The actin-organizing formin protein Fhod3 is required for postnatal development and functional maintenance of the adult heart in mice

Cardiac development and function require actin–myosin interactions in the sarcomere, a highly organized contractile structure. Sarcomere assembly mediated by formin homology 2 domain-containing 3 (Fhod3), a member of formins that directs formation of straight actin filaments, is essential for embryonic cardiogenesis. However, the role of Fhod3 in the neonatal and adult stages has remained unknown. Here, we generated floxed Fhod3 mice to bypass the embryonic lethality of an Fhod3 knockout (KO). Perinatal KO of Fhod3 in the heart caused juvenile lethality at around day 10 after birth with enlarged hearts composed of severely impaired myofibrils, indicating that Fhod3 is crucial for postnatal heart development. Tamoxifen-induced conditional KO of Fhod3 in the adult heart neither led to lethal effects nor did it affect sarcomere structure and localization of sarcomere components. However, adult Fhod3-deleted mice exhibited a slight cardiomegaly and mild impairment of cardiac function, conditions that were sustained over 1 year without compensation during aging. In addition to these age-related changes, systemic stimulation with the α1-adrenergic receptor agonist phenylephrine, which induces sustained hypertension and hypertrophy development, induced expression of fetal cardiac genes that was more pronounced in adult Fhod3-deleted mice than in the control mice, suggesting that Fhod3 modulates hypertrophic changes in the adult heart. We conclude that Fhod3 plays a crucial role in both postnatal cardiac development and functional maintenance of the adult heart.

Cardiac development and function require actin-myosin interactions in the sarcomere, a highly organized contractile structure. Sarcomere assembly mediated by formin homology 2 domain-containing 3 (Fhod3), a member of formins that directs formation of straight actin filaments, is essential for embryonic cardiogenesis. However, the role of Fhod3 in the neonatal and adult stages has remained unknown. Here, we generated floxed Fhod3 mice to bypass the embryonic lethality of an Fhod3 knockout (KO). Perinatal KO of Fhod3 in the heart caused juvenile lethality at around day 10 after birth with enlarged hearts composed of severely impaired myofibrils, indicating that Fhod3 is crucial for postnatal heart development. Tamoxifeninduced conditional KO of Fhod3 in the adult heart neither led to lethal effects nor did it affect sarcomere structure and localization of sarcomere components. However, adult Fhod3-deleted mice exhibited a slight cardiomegaly and mild impairment of cardiac function, conditions that were sustained over 1 year without compensation during aging. In addition to these agerelated changes, systemic stimulation with the ␣1-adrenergic receptor agonist phenylephrine, which induces sustained hypertension and hypertrophy development, induced expression of fetal cardiac genes that was more pronounced in adult Fhod3deleted mice than in the control mice, suggesting that Fhod3 modulates hypertrophic changes in the adult heart. We conclude that Fhod3 plays a crucial role in both postnatal cardiac development and functional maintenance of the adult heart.
Normal cardiac function requires the accurately regulated actin-myosin interaction, which can be achieved by the precisely assembled and maintained sarcomere, the highly organized arrays of thin actin filaments and thick myosin filaments (1). During sarcomere assembly in the embryonic heart, actin cytoskeleton undergoes dynamic rearrangement by action of various actin-binding proteins (2), including tropomodulin, troponin T, ␣-tropomyosin, and formins; genetic deletion of these proteins causes failure of heart development and thereby results in embryonic lethality (3)(4)(5)(6)(7).
The structural maintenance of mature sarcomeres is also required for normal cardiac function. Although it was thought that actin filaments in mature muscle cells are relatively stable and do not easily turn over, recent studies have revealed dynamic exchange of cardiac actin thin filaments, i.e. a free actin is incorporated into actin filaments in mature sarcomeres (8,9). It remains uncertain, however, how actin is exchanged in and out of thin actin filaments in the heart under continuously beating conditions. Because actin filaments in the sarcomere are repetitively pulled into the array of myosin filaments by the periodic actin-myosin interaction, unregulated actin turnover would be directly linked to cardiac dysfunction. Under beating conditions, cardiomyocytes can increase their cell volume by expanding the contractile unit sarcomere in response to physiological demands and pathological changes such as hypertension (10). Although the two actin dynamics regulators Wdr1 (11) and Lmod2 (12) are known to participate in postnatal cardiac development and maintenance of adult hearts, the regulatory mechanism for actin dynamics during development and maintenance of the heart has largely remained to be elucidated (2).
The formin family proteins are structurally characterized by the presence of the formin homology (FH) 3 domains 1 and 2 and constitute a group of actin nucleation factors that play piv-otal roles in controlling actin polymerization (13)(14)(15). The FH2 domain directly binds to two actin molecules to facilitate actin filament nucleation and remains associated with the barbed end of the filament to promote polymerization, which is accelerated by FH1-mediated recruitment of profilin complexed with an actin monomer (16). Through cooperation of the FH1 and FH2 domains, formins direct formation of straight actin filaments, thereby modulating the actin dynamics in various actin structures, such as lamellipodia, filopodia, and contractile fibers (17). Recent studies have revealed that formins, including mDia1, Daam1, and Fmn2, are critical for morphogenesis and organogenesis during development (18 -20).
Fhod3 (previously known as Fhos2), a formin that is abundantly expressed in the heart (21), plays an essential role in the regulation of actin assembly in cardiac myofibrils (22)(23)(24). We have recently shown that genetic deletion of Fhod3 in mice confers embryonic lethality with defects in cardiogenesis (6). In Fhod3-null embryos, premyofibrils are formed once but fail to mature, suggesting that Fhod3 is essential for myofibrillogenesis, particularly for myofibril maturation. Indeed, Fhod3 accumulates at the central region of the sarcomere before myofibrillogenesis is completed, and its actin-assembling activity is required for myofibril maturation (25). However, the involvement of Fhod3 in postnatal cardiac development is unknown. Similarly, the role of Fhod3 in sarcomere maintenance in the fully-developed heart remains to be elucidated, although its significance can be expected from recent reports showing that some genetic variants of the Fhod3 gene are associated with human adult-onset cardiomyopathy (26,27).
Here we have generated a floxed allele of Fhod3 (Fhod3 flox ) for conditional knock-out of Fhod3. By crossing Fhod3 flox mice with muscle creatine kinase (MCK)-Cre mice and ␣-myosin heavy chain (␣MHC)-MerCreMer mice, we examined the effect of Fhod3 depletion at perinatal and adult stages, respectively. In addition, by continuous infusion of phenylephrine, an ␣ 1 -adrenergic receptor agonist, we evaluated the role of Fhod3 in modulation of hypertrophic response.

