Circulation Journal ORIGINAL ARTICLE Circ J 2019; 83: 1737 – 1746 doi: 10.1253/circj.CJ-19-0116 Molecular Cardiology

Familial Left Ventricular Non-Compaction Is Associated With a Rare p.V407I Variant in Bone Morphogenetic 10

Keiichi Hirono, MD, PhD; Kazuyoshi Saito, MD, PhD; Undral Munkhsaikhan, MD; Fuyi Xu, PhD; Ce Wang, MD; Lu Lu, MD, PhD; Fukiko Ichida, MD, PhD; Jeffrey A. Towbin, MD; Enkhsaikhan Purevjav, MD, PhD

Background: Left ventricular non-compaction (LVNC) is a heritable cardiomyopathy characterized by hypertrabeculation, inter- trabecular recesses and thin compact myocardium, but the genetic basis and mechanisms remain unclear. This study identified novel LVNC-associated mutations in NOTCH-dependent and investigated their mutational effects.

Methods and Results: High-resolution melting screening was performed in 230 individuals with LVNC, followed by whole exome and Sanger sequencing of available family members. Dimerization of bone morphogenetic protein 10 (BMP10) and its binding to BMP receptors (BMPRs) were evaluated. Cellular differentiation, proliferation and tolerance to mechanical stretch were assessed in H9C2 cardiomyoblasts, expressing wild-type (WT) or mutant BMP10 delivered by adenoviral vectors. Rare variants, p.W143*-NRG1 and p.V407I-BMP10, were identified in 2 unrelated probands and their affected family members. Although dimerization of mutant V407I-BMP10 was preserved like WT-BMP10, V407I-BMP10 pulled BMPR1a and BMPR2 receptors more weakly compared with WT-BMP10. On comparative expression and siRNA analysis, expressed BMPR1a and BMPR2 receptors were responsive to BMP10 treatment in H9C2 cardiomyoblasts. Expression of V407I-BMP10 resulted in a significantly lower rate of proliferation in H9C2 cells compared with WT-BMP10. Cyclic stretch resulted in destruction and death of V407I-BMP10 cells.

Conclusions: The W143*-NRG1 and V470I-BMP10 variants are associated with LVNC. Impaired BMPR-binding ability, perturbed proliferation and differentiation processes and intolerance to stretch in V407I-BMP10 mutant cardiomyoblasts may underlie myocar- dial non-compaction.

Key Words: Bone morphogenetic protein 10 (BMP10); Cardiomyocyte differentiation; Left ventricular non-compaction; Proliferation; Stretch

eft ventricular non-compaction (LVNC) is a heri- phenotypes.2 Genetically, autosomal dominant and X-linked table cardiomyopathy characterized by myocardial recessive inheritance are reported, with familial pattern hypertrabeculation, inter-trabecular recesses, and present in approximately 40% of LVNC cases.1,4 Despite L 1 thin compacted LV wall. LVNC occurs in 0.05% or this, only a few LVNC-associated mutations have been 1/2,000 of adults.2 Clinically, LVNC is heterogeneous, cat- reported in LBD3/ZASP, DTNA, TAZ/G4.5, LMNA, egorized by ventricular size, thickness, function, atrial size, MYH7, ACTC, TPM1 and TNNT2, indicating that genetic rhythm, outcomes and whether there is associated congeni- etiologies and underlying mutation-induced mechanisms tal heart disease (CHD).3 Progressive cardiac dysfunction, remain largely unexplored.1,4 arrhythmias, or thromboembolism are common manifes- Imbalanced myocardial development regulated by tations. Evidence of clinical LVNC coinciding with other NOTCH signaling is widely accepted as associated with types of cardiomyopathies (dilated [DCM], hypertrophic, LVNC.5 Bone morphogenetic protein 10 (BMP10), a cardiac- restrictive or arrhythmogenic) and CHD suggests multiple specific ligand belonging to the transforming growth factor-β mechanisms that may interact, culminating in overlapping superfamily,6 is expressed by endothelial and myoblast cells

