Correlation of benign incidental findings seen on whole-body PET-CT with knee MRI: patterns of 18F-FDG avidity, intra-articular pathology, and bone marrow edema lesions.

OBJECTIVES: To correlate patterns of 18F-FDG uptake on whole-body PET-CT with MR findings and compare the degree of FDG activity between symptomatic and asymptomatic knees. MATERIALS AND METHODS: Retrospective database query was performed using codes for knee MRI as well as whole-body PET-CT. Patients with malignant disease involving the knee or hardware were excluded. Patients who had both studies performed within 1 year between 2012 and 2017 were included for analysis. Knee joint osteoarthrosis, meniscal and ligamentous integrity, presence of joint effusion, and synovitis were assessed and recorded. Bone marrow edema lesions (BMELs) were identified, segmented, and analyzed using volumetric analysis. SUVmax was assessed over the suprapatellar joint space, intercondylar notch and Hoffa's fat pad. Symptomatic and asymptomatic knees were compared in patients with unilateral symptoms. RESULTS: Twenty-two cases (20 patients) with mean age 63.3 years (range, 36-91 years) were included. Two patients had bilateral pain. The most FDG avid regions in both symptomatic and asymptomatic knees were the intercondylar notch (SUVmax = 1.84 vs. 1.51), followed by suprapatellar pouch (SUVmax = 1.74 vs. 1.29) and Hoffa's fat pad (SUVmax = 1.01 vs. 0.87). SUVmax was significantly associated with cartilage loss (mean modified Outerbridge score) (r = 0.60, p = 0.003) and degree of synovitis (r = 0.48, p = 0023). Overall, mean SUVmax was significantly higher in the presence of a meniscal tear (1.83 ± 0.67 vs. 1.22 ± 0.40, p = 0.030). Nine patients had BMELs (volume: range = 0.6-27.8, mean = 7.79) however there was no significant association between BMEL volume and SUVmax. CONCLUSIONS: Higher FDG activity correlates with intra-articular derangement and the intercondylar notch represents the most metabolically active region of the knee.

Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

Use of a web-based image reporting and tracking system for assessing abdominal imaging examination quality issues in a single practice.

This article presents our local experience in the implementation of a real-time web-based system for reporting and tracking quality issues relating to abdominal imaging examinations. This system allows radiologists to electronically submit examination quality issues during clinical readouts. The submitted information is e-mailed to a designate for the given modality for further follow-up; the designate may subsequently enter text describing their response or action taken, which is e-mailed back to the radiologist. Review of 558 entries over a 6-year period demonstrated documentation of a broad range of examination quality issues, including specific issues relating to protocol deviation, post-processing errors, positioning errors, artifacts, and IT concerns. The most common issues varied among US, CT, MRI, radiography, and fluoroscopy. In addition, the most common issues resulting in a patient recall for repeat imaging (generally related to protocol deviation in MRI and US) were identified. In addition to submitting quality problems, radiologists also commonly used the tool to provide recognition of a well-performed examination. An electronic log of actions taken in response to radiologists' submissions indicated that both positive and negative feedback were commonly communicated to the performing technologist. Information generated using the tool can be used to guide subsequent quality improvement initiatives within a practice, including continued protocol standardization as well as education of technologists in the optimization of abdominal imaging examinations.

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1µ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and µ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1µ non-muscle isoform protein expression before and during cardiac differentiation.

A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.

