N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. N-RAP is concentrated in myofibril precursors during sarcomere assembly in cultured cardiomyocytes and early embryos. Distinct regions of N-RAP bind specific cytoskeletal proteins, and overexpression of these regions in cultured chick cardiomyocytes inhibits or arrests myofibril assembly. Thus, we hypothesize that N-RAP is a scaffolding protein that coordinates organization of alpha-actinin and actin in the initial premyofibril complexes that form at the cell periphery, and that deletion of N-RAP will yield cardiomyocytes incapable of assembling mature myofibrils. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by ~70% within two days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching ~50% of control levels within six days. In contrast, nebulin transcripts, which encode the protein with the closest homology to N-RAP repeats targeted by the N-RAP siRNA, were unaffected, demonstrating the extremely specific nature of RNA interference. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations, while cell spreading was not affected. The effect of N-RAP knockdown on alpha-actinin organization was partially rescued by expression of N-RAP-LIM-IB, a deletion mutant containing the N-RAP LIM domain and simple repeats, but omitting the super repeats. This construct allows normal assembly of alpha-actinin into mature striations, but disrupts sarcomeric actin organization. N-RAP-LIM-IB expression prevented N-RAP knockdown by siRNA at both the mRNA and protein levels. Therefore, the mechanism by which N-RAP-LIM-IB rescues myofibril assembly is likely to be by its effect on endogenous N-RAP expression. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP knockdown, and alpha-actinin, Krp-1, and sarcomeric actin protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days six and seven, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that myofibril assembly is closely linked to N-RAP protein levels, that N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms, and that the levels of muscle-specific actin and myosin isoforms can be independently regulated.? ? Much of the information on N-RAP?s probable role as an assembly catalyst comes from targeting and localization studies in cells visualized after fixation. We are using time-lapse microscopy of living cardiomyocytes to explore the dynamics of premyofibril assembly. Preliminary experiments have demonstrated dynamic organization of full-length GFP-N-RAP into punctate dots at the periphery of cardiomyocytes and their disappearance from these structures in time intervals similar to that seen for alpha-actinin organization into premyofibrils in spreading chick cardiomyocytes. Delivery of labeled proteins using the ProVectin reagent has also been effectively demonstrated. The study is expected to uncover the sequential steps involved in the association of N-RAP with alpha-actinin and actin during myofibrillogenesis in real time.? ? Our discovery of N-RAP and accumulation of evidence that N-RAP catalyzes the first steps in premyofibril assembly have led us to hypothesize that other proteins catalyze subsequent steps in assembly. This hypothesis predicts that knockdown of downstream assembly catalysts will lead to accumulation of intermediates in the myofibril assembly pathway. Our previous discovery that Krp1 binds N-RAP repeats but is not localized until late in the assembly process and is not a component of mature sarcomeres makes this protein an ideal candidate for a catalyst of myofibril assembly that functions downstream from N-RAP. Krp1 is a skeletal- and cardiac-muscle-specific member of the kelch superfamily of proteins. In cultured embryonic chick cardiomyocytes, we found Krp1 at the periphery of mature myofibrils which appeared to be joining laterally with narrow myofibrils. This lateral fusion process is responsible for transforming myofibril precursors into mature myofibrils with broad Z-lines. We hypothesize that Krp1 is involved late in myofibril assembly, and may catalyze the lateral fusion of myofibril precursors. To elucidate the role of Krp1 in myofibril assembly, endogenous Krp1 expression was reduced using RNA interference. Chemically synthesized siRNA against a Krp1 target sequence was transfected into primary mouse embryonic cardiomyocytes, and Krp1 expression was determined by quantitative PCR and immunoblotting. Krp1 transcript levels were reduced over 70% within 48 hours post-transfection compared to mock- and scrambled siRNA-transfected control cells, and Krp1 protein levels were decreased 50% by 72 hours. In contrast, levels of sarcomeric alpha-actin and muscle myosin were unaffected. Alpha-actinin organization into mature striations was used as a measure of myofibril content and assessed by confocal microscopy and morphometric analysis. Cardiomyocytes transfected with scrambled siRNA and Krp1 siRNA contained mean myofibril areas corresponding to 54% and 29% of total cell area, respectively, indicating that Krp1 is required for myofibril assembly and/or maintenance of mature myofibrils. In control cells transfected with scrambled siRNA, many broad Z-lines were observed with 1.8 to 2.2 micron periodicity. After Krp1 knockdown, broad Z-lines were often absent and alpha-actinin was organized into continuous fibers, aperiodic dots, or narrow bands or dots with 1.8 to 2.2 micron periodicity. The appearance of numerous punctate alpha-actinin dots with mature sarcomere spacings after Krp1 knockdown suggests blockade of an assembly step following punctate Z-body assembly. These data are consistent with a role for Krp1 late in myofibril assembly.? ? In striated muscles, the giant nebulin polypeptide extends the length of the actin filaments, and nebulin size has been correlated with actin filament lengths in muscles from different species. Recent studies have demonstrated the presence of nebulin in cardiac muscle, but at levels more than 100-fold less than in skeletal muscle, in which each sarcomeric actin filament contains two nebulin polypeptides. A recent study demonstrated that nebulin knockdown leads to loss of actin filament length regulation in cardiomyocytes, providing functional evidence for the regulation of actin filament lengths by nebulin in cardiac muscle. I have proposed some testable models by which substoichiometric levels of nebulin may effectively regulate the distribution of actin filament lengths in cardiac muscle. These include dynamically exchanging substoichiometric populations of nebulin rulers, as well as a scanning ruler model of actin filament length regulation in which nebulin rotates through an angle to detect and cap actin filament ends by recruiting tropomodulin binding.
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