© 2019. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019) 12, dmm041103. doi:10.1242/dmm.041103

RESEARCH ARTICLE Perturbation of the /MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients Yuta Higashikuse1,2, Nishant Mittal1, Takuro Arimura3,*, Sung Han Yoon4, Mayumi Oda1, Hirokazu Enomoto1, Ruri Kaneda1, Fumiyuki Hattori1, Takeshi Suzuki2, Atsushi Kawakami5, Alexander Gasch6, Tetsushi Furukawa7, Siegfried Labeit6, Keiichi Fukuda1, Akinori Kimura3 and Shinji Makino1,8,‡

ABSTRACT INTRODUCTION Hypertrophic cardiomyopathy (HCM) is a hereditary disease Zebrafish is well established as a model vertebrate in function characterized by cardiac hypertrophy with diastolic dysfunction. studies (Seeley et al., 2007; Xu et al., 2002). The medaka fish, Gene mutations causing HCM have been found in about half of Oryzias latipes, recently emerged as another important such model HCM patients, while the genetic etiology and pathogenesis remain system because of its smaller genome size and larger genome unknown for many cases of HCM. To identify novel mechanisms diversity (Kasahara et al., 2007). In particular, medaka is an excellent underlying HCM pathogenesis, we generated a cardiovascular- model for studying the cardiovascular system because the embryos mutant medaka fish, non-spring (nsh), which showed are transparent and able to tolerate an absence of blood flow under diastolic dysfunction and hypertrophic myocardium. The nsh sufficient oxygen delivery by diffusion (Burggren and Pinder, 1991). homozygotes had fewer myofibrils, disrupted and It is therefore possible to attribute cardiac phenotype directly to expressed pathologically stiffer titin isoforms. In addition, the nsh particular by ruling out the secondary effect of hypoxia. heterozygotes showed M-line disassembly that is similar to the Hypertrophic cardiomyopathy (HCM) is characterized by cardiac pathological changes found in HCM. Positional cloning revealed a hypertrophy accompanied by myofibrillar disarray and diastolic in an immunoglobulin (Ig) domain located in the M- dysfunction. The first HCM-causing gene identified was a missense β line–A-band transition zone of titin. Screening of mutations in 96 mutation in the cardiac - heavy chain gene (Geisterfer- unrelated patients with familial HCM, who had no previously implicated Lowrance et al., 1990). Such causative mutations were also mutations in known sarcomeric gene candidates, identified two identified in genes encoding contractile apparatus of , α mutations in Ig domains close to the M-line region of titin. In vitro such as -, cardiac T and cardiac myosin- studies revealed that the mutations found both in medaka fish and in binding -C (Ho and Seidman, 2006), whereby HCM was familial HCM increased binding of titin to muscle-specific ring finger thought to result from compensation for dysregulated sarcomeric protein 1 (MURF1) and enhanced titin degradation by ubiquitination. contractile function. An HCM-associated mutation (Gerull et al., These findings implicate an impaired interaction between titin and 2002; Satoh et al., 1999; Itoh-Satoh et al., 2002) identified in the MURF1 as a novel mechanism underlying the pathogenesis of HCM. titin gene (TTN) was the first causative gene not encoding contractile apparatus, which implied the existence of a novel pathogenesis without the compensation of systolic dysfunction. KEY WORDS: Genetics, Familial hypertrophic cardiomyopathy, Titin, Titin is the largest known single protein and, after myosin and Ubiquitin, Myocardial stiffness , the third most abundant protein of vertebrate striated muscle. Titin connects the Z-disc to the M-line region, where titin filaments extending from the opposite sides of half-sarcomeres overlap 1Department of Cardiology, Keio University School of Medicine, 35-Shinanomachi (Ojima et al., 2005; LeWinter and Granzier, 2010). Titin provides a Shinjuku-ku, Tokyo 160-8582, Japan. 2Division of Basic Biological Sciences, scaffold for the assembly of thick and thin filaments (Gregorio et al., Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan. 3Laboratory of Genome Diversity, Graduate School of Biomedical Science, Tokyo Medical and 1999), and provides passive stiffness during diastole (Granzier and Dental University, Tokyo 113-8510, Japan. 4Department of Interventional Irving, 1995). Recently, titin was proposed to function as a Cardiology, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, AHSP A9229, sarcomeric stretch sensor that regulates muscle protein expression Los Angeles, CA 90048, USA. 5Department of Biological Information, Tokyo Institute of Technology, Yokohama 226-8501, Japan. 6Department of Integrative and turnover (Lange et al., 2006, 2005; Miller et al., 2004). Titin Pathophysiology, Medical Faculty Mannheim, Mannheim 68167, Germany. carries a kinase domain at the M-line–A-band transition zone, and 7Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan. 8Keio University Health adjacent to the kinase domain is a binding site for muscle-specific Centre, 35-Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan. ring finger protein 1 (MURF1) (Centner et al., 2001), where the *Present address: Joint Faculty of Veterinary Medicine, Kagoshima University, mechanical load on the sarcomere is transmitted through titin and its Kagoshima 890-0065, Japan. interaction partners to the nucleus. Functional alteration due to the ‡Author for correspondence ([email protected]) TTN mutations may increase passive tension upon stretching of the sarcomere (Satoh et al., 1999; Kimura, 2008). Titin is extensively S.M., 0000-0003-3365-3369 modular in structure and can be thought of as a microscopic necklace This is an Open Access article distributed under the terms of the Creative Commons Attribution comprising up to 166 copies of 90- to 95-amino-acid-residue repeats License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. from the immunoglobulin (Ig) domains. Generally, the Ig domains located in the highly repetitive A-band

Received 1 July 2019; Accepted 25 September 2019 region of titin share high structural similarity (Müller et al., 2007). Disease Models & Mechanisms

