Somatic Events Modify Hypertrophic Cardiomyopathy Pathology and Link Hypertrophy to Arrhythmia

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 vulnerability 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 provocative 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 premature 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 hematoxylin 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 experienced 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, subendocardial, 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 magnification) by using a custom-made program generated on Heart Isolation and High-Resolution Optical Mapping. Mice were MATLAB 6.1. Three-dimensional heart images were generated by treated with heparin (500 units͞kg) to prevent intracardiac blood computerized assembly of 15 sections (oriented from the base to coagulation, anesthetized with CO2, and euthanized by cervical the apex) with fibrosis (blue) and myocardial tissue (red) shown. dislocation; hearts were surgically removed by a thoracotomy. The maximum fibrosis observed on any section was calculated While fully immersed in oxygenated (95% O2͞5% CO2) Tyrode’s as the area occupied by blue-stained connective tissue divided solution (114 mM NaCl, 25 mM NaHCO3, 10 mM dextrose, 4.6 by the areas occupied by connective tissue plus cardiac myo- mM KCl, 1.5 mM CaCl , 1.2 mM Na PO , 0.7 mM MgCl ), the ϫ 2 2 4 2 cytes 100. Total fibrosis within the heart was calculated from aorta was cannulated and Langendorff-perfused at a constant the sum of fibrosis on all sections divided by the sum of the areas pressure at 37°C. Hearts were subsequently stained with the ϫ occupied by connective tissue plus cardiac myocytes 100. voltage-sensitive dye (Di-4-ANEPPS), and high-resolution op- Intramural vessels, perivascular collagen, endocardium, and tical mapping studies were performed as described in ref. 28. trabeculae were excluded. Recordings were made in the bin mode, which allows for an array Myofiber disarray was identified by using criteria described by of 64 ϫ 64 pixels to be acquired at 947 frames per s with 12-bit Maron et al. (27). Myocardial areas were considered disorga- resolution in the absence of any pharmacological or mechanical nized when adjacent longitudinally cut muscle cells were aligned motion-reduction techniques. Epicardial conduction velocity perpendicularly or obliquely to each other, forming a ‘‘pinwheel’’ measurements were obtained by pacing the right ventricle and configuration (type I-A disorganization). Semiquantitative as- LV directly with a silver monopolar electrode at a basic cycle sessment of myocyte disarray (scored as present or absent) was length of 100 ms with 4-ms stimuli at two times diastolic made by one observer (C.M.W.), who examined three fields threshold. Activity during pacing was recorded for 2 s. Optical within the left ventricular free wall and three fields within the movies were signal-averaged, and conduction velocities were interventricular septum on each of 10–12 hematoxylin and calculated as described in refs. 28 and 29. eosin-stained sections (ϫ400 magnification). The percentage of myocyte disarray was calculated by dividing the number of fields Results and Discussion with disarray by the total number of fields examined (Ϸ60 per Four young (8–12 weeks old) and 16 adult (Ͼ30 weeks old) male heart). To assess for inter-observer variability, two independent MHC403/ϩ mice and 5 adult age-matched WT inbred 129SvEv observers (C.M.W. and I.P.G.M.) scored disarray and ranked mice that were housed in a single viral antibody-free facility were five mutant hearts. The rank order of myocyte disarray in these studied. With increasing age MHC403/ϩ mice developed cardiac five mutant hearts was the same by each observer, and the hypertrophy, and by age 20–30 weeks hypertrophy was uniformly percentage of myocyte disarray differed by 13%. present (Table 1; maximum LVWT). The range of maximum

18124 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509145102 Wolf et al. Downloaded by guest on September 30, 2021 Table 1. Cardiac morphology, ﬁbrosis, and disarray in WT and MHC403/؉ mice MHC403/ϩ arrhythmia