Received February 7, 2019; revised manuscript received May 14, 2019; accepted May 21, 2019; J-STAGE Advance Publication released online June 26, 2019 Time for primary review: 56 days Department of Pediatrics, Graduate School of Medicine, University of Toyama, Toyama (K.H., K.S., C.W., F.I.), Japan; The Heart Institute, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH (K.H., K.S., J.A.T., E.P.); The Heart Institute, Department of Pediatrics (K.S., U.M., J.A.T., E.P.), Department of Genetics, Genomics and Informatics (F.X., L.L.), University of Tennessee Health Science Center, Memphis, TN; Children’s Foundation Research Institute, Le Bonheur Children’s Hospital Memphis, TN (U.M., J.A.T.); and Pediatric Cardiology, St. Jude Children’s Research Hospital, Memphis, TN (J.A.T.), USA The first two authors contributed equally to this work (K.H., K.S.). Mailing address: Enkhsaikhan Purevjav, MD, PhD, The Heart Institute, Department of Pediatrics, University of Tennessee Health Science Center, 71 S Manassas Street, Room 430K, Memphis, TN 38103, USA. E-mail: [email protected] ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Circulation Journal Vol.83, August 2019 1738 HIRONO K et al. briefly and only in embryonic myocardium at E9.0–E13.5 Cell Culture and Animals and its expression (pre-pro-mRNA→pro-mRNA→pro- H9C2, HEK293F and C2C12 cells were obtained from the BMP10) is regulated by NOTCH signaling in a spatiotem- American Type Culture Collection. HEK293F cells were poral and dosage-dependent manner.7 Secreted into the used for purification of BMP10 protein and immunopre- cytoplasm, pro-BMP10 protein dimerizes and forms a cipitation (IP), while H9C2 and C2C12 were used for mature ligand-homodimer, which is capable of binding to siRNA knockdown, adenoviral infection, cellular prolifera- BMP receptors (BMPRs). In cardiomyocytes, BMPR con- tion and stretch studies. All cell lines were maintained in sist of activin A receptors (ACVR) and activin receptor- Dulbecco’s modified Eagle’s medium (DMEM) containing like kinases (ALKs). Receptors BMPR1a (ALK3), BMPR1b 10% fetal bovine serum (FBS), 100 units/mL penicillin G and (ALK6) and BMPR2 are specific to BMP10. Binding BMP10 100 μg/mL streptomycin. All animal work was conducted to its BMPRs regulates the downstream SMAD1/5/8 according the protocols approved by Institutional Animal (canonical) and MAPK (non-canonical) pathways, subse- Care and Use Committee (IACUC), Cincinnati Children’s quently activating cardiogenic factors (NKX2-5, MEF2c, Hospital Medical Center (CCHMC). TBX20) and inhibiting CDKN1c/p57-kip2.8 Ablation of Bmp10 in mice resulted in hypoplastic ventricular walls and Site-Directed Mutagenesis, Protein Production and embryonic death at E10.5.9 In contrast, BMP10 overexpres- Purification sion caused LVNC and ventricular septal defect in vivo,10 The human BMP10 cDNA (1.5 kb) was cloned into a highlighting its importance in the development of LVNC. pcDNA3.1/Myc-His expression vector (Invitrogen) and the We performed screening of NOTCH-dependent genes in V407I-BMP10 variant was introduced using site-directed 230 individuals with LVNC and their family members, and mutagenesis. Then, WT or mutant BMP10 expressed in identified 2 very rare heterozygous variants: missense HEK293F cells was extracted using the FreestyleTM 293 c.1219G>A (p.V407I) in BMP10 and nonsense c.661G>A system (Invitrogen), as described in Supplementary Materials. (p.W143*) in neuregulin1 (NRG1). In this study, we selected V407I-BMP10 identified in a proband with isolated LVNC IP and Immunoblotting and her mother with LVNC/DCM for molecular and cel- HEK293F cells were transfected/co-transfected with WT lular studies. To test whether V407I-BMP10 is associated or mutant BMP10 (pcDNA3.1/Myc-His) and/or BMPR with LVNC, we investigated V407I-mutation effects on (pcDNA-I) constructs. Forty-eight hours after transfec- BMP10 by analyzing dimerization of wild-type (WT) and tion, cells were collected and cell lysates were processed V407I-BMP10 and their binding to BMPRs. Then, with IP and Co-IP and immunoblotted with the anti- effects of V407I-BMP10 on cellular proliferation and dif- FLAG-M2, anti-Myc-9E10, anti-HA-3F10 (Boehringer ferentiation and cyclic stretch tolerance were tested in Mannheim), or anti-phosphoserine antibodies (Zymed) as H9C2 cardiomyoblasts. Based on the results, we propose described in Supplementary Materials. Data were analyzed that the V407I-BMP10 variant is associated with LVNC, using results from 3 pull-down experiments. and that further in vivo studies are needed to comprehen- sively understand its roles in LVNC pathogenesis. Real-Time Quantitative Polymerase Chain Reaction (qPCR) and siRNA Knockdown Methods We first performed qPCR to assess expression of BMP10, BMPR and GAPDH (control) in H9C2 cells in triplicates Human Subjects and Clinical Diagnostic Criteria at least twice (Prism7500, Applied Biosystems). Then, After obtaining informed consent, patients and healthy BMPR siRNA knockdown in H9C2 cells followed by control individuals were recruited in this study as described BMP10 treatment was used to test whether H9C2 cardio- in Supplementary Materials. Clinical evaluation of probands myoblasts can serve as an appropriate in vitro model of and their first-degree relatives and collection of family history BMP10 and BMPR interactions. Experimental details and were performed. Blood was collected from 230 patients analysis are described in Supplementary Materials. and their relatives (Latino, n=43; Caucasian, n=52; Asian, n=100; African, n=35; female, n=118; male, n=112; fami- Adenoviral Construction, Cellular Differentiation and Cell lies, n=73) and 272 ethnicity-matched controls. Genomic Proliferation Assay DNA was extracted and biobanked in the Pediatric Car- Adenoviral vectors containing mature WT- or V407I- diomyopathy Specimen Repository (PCSR). BMP10 were constructed, and infection of H9C2 cells was performed. The rate of cellular proliferation of the H9C2 High-Resolution Melting (HRM) Analysis, Whole Exome cells was then assessed using the Vybrand® MTT Cell Sequencing (WES) and Sanger Sequencing Proliferation Assay Kit (ThermoFisher) on days 1, 3, and To effectively select LVNC candidate genes, we first 5 after adenoviral infection as described in Supplementary selected genes using an algorithm based on association with Materials. Absorbance at 570 nm was measured using NOTCH signaling followed by Protein Interaction Network SynergyH1 reader (BioTek). Analysis2 (PINA2) at http://cbg.garvan.unsw.edu.au/pina/. Then, 50 genes underwent HRM using ABI7500fast (Applied Immunohistochemistry and Cell Death Analysis Biosystems). We performed WES in a proband carrying H9C2 cells grown in 10% FBS (proliferation condition) or the V407I-BMP10 variant. All non-synonymous or non- 2% heat inactivated horse serum (HIHS, differentiation sense variants with minor allele frequency (MAF) <0.01 condition) on chamber slides (Lab-Tek) were infected with were prioritized using in silico algorithms. Then, the exons WT-BMP10 or V407I-BMP10 vectors. Immunohisto- containing the deleterious variant were amplified in an chemistry using anti-HA (Sigma), anti-BMPR1a and anti- exon-by-exon manner using the 3730xl Analyzer (Applied BMPR2 (Santa Cruz), anti-SMAD1/5/8 (Cell Signaling), Biosystems). Experimental details of HRM, WES and bioin- anti-a-actinin2 (Invitrogen) antibodies and 0.5 μg/mL formatics analysis are described in Supplementary Materials. diamidino-2-phenylindole (DAPI; Sigma) was performed