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However, a subset of Ig domains close to the titin kinase has unique units of the WT cardiomyocytes at 5 dpf showed highly organized, structural features. For instance, Ig-A169 provides a binding site for precisely aligned thick and thin , flanked by Z-discs MURF1 (Centner et al., 2001; Müller et al., 2007), a sarcomere- and M-lines (Fig. 1L). In the nsh homozygotes, cardiomyocytes associated E3 ubiquitin ligase that conjugates ubiquitin to appeared abnormal with loosely arranged contractile filaments, destined for proteolysis (Bodine et al., 2001; Kedar et al., 2004). where thick and thin filaments were disassembled and Z-discs were MURF1 belongs to the MURF family of proteins, which are not parallel (Fig. 1M). We next analyzed the sarcomeric structure of transcribed from three genes, form heterodimers (Centner et al., adult cardiomyocytes in the WT and nsh heterozygotes (Fig. 1N,O). 2001) and interact with titin kinase. MURFs have also been Expression of sarcomeric α- was maintained in the nsh proposed to regulate protein degradation and gene expression in heterozygotes, although with fewer striations and patchy staining muscle tissues (Lange et al., 2005). (Fig. 1O). Transmission electron microscopy demonstrated ruptured Here, we identified the non-spring heart (nsh) medaka fish, a sarcomeric units with irregular and widened Z-discs and M-lines in cardiovascular mutant with a missense mutation in an Ig domain the cardiomyocytes of nsh heterozygotes (Fig. 1P,Q). We have located at the M-line–A-band transition zone of titin. The nsh fish analyzed body:heart weight ratio in 3- to 4-month-old adult medaka show diastolic dysfunction and hypertrophic heart. To decipher the fish. The of nsh heterozygotes were heavier than WT hearts in roles of titin in these pathological effects, we focused on the M-line– relation to body weight (P=0.01, n=24) (Fig. S2). The nsh mutants A-band transition zone and identified two mutations in other Ig did not show skeletal paralysis; however, the nsh homozygous domains of titin in familial HCM patients. These titin mutations embryos had a slightly smaller body, and skeletal myocytes both increased the binding of titin to MURF1 and enhanced the demonstrated severely disturbed sarcomeric integrity with ruptured degradation of titin by ubiquitination. This is the first report myofibrils (Fig. S3). Unlike in WT, staining for α-actinin was patchy in suggesting that the impaired binding of MURF1 to titin is a newly the nsh heterozygotes (Fig. S3). By transmission electron microscopy, identified pathogenesis causing HCM. the skeletal myofibrils of nsh heterozygotes demonstrated occasionally disrupted M-lines, although they displayed well-formed Z-discs RESULTS and apparently mature myofibrils (Fig. S3). Body size, movement, Medaka nsh mutants showed hypertrophic heart and lifespan and fertility of the nsh heterozygotes were not affected (data disrupted sarcomeric structure not shown), although the abnormal sarcomeric structure was inherited In a previous screening for N-ethyl-N-nitrosourea (ENU)-induced as an autosomal dominant trait. mutations of medaka fish, we identified several mutants with abnormal heart development phenotypes (Taneda et al., 2010). TTN is the nsh gene Among these, one mutant showed rigid hearts with hypertrophy. To identify the gene responsible for the nsh phenotype, we carried Because the mutant heart had lost elasticity and beat in a rigid out positional cloning. We scored 2016 fishes from nsh×nsh/+ manner, we designated it as non-spring heart (nsh). The nsh crosses and found that the nsh phenotype was linked to the medaka embryos were indistinguishable from their wild-type (WT) siblings linkage group 21 (LG 21). Single-strand conformation up to the heart tube stage (48 h), and there was no difference in the polymorphism markers from introns 193 to 194 and the 3′UTR of heart rate. However, after 72 h, the nsh hearts showed increased TTN confined the nsh to a 19.0-kb critical region (Fig. 2A). thickening of the myocardial wall, especially in the atrium and even TTN was the only transcript identified in the nsh critical region. without blood flow (Fig. 1A,B). Despite these abnormalities, the Analysis of cDNAs from the nsh mutant and several WT strains mutant embryos survived until 8 days post-fertilization (dpf), a revealed an adenine-to-thymine transversion causing the stage around hatching, in a relatively healthy condition without substitution of D ()-23186 to V (valine) in the nsh forming pericardial edemas. titin (Fig. 2B). Medaka titin is encoded by a single gene composed Blood flow was very slow or clogged around 3 dpf in the mutants of 219 exons, and the nsh missense mutation was localized to TTN due to defects in diastole (see Movies 1,2). However, hemoglobin exon 204 near the MURF1-binding region that encodes an Ig activity was normal in both WT embryos and nsh mutants at 3 dpf domain (Fig. 2C). Alignment of titin from several species revealed based on an o-dianisidine staining analysis (data not shown). After that the nsh mutation occurred at an evolutionary highly conserved crossing with friend leukemia integration 1 transcription factor amino acid residue (Fig. 2D). ( fli1)-green fluorescent protein (GFP) transgenic medaka (Fig. S1), alkaline phosphatase staining analysis revealed normal vascular Protein turnover of the mutated-Ig-domain titin at the M-line– endothelial cells at 3 dpf and normal blood vessel formation in the A-band transition zone was faster than that of WT-Ig-domain nsh mutants. To estimate the number of cardiomyocytes, we crossed titin the nsh mutants with a transgenic medaka [Tg(cmlc2:DsRed2-nuc)] To investigate the molecular mechanisms of TTN mutation as the expressing DsRed in the cardiomyocyte nuclei (Fig. 1C,D). We nsh gene, we performed reverse-transcriptase PCR (RT-PCR) counted the nuclei of cardiomyocytes using confocal images and analysis of the TTN N-terminal, A-I junction, exon 204 and the titin found no difference in the numbers between WT and nsh mutants at kinase domain in WT and nsh mutant embryos at 3 dpf. This 3 dpf (Fig. 1C-E). analysis revealed unchanged expression levels of TTN mRNA in the In situ hybridization with cardiac-differentiation markers – atrial nsh mutant compared to WT (Fig. 3A). and ventricular myosin heavy chains and cardiac myosin light Titin isoform shifts have been reported in pathological conditions chain – demonstrated normal expression of sarcomeric proteins in (Lahmers et al., 2004; Neagoe et al., 2002; Opitz et al., 2004; the nsh mutant (Fig. 1F-I). A cardiac stretch marker, brain Warren et al., 2004; Makarenko et al., 2004). In cardiac muscle, the natriuretic peptide (bnp), is normally expressed at 3 dpf in the elastic region expresses two types of titin isoform, termed N2B and WT ventricle, but its expression was increased in the atrium of nsh N2BA, which arose from alternative splicing and result in titin mutants (Fig. 1J,K). molecules that contain either a unique N2B element and short We then examined the sarcomeric structures of embryonic PEVK region (N2B isoform) or unique N2B and N2A elements and cardiomyocytes by transmission electron microscopy. Sarcomeric a long PEVK region (N2BA isoform) (Miller et al., 2004). Disease Models & Mechanisms

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Fig. 1. Hypertrophic myocardium and disrupted sarcomeric structure in the medaka nsh mutant heart. (A,B) Morphology of the wild-type (WT) and nsh mutant heart at 3 days post fertilization (dpf). The mutant showed hypertrophic heart, especially in the atrium (double-headed arrow). The nsh heart lost elasticity and showed increased passive stiffness. Due to diastolic inability (see Movie 2), the blood flow was very slow or clogged around 3 dpf. (C,D) Confocal z-stacked images of the WT and nsh mutant hearts at 3 dpf, in which cardiomyocytes were visualized by the Tg(cmlc2: DsRed2-nuc) signal. These reconstructed confocal images show approximately half of the total cardiomyocytes. (E) Quantification of cardiomyocytes using the transgenic line. Cell counting was performed on the optical slices to attain an accurate count. There was no difference in the number of cardiomyocytes between the WT and nsh mutant hearts. NS, statistically not significant (n=5 for WT and nsh). (F-K) In situ RNA hybridization analysis showing cardiac 2 (cmlc2) (F,G), and atrial and ventricular myosin heavy chains (amhc and vmhc, respectively) (H,I) in the WT and nsh mutant at 3 dpf. Normal sarcomeric protein expression was observed in the nsh mutant. (J,K) Strong expression of brain natriuretic peptide (bnp; arrowheads) was detected at 3 dpf in the WT ventricle (J), whereas bnp expression was strong in the atrium of nsh mutants (K). (L,M) Transmission electron microscopic findings of medaka embryonic cardiomyocytes at 5 dpf. Myofibrillar arrays were evident in the WT heart (L), but myofibrils were reduced with loosely arranged contractile filaments in the nsh mutant (nsh −/−)(M). Thick-filament structures and integrity of the M-line region were disrupted, and Z-discs were not parallel. (N,O) Medaka adult cardiac myocytes in the WT and nsh heterozygotes (nsh +/−) were stained for α-actinin. Expression of sarcomeric α-actinin was maintained in the nsh heterozygous mutant, but fewer striations and patchy staining was observed (O). (P,Q) Transmission electron microscopy showed the rupture of sarcomeric units, as well as irregularand widened Z-discs and M-lines in cardiomyocytes of the nsh heterozygotes. The integrity of the thick filaments is disrupted (Q) Z-discs (arrowheads) and M-lines (arrows) are indicated. a, atrium; v, ventricle; Myo, myocardium. Scale bars: 50 µm (A-D),100 µm (F-K), 1 µm (L,M), 5 µm (N,O), 0.5 µm (P,Q).

Predominant expression of the N2B isoform results in stiffer cardiac myocardium under conditions of diastolic heart failure is lower than tissue, while the expression of the N2BA isoform results in more that observed during systolic heart failure (van Heerebeek et al., compliant cardiac myofibrils (Wu et al., 2002). Changes in the 2006). We detected different titin N2B isoform shifts between the relative proportions of N2B and N2BA was paralleled by changes in WT and nsh mutant titin in the nsh heart, which would be associated diastolic stiffness (Ouzounian et al., 2008). The N2BA:N2B ratio in with less elasticity and increased passive stiffness (Fig. 3B). Disease Models & Mechanisms

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Fig. 2. TTN is the nsh gene. (A) Genetic mapping of the nsh locus on the medaka linkage group 21 (LG 21). To define the nsh gene, we scored 2016 fish from nsh/+×nsh/+ mapping crosses. Single-strand conformation polymorphism markers from intron 193-194 and the 3′UTR of TTN confined nsh to a 19.0-kb critical region. (B) DNA sequence analysis of cDNAs from the nsh and WT strains revealed an adenine-to-thymine mutation in nsh, causing D (aspartic acid)-23186 to V (valine) mutation (red arrow), located in exon 204. (C) Modular structure of the M-line–A-band transition zone of titin contains a binding site for muscle-specific ring finger protein 1 (MURF1). The missense mutation identified in nsh was located in an Ig domain near this site. (D) Clustal W alignment of human, mouse, zebrafish and medaka TTN sequences. Medaka titin and human titin proteins share 62% similarity. GenBank or Ensembl accession numbers used for the analysis are as follows: human TTN (NM_133378), mouse Ttn (NP_035782), zebrafish ttna (DQ649453) and medaka TTN (ENSORLT00000022736). Alignment of titin protein from several species along with the nsh mutation showed that the mutation affected the evolutionary conserved amino acid residue (red asterisk). Identical and similar amino acids are indicated by dark blue and light blue boxes, respectively.

