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1Department of Molecular Pathogenesis, Medical Research Institute, and 2Laboratory of Genome Diversity, School of Biomedical Science, Tokyo Medical and Dental University, Tokyo; 3Department of Internal Medicine and Cardiology, Kitasato University School of Medicine, Kanagawa; 4Tokyo Metropolitan Geriatric Hospital, Tokyo; 5Nagasaki Medical Center, National Hospital Organization, Nagasaki; 6Department of Medicine and Geriatrics, Kochi University School of Medicine, Kochi; and 7Department of Geriatric Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
Submitted 13 March 2007 ; accepted in final form 11 September 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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bone morphogenetic protein-10; wall stress; Z disc; cardiomyopathy
-actinin, and plays pivotal roles in sarcomere assembly, force generation, and force transmission (16). A functional key molecule of the Z disc is a 19-kDa protein, titin-cap (Tcap)/telethonin, whose interaction with titin, muscle Lin 11, Isl-1, and Mec-3 (LIM) protein (MLP), and mini K+ channel (minK) appears to be a crucial; it acts as an adaptor protein for the assembly of biomechanically active signaling complexes (8, 10, 11). Tcap binds to MLP, and mutations in these molecules cause dilated cardiomyopathy (DCM) (11, 16), a heart muscle disease characterized by dilatation and impaired contraction of left ventricle (LV). Tcap also binds to a member of transforming growth factor-β (TGF-β), myostatin (also known as growth differentiation factor, GDF8), within skeletal myoblasts and regulates skeletal muscle growth (23), and a myostatin mutation causes muscular disease in humans (28). These observations implied that abnormalities in the stretch-sensing Z disc are one of the molecular mechanisms involved in the skeletal and heart muscle disorders. Previous studies showed that knockout mice of Nkx2.5 or FKBP12 developed myocardial hypertrophy with excessive trabeculation, where the expression of a cardiac-restricted morphogen, bone morphogenetic protein-10 (BMP10), was upregulated, since BMP10 is negatively regulated by Nkx2.5 and FKBP12 (4, 25, 30). BMP10 is a member of the TGF-β family and translated as a precursor protein (preproprotein) with NH2-terminal hydrophobic leader peptide followed by prodomain and the mature COOH-terminal region that is released after the cleavage at multibasic motif RXRR (22, 31). In clear contrast to the mice with overexpression of BMP10, mice deficient for BMP10 showed hypoplastic and thin ventricles with little trabeculation (4). These data strongly suggest that BMP10 may promote the growth and lineage specification of cardiomyocytes, and its mutation, if any, would affect cardiac function and lead to heart diseases.
In this study, we investigated the role of BMP10 in hypertensive heart disease. We demonstrated that BMP10 was upregulated during the hypertension-induced cardiac hypertrophy in a rat model. We also found that BMP10 bound and colocalized with Tcap. A variant of BMP10, Thr326Ile, was found in patients with hypertensive DCM, and the variant reduced the binding to Tcap and, in turn, augmented the extracellular secretion of BMP10 and promoted the hypertrophy of cardiomyocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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RT-PCR and real-time PCR. Total RNA and protein were isolated simultaneously from LV by Trizol (Invitrogen), and mRNA was reverse transcribed with oligo(dT) and Superscript II (Invitrogen). Homogenized tissue pellets were precipitated by isopropanol and washed with 0.3 M guanidine HCl-95% ethanol. Protein extracts were obtained by resuspending the pellets in the lysis buffer as described below. Steady-state level of BMP10 mRNA was measured by real-time PCR (Bio-Rad) with SYBR Green (Applied Biosystems). Three replicates of each sample were amplified for BMP10 and repeated two times with the RNA from at least two animals in each group. All data were standardized by GAPDH expression. Human RNA samples were purchased from Clontech. Sequences of primers used in this study are available on request.
