Nonmuscle Myosin IIB & N-RAP: We previously showed that the muscle protein N-RAP controls assembly of alpha-actinin and actin into I-Z-I structures, providing a scaffold for premyofibril assembly. N-RAP knockdown by RNA interference inhibited myofibril assembly and decreased levels of nonmuscle myosin heavy chain IIB (nmMHCIIB) by post-transcriptional mechanisms. Others have proposed that nmMHCIIB is required for sarcomere formation in cardiac myocytes. To investigate its role in myofibrillogenesis and its relationship with N-RAP, we specifically decreased nmMHCIIB in cultured cardiomyocytes using RNA interference initiated by transfection with specific small inhibitory RNA duplexes (siRNAs). nmMHCIIB transcript and protein levels decreased 90% compared with mock transfected cells by 1 and 3 days post transfection, respectively, and both gradually recovered to normal levels after 8 days. nmMHCIIB knockdown resulted in a slow decrease in N-RAP protein levels over 6 days with no change in N-RAP transcript levels. N-RAP levels markedly increased after treatment with the proteasome inhibitor MG132, suggesting that decreased N-RAP levels after nmMHCIIB knockdown may be due to effects on N-RAP ubiquitination and degradation by proteasomes. Alpha-actinin organization into mature striations was used as a measure of myofibril content and assessed by confocal microscopy and morphometric analysis. Cell areas and myofibril areas were unaffected by nmMHCIIB knockdown at 3 days post-transfection, when nmMHCIIB levels were low but N-RAP levels were normal. However, additional cell spreading was halted by nmMHCIIB knockdown, while myofibril accumulation rates slowed as N-RAP levels decreased. The existing myofibrils were normal, as assessed by staining for alpha-actinin, actin, muscle myosin, and myomesin. As nmMHCIIB and N-RAP levels spontaneously returned to normal between 6 and 8 days, cell spreading and myofibril accumulation resumed. The results show that nmMHCIIB is required for cell spreading but plays no role in myofibril maintenance in mouse cardiomyocytes, while N-RAP levels are closely linked to myofibril assembly.? ? Krp1: Krp1 is a kelch-repeat protein hypothesized to promote myofibril assembly through interaction with N-RAP and actin. We studied the distribution and function of Krp1 in cultured embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant proportion of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation. Cells were otherwise healthy as indicated by lack of apoptosis, normal spreading over time in culture, and retention of intact organelles assessed by electron microscopy. Instead of accumulating mature myofibrils, Krp1 siRNA-transfected cardiomyocytes were often filled with periodically spaced structures resembling newly forming myofibrils. Confocal microscopy of cells stained for actin, myosin, and myomesin demonstrated that these structures contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, and electron microscopy showed normal thick and thin filaments. However, fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the previously reported myofibril disassembly pathway observed during cell division in the developing heart. These data indicate 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 addition, myofibril disassembly can occur by two distinct pathways in cardiomyocytes.? ? Time-lapse Imaging of Myofibril Assembly: Experimental studies on fixed cells from our lab have shown that N-RAP is an assembly scaffold during myofibrillogenesis. The present study aims to understand the details of N-RAP's role in myofibril assembly through time-lapse microscopic imaging of fluorescently labeled proteins introduced into cultured cardiomyocytes. Various conditions were explored to overcome technical difficulties inherent in this type of experiment: Primary cardiomyocytes isolated from day 17 mouse embryos were compared with the HL-1 atrial cell line, and fluorescent protein delivery via plasmid transfection and direct proteinfection were compared. We found that immortalized HL-1 atrial cells accumulate few myofibrils due to constant assembly and disassembly associated with cell division, making this system unsuitable for studying assembly. In contrast, primary embryonic mouse cardiomyocytes accumulate additional myofibrils over several days in culture, constituting an excellent system in which to study dynamic aspects of myofibril assembly. Delivery of alexa-fluor 555 labeled alpha-actinin through proteinfection using Provectin reagent led to protein aggregation and trafficking, but only minimal incorporation into myofibrils. In contrast, promising results have been obtained with standard transfection of plasmids encoding recombinant fluorescently tagged proteins. In preliminary experiments, YFP-alpha-actinin formed faint separated spots that aligned into striations and intensified over a two hour period, and GFP-N-RAP organized into punctate structures resembling myofibril precursors. Since GFP-N-RAP and YFP-alpha-actinin have overlapping emission spectra, we have created a plasmid construct to express mCherry tagged N-RAP for simultaneous confocal imaging of both alpha-actinin and N-RAP. The study is expected to uncover the sequential steps involved in the association of N-RAP with alpha-actinin during myofibrillogenesis in real time.
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