Mutations in the TTN gene and protein have also been linked to Next, we examined the faster turnover of the nsh-Ig titin disorders of skeletal muscles (Ojima et al., 2005; Gregorio et al., domain, focusing on the ubiquitin proteasome (UPS) system 1999). To analyze the expression of titin proteins in the nsh mutant, because of the proximity of the nsh mutation to the MURF1- we conducted indirect immunofluorescence on the skeletal muscle binding site. We found that the nsh-Ig-domain protein was of 3 dpf embryos using an antibody to the A-I junction portion of degraded faster via the UPS-dependent pathway in vitro than the titin. In WT, titin appeared in a regularly striated pattern between the WT Ig domain (Fig. 3K), and that this effect was rescued by the sarcomeres of adjacent myofibrils (Fig. 3C). However, the nsh UPS inhibitor epoxomicin (Fig. 3K), indicating that the turnover mutants showed severely decreased titin immunofluorescence at the of this specific titin Ig domain is controlled by the UPS. This A-I junction (Fig. 3D). Analysis of the titin C-terminal region and α- regulation mechanism could involve MURF1 because it is thought actinin staining demonstrated an altered muscle organization, but to act in muscle as a rate-limiting E3 ligase to initiate UPS- there was no apparent decrease in expression of the titin C-terminal mediated degradation. portion (Fig. 3E,F; Fig. S2). These findings indicated that certain titin isoforms at the A-I junction were selectively perturbed. Medaka nsh mutants showed rigid hearts with normal To further investigate the dominant-negative effect of mutant heart rhythm TTN in vivo, we overexpressed the Ig domain of TTN (GFP-tagged We quantified cardiac systolic and diastolic motion of WT and nsh WT-TTN-A156 or E29783V-TTN-A156 construct, which was the mutants (see Movies 1,2) using high-speed video imaging and equivalent of the D23186V mutation in medaka) (Fig. 3G-J) by analysis with the motion vector prediction (MVP) method as injecting mRNA corresponding to exon 204, in which the described in the Materials and Methods section. This method could missense mutation was found, fused with the GFP fragment into extract contraction and relaxation motions of cardiomyocytes non- one-cell-stage medaka embryos. Overexpression of neither the invasively, and evaluate characteristics such as beating rate, WT nor nsh Ig domain induced a specific cardiac phenotype orientation of contraction, beating homogeneity and wave (n=200 in each case). However, in the nsh-Ig-domain-injected propagation of contraction (da Silva Lopes et al., 2011). As medaka embryos, the GFP signal disappeared faster than in the shown in Fig. 4B,C, maximum systolic and diastolic velocities of WT-Ig-domain-injected embryos (Fig. 3G-J), demonstrating that atrium were decreased from ∼183 μm/s in WT embryos to ∼64 μm/s the nsh Ig domain was susceptible to degradation and/or was in nsh mutants, and from ∼140 μm/s in WT embryos to ∼35 μm/s in unstable in vivo.Thensh-Ig-domain fragments could not be nsh mutants, respectively. Similarly, the maximum systolic and observed 2 days after the injection, before the heart began beating, diastolic velocities of ventricles were decreased from ∼189 μm/s in and this could explain why we could not replicate the nsh WT embryos to ∼29 μm/s in nsh mutants, and from ∼136 μm/s in phenotype in the injected embryos (Fig. 3G-J). WT embryos to ∼22 μm/s in nsh mutant embryos, respectively. Disease Models & Mechanisms

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Fig. 3. The mutant TTN Ig domain binds more strongly to MURF1 and is degraded faster than wild type, both in vitro and in vivo. (A) RT-PCR analysis of TTN for the N-terminal portion, A-I junction region, exon 204 and titin kinase domain in the WT and nsh mutant embryos at 3 dpf. Despite the presence of a missense mutation, the level of TTN mRNAs for these portions in the nsh mutant was similar to that in the WT. The ef1α transcript was used as a control. (B) RT-PCR analysis of titin N2B isoforms in the WT and nsh mutants. N2B isoforms were increased. (C-F) Embryonic skeletal muscle of the WT and nsh mutant at 3 dpf were stained with antibodies to A-I junction titin (C,D) and C-terminal titin (E,F). Despite the similar expression level of TTN mRNA, expression of titin containing the A-I junction isoform was reduced (D). Sarcomeric structures were disrupted in the nsh mutant (F). (G-J) WT and mutant (E29783V which was the equivalent of D23186V mutation in medaka) Ig fragments mRNA injection into medaka embryos. Despite the same amount of mRNA injection, mutant (E29783V) Ig fragments degraded faster than WT TTN Ig fragments. (K) Western blots (WBs) of HEK293FT cells transfected with HA-tagged WT and mutant (E29783V) Ig fragments with or without a proteosome inhibitor, epoxomicin. Myc-tagged ubiquitin was co-transfected to visualize ubiquitination. WBs of anti-HA IPs and control normal IgG IPs are shown. Note that the myc-labeled large molecules in mutant TTN transfectants were present only in the presence of epoxomicin. (L) GFP-tagged TTN-A156 domain co-precipitated with myc-tagged MURF1 are shown (top panel). Expression levels of GFP- tagged TTN-A156 (middle panel) and myc- tagged MURF1 (lower panel) was confirmed by WB of whole-cell lysates. Binding pairs were WT or E29783V mutant TTN-A156 in combination with MURF1. Dashes indicate no myc-tagged proteins (transfected only with pCMV-Tag3). (M) Densitometric data obtained from the co-IP assays. Bar graphs indicate the amount of IPs normalized to the input amount of GFP-TTN. Value for WT TTN-A156 with MURF1 was arbitrarily defined as 1.00 arbitrary unit (AU). Data are represented as means±s.e.m. (n=10). **P<0.001 versus WT. Scale bars: 50 µm.

However, the heart rate between WT and nsh mutants was not morpholino knockdown of MURF1. After the injection of changed significantly (Fig. 4B,C). These data demonstrated that the morpholino, we analyzed cardiac movement by motion vector nsh mutated gene was not required for the establishment of cardiac analysis. The maximum systolic and diastolic velocities of both the rhythm, but rather for control of cardiac elasticity (Fig. 4B). As we atrium and ventricle was increased when nsh mutant embryos were had previously shown that the nsh-Ig-domain protein was degraded microinjected with MURF1 morpholino, suggesting that cardiac faster than the WT Ig domain (Fig. 3K), we hypothesized that elasticity was partially recovered (Fig. 4B,C). However, we still cardiac rigidity might be due to excessive degradation of titin. To observed cardiac hypertrophy in MURF1-morpholino-injected nsh confirm our hypothesis, we performed a rescue experiment with embryos. This may be because the effects of morpholinos generally Disease Models & Mechanisms

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Fig. 4. Medaka nsh mutants showed rigid hearts with normal heart rhythm. The beating motions of medaka atrium and ventricle were captured with a high- speed camera and were analyzed with the motion vector prediction (MVP) method. (A) Phase-contrast images of medaka embryos of WT (left), nsh mutant (middle) and nsh mutant+MURF1 morpholino (right). The yellow squares in the images indicate the regions of interest (ROI) employed for the motion vector calculation of atrium wall. (B) Typical beating profile of atrium observed for WT (left), nsh mutant (middle) and nsh mutant+MURF1 morpholino (right). Motion velocity (the vertical scale) represents averaged motion vector length in the ROI. In both figures, each large peak followed by a small peak represents a single beat of atrium. Since we obtained the vector lengths simply from the square root of x2+y2 (x and y represent the vector components), the systolic and diastolic peaks both had positive values. (C) Maximum velocities observed for systolic and diastolic motions in WT, nsh mutant and nsh mutant+MURF1 morpholino are shown for atrium and ventricle. Systolic and diastolic velocities were decreased in nsh mutants and were partially rescued by MURF1 morpholino, while the heart rate was not changed. Data are represented as means±s.e.m. (n=3). only persist up to 3-4 dpf and the cardiac hypertrophy phenotype is phenotype in turn underscored the probable key role of titin in developed at a later stage in nsh mutant embryos. regulating and maintaining cardiac muscle elasticity. The next Next, we investigated the binding between MURF1 and the Ig logical step would have been to rescue the nsh phenotype by domain with or without the mutations using the coimmunoprecipitations injecting full-length TTN mRNA. However, since the medaka TTN (co-IPs) assays, and found that co-IPs from cells transfected with the gene is very large, we were unable to clone the full-length cDNA myc-tagged MURF1 construct in combination with the GFP-tagged (∼15 kb) to perform the rescue experiments, and as a result there WT-TTN-A156 or E29783V-TTN-A156 construct revealed that the remains a chance that the wrong variant has been identified. A156 domain bound MURF1 and that the E29783V mutation significantly increased the binding (9.23±2.11 AU; P<0.001) Mutational analysis of the human TTN gene around the (Fig. 3L,M). Notably, the expression level of mutant-TTN-A156 MURF1-binding domain in patients with familial HCM was lower than that of WT-TTN-A156 (Fig. 3L). The proximity of the nsh mutation to the titin-MURF1 binding Taken together, our data indicate that the Ig domain of the domain and the cardiac phenotype in mutant medaka implicated the M-line–A-band transition zone of titin plays an important role in titin M-line–A-band transition zone as a candidate region for maintaining and organizing the myofibril structure. The severely mutations associated with HCM in humans. We therefore analyzed reduced titin protein levels in combination with the genetic linkage a total of 96 genetically unrelated Japanese patients with familial data implicated the TTN mutation as being causative of the nsh HCM who carried apparent family history of HCM consistent with phenotype. The alternations in N2B/N2BA titin isoforms in the nsh an autosomal dominant trait. They were diagnosed with HCM Disease Models & Mechanisms

