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

Abstract

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.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 ...

Dilated Cardiomyopathy-Associated <i>FHOD3</i> Variant Impairs the Ability to Induce Activation of Transcription Factor Serum Response Factor

“…3 The I1127A substitution was demonstrated to reduce SRF reporter gene activity by 90% in this study. In contrast, the DCM-associated variation reduced SRF activation by only 10%.…”

“…Because FHOD3 is known to localize at the A-band, 3 we analyzed cellular localization of FHOD3-WT or DCM-associated FHOD3 variant. NRCs were transfected with Flag-tagged WT or variant Fhod3 construct, co-immunostained for Flag-tag and a Z-disc marker, cardiac α-actinin, and examined under confocal microscopy, which showed Flag-FHOD3-WT mainly localizing at the A-band in NRCs (Figures 3A-C) as reported previously, 3 and no apparent change in the localization of the DCM-associated variant type Flag-FHOD3 (Figures 3D-F), suggesting that overexpression of variant FHOD3 was neither deleterious to maintaining sarcomere structure nor causative of drastic change in the cellular localization of FHOD3.…”

Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

“…In muscle cells, the specific mechanisms for actin nucleation and maintenance in sarcomeres were poorly understood (8). Recent studies have uncovered actin nucleation activities of the nebulin-N-WASP complex (9) and of formin proteins FHOD3 (10)(11)(12), mDia2, DAAM, FMNL1, and FMNL2 (13,14) in sarcomeres. In particular, leiomodin (Lmod) has been identified as a class of potent tandem-G-actin-binding nucleators in muscle cells (15,16); Lmod1 is found in smooth muscle of many human tissues, and Lmod2 and Lmod3 are found in cardiac and skeletal muscle (17).…”

Insights into the effects of disease‐causing mutations in human actins

“…The human formin FHOD1 has been shown to be important for normal smooth muscle development [Mi-Mi et al, 2012], and an altered interaction of the TAAD mutant actin with FHOD1 could be a significant factor in the development of the aortic aneurysms associated with this disease. Another formin, FHOD3, has been associated with proper development and function of striated muscle, both skeletal and cardiac [Taniguchi et al, 2009;Iskratsch and Ehler, 2011;Kan-o et al, 2012;Iskratsch et al, 2010Iskratsch et al, , 2013, and this results suggests that an altered interaction of this formin with mutant actins could give rise to pathogenic states in both skeletal and cardiac muscle. In a third example, a mutation in the formin homologue DIAPH3, analogous to the yeast formin Bni1, has been reported to cause deafness [Schoen et al, 2010[Schoen et al, , 2013, again suggesting that in the case of the deafness-causing actin mutations, an altered actin/formin interaction might be involved.…”