clinical Research Institute, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lydia Coulter Kwee 1 Duke Molecular Physiology Institute, Duke University School of Medicine , Durham, NC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: lydia.kwee{at}duke.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract The heart is a metabolically active organ with high energy demands that depend on mitochondrial oxidative phosphorylation, however as the heart begins to fail there is metabolic inflexibility and changes in mitochondrial function. Epigenetic changes such as DNA methylation can modify the transcription of genes essential for normal mitochondrial function, however this has not been studied in Stage B or pre-heart failure (HF). In 752 participants from the Project Baseline Health Study, we tested differences in DNA methylation in mitochondrial-related genes for association with Stage B HF compared to Stage A (at-risk for HF). We then validated these findings in 918 participants from the CATHGEN study for risk of incident HF hospitalization. One region on chromosome 13 overlapping with the cysteinyl-tRNA synthetase 2 ( CARS2 ) gene, containing five cytosine-phosphate-guanine (CpG) probes, was significantly associated with a small mean regional increase in DNA methylation in Stage B HF (0.63%, Stouffer p=0.005). In CATHGEN, one CARS2 probe, cg08289839, was significantly associated with time-to-incident HF hospitalization (adjusted HR 1.74 [95% CI 1.18-2.56], FDR p=0.02). In this study of epigenetic changes of mitochondrial genes, these results indicate that CARS2 DNAm may play a role in HF pathogenesis and should be studied in future HF research. A 2021 consensus statement defined four stages of heart failure (HF): at-risk for HF (Stage A), pre-HF (Stage B), HF (Stage C) and advanced HF (Stage D), however, there is limited understanding of mechanisms by which individuals transition from Stage A to Stage B and to then symptomatic HF 1 . The heart’s high energy demands are dependent on mitochondrial oxidative phosphorylation of fatty acids for the majority of ATP generation, while HF is characterized by metabolic inflexibility and increased reliance on alternative fuel substrates. Epigenetic changes such as DNA methylation (DNAm) can modify transcription of genes central to mitochondrial function, thereby impacting downstream cellular and metabolic function. DNAm changes have been identified in myocardial tissues of subjects with end-stage HF, but whether mitochondrial-related DNAm may signal early molecular changes in Stage B HF has not been studied 2 . Here, we investigate the role of DNAm in mitochondrial-related genes in early-stage HF and in risk of incident HF hospitalization. The Project Baseline Health Study (PBHS) recruited 2502 participants, of which 752 had either Stage A or Stage B HF along with peripheral blood DNAm profiling from the Illumina EPIC array 3 . Mean age was 54.4 ±15.8 years, 46.9% of participants were male and 39.9% self-identified as non-white race. 509 participants were at-risk for HF (Stage A) defined as history of hypertension, prevalent cardiovascular disease, diabetes, obesity, chronic kidney disease, prior exposure to cardiotoxins or family history of cardiomyopathy without structural heart disease. 243 had Stage B HF, defined as subjects without current or prior signs or symptoms of HF, but with at least one of the stage A HF characteristics, along with evidence of structural heart disease including left ventricular mass, hypertrophy, chamber enlargement, valvular heart disease or systolic or diastolic cardiac dysfunction on echocardiography 4 . We considered 25,774 cytosine-phosphate-guanine (CpG) probes in 1,192 mitochondrial-related genes identified from MitoCarta 3.0, Harmonizome 3.0, the PANTHER database and genes previously described to have DNAm changes or variable expression in HF 2 . Linear regression was used to test the association of Stage B (vs. stage A) HF with DNAm using the limma package for individual probes and DMRcate for region-based analysis (R v4.4.2). Models were adjusted for age, self-reported race, sex, body mass index, systolic blood pressure, creatinine, hypertension, diabetes, coronary disease and cell proportions derived from a complete blood count. Significance was considered at a false discovery rate (FDR) p<0.05 for both individual probe and region-based analyses. Given that hypermethylation is a hallmark of cancer, sensitivity analyses were also performed excluding the 104 participants with a history of cancer. We then tested the association of significant findings with echocardiographic criteria defining Stage B HF. We validated significant findings in the CATHGEN cohort. Although CATHGEN does not have stage B HF phenotyping available, we tested for association between DNAm and time-to-incident HF hospitalization using Cox proportional hazard models in 918 participants without prevalent HF, adjusted for the above covariates. Informed consent was obtained from all participants enrolled in PBHS in accordance with the Helsinki declaration. The study was approved by a central Institutional Review Board (the WCG IRB; approval tracking number 