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Fig. 5. Mutations in the Ig domains of titin are associated with HCM. (A) Schematic structure of titin at the C-terminal A-band and M-line region. C-terminal ends of titin meet in the M-line. Arrows indicate the position of two mutations found in familial HCM. The nsh missense mutation corresponds to exon 297 of human titin. Fn-3 indicates fibronectin type 3. (B,C) Pedigrees of Japanese families with HCM; affected and unaffected members are indicated by black and white symbols, respectively. Upon screening for mutations in exons 296-307 of TTN, which encode for the A-band region of titin, including the titin-kinase domain, two disease-associated mutations, S30186A (B) and D30994N (C), were identified. The latter mutation is located at the edge of the M-line region of titin and within a binding site for MURF1. Both mutations were found in the Ig domains of titin as indicated by red boxes in A. The proband patient of each family is indicated by the arrow. Presence (+) of the mutations is noted. according to the diagnostic criteria as described previously (Arimura MURF1. It should be noted here that all three mutations found in the et al., 2009). They had been analyzed for mutations in the known medaka nsh mutant and human HCM patients were located in the Ig disease genes for HCM, i.e. genes for contractile elements (MYH7, domains (Fig. 5A). TNNT2, MYBPC3, TNNI3, MYL2, MYL3, ACTC1, TNNC1), Z-disc elements and I-band elements, and no disease-associated Altered binding of titin to MURF1 caused by the TTN mutation was found (Arimura et al., 2009). Control subjects were mutations 400 healthy Japanese individuals selected at random from non-HCM Based on the D30994N mutation being located in the MURF1- patients at Tokyo Medical and Dental Hospital. binding Ig domain of titin, A168-170, we investigated a possible Exon 204 of medaka TTN, containing the nsh mutation, functional change in binding to MURF1 due to the mutation. corresponds to exon 297 of human TTN; thus, we searched for GFP-tagged WT- or D30994N-TTN-A168-170 construct was mutations in the patients by direct sequencing of exons 296-307 of co-transfected with a myc-tagged MURF1 construct into COS-7 TTN (GenBank accession no. NM_133378), which encoded for the cells. Western blot (WB) analysis of co-IPs of the transfected cells M-line–A-band transition region of titin, including the titin kinase using anti-myc antibody demonstrated that the D30994N mutation domain. We identified two disease-associated missense mutations significantly increased binding to MURF1 (1.58±0.19 AU; P<0.01) (Fig. 5A): TCT→GCT, leading to the replacement of with (Fig. 6A,B). The A168-170 domain contains two Ig domains, A168 alanine at position 30186 (S30186A) in exon 301 (Fig. 5B), and and A169, of which the A169 domain is the predominant binder GAT→AAT, with the exchange of aspartic acid for asparagine at of MURF1 (Müller et al., 2007), despite the D30994N mutation position 30994 (D30994N) in exon 306 (Fig. 5C). The proband being located in the A168 domain. To further characterize the patients and their affected relatives were heterozygous for the increased binding of titin to MURF1 in the presence of mutations, and both mutations affected highly conserved amino the D30994N mutation, we performed additional WB analyses acids. Quite interestingly, the latter mutation in the M-line–A-band of co-IPs from the cells transfected with the myc-tagged MURF1 transition zone of titin was mapped within the binding site for construct in combination with GFP-tagged WT-TTN-A168, Disease Models & Mechanisms

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Fig. 6. Altered binding of titin and MURF1 caused by the TTN mutations. (A) GFP-tagged TTN-A168-170 domain co-immunoprecipitated with myc-tagged MURF1 is shown (top panel). Expression levels of GFP-tagged TTN-A168-170 (middle panel) and myc-tagged MURF1 (lower panel) were evaluated by WB of whole-cell lysates. Binding pairs were WT or D30994N mutant TTN-A168-170 in combination with MURF1. Dashes indicate no GFP-tagged or myc-tagged proteins (transfected only with pEGFP-C1 or pCMV-Tag3, respectively). (B) Densitometric data obtained from the co-IP assays. Bar graph indicates the amount of precipitate normalized to the input amount of GFP-TTN. Value for WT TTN-A168-170 with MURF1 was arbitrarily defined as 1.00 AU. *P<0.01 versus WT. (C) GFP-tagged TTN-A168 or TTN-A169 domains co-immunoprecipitated with myc-tagged MURF1 are shown (top panel). Expression levels of GFP-tagged TTN-A168 or TTN-A169 (middle panel) and myc-tagged MURF1 (lower panel) were evaluated by WB of whole-cell lysates. Binding pairs were WT or D30994N mutant TTN-A168 in combination with MURF1, and WT TTN-A169 in combination with MURF1. (D) Densitometric data obtained from the co-IP assays. Bar graph indicates the amount of precipitate normalized to the input amount of GFP-TTN. Value for WT TTN-A168 with MURF1 was arbitrarily defined as 1.00 AU. Data are represented as means±s.e.m. (n=10). *P<0.01 versus WT. (E) GFP-tagged TTN-A160-161 domains co-precipitated with myc-tagged MURF1 are shown (top panel). Expression levels of GFP-tagged TTN-A160-161 (middle panel) and myc-tagged MURF1 (lower panel) was confirmed by WB of whole-cell lysates. Binding pairs were WT or S30186A mutant TTN-A160-161 in combination with MURF1. Dashes indicate no myc-tagged proteins (transfected only with pCMV-Tag3). (F) Densitometric data obtained from the co-IP assays. Bar graph indicates the amount of IPs normalized to the input amount of GFP-TTN. Value for WT TTN-A160-161 with MURF1 was arbitrarily defined as 1.00 AU. Data are represented as means±s.e.m. (n=10). **P<0.001 versus WT. (G) Titin A168-170 was ubiquitinated by MURF1 and MURF2. The titin fragment A168-170 containing the MURF1-binding site was used as a substrate for in vitro ubiquitination assays. Addition of recombinant MURF1 or MURF2 catalyzes multi-ubiquitination of titin fragments. Lanes: Co, control (no E3 ligase added); M1, inclusion of MURF1;

M2, inclusion of MURF2. After incubation, A168-170 fragments were detected by WB, using specific antibodies to titin A169 (left), and A168-170 (right). Disease Models & Mechanisms

