Myofibril Assembly Visualized in Living Cardiomyocytes: Experimental studies on fixed cells from our laboratory have shown that N-RAP is a striated muscle-specific scaffolding protein found in developing myofibrils. Previous studies suggest that N-RAP organizes alpha-actinin and actin into symetrical I-Z-I structures, with the barbed ends of actin filaments anchored at a central Z-body or Z-line. The present study aims to understand the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic cardiomyocytes expressing fluorescently-tagged proteins. We have created a plasmid construct designed to express mCherry tagged N-RAP for simultaneous confocal imaging of N-RAP with either recombinant YFP-alpha-actinin or recombinant YFP-actin. Primary cardiomyocyte cultures were prepared from day 7 chick embryos and double transfected with EYFP-alpha-actinin or EYFP-actin (generous gifts from Dr. Joseph Sanger, SUNY Upstate Medical University) and mCherry-N-RAP plasmids. 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 the N-RAP fluorescence exhibited 1 micron periodicity before alpha-actinin binding. The alpha-actinin dots were often initially organized along the N-RAP fibrils with submicron longitudinal spacings, but sometimes exhibited initial periodic longitudinal spacings of 1.5-2.0 microns, similar to the sarcomere spacing in mature myofibrils. 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 exhanged during 10-20 minutes after photobleaching, but this value increased to 70% during myofibril maturation. Simultaneously imaging mCherry-N-RAP and YFP-actin revealed that newly formed actin fibrils incorporate N-RAP with a short delay. The results show that N-RAP assembles into an actin containing scaffold; that alpha-actinin is recruited to the complex following N-RAP incorporation; that periodicity is established in the premyofibril 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.[unreadable] [unreadable] The results of this study are consistent with N-RAP scaffolding of I-Z-I assembly during myofibrillogenesis, but disprove some details of our previously published model of this process. We propose a new molecular model of N-RAP function consistent with the order of events observed in the present study: (1) Actin filaments form. (2) Antiparallel N-RAP dimers cross-link actin filaments in an antiparallel orientation. (3) alpha-actinin binds to N-RAP and cross-links the actin filaments. (4) Krp1 promotes lateral fusion of the Z-bodies to form Z-lines, and N-RAP leaves the complex.[unreadable] [unreadable] Alternative Splicing of N-RAP during Mouse Skeletal Muscle Development: Genetic mouse models of myotonic dystrophy exhibit a specific failure of post-natal alternative splicing transitions in a wide variety of genes, including N-RAP. To establish the pattern of N-RAP alternative splicing during normal muscle development, we studied N-RAP alternative splicing and protein localization 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 &#945;-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.[unreadable] [unreadable] Role of Krp1 in Myofibril Assembly: Krp1 is a kelch-repeat protein hypothesized to promote myofibril assembly through interaction with N-RAP and actin. Our previous work demonstrated that myofibril maturation was interrupted by Krp1 knockdown in cultured cardiomyocytes, leading to the accumulation of very thin myofibrils and their subsequent degradation. We concluded that Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils but is not necessary for longitudinal organization of actin and myosin filaments. In order to discover the molecular mechanism by which Krp1 promotes lateral fusion of myofibril assembly intermediates, we intend to study the targeting and binding properties of individual regions of Krp1. To that end we have subcloned the Krp1 kelch repeats, the Krp1 BTB domain, and the Krp1 BACK domain into vectors for expression as YFP-fusion proteins and GST fusion proteins for cell biological and biochemical studies.[unreadable] [unreadable] Creation of N-RAP Overexpressing Transgenic Mice: Our previous collaborative work has shown that N-RAP expression increases in genetic mouse models of dilated cardiomyopathy, and that the increased N-RAP expression in these animals occurs shortly after birth and precedes other molecular changes in the heart. We aim to directly test the effects of N-RAP overexpression by creating transgenic mouse lines that overexpress N-RAP in heart and skeletal muscle tissues using the well-characterized tet-off system.[unreadable] [unreadable] We have cloned full length N-RAP cDNA (both N-RAP-s and N-RAP-c isoforms) into the pTRE2hyg2-myc tet-responsive vector and verified expression of the full length myc-tagged N-RAP proteins in cultured cells constitutively expressing the tetracycline transactivator (tTA) protein. Transgenic founder mice were produced for each N-RAP isoform, and the tet-responsive N-RAP transgenic mice were mated with cardiac-specific tTA expressing mice. We discovered that matings of male tTA mice with female myc-N-RAP mice produced few offspring. In contrast, normal sized litters were produced by female tTA mice mated with male myc-N-RAP mice. In initial experiments, doxycycline was used to repress transgene expression during prenatal and postnatal development. However, we discovered that animals treated with doxycycline from conception until 3 weeks after birth did not exhibit transgene expression after doxycycline withdrawal. In contrast, screening double transgenic animals for myc-N-RAP expression without exposure to doxycycline has identified several founder lines that exhibit cardiac-specific expression of the N-RAP transgene at both the mRNA and protein level. Interestingly, a doxycycline-treated animal from one of these newly identified positive lines had failed to express the N-RAP transgene 19 weeks after doxycline withdrawal. We conclude that we have successfuly produced cardiac-specific tet-responsive N-RAP expressing transgenic animals, and that the effects of doxycycline repression of transgene activity are extraordinarily long-lived. We are continuing to screen founder lines in order to propagate several independent transgenic lines for each N-RAP isoform. We are also characterizing the animals for transgene expression levels and cardiac phenotypes using histological and echocardiographic methods.