Cardiac-specific deletion of Fhod3 in perinatal mice
We have previously reported that Fhod3 is indispensable for cardiogenesis, especially for myofibrillogenesis (6). In contrast, the role of Fhod3 in the fully-developed heart has remained elusive, although the Fhod3 mRNA is abundant in the adult heart as well as in the fetal heart ( Fig. 1A) (21,24). To address the question, we first tested the protein level of Fhod3 in various developmental stages of mice. Immunoblot analysis revealed that the Fhod3 protein was expressed in the heart at comparable levels throughout the lifetime from the embryo to the senior adult (Fig. 1B). The expression of Fhod3 is mostly restricted to the heart (Fig. 1, C and D). To investigate how Fhod3 functions in the developed heart, we generated the Fhod3 flox allele by introduction of loxP sites into the introns surrounding the exon 18 ( Fig. 2), which encodes the entire FH1 and the first part of FH2 domains (the core domains for actin polymerization). Cremediated recombination leads to deletion of the exon 18 and introduces a frameshift mutation, thereby resulting in a premature stop codon 7 amino acids downstream from the deletion.
To delete Fhod3 from the neonatal heart, we crossed mice carrying the Fhod3 flox allele to MCK-Cre mice, which express Cre recombinase in the heart, skeletal muscle, and some types of the smooth muscle from the late-embryonic stage to adulthood (28,29). Because Fhod3 is hardly expressed in the skeletal muscle and vascular smooth muscle ( Fig. 1, C-E) (21,24), MCK-Cre-mediated recombination is expected to induce a cardiac-specific depletion of the Fhod3 protein in the perinatal period. Perinatal Fhod3 cKO mice (Fhod3 flox/Ϫ ; MCK-Cre ϩ ), in which the Cre-mediated recombination occurred around birth (Fig. 3A), were born in the expected Mendelian ratio (25%) and were indistinguishable in appearance from control littermates at birth. However these mice died by around postnatal day (P) 15 (Fig. 3B). As demonstrated by immunoblot analysis of heart extracts (Fig. 3C), the Fhod3 protein in Fhod3 cKO mice (Fhod3 flox/Ϫ ;MCK-Cre ϩ ) began to decrease from around birth and became nearly undetectable at P8. Macroscopically, Fhod3 cKO neonates began to exhibit growth retardation at around P6, and the difference in the body size between Fhod3 cKO and control neonates became apparent with time (Fig. 3, D and E). In contrast, the heart of Fhod3 cKO neonates was markedly enlarged in comparison with that of control ones (Fig. 3D), suggesting that lethality is associated with cardiac defects.

Depletion of Fhod3 in the neonatal heart induces disruption of the sarcomere structure
When myocardial sections prepared from Fhod3 cKO mice were stained with hematoxylin and eosin (Fig. 4A), they showed A, total RNAs prepared from mouse heart at the indicated postnatal days were analyzed by reverse transcription-PCR with specific primers for Fhod3 and GAPDH. RT, reverse transcriptase. B, proteins prepared from the heart of wild-type mice at the indicated age were analyzed by immunoblot with anti-Fhod3-C20, anti-␣tubulin, anti-GAPDH, and anti-␤-actin antibodies. The loading amount was verified by myosin heavy chain bands stained with fast green. Positions for marker proteins are indicated in kDa. C, proteins prepared from the indicated tissues of wild-type mice at 16 weeks of age were analyzed by immunoblot with anti-Fhod3-C20 and anti-GAPDH antibodies. Positions for marker proteins are indicated in kDa. D and E, lacZ staining of the heart (D, top view) and aorta (E, lateral view) of Fhod3 ϩ/ϩ and Fhod3 ϩ/Ϫ (Fhod3 ϩ/lacZ ) mice at 30 weeks. Roots of aortic and pulmonary arteries were indicated by arrowheads and arrows, respectively. Scale bars, 2 mm.