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D30994N-TTN-A168 or WT-TTN-A169 constructs. WT-TTN- et al., 1999), and that passive forces maintain the correct localization A169 showed significantly higher binding to MURF1 than of M-lines and tether various signaling and structural molecules WT-TTN-A168 (6.69±1.53 AU; P<0.01)(Fig.6C,D).Onthe (LeWinter and Granzier, 2010). Multiple interacting proteins other hand, the D30994N mutation also significantly increased integrate titin into the sarcomere and modulate the titin filament the binding of the A168 domain to MURF1 (3.06±0.68 AU; via links to basic cellular functions, including trophic signaling and P<0.01) (Fig. 6C,D). energy balance. Because the other mutation (S30186A) also located in the Ig Herman et al. (2012) recently showed by next-generation domain (A160), we also investigated the binding between MURF1 sequencing that TTN-truncating mutations are the most common and the A160 Ig domain with or without the mutation using the co- known genetic cause of (DCM). Their IP assays. The co-IPs from cells transfected with the myc-tagged findings revealed that mutations associated with DCM were MURF1 construct in combination with GFP-tagged WT- or overrepresented in the titin A-band, but were absent around the S30186A-TTN-A160-161 construct revealed that the A160-161 M-band regions of titin. One may therefore speculate that some domains also bound MURF1 and that the S30186A mutation truncated titin peptides are degraded via the RNA- and protein- significantly increased the binding (3.11±0.71 AU; P<0.001) surveillance pathways in DCM. The consequently decreased titin (Fig. 6E,F). Notably, the expression level of mutant-TTN-A160- levels could limit sarcomere formation, resulting in cardiac 161 was lower than that of WT-TTN-A160-161 (Fig. 6E). These dysfunction. However, this is not the case for previously reported data suggested that mutations in the Ig domains of the M-line–A- TTN mutations that delete only part of the M-band portion of titin band transition zone enhanced the interactions with MURF1. This (Carmignac et al., 2007); immunohistochemical studies showed that raises an interesting possibility that the association of MURFs with some of these C-terminal-truncated titin proteins are integrated into the M-line regions could link ubiquitin-transfer pathways to the titin the sarcomere. If more-proximal TTN-truncating mutations caused filament system, which may relate to the physiological turnover of DCM through haploinsufficiency, the distribution of such mutations titin in myofibrils. To test this hypothesis, we performed an in vitro would be rather uniform across the susceptible portion of titin. ubiquitination assay of titin fragment. The titin fragment A168-170, The current study of our HCM families found point mutations containing the MURF1-binding site, was used as a substrate. around the M-line–A-band transition zone, which is implicated in Addition of recombinant MURF1 or MURF2 indeed catalyzed sensing and modulating sarcomeric force. These mutations also multi-ubiquitination of titin fragment (Fig. 6G). Moreover, to induced high binding affinity between titin and MURF1. Using the confirm this idea, we also performed immuno-electron microscopy medaka fish and our in vitro system, we showed that mutated titin with anti-ubiquitin antibodies in WT and MURF1-knockout mice fragments are degraded faster by ubiquitination. This study (Bodine et al., 2001). Ubiquitin signals were detected at the Z-line, indicates that titin proteins found in families with HCM, as in the central I-band and M-line regions in WT sarcomere. It was hard to C-terminal titin truncations studied, are integrated into sarcomeres quantify the signals; however, ubiquitin seemed to be reduced in and cause HCM via a dominant-negative mechanism. Our results MURF1-knockout mice at the M-line region (data not shown). indicated that titin protein mutations in HCM families could be Endogenous MURF1 levels were tightly regulated (Centner et al., incorporated into the sarcomere and impair MURF1 binding, 2001), in combination with ubiquitination of the titin A168-170 resulting in HCM through disturbance of the normal turnover of titin fragment by MURFs (Fig. 6G), and increased binding of titin and and abnormal sarcomere stiffness. MURF1 (Fig. 3L; A,C,E) suggested that the interactions of MURF1 Until now, altered binding of MURF1 to titin has not been with titin are critical for maintaining the stability of titin. identified to be closely associated with human HCM or diastolic dysfunction. In this study, we analyzed exons 296-307 (M-line–A DISCUSSION band transition zone) of TTN for mutations in patients with familial We generated a cardiovascular-mutant nsh medaka fish by ENU HCM and identified two disease-associated missense mutations in mutagenesis that developed hypertrophy and diastolic dysfunction. the Ig domains. One of the TTN mutations in familial HCM was in a We identified a causative mutation in a region of titin around the binding site for MURF1. Although MURF1 binds to the Ig domain MURF1-binding site. The nsh homozygous fish showed loosely of the M-line–A-band transition region, A168-170, MURF1 was arranged contractile units, and died around 8 dpf. The nsh not considered to target titin for degradation (Centner et al., 2001). heterozygotes showed normal lifespan and fertility, but they Data in this study and previous studies indicate that MURF1 may exhibited M-line disassembly in myofibrils, indicating that the regulate the turnover of critical sarcomeric proteins (Kedar et al., phenotype of M-line disassembly was inherited as a dominant trait. 2004) including some titin isoforms (Bodine et al., 2001; Due to the extremely large size and presence of multiple McElhinny et al., 2002). We also found that all the TTN mutations isoforms, it has been difficult to study the structure and function of associated with HCM both in medaka and human in the Ig domains of titin. A knock-in mouse of eGFP to the titin gene showed that the M-line–A-band transition zone increased the binding of MURF1 sarcomeric titin is more dynamic than previously suggested (da to titin (Fig. 6A,C,E). In addition, the TTN constructs carrying the Silva Lopes et al., 2011). The large cardiac titin N2BA isoform is HCM-associated mutations expressed less protein when they were rapidly replaced by the smaller N2B isoform both after birth and injected into medaka embryos and transfected into cells (Fig. 3G-J). with re-expression of the fetal gene program in cardiac pathological Although the molecular mechanisms underlying this reduced conditions (Lahmers et al., 2004; Neagoe et al., 2002; Opitz et al., expression remain to be clarified, it seems to be caused by 2004; Warren et al., 2004; Makarenko et al., 2004). The isoform accelerated degradation by the ubiquitin proteasome pathway switch plays an important role in adjusting diastolic function. It has (Figs 3K and 6G). Further, the knockdown of MURF1 in nsh been used with knockout technology to gain insight into the roles of mutants partially recovered the cardiac systolic and diastolic velocity. titin in sarcomere assembly, stability and mechanotransduction These studies therefore suggests the inhibition of MURF1 function as during muscle pathophysiology (Weinert et al., 2006; Radke et al., a therapeutic intervention for HCM patients. 2007; Granzier et al., 2009). Based on these studies, it was proposed In summary, we have identified TTN mutations that increased the that titin provides a scaffold for the developing sarcomere (Gregorio binding of titin to MURF1 in a medaka model and in patients with Disease Models & Mechanisms