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Figure 1. Mutational analysis of patients with left ventricular non-compaction (LVNC). (A,B) Chromatographs of direct sequencing and pedigrees of the LVNC families. (+) Proband and affected family members diagnosed with LVNC carrying (A) the c.1219G>A (V407I-BMP10) or (B) c.661G>A (W143*-NRG1) variants. Half black circle, female family member with a heterozygote variant. (−) Healthy family member negative for the variant. (C) Structure of bone morphogenetic protein 10 (BMP10). Mature BMP10 forms 2 domains: α and β. The β-domain has 2 finger-shaped convex parts, which bind to BMPR2. Red arrow, location of the V407I variant in exon 2. BMPR, BMP receptor.

on days 0, 1, 3 and 5 as described in Supplementary Materials. (82.2%) had isolated LVNC (Supplementary Table 1). LVNC accompanied by cardiomyopathy, CHD or other systemic Cyclic Stretch diseases accounted for 17.8%. Due to the large size of the H9C2 cells were infected with WT-BMP10 or V407I- patient cohort, we first performed HRM analysis on 50 BMP10 vectors (multiplicity of infection or MOI=5). After NOTCH-associated candidate genes (Supplementary Table 2). 48 h, H9C2 cells were plated on 6-well BioFlex plates in HRM identified 2 very rare heterozygous variants in BMP10 DMEM containing 10% FBS. When cells reached 80% and NGR1. A missense variant c.1219G>A (p.V407I) in confuency, they underwent cyclic 10% mechanical stretch BMP10 (rs148561995, MAF=0.00007783) was identified for 1 h/day for 3 consecutive days using FlexCell5000 system in a proband with isolated LVNC and her mother with (FlexCell International) as previously described.11 On days LVNC/DCM from a Latino family (Figure 1A). The sec- 1 and 3, the cells were fixed, immunostained and visualized ond variant, c.661G>A (p.W143*) in NRG1 (rs913880282, using a Zeiss710 confocal microscope. MAF=0.00002) was identified in a proband and his mother with isolated LVNC, while the proband’s unaffected brother Statistical Analysis was negative (Figure 1B). The W143*-NRG1 variant resulted Student t-test, 2-way ANOVA or repeated-measures in premature termination of NRG1 type II (NG_012005.2), ANOVA over time were used to analyze data, and P<0.05 resulting in ablation of glial growth factor-2 (GGF2), the was considered significant. precursor pro-NRG1 expressed in many tissues, including the heart.12 Notably, a GGF2-knockout mouse model has Results demonstrated abnormalities in cardiac and neuron devel- opment, supporting that p.W143*-NRG1 is an LVNC- Patient Cohort and Mutational Analysis associated variant.13 On analysis of the 230 individuals with LVNC, the majority Therefore, we selected the V407I-BMP10 variant for

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Table. Genes Re-Sequenced on Sanger Sequencing Gene dbSNP AA change Proband Mother Father 1 ADAMTSL3 rs143157214 NM_001301110:exon19:c.G2441A:p.C814Y No No No 2 ARHGEF11 – NM_014784:exon15:c.C1168T:p.R390C No No No 3 ATP5SL* rs185136417* NM_001167870:exon4:c.C376T:p.R126W* YES* YES* No 4 BMP10* rs148561995* NM_014482:exon2:c.G1219A:p.V407I* YES* YES* No 5 CTNNA2* rs373818447* NM_001164883:exon3:c.C289T:p.R97C* YES* YES* No 6 FHOD3 – NM_001281739:exon5:c.G455A:p.C152Y No No No 7 NDUFB11* – NM_001135998:exon3:c.G361A:p.E121K* YES* YES* No 8 NRP1* rs117525057* NM_001024628:exon9:c.C1571T:p.S524L* YES* YES* No 9 PCYOX1 rs763063296 NM_016297:exon6:c.C1418T:p.A473V YES No YES 10 SLC25A5 rs200550329 NM_001152:exon2:c.G413A:p.R138H No No No 11 SLC25A5 rs753913830 NM_001152:exon2:c.G361T:p.G121C No No No 12 SLC25A5 rs199707714 NM_001152:exon2:c.G352A:p.A118T No No No 13 ULK4 rs35833603 NM_001322500:exon5:c.C417G:p.N139K No No No *Found in the proband and her affected mother. AA, amino acid; dbSNP, The Single Nucleotide Polymorphism Database; No, negative for a variant; YES, positive for a variant.

Figure 2. Dimerization and receptor-binding of wild-type (WT) and mutant bone morphogenetic protein 10 (BMP10). (A) Western blotting of WT-rhBMP10 and V407I-rhBMP10. First lower arrow, predicted 12.2-kDa mature rhBMP10 monomer. Upper arrows, dimers and polymers of rhBMP10. C, control; M, molecular weight marker; V407I, mutant rhBMP10. (B) Immunoprecipitation (IP) and co-IP of BMP10 and BMP receptors (BMPRs). (Upper panel) Combinations of WT- or V407I-BMP10 fused with GFP (green fluorescent protein) and receptors, BMPR1a or BMPR2, fused with FLAG epitope. (+) Expression of a construct. (Lower panel) Western blotting. IB, immunoblotting. Arrow and asterisk, increase in intensity of the band, representing the amount of BMP10 pulled down by BMPR1 and BMPR2-FLAG, respectively. (C) Relative ratio of signal strength of WT or V407I BMP10 pulled by BMPR1a or BMPR2. Average ratios of BMP10-GFP to BMPR1-FLAG or BMPR2-FLAG from 3 pull-down experiments, quantified using ImageJ. *P<0.05.

