Mammalian Formin Fhod3 Regulates Actin Assembly and Sarcomere Organization in Striated Muscles

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells. Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells. In striated muscle, thin actin filaments and thick filaments of myosin are highly organized to form myofibrils (1Clark K.A. McElhinny A.S. Beckerle M.C. Gregorio C.C. Annu. Rev. Cell Dev. Biol. 2002; 18: 637-706Crossref PubMed Scopus (474) Google Scholar) (Fig. 1A). During myofibrillogenesis, actin cytoskeleton undergoes dynamic remodeling to produce uniform lengths of straight filaments packaged in the sarcomere, a contractile unit of myofibrils (2Gregorio C.C. Antin P.B. Trends Cell Biol. 2000; 10: 355-362Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 3Sanger J.W. Kang S. Siebrands C.C. Freeman N. Du A. Wang J. Stout A.L. Sanger J.M. J. Muscle Res. Cell. Motil. 2005; 26: 343-354Crossref PubMed Scopus (154) Google Scholar, 4Littlefield R.S. Fowler V.M. Semin. Cell Dev. Biol. 2008; 19: 511-519Crossref PubMed Scopus (94) Google Scholar). In nascent sarcomeres, a filamentous actin-containing structure, referred to as the Z-body or I-Z-I structure, emerges as a precursor of the Z-line that anchors actin filaments. Subsequent alignment of the precursors leads to formation of a striated pattern of the Z-line, and myosin filaments are incorporated between Z-lines. Finally, the M-line that serves as an anchoring site for myosin filaments becomes visible; the appearance is accompanied by alignment of the unanchored end of actin filaments (5Markwald R.R. J. Mol. Cell. Cardiol. 1973; 5: 341-350Abstract Full Text PDF PubMed Scopus (68) Google Scholar). Thus, the mature distribution pattern of actin filaments is constructed at the final step in myofibril assembly, indicating that actin filaments continue to develop throughout myofibrillogenesis. However, the regulation of actin dynamics in this process has remained poorly understood. In nonmuscle cells, organization of actin cytoskeleton is achieved by two major actin nucleating-polymerizing systems, formins and the Arp2/3 complex, with the former producing long straight actin filaments and the latter producing branched actin network (6Pollard T.D. Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 451-477Crossref PubMed Scopus (742) Google Scholar, 7Chhabra E.S. Higgs H.N. Nat. Cell Biol. 2007; 9: 1110-1121Crossref PubMed Scopus (572) Google Scholar). Because an unbranched straight actin filament is the major form in striated muscle cells, it is possible that a formin family protein serves as the key regulator of actin dynamics in myofibrils. Formins are characterized by the presence of two conserved regions, the formin homology 1 and 2 domains (FH1 and FH2 domains, respectively) 2The abbreviations used are: FH1 and FH2formin homology 1 and 2, respectivelyDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumGFPgreen fluorescent proteinHAhemagglutininsiRNAsmall interfering RNA. (8Kovar D.R. Curr. Opin. Cell Biol. 2006; 18: 11-17Crossref PubMed Scopus (196) Google Scholar, 9Goode B.L. Eck M.J. Annu. Rev. Biochem. 2007; 76: 593-627Crossref PubMed Scopus (614) Google Scholar). The FH2 domain associates with the barbed end of an actin filament and promotes actin nucleation and polymerization. The FH2 domain continues to associate with the barbed end during polymerization; this processive association protects the growing barbed end from capping proteins that inhibit actin elongation. The FH1 domain, located N-terminally to the FH2 domain, accelerates the FH2-mediated actin elongation via recruiting profilin complexed with an actin monomer. Through cooperation of the FH1 and FH2 domains, formins produce long straight actin filaments even in the presence of capping proteins. Here, we focused on the role of the mammalian formin Fhod3 (previously designated as Fhos2L), which is expressed predominantly in