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.In striated muscle, thin actin filaments and thick filaments of myosin are highly organized to form myofibrils (1) (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 (2–4). 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 (5). 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 (6, 7). 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.Open in a separate windowFIGURE 1.Localization of Fhod3 in cultured rat cardiomyocytes. A, shown is a representation of the sarcomere structure (upper panel) and relative localization of Fhod3 and other sarcomeric proteins from B–D (lower panel). B–D, neonatal rat cardiomyocytes were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green) (B), myomesin (green) (C), or phalloidin (green) (D). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Scale bar, 10 μm.Formins are characterized by the presence of two conserved regions, the formin homology 1 and 2 domains (FH1 and FH2 domains, respectively)² (8, 9). 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 (10), in actin assembly in myofibrils.     

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.     

A dual role of the GTPase Rac in cardiac differentiation of stem cells

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.     

E-Tmod capping of actin filaments at the slow-growing end is required to establish mouse embryonic circulation

Tropomodulins are a family of proteins that cap the slow-growing end of actin filaments. Erythrocyte tropomodulin (E-Tmod) stabilizes short actin protofilaments in erythrocytes and caps longer sarcomeric actin filaments in striated muscles. We report the knockin of the beta-galactosidase gene (LacZ) under the control of the endogenous E-Tmod promoter and the knockout of E-Tmod in mouse embryonic stem cells. E-Tmod(-/-) embryos die around embryonic day 10 and exhibit a noncontractile heart tube with disorganized myofibrils and underdevelopment of the right ventricle, accumulation of mechanically weakened primitive erythroid cells in the yolk sac, and failure of primary capillary plexuses to remodel into vitelline vessels, all required to establish blood circulation between the yolk sac and the embryo proper. We propose a hemodynamic "plexus channel selection" mechanism as the basis for vitelline vascular remodeling. The defects in cardiac contractility, vitelline circulation, and hematopoiesis reflect an essential role for E-Tmod capping of the actin filaments in both assembly of cardiac sarcomeres and of the membrane skeleton in erythroid cells that is not compensated for by other proteins.     

Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation

Myofibrillogenesis - sarcomeres - mouse embryonic stem cells - cardiomyocytes - beta1 integrin Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle-, atrium- and pacemaker-like cell types characterized by developmentally controlled expression of cardiac-specific genes, structural proteins and ion channels. Using this model system, we show here, (I) that during cardiac myofibrillogenesis sarcomeric proteins are organized in a developmentally regulated manner following the order: titin (Z-disk), alpha-actinin, myomesin, titin (M-band), myosin heavy chain, alpha-actin, cardiac troponin T and M-protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I-Z-I complexes is developmentally delayed with respect to A-band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild-type, as well as of beta1 integrin-deficient (beta1-/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild-type and in beta1-/- cardiac cells. The data demonstrate the plasticity of cardiogenesis during the differentiation of wild-type and of genetically modified ES cells.     

Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.     

Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.     

ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.     

Angiotensin II stimulates α-skeletal actin expression in cadiomyocytes in vitro and in vivo in the absence of hypertension

Using a specific alpha-skeletal actin antibody, we have previously shown, that during hypertension-associated cardiac hypertrophy in the rat, the expression of alpha-skeletal actin in the myocardium is increased, but maintains focal distribution, compared to normotensive animals. In the present study, we have investigated whether alpha-skeletal actin expression can be induced in the absence of hypertension. For this purpose, we have examined transgenic mice overexpressing angiotensinogen exclusively in the heart. These animals are characterized by high cardiac angiotensin II levels and cardiac hypertrophy accompanied or not by high blood pressure depending on their genetic background, i.e. presence of one or two renin genes. Alpha-skeletal actin levels were highly increased in transgenic compared to wild-type myocardium independently of the number of renin genes, indicating that angiotensin II can stimulate alpha-skeletal actin expression in normotensive animals. Additional in vitro experiments using cultured mouse and rat cardiomyocytes showed that angiotension II not only increases alpha-skeletal actin expression but also induces an increase of its incorporation within II-bands compared to control cardiomyocytes. Angiotensin II increases also the expression of alpha-smooth muscle actin in sarcomeres of cardiomyocytes as well as in fibroblastic cells present within the culture.     