Myofibrils in vertebrate skeletal muscle are organized in aligned arrays of filaments formed from multiple protein components. Despite considerable information describing individual proteins, how they assemble de novo into mature myofibrils is still a challenge. Studies in our lab of sarcomeric protein localization during myofibril assembly led us to propose a three-step progression: premyofibrils to nascent myofibrils, culminating in mature myofibrils. Premyofibrils, forming at the spreading edges of muscle cells, are composed of minisarcomeres containing small bands of non-muscle myosin II filaments alternating with muscle-specific α-actinin Z-Bodies attached to barbed ends of actin filaments, establishing bipolar F-actin arrangements in sarcomeres. Assembly of nascent myofibrils occurs with addition of muscle-specific myosin II, F-actin, titin, and the alignment of Z-Bodies in adjacent fibrils to form beaded Z-Bands. Muscle-specific myosin II filaments in nascent myofibrils appear in an overlapping arrangement when viewed with wide-field and confocal microscopes. In mature myofibrils, non-muscle myosin II is absent, and M-Band proteins localize to the muscle myosin II filaments, aiding their alignment by cross-linking them into A-Bands. Super-resolution microscopy (SIM and STED) revealed muscle myosin II in mini-A-Bands in nascent myofibrils. In contrast to previous reports that vertebrate muscle myosin thick filaments form at their final 1.6 µm lengths, mini-A-Bands are first detected at a length of about 0.4 µm, and gradually increase four-fold in length to 1.6 µm in mature myofibrils. These new discoveries in avian skeletal muscle cells share a common characteristic with invertebrate muscles where some A-Bands can grow to lengths reaching 25 µm.

Tropomyosin Isoform Diversity in the Cynomolgus Monkey Heart and Skeletal Muscles Compared to Human Tissues.

Old world monkeys separated from the great apes, including the ancestor of humans, about 25 million years ago, but most of the genes in humans and various nonhuman primates are quite similar even though their anatomical appearances are quite different. Like other mammals, primates have four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing. Only TPM1 produces two sarcomeric isoforms (TPM1α and TPM1κ), and TPM2, TPM3, and TPM4 each generate one sarcomeric isoform. We have cloned and sequenced TPM1α, TPM1κ, TPM2α, TPM3α, and TPM4α with RNA from cynomolgus (Cyn) monkey hearts and skeletal muscle. We believe this is the first report of directly cloning and sequencing of these monkey transcripts. In the Cyn monkey heart, the rank order of TPM isoform expression is TPM1α > TPM2α > TPM1κ > TPM3α > TPM4α. In the Cyn monkey skeletal muscle, the rank order of expression is TPM1α > TPM2α > TPM3α > TPM1κ > TPM4α. The major differences in the human heart are the increased expression of TPM1κ, although TPM1α is still the dominant transcript. In the Cyn monkey heart, the only sarcomeric TPM isoform at the protein level is TPM1α. This is in contrast to human hearts where TPM1α is the major sarcomeric isoform but a lower quantity of TPM1κ, TPM2α, and TPM3α is also detected at the protein level. These differences of tropomyosin and/or other cardiac protein expression in human and Cyn monkey hearts may reflect the differences in physiological activities in daily life.

STED analysis reveals the organization of nonmuscle muscle II, muscle myosin II, and F-actin in nascent myofibrils.

A three-step model has been proposed to describe myofibril assembly in vertebrate cardiac and skeletal muscle cells beginning with premyofibrils, followed by nascent myofibrils, and ending as mature myofibrils (reviewed in Sanger, Wang, et al. (2017). Assembly and maintenance of myofibrils in striated muscle. Handbook of Experimental Pharmacology 235, 39-75; Wang, Fan, (2020). Myofibril assembly and the roles of the ubiquitin proteasome system. Cytoskeleton 77, 456-479). Premyofibrils are composed of minisarcomeres that contain nonmuscle myosin II filaments interdigitating with actin filaments embedded at their barbed ends in muscle-specific alpha-actinin-rich Z-bodies. Sarcomeres in mature myofibrils have filaments of muscle myosin II that interact with actin filaments that are attached to muscle alpha-actinin in Z-bands. Nascent myofibrils, the transitional step between premyofibrils and mature myofibrils, possess two types of myosins II, that is, nonmuscle myosin II and muscle myosin II. The relationship of these two different myosins II in nascent myofibrils, however, is not clear. Stimulated emission depletion (STED) microscopic analyses of nascent myofibrils in both embryonic chick cardiomyocytes, and hiPSC-derived cardiomyocytes revealed that nonmuscle myosin II is in the middle of the nascent myofibril, surrounded by overlapping muscle myosin II filaments at the periphery, and non-striated filamentous actin is present in the nascent myofibril. These findings support the original three-step model of myofibril assembly proposed by Rhee, Sanger, and Sanger, (1994). The premyofibrils: Evidence for its role in myofibrillogenesis. Cell Motility and the Cytoskeleton 28, 1-24.