the heart (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar), in actin assembly in myofibrils. formin homology 1 and 2, respectively Dulbecco's modified Eagle's medium fetal calf serum green fluorescent protein hemagglutinin small interfering RNA. The cDNA fragments encoding mouse Fhod3 of 1578 amino acids and its spliced variant Fhod3S with deletion of amino acids 401–551 (designated as Fhos2L and Fhod2S, respectively, in our previous paper (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar)) and human Fhod1 were prepared as described previously (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar, 11Takeya R. Sumimoto H. J. Cell Sci. 2003; 116: 4567-4575Crossref PubMed Scopus (47) Google Scholar). The cDNA encoding mouse Fhod3-ΔN (amino acids 931–1578) was constructed by PCR using the cDNA encoding mouse Fhod3. The cDNA encoding mDia1-FH1FH2 (amino acids 549–1175) was cloned by PCR using the expressed sequence tag clone MGC:86169 as a template. The cDNA for human profilin IIa was cloned by PCR using human brain cDNAs from the Human Multiple Tissue cDNA Panel (BD Biosciences) as a template. Mutations leading to the indicated amino acid substitutions were introduced by PCR-mediated sited-directed mutagenesis. The DNA fragments were ligated to pEGFP-C1 (Clontech) or pEF-BOS for expression in HeLa cells as an N-terminally green fluorescent protein (GFP)-tagged protein or for expression in HEK-293F cells as an N-terminally FLAG-tagged protein, respectively. For expression of Fhod3 and their mutants as HA-tagged proteins in primary cultures of neonatal rat cardiomyocytes, the adenoviruses encoding HA-tagged mouse Fhod3 and their mutants were constructed by the Adeno-XTM Expression System (Clontech) according to the manufacturer's instructions. All constructs were sequenced for confirmation of their identities. Affinity-purified rabbit polyclonal antibodies specific for Fhod3 (anti-Fhod3-(650–802), anti-Fhod3-(873–974), and anti-Fhod3-(C-20)) were prepared as described previously (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar). Monoclonal antibodies were purchased from commercial sources as indicated: clone EA-53 against α-actinin (Sigma); clone 3-48 against cardiac myosin heavy chain (Abcam); clone Alpha-Sr-1 against sarcomeric actin (Dako); clone 16B12 against HA (Covance). Goat polyclonal antibodies against myomesin-1 (C-16) were purchased from Santa Cruz Biotechnology. Primary cultures of cardiac myocytes were prepared from ventricles of neonatal Sprague-Dawley rats according to the method of Simpson (12Simpson P. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (408) Google Scholar) with minor modifications (13Suematsu N. Tsutsui H. Wen J. Kang D. Ikeuchi M. Ide T. Hayashidani S. Shiomi T. Kubota T. Hamasaki N. Takeshita A. Circulation. 2003; 107: 1418-1423Crossref PubMed Scopus (348) Google Scholar). Briefly, hearts were isolated from 1-day-old postnatal Sprague-Dawley rats, trisected, and then incubated with trypsin (50 μg/ml, Worthington TRLS) in Hanks' balanced saline solution buffer (137 mm NaCl, 5.3 mm KCl, 0.34 mm Na2HPO4, 0.44 mm KH2PO4, 4.2 mm NaHCO3, and 5.6 mm glucose) overnight at 4 °C. The next day, they were digested with collagenase type II (300 units/ml, Worthington CLS2) on a shaking instrument (140 rpm) for 20 min at 37 °C. Cells were preplated for 70 min into 100-mm culture dishes in DMEM supplemented with 10% FCS to reduce the number of nonmyocytes. Cells that were not attached to the dishes were plated and cultured in DMEM with 10% FCS. The purity of myocytes in the culture was >80%, which was estimated by immunofluorescent staining with an anti-sarcomeric actin antibody. Transfection of cardiomyocytes with the adenovirus encoding Fhod3 was performed 24 h after plating. After incubation with the adenovirus for 60 min, cells were cultured in DMEM with 10% FCS for another 48 h and used for the experiments. In