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1.  Cardiac-Specific NRAP Overexpression Causes Right Ventricular Dysfunction in Mice 
Experimental cell research  2011;317(8):1226-1237.
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.
PMCID: PMC3070810  PMID: 21276443
NRAP; cardiomyopathy; heart; intercalated disk; transgenic; tetracycline-controlled transactivator
2.  Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle 
Biophysical reviews  2011;3(1):25-32.
Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.
PMCID: PMC3110075  PMID: 21666840
Myofibrillogenesis; N-RAP; Krp1; Hsp90; Hsc70; UNC-45
3.  Myofibril Assembly Visualized by Imaging N-RAP, Alpha-Actinin, and Actin in Living Cardiomyocytes 
Experimental cell research  2009;315(12):2126-2139.
N-RAP is a striated muscle-specific scaffolding protein that organizes α-actinin and actin into symetrical 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 α-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of α-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 α-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 α-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 minutes after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before α-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.
PMCID: PMC2742992  PMID: 19233165
myofibrillogenesis; heart; sarcomere
4.  Expression and Alternative Splicing of N-RAP during Mouse Skeletal Muscle Development 
Cell motility and the cytoskeleton  2008;65(12):945-954.
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 three 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 α-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.
PMCID: PMC2754055  PMID: 18792955
myofibrillogenesis; myotube; sarcomere
5.  Krp1 (Sarcosin) Promotes Lateral Fusion of Myofibril Assembly Intermediates in Cultured Mouse Cardiomyocytes 
Experimental cell research  2008;314(5):1177-1191.
Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch-repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly.
PMCID: PMC2275804  PMID: 18178185
kelch; heart; myofibrillogenesis; α-actinin; actin; myosin
6.  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 α-actinin and actin assembly during myofibrillogenesis, and we quantitated myofibril accumulation by morphometric analysis of α-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.
PMCID: PMC2593092  PMID: 18615632
myofibrillogenesis; RNA interference; heart; sarcomere
7.  Alterations at the Intercalated Disk Associated with the Absence of Muscle Lim Protein 
The Journal of Cell Biology  2001;153(4):763-772.
In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain– containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.
PMCID: PMC2192386  PMID: 11352937
dilated cardiomyopathy; N-RAP; tropomodulin; adherens junction; gap junction

Results 1-7 (7)