Comparison of incorporation of wild type and mutated actins into sarcomeres in skeletal muscle cells: A fluorescence recovery after photobleaching study.

The α-actin mutation G15R in the nucleotide-binding pocket of skeletal muscle, causes severe actin myopathy in human skeletal muscles. Expressed in cultured embryonic quail skeletal myotubes, YFP-G15R-α-actin incorporates in sarcomeres in a pattern indistinguishable from wildtype YFP-α-actin. However, patches of YFP-G15R-α-actin form, resembling those in patients. Analyses with FRAP of incorporation of YFP-G15R-α-actin showed major differences between fast-exchanging plus ends of overlapping actin filaments in Z-bands, versus slow exchanging ends of overlapping thin filaments in the middle of sarcomeres. Wildtype skeletal muscle YFP-α-actin shows a faster rate of incorporation at plus ends of F-actin than at their minus ends. Incorporation of YFP-G15R-α-actin molecules is reduced at plus ends, increased at minus ends. The same relationship of wildtype YFP-α-actin incorporation is seen in myofibrils treated with cytochalasin-D: decreased dynamics at plus ends, increased dynamics at minus ends, and F-actin aggregates. Speculation: imbalance of normal polarized assembly of F-actin creates excess monomers that form F-actin aggregates. Two other severe skeletal muscle YFP-α-actin mutations (H40Y and V163L) not in the nucleotide pocket do not affect actin dynamics, and lack F-actin aggregates. These results indicate that normal α-actin plus and minus end dynamics are needed to maintain actin filament stability, and avoid F-actin patches.

Inhibitors of the ubiquitin proteasome system block myofibril assembly in cardiomyocytes derived from chick embryos and human pluripotent stem cells.

Details of sarcomeric protein assembly during de novo myofibril formation closely resemble myofibrillogenesis in skeletal and cardiac myocytes in birds, rodents, and zebrafish. The arrangement of proteins during myofibrillogenesis follows a three-step process: beginning with premyofibrils, followed by nascent myofibrils, and concluding with mature myofibrils (reviewed in Sanger et al., 2017). Assembly and maintenance of myofibrils in living muscle cells. In: Handbook of experimental pharmacology, 2017 [pp. 39-75]. Our aim is to determine if the same pathway is followed in human cardiomyocytes derived from human inducible pluripotent stem cells. We found that the human cardiomyocytes developed patterns of protein organization identical to the three-step series seen in the model organisms cited above. Further experiments showed that myofibril assembly can be blocked at the nascent myofibril by five different inhibitors of the Ubiquitin Proteasome System (UPS) stage in both avian and human cardiomyocytes. With the exception of Carfilzomib, removal of the UPS inhibitors allows nascent myofibrils to proceed to mature myofibrils. Some proteasomal inhibitors, such as Bortezomib and Carfilzomib, used to treat multiple myeloma patients, have off-target effects of damage to hearts in three to 6 % of these patients. These cardiovascular adverse events may result from prevention of mature myofibril formation in the cardiomyocytes. In summary, our results support a common three-step model for the formation of myofibrils ranging from avian to human cardiomyocytes. The Ubiquitin Proteasome System is required for progression from nascent myofibrils to mature myofibrils. Our experiments suggest a possible explanation for the cardiac and skeletal muscle off-target effects reported in multiple myeloma patients treated with proteasome inhibitors.

Identification of a novel TPM4 isoform transcript and comparison to the expression of other tropomyosin isoforms in bovine cardiac and skeletal muscles.

