Expression and Subcellular Localization of Mammalian Formin Fhod3 in the Embryonic and Adult Heart

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain

Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis.

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain

Mammalian formin Fhod3 plays an essential role in cardiogenesis by organizing myofibrillogenesis

Summary Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3−/− mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3−/− embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development

Localization of Fhod3 in the adult heart.

<p>(A) Glycerinated fibers from adult mouse hearts were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 5 µm. (B) Sections of adult mouse hearts were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green) followed by phalloidin staining (not shown in merge). For Fhod3 staining, the anti-Fhod3-(650–802), the anti-Fhod3-(873–974), and the anti-Fhod3-(C-20) polyclonal antibodies were used. The anti-Fhod3-(C-20) antibodies used here were pre-adsorbed with an acetone powder of mouse embryonic fibroblast derived from Fhod3 knockout mice. For details, see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034765#s4" target="_blank">Materials and Methods</a>”. Bar, 2 µm. (C) Sections of tissues from a left ventricle of a human heart were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 2 µm. (D) Sections of tissues from a left ventricle of two patients (#1 and #2) with idiopathic dilated cardiomyopathy (DCM) and a patients with idiopathic hypertrophic cardiomyopathy (HCM) were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 2 µm.</p

Localization of Fhod3 in the heart of Fhod3 transgenic mice.

<p>(A) Sections of adult hearts from transgenic mice that specifically express a high amount of the T(D/E)₅XE-region-deficient Fhod3 protein in the heart were subjected to immunofluorescent double staining for Fhod3 (red) and α-actinin (green), followed by phalloidin staining. For Fhod3 staining, the anti-Fhod3-(C-20) (top panels), the anti-Fhod3-(650–802) (middle panels), or the anti-Fhod3-(873–974) (bottom panels) polyclonal antibodies were used. Bar, 2 µm. (B) Sections of adult hearts from Fhod3 transgenic mice were subjected to immunofluorescent double staining for Fhod3 (red) and myomesin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 2 µm. (C) Magnified image of single myofibril from immunostained sections of adult hearts of Fhod3 transgenic mice. Sections were stained for α-actinin (green), phalloidin (blue), and Fhod3 or Tmod1 (red). For Fhod3 staining, the anti-Fhod3-(650–802) (top panels), the anti-Fhod3-(873–974) (2nd panels), and the anti-Fhod3-(C-20) (3rd panels) polyclonal antibodies were used. Bar, 2 µm. (D) Fluorescence intensity profiles in a line scan of sarcomeres. Line scan profiles of fluorescence intensities for the anti-α-actinin antibody (green) and the anti-Fhod3-(C-20) or anti-Tmod antibodies (red) are generated from immunofluorescent images shown in <i>C</i>. (E) Distance between the Z-line and Fhod3 and that between the Z-line and Tmod. The distance of the fluorescence peak of Fhod3 or Tmod from that of α-actinin are measured on immunofluorescent images. Box-and-whisker plots indicate 25th percentile (bottom line), median (middle line), 75th percentile (top line), and nearest observations within 1.5 times the interquartile range (whiskers). *, <i>P</i><0.001, Welch's <i>t</i> test. (F) Magnified image of single myofibril from immunostained sections of adult hearts of Fhod3 transgenic mice. Sections were stained for Fhod3 (red), Tmod1 (green), and phalloidin (blue). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. (G) Ultrastructural localization of Fhod3. Ultrathin cryosections of adult hearts from Fhod3 transgenic mice were immunolabeled using the anti-Fhod3-(650–802) antibodies, and labeling was detected using gold-conjugated secondary antibodies.</p

Localization of Fhod3 in the embryonic heart.

<p>(A) Embryonic mouse cardiomyocytes were subjected to immunofluorescent double staining for endogenous Fhod3 (green) and α-actinin (red). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 5 µm. (B) Magnified image of single myofibril from immunostained embryonic mouse cardiomyocytes. (C) Sections of mouse embryonic hearts were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(C-20) (top panels), the anti-Fhod3-(650–802) (middle panels), and the anti-Fhod3-(873–974) (bottom panels) polyclonal antibodies were used. Bar, 2 µm.</p

Effect of the T(D/E)₅XE region of Fhod3 on actin assembly.

<p>(A) Effect of the Fhod3 T(D/E)₅XE region encoded by exon 25 on <i>in vivo</i> actin assembly. HeLa cells were transfected with plasmids encoding Fhod3-ΔN (wt) (amino acids 931–1,586) with or without the T(D/E)₅XE region in the FH2 domain; or plasmids encoding Fhod3-ΔN (I1127A) with or without the T(D/E)₅XE region. Cells were fixed and visualized by GFP-fluorescence (green) or phalloidin staining (red). Scale bar, 10 µm. (B) SDS-PAGE analysis of purified proteins used in an actin polymerization assay. Purified proteins were subjected to 10% SDS-PAGE and stained with <i>Coomassie Brilliant Blue</i>. Positions for marker proteins are indicated in kDa. (C) Effect of the T(D/E)₅XE region of Fhod3 on <i>in vitro</i> actin assembly. G-actin (10% pyrene-labeled) at 2 µM was incubated with 50 nM Fhod3-T(D/E)₅XE(+)-ΔN (wt), Fhod3-T(D/E)₅XE(+)-ΔN (I1127A), Fhod3-T(D/E)₅XE(−)-ΔN (wt), or mDia1-FH1FH2 (amino acids 549–1,175) in the presence of 2 µM profilin I.</p

Expression of alternatively spliced variants of Fhod3.

<p>The RT-PCR fragments amplified using specific primers flanking exon 25 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034765#pone-0034765-g001" target="_blank">Figure 1B</a>) are subcloned and subjected to sequencing analysis. The number in parenthesis indicates the percentage of each variant in the indicated tissue.</p>*<p>N.D. not determined.</p

Expression of Fhod3 isoforms in the embryonic tissues.

<p>(A) Exon structure of the moue Fhod3 gene. Alternative splicing exons are indicated by gray boxes. (B) Schematic presentation of domain structure of mouse Fhod3. The alternative splicing regions are in black boxes, and primers for RT-PCR analysis are indicated by arrows. (C) Specific primers designed for isoforms derived from the alternative splicing in the C-terminal FH2 domain. The primers ‘S6’ and ‘S7’ are specific for Fhod3 mRNAs with and without exon 25 that encodes the T(D/E)₅XE region (boxed in black), respectively. The primer ‘S8’ is common for both variants. (D) Tissue-specific expression of the “T(D/E)₅XE” exon in mouse embryos. The RT-PCR products using specific primers (shown in B and C) were subjected to agarose-gel electrophoresis. sk. muscle, skeletal muscle. (E) Tissue-specific expression of splicing variants lacking exons 11 and 12. The RT-PCR products (shown in B) were subjected to agarose-gel electrophoresis.</p

Schematic representation of the sarcomeric structure at embryonic stages.

<p>Relative localization of Fhod3 to α-actinin and F-actin shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148472#pone.0148472.g003" target="_blank">3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148472#pone.0148472.g005" target="_blank">5</a> is schematically represented. F-actin content is represented in gray tones. Arrowheads indicate the center of the sarcomere.</p