RESEARCH | REPORTS HEART DISEASE significant difference between wild-type (WT) and TTNtv iPS-CMs (fig. S2D). Because three- dimensional (3D) CMTs (Fig. 1B) better reca- pitulate native cardiomyocyte architecture and Titin mutations in iPS cells define mechanics, improving sarcomere alignment, ex- pression of contractile proteins, and iPS-CMs sarcomere insufficiency as a cause of maturity (6, 11, 12), we assessed the contractile function of iPS-CMTs containing WT or mutant dilated cardiomyopathy iPS-CMs. We observed minor variation in con- tractile function between biological replicates of CMTs or between CMTs made from independent John T. Hinson,1*† Anant Chopra,2,3† Navid Nafissi,4 William J. Polacheck,2,3 clones from the same patient (fig. S2, E and F). Craig C. Benson,5 Sandra Swist,6 Joshua Gorham,4 Luhan Yang,3,4 Sebastian Schafer,7 4 1,4,8 4 7,9 However, CMTs expressing either A-band TTNtv Calvin C. Sheng, Alireza Haghighi, Jason Homsy, Norbert Hubner, − or W976R+/ variants exhibited less than half the George Church,3,4 Stuart A. Cook,10,11 Wolfgang A. Linke,6 Christopher S. Chen,2,3‡ contractile force (Fig. 1, C and D, and movies S1 4‡ 1,4,8 ‡ J. G. Seidman, Christine E. Seidman * to S6) or the stress (force normalized to tissue area) (fig. S2I) generated by pWTs, and function Human mutations that truncate the massive sarcomere protein titin [TTN-truncating did not improve over time (fig. S2G). As static variants (TTNtvs)] are the most common genetic cause for dilated cardiomyopathy (DCM), force was similar in pWT and pP22582fs+/− CMTs a major cause of heart failure and premature death. Here we show that cardiac (fig. S2H), we conclude that the contractile deficits microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful observed in mutant CMTs are not due to non- system for evaluating the pathogenicity of titin gene variants. We found that certain myocyte factors. In addition, the comparable force missense mutations, like TTNtvs, diminish contractile performance and are pathogenic. By deficits observed in CMTs with both A-band combining functional analyses with RNA sequencing, we explain why truncations in the TTNtvs and the Z/I junction missense mutation A-band domain of TTN cause DCM, whereas truncations in the I band are better demonstrate that W976R+/− is a pathogenic TTN – tolerated. Finally, we demonstrate that mutant titin protein in iPS cell derived missense mutation. cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and To ensure that the observed functional abnor- b-adrenergic stress, and attenuated growth factor and cell signaling activation. Our malities did not reflect background genetic dif- on September 2, 2015 findings indicate that titin mutations cause DCM by disrupting critical linkages between ferences in patient-derived iPS-CMs, we also sarcomerogenesis and adaptive remodeling. introduced TTNtvs into an independent, iso- genic iPS cell using scarless, CRISPR (clustered ilated cardiomyopathy (DCM) is charac- the Z disk; (ii) a distensible I band (~1 MD) com- regularly interspaced short palindromic repeats)/ terized by progressive left ventricular dila- posed of repeating immunoglobulin-like domains CAS9 technology (5)totargettheI-orA-band tion; systolic dysfunction; and, ultimately, and disordered regions; (iii) an inextensible, thick exons (Fig. 1A and table S1B). Mutant isogenic D heart failure. Occurring in 1 of 250 adults filament–binding A band (~2 MD); and (iv) a iPS cell lines (labeled with “c” preceding geno- (1), DCM arises from underlying cardio- carboxyl M band with a kinase domain. TTNtvs type) were differentiated into iPS-CMs and incor- vascular conditions or as a primary genetic dis- have been identified in each protein segment, porated into CMTs. cN22577fs+/− creates an A-band www.sciencemag.org order. We recently identified dominant mutations but TTNtvs in DCM patients are markedly en- TTNtv in exon 322 (similar to patient-derived that truncate the sarcomere protein titin [TTN- riched in the A band (2, 3). In addition, numer- pP22582fs+/−) (Fig. 1A). cN22577fs+/− CMTs had truncating variants (TTNtvs)] as the most com- ous rare missense variants in TTN have been significantly reduced contractile force (2.19 mN) mon genetic cause of DCM, occurring in ~20% of identified, most with unknown medical impor- compared with isogenic cWT-CMTs (Fig. 1E) familial or sporadic cases (2). tance (2, 3). Both TTN’s size and a general in- (P < 0.003), but not to the extent observed in TTN is a massive protein that spans half of the complete knowledge of the protein’sfunctionin pA22352fs+/− or pP22582fs+/− CMTs (0.767 and sarcomere (1 mm) and includes >34,000 amino cardiomyocyte biology have hindered