WT MHC403/ϩ P* Inducible Not Inducible P**

No. of mice 5 16 11 5 Age, weeks 71 Ϯ 23 57 Ϯ 21 NS 56 Ϯ 15 59 Ϯ 32 NS Total ﬁbrosis 0.17 Ϯ 0.24 0.98 Ϯ 0.75 0.001 1.10 Ϯ 0.86 0.72 Ϯ 0.39 NS Max ﬁbrosis 0.66 Ϯ 0.68 2.58 Ϯ 1.85 0.002 2.85 Ϯ 2.0 1.99 Ϯ 1.6 NS Myocyte disarray 8.07 Ϯ 4.28 40.07 Ϯ 11.02 Ͻ0.0001 38.89 Ϯ 10 41.01 Ϯ 13 NS Maximum LVWT, mm 0.85 Ϯ 0.05 1.17 Ϯ 0.13 Ͻ0.001 1.29 Ϯ 0.09 1.1 Ϯ 0.09 0.008 LVEDD, mm 3.29 Ϯ 0.3 2.70 Ϯ 0.33 0.002 2.37 Ϯ 0.29 2.87 Ϯ 0.20 0.005 FS, % 51.6 Ϯ 6.7 64.25 Ϯ 10.88 0.005 73.8 Ϯ 11 59.5 Ϯ 8 0.024 CV min, m/s 0.43 Ϯ 0.10 0.41 Ϯ 0.10 NS 0.34 Ϯ 0.13 0.46 Ϯ 0.11 NS CV max, m/s 0.69 Ϯ 0.14 0.76 Ϯ 0.13 NS 0.75 Ϯ 0.10 0.85 Ϯ 0.13 NS Anisotropic ratio 1.63 Ϯ 0.22 1.99 Ϯ 0.56 NS 2.37 Ϯ 0.61 1.98 Ϯ 0.68 NS

Values reflect means Ϯ standard deviation. P*, t test comparison of WT vs. MHC403/ϩ mice; P**, t test comparison of MHC403/ϩ mice with inducible arrhythmias versus MHC403/ϩ mice without inducible arrhythmias; NS, not significant; Max fibrosis, maximum fibrosis on any heart section; Myocyte disarray, total disarrayed area͞total LV area; LVEDD, left ventricular end diastolic diameter; FS, fractional shortening; CV min, minimal signal conduction velocity; CV max, maximal signal conduction velocity.