the case of sequential transfection with the adenovirus and siRNA, cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen) immediately after incubation with the adenovirus for 60 min. After the sequential transfection, the cells were cultured for 48 h and used for the experiments. HeLa cells were cultured in DMEM supplemented with 10% FCS. HeLa cells were transfected with plasmids using Lipofectamine and cultured for 3 h. After the addition of DMEM containing 10% FCS, cells were cultured for another 13 h. Cells were fixed in 100% methanol for 10 min at −20 °C and blocked with phosphate-buffered saline (137 mm NaCl, 2.68 mm KCl, 8.1 mm Na2HPO4, and 1.47 mm KH2PO4, pH 7.4) containing 3% bovine serum albumin for 60 min. In the case of phalloidin staining, cells were fixed in 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 4 min, and blocked with phosphate-buffered saline containing 3% bovine serum albumin for 60 min. Indirect immunofluorescence analysis was performed using anti-Fhod3, anti-α-actinin, anti-myomesin, and anti-sarcomeric actin antibodies as primary antibodies, and Alexa Fluor 488-labeled anti-rabbit, anti-mouse, and anti-goat antibodies (Invitrogen) were used as secondary antibodies. For F-actin staining, Texas Red-X phalloidin (Invitrogen) was used. Images were taken with a microscope (Axiovert 200; Carl Zeiss MicroImaging) coupled to a camera (Axiocam HRm; Carl Zeiss MicroImaging), with the exception of those shown in Fig. 1, B and C, and supplemental Fig. S1, A and B, which were acquired and processed by deconvolution using a DeltaVision RT imaging system (Applied Precision). Double-stranded siRNAs targeting Fhod3 were synthesized as a 25-nucleotide modified synthetic RNA (StealthTM RNAi; Invitrogen). The sequences were as follows: Fhod3-1(3212) (sense), 5′-CGGUAAUUUAUUGGCUUCUCCUGUG-3′; Fhod3-1(3212) (antisense), 5′-CACAGGAGAAGCCAAUAAAUUACCG-3′; Fhod3-2(3429) (sense), 5′-GAGCACCUGUUUGAGUCCAAGUCUA-3′; Fhod3-2(3429) (antisense), 5′-UAGACUUGGACUCAAACAGGUGCUC-3′; Fhod3-3(U1) (sense), 5′-CGCAUUGACAUGGAUCUCCAGAAAU-3′; Fhod3-3(U1) (antisense), 5′-AUUUCUGGAGAUCCAUGUCAAUGCG-3′; Fhod3-4(U3) (sense), 5′-UCUCCAGAAAUCUCCUGCAUGUAUU-3′; and Fhod3-4(U3) (antisense), 5′-AAUACAUGCAGGAGAUUUCUGGAGA-3′. Fhod3-1 and Fhod3-2 were designed for a coding region, and Fhod3-3 and Fhod3-4 were for a 3′-untranslated region of Fhod3. As a negative control for Fhod3 siRNAs, Low GC Duplex of StealthTM RNAi negative control duplexes (Invitrogen) were used. Transfection of cardiomyocytes with siRNA was performed using Lipofectamine 2000, according to the manufacturer's protocol. After transfection, cells were cultured for 48 h and used for the experiments. Cells were broken with a lysis buffer (10% glycerol, 135 mm NaCl, 5 mm EDTA, and 20 mm Hepes, pH 7.4) containing 1% Triton X-100. The lysates were applied to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was probed with the anti-Fhod3 polyclonal antibodies or anti-HA monoclonal antibody. For preparation of Fhod3 and mDia1 proteins, FreeStyle HEK-293F cells (Invitrogen) were transfected with an expression vector encoding the respective cDNAs. The transfected cells were broken with the lysis buffer, and the lysate was precipitated with an anti-FLAG antibody (M2)-conjugated agarose (Sigma). Proteins eluted with FLAG peptide (200 μg/ml) in X buffer (2 mm MgCl2, 100 mm KCl, 0.1 mm CaCl2, 5 mm EGTA, 1 mm dithiothreitol, and 10 mm Hepes, pH 7.9) were used for an actin polymerization assay as described below. Human profilin IIa were expressed in Escherichia coli as a glutathione S-transferase fusion protein, purified by glutathione-Sepharose 4B (Amersham Biosciences), and cleaved with PreScission protease (Amersham Biosciences). Spectrin-actin seeds were isolated from erythrocyte ghosts by the method of Lin and Lin (14Lin D.C. Lin S. Anal. Biochem. 