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Figure 3. Expression of bone morphogenetic protein receptor (BMPRs) in (A) the left (LV) of 3-month-old mice and (B) H9C2 cardiomyoblasts on real-time quantitative polymerase chain reaction (qPCR). (C) Expression of BMPRs under siRNA knock- down conditions without (0 ng/mL) or with BMP10 (50 ng/mL) in H9C2 cells. *P<0.05.

further genetic studies. This variant has been reported as a UNIPROT). ATP5SL and NDUFB11 encode the compo- rare single-nucleotide polymorphism (SNP) in Latino nents of mitochondrial complex 1. Interestingly, the same (MAF=0.0003), Asian (MAF=0.00006) and non-Finnish variant c.361G>A (p.E121K) in NDUFB11 identified in European ethnicity (MAF=0.000007; https://gnomad. the present patients was previously reported in a prema- broadinstitute.org/variant/2-69092819-C-T), while no SNP ture male infant carrying hemizygous E121K-NDUFB11 have been reported in other races. Interestingly, high MAF on the X , who died soon after birth due to (0.03) was reported in the Puerto Rican population only mitochondrial complex I deficiency (MCID).14 Most patients (http://grch37.ensembl.org), although no reports on LVNC with MCID do not survive beyond early childhood, frequency are available in this region. In this study, we found whereas the present patients carrying the heterozygous no V407I-BMP10 in the father or in 272 Latino non-Puerto E121K-NDUFB11 had no evidence of metabolic disorder. Rican unrelated control individuals (546 alleles). Thus, to NRP1 is reported to play roles in angiogenesis, axon guid- discover other potential LVNC-associated variants, we per- ance, and cell survival, and CTNNA2 may function as a formed WES using the proband’s genomic DNA. A total cadherin receptor and cytoskeleton linker in the nervous of 8.6 Gb of raw sequence data were generated, of which system. Therefore, we excluded ATP5SL, NDUFB11, 98.64% was mapped onto the human reference genome CTNNA2 and NRP1 from further studies. Based on the with a 93X median read depth. Of the coding variants, very low MAF in the non-Puerto Rican Latino population 11,378 synonymous, 10,468 non-synonymous, 94 stop- and potential deleterious effects on BMP10 function in gain, and 11 stop-loss variants were identified. After variant silico (Supplementary Table 4), we hypothesized that V407I- prioritization by SIFT, Polyphen2, LRT, MutationTaster, BMP10 is associated with LVNC, and finally selected this FATHMM, PROVEAN, and MetaSVM, 54 deleterious variant for further investigation on protein and cellular variants in 49 genes were retained (Supplementary Table 3). levels. Then, based on 2 criteria, that is, whether the gene is expressed in the heart and whether there is functional evi- Preserved Dimerization and Polymerization of Mutant dence of cardiovascular disease according to DAVID V407I-BMP10 (https://david.ncifcrf.gov/) and VarElect (https://ve.genecards. The V407I variant (Figure 1C, red arrow) is located at the org/), we selected 13 variants (Table) for resequencing, and C-terminus of BMP10, which is responsible for binding to identified 5 deleterious variants in ATP5SL, BMP10, BMPR2. The BMP10-monomer has a structure of “a wrist CTNNA, NDUFB11, and NRP1, which were co-segregated with 2 fingers”. This means that the N-terminus forms the in the proband and her mother and negative in her father. first finger-shaped strand, the rod-domain forms the Then, we compared tissue expression, biological conse- α-helix (wrist) and the C-terminus forms the second finger- quences, structural and functional features of those 5 vari- tip strand. The BMP10 monomers of 12.2 kDa form a ants using online sources (NCBI, ExAC, OMIM, GAD, dimer, stabilized by a disulphide bond, and only dimerized

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Figure 4. Immunohistochemistry of H9C2 cells cultured in proliferating conditions (10% fetal bovine serum). Immunostaining was performed on days 0, 1, 3 and 5. Green, bone morphogenetic protein 10 (BMP10); red, α-actinin2; yellow, overlap of BMP10 and α-actinin2; blue, DAPI; bars, 10 µm. Insets, detailed view of the cytoplasm in the squares. (i) Arrows, dotted expression of V407I- BMP10. WT, wild type.

mature BMP10 bind the BMPR.6 Therefore, we first puri- compared with the other 3 lanes, suggesting that WT- fied recombinant human WT-BMP10 and V407I-BMP10 BMP10 has stronger binding to BMPR1a compared with proteins, blotted and detected using His-Tag to determine V407I-BMP10. Furthermore, the intensity of bands corre- whether V407I alters the dimerization of BMP10. As sponding to the pulled BMPR2-FLAG by FLAG was shown in Figure 2A, His blotting patterns were identical in significantly higher in V407I-BMP10 cells (Figure 2B asterisk) the WT-BMP10 and V407I-BMP10 lanes, with the lowest than in WT-BMP10 cells. This suggests the possibility of a molecular weight bands of approximately 18 kDa repre- higher concentration of ligand-free BMPR2 in mutant cells senting monomers, the higher bands of approximately available for FLAG pulling. Thus, we quantified the ratio 37 kDa representing dimers, and the approximately 75 kDa of band intensity corresponding to BMP10-GFP to the bands representing polymers, suggestive of the ability of intensity of BMPR1a or BMPR2 from 3 independent pull- V407I-BMP10 to form dimers similar to WT-BMP10. down experiments (Figure 2C), and noted a lower ratio in V407I-BMP10 compared with WT-BMP10. We therefore Pull-Down of BMPR1a and BMPR2 by V407I-BMP10 Is assumed that V407I-BMP10 had impaired receptor-binding Decreased ability, and further investigated the effects using cellular Given that dimerization was preserved in V407I-BMP10, models. binding of mutant BMP10 to BMPRs was expected. We further hypothesized that mutation may have effects on H9C2 Cells Express BMP10 and BMPRs Similar to Mouse BMP10 binding to BMPR2 specifically. To test if the pulling Embryonic Hearts of receptors by V407I-BMP10 is altered, we tagged WT- BMP10 expression regulates the balanced proliferation BMP10 or V407I-BMP10 with GFP (green fuorescent and differentiation processes in myoblasts during the par- protein), and BMPR1a or BMPR2 with FLAG epitope. ticular embryonic period when myocardium develops under Equal amounts of each construct were transfected into mechanical and shear stretch,15 and BMP10-BMPR inter- HEK293F cells and IP and Co-IP were performed. As actions specifically reduce proliferation of cardiomyoblasts shown in Figure 2B, pulling of BMPR1a-FLAG by FLAG in an autocrine and paracrine manner. To corroborate, we was identical in WT- and V407I-BMP10 co-transfected first determined the BMP10 and BMPRs co-expression cells. Bands of WT-BMP10-GFP pulled by FLAG pattern in mouse embryonic hearts from E6.5 to E16.5 using (Figure 2B arrowhead), however, had higher intensity qPCR. We found that expression of BMP10 was signifi-