traditional 1.001 mN, respectively; P < 0.02). Tissue stress acids within four functionally distinct segments approaches for elucidating why some TTN muta- was similarly reduced in TTNtv CMTs compared (Fig. 1A): (i) an N-terminus that is anchored at tions produce clinical phenotypes. To address to WT CMTs (fig. S2J). These data confirm that Downloaded from this, we harnessed recent advances in stem cell A-band TTNtvs markedly reduced contractile reprogramming (4), gene editing (5), and tissue function in both patient-derived and isogenic 1 ’ Division of Cardiovascular Medicine, Brigham and Women s engineering (6) to produce human cardiac micro- CMTs and raise the possibility that the genetic Hospital, Boston, MA 02115, USA. 2Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA. tissue (CMT) models of TTNtvs. background can modify the functional severity of 3The Wyss Institute for Biologically Inspired Engineering at We generated human induced pluripotent stem TTNtvs. Harvard University, Boston, MA 02115, USA. 4Department of cell–derived cardiomyocytes (iPS-CMs) from pa- Premature protein truncation at any location Genetics, Harvard Medical School, Boston, MA 02115, USA. tients (labeled with “p” preceding genotype) (see within a molecule is generally assumed to result 5Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA. 6Department of supplementary materials and methods). Cryo- in loss of function and comparable deleterious Cardiovascular Physiology, Ruhr University Bochum, MA preserved blood samples from one unaffected consequences. However, I-band TTNtvs have 3/56 D-44780, Bochum, Germany. 7Cardiovascular and and three DCM patients with dominant TTN been identified in healthy individuals and in Metabolic Sciences, Max Delbrück Center for Molecular mutations (Fig. 1A and table S1A) were repro- the general population without DCM (2, 3). Two Medicine, Berlin, Germany. 8Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. 9DZHK (German grammed, and high-quality iPS cell clones (fig. models have been proposed to explain this dichot- Center for Cardiovascular Research), Partner Site Berlin, S1, A to C) were expanded, differentiated (7), and omy: (i) Alternative splicing excludes I-band Berlin, Germany. 10National Institute for Health Research enriched by metabolic selection (8)toachieve exons from most mature TTN transcripts and (NIHR) Biomedical Research Unit in Cardiovascular Disease cultures with >90% iPS-CMs (fig. S2, A to C). thereby reduces the functional consequences of at Royal Brompton and Harefield National Health Service (NHS) Foundation Trust, Imperial College London, London, We produced iPS-CMs with two A-band TTNtvs I-band TTNtvs, whereas the inclusion of A-band +/− +/− UK. 11National Heart Centre and Duke–National University, (pA22352fs or pP22582fs ) and a missense exons with TTNtvs is deleterious. (ii) A-band TTNtv Singapore, Singapore. mutation (pW976R+/−) within the Z/I junction transcripts produce longer, stable mutant pro- *Corresponding author. E-mail: [email protected] (J.T.H.); that cosegregated with DCM in a large family (9). teins with dominant negative effects on sarco- [email protected] (C.E.S.) †These authors contributed equally to this work. ‡These authors contributed Single-cell assays of contractile function on mi- mere biology. To distinguish these models, we equally to this work. croarray post detectors (mPADS) (10)showedno compared the functional consequences of isogenic 982 28 AUGUST 2015 • VOL 349 ISSUE 6251 sciencemag.org SCIENCE RESEARCH | REPORTS heterozygous (cV6382fs+/−) and homozygous nant mechanism for reduced penetrance of I- (9D10 recognizes the N-terminus of TTNtvs) (Fig. (cV6382fs−/−) I-band TTNtvs in exon 66 to A-band band TTNtvs. 2A) and a-actinin (Fig. 2, B and C), we identified TTNtvs (cN22577fs+/−,cN22577fs−/−, cT33520fs−/−). As TTN is responsible for sensing and respond- well-formed parallel arrays of repeating sarcomeres We used RNA sequencing (RNA-seq) analyses to ing to myocardial stresses (13), we posited that in myofibrils from WT iPS-CMs. pP22582fs+/− assess TTN RNA splicing in iPS-CMs and to com- TTNtv mutants would exhibit aberrant stress iPS-CMs had fewer myofibrils and abnormal, ir- pare our findings with normal adult left ventricle responses. To model low and high mechanical regular sarcomeres, a phenotype that was even (LV) tissue. TTN transcripts from iPS-CMs incor- load, we assessed contractile performance of more pronounced in homozygous cT33520fs−/− poratedmoreI-bandexonsthandidadultLV pWT and pP22582fs+/− CMTsgrownonflexible iPS-CMs (Fig. 2, A and D, and fig. S4A). Sarco- tissue (fig. S3, A and B). More than 80% of iPS- (0.2 mN/mm) and rigid (0.45 mN/mm) cantilevers (Fig.