Construct preparation. Full-length or COOH-terminal 321-bp fragments (mature region, 952nt–1272nt of GenBank accession no. NM014482) of human BMP10 cDNA were obtained from heart cDNA library (Clontech) by RT-PCR with oligonucleotide primers, full-Nde-BMP-Fw (5'-CATATGGGCTCTCTGGTCCTGACACTG-3') and Xma-BMP-Rv (5'-CCCGGGCTATCTACAGCCACATTCGG-3'), mature-Nde-BMP-Fw (5'-CATATGAACGCCAAAGGAAACTACTG-3') and Xma-BMP-Rv, or mature-Nde-BMP-Fw and Xho-BMP-Rv (5'-CTCGAGTCTACAGCCACATTCGGAGA-3'), respectively. These cDNAs were TA cloned into pCR2.1 (Invitrogen). Underlined letters in primer sequences correspond to restriction enzyme sites. To introduce the Thr326Ile variation into cloned BMP10, we used primer-mediated mutagenesis (13) with oligonucleotide primers, BMP-T326I-Fw (5'-ACTGTAAGAGGATCCCGCTC-3') and BMP-T326I-Rv (5'-AGTTTCCTTTGGCGTTCCTT-3'), and the obtained fragment was cloned into pCR2.1. Full-length human TCAP cDNA was obtained with oligonucleotide primers, Eco-TCAP-Fw (5'-GAATTCATGGCTACCTCAGAGCTG-3') and Bam-TCAP-Rv (5'-GGATCCGCCTCTCTGTGCTTCCTG-3'). Each BMP10 cDNA insert from pCR2.1 was subcloned into pGADT7 (Clontech), while the human TCAP cDNA was subcloned into pGBKT7 (Clontech). To generate enhanced green fluorescent protein (EGFP)-tagged expression vectors, NdeI/XmaI-digested inserts from pCR2.1 plasmids containing wild-type (WT) or variant BMP10 were blunt ended by T4 DNA polymerase (TOYOBO) and subcloned into pEGFP-C1 (Clontech) at the SmaI site. Human TCAP cDNA was also subcloned into Flag-pcDNA vector (Sigma-Aldrich). All the plasmid clones were sequenced to ensure that no errors were introduced during the PCR and cloning procedures.
Yeast two-hybrid assays. Saccharomyces cerevisiae (Y187 strain) were transformed with Tcap-pGBKT7 and BMP10-pGADT7 constructs on selective plates lacking leucine and tryptophan. Transformants grown after incubation for 4–5 days at 30°C were measured for β-galactosidase (β-gal) activity with o-nitrophenyl-β-D-galactopyranoside as substrate in independent transformants (n = 18) as described previously (2, 11).
Immunoblotting and immunoprecipitation.
For transient transfection, COS7 cells (American Type Culture Collection) were maintained in DMEM (Sigma) containing 10% fetal bovine serum (FBS; Hyclone) with 1% penicillin-streptomycin (Gibco) and transfected with 1 µg of Flag-Tcap vector, 4 µg of EGFP vector, and 10 µl of Lipofectamine 2000 (Invitrogen). Thirty-six hours after the transfection, conditioned medium were stocked for the hypertrophy assays, and cells were harvested in lysis buffer (50 mM Tris·HCl, pH 7.5, 250 mM NaCl, 0.5% Nonidet P-40). The protein content was measured by the assay reagent (Bio-Rad). Flag-tagged proteins were incubated with 4 µg of anti-Flag antibody (Sigma-Aldrich, clone M2) overnight, trapped by protein G beads (Amersham). After denaturing of the immunoprecipitants or tissue lysates in a reduced condition with 2% β-mercaptoethanol, samples were subjected to 6 or 13.5% SDS-PAGE. Proteins transferred to polyvinylidene fluoride filters (Millipore) were blocked with 5% nonfat dry milk. Filters incubated with appropriate primary antibodies were reacted with secondary antibodies labeled with horseradish peroxidase (Amersham Pharmacia Biotech) and visualized with enhanced chemiluminescence reagent (NEN). Signals of proteins were quantified by densitometry using NIH IMAGE v.1.59. Protein levels of EGFP-BMP10 were normalized relative to those of Tcap. Monoclonal antibodies for β-myosin heavy chain (MAB1628), -actinin (EA-53), or EGFP (JL-8) were purchased from CHEMICON, Sigma-Aldrich, and Clontech, respectively.
Recombinant protein and antibody production.