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Figure 5. Rates of proliferation in H9C2 cells, expressing wild-type (WT)- or V407I-bone morphogenetic protein 10 (BMP10). Proliferating condition, 10% fetal bovine serum (FBS) and dotted lines. Differentiating condition, 2% heat inactivated horse serum (HIHS) and solid lines. *P<0.05. The data are standardized according to the control condition on day 1.

cantly increased at E10.5 and E15.5-E16.5 (Supplementary proliferation and differentiation. Figure 1). Slight increase of BMPR1a was seen at E11.5 and E16.5. BMPR2 was increased at E10.5 and remained V407I-BMP10 Form Aggregates in H9C2 Cells high throughout E15.5-E16.5, with simultaneous increase To visualize BMP10, BMPR1a and BMPR2 expression, in BMP10, BMPR1a and BMPR2 expression. Other BMPRs we infected H9C2 cells with adenoviral vectors expressing (ALK1, ALK2, BMPR1b/ALK6, ACVR2a and ACVR2b) WT-BMP10 or V407I-BMP10, and then carried out immu- had no difference in expression levels (Supplementary nohistochemical analysis followed by confocal microscopy. Figures 1,2), suggesting that BMPR1a and BMPR2 are We found that WT-BMP10, V407I-BMP10, BMPR1a and indeed the main receptors that respond to BMP10 increase. BMPR2 were all detectable after 24 h of infection at base- Although the effects of BMP10 are specifically perceived line (data not shown). To determine the long-term effects in cardiac myoblasts, most in vitro BMP10 studies have of V407I-BMP10 on cellular processes, we continued cul- been performed in endothelial cells.16 Thus, we next turing the infected cells for 5 days in 10% FBS media, and required an appropriate immature myoblast cell line that evaluated the co-expression of BMP10 and α-actinin2 used can experimentally resemble proliferation and differentia- to assess the cytoskeletal assembly, as described previously.11 tion processes and effectively facilitate mechanical stretch On day 0, no BMP10 was detected in any cells, while in vitro. We have previously shown that H9C2 cells α-actinin2 was seen mainly in the periphery of H9C2 myo- mimicked the responses to differentiation and cyclic blasts in all groups (Figure 4a–c). On day 1, defuse cyto- stretch, similar to primary rat neonatal cardiomyocytes.11 plasmic expression of BMP10 (green) and α-actinin2 (red) Thus, we examined whether H9C2 cells can serve as a was detectable in WT-BMP10 (Figure 4e) and V407I- suitable model for V407I-BMP10 studies. First, we com- BMP10 (Figure 4f) cells. On day 3, control cells (Figure 4g) pared the expression levels of BMP10 and BMPRs in retained a myoblast-like appearance with no BMP10 expres- H9C2 cells and adult (3-month-old) murine myocardium. sion. The WT-BMP10 cells (Figure 4h) were elongated, and qPCR indicated similar expression patterns of all 7 BMPRs diffuse cytoplasmic and nuclear expression of BMP10 was in H9C2 cells (Figure 3A) and murine LV (Figure 3B), and noted. Expression of α-actinin2 was seen in the cellular we found that H9C2 cells and murine LV highly expressed periphery and throughout the cytoplasm, with a tendency BMPR1a and BMPR2. Then, we tested whether BMPR toward a striation-like arrangement. The V407I-BMP10 cells respond to BMP10 alterations. For this, we siRNA (Figure 4i) in contrast, retained a round myoblast-like shape knocked-down all ALKs/BMPRs in H9C2 cells. Then, the with V407I-BMP10 seen as dotted spots, suggesting the pos- cells were treated without (0 ng/mL) or with BMP10 sibility of mutant BMP10 aggregates formed in the cytoplasm (50 ng/mL) and expression of ALK and BMPR were (Figure 4i arrows, Lower right panel). Scattered α-actinin2 analyzed (Figure 3C). BMPR1a (ALK3) and BMPR2 expression was seen in the cytoplasm compared with WT- (asterisks) continued being significantly downregulated in BMP10 cells. On day 5, control cells (Figure 4j) continued the presence of BMP10, unlike other receptors. Taken being seen as myoblast-like cells, similar to days 0–3. Diffuse together, BMPR1a and BMPR2 are expressed in H9C2 cytoplasmic α-actinin2 overlapped with BMP10 in WT cells cells similar to ventricular myocardium, and are specifically (Figure 4k). In contrast, V407I-BMP10 cells contained vast responsive to BMP10, suggesting that H9C2 myoblasts can vacuoles in the cytoplasm and nuclei. Mutant BMP10 was be utilized in vitro to determine V407I-induced effects on mainly localized in nuclear and perinuclear areas, whereas

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Figure 6. (A) Immunohistochemistry of H9C2 cells under stretch conditions (cyclic 10% biaxial stretch). Immunostaining was performed on day 1 and 3 after cyclic stretch. Green, α-actinin2; red, Smad1/5/8; yellow, overlap of α-actinin2 and SMAD1/5/8; blue, DAPI; bars, 10 µm. (Right-hand panels) Ratio of cell nuclei fragmentation and chromatin condensation to total number of DAPI-positive nuclei for (Upper panel) control, (Middle panel) wild-type (WT) or (Lower panel) mutant H9C2 cells in response to stretch on day (D) 0, D1, D3. ***P<0.001.