LVWT was 1.00–1.40 mm for inbred adult MHC403/ϩ mice and MHC403/ϩ adult hearts (e.g., Fig. 2B) had very little fibrosis, 0.80–0.92 mm for inbred WT mice. The end-diastolic LV comparable to WT hearts, whereas 14 mutant mice (e.g., Fig. 2 dimensions were significantly smaller, and fractional shortening C and D) had significantly more fibrosis than WT mice. was increased in adult MHC403/ϩ mice compared with age- The location and pattern of the fibrotic regions varied con- matched WT mice, findings that are consistent with increased siderably among MHC403/ϩ hearts. Although the total amounts cardiac contractility, as has been previously reported in outbred of fibrosis in two MHC403/ϩ hearts was insufficient (Ͻ0.1%) to ϩ MHC403/ mice (refs. 19, 20, and 30 and Table 1). Cardiac assess distribution, among other mutant mice fibrosis was found ϩ histopathology (Fig. 1) of inbred MHC403/ mice was similar to in subepicardial, subendocardial, and intramural portions of the that described previously for mice carrying the Arg403Gln left ventricular free wall as well as the interventricular septal missense mutation on an outbred background (19, 22). wall. Fibrosis was diffuse in four MHC403/ϩ hearts (Fig. 1D), Mice underwent standard electrophysiologic testing and pro- focal in six mutant hearts (Fig. 1 E and F), and both diffuse and grammed electrical pacing to identify inducible arrhythmias (21, 24, focal in four mutant hearts. Focal fibrosis was identified in 25). Ventricular tachyarrhythmia, defined by more than five ven- variable locations, including an extended area from the subval- ͞ ͞ tricular beats, was induced in 25% (1 4) of young and 69% (11 16) vular region to the apex (Fig. 2C) and a confined area in the 403/ϩ of adult inbred MHC mice by rapid ventricular pacing at a cycle subvalvular region (Fig. 2D). Ն length of 50 ms, or by double and triple extra-stimuli with a Neither the total myocardial fibrosis nor the maximal fibrosis Ն coupling interval of 50 ms. Induced ventricular tachyarrhythmias per section in MHC403/ϩ hearts correlated with inducible ar- lasted 2.1 Ϯ 3.3 s (range ϭ 0.2–22 s) and were polymorphic in 9͞16 ͞ rhythmias produced by in vivo electrophysiologic studies (Table mice and monomorphic degrading to polymorphic in 2 16 mice. No 1). Furthermore, the location and pattern of fibrosis in ventricular fibrillation or atrial arrhythmias were induced. Consis- MHC403/ϩ LVs were the same in mice with and without inducible tent with previous studies (21, 22, 24, 25), no arrhythmias were arrhythmias; diffuse, focal, or composite fibrosis patterns were induced in WT mice. seen equally in these study groups (Fig. 2; P ϭ not statistically Left ventricular conduction velocities were assessed in 16 403/ϩ ϩ significant; data not shown). For example, one MHC mouse independent adult MHC403/ and 5 WT mice by high-resolution with long runs of inducible ventricular tachycardia had almost no optical mapping. Conduction velocities in both transverse (min- fibrosis (Fig. 2B). Two MHC403/ϩ mice with inducible ventricular imal signal conduction velocity) and longitudinal (maximal signal conduction velocity) directions and the anisotropic ratio tachycardia had different amounts and locations of fibrosis (Fig. (maximal signal conduction velocity͞minimal signal conduction 2 C and D). Regression analyses of the total myocardial fibrosis velocity), a measurement of conduction pattern, were similar in or maximum fibrosis per section did not indicate a relationship 2 ϭ MHC403/ϩ compared with WT hearts, when paced at 100 ms to the minimal signal conduction velocity (r 0.11), the 2 ϭ cycle lengths to approximate physiologic heart rates of 600 beats maximal signal conduction velocity (r 0.15), or the anisotropic 2 ϭ per minute (Table 1). Among mutant mice, neither conduction ratio (r 0.01). velocities nor anisotropic ratios correlated with susceptibility Myocyte disarray occurred significantly more in microscopic 403/ϩ to inducible arrhythmias from programmed electrical pacing fields from young (29%) and adult (40%) mutant MHC than (Table 1). from WT (8%) hearts (Table 1 and Fig. 1 A and B). Disorganized A comparison of the histopathology throughout the entire myocyte architecture in mutant specimens usually clustered in myocardium demonstrated greater fibrosis in inbred MHC403/ϩ the LV free wall or interventricular septum. No association was mice than in WT mice (Table 1 and Fig. 2). Young MHC403/ϩ observed between the extent or the location of myocyte disarray hearts displayed only 2-fold more maximum and total fibrosis in mutant mice and inducible arrhythmias (Table 1). Myocyte than the WT hearts (data not shown), but with increasing age the disarray also did not correlate with transverse and longitudinal fibrosis in mutant mice increased. The maximum fibrosis per conduction velocities or the anisotropic ratio (data not shown). ϩ section was 4-fold greater and total myocardial fibrosis content The MHC403/ mice with inducible arrhythmias had signifi- was 5-fold greater in adult MHC403/ϩ than in WT hearts. Despite cantly greater LVWT and significantly greater hypercontractility the identical genetic background of MHC403/ϩ mutant mice, the than did MHC403/ϩ mice without inducible arrhythmias (Table total amount of fibrosis within each heart varied broadly, ranging 1). Regression analysis showed no correlation between LVWT from 0.04% to 2.52% of heart area (Table 1 and Fig. 2 B–D). Two and the amount of fibrosis or myocyte disarray (data not shown), MEDICAL SCIENCES