In mammals, there are four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) each of which generate a multitude of alternatively spliced mRNAs. TPM isoform diversity in bovine unlike in humans are not well characterized. The purpose of this investigation is to perform an extensive analysis of the expression of both transcripts and corresponding protein of sarcomeric TPMs in bovine strated muscles. We have cloned and sequenced the transcripts of the sarcomeric isoform of the TPM4 gene designated as TPM4α as well as a new splice variant TPM4ε from bovine striated muscles. Additionally, we have determined the expression of various sarcomeric TPM isoforms and TPM4ε in bovine heart and skeletal muscles. Relative expression as well as absolute copy number determination by qRT-PCR suggests that TPM1α expression is significantly higher in bovine cardiac muscle, whereas TPM2α is higher in skeletal muscle. The relative expression of TPM3α in bovine heart and skeletal muscle is very similar. The relative expression of TPM4α and TPM4ε is higher in bovine heart and skeletal muscle, respectively. We have evaluated the protein expression levels of various TPM isoforms by 2D western blot analyses in commercially available protein extracts of heart and skeletal muscles with the CH1 monoclonal antibody against TPM. Protein from each CH1-positive spot was extracted for LC-MS/MS analyses, which show that bovine heart extract contains 91.66% TPM1 and 8.33% TPM2, whereas skeletal muscle extract contains 57% TPM1 and 42.87% TPM2. We have failed to detect the presence of unique peptide(s) for TPM3α, TPM4α, and TPM4ε.

Effect of MG-132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes.

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

Assembly and dynamics of myofibrils.

We review some of the problems in determining how myofibrils may be assembled and just as importantly how this contractile structure may be renewed by sarcomeric proteins moving between the sarcomere and the cytoplasm. We also address in this personal review the recent evidence that indicates that the assembly and dynamics of myofibrils are conserved whether the cells are analyzed in situ or in tissue culture conditions. We suggest that myofibrillogenesis is a fundamentally conserved process, comparable to protein synthesis, mitosis, or cytokinesis, whether examined in situ or in vitro.

Myofibrillogenesis in skeletal muscle cells in zebrafish.

The "premyofibril" model of myofibrillogenesis, based on observations in cultured avian muscle cells, proposes that mature myofibrils are preceded by two intermediary structures: premyofibrils and nascent myofibrils. To determine if this model applies to zebrafish skeletal muscle development, we stained developing embryos with antibodies to sarcomeric alpha-actinin and myosin II. In the youngest muscle cells, sarcomeric alpha-actinin and non-muscle myosin II were each localized in linear arrays of small bands that resembled the premyofibrils in avian myocytes. The distribution of muscle-specific myosin II began as scattered short filaments followed in time by overlapping bundles of filaments and organized A-bands in the older somites. Alpha-actinin organization changed from small z-bodies to beaded Z-bands and ordered Z-bands in myofibrils that extended the length of the elongating somites. In older somites with mature myofibrils, premyofibrils were also present at the ends of the mature myofibrils, suggesting that as the cells and somites grew longer, premyofibrils were involved in the elongation of existing mature myofibrils. Fluorescence Recovery After Photobleaching showed that the exchange of proteins (actin, alpha-actinin, FATZ, myotilin and telethonin) between sarcoplasm and the Z-bands of mature myofibrils in zebrafish resembled that seen for the same proteins in cultured avian myotubes, suggesting that myofibril assembly and maintenance in zebrafish share common properties with avian muscle. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Sarcomeric TPM3 expression in human heart and skeletal muscle.

In mammals, four tropomyosin genes TPM1, TPM2, TPM3, and TPM4 are known. One isoform of the TPM3 gene, encoding 285 amino acid residues designated as TPM3α, has been reported. TPM3α protein expression in human hearts is not definitively established. We have cloned from human heart and skeletal muscle transcripts of TPM3α and three novel TPM3 isoforms, TPM3ν, TPM3ξ, and TPM3ο. TPM3ν and TPM3ο are alternatively spliced RNAs with different 3'-UTRs encoding an identical novel protein with 285 amino acid differing from TPM3α and TPM3ξ in exon 6 only. TPM3α and TPM3ξ, which have different 3'UTRs, also encode an identical protein. qRT-PCR data show that the transcripts of TPM3α, TPM3ν, TPM3ξ, and TPM3ο are expressed in both heart and skeletal muscle. We have evaluated the expression of various TPM proteins in fetal and adult human hearts, and also in skeletal muscle samples. Western blots using CG3 antibody show a stronger signal of TPM3 protein in fetal heart and adult skeletal muscle compared to adult heart. LC-MS/MS studies with the protein