α-actinin2 was seen mainly in the cellular periphery (Figure 4l). V407I-BMP10 Decreases Proliferation of Differentiating Taken together, the experiments demonstrated that V407I H9C2 Cells causes alterations in BMP10 and α-actinin2 expression and Given that BMP10 induced proliferation in cardiomyo- localization. blasts,17 we then compared the rate of proliferation in WT- BMP10 and V407I-BMP10 myoblasts grown in 10% FBS Effects of BMP10 in Differentiating H9C2 Cells (proliferating condition) and 2% HIHS (differentiating To examine whether V407I affects cellular differentiation condition). Cells were cultured for 5 days and the rates of and proliferation processes, we carried out differentiation of MTT absorption measured on days 1, 3 and 5 were equated H9C2 cells with HIHS media for 5 days. On day 1, α-actinin2 to that of the control cells on day 1 (Figure 5). On day 1, expression was seen in the periphery and cytoplasm of WT- the rate of MTT absorption was similar in all cell groups. BMP10 and V407I-BMP10 cells (Supplementary Figure 3). On day 3, the rate of MMT absorption was equally increased In WT cells, BMP10 was diffusely expressed (Supplementary in uninfected control, WT or mutant cells in proliferating Figure 3b), while V407I-BMP10 formed aggregates in mutant conditions (Figure 5, dashed lines), indicating an increase cells (Supplementary Figure 3c). On day 3, WT- and V407I- in proliferation. In differentiating conditions (Figure 5, BMP10 cells (Supplementary Figure 3e,f) appeared elongated, solid lines), only WT-BMP10 cells preserved a high prolif- diffusely expressing BMP10 and α-actinin2 throughout the eration rate similar to that in proliferating conditions, sug- cytoplasm, suggesting that they are undergoing successful gesting that WT-BMP10 may transiently promote cellular differentiation. On day 5, WT-BMP10 and V407I-BMP10 proliferation during cardiomyoblast differentiation. In con- cells continued being elongated and expressing diffuse cyto- trast, mutant cells had a significantly lower proliferation plasmic α-actinin2. Taken together, this indicates that V407I- rate compared with control and WT-BMP10 cells (P<0.05), BMP10 did not affect differentiation of H9C2 cells (days suggesting that the V407I-BMP10 mutation may inhibit 3–5), despite the V407I-BMP10 aggregates seen on day 1. proliferation of differentiating cardiomyoblasts. On day 5,