Role of Fhod3 in the neonatal and adult heart
signs of myofibrillar degeneration such as wavy appearance of myofibrils and myocyte swelling with an increase in vacuoles. To further investigate myocardial changes associated with depletion of Fhod3, we performed immunofluorescence staining for Fhod3 and sarcomeric ␣-actinin, a marker for myofibril assembly, and we found that Fhod3 was not detected at the protein level (Fig. 4B). Regularly-striated alignment of sarcomeric ␣-actinin was largely maintained in Fhod3-deleted myofibrils, although some myofibrils had abnormal ␣-actinin signals, which are aggregated or continuous along the sarcolemma. F-actin staining exhibited more pronounced myofibrillar changes with continuous aggregates of F-actin with ␣-actinin, a protein necessary for F-actin attachment to the Z-lines in cardiac sarcomeres (Fig. 4C). Transmission electron microscopic analysis also showed breakdown of sarcomere structure in the neonatal heart of Fhod3 cKO mice (Fig. 4, D and E). In contrast, cardiac sarcomeres in Fhod3 cKO mice were normally developed before birth (Fig. 4F). Thus, neonatal depletion of Fhod3 likely disrupts the sarcomere which has been once properly formed before birth, indicating that Fhod3 participates in postnatal development and maintenance of the cardiac sarcomere.
However, the sarcomeric structure of the skeletal muscle showed no obvious differences between Fhod3 cKO and control neonates (Fig. 4G), supporting the idea that lethality is primarily associated with defects in the cardiac sarcomere. We further investigated the phosphorylation status of Fhod3 in the neonatal heart, because it has been reported that the activity of Fhod3 is regulated by phosphorylation (30). As shown in Fig.  4H, only weak bands of phosphorylated Fhod3 were detected both in the neonatal and adult heart under conditions where cMyBP-C, a sarcomeric phosphoprotein (31), was substantially phosphorylated.

Cardiac-specific deletion of Fhod3 in adult mice
Fhod3 plays a crucial role in not only the embryonic heart (6) but also the neonatal heart (Figs. 3 and 4). However, it was difficult to assess whether Fhod3 functions in the fully-developed adult heart by using MCK-Cre-mediated cKO mice, because Fhod3 cKO mice did not fully survive until adulthood (Fig. 3). To delineate the role of Fhod3 in the adult heart, we crossed mice carrying the Fhod3 flox allele with transgenic mice expressing MerCreMer under the control of the ␣MHC promoter (32), thereby generating mice with the Fhod3 alleles that can be deleted specifically in the adult heart by administration of TAM. Fhod3 flox/Ϫ ;␣MHC-MerCreMer ϩ mice were born at the expected Mendelian frequency and were indistinguishable in appearance from control littermates.
To delete Fhod3 specifically in the adult heart, 4 -6-week-old mice were orally administered TAM for 2 weeks. The occurrence of Cre-mediated, heart-specific recombination was con- gene is represented as a yellow box. Green and blue bars indicate probes used for Southern blot analysis, and expected sized of fragments obtained after SpeI or BstEII digestion are indicated in base pairs. Small black arrows indicate primers for PCR genotyping. B, PCR analyses of tail DNAs from wild-type and heterozygous mice for the targeted allele. The PCR product from the wild-type allele was 329 bp in length, and the PCR products from the targeted allele were 387 and 645 bp in length. C, Southern blot analyses of tail DNAs from the wild-type and heterozygous mice for the targeted allele. SpeI-digested DNAs and BstEII-digested DNAs were probed with 5Ј-probe (green in A) and 3Ј-probe (blue in A), respectively. The wild-type allele was detected as 12.2-and 12.1-kb fragments by 5Ј-and 3Ј-probes, respectively. The targeted allele was detected as 8.4-and 14.1-kb fragments by 5Ј-and 3Ј-probes, respectively.

Role of Fhod3 in the neonatal and adult heart
firmed by PCR analysis using genomic DNAs extracted from the heart and kidney in adult mice treated with TAM; the recombinant allele was detected only in the heart of Cre-positive mice but not in the kidney of Cre-positive mice or the heart of Cre-negative mice (Fig. 5A).
The protein expression level of Fhod3 in Fhod3 iKO mice was reduced to ϳ5% or less of that of wild-type mice, as demonstrated by immunoblot analysis of lysates of isolated left ventricular tissue (Fig. 5, B and C). Thus, TAM administration effectively induces Cre-mediated, cardiac-specific recombination of the Fhod3 allele to significantly reduce the protein expression level of Fhod3 in the adult heart.