Expression of nebulette during early cardiac development

Nebulette, a cardiac homologue of nebulin, colocalizes with alpha-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of nebulette in myofibrillogenesis, we have analyzed the expression of nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa nebulette in heart rudiments derived from stage 9-10 using the anti-nebulin mAb, N114, or polyclonal anti-nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-nebulette and anti-alpha-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of approximately 1.0 microm indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that nebulette may play an important role in the formation of these structures.     

Contribution of myosin rod protein to the structural organization of adult and embryonic muscles in Drosophila

Myosin rod protein (MRP) is a naturally occurring 155 kDa protein in Drosophila that includes the myosin heavy chain (MHC) rod domain, but contains a unique 77 amino acid residue N-terminal region that replaces the motor and light chain-binding domains of S1. MRP is a major component of myofilaments in certain direct flight muscles (DFMs) and it is present in other somatic, cardiac and visceral muscles in adults, larvae and embryos, where it is coexpressed and polymerized into thick filaments along with MHC. DFM49 has a relatively high content of MRP, and is characterized by an unusually disordered myofibrillar ultrastructure, which has been attributed to lack of cross-bridges in the filament regions containing MRP. Here, we characterize in detail the structural organization of myofibrils in adult and embryonic Drosophila muscles containing various MRP/MHC ratios and in embryos carrying a null mutation for the single MHC gene. We examined MRP in embryonic body wall and intestinal muscles as well as in DFMs with consistent findings. In DFMs numbers 49, 53 and 55, MRP is expressed at a high level relative to MHC and is associated with disorder in the positioning of thin filaments relative to thick filaments in the areas of overlap. Embryos that express MRP in the absence of MHC form thick filaments that participate in the assembly of sarcomeres, suggesting that myofibrillogenesis does not depend on strong myosin-actin interactions. Further, although thick filaments are not well ordered, the relative positioning of thin filaments is fairly regular in MRP-only containing sarcomeres, confirming the hypothesis that the observed disorder in MRP/MHC containing wild-type muscles is due to the combined action between the functional behavior of MRP and MHC myosin heads. Our findings support the conclusion that MRP has an active function to modulate the contractile activity of muscles in which it is expressed.     

Actin in striated muscle: recent insights into assembly and maintenance

Striated muscle cells are characterised by a para-crystalline arrangement of their contractile proteins actin and myosin in sarcomeres, the basic unit of the myofibrils. A multitude of proteins is required to build and maintain the structure of this regular arrangement as well as to ensure regulation of contraction and to respond to alterations in demand. This review focuses on the actin filaments (also called thin filaments) of the sarcomere and will discuss how they are assembled during myofibrillogenesis and in hypertrophy and how their integrity is maintained in the working myocardium.     

Proliferation of embryonic cardiomyocytes in zebrafish requires the sodium channel scn5Lab

In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of precardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti‐scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 h post‐fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU‐positive cardiomyocytes in embryos subjected to anti‐scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti‐tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti‐tnnt2 and anti‐scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis. genesis 51:562–574. © 2013 Wiley Periodicals, Inc.     

Cardiac myofibrillogenesis inside intact embryonic hearts

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 μm filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.     

Immunoelectron microscopic localization of alpha-actinin and actin in embryonic hamster heart cells

Myofibrillogenesis in developing cardiac cells of the Syrian hamster from early embryonic stages through newborn was studied by electron microscopy, immunofluorescence microscopy and immunoelectron microscopy. alpha-Actinin and actin were localized at light and electron microscopic levels in embryonic heart cells which had been fixed in a periodate-lysine-paraformaldehyde or a glutaraldehyde-formaldehyde mixture, and embedded in Lowicryl K4M. Indirect staining methods were used for immunofluorescence staining of thick sections and immunoferritin staining of thin sections. The earliest evidence of myofibrillogenesis in embryonic myocardial cells was the presence of many randomly arranged thin (6 nm) filaments and a few scattered thick filaments (15 nm) near the plasma membrane. alpha-Actinin was detected in a semi-continuous, diffuse layer in some portions of the cell just beneath the plasma membrane in association with the filamentous collections. Later in development, alpha-actinin coalesced into Z-plaques at the membrane as the filaments arranged into parallel arrays. Actin was localized in the thin filaments as expected. In later stages of development, alpha-actinin was observed at the Z-lines and intercalated discs of the mature myofibrils while actin was localized at both the I-band and Z-line. Our results suggest that myofibrillogenesis is initiated at the plasma membrane and that Z-plaques are precursors of myofibrillar Z-bands and may serve as organizing centers for myofibrillogenesis in developing cardiomyocytes.