20170163, work order number 1-1506365-1) and IRBs at each of the participating institutions (Stanford University, Duke University, and the California Health and Longevity Institute). Baseline characteristics of the participants included in these analyses are shown in Table 1 . No individual mitochondrial-related CpGs were associated with Stage B HF in PBHS after FDR adjustment ( Supplemental Table 1 ). In region-based analysis, a 706bp region on chromosome 13 overlapping with the cysteinyl-tRNA synthetase 2 ( CARS2 ) gene, which contains five CpGs, was significantly associated with Stage B HF (Stouffer p=0.005), although the mean regional increase in DNAm was small (0.63%) ( Supplemental Table 2 ). In sensitivity analyses, excluding participants with history of cancer strengthened the association between the CARS2 region and Stage B HF (p=2.1×10 −7 ) ( Supplemental Table 3, Supplemental Table 4 ). There were no significant associations between the five CARS2 CpG probes and the individual structural and functional components of Stage B HF ( Supplemental Table 5, Supplemental Table 6 ). In CATHGEN, baseline characteristics are shown in Supplemental Table 7 . 254/918 CATHGEN participants had an incident HF hospitalization event. One of the probes in the CARS2 region, cg08289839, was significantly associated with time-to-incident HF hospitalization: adjusted HR 1.74 (95% CI 1.18-2.56), FDR p=0.02 ( Supplemental Table 8 ). View this table: View inline View popup Download powerpoint Table 1. Baseline Participant Characteristics Here, in a study of epigenetic changes of mitochondrial genes, we have shown that CARS2 DNAm may play a role in HF pathogenesis, as suggested by its association with Stage B HF and HF incidence ( Figure 1 ). CARS2 encodes an aminoacyl-tRNA synthetase that is transcribed by the nuclear genome but exclusively targets mitochondria, where it is imported and enzymatically catalyzes the ligation of cysteine to tRNA molecules. CARS2 is ubiquitously expressed in the heart and many other tissues, and variants in CARS2 have been implicated in mitochondrial epileptic encephalopathy, whereas variants in other aminoacyl-tRNA synthetases and nuclear encoded proteins impacting mitochondrial translation have been associated with cardiomyopathy 5 . Here we have used peripheral blood DNAm as a circulating surrogate for myocardial changes in Stage B HF, as unfortunately, access to myocardial tissue in early HF is likely not feasible. Further studies should evaluate the role of epigenetic changes in genes essential to mitochondrial and metabolic function across the spectrum of stages of HF in large scale human cohorts. Download figure Open in new tab Figure 1. CARS2 Hypermethylation in Stage B and Incident HF hospitalization. A. Conceptual model exploring the epigenetic changes that may prompt the transition from Stage A HF characterized by cardiovascular and metabolic risk factors to Stage B HF. Mitochondrial promoter hypermethylation of CARS2 which serves an essential tRNA-synthetase role for mitochondrial protein translation could lead to subsequent metabolic and mitochondrial dysfunction and ultimately subclinical cardiac dysfunction and progressive HF. B. Kaplan-Meier curve by tertile of cg08289839 methylation in CARS2 in CATHGEN cohort for time-to-HF hospitalization. HF, heart failure; CARS2 , cysteinyl-tRNA synthetase 2. Data Availability The deidentified PBHS data corresponding to this study are available upon request for the purpose of examining its reproducibility. Requests are subject to approval by PBHS governance. Funding The Project Baseline Health Study and this analysis were funded by Verily Life Sciences, South San Francisco, California. J.A.R is supported by 1K38HL175026. K.K. is supported by 1F31HL175914-01. Ethics Statement The study was approved by the Duke University and Stanford University Institutional Review Boards. Informed consent was obtained from all participants enrolled in the Project Baseline Health Study in accordance with the Declaration of Helsinki. ClinicalTrials.gov Identifier NCT03154346, https://clinicaltrials.gov/ct2/show/NCT03154346 Disclosure of Conflicts of Interest All authors acknowledge institutional research grants from Verily Life Sciences. KM reports grants from Verily, Afferent, the American Heart Association (AHA), Cardiva Medical Inc, Gilead, Luitpold, Medtronic, Merck, Eidos, Ferring, Apple Inc, Sanifit, and St. Jude; grants and personal fees from Amgen, AstraZeneca, Bayer, CSL Behring, Johnson & Johnson, Novartis, and Sanofi; and personal fees from Anthos, Applied Therapeutics, Elsevier, Inova, Intermountain Health, Medscape, Mount Sinai, Mundi Pharma, Myokardia, Novo Nordisk, Otsuka, Portola, SmartMedics, and Theravance outside the submitted work. FR reports grants from the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (R01HL168188; R01HL167974; R01HL169345), American Heart Association/Harold Amos Medical Faculty Development program, and Doris Duke Foundation (Grant #2022051); equity from Carta Healthcare and HealthPals; and consulting fees from HealthPals, Novartis, NovoNordisk, Esperion Therapeutics, Movano Health, Kento Health, Inclusive