Depletion of Fhod3 in the adult heart does not lead to lethality
Despite the reduction of Fhod3 in the heart from TAM-treated iKO mice, no significant difference in the long-term survival up to 60 weeks was observed among all the genotype groups (Fig. 5C). Some mice died during the 2 weeks of TAM administration, probably due to acute cardiotoxicity (33). Adult Fhod3-deleted mice showed a slightly higher mortality rate than other genotype mice but with no statistical significance. We next evaluated the effect of Fhod3 depletion in the adult heart at 8 -10 weeks after TAM administration; the period was selected because the transient impairment of cardiac function is known to be fully recovered until 4 weeks after the cessation of TAM administration (34).

Role of Fhod3 in the neonatal and adult heart
The size of the adult heart in TAM-treated Fhod3 iKO mice was slightly larger than that in control mice (Fig. 5E), which was supported by histological analysis (Fig. 5F). Left ventricular weight-to-body weight ratio of adult Fhod3-deleted mice was slightly but significantly higher than that of control mice, whereas there was no difference in kidney weight-to-body weight ratio between adult Fhod3-deleted and control mice (Fig. 5G). On the other hand, we were not able to detect any compensatory increase in the expression of Fhod1, the only other member of the Fhod subfamily (Fig. 5H). Thus deletion of Fhod3 in the adult heart confers a slight cardiomegaly.

Depletion of Fhod3 in the adult heart does not cause any detectable changes in the sarcomere structure
We next investigated the effect of Fhod3 depletion in the adult heart on the sarcomeric structure. In contrast to the finding that the neonatal heart of Fhod3 cKO mice shows disrupted structures of sarcomeres (Fig. 4, B-E), the adult heart of TAM-

Role of Fhod3 in the neonatal and adult heart
treated Fhod3 iKO mice showed apparently normal sarcomeric structures (Fig. 6). There were no significant differences in sarcomeric structures, including the Z-line marked with the antibody to ␣-actinin (Fig. 6, A and B), thin actin filaments stained with phalloidin ( Fig. 6, B and C), and thick myosin filaments visualized with the antibody to myosin-binding protein C (MyBPC) (Fig. 6D), albeit specific signals for Fhod3 were absent in the Fhod3-deleted adult heart (Fig. 6, A, C, and D). Immunohistochemical staining of tropomodulin, a protein that caps actin filament at the pointed end, showed no detectable differences between adult Fhod3-deleted and control hearts (Fig. 6E), suggesting that the length of thin actin filaments and the capping state of pointed ends are unaffected. Intact sarcomeres in the Fhod3-deleted adult heart were also observed by transmission electron microscopy (Fig. 6F). Furthermore, intercalated disc structure, which is disrupted in several mouse models of dilated cardiomyopathy (35), was not altered in the Fhod3-deleted adult heart, as estimated by immunohistochemical staining of vinculin, a component of intercalated discs, and by transmission electron microscopy ( Fig. 6, G and H). Thus, adult Fhod3 deletion hardly affects the structure of sarcomeres and intercalated discs.

Mice lacking cardiac Fhod3 exhibit prolonged morphological and functional changes at advanced ages
Morphological and functional characteristics of Fhod3 cKO mice were followed until 1 year of age for evaluation of late effects of Fhod3 deletion in the adult heart. As shown in Fig. 7A, the left ventricular chamber of aged Fhod3-deleted mice was slightly enlarged, suggesting that structural changes induced by Fhod3 deletion was sustained but not corrected over a follow-up period of 1 year. Consistent with these findings, ventricular dilation with thinned walls was observed in the heart of aged Fhod3-deleted mice by echocardiographic measurement of left ventricular dimensions and posterior wall thickness (Fig.  7B). The measurement also revealed significant reduction of left ventricular ejection fraction in aged Fhod3-deleted mice (Fig. 7B). These macroscopic anatomical changes suggest that cardiac remodeling is induced by Fhod3 depletion in the adult heart. To test the possibility, we performed quantitative realtime PCR analysis to examine activation of cardiac remodelingassociated fetal genes that encode atrial natriuretic factor (ANF), B-type natriuretic peptide (BNP), and ␤-myosin heavy chain (␤MHC). As shown in Fig. 7C, mRNA expression of the three genes in aged Fhod3-deleted mice was elevated when compared with those in age-matched control mice, although the increase in ANF mRNA was not statistically significant. Picrosirius red staining revealed that fibrotic changes such as accumulation of collagen I or collagen III also occurred in the heart of aged Fhod3-deleted mice (Fig. 7D), which is consistent with cardiac increase in mRNA for the genes encoding the ␣1 chain of collagen I (Col1a1) as demonstrated by real-time PCR analysis (Fig. 7E). Thus, morphological changes and functional impairment by depletion of Fhod3 are only slightly induced but not compensated during aging, which leads to ventricular dilation with wall thinning and fibrosis at advanced age.

