Somatic events modify hypertrophic cardiomyopathy pathology and link hypertrophy to arrhythmia Cordula M. Wolf*†, Ivan P. G. Moskowitz†‡§¶, Scott Arno‡, Dorothy M. Branco*, Christopher Semsarian‡§ʈ, Scott A. Bernstein**, Michael Peterson‡¶, Michael Maida‡, Gregory E. Morley**, Glenn Fishman**, Charles I. Berul*, Christine E. Seidman‡§††‡‡, and J. G. Seidman‡§†† Departments of *Cardiology and ¶Pathology, Children’s Hospital, Boston, MA 02115; ‡Department of Genetics, Harvard Medical School, Boston, MA 02115; §Howard Hughes Medical Institute, Boston, MA 02115; and **Department of Cardiology, New York University School of Medicine, New York, NY 10010 Contributed by Christine E. Seidman, October 19, 2005 Sarcomere protein gene mutations cause hypertrophic cardiomy- many HCM patients who succumb to ventricular arrhythmias all opathy (HCM), a disease with distinctive histopathology and in- of these risk factors are absent (11–13). creased susceptibility to cardiac arrhythmias and risk for sudden Increased myocardial fibrosis and abnormal myocyte archi- death. Myocyte disarray (disorganized cell–cell contact) and car- tecture are associated with arrhythmia vulnerability in many diac fibrosis, the prototypic but protean features of HCM histopa- cardiovascular diseases (14–16), and these parameters are hy- thology, are presumed triggers for ventricular arrhythmias that pothesized to also increase sudden death risk in HCM (12, 17). precipitate sudden death events. To assess relationships between In support of this suggestion, histopathological studies of HCM arrhythmias and HCM pathology without confounding human hearts in some sudden death victims have demonstrated remark- variables, such as genetic heterogeneity of disease-causing muta- able amounts of fibrosis and myocyte disarray (18). However, tions, background genotypes, and lifestyles, we studied cardiac limited sample numbers, incomplete anatomic analyses, and lack electrophysiology, hypertrophy, and histopathology in mice engi- of relevant control specimens have confounded a rigorous neered to carry an HCM mutation. Both genetically outbred and investigation of the relationship among myocardial fibrosis, inbred HCM mice had variable susceptibility to arrhythmias, dif- myocyte disarray, and arrhythmias or sudden death in HCM. ferences in ventricular hypertrophy, and variable amounts and Furthermore, human pathologic studies have failed to take into account the different genetic causes of HCM or variance in distribution of histopathology. Among inbred HCM mice, neither background genotype and lifestyles that may also influence the extent nor location of myocyte disarray or cardiac fibrosis sudden death risk. correlated with ex vivo signal conduction properties or in vivo Mice engineered to carry the MHC mutation Arg403Gln electrophysiologically stimulated arrhythmias. In contrast, the (designated MHC403/ϩ) provide a relevant model for assessing amount of ventricular hypertrophy was significantly associated factors that influence disease expression and arrhythmia vulner- with increased arrhythmia susceptibility. These data demonstrate ability in HCM. Initial studies of genetically outbred MHC403/ϩ that distinct somatic events contribute to variable HCM pathology mice demonstrated variable degrees of hypertrophy and histo- and that cardiac hypertrophy, more than fibrosis or disarray, pathology (19–22). To control for the influence of background correlates with arrhythmic risk. We suggest that a shared pathway genotype on disease expression, we bred the MHC403/ϩ allele triggered by sarcomere gene mutations links cardiac hypertrophy into the inbred 129SvEv genetic background and investigated the and arrhythmias in HCM. relationship between histopathology and arrhythmia vulnerabil- ity in hypertrophic mice using in vivo and ex vivo electrophysi- fibrosis ͉ somatic modifiers ͉ disarray ͉ electrophysiology ͉ left ventrical ological studies (23, 24). wall thickness Methods 403/ϩ arcomere protein gene mutations cause hypertrophic car- Mice. MHC mice were produced by homologous recombi- 403/ϩ diomyopathy (HCM) (1, 2), an autosomal dominant disorder nation as described (19). The MHC allele was bred onto the S 129SvEv genetic background and maintained in that background with increased left ventricular wall thickness (LVWT; hypertro- Ͼ phy) and histopathologic findings of myocyte enlargement, for 10 generations. Mice were housed as prescribed by the disarray, and increased cardiac fibrosis. Clinical signs and symp- Association for Assessment and Accreditation of Laboratory toms of disease include shortness of breath, angina, palpitations, Animal Care. The Standing Committee on Animals at Harvard Medical School approved all experimental protocols used in this