Circulation Journal Vol.83, August 2019 Myocardial Non-Compaction and BMP10 Mutation 1745 no change in proliferation rates is seen proliferating cells, and embryonic lethality of Bmp10-KO mice at E10.5.23 A while proliferation significantly decreased in all cell groups similar phenotype was seen in Bmpr1a-deficient mice, in the differentiating condition, with the lowest seen in suggesting the importance of BMP10 and BMPR1a in mutant cells. Taken together, we demonstrated no effects BMP10 signaling.24 of WT-BMP10 or V407I-BMP10 on proliferation of H9C2 The aim of this study was to identify novel NOTCH- cells at baseline. In differentiating conditions, WT-BMP10 associated gene mutations in patients with LVNC and clar- transiently promoted the proliferation up to day 3, while ify mutation-induced mechanism(s) using genetic, molecular V407I-BMP10 steadily inhibited the proliferation of dif- and in vitro studies. We screened 50 NOTCH-associated ferentiating cells. genes in 230 patients with LVNC and identified p.W143*- NRG1 and p.V407I-BMP10 variants. Based on results of V407I-BMP10 Cells Are Intolerant to Cyclic Stretch WES of a proband and direct sequencing in family mem- Maturation of the multi-layered compacted myocardium bers, we propose that the V407I-BMP10 variant is poten- occurs under mechanical and shear stretch created by cyclic tially associated with the LVNC phenotype in this family. contractions and blood fow.18 Thus, we tested whether Given that only mature BMP10 dimer retains an ability to V407I-BMP10 alters cellular tolerance to mechanical cyclic bind BMPR,23 we tested whether the mutant V407I- stretch by applying biaxial 10% cyclic stretch to H9C2 cells, BMP10 dimerizes, and, if so, whether it binds to BMPRs. followed by immunohistochemistry using anti-α-actinin2 We showed that V407I-BMP10 may form dimers to interact to observe the cytoskeletal assembly19 and anti-SMAD1/5/8 with BMPR, but that the binding abilities of mutant to assess stretch-associated responses.20 On day 1, uninfected BMP10 to BMPR1a and BMPR2 were decreased compared control cells were round and resisted cyclic stretch well, with that of WT-BMP10. Thus, we further investigated the diffusely expressing α-actinin2 (Figure 6a). No SMAD1/5/8 effects of V407I-BMP10 on proliferation and differentiation expression was detected in uninfected cells. In contrast, in the cellular model. WT-BMP10 and V407I-BMP10 cells (Figure 6b,c) were small, First, we examined if rat ventricular H9C2 cardiomyo- elongated and separated from each other. We assumed that blasts can serve as an appropriate model for BMP10 studies. this is due to adenoviral infection. Expression of Smad1/5/8 For this purpose, we performed comparative gene expres- was detected in both WT- and V407I-BMP10 cells. On day sion analysis of BMP10 and BMPRs in mouse embryonic 3, control cells continued resisting cyclic stretch and formed heart, adult LV myocardium, H9C2 cells and C2C12 myo- cell clusters with enlarged and elongated cells, expressing blasts followed by siRNA knockdown of BMPRs in H9C2 cytoplasmic α-actinin2 (green, Figure 6d). Smad1/5/8 was and C2C12 cells. We found that BMPR1a and BMPR2 expressed as well, suggestive of stretch-associated activa- responded selectively to BMP10 in H9C2 cells, but not in tion of the BMP10-downstream pathway. WT-BMP10 cells C2C12 myoblasts derived from skeletal muscle. Thus, we (Figure 6e) were also elongated but separated from each used H9C2 cells and found several important mutation- other and expressed α-actinin2, suggesting the ability to resist induced effects. We showed that V407I-BMP10 is expressed the mechanical stretch. In contrast, mutant cells (Figure 6f) as dotted spots, unlike the diffuse expression of WT-BMP10, were distorted, damaged and most of them underwent cell suggesting that mutant protein may form aggregates in the death. To evaluate cell death, we counted the number of cytosol. We also found that mutant cells contained vast DAPI-stained nuclei per field and assessed nuclear mor- cytosolic and nuclei vacuoles, suggesting that V407I may phology (fragmentation and chromatin condensation) as affect endosomal trafficking of BMP10. BMP-induced described previously,21 and found a significant reduction in SMAD phosphorylation in early endosomes and spatial the number of DAPI-stained nuclei and an increase in the BMP/SMAD segregation due to inhibition of dynamin- percentage of damaged nuclei in mutants from day 0 (9%) dependent endocytosis have been shown to affect differen- to day 1 (52%) and 3 (70%) in response to stretch (Figure 6, tiation in C2C12 cells.20 This suggests that an advanced Right lower panel) compared with the control cells (4%, 4% investigation of how the V407I-BMP10 affects endocytic to 7%, Right upper panel) or WT cells (6%, 8% to 11%, pathways may clarify the mechanisms of the vacuole accu- Right middle panels), respectively. These data suggest the mulation in the present mutant cells. detrimental effects of V407I-BMP10 on cellular tolerance Regarding cellular proliferation, we showed that over- to mechanical stretch. expression of WT-BMP10 transiently preserved a high rate of proliferation in differentiating H9C2 cells, consistent Discussion with previously observed enhanced cardiomyocyte prolif- eration in embryonic myocardium in response to increased The genetic basis of the LVNC phenotype is extremely het- expression of BMP10.22 In contrast, differentiating mutant erogeneous, and the abnormal process of maturation and cells had a persistently suppressed rate of proliferation, compaction in the last stage of embryonic endomyocardial suggestive of inhibitory effects of mutant V407I-BMP10 on morphogenesis is a widely accepted hypothesis for the devel- the proliferation process. During differentiation, myofila- opment of LVNC.2,9 BMP10 is one of the critical NOTCH- ment organization is regulated by α-actinin2, which links associated molecules involved in embryonic cardiomyoblast the membrane-associated proteins with actin, initiating the proliferation and differentiation.7,16 An increase in BMP10 assembly of primitive Z-bodies.19 In the present study, expression enhances cardiomyocyte proliferation in embry- immunochemistry demonstrated diffuse cytoplasmic over- onic myocardium, resulting in hypertrabeculation and non- lapping of WT-BMP10 with α-actinin2 in H9C2 myoblasts. compaction of ventricular walls via direct upregulation of Conversely, we observed less overlap between V407I-BMP10 Tbx20 and Hey2 and downregulation of p57kip2.8,22 In the and α-actinin2 due to the fact that α-actinin2 remained at postnatal myocardium, persistent overexpression of BMP10 the cellular periphery, suggestive of disturbed cytoskeletal has been shown to prevent cardiomyocyte hypertrophic differentiation in mutant cells. growth.10 In contrast, BMP10 deficiency in embryonic heart Finally, development of the compacted myocardium results in the development of a hypoplastic ventricular wall occurs in the mid-gestational period of cardiac develop-