Mice lacking cardiac Fhod3 show a hypertrophic response to phenylephrine infusion but exhibit no substantial changes in sarcomeric structures
A modest but significant cardiac dysfunction in adult Fhod3deleted mice (Fig. 7) may implicate that Fhod3 regulates cardiac function under pathological conditions. To test the possibility, we treated adult Fhod3-deleted and control mice with phenylephrine, an ␣ 1 -adrenergic agonist that induces sustained hypertension and development of hypertrophy (36). Infusion of phenylephrine using osmotic mini-pumps for 2 weeks led to the death of a minor portion of mice probably due to intolerance of acute hemodynamic changes, whereas mice without the infusion survived uneventfully (Fig. 8A). The mortality of phenylephrine-infused Fhod3 cKO mice was not significantly different from that of phenylephrine-infused control mice. At the time point after continuous phenylephrine infusion for 2 weeks, the heart of treated mice markedly enlarged (Fig. 8, B and C). Consistent with this, the ratios of left ventricular to body weight in the phenylephrine-infused mice were significantly higher when compared with those in mice without phenylephrine infusion (Fig. 8D). There were no significant differences in the ratios between Fhod3-deleted and control mice after phenylephrine infusion. However, the heart in phenylephrine-treated Fhod3deleted mice showed a tendency to be more enlarged than that of phenylephrine-treated control mice, raising the possibility that Fhod3 is involved in modulation of hypertrophic processes.
To test this, we investigated whether Fhod3 expression is altered under hypertrophic conditions. As shown by immuno-

Role of Fhod3 in the neonatal and adult heart
blot analysis (Fig. 9A), the amount of Fhod3 in the heart was not up-regulated in phenylephrine-treated mice, whereas phenylephrine treatment markedly increased ␤MHC expression in both Fhod3-deleted and control mice. Immunofluorescence staining confirmed the phenylephrine-induced accumulation of ␤MHC in the Fhod3-deleted heart (Fig. 9B), indicating that phenylephrine treatment indeed induces hypertrophic changes in Fhod3-deleted mice as well as control mice. Phenylephrine infusion did not induce significant changes in sarcomeric structures in both Fhod3-deleted and control mice, as demonstrated by immunofluorescence staining (Fig. 9C) and by transmission electron microscopy (Fig. 9D). Thus, the adult Fhod3-deleted heart is sensitive to phenylephrine infusion, although Fhod3 deletion does not induce detectable changes in sarcomeric structures even after phenylephrine treatment.

Deletion of Fhod3 in the adult heart negatively affects cardiac function and promotes hypertrophic responses
We finally examined the effect of Fhod3 depletion on cardiac function under hypertrophic conditions by echocardiography. Before phenylephrine treatment, the left ventricular chamber in adult Fhod3-deleted mice was enlarged compared with that

Role of Fhod3 in the neonatal and adult heart
in control mice (Fig. 10A), and the left ventricular function was significantly impaired in adult Fhod3-deleted mice (Fig. 10B). These observations are consistent with the present macroscopic and histological analyses showing that Fhod3 deletion in the adult heart induces slight but significant cardiomegaly (Fig.  5, E-G). After 2 weeks of phenylephrine infusion, left ventricular chamber enlargement and functional impairment were further developed in both Fhod3-deleted and control mice (Fig.  10, A and B), although some of the parameter differences were not statistically significant. At the same time, wall thickening and increments in left ventricular mass were also observed in both Fhod3-deleted and control mice (Fig. 10C), indicating that Fhod3-deleted mice have an ability to induce hypertrophy. Consistent with this, the cell width of individual cardiomyocytes was increased in response to phenylephrine treatment in the heart of Fhod3-deleted mice (Fig. 10D), although the extent in Fhod3-deleted mice tended to be smaller than that in control mice. Next, we tested the effect of phenylephrine on expression of cardiac remodeling-associated fetal genes relative to that of Gapdh. It should be noted that Gapdh expression is not affected by phenylephrine treatment (Fig. 9A) (37). As shown in Fig.  10E, phenylephrine treatment enhanced the expression of mRNAs for ANF, BNP, and ␤MHC in the heart of Fhod3deleted mice more profoundly than that of control mice. Thus, Fhod3 is likely able to modulate cardiac hypertrophic responses.

Discussion
In this study, we show that Fhod3 plays a crucial role in postnatal development of the heart; depletion of Fhod3 in the neonatal heart results in juvenile lethality associated with cardiac defects (Figs. 3 and 4). In embryos, Fhod3 is dispensable for initiation of spontaneous contraction of the developing heart, composed of premyofibrils, but it is absolutely required for subsequent myofibril maturation in cardiogenesis (6), indicating that Fhod3 is responsible for expanding the basic contractile unit called sarcomere during embryonic heart development (6,25). During the early postnatal period, greater circulatory demands of the growing organism induce a 2-fold or more increase in left ventricular mass, which mainly results from an increase in the size of cardiomyocytes with expansion of their contractile apparatus (38,39). It is therefore likely that Fhod3, an actin-assembling factor (22), functions via expanding the sarcomere during heart development in neonates as well as embryos. In addition to sarcomere expansion, Fhod3 may also be involved in maintenance of sarcomere structure in the neonatal stage, because myofibrils once formed during embryonic cardiogenesis become degenerated after birth in perinatally Fhod3-deleted mice (Fig. 4). Thus Fhod3-mediated actin assembly appears to contribute to postnatal cardiac development via expansion and maintenance of the contractile apparatus sarcomere.
In contrast to the life-threatening effect of embryonic and perinatal deletion of Fhod3, its deletion in the adult stage does not affect mortality (Fig. 5). This is consistent with the present finding that sarcomeric structures are largely maintained in the adult heart of Fhod3-deleted mice (Fig. 6). A weak but significant cardiac remodeling, however, occurs with aging in adult Fhod3-deleted mice, as indicated by macroscopic and functional changes, signs of fibrosis, and expression of the cardiac remodeling-associated fetal genes (Fig. 7). Furthermore, systemic infusion of the ␣ 1 -stimulant phenylephrine induces more

