Cardiac Ankyrin Repeat Protein Gene (ANKRD1) Mutations in Hypertrophic Cardiomyopathy
Takuro Arimura, DVM, PhD*,
J. Martijn Bos, MD ,
Akinori Sato, MD*,
Toru Kubo, MD, PhD ,
Hiroshi Okamoto, MD, PhD ,
Hirofumi Nishi, MD, PhD||,
Haruhito Harada, MD, PhD¶,
Yoshinori Koga, MD, PhD¶,
Mousumi Moulik, MD#,
Yoshinori L. Doi, MD, PhD,
Jeffrey A. Towbin, MD**,
Michael J. Ackerman, MD, PhD and
Akinori Kimura, MD, PhD,*
* Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Departments of Medicine, Pediatrics, and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota
Department of Medicine and Geriatrics, Kochi Medical School, Kochi, Japan
Division of Cardiovascular Medicine, Nishi Sapporo National Hospital, Sapporo, Japan
|| Nishi Hospital, Omuta, Japan
¶ Division of Cardiovascular Disease, Kurume University Medical Center, Kurume, Japan
# Department of Pediatrics, Division of Cardiology, University of Texas Medical School Houston, Houston, Texas
** Heart Institute, Department of Pediatrics and Pediatric Cardiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
Laboratory of Genome Diversity, School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan
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Figure 1 Mutational Analyses of ANKRD1 and TTN in HCM
(A) Sequence variations found in ANKRD1. Single-letter code was used to indicate the amino acid residue. Solid boxes represent protein coding region corresponding to exons 1 to 9. Dotted boxes indicate ankyrin repeat domains encoded by exons 5 to 8. (B) Sequence variations found in TTN. Solid boxes represent Ig domains corresponding to exons 98, 99, and 102 to 104. Dotted boxes indicate tyrosine-rich motif encoded by exons 99 to 101. Pedigrees of hypertrophic cardiomyopathy (HCM) families with (C) the ANKRD1 T123M (CM 1288 family) and (D) the TTN R8604Q (CM 1480 family). Filled squares and circles indicate affected male and female patients, respectively. Open squares and circles represent unaffected or unexamined male and female patients with HCM, respectively. An arrow indicates the proband patient. Presence (+) or absence (–) of the mutations is noted.
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Figure 2 Binding of CARP to TTN and MYPN
Binding of cardiac ankyrin repeat protein (CARP) to titin/connectin (TTN) or myopalladin (MYPN) was analyzed by coimmunoprecipitation (co-IP) assays. (A) Myc-tagged CARPs coprecipitated with GFP-tagged TTN-N2A domain were shown (top panel). Expressions of GFP-tagged TTN- N2A (middle panel) and myc-tagged CARP (lower panel) were confirmed by immunoblotting of whole cell supernatants. Binding pairs were wild-type (WT) CARP in combination with WT, I8474T, R8500H, or R8604Q mutant TTN-N2A , or WT TTN-N2A with WT, P52A, T123M, or I280V mutant CARP. Dashes indicate no GFP- or myc-tagged proteins (transfected only with pEGFP-C1 or pCMV-Tag3 vectors, respectively). (B) Densitometric data obtained in the co-IP assay. Data for WT CARP with WT TTN-N2A were arbitrarily defined as 1.00 arbitrary unit (AU). Data are represented as means ± SEM (n = 6 for each case). *p < 0.05 versus WT; **p < 0.01 versus WT; ***p < 0.001 versus WT. (C) Myc-tagged CARP coprecipitated with GFP-tagged full-length MYPN was detected by immunoblotting using anti-myc antibody (top panel). Expressed amounts of GFP-tagged MYPN (middle panel) and myc-tagged CARP (lower panel) were confirmed as in (A). Binding pairs were full-length WT-MYPN with WT, P52A, T123M, or I280V mutant CARP. (D) Densitometric analysis of myc-blotting data in (C). Data were arbitrarily represented as intensities, and that for WT CARP with full length or N-terminal half WT MYPN were defined as 1.00 AU. Data are expressed as means ± SEM (n = 9 for each case). **p < 0.01 versus WT; ***p < 0.001 versus WT.
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Figure 3 Distribution Myc-Tagged CARP in Immature Rat Cardiomyocytes
Neonatal rat cardiomyocytes transfected with myc-tagged wild-type (WT) (A to C) or mutant P52A (D to F), T123M (G to I), or I280V (J to L) cardiac ankyrin repeat protein (CARP) constructs were fixed 18 h after the transfection, and stained with 4'6-diamidino-2-phenylindole (DAPI) and anti– -actinin antibody followed by secondary antibody (B, E, H, K). Merged images (C, F, I, L) are shown. In the immature cardiomyocytes showing nascent myofibrils with Z bodies (Z-disc precursors), myc-tagged CARPs were preferentially localized to the nucleus, and mutant CARP showed relatively low expression in the cytoplasm. Scale bar = 10 µm.
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Figure 4 Distribution of Myc-Tagged CARP in Mature Rat Cardiomyocytes
Neonatal rat cardiomyocytes transfected with myc-tagged wild-type (WT) (A to C) or mutant P52A (D to F), T123M (G to I), or I280V (J to L) cardiac ankyrin repeat protein (CARP) constructs were fixed 48 h after the transfection, and stained with 4'6-diamidino-2-phenylindole (DAPI) and anti–-actinin antibody followed by secondary antibody (B, E, H, K). Merged images (C, F, I, L) are shown. In the mature cardiomyocytes showing myofibrils with Z-discs, normal localization of myc-tagged WT CARP at the Z-discs was observed (A to C). In contrast, myc-tagged mutant CARP proteins showed intense localization at the I-discs (colocalization with -actinin) and diffused localization in the cytoplasm (D to F, G to I, J to L). In addition, myc-tagged mutant CARPs expressed at high levels around the nuclear membrane (white arrows) and/or in the nucleus (white arrowheads). Scale bar = 10 µm.
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