Health, Edwards, Arrowhead Pharmaceuticals, HeartFlow, iRhythm, Amgen, and Cleerly Health outside the submitted work. AH reports grants and personal fees from AstraZeneca, Amgen, Bayer, Merck, and Novartis; and personal fees from Boston Scientific outside the submitted work. The other authors have no conflicts of interest to disclose. Data Sharing Statement The deidentified PBHS data corresponding to this study are available upon request for the purpose of examining its reproducibility. Requests are subject to approval by PBHS governance. ACKNOWLEDGMENTS We would like to thank the Project Baseline Health Study and CATHGEN participants whose data contributed to this work. Footnotes Included demographic table for CATHGEN participants (Supplemental Table 7) References 1. ↵ Bozkurt B , Coats AJS , Tsutsui H , et al. Universal definition and classification of heart failure: a report of the Heart Failure Society of America, Heart Failure Association of the European Society of Cardiology, Japanese Heart Failure Society and Writing Committee of the Universal Definition of Heart Failure: Endorsed by the Canadian Heart Failure Society, Heart Failure Association of India, Cardiac Society of Australia and New Zealand, and Chinese Heart Failure Association . Eur J Heart Fail . Mar 2021 ; 23 ( 3 ): 352 – 380 . doi: 10.1002/ejhf.2115 OpenUrl CrossRef PubMed 2. ↵ Pepin ME , Drakos S , Ha CM , et al. DNA methylation reprograms cardiac metabolic gene expression in end-stage human heart failure . Am J Physiol Heart Circ Physiol . Oct 1 2019 ; 317 ( 4 ): H674 – H684 . doi: 10.1152/ajpheart.00016.2019 OpenUrl CrossRef PubMed 3. ↵ Arges K , Assimes T , Bajaj V , et al. The Project Baseline Health Study: a step towards a broader mission to map human health . NPJ Digit Med . 2020 ; 3 : 84 . doi: 10.1038/s41746-020-0290-y OpenUrl CrossRef PubMed 4. ↵ Cauwenberghs N , Haddad F , Daubert MA , et al. Clinical and Echocardiographic Diversity Associated With Physical Fitness in the Project Baseline Health Study: Implications for Heart Failure Staging . J Card Fail . Nov 2023 ; 29 ( 11 ): 1477 – 1489 . doi: 10.1016/j.cardfail.2023.04.008 OpenUrl CrossRef PubMed 5. ↵ Coughlin CR , 2nd, Scharer GH , Friederich MW , et al. Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder . J Med Genet . Aug 2015 ; 52 ( 8 ): 532 – 40 . doi: 10.1136/jmedgenet-2015-103049 OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted May 03, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following CARS2 Hypermethylation Is a Risk Factor for Stage B Heart Failure and Hospitalizations in the Project Baseline Health Study Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share CARS 2 Hypermethylation Is a Risk Factor for Stage B Heart Failure and Hospitalizations in the Project Baseline Health Study Jessica A. Regan , Jordan Franklin , Kalyani Kottilil , Nicholas Cauwenberghs , Kenneth W. Mahaffey , Pamela S. Douglas , Fatima Rodriguez , Francois Haddad , Adrian F. Hernandez , Svati H. Shah , Lydia Coulter Kwee , the Project Baseline Health Study Group medRxiv 2024.12.20.24319449; doi: https://doi.org/10.1101/2024.12.20.24319449 Share This Article: Copy Citation Tools CARS 2 Hypermethylation Is a Risk Factor for Stage B Heart Failure and Hospitalizations in the Project Baseline Health Study Jessica A. Regan , Jordan Franklin , Kalyani Kottilil , Nicholas Cauwenberghs , Kenneth W. Mahaffey , Pamela S. Douglas , Fatima Rodriguez , Francois Haddad , Adrian F. Hernandez , Svati H. Shah , Lydia Coulter Kwee , the Project Baseline Health Study Group medRxiv 2024.12.20.24319449; doi: https://doi.org/10.1101/2024.12.20.24319449 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cardiovascular Medicine Subject Areas All Articles Addiction Medicine (573) Allergy and Immunology (865) Anesthesia (302) Cardiovascular Medicine (4453) Dentistry and Oral Medicine (444) Dermatology (383) Emergency Medicine (609) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1515) Epidemiology (15242) Forensic Medicine (30) Gastroenterology (1131) Genetic and Genomic Medicine (6615) Geriatric Medicine (669) Health Economics (1001) Health Informatics (4552) Health Policy (1372) Health Systems and Quality Improvement (1614) Hematology (543) HIV/AIDS (1270) Infectious Diseases (except HIV/AIDS) (15929) Intensive Care and Critical Care Medicine (1106) Medical Education (624) Medical Ethics (147) Nephrology (670) Neurology (6625) Nursing (346) Nutrition (999) Obstetrics and Gynecology (1148) Occupational and Environmental Health (957) Oncology (3344) Ophthalmology (979) Orthopedics (369) Otolaryngology (421) Pain Medicine (436) Palliative Medicine (130) Pathology (665) Pediatrics (1696) Pharmacology and Therapeutics (693) Primary Care Research (714) Psychiatry and Clinical Psychology (5461) Public and Global Health (9252) Radiology and Imaging (2207) Rehabilitation Medicine and Physical Therapy (1371) Respiratory Medicine (1197) Rheumatology (597) Sexual and Reproductive Health (715) Sports Medicine (530) Surgery (714) Toxicology (99) Transplantation (289) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a02de7b2d99e4807',t:'MTc3OTk3ODA3OQ=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();