1980; 103: 316-322Crossref PubMed Scopus (12) Google Scholar). Pyrene-actin polymerization assays were performed in X buffer as described previously (15Suetsugu S. Miki H. Takenawa T. J. Biol. Chem. 2001; 276: 33175-33180Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, G-actin (10% pyrene-labeled) was prepared in G buffer (5 mm Tris-HCl, pH 8.0, 0.2 mm CaCl2, 0.2 mm ATP, 0.2 mm dithiothreitol) and centrifuged at 100,000 × g for 60 min at 4 °C for the removal of residual F-actin. Polymerization reactions were performed in 100 μl of X buffer containing 2 μm actin (10% pyrene-labeled) or 0.5 μm actin (20% pyrene-labeled), and 25 nm formin proteins. All reaction components except the actin were mixed in X buffer, and the reaction was started by the addition of actin. Fluorescence changes (excitation wavelength of 365 nm; emission wavelength of 430 nm) were measured using the microplate reader FlexStation3 (Molecular Devices). shown previously in the fetal rat Fhod3 in a striated pattern (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar). the of Fhod3 with to sarcomeric proteins, we neonatal rat cardiomyocytes for Fhod3 and the Z-line staining with the antibodies that cardiac muscle Fhod3 localizes to a of between the (Fig. was with another two anti-Fhod3 polyclonal the and Fig. expressed Fhod3 as between the Fig. next the of Fhod3 with to the located at the of a sarcomere. staining for Fhod3 and the sarcomeric M-line protein J. Cell Biol. PubMed Scopus Google Scholar) that the M-line was in a of Fhod3 (Fig. staining of F-actin and Fhod3 that Fhod3 (Fig. as as Fhod3 Fig. localizes at of the thin filaments. Thus, Fhod3 in cardiac myocytes is located on thin actin the of the sarcomere (Fig. 1A). In of cells Fhod3, striated myofibrils not throughout the but a to actin filament that striated pattern of of the myofibril organization Fig. These are in cells a variant expressed predominantly in the and brain (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar) Fig. B and the that Fhod3 a role in sarcomere the role of Fhod3 in sarcomeric we Fhod3 in rat cardiomyocytes using siRNAs Transfection of cardiomyocytes with siRNAs to a in Fhod3 at the protein (Fig. 2, A and The of siRNAs to specific to Fhod3 the of myosin heavy a sarcomeric protein, was not (Fig. In cells, sarcomere organization was the α-actinin in a which is in marked with the striated pattern of α-actinin expression in control cells (Fig. 2, A and C, and supplemental Fig. of Fhod3 in of phalloidin staining, indicating that actin filament formation is blocked (Fig. control cardiac myocytes a with myofibrils the of cells, cardiomyocytes a Thus, depletion of Fhod3 a of myocardial the of Fhod3 in sarcomere we transfected the cardiomyocytes with Fhod3 siRNA to a 3′-untranslated region, and with the adenovirus encoding the wild-type Fhod3 protein, which the 3′-untranslated region, and from the cDNA was to by the Expression of wild-type Fhod3 rescued the of Fhod3 siRNA on sarcomere organization (Fig. A and that Fhod3 is for sarcomere by Fhod3 is by expression of the wild-type Fhod3 but not by that of mutant proteins in the actin assembly cardiomyocytes were transfected with the adenovirus encoding HA-tagged wild-type or mutant Fhod3 and with Fhod3 siRNA 4 and cultured for 48 h. Cells were fixed and with the antibodies against anti-HA and α-actinin 10 B, cells with sarcomere organization were and the from are expressed as the as in Fig. C, the protein of expressed HA-tagged Fhod3 was by analysis using the anti-HA next the sarcomere organization its actin assembly a to as an form of a of the by the region (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar), formation of in HeLa cells (Fig. has shown that substitution of for or for in the FH2 domain of the formin the D. B.L. Eck M.J. Cell. 116: Full Text Full Text PDF PubMed Scopus Google Scholar, T. D.R. M. 2005; PubMed Scopus Google Scholar). The substitution in the Fhod3 FH2 domain, or in a of formation (Fig. A and indicating that substitutions the actin assembly of Fhod3. In to the wild-type Fhod3, or in an form to the formation of the striated pattern of α-actinin (Fig. A and mutant proteins were expressed in the cells (Fig. F-actin organization in a striated pattern was not by expression of or in cells of Fhod3 Fig. These findings that the Fhod3 FH2-mediated actin assembly is critical for sarcomere organization in myofibrils. Finally, we the of Fhod3 proteins purified Fig. to actin in As shown in Fig. Fhod3-ΔN assembly of actin. The was by substitution of for in Fhod3. the of an protein that accelerates actin elongation from by actin to the FH1 domain of formins D.R. A.L. T.D. J. Cell Biol. 2003; PubMed Scopus Google Scholar, S. D. D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). in the presence of Fhod3 not but actin assembly, mDia1 (Fig. the Fhod3 actin assembly, we actin assembly in the presence of 0.5 μm G-actin (Fig. the actin elongation is from the barbed The wild-type Fhod3 the elongation from barbed it was by Thus, Fhod3 associates the barbed end of actin filaments and regulates the elongation of actin filaments. As shown in the Fhod3 in cardiomyocytes localizes to thin actin to the of the sarcomere. Because actin filaments in mature myofibrils their the M-line at the of the sarcomere, Fhod3 to associate with actin filaments the end (Fig. 1A). the it that Fhod3 at the barbed end of actin filaments in a to that of the residues and in the FH2 domain of are critical for to the of an actin T. D.R. M. 2005; PubMed Scopus Google Scholar), and conserved in the Fhod3 FH2 domain (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar). As Fhod3 regulates the elongation from the barbed end (Fig. Thus, Fhod3 by with the barbed end of actin filaments. The region where Fhod3 localizes in cells containing the sarcomere the of the between the and M-line in the to from the site where Fhod3 has to form actin filaments. The that the a spliced variant that amino acids in the region (10Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar), is not in to Fhod3 (Fig. and supplemental Fig. that the region the FH2 domain is for the of Fhod3 the of the sarcomere. In cardiomyocytes, Fhod3S the of α-actinin (Fig. Fhod3S promotes F-actin but in a striated pattern and Thus, the of Fhod3 to in sarcomere is possible that the barbed with which Fhod3 associates are to the region of the sarcomere. of in muscle cells has that actin dynamics at the the of the sarcomere R. A. Fowler V.M. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). filaments at their barbed by Fhod3 are incorporated to the end of thin filaments barbed the of the sarcomere. In this it that actin filaments by the formin in the presence of is a major of the thin filaments in myofibrils D.R. Mol. Biol. Cell. PubMed Scopus Google Scholar). are to the for of Fhod3. The that Fhod3 a role in the sarcomere organization of cardiomyocytes, which its actin assembly is known in mature and barbed of the thin filaments are and the barbed by and the by (1Clark K.A. McElhinny A.S. Beckerle M.C. Gregorio C.C. Annu. Rev. Cell Dev. Biol. 2002; 18: 637-706Crossref PubMed Scopus (474) Google Scholar). These two capping proteins shown to for organization of the sarcomere J. Cell Biol. PubMed Scopus Google Scholar, C.C. A. M. Fowler V.M. PubMed Scopus Google Scholar). the actin polymerization is for J. Sci. PubMed Scopus Google Scholar). 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Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) and mammalian D. M. A. P. T.D. R. 2008; PubMed Scopus Google Scholar) as of actin polymerization at in muscle cells. Fhod3 actin assembly and in in with The findings not on a role of Fhod3 in cardiomyocytes but into the regulation of actin dynamics during myofibrillogenesis. for on primary cultures of neonatal rat Watanabe and for and and for and for the from the of with