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HCM. There are several pathways underlying the pathogenesis of Immunohistochemistry HCM, including abnormal power generation, power transmission, Medaka embryos were fixed in 4% PFA/PBS overnight at 4°C. Following calcium sensitivity, energy homeostasis and stretch response. fixation and removal of the egg chorion with forceps, samples were MURF1 may be involved with a novel pathway responsible for cryoprotected in sucrose at 4°C overnight, embedded in OCT compound − both the regulation of myofibril turnover and isoform shift as well as (Sakura Finetec) and snap-frozen in hexane at 80°C. The sections were incubated in primary antibodies (see below) overnight at 4°C, washed three muscle gene expression, because MURF1 exists in multiple times for 5 min each in PBS and then incubated in the appropriate secondary locations within the cell, including the cytosol, sarcomere and antibodies (Invitrogen) for 1 h at room temperature. Stained samples nucleus. Our observations suggest that pathogenic mechanisms were observed by confocal laser-scanning microscopy (LSM510; Carl underlying HCM cases that are related to mutations in the titin M- Zeiss, Jena, Germany). The following primary antibodies were used at line–A-band transition zone might be a consequence of abnormal the indicated dilutions: anti-α-actinin (Sigma-Aldrich; A7811) at 1:300, protein turnover and impaired adjustment of diastolic function anti-A-I junction Titin [T11 (Sigma-Aldrich; T9030)] at 1:100, and anti-C- regulated by titin and MURF1. terminal Titin [Titin H-300 (Santa Cruz Biotechnology; sc-28536)] at 1:100. Secondary antibodies were goat anti-rabbit IgG-FITC (sc-2012, 1:100) and goat anti-mouse IgG H&L–Alexa-Fluor-488 (ab150113, 1:100). MATERIALS AND METHODS Fish strains and maintenance High-speed video microscopy Medaka fish (Oryzias latipes) were maintained at 28.5°C in a recirculating Medaka embryos were placed in a bottom of the well (24-multiwell plate) system with a 14-h day and 10-h night cycle. The Qurt strain, derived from and incubated with 500 μl of medaka Ringer’s solution. Embryos the southern Japanese population, was used for the genetic screen. Another were arranged to direct their heart towards the bottom of the plate strain, Kaga, which came from the northern population in Japan, was used so that the cross-sectional heart images for each embryo can be for the mapping cross. The medaka heart mutant non-spring heart (nsh)was observed from a similar direction. A high-speed digital CMOS camera isolated by mutational screening (Sakamoto et al., 2004). Fertilized eggs (KP-FM400WCL, Hitachi Kokusai Electric, Tokyo, Japan) was mounted were collected and then incubated at 28.5°C in medaka Ringer’s solution on an inverted microscope (Eclipse TE2000, Nikon, Tokyo, Japan), (0.65% NaCl, 0.04% KCl, 0.011% CaCl2, 0.01% MgSO4, 0.01% NaHCO3 which was equipped with an xy scanning stage (Bios-T, Sigma Koki, and 0.0001% Methylene Blue). Developmental stages were determined by Tokyo, Japan). The microscope was also equipped with a stage-top mini- their morphology according to the medaka stage table described by incubator (WSKM, Tokai Hit, Shizuoka, Japan), which maintained the Iwamatsu (2004). The fli1-GFP transgenic line was kindly gifted by temperature of the culture plate at 28.5°C. Videos of medaka embryos Dr A. Kudo (Moriyama et al., 2010). All experimental procedures and were recorded as sequential phase-contrast images with a 10× or 20× protocols were approved by the Animal Care and Use Committee of Keio objective at a frame rate of 150 fps, a resolution of 2048×2048 pixels and University and conformed to the current NIH Guidelines for the Care and a depth of 8 bits. Use of Laboratory Animals. Evaluation of beating heart with MVP algorithm Counting cardiomyocytes in vivo To evaluate cardiac systolic and diastolic motion of the medaka heart from The DsRed2-nuc gene under control of the zebrafish cardiac myosin light the high-speed video imaging of the embryos, we have applied the MVP chain 2 (cmlc2) promoter transgenic strain (Taneda et al., 2010) was used for method as previously reported (Hayakawa et al., 2012; Ghanbari, 1990). scoring cardiomyocyte numbers. The WT and nsh homozygous hearts Briefly, each frame was divided into square blocks of N×N pixels (Fig. S4). carrying Tg(cmlc: DsRed2-nuc) were dissected, fixed with 4% Then, for a maximum motion displacement of w pixels per frame, the paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at current block of pixels was matched to the corresponding block at the same 4°C, and then mounted in 1% agarose gel. We routinely prepared 50-100 coordinates in the previous frame within a square window of width N+2w. optical sections from one heart sample, and counted cells on the merged (Optimal values of N and w for the cardiac motion detection may vary with images from one in every 25 sections. Data were presented as mean±s.e.m. the observation magnification and resolution of the employed camera. Here, Student’s t-test was used to test statistical significance. we set N=16 and w=4. These values were determined empirically based on the throughput speed of calculation and accuracy of the block-matching in situ Whole-mount RNA hybridization (ISH) analysis detection.) The best match on the basis of a matching criterion yielded the Whole-mount ISH analysis was performed as described previously (Thisse displacement. The mean absolute error (MAE) was used as the matching and Thisse, 2008). After staining, tissues were fixed with 4% PFA in PBS criterion. The matching function is given by for color preservation, equilibrated with 80% glycerol and then mounted on slide glass for microscopic observation. The following primers were used 1 XN XN ′ Mði; jÞ¼ j ftðm;nÞft1ðm þ i;n þ jÞj w i; j w; for cloning the probes by PCR: for cmlc2,F5-AATGTCTTTTCCATGT- N 2 TTGAGC-3′ and R 5′-CTCCTCTTTCTCATCCCCATG-3′; for atrium m¼1 n¼1 myosin heavy chain (amhc), F 5′-TGCACTGATGGCTGAATTTG-3′ where ft(m,n) represents the intensity at coordinates (m,n) in the current and R 5′-ACTTGATCTACACCTTGGCC-3′; for ventricular myosin block of N×N pixels and ft-1(m+i,n+j) represents the intensity at new heavy chain (vmhc),F5′-GCTGAGATGTCCGTGTATGGTGC-3′ and R coordinates (m+i,n+j) in the corresponding block in the previous frame. 5′-GCTCCTCACGAGGCCTCTGCTTG-3′; and for brain natriuretic We performed this calculation for every 4×4 pixels in the region of interest peptide (bnp), F 5′-GATCCATCCATCCATCATCC-3′ and R 5′-TGATA- (ROI). CTTTAAAGACACAATGTCCAA-3′. The PCR products were cloned into standard vectors and used to prepare the digoxigenin (dig)-labeled RNA probes. Genetic mapping and positional cloning Genetic mapping was performed as described previously (Kimura et al., Histology and electron microscopy 2005). Briefly, an nsh mutant on the Qurt genetic background was crossed We fixed medaka embryos with 4% PFA in PBS overnight at 4°C, before with the Kaga strain to obtain F1 offspring, which were then crossed to dehydrating them in 70% ethanol, mounting in paraffin and sectioning at obtain the nsh mutant embryos for mapping. A pooled bulk segregation 5 µm thickness. Sections were stained with hematoxylin and eosin (H&E) analysis was performed to localize the nsh mutation on the medaka linkage and viewed using a BIOREVO optical microscope (BZ-9000; Keyence, group (Kimura et al., 2005). Further mapping was also done using custom- Japan). For transmission electron microscopy, samples were prepared as designed markers based on the sequence polymorphisms that differed described previously (Rottbauer et al., 2001), sectioned using an RMC between the Qurt and Kaga strains. The following primers and restriction MT6000 ultramicrotome and visualized using the JEM-1230 (JEOL) enzymes were used for the mapping: for TTN exon 90, F 5′-AACTTCAC- electron microscope. AGGCTTCAGGTC-3′ and R 5′-CAGCATCTGTGACGTAGCTC-3′ Disease Models & Mechanisms