Role of Fhod3 in the neonatal and adult heart
severe hypertrophic processes in the heart of adult Fhod3-deleted mice than in the Fhod3-expressing heart (Figs. 8 -10), indicating that Fhod3 is involved in hypertrophic responses to cardiac stress in the adult heart. Although juvenile lethality is induced by perinatal deletion of Fhod3 (Fig. 3), Fhod3 knockout in adult mice does not result in a significant increase in morbidity after phenylephrine treatment (Fig. 8). Thus, Fhod3 plays a major role in neonatal cardiac development but not in stress-induced cardiac hypertrophy during adulthood. The difference is likely due to the fact that cardiomyocytes increase their size more than 2-fold under physiological conditions in the neonatal stage (40), but the cell size grows to a limited extent in response to pathological stimuli in the adult (39). The greater increase in cardiomyocyte size during neonatal cardiac development appears to explain the reason for the greater dependence on Fhod3, because this actin-assembling factor is crucial for cardiac sarcomere expansion, which leads to an increase in cardiomyocyte size, as discussed above.
Although this study supports the idea that Fhod3 regulates actin assembly for sarcomere integrity in the heart (6,21,24), the molecular mechanism underlying the regulation remains largely unknown. The precise site where Fhod3 functions in the cardiac sarcomere is a controversial issue. We have provided several lines of evidence showing that Fhod3 localizes to the center of sarcomeres, specifically the zone of the thin actin filaments that overlap the thick myosin filaments; localization of endogenous Fhod3 to this specific zone in the embryonic and adult heart (24, 25) as well as in cultured cardiomyocytes (22) is demonstrated by immunochemical analysis using three anti-Fhod3 antibodies, each recognizing distinct regions of Fhod3 (21); and the same localization of HA-tagged Fhod3 is observed using an anti-HA antibody (22). In contrast, other groups have reported that Fhod3 localizes to the Z-line, where the barbed ends of actin filaments are anchored, in the adult heart (23,30,41). Although both patterns of Fhod3 localization are possible, this study strengthens our conclusion by confirming the authenticity of the three distinct antibodies to Fhod3; their signals disappear from the zone containing both thin and thick filaments in neonatal and adult Fhod3-deleted hearts (Figs. 4B,  6D, and 9C). Further studies are required to address the question how Fhod3 functions at the zone of the actin filaments that is superimposed by the myosin filaments.
According to a recent assessment (41), the Fhod3 mRNA is expressed abundantly in the embryonic stage but not after birth. In contrast, the Fhod3 mRNA is similarly detected in the postnatal and adult heart in this study (Fig. 1A). Although the reason for the discrepancy is presently unknown, the protein level of Fhod3 in the heart does not change significantly throughout the lifetime from the embryo to the senior adult (Fig. 1B), consistent with the critical role of Fhod3 after birth. Besides Fhod3, other formin family proteins also seem to participate in regulation of actin dynamics in cardiomyocytes (41). Actin polymerization by formins is generally accelerated via FH1 domain-mediated recruitment of the profilin-actin complexes (16), which is consistent with the recent finding that profilin-1 participates in actin dynamics in cardiomyocytes,

Role of Fhod3 in the neonatal and adult heart
including sarcomeric organization and cell size control (42), probably via cooperation with formins. Daam1, another major cardiac formin (41), has been reported to be required for myocardial maturation and sarcomere assembly (7,43). In contrast to the localization of Fhod3 at the central region of sarcomeres, mouse Daam1 in the cardiomyocytes mainly localizes to the cell membrane not in a sarcomeric pattern (7), suggesting that Daam1 and Fhod3 regulate cardiac actin dynamics in a different manner.
In contrast to the failure of the ␣ 1 -adrenergic agonist phenylephrine to induce expression of Fhod3 in the heart (Fig. 9A), Zhou et al. (44) have very recently reported that treatment with angiotensin II increases Fhod3 expression, and ROCK2-mediated phosphorylation of Fhod3 participates in the angiotensin II-induced hypertrophy. Although neonatal cardiac development requires Fhod3 but does not seem to involve its phosphorylation, because Fhod3 is only marginally phosphorylated in the developing neonatal hearts (Fig. 4H). Future studies are needed to evaluate the contribution of Fhod3 and its phosphorylation to the development of cardiac hypertrophy under various pathological conditions.
In conclusion, we report here that Fhod3 plays an important role in neonatal cardiac development and in functional maintenance and stress-induced hypertrophic response of the developed heart. Taken together with the recent findings that Fhod3 variants are associated with hypertrophic and dilated cardiomyopathy (26,27), Fhod3 might represent a potential therapeutic target for cardiac disease associated with failure in myofibril maintenance.

