N-RAP scaffolds I-Z-I assembly during myofibrillogenesis in cultured chick cardiomyocytes

Abstract

N-RAP is a muscle-specific protein with an N-terminal LIM domain (LIM), C-terminal actin-binding super repeats homologous to nebulin (SR) and nebulin-related simple repeats (IB) in between the two. Based on biochemical data, immunofluorescence analysis of cultured embryonic chick cardiomyocytes and the targeting and phenotypic effects of these individual GFP-tagged regions of N-RAP, we proposed a novel model for the initiation of myofibril assembly in which N-RAP organizes α-actinin and actin into the premyofibril I-Z-I complexes. We tested the proposed model by expressing deletion mutants of N-RAP (i.e. constructs containing two of the three regions of N-RAP) in chick cardiomyocytes and observing the effects on α-actinin and actin organization into mature sarcomeres. Although individually expressing either the LIM, IB, or SR regions of N-RAP inhibited α-actinin assembly into Z-lines, expression of either the LIM-IB fusion or the IB-SR fusion permitted normal α-actinin organization. In contrast, the LIM-SR fusion (LIM-SR) inhibited α-actinin organization into Z-lines, indicating that the IB region is critical for Z-line assembly. While permitting normal Z-line assembly, LIM-IB and IB-SR decreased sarcomeric actin staining intensity; however, the effects of LIM-IB on actin assembly were significantly more severe, as estimated both by morphological assessment and by quantitative measurement of actin staining intensity. In addition, LIM-IB was consistently retained in mature Z-lines, while mature Z-lines without significant IB-SR incorporation were often observed. We conclude that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes, and that they also play an important role in removal of the N-RAP scaffold from the completed myofibrillar structure. This work strongly supports the N-RAP scaffolding model of premyofibril assembly.

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

“…Digital images were obtained using Zeiss LSM software. Morphometric analysis was performed using Image J software as previously described [24,25].…”

“…Therefore, conclusions regarding the effects of Krp1 knockdown on myofibril assembly can only be made based on quantitative analysis from many cells. The morphometric analysis of striated α-actinin provides a reproducible measure of Zline assembly (figure 5B) [24][25][26], while assignment of cardiomyocytes according to patterns of α-actinin organization yields additional information concerning the organization of this protein at different times ( figure 7). Using these quantitative assays, we examined the effects of Krp1 knockdown on accumulation of mature Z-lines and tracked the accumulation of normally low abundance assembly and disassembly intermediates in the cardiomyocyte population.…”

“…These include N-RAP, a LIM domain and nebulin repeat protein found in the premyofibril regions of cardiomyocytes and at the Zlines and M-lines of newly formed myofibrils [14,22,23], and Krp1, which binds N-RAP and is found between laterally fusing myofibrils in spreading chick cardiomyocytes [14]. During myofibril assembly, N-RAP organizes actin and α-actinin into I-Z-I structures (symmetrical actin filaments with their barbed ends anchored at a central Z-body or Z-line containing α-actinin) [24,25], while Krp1 has been hypothesized to promote lateral fusion of premyofibril structures [14].…”

N-RAP expression during mouse heart development

“…Although subcellular localization and biochemical copurification from adult muscles suggested that N-RAP may link the ends of myofibrils to membrane-associated protein complexes, studies in cultured embryonic cardiomyocytes have implicated N-RAP in myofibril assembly (Carroll and Horowits, 2000;Carroll et al, 2001Carroll et al, , 2004Lu et al, 2003). These studies showed that N-RAP localizes with the earliest myofibril precursors that originate near the membrane (Carroll and Horowits, 2000;Lu et al, 2003).…”

Molecular mechanisms of mechanosensing in muscle development

“…Studies in cell culture have established that sarcomere assembly is a stepwise program that initiates at the basal surface of the sarcolemma, at which sarcomere precursors are tethered before their transition to mature striated myofibrils (Rhee et al, 1994;Dabiri et al, 1997;Carroll et al, 2004). Thus, sarcolemmal adhesions appear to contribute to sarcomerogenesis by providing a scaffold for assembly.…”