Wolf et al. PNAS ͉ December 13, 2005 ͉ vol. 102 ͉ no. 50 ͉ 18125 Downloaded by guest on September 30, 2021 Fig. 2. Assessment of fibrosis and arrhythmias in MHC403/ϩ hearts. (A) Schematic of serial reconstruction of hearts from digitalized images of 10–15 sections stained with Masson trichrome. (B–D) Quantity and distribution of cardiac fibrosis (red) in three MHC403/ϩ hearts identified by 3D reconstruction and corresponding lead I surface ECG recorded during right ventricle programmed pacing (cycle length ϭ 200 ms in B and 50 ms in C and D). (B) The heart of a 91-week-old MHC403/ϩ mouse with 0.04% cardiac fibrosis and reproducible long runs of ventricular tachycardia (duration ϭ 18 s). (C) The heart of a 63-week-old MHC403/ϩ mouse contains 1.45% fibrosis, distributed in a bundled pattern, located in the subepicardial of the LV free wall and short runs of ventricular tachycardia (Ͻ1 s). (D) The heart of a 54-week-old MHC403/ϩ mouse with 1.13% fibrosis, distributed in a bundled and patchy pattern, located in the subepicardial LV free wall and subendocardial interventricular septum and reproducible ventricular tachycardia (5.6 s).

indicating that myocyte hypertrophy primarily accounted for ability to secondary insults that increased focal myocyte death increased ventricular wall dimensions. and fibrosis (Fig. 3). Surprisingly, these second hit events are Because genetically identical MHC403/ϩ mice that were housed neither genetically programmed nor clearly related to local in a uniform environment exhibited variable amounts of hyper- hemodynamic parameters or mechanical strain, given the vari- trophy, fibrosis, myocyte disarray, and susceptibility to induced able anatomic extent and location of focal fibrosis found in arrhythmias, we conclude that the broad range of responses to inbred mutant mice. a sarcomere protein gene reflects in part stochastic, somatic Local ischemia is one potential stimulant for focal myocyte events. Variation in the amount and distribution of cardiac death, and, although the hemodynamics of coronary blood flow fibrosis was illustrative of this hypothesis. Focal cardiac fibrosis would predict greater susceptibility of myocytes located within occurs when myocytes die and are replaced by fibroblasts, the subendocardial layer of the ventricle, this was not observed. whereas diffuse fibrosis reflects increased collagen expression by Other stimuli that might account for stochastic development of interstitial fibroblasts that are dispersed between myocytes (31). focal areas of fibrosis in HCM hearts might include local Whereas the diffuse pattern of fibrosis observed in 8͞16 adult fluctuations in hormones and͞or ions that influence myocyte MHC403/ϩ hearts was consistent with an enhanced overall vul- signal transduction; changes in these signals could trigger cell nerability of mutant myocytes to premature death, 10 mutant death and produce a nidus of biochemical factors that promote adult MHC403/ϩ hearts displayed prominent foci of fibrosis (4 of additional death of surrounding myocytes. these also had diffuse fibrosis), a finding that suggests an Myocyte disarray in inbred MHC403/ϩ mice also varied in additional trigger for myocyte death. Given the underlying amount and distribution and did not correlate with either the genetic cause of HCM, a ‘‘two-hit’’ process aptly models these total amount of fibrosis (r2 ϭ 0.12) or the maximum fibrosis data, in which a primary sarcomere mutation enhances vulner- present in sections (r2 ϭ 0.18; data not shown). Although the