Circulation Journal Vol.83, August 2019 1746 HIRONO K et al. ment, under constant mechanical and shear stretch created 5. Luxan G, Casanova JC, Martinez-Poveda B, Prados B, D’Amato by cyclic contractions and blood fow.18 Therefore, we also G, MacGrogan D, et al. Mutations in the NOTCH pathway tested the impact of mechanical cyclic stretch in H9C2 regulator MIB1 cause left ventricular noncompaction cardiomy- opathy. Nat Med 2013; 19: 193 – 201. single cell cultures and observed detrimental effects of the 6. Neuhaus H, Rosen V, Thies RS. Heart specific expression of mouse mutant V407I-BMP10 on cellular tolerance to cyclic stretch. BMP-10 a novel member of the TGF-beta superfamily. Mech Dev We have therefore demonstrated that the V407I variant in 1999; 80: 181 – 184. BMP10 disturbed protein (reduced receptor binding), 7. Grego-Bessa J, Luna-Zurita L, del Monte G, Bolos V, Melgar P, Arandilla A, et al. Notch signaling is essential for ventricular expression/trafficking (forming dotted spots) and cellular chamber development. Dev Cell 2007; 12: 415 – 429. function (vacuoles in the cytoplasm and nuclei, altered 8. Huang J, Elicker J, Bowens N, Liu X, Cheng L, Cappola TP, et proliferation/differentiation and intolerance to mechanical al. Myocardin regulates BMP10 expression and is required for stretch). heart development. J Clin Invest 2012; 122: 3678 – 3691. 9. Chen H, Zhang W, Li D, Cordes TM, Mark Payne R, Shou W. Analysis of ventricular hypertrabeculation and noncompaction Conclusions using genetically engineered mouse models. Pediatr Cardiol 2009; 30: 626 – 634. The V407I-BMP10 variant is most likely associated with 10. Chen H, Yong W, Ren S, Shen W, He Y, Cox KA, et al. Overex- pression of bone morphogenetic protein 10 in myocardium dis- LVNC and may contribute to detrimental alterations in rupts cardiac postnatal hypertrophic growth. J Biol Chem 2006; cardiac myocytes, maturing under contractile and shear 281: 27481 – 27491. stretch. These effects are potentially associated with decreased 11. Purevjav E, Varela J, Morgado M, Kearney DL, Li H, Taylor binding of mutant V407I-BMP10 to both BMPR1a and MD, et al. Nebulette mutations are associated with dilated car- diomyopathy and endocardial fibroelastosis. J Am Coll Cardiol BMPR2 receptors. 2010; 56: 1493 – 1502. 12. Falls DL. Neuregulins: Functions, forms, and signaling strate- Study Limitations gies. Exp Cell Res 2003; 284: 14 – 30. The present genetic studies identified the V407I-BMP10 13. Kramer R, Bucay N, Kane DJ, Martin LE, Tarpley JE, Theill variant in a Latino family with a history of LVNC. The LE. Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development. Proc Natl Acad Sci USA molecular studies showed promising data on disturbed 1996; 93: 4833 – 4838. BMP10 protein functions and associated cellular processes 14. Kohda M, Tokuzawa Y, Kishita Y, Nyuzuki H, Moriyama Y, in vitro, but we were unable to fully analyze genotype–phe- Mizuno Y, et al. A comprehensive genomic analysis reveals the notype correlations in the extended family, because the genetic landscape of mitochondrial respiratory chain complex deficiencies.PLoS Genet 2016; 12: e1005679. maternal relatives were not available for genetic screening. 15. Zhang W, Chen H, Qu X, Chang CP, Shou W. Molecular mech- In addition, not all protein interactions and physiological anism of ventricular trabeculation/compaction and the pathogen- consequences were studied, and definitive pathogenetic esis of the left ventricular noncompaction cardiomyopathy (LVNC). mechanisms are still lacking. We emphasize that the cre- Am J Med Genet C Semin Med Genet 2013; 163C: 144 – 156. 16. Cai J, Pardali E, Sanchez-Duffhues G, ten Dijke P. BMP signal- ation of a mutant animal model of the V407I-BMP10 ing in vascular diseases. FEBS Lett 2012; 586: 1993 – 2002. variant will greatly contribute to the understanding of the 17. Sun L, Yu J, Qi S, Hao Y, Liu Y, Li Z. Bone morphogenetic pathogenesis and pathophysiology of LVNC. protein-10 induces cardiomyocyte proliferation and improves cardiac function after myocardial infarction. J Cell Biochem 2014; Acknowledgments 115: 1868 – 1876. 18. Ji RP, Phoon CK, Aristizabal O, McGrath KE, Palis J, Turnbull The authors acknowledge Dr. Akinori Kimura (Tokyo Medical and DH. Onset of cardiac function during early mouse embryogenesis Dental University, Tokyo, Japan) for providing BMP10 and BMPR coincides with entry of primitive erythroblasts into the embryo constructs; Dr. Yukiko Hata (University of Toyama, Toyama, Japan) proper. Circ Res 2003; 92: 133 – 135. for assisting in in silico analysis; and N. Scott Blair, BS and Bishoy 19. Otey CA, Carpen O. Alpha-actinin revisited: A fresh look at an Sadek, MS (CCHMC) for their assistance in obtaining adenoviral old player. Cell Motil Cytoskeleton 2004; 58: 104 – 111. vectors and qPCR experiments. 20. Heining E, Bhushan R, Paarmann P, Henis YI, Knaus P. Spatial segregation of BMP/Smad signaling affects osteoblast differen- Disclosures tiation in C2C12 cells. PLoS One 2011; 6: e25163. 21. Cummings BS, Wills LP, Schnellmann RG. Measurement of cell The authors declare no conficts of interest. death in Mammalian cells. Curr Protoc Pharmacol 2012; 56: 12.8.1 – 12.8.24. Funding 22. Zhang W, Chen H, Wang Y, Yong W, Zhu W, Liu Y, et al. Tbx20 is a downstream mediator for bone This study was supported by National Institutes of Health HL53392 morphogenetic protein-10 in regulating cardiac ventricular wall and HL087000 R01 grants (J.A.T.), Johnson & Johnson Codman development and function. J Biol Chem 2011; 286: 36820 – 36829. Neurovascular Fellowship for Pediatric Interventional Cardiology, 23. Chen H, Shi S, Acosta L, Li W, Lu J, Bao S, et al. BMP10 is Japan (K.S.), Takeda Science Foundation, Japan (K.H.). essential for maintaining cardiac growth during murine cardio- genesis. Development 2004; 131: 2219 – 2231. References 24. Nomura-Kitabayashi A, Phoon CK, Kishigami S, Rosenthal J, 1. Towbin JA. Inherited cardiomyopathies. Circ J 2014; 78: 2347 – Yamauchi Y, Abe K, et al. Outfow tract cushions perform a 2356. critical valve-like function in the early embryonic heart requiring 2. Towbin JA, Jefferies JL. Cardiomyopathies due to left ventricular BMPRIA-mediated signaling in cardiac neural crest. Am J 297: noncompaction, mitochondrial and storage diseases, and inborn Physiol Heart Circ Physiol 2009; H1617 – H1628. errors of metabolism. Circ Res 2017; 121: 838 – 854. 3. Gati S, Rajani R, Carr-White GS, Chambers JB. Adult left ven- Supplementary Files tricular noncompaction: Reappraisal of current diagnostic imag- ing modalities. JACC Cardiovasc Imaging 2014; 7: 1266 – 1275. Please find supplementary file(s); 4. 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Circulation Journal Vol.83, August 2019