Generation of Fhod3 conditional knock-out mice
Mice carrying the Fhod3 floxed allele (accession no. CDB0927K) were generated according to the protocols of RIKEN Center for Life Science Technologies. In brief, the 5Ј-ho- Figure 8. Continuous infusion of phenylephrine induces hypertrophic changes in the Fhod3-deleted heart. A, survival curves after continuous infusion of phenylephrine (PE) for 2 weeks. TAM-treated Fhod3 iKO and TAM-treated control littermate mice were infused with PE (100 mg/kg/day) or saline for 2 weeks. B, representative images of hearts of TAM-treated Fhod3 iKO and TAM-treated control littermate mice after PE infusion. These experiments have been repeated three times with similar results. C, histological analyses of hearts of TAM-treated Fhod3 iKO (Fhod3 flox/Ϫ ;MerCreMer ϩ , TAM(ϩ)) and TAM-treated control littermate (Fhod3 flox/Ϫ ;MerCreMer Ϫ , TAM(ϩ)) mice after PE infusion. Longitudinal sections of hearts were stained with hematoxylin and eosin. Scale bars, 3 mm. D, heart-to-body weight and kidney-to-body weight ratios after PE infusion were determined. †, p Ͻ 0.01; ‡, p Ͻ 0.001.
Mice heterozygous for the constitutive null Fhod3 allele (Fhod3 ϩ/Ϫ ) were generated by replacement of exon 1 with LacZ as described previously (6). Transgenic mice expressing Cre recombinase under the control of muscle myosin kinase (MCK) promoter (MCK-Cre mice, B6.Cg-Tg(Ckmm-cre)5Khn/J; 006475) and transgenic mice expressing Cre recombinase fused to two mutated estrogen receptors under the control of ␣-my-
All experimental protocols were approved by the Animal Care and Use Committee of Kyushu University (Permit No. A26-102) and the Animal Care and Use Committee of Miyazaki University (Permit No. 2014-526-3). All mice were housed and maintained in a specific pathogen-free animal facility at Kyushu University, and all efforts were made to minimize the number of animals used and their suffering. All experiments were performed in strict accordance with the guidelines for Proper Conduct of Animal Experiments (Science Council of Japan) and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Tamoxifen administration
TAM pellets containing 400 mg of TAM citrate per kg diet (daily administration of TAM of ϳ40 mg per kg body weight) was obtained from Oriental Yeast Co., Ltd. After TAM administration of 2 weeks, the mice were fed with standard diet over 4 weeks to avoid interference with transient Cre expression and were used on all experiments.

Role of Fhod3 in the neonatal and adult heart
clone 6C5 against GAPDH from Chemicon. Rabbit polyclonal antibodies against tropomodulin1 were purchased from ProteinTech Group; and rabbit polyclonal anti-phosphoserine antibodies were from Chemicon. Alexa Fluor 488-conjugated F(abЈ) 2 fragment of anti-mouse IgG and Alexa Fluor 555-conjugated F(abЈ) 2 fragment of anti-rabbit IgG were purchased from Cell Signaling Technology.

Immunoblot analysis
Immunoblot analysis was performed as described previously (25). Briefly, the hearts of neonates were homogenized and sonicated at 4°C in a lysis buffer composed of 10% glycerol, 135 mM NaCl, 5 mM EDTA, and 20 mM Hepes, pH 7.4, containing protease inhibitor mixture (Sigma). In the case of adult mice, the ventricular tissue samples of mice were snapfrozen, crushed using SK-Mill (SK-100, FUNAKOSHI), and dissolved in a buffer composed of 9 M urea, 2% SDS, 2% Triton X-100, 1% dithiothreitol, and 10 mM Tris-HCl, pH 6.8, containing Protease Inhibitor Mixture (Sigma). The lysates were applied to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). Protein transfer was immediately confirmed by fast green staining (loading). The membrane was probed with the antibody, followed by development using ECL-plus (GE Healthcare) for visualization of the antibodies.

In vivo phosphorylation assay
Immunoprecipitation was performed, as described previously (47), with minor modifications. Briefly, the heart tissues were snap-frozen, crushed using SK-Mill (SK-100, FUNAKOSHI), and dissolved in a lysis buffer (10% glycerol, 135 mM NaCl, 5 mM EDTA, and 20 mM Hepes, pH 7.4) containing 0.1% Triton X-100, Protease Inhibitor Mixture (Sigma), and PhosSTOP phosphatase inhibitor mixture (Sigma). The lysates were precipitated with the anti-Fhod3-C20 or anti-cMyBP-C antibodies in the presence of protein G-magnetic beads. After washing three times with the lysis buffer, the precipitants were applied to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with the anti-phosphoserine, anti-Fhod3-C20, or anti-cMyBP-C antibodies, followed by development using ECL-prime (GE Healthcare) for visualization of the antibodies.