The mature BMP10 cDNA that had NdeI/XhoI restriction sites in pCR2.1 was subcloned into pET23a (Novagen) and transformed into Escherichia coli (BL21 Gold DE3, Stratagene). Transformed bacteria were inoculated into 100 ml of culture at 37°C for 
2–3 h with vigorous shaking. After optical density at 600 nm (OD600) reached 0.6–1.0, isopropyl-thio-β-D-galactopyranoside was added at a final concentration of 0.4 mM, and the culture was continued for an additional 4 h to induce the expression of COOH-terminally His6-tagged protein. The recombinant BMP10 protein was purified under denaturing conditions in 6 M urea by use of nickel-nitriloaminetriacetic acid resin (Qiagen) and dialyzed with decreasing concentrations of urea, by 0.5 M every 6 h. Finally, protein was dialyzed against PBS containing 0.1% sodium acetate at 4°C overnight. By immunization of mice with the recombinant BMP10, a monoclonal antibody was raised by use of standard techniques. Hybridoma cells secreting anti-BMP10 antibodies were screened by ELISA to recombinant BMP10, and the specificity of antibody was determined by use of various recombinant BMP10 proteins (data not shown).
Preparation of neonatal rat ventricular cardiomyocytes. Neonatal cardiomyocytes were isolated from 1- or 2-day-old Sprague-Dawley rat hearts by mincing and repetitive digestion with 0.2% collagenase type II (Worthington) and then purified by discontinuous gradient method with Percoll (20). Cells were plated at a density of 2 x 105 cells/ml on the collagen I-coated eight-well coverslips (Becton Dickinson) in DMEM (Sigma-Aldrich) containing 10% FBS (Hyclone) and 1% penicillin-streptomycin for 24 h.
Localization and effect of BMP10 on cardiomyocytes. For transient transfection into neonatal cardiomyocytes, culture medium was replaced with 10% FBS-DMEM without antibiotic, and 1 µg of EGFP constructs was overlaid onto each well with 2 µl of Lipofectamine 2000. Twenty-four hours after the transfection, cardiomyocytes were stained as described below. For the hypertrophy assays, isolated cardiomyocytes maintained in 10% FBS-DMEM for 1 day were replaced with 1% FBS-DMEM. Aliquots of conditioned medium from COS7 cells cotransfected with Flag-Tcap and either EGFP-alone control, WT-BMP10-EGFP, or variant BMP10-EGFP were added to culture medium of cardiomyocytes, where a final concentration of added conditioned medium was 0.2, 1, or 5% of total culture volume. Forty-eight hours after the addition of conditioned medium, cardiomyocytes were washed, fixed, and stained as described below.
Immunofluorescence analysis.
Cardiomyocytes on the coverslips were fixed with 4% formaldehyde in PBS for 10 min and incubated with PBS containing 5% bovine serum albumin for 1 h. The cells were treated with anti--actinin (1:800, Sigma-Aldrich) or anti-Tcap (1:100, Pharmingen) antibody and incubated with either FITC-labeled (Dako) or tetramethyl rhodamine isothiocyanate-labeled anti-mouse antibody (Zymed). For staining F-actin, cells were incubated with rhodamine-labeled phalloidin (Molecular Probe). Stained cells were mounted in Vectashield (Vector) and investigated under a laser confocal microscope (Axioplan2 MOT; Carl Zeiss) using the x40 or x63 objective. Micrographs were recorded as digital images on a charge-coupled device camera and processed for presentation using Adobe Photoshop 4.0. Randomly selected images were captured, and cell area was semiautomatically measured for 50 cells using NIH IMAGE v.1.59.
Human subjects. To search for variations in human BMP10, we examined 36 familial and 97 nonfamilial cases with idiopathic DCM and 46 cases with hypertensive DCM (HT-DCM). DCM was diagnosed by echocardiography, coronary angiography, and endomyocardial biopsy (2, 11). All idiopathic DCM patients were confirmed as not having systemic diseases or disease-causing mutations for cardiomyopathies (2, 11). HT-DCM was defined as hypertension-induced DCM where systolic dysfunction was not improved despite intensive medical care and blood pressure control. Presence of sequence variation was tested in 288 unrelated healthy Japanese selected at random and 1,382 consecutive autopsied elderly individuals who were confirmed to be without HT-DCM. Autopsied heart tissues from two individuals without heart disease were investigated for BMP10 expression by immunostaining. The study protocol was approved by the Ethics Reviewing Committee of the Medical Research Institute, Tokyo Medical and Dental University, and that of the Tokyo Metropolitan Geriatric Hospital.