18126 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509145102 Wolf et al. Downloaded by guest on September 30, 2021 between HCM histopathology and arrhythmogenicity than was previously appreciated. The variable risk for sudden death associated with cardiac troponin T gene mutations is illustrative of this complexity; both human HCM patients (11, 17) and mouse models (34, 35) with one of these mutations exhibit little myocardial fibrosis but increased arrhythmogenicity. The extent of left ventricular hypertrophy and increased contractility in MHC403/ϩ mice was informative for susceptibility to induced arrhythmias. Notably, an association between hypertrophy and arrhythmia was identified despite only mild to moderate increases in LVWT in MHC403/ϩ mice. The mechanism by which myocyte enlargement increases arrhythmic risk may relate to changes in intrinsic automaticity, in that other experimental models of hypertrophied myocytes exhibit reexpression of pacemaker cur- Fig. 3. A two-hit model to explain variability in HCM pathology. Incorpo- rents (36) and enhanced prolongation of the action potential by a ration of mutant proteins produces sarcomere defects that trigger abnormal down-regulation of the main outward current Ito (37, 38). Calcium Ca2ϩ signaling that stimulates myocyte hypertrophy and myocyte disarray and signaling may also play a role in arrhythmia vulnerability, both increases cardiac fibrosis (19, 20). The severity of HCM pathology is modified because calcium handling is directly perturbed by sarcomere mu- by ‘‘second hits’’ or somatic events (potentially exercise, diet, and local he- tations (26) and because calcium cycling increases with enhanced modynamic factors) that exacerbate hypertrophy, disarray, and fibrosis. An contractility (39, 40). However, given that high-resolution optical association between even modest increases in LVWT and arrhythmia vulnerability in MHC403/ϩ mice suggests a shared mechanistic pathway. mapping defined comparable conduction velocities and anisotropic ratios in the hearts of MHC403/ϩ and WT mice, we presume that any electrophysiologic abnormalities in mutant hearts are focal and causes of local disorganized myocyte architecture are unknown, produce small reentrant circuits or areas of increased susceptibility these data indicate that myocardial disarray and fibrosis, two to triggered automaticity in HCM mice that, if propagated through- characteristic pathologic findings of HCM, may arise by inde- out the myocardium, could cause arrhythmic sudden death. pendent mechanisms. We conclude that the three cardinal manifestations of HCM Studies of human HCM have hypothesized that cardiac fibro- histopathology (cardiac hypertrophy, myocyte fibrosis, and dis- sis and͞or myocyte disarray increase susceptibility to ventricular array) reflect independent pathologic processes within myocytes arrhythmias and increase risk for sudden death (9, 10, 32, 33). carrying a sarcomere gene mutation (Fig. 3). The severity of two However, we identified no correlation between the amount components of this triad, fibrosis and disarray, is substantially and͞or pattern of fibrosis or the quantity of myocyte disarray in influenced by unknown somatic factors. Myocyte hypertrophy, ϩ MHC403/ mice and the propensity for arrhythmia as assessed by although genetically programmed, is also influenced by other two independent techniques, ex vivo high-resolution optical factors that also increased susceptibility to ventricular arrhyth- mapping and in vivo electrophysiologic study (Fig. 2). Almost mias. Although direct extrapolation from genetically inbred ϩ one-third of MHC403/ mice were resistant to cardiac arrhyth- mouse models of HCM to the human condition is difficult, these mias and had anisotropic ratios comparable to WT mice, even observations imply that therapeutic approaches that mitigate when displaying a significant amount of fibrosis (Table 1 and even moderate myocyte growth in HCM may also attenuate the data not shown). Sites of fibrosis did not disrupt normal elec- risk for cardiac arrhythmia. Identification of somatic factors that trical currents, initiate novel currents, or trigger ventricular influence cardiac responses to a sarcomere protein gene muta- tachycardia. Our data do not exclude the possibility of very small tion may provide other strategies to limit histopathology and amounts of fibrosis within a specific myocardial locus that might attenuate important adverse clinical events in HCM. initiate narrow reentrant pathways; highly selective differences in conduction velocity would not be detected by the overall This work was supported by grants to J.G.S., C.E.S., and I.P.G.M. from conduction velocities assessed here. the Howard Hughes Medical Institute and the National Institutes of Although many issues limit direct extrapolation of these data to Health. C.M.W. was supported in part by a grant from the Boston human disease, our findings suggest a more complex relationship Children’s Heart Foundation.

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