Shajia Lu

New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly

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. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: α-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB). We detected significant α-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with α-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly.

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

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.

N‐RAP expression during mouse heart development

N‐RAP gene expression and N‐RAP localization were studied during mouse heart development using semiquantitative reverse transcriptase‐polymerase chain reaction and immunofluorescence. N‐RAP mRNA was detected at embryonic day (E) 10.5, significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth. In E9.5–10.5 heart tissue, N‐RAP protein was primarily associated with developing premyofibril structures containing α‐actinin, as well as with the Z‐lines and M‐lines of more‐mature myofibrils. In contrast, N‐cadherin was concentrated in patches at the periphery of the cardiomyocytes. N‐RAP labeling markedly increased between E10.5 and E16.5; almost all of the up‐regulated N‐RAP was associated with intercalated disk structures, and the proportion of mature sarcomeres containing N‐RAP decreased. In adult hearts, specific N‐RAP staining was only observed at the intercalated disks and was not found in the sarcomeres. The results are consistent with N‐RAP functioning as a catalytic scaffolding molecule, with low levels of the scaffold being sufficient to repetitively catalyze key steps in myofibril assembly. Developmental Dynamics 233:201–212, 2005.

Expression and alternative splicing of N-RAP during mouse skeletal muscle development.

N-RAP alternative splicing and protein localization were studied in developing skeletal muscle tissue from pre- and postnatal mice and in fusing primary myotubes in culture. Messages encoding N-RAP-s and N-RAP-c, the predominant isoforms of N-RAP detected in adult skeletal muscle and heart, respectively, were present in a 5:1 ratio in skeletal muscle isolated from E16.5 embryos. N-RAP-s mRNA levels increased three-fold over the first 3 weeks of postnatal development, while N-RAP-c mRNA levels remained low. N-RAP alternative splicing during myotube differentiation in culture was similar to the pattern observed in embryonic and neonatal muscle, with N-RAP-s expression increasing and N-RAP-c mRNA levels remaining low. In both developing skeletal muscle and cultured myotubes, N-RAP protein was primarily associated with developing myofibrillar structures containing alpha-actinin, but was not present in mature myofibrils. The results establish that N-RAP-s is the predominant spliced form of N-RAP present throughout skeletal muscle development.

Role of Nonmuscle Myosin IIB and N-RAP in Cell Spreading and Myofibril Assembly in Primary Mouse Cardiomyocytes

We investigated the role of nonmuscle myosin heavy chain (NMHC) IIB in cultured embryonic mouse cardiomyocytes by specific knockdown using RNA interference. NMHC IIB protein levels decreased 90% compared with mock-transfected cells by 3 days post transfection. NMHC IIB knockdown resulted in a slow decrease in N-RAP protein levels over 6 days with no change in N-RAP transcript levels. N-RAP is a scaffold for alpha-actinin and actin assembly during myofibrillogenesis, and we quantitated myofibril accumulation by morphometric analysis of alpha-actinin organization. Between 3 and 6 days, NMHC IIB knockdown was accompanied by the abolishment of cardiomyocyte spreading. During this period the rate of myofibril accumulation steadily decreased, correlating with the slowly decreasing levels of N-RAP. Between 6 and 8 days NMHC IIB and N-RAP protein levels recovered, and cardiomyocyte spreading and myofibril accumulation resumed. Inhibition of proteasome function using MG132 led to accumulation of excess N-RAP, and the secondary decrease in N-RAP that otherwise accompanied NMHC IIB knockdown was abolished. The results show that NMHC IIB knockdown led to decreased N-RAP levels through proteasome-mediated degradation. Furthermore, these proteins have distinct functional roles, with NMHC IIB playing a role in cardiomyocyte spreading and N-RAP functioning in myofibril assembly.

Cardiac-specific NRAP overexpression causes right ventricular dysfunction in mice.

The muscle-specific protein NRAP is concentrated at cardiac intercalated disks, plays a role in myofibril assembly, and is upregulated early in mouse models of dilated cardiomyopathy. Using a tet-off system, we developed novel transgenic lines exhibiting cardiac-specific NRAP overexpression ~2.5 times greater than normal. At 40-50 weeks, NRAP overexpression resulted in dilation and decreased ejection fraction in the right ventricle, with little effect on the left ventricle. Expression of transcripts encoding brain natriuretic peptide and skeletal α-actin was increased by cardiac-specific NRAP overexpression, indicative of a cardiomyopathic response. NRAP overexpression did not alter the levels or organization of N-cadherin and connexin-43. The results show that chronic NRAP overexpression in the mouse leads to right ventricular cardiomyopathy by 10 months, but that the early NRAP upregulation previously observed in some mouse models of dilated cardiomyopathy is unlikely to account for the remodeling of intercalated disks and left ventricular dysfunction observed in those cases.

Cardiac Specific Overexpression of N-RAP in Transgenic Mice

The muscle specific protein NRAP plays a role in myofibril assembly and is upregulated in mouse models of dilated cardiomyopathy. We sought to determine if increased N-RAP expression would directly lead to a cardiomyopathy phenotype. Novel transgenic lines were developed using the tet-off system with transgenic N-RAP expression requiring the tetracycline transactivator (tTA). tTA was introduced by mating the N-RAP transgenic animals with well-characterized animals carrying the tTA transgene controlled by the cardiac specific alpha-myosin heavy chain promoter. Multiple founder lines were examined and lines showing the most significant increase in NRAP expression were used for further investigation. N-RAP expression in theses animals was up to 2.5 times greater than control littermates as determined by western blot analysis. Histological examination of hearts from ∼12 week old transgenic mice showed no structural defects compared to control littermates. Additionally, examination of these hearts by immunofluorescence microscopy revealed normal myofibrillar structure and localization of the transgenic protein to intercalated disks, as normally seen with the endogenous protein. Protein markers for cardiomyopathy were examined by qPCR and revealed no difference between non-transgenic and transgenic animals. Echocardiography and magnetic resonance imaging of the N-RAP transgenic animals revealed no significant structural or functional differences when compared to control littermates at 12 weeks of age. Based on these data, it does not appear that overexpression of N-RAP directly leads to an observable cardiac phenotype. The alternative hypothesis that upregulation of N-RAP in dilated cardiomyopathy is compensatory remains to explored.