Search for genomic variation in the human BMP10 gene. Genomic DNA was extracted from peripheral blood or autopsied samples (21), and all exons of human BMP10 gene including partial 5'- and 3'-untranslated region and intron were amplified by PCR with primers (sequences are available on request). The PCR products were analyzed by the single-strand conformation polymorphism (SSCP) method (15), and when abnormal patterns were observed, they were cloned into pCR2.1 (Invitrogen); 10 independent clones were sequenced on both strands to verify the sequence variations. Presence of the Thr326Ile variant was confirmed by digestion of the PCR products by BamHI.
Statistical analysis. The frequency of the BMP10 variant in the HT-DCM patients was compared with that in the control group. Odds ratio was calculated to estimate the strength of association from a 2 x 2 table, and statistical significance was calculated by Fishers exact probability test. Numerical assay data are expressed as means ± SD. Student's-t test was used to estimate statistical significance of the variance with a significance level of P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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Colocalization and binding of BMP10 with Tcap. No data are available for the cytoplasmic localization and binding partner of BMP10, but myostatin, another TGF-β family protein that is specifically expressed in the skeletal muscle, is reported to bind Tcap (23). Because we found that BMP10 localized to the Z disc, and its expression is confined to the cardiac muscle, we investigated whether BMP10 could bind Tcap. EGFP-tagged mature BMP10 was transiently expressed in neonatal cardiomyocytes followed by the immunostaining with anti-Tcap antibody. Control cells transfected with EGFP-alone construct exhibited a diffuse expression of EGFP (Fig. 2Ba) not colocalized with endogenous Tcap at the Z disc (Fig. 2B, b and c). When mature BMP10-EGFP was introduced, its expression showed a striated pattern (Fig. 2B, d and f) in colocalization with Tcap (Fig. 2Be).
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Direct interaction of BMP10 with Tcap was further ascertained by immunoprecipitation analysis (Fig. 2C). When COS7 cells were cotransfected with Tcap-Flag and EGFP-alone vector, EGFP protein was not detected in the fraction precipitated by anti-Flag (Fig. 2C,top,lane 1). From COS cells transfected with Tcap-flag and BMP10-EGFP constructs, BMP10-EGFP protein was not detected in the fraction precipitated by irrelevant IgG (Fig. 2C, top, lane 2). In clear contrast, BMP10-EGFP was coprecipitated with Tcap-Flag, i.e., BMP10-EGFP was found in the fraction precipitated with anti-Flag (Fig. 2C, top, lane 3). These data suggested that localization of BMP10 at the Z disc was mediated by its binding to Tcap. Aside from the role in sarcomere assembly at the Z disc, Tcap also links the Z disc and sarcolemmal T-tubule via interacting with minK, a subunit of the minK/KvLQT1 channel complex (8), and thus Tcap may play a role in electrical feedback for the mechanical stretch. The subcellular localization of BMP10 at the Z disc and its interaction with Tcap imply that BMP10 might be involved in the sensing mechanism of increased wall stress.
Hypertrophic effect of BMP10 on neonatal rat cardiomyocytes.