Histological analysis
Histological analysis was performed as described previously (6). Briefly, mice were sacrificed via cervical dislocation, and the whole heart was harvested. The harvested tissues were fixed by immersion in a solution containing 3.7% formaldehyde in PBS. The fixed tissues were dehydrated in ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or picrosirius red. Images were taken with BZ-9000 microscope (Keyence). For estimation of fibrosis level, picrosirius redstained sections were examined with a microscope (BX-51; Olympus) coupled to an image processing system (NIS-Element-D3.2; Nikon) and semi-quantified using an image analysis software (WinRoof; Mitani).

Immunofluorescence staining
Immunofluorescence staining was performed according to the previously described method (6) with minor modifications. Briefly, mice were deeply anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg/body weight) and sevoflurane inhalation. After exposure of the heart, PEM buffer (1 mM EGTA, 1 mM MgCl 2 , and 100 mM PIPES, pH 6.9) containing 100 mM 2,3-butanedione monoxime (BDM) was administered from the left ventricular apex, followed by the administration of 3.7% formaldehyde in the PEM buffer. In the case of preparation of heart tissues, for facilitation of selective coronary perfusion, the ascending aorta was clamped with a hemostat, and the right atrium was clipped with surgical scissors before the perfusion. The perfused tissues were removed from the deceased mice, cut into small pieces, and immersed for 90 min at 4°C in the same fixative. The fixed tissues were washed in PBS, subjected to osmotic dehydration overnight at 4°C in 30% sucrose, and embedded in OCT compound (Sakura Finetek). The blocks were frozen and cut into 5-m sections using a cryostat (HM550; Thermo Fisher Scientific). Sections were then washed with PBS containing 0.1% Triton X-100 and blocked with a blocking buffer (Blocking One Histo; Nacalai Tesque) for 5 min at room temperature. Sections were labeled overnight at 4°C with primary antibodies diluted in a dilution buffer (PBS containing 3% bovine serum albumin, 2% goat serum, and 0.1% Triton X-100) and then labeled for 2 h at room temperature with a fluorescein-conjugated secondary antibody mixture in the same buffer. For Fhod3 immunofluorescence staining, anti-Fhod3-(650 -802) antibody was used. Actin filaments were stained with Alexa Fluor 488 phalloidin (Invitrogen). Nuclei were stained with Hoechst 33342 (Invitrogen). Cell membranes were stained with FITC-labeled WGA (J-OIL MILLS). Images were taken with LSM700 or LSM780 confocal scanning laser microscope (Carl Zeiss MicroImaging).

Phenylephrine administration
Continuous subcutaneous infusion of phenylephrine (100 mg/kg/day) or saline was conducted with Alzet osmotic minipumps (model 2002, DURECT Corp.). Mice were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg/ body weight) and sevoflurane inhalation, and then the pump was subcutaneously implanted at the back. After continuous administration for 2 weeks, the mice were analyzed.

Transmission electron microscopic analysis
Transmission electron microscopy of thin sections was performed according to the previously described method (6) with minor modifications. Briefly, mice were deeply anesthetized with an intraperitoneal injection of pentobarbital and sevoflurane inhalation. After exposure of the heart, the PEM buffer containing 100 mM BDM was administered from the left ventricular apex, followed by the administration of a fix buffer (2.5% glutaraldehyde, 0.1 M sucrose, 3 mM CaCl 2 , and 0.1 M sodium cacodylate, pH 7.4) for 1 h, followed by rinse in PBS overnight at 4°C. Then the heart was postfixed for 1 h with 1% OsO 4 , dehydrated in ethanol and propylene oxide, and embedded in the Epon 812 resin. Thin sections containing the heart were stained for 5 min with 2% uranyl acetate and for 10 min

Role of Fhod3 in the neonatal and adult heart
with Sato's lead mixture and then examined with a JEM-2000EX (JOEL).

Echocardiography
Serial echocardiographic examinations were performed non-invasively using a Vevo 2100 (Visual Sonics). Under anesthesia with 1-2% isoflurane inhalation, two-dimensional targeted M-mode images were obtained from the short-axis view at the level of papillary muscles. Echocardiographic images were analyzed by the analysis software of Vevo 2100. Left ventricular diastolic and systolic diameters (LVDd and LVDs) and end diastolic posterior wall thickness were measured following the guidelines of American Society of Echocardiography. The left ventricular ejection fraction (LVEF) was calculated according to the Teichholz method, and the left ventricular mass was calculated by the Devereux formula.

Statistical analysis
All data were expressed as mean Ϯ S.E. Two groups were compared by paired or unpaired Student's t test. Multiple groups were compared by analysis of variance followed by post hoc Tukey test. A p value of Ͻ0.05 was considered to be statistically significant. GraphPad Prism 5.0 (GraphPad Software Inc., San Diego) was used for all statistical analysis.