Abstract
Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.
Highlights
Myofibrils, a contractile structure in striated muscles, are composed of functional repeating units called sarcomeres, which are highly organized arrays of thin actin filaments and myosinbased thick filaments [1]
Overexpression is a useful approach to identifying Fhod3 localization; we have previously shown that Fhod3 protein expressed in a transgenic manner distributes in the same pattern as the endogenous protein, but more intensely [19]
Morphological analysis of embryos revealed that Fhod3Tg(β-myosin heavy chain (MHC)-Fhod3IA) embryos (Fhod3+/+; Tg(IA)+) at E9.5 were comparable to nontransgenic wild-type embryos (Fhod3+/+) in size and gross morphology, appearing remarkably normal (Fig 1A), the exogenous Fhod3 protein was already abundantly expressed at this time point (S1 Fig)
Summary
Myofibrils, a contractile structure in striated muscles, are composed of functional repeating units called sarcomeres, which are highly organized arrays of thin actin filaments and myosinbased thick filaments [1]. Actin filaments are dynamically organized into highly ordered mature structures from an irregularly-oriented state with a striking increase of their content [2,3]. Mechanisms for the regulation of actin dynamics during myofibrillogenesis have remained largely unknown, various actin-binding proteins, including tropomodulin (Tmod), troponin T, and α-tropomyosin are known to contribute to this process [4,5,6]. A member of the formin family proteins, is another probable candidate for a key regulator of actin dynamics during myofibrillogenesis. Structurally characterized by the presence of the formin-homology domains 1 and 2 (FH1 and FH2), play pivotal roles in remodeling the actin and microtubule cytoskeletons [7,8,9]. Through cooperation of the FH1 and FH2 domains, formins contribute to various biological functions via regulation of actin dynamics. Recent studies using genetically engineered animals revealed that various formins play critical roles in morphogenesis and organogenesis during development [11,12]
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