Because the sensing of wall stress is an initial phase of the hypertrophic response in the heart, the binding of BMP10 to Tcap, similar to myostatin, suggested that BMP10 might function as a prohypertrophic hormone. To test this hypothesis, aliquots of conditioned medium obtained from transfected cells (as shown in Fig. 2C) were added to cultured medium of neonatal rat cardiomyocytes, and the cells were stained with anti--actinin. As shown in Fig. 3, cardiomyocytes cultured with the conditioned medium of BMP10-transfected cells were enlarged compared with those cultured with the control conditioned medium (Fig. 3A, d and a, respectively). It was observed in the cardiomyocytes cultured with control medium that -actinin, a marker for the Z disc, represented a dot-like pattern, and stress fiber-like organization still remained (Fig. 3Ab), whereas phalloidin staining (F-actin) demonstrated a filamentous, unpolymerized pattern (Fig. 3Ac). These features of sarcomeric structure were those observed for the immature cardiomyocytes (1, 7). In contrast, conditioned medium from BMP10-transfected cells induced polymerized, typical cross-striated assembly of -actinin and F-actin (Fig. 3B, e and f, respectively), suggesting that BMP10 secreted from the transfected cells promoted cellular hypertrophy and matured sarcomeric assembly in neonatal cardiomyocytes.
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-actinin-stained cardiomyocytes was measured. As shown in Fig. 3B, BMP10 conditioned medium induced significant hypertrophy of neonatal cardiomyocytes compared with control medium (946.6 ± 122.4 vs. 611.1 ± 94.3 µm2, respectively, n = 3, *P < 0.05). Two explanations were possible as to how BMP10 might exert the hypertrophic effect over cardiomyocytes. One was that secreted BMP10 directly promoted the hypertrophy of cardiomyocytes. The other was that an unknown secreted peptide induced by BMP10 indirectly stimulated the hypertrophy in a dependent manner by Nkx2.5 and/or myocyte enhancer factor-2C transcriptional controls (4, 25). Whichever it might be, our observations suggested that BMP10 sufficiently promoted the hypertrophy of neonatal cardiomyocytes, and, in turn, BMP10 played a hypertrophic role in the hypertensive heart. Association of Thr326Ile variation with hypertensive cardiomyopathy. About 5% of hypertensive patients eventually developed systolic dysfunction despite having blood pressure similar to others who did not develop it, suggesting that genetic factors might play pivotal roles in the transition from compensated hypertrophy to heart failure (17). This systolic dysfunction status is thus called HT-DCM. Because this study implicated the involvement of BMP10 in the hypertrophic response in hypertensive rats, BMP10 might play a role in the pathogenesis of human heart disease such as HT-DCM. To investigate the possibility, we searched for sequence variations in the human BMP10 gene. An SSCP analysis was performed in 46 HT-DCM patients, 133 patients with idiopathic DCM who were not hypertensive, and 288 healthy controls. Three variations were found in the patients: a C-to-T transition at +26 in 5'-untranslated region, a GAT-to-GAC change at codon 242 in exon 2 (synonymous polymorphism), and an ACC-to-ATC change at codon 326, which substitutes threonine into isoleucine (hereafter Thr326Ile, codon nos. are from GenBank accession no. NP055297). The former two were common polymorphisms, because their frequencies in the DCM patients and controls were not significantly different. In contrast, the Thr326Ile variation was found in two HT-DCM patients (4.3%; Table 1). The variant was found in the father of an HT-DCM patient who also suffered from hypertensive cardiomyopathy. In contrast, the variation was not found in 133 idiopathic DCM patients and was detected in only 1 of 288 healthy controls (0.0 and 0.35%; Table 1). We further confirmed the significant association of this Thr326Ile variation with HT-DCM by analyzing a larger population. In the 1,382 elderly consecutive autopsied cases diagnosed as negative for DCM, 616 had hypertension and 766 did not (Table 1). One variant was found in hypertensive cases and one in nonhypertensive cases (0.16 and 0.13%, respectively; Table 1). Therefore, the variant was rare in Japanese but significantly more prevalent in the HT-DCM patients than in the others (Table 1). Representative data of the SSCP analysis, DNA sequencing, and BamHI digestion pattern are shown in Fig. 4, A–C, respectively.
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Reduced interaction of Thr326Ile BMP10 with Tcap.