syncope, and, most importantly, sudden death. Cardiac arrhyth- study. mias cause both syncope and sudden death in HCM (3). A missense mutation in codon 403 (Arg403Gln) of the myosin Electrocardiography, Electrophysiology, and Echocardiography Stud- heavy chain (MHC) gene is associated with particularly severe ies. Measurements of electrocardiographic and electrophysi- HCM histopathology, early onset of symptoms, and a high risk ologic parameters and provocative electrophysiologic testing of sudden death (4, 5). However, even among HCM patients with this mutation there is variability in both the anatomic pathology and clinical course, an observation interpreted to indicate that Conflict of interest statement: No conflicts declared. different genetic backgrounds and environmental stimuli influ- Freely available online through the PNAS open access option. ence disease expression (1, 6, 7). Elucidation of the contribution Abbreviations: HCM, hypertrophic cardiomyopathy; LV, left ventricle; LVWT, left ventric- that primary responses to a sarcomere gene mutation and ular wall thickness; MHC, myosin heavy chain. disease-modifying factors have on clinically important manifes- †C.M.W. and I.P.G.M. contributed equally to this work. tations of HCM, such as arrhythmia vulnerability and sudden ʈPresent address: Molecular Cardiology Group, Centenary Institute, Sydney, NSW 2042, death, has been difficult from patient studies. At present only Australia. four parameters are recognized to convey increased risk for ††C.E.S. and J.G.S. contributed equally to this work. these serious HCM sequela: specific gene mutations including ‡‡To whom correspondence should be addressed at: Department of Genetics, Harvard MHC Arg403Gln (8), massive ventricular hypertrophy defined as Medical School, Room 256 NRB, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: LVWT Ͼ30 mm (9), family history of sudden death, and [email protected]. MEDICAL SCIENCES abnormal hemodynamic response to exercise (10). However, in © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509145102 PNAS ͉ December 13, 2005 ͉ vol. 102 ͉ no. 50 ͉ 18123–18128 Downloaded by guest on September 30, 2021 were performed as described (21, 24). Electrophysiological parameters were measured at baseline, and no drugs were administered during electrophysiologic studies. Based on pro- vocative electrophysiologic testing, mice were scored as not inducible, inducible of a minor (lasting Ͻ1 s) arrhythmia, or inducible of a major (lasting Ͼ1 s) arrhythmia. Induced prema- ture ventricular contractions or couplets were not considered as induced arrhythmia. Echocardiography was performed with a Hewlett–Packard SONOS 4500 echocardiograph by using a 12-Mz probe as described previously (20, 25, 26) on anesthetized mice with a heart rates Ͼ450 beats per minute. One experienced observer (C.S.) analyzed all studies. Reproducibility of echocardiographic data has been described (22, 26). Histopathology: Quantification of Disarray and Fibrosis. Hearts were quickly excised, washed in PBS, fixed in 4% formaldehyde, and embedded in paraffin as described (20, 25). Embedded hearts were serially sectioned every 50 ␮m from apex to base in a transverse plane. Adjacent sections were stained with hematox- ylin and eosin to assess myocyte disarray and hypertrophy and with Masson trichrome to assess collagen deposition resulting from fibrosis. Fibrosis was independently scored by two experi- enced observers (C.M.W. and I.P.G.M.) in a subset of five MHC403/ϩ and three WT littermates without knowledge of genotype. Inter-observer variability was 7% for fibrosis content. All data presented were obtained by one observer (C.M.W.). The pattern of fibrosis (diffuse, patchy, or bundled), the location of fibrosis within the myocardium (subepicardial, subendocar- dial, subepi- and subendocardial, or intramural), and the distri- Fig. 1. A comparison of histopathologic sections from WT (A and C) and bution within the left ventricle (LV) (free wall, interventricular MHC403/ϩ (B and D–F) mice stained with hematoxylin and eosin (A and B) and septum, or ventricular free wall and interventricular septum) was Masson trichrome (C–F). Normal myofibers (A) without fibrosis (C; note ab- also scored. sence of blue stained collagen) are found in WT mice. Histopathology of Total myocardial fibrosis was assessed in two ways. Fifteen to MHC403/ϩ mice shows severe myocyte disarray (B) and diffuse (D) and focal (E 20 serial sections of the LV and right ventricle were taken at and F) patterns of fibrosis. (Scale bars, 400 ␮minA and B, 200 ␮minE and F, 50-␮m intervals (Fig. 1) and stained with Masson trichrome, and and 100 ␮minC and D.) fibrosis content was assessed from digitized images (ϫ25 mag- nification) by using a custom-made