ABSTRACT
N-RAP is a striated muscle-specific scaffolding protein that organizes alpha-actinin and actin into symmetrical I-Z-I structures in developing myofibrils. Here we determined the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either alpha-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of alpha-actinin subsequently organizing along these structures. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The alpha-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the alpha-actinin dynamically exchanged during all stages of myofibril assembly. In contrast, less than 20% of the N-RAP in premyofibrils was exchanged during 10-20 min after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before alpha-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.
Subject(s)
Actinin/metabolism , Actins/metabolism , Muscle Proteins/metabolism , Myocytes, Cardiac/physiology , Myofibrils/physiology , Animals , Cells, Cultured , Chick Embryo , Humans , Myocytes, Cardiac/ultrastructure , Protein BindingABSTRACT
BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK-3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/beta-catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells-c1 (NFATc1), both key regulators of valve development. Li-exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK-3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/beta-catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up-regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK-3 directly to augment Wnt signaling.
Subject(s)
Antipsychotic Agents/adverse effects , Gastrulation/drug effects , Heart Valves/abnormalities , Lithium/adverse effects , NFATC Transcription Factors , Wnt Proteins , Animals , Antipsychotic Agents/administration & dosage , Chick Embryo , Gene Expression Regulation, Developmental , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Heart/drug effects , Heart/embryology , Heart Defects, Congenital/embryology , Heart Valves/drug effects , Heart Valves/embryology , Humans , Lithium/administration & dosage , Mice , Myocardium/cytology , Myocardium/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Neural Crest/drug effects , Wnt Proteins/genetics , Wnt Proteins/metabolismABSTRACT
Members of both Wnt and bone morphogenetic protein (BMP) families of signaling molecules are important in heart development. We previously demonstrated that beta-catenin, a key downstream intermediary of the canonical Wnt signaling pathway, delineates the dorsal boundary of the cardiac compartments in an anteroposterior progression. We hypothesized the progression involves canonical Wnt signaling and reflects development of the primary body axis of the embryo. A similar anteroposterior signaling wave leading to cardiac cell specification involves inductive signaling by BMP-2 synthesized by the underlying endoderm in anterior bilateral regions. Any molecule that disrupts the normal balance of Wnt and BMP concentrations within the heart field may be expected to affect early heart development. The canonical Wnt signaling step mimicked by lithium involves inactivation of glycogen synthase kinase-3beta (GSK-3beta; Klein and Melton [1996] Proc. Natl. Acad. Sci. U. S. A. 93:8455-8459). We show that lithium, Wnt-3A, and an inhibitor of GSK-3beta, SB415286, affect early heart development at the cardiac specification stages. We demonstrate that normal expression patterns of key signaling molecules as Notch-1 and Dkk-1 are altered in the anterior mesoderm within the heart fields by a one-time exposure to lithium, or by noggin inhibition of BMP, at Hamburger and Hamilton (HH) stage 3 during chick embryonic development. The severity of developmental defects is greatest with exposure to lithium or Wnt-3A at HH stage 3 and decreases at HH stage 4. Taken together, our results demonstrate that there are temporal-specific responses and differential sensitivities to lithium/Wnt-3A exposure during early heart development.