To demonstrate the possible functional alteration caused by the Thr326Ile variant, we investigated the amounts of extracellular BMP10 by direct immunoblotting of conditioned medium from COS7 cells transfected with Tcap and BMP10. As shown in Fig. 5, contents of bovine IgG were comparable among the conditioned media, indicating that equal amounts of samples were loaded. In the control medium, EGFP was not detected (Fig. 5A, lane 1). In contrast, the Thr326Ile variant increased extracellular secretion compared with WT-BMP10 (Fig. 5A, lanes 2 and 3, respectively; 2.1 ± 0.1-fold over WT-BMP10 medium). To reveal the functional change caused by the Thr326Ile variation, these conditioned media were added to the culture media for neonatal rat cardiomyocytes, and the area size of -actinin-stained cardiomyocytes was measured. Interestingly, the hypertrophic effect of adding 0.2% conditioned medium from cells transfected with the variant BMP10 construct was larger than that from cells transfected with WT-BMP10 (Fig. 5B; 696.5 ± 119.6 vs. 611.1 ± 94.3 µm2, n = 3, P < 0.05). Similar results were observed for addition of 1% conditioned medium (Fig. 5B; 884.6 ± 138.1 vs. 693.1 ± 130.4 µm2, n = 3, P < 0.05). These observations suggested that the hypertrophic stimulation was proportional to the concentration of active BMP10 in the conditioned medium, and that the variant BMP10 might have a hypertrophic activity equivalent to that of WT-BMP10, except for its increased secretion efficacy. This was similar to a finding that Tcap could reduce the extracellular secretion of endogenous myostatin by retaining it within the myoblasts (23). To investigate whether the retention of BMP10 within the cells might be altered by the variation, cell lysates used in the transfection experiments as shown in Fig. 2B were immunoblotted with anti-EGFP (to detect BMP10-EGFP proteins). As expected, the intracellular amount of BMP10 was significantly reduced by the Thr326Ile variation (compare lanes 3 and 4 in Fig. 5C; 59.5 ± 8.9% vs. WT, n = 3, P < 0.05).
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Cleaved prodomains of myostatin, BMP11, and BMP9 were reported to form a noncovalent link with their mature region (3, 9, 32), and prodomain/ligand complexes were found in blood circulation. Thr326Ile variation in the mature region of BMP10 seems not to interfere with the binding of ligands with receptors, judging from the crystal structure models of mature BMP9 in association with type I and II receptors (3). Also, this variation is assumed not to affect the cleavage of the mature region, as codon 326 exists outside the RXRR cleavage site. Interestingly, the NH2-terminal regions of mature BMPs are supposed to have acquired different properties of their own diffusions within the extracellular matrix through editing during evolution (24). The NH2-terminal region of mature BMP10 may also have the regulatory mechanisms for the concentration gradient through the association with Tcap. In addition, at the NH2 terminus of mature BMP9, myostatin and BMP10, caveolin scaffolding domain (CSD; 
XXXXXX, where represents an aromatic amino acid such as tryptophan, phenylalanine, or tyrosine) is located (3). CSD is reported to be involved in the receptor-independent internalization of secreted insulin-like growth factor binding protein-3 (18). Tcap binding affinity might somehow affect the CSD-mediated ligand internalization of BMP10. These important issues are beyond the scope of this study, and their clarification will require further investigation.
A transgenic mouse model with cardiac-specific overexpression of BMP10 has been reported in which the heart was small because postnatal cardiac hypertrophy was disrupted (5). This is not consistent with the findings in this study. The underlying mechanisms remain unknown, but the overexpression of BMP10 early in the postnatal period modifies the course of physiological hypertrophy. Because hypertensive cardiac hypertrophy is a pathological hypertrophy, the mechanism of which is different from that of physiological hypertrophy (19), the role of BMP10 might be different in physiological and pathological cardiac hypertrophy.
In summary, we showed that BMP10 possessed prohypertrophic activity and was upregulated in hypertensive cardiac hypertrophy in a manner similar to that of the other prohypertrophic hormones. We also demonstrated that BMP10 interacted and colocalized with Tcap at the Z disc. A variation of BMP10, Thr326Ile, associated with HT-DCM reduced the binding to Tcap and, in turn, augmented the extracellular secretion of BMP10, thereby enhancing the hypertrophy of cardiomyocytes. The BMP10 variant may be a promoting factor of cardiac hypertrophy to systolic dysfunction in heart failure. This is the first report suggesting the involvement of BMP10 in hypertensive cardiomyopathy in humans.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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