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(HaeIII); for TTN intron 193-194, F 5′-CCGTGTAACCAACCTCACTG-3′ (3F10, Roche) or normal IgG (GE Healthcare) for 3 h and combined with and R 5′-GGCTTGGTCCATTCAAGTGT-3′ (HaeIII); for TTN 3′UTR, 20 µl protein G PLUS-Agarose (Santa Cruz Biotechnology). The lysates F5′-CAGGAATTGGTCCTGAAAGG-3′ and R 5′-TCGACTTGGGTG- with agarose-bead complexes were incubated overnight with rotation. The TTTTCAGA-3′; for LG 21_30057841, F 5′-AATAGGTTGAGGCTGC- bead complexes were washed in TGN four times and twice in PBS. The ATGG-3′ and R 5′-TTCACTGGTTCTTGGCGTTC-3′ (HaeIII). The beads were suspended in 2×Laemmli sample buffer (Bio-Rad) containing reference coding sequence of TTN cDNA was in the Ensembl DNA 15% 2-mercaptoethanol and boiled for 10 min. sequence database under the accession number ENSORLT00000022736. Western blotting RT-PCR analysis Cellular lysates and IPs were separated by SDS-PAGE and transferred to Hearts were harvested from WT and nsh mutant embryos (n=50 for each) at polyvinylidene fluoride (PVDF) membranes. After a pre-incubation with 3 dpf. Total RNA was extracted using Trizol reagent (Invitrogen). For RT- 3% skim milk in Tris-buffered saline (TBS), the membrane was incubated PCR analysis, we carried out oligo-dT primed reverse transcription on with primary antibody overnight. After TBS washing, the membrane was aliquots (1 µg) of total RNA and used 1/10 of the single-strand cDNA incubated with an appropriate secondary antibody {anti-rabbit or anti- products for each PCR amplification. Gene-specific primers were designed mouse IgG HRP-conjugated Ab (1:10,000 in 3% skim milk in TBS; GE based on published sequences. ef1α was used as an internal control. The Healthcare), mouse TrueBlot ULTRA: anti-mouse Ig HRP [1:1000 in 3% following primers were used for RT-PCR analysis: for N-terminal TTN, skim milk in TBS containing 0.1% Tween-20 (TBST)]} for 1 h. After five F5′-GCTCTGCACAAAGAGCTGTC-3′ and R 5′-GCAAGATGTGCTG- more washes in TBST, signals were visualized using the Chemi-Lumi One ATGGTTC-3′; for A-I junction TTN,F5′-GCAAGCCAAGAACAAGTT- Super (Nacalai) and Luminescent Image Analyzer LAS-3000 mini TGA-3′ and R 5′-AGAAAAGGTCGGGACAGAGG-3′;forTTN exon 204, (Fujifilm, Tokyo, Japan). F5′-TTGTTCATGTCCATCGTGGT-3′ and R 5′-GCTCCTCTGGAGTTG- ATTGC-3′; for titin kinase domain, F 5′-AATGTTGCAAGGCACAAAAA-3′ Antibodies and R 5′-AGCCTCAAGGCTAATGTCCT-3′;foref1α,F5′-ATCGTTGCT- We used the following primary antibodies: first antibodies: anti-Flag GCTGGTGTTG-3′ and R 5′-AGGCGATGTGAGCTGTGTG-3′; for N2B (1:1000 in 5% BSA/TBST, M2, F7425, Sigma-Aldrich), anti-HA (1:2000 in isoform, F 5′-TATGCCACAACAGCAGGAGA-3′ and R 5′-AAGCCTT- 3% skim milk/TBST, clone HA-7, H9658 Sigma-Aldrich), myc (1:400 in GCAGGTGTATTCG-3′; for N2BA isoform, F 5′-TATGCCACAACAG- 5% BSA/TBST, clone 9E10, sc-40, Santa Cruz Biotechnology) and CAGGAGA-3′ and R 5′-TTTGGTCTTTGCAGCCTCTT-3′. GAPDH (1:1000 in 5% BSA/TBST, 14C10, rabbit mAb; Cell Signaling Technology); second antibodies: ECL anti-mouse IgG, peroxidase-linked Overexpression of TTN Ig domain species-specific F(ab’)2 fragment (from sheep) (1:10,000 in 3% skim milk/ AWTTTN cDNA fragment covering A156 (from bp89426 to bp89711 of TBST, NA9310; GE Healthcare), ECL anti-rabbit IgG, peroxidase-linked NM_133378 corresponding to aa29735-aa29829) or TTN mutants carrying species-specific F(ab’)2 fragment (from donkey) (1:10,000 in 3% skim the A-to-T change (E29783V mutation equivalent of the medaka mutation milk/TBST, NA9340; GE Healthcare), and mouse TrueBlot ULTRA: anti- D23186V) was cloned into the pCS2+ expression vector (Turner and mouse Ig HRP (1:1000 in 3% skim milk/TBST; eBioscience). IPs used the Weintraub, 1994). A capped RNA linked to the cDNA for GFP was anti-HA high-affinity (3F10; Roche) normal mouse IgG (GE Healthcare). synthesized by an MMESSAGE mMACHINE Kit (Ambion) and injected into the cytoplasm of one-cell-stage embryos at a final concentration of Clinical subjects 500 ng/µl. We studied 96 genetically unrelated Japanese patients with HCM, who carried apparent family history of HCM consistent with an autosomal Cloning of titin fragment dominant trait. They were diagnosed with HCM according to the diagnostic We prepared fragments of human TTN by PCR from a TTN A156 Ig domain criteria as described previously (Arimura et al., 2009). They had been construct as templates. AWT TTN cDNA fragment covering the A156 (from analyzed for mutations in the known disease genes for HCM, i.e. genes for bp89426 to bp89711 of NM_133378 corresponding to aa29735-aa29829) contractile elements, Z-disc elements and I band elements, and no disease- domain was obtained by PCR from cDNAs prepared from total heart RNA, associated mutation was found (Arimura et al., 2009). Control subjects were and the A-to-T substitution mutant (E29783V mutation equivalent of the 400 healthy Japanese individuals selected at random from non-HCM medaka mutation D23186V) was created by the primer-mediated patients at Tokyo Medical and Dental Hospital. Blood samples were mutagenesis method. obtained from the subjects after written informed consent and the research protocol was approved by the Ethics Review Committee of Medical Constructs for epitope-tagged proteins Research Institute, Tokyo Medical and Dental University, Japan. The cDNA fragments of WT and mutant TTN were cloned into 3HA-tagged pcDNA3-NTAP. Full-length MURF1 fragment in a Myc-tagged pCMV- Mutational analysis Tag3B construct (McElhinny et al., 2002) was cloned into Flag-tagged Genomic DNA extracted from blood samples of each individual was pcDNA3-NTAP. The Myc-tagged ubiquitin construct was a kind gift from subjected to PCR in an exon-by-exon manner by using primer pairs specific Dr Nakada (Osaka University, Osaka, Japan). These constructs were to exons 296-307 of TTN (GenBank accession no. NM_133378), which sequenced to ensure that no errors were introduced. encode for titin at the M-line–A-band transition zone including the titin- kinase domain. Sequences of primers and PCR conditions used in this study Cellular transfection, protein extraction and are listed in Table S1. PCR products were analyzed by direct sequencing immunoprecipitation using Big Dye Terminator chemistry (Applied Biosystems) and an ABI3100 A total of 8 µg of plasmid DNA was transfected into semi-confluent DNA analyzer (Applied Biosystems). HEK293FT cells on 6-cm-diameter culture dishes using Lipofectamine 2000 (Invitrogen). Epoxomicin or DMSO was added to the culture 4 h Co-immunoprecipitation assay before lysate preparation. At 24 h after transfection, cells were lysed in We used cDNA fragments of human TTN and MURF1 by PCR from TTN 400 µl RIPA buffer [25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, A156, A160-161 and A168-170 Ig-like domain constructs and a MURF1 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] with full-length construct, respectively, as templates for PCR. A WT TTN protease inhibitor, incubated on ice for 30 min and centrifuged at cDNA fragment covering the A156 (from bp89426 to bp89711 of 15,000 rpm (16,000 g) for 30 min. Supernatants were subjected to WB NM_133378 corresponding to aa29735-aa29829), A160-161 (from bp90626 and immunoprecipitation (IP). For IP, lysates were diluted 1:10 using TGN to bp91202 corresponding to aa30134-aa30326), A168 (from bp93006 to buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% Tween-20, 0.3% NP40) bp93287 corresponding to aa30928-aa31021), A169 (from bp93288 to and precleaned by protein G PLUS-Agarose (Santa Cruz Biotechnology). bp93581 corresponding to aa31022-aa31119) or A168-A170 (from bp93006

The precleaned lysates were incubated with 1 µg anti-HA high-affinity to bp93908 corresponding to aa30928-aa31228) domains was obtained by PCR, Disease Models & Mechanisms

11 RESEARCH ARTICLE Disease Models & Mechanisms (2019) 12, dmm041103. doi:10.1242/dmm.041103 and TTN mutants carrying A-to-T (E29783V mutation equivalent of the medaka The NOVARTIS Foundation (Japan) for the Promotion of Science, Japan Heart mutation D23186V), T-to-G (an HCM-associated S30186A mutation) or T-to- Foundation, the Institute of Life Science, KEIO Gijuku Academic Development A (another HCM-associated D30994N mutation) substitutions were created by Funds and Keio Kanrinmaru project, Japan. the primer-mediated mutagenesis method. A full-length MURF1 cDNA fragment spanning from bp137 to bp1198 of GenBank Accession No. Supplementary information NM_032588 (corresponding to aa1-aa354) was obtained by PCR. The TTN Supplementary information available online at http://dmm.biologists.org/lookup/doi/10.1242/dmm.041103.supplemental cDNA fragment was cloned into pEGFP-C1 (Clontech, CA, USA), while the MURF1 cDNA fragment was cloned into myc-tagged pCMV-Tag3 (Stratagene, References CA, USA). These constructs were sequenced to ensure that no errors were Arimura, T., Bos, J. M., Sato, A., Kubo, T., Okamoto, H., Nishi, H., Harada, H., introduced. Koga, Y., Moulik, M., Doi, Y. L. et al. (2009). Cardiac repeat protein gene Cellular transfection and protein extraction were performed as described (ANKRD1) mutations in hypertrophic cardiomyopathy. J. Am. Coll. Cardiol. 54, previously (Arimura et al., 2009) and co-IP assays were done using the 334-342. doi:10.1016/j.jacc.2008.12.082 Catch and Release v2.0 Reversible Immunoprecipitation System according Bodine, S. C., Latres, E., Baumhueter, S., Lai, V. K., Nunez, L., Clarke, B. A., ’ Poueymirou, W. T., Panaro, F. J., Na, E., Dharmarajan, K. et al. (2001). to the manufacturer s instructions. IPs were separated by SDS-PAGE and Identification of ubiquitin ligases required for skeletal muscle atrophy. Science transferred to nitrocellulose membrane. After a pre-incubation with 3% skim 294, 1704-1708. doi:10.1126/science.1065874 milk in PBS, the membrane was incubated with primary rabbit anti-myc Burggren, W. W. and Pinder, A. W. (1991). Ontogeny of cardiovascular and polyclonal or mouse anti-GFP monoclonal Ab (1:100, Santa Cruz respiratory physiology in lower vertebrates. Annu. Rev. Physiol. 53, 107-135. Biotechnology), and with secondary goat anti-rabbit (for polyclonal Ab) doi:10.1146/annurev.ph.53.030191.000543 or rabbit anti-mouse (for monoclonal Ab) IgG HRP-conjugated Ab (1:2000, Carmignac, V., Salih, M. A., Quijano-Roy, S., Marchand, S., Al Rayess, M. M., Mukhtar, M. M., Urtizberea, J. A., Labeit, S., Guicheney, P., Leturcq, F. et al. Dako A/S, Glostrup, Denmark). Signals were visualized by Immobilon (2007). C-terminal titin deletions cause a novel early-onset myopathy with fatal Western Chemiluminescent HRP Substrate (Millipore, MA, USA) and cardiomyopathy. Ann. Neurol. 61, 340-351. doi:10.1002/ana.21089 Luminescent Image Analyzer LAS-3000mini (Fujifilm, Tokyo, Japan), and Centner, T., Yano, J., Kimura, E., McElhinny, A. S., Pelin, K., Witt, C. C., Bang, their densities were quantified using Multi Gauge ver3.0 (Fujifilm, Tokyo, M.-L., Trombitas, K., Granzier, H., Gregorio, C. C. et al. (2001). Identification of Japan). Numerical data were expressed as means±s.e.m. Statistical differences muscle specific ring finger proteins as potential regulators of the titin kinase were analyzed using Student’s t-test for paired values. A P-value of less than domain. J. Mol. Biol. 306, 717-726. doi:10.1006/jmbi.2001.4448 Da Silva Lopes, K., Pietas, A., Radke, M. H. and Gotthardt, M. (2011). Titin 0.05 was considered to be statistically significant. visualization in real time reveals an unexpected level of mobility within and between sarcomeres. J. Cell Biol. 193, 785-798. doi:10.1083/jcb.201010099 Geisterfer-Lowrance, A. A., Kass, S., Tanigawa, G., Vosberg, H.-P., McKenna, In vitro ubiquitination assay W., Seidman, C. E. and Seidman, J. G. (1990). A molecular basis for familial In vitro ubiquitination reactions contained as E1 ligase 100 nM UBE1 hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense (Boston Biochem), and 1 µM UbcH5c as E2 ligase (Boston Biochem), mutation. Cell 62, 999-1006. doi:10.1016/0092-8674(90)90274-I 150 µM ubiquitin, 4 mM ATP and 2.5 µM recombinant titin A168-170 Gerull, B., Gramlich, M., Atherton, J., McNabb, M., Trombita¡S,̃ K., Sasse- fragment. Reactions were started by addition of 2 µM MURF1 or Klaassen, S., Seidman, J. G., Seidman, C., Granzier, H., Labeit, S. et al. (2002). Mutations of TTN, encoding the giant muscle filament titin, cause familial MURF2, respectively. After incubation for 4 h at room temperature, dilated cardiomyopathy. Nat. Genet. 30, 201-204. doi:10.1038/ng815 sampleswereboiledinSDSloadingbuffer, and products were separated Ghanbari, M. (1990). The cross-search algorithm for motion estimation. IEEE Trans. by SDS-PAGE and blotted onto nitrocellulose membranes. Titin-specific Commun. 38, 950. doi:10.1109/26.57512 products were detected by using antibodies directed to titin A169 or titin Granzier, H. L. and Irving, T. C. (1995). Passive tension in cardiac muscle: A168-170. contribution of collagen, titin, , and intermediate filaments. Biophys. J. 68, 1027-1044. doi:10.1016/S0006-3495(95)80278-X Granzier, H. L., Radke, M. H., Peng, J., Westermann, D., Nelson, O. L., Rost, K., Acknowledgements King, N. M. P., Yu, Q., Tschöpe, C., Mcnabb, M. et al. (2009). Truncation of titin’s We thank the National Bio Resource Project for supplying medaka strains and other elastic PEVK region leads to cardiomyopathy with diastolic dysfunction. Circ. Res. support. We thank Dr T. Hayakawa and T. Kunihiro from Sony Corporation for their 105, 557-564. doi:10.1161/CIRCRESAHA.109.200964 technical help in the high-speed video recording of medaka embryos and in the Gregorio, C. C., Granzier, H., Sorimachi, H. and Labeit, S. (1999). Muscle image analysis using the MVP method. We are also grateful to Ms Ono for assembly: a titanic achievement? Curr. Opin. Cell Biol. 11, 18-25. doi:10.1016/ maintenance of medaka. S0955-0674(99)80003-9 Hayakawa, T., Kunihiro, T., Dowaki, S., Uno, H., Matsui, E., Uchida, M., Competing interests Kobayashi, S., Yasuda, A., Shimizu, T. and Okano, T. (2012). Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion The authors declare no competing or financial interests. vector analysis. Tissue Eng. Part C Methods 18, 21-32. doi:10.1089/ten.tec.2011. 0273 Author contributions Herman, D. S., Lam, L., Taylor, M. R., Wang, L., Teekakirikul, P., Christodoulou, Conceptualization: N.M., T.A., S.H.Y., M.O., H.E., R.K., F.H., T.S., A. Kawakami, D., Conner, L., Depalma, S. R., Mcdonough, B., Sparks, E. et al. (2012). A.G., T.F., S.L., S.M.; Methodology: Y.H., M.O., S.L., S.M.; Validation: S.M.; Formal Truncations of titin causing dilated cardiomyopathy. N. Engl. J. Med. 366, analysis: Y.H., S.H.Y.; Investigation: Y.H., N.M., T.A., S.H.Y., M.O.; Resources: 619-628. doi:10.1056/NEJMoa1110186 A. Kawakami; Data curation: F.H., S.M.; Writing - original draft: Y.H., N.M., R.K., Ho, C. Y. and Seidman, C. E. (2006). A contemporary approach to hypertrophic A.G., S.L., S.M.; Writing - review & editing: N.M., H.E., F.H., T.S., A. Kimura, S.M.; cardiomyopathy. Circulation 113, e858-e862. doi:10.1161/CIRCULATIONAHA. Visualization: R.K., A.G., S.M.; Supervision: M.O., R.K., T.S., A. Kawakami, T.F., 105.591982 K.F., A. Kimura, S.M.; Project administration: T.S., A.G., K.F., A. Kimura, S.M.; Itoh-Satoh, M., Hayashi, T., Nishi, H., Koga, Y., Arimura, T., Koyanagi, T., Funding acquisition: T.S., T.F., K.F., A. Kimura, S.M. Takahashi, M., Hohda, S., Ueda, K., Nouchi, T. et al. (2002). Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem. Biophys. Res. Funding Commun. 291, 385-393. doi:10.1006/bbrc.2002.6448 Iwamatsu, T. (2004). Stages of normal development in the medaka Oryzias latipes. This study was supported in part by the program for Grant-in-Aid for Scientific Mech. Dev. 121, 605-618. doi:10.1016/j.mod.2004.03.012 Research (21390248, 24390248, 16H05305, 25670397, 25293096 and Kasahara, M., Naruse, K., Sasaki, S., Nakatani, Y., Qu, W., Ahsan, B., Yamada, 19590832) from the Ministry of Education, Culture, Sports, Science and T., Nagayasu, Y., Doi, K., Kasai, Y. et al. (2007). The medaka draft genome and Technology of Japan, a research grant for Idiopathic Cardiomyopathy from the insights into vertebrate genome evolution. Nature 447, 714-719. doi:10.1038/ Ministry of Health, Labour and Welfare, Japan, and a grant for Basic Scientific nature05846 Cooperation Program between Japan and Korea from the Japan Society for the Kedar, V., McDonough, H., Arya, R., Li, H.-H., Rockman, H. A. and Patterson, C. Promotion of Science. It was also supported by the Joint Usage/Research (2004). Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades Program of Medical Research Institute, a follow-up grant from the Tokyo cardiac . Proc. Natl. Acad. Sci. USA 101, 18135-18140. doi:10.1073/ Medical and Dental University and Association Française contre les Myopathies pnas.0404341102 (Grant No. 15261). This investigation was also supported in parts by Kimura, A. (2008). Molecular etiology and pathogenesis of hereditary

The Mochida Memorial Foundation for Medical and Pharmaceutical Research, cardiomyopathy. Circ. J. 72 Suppl. A, A38-A48. doi:10.1253/circj.CJ-08-0050 Disease Models & Mechanisms

12 RESEARCH ARTICLE Disease Models & Mechanisms (2019) 12, dmm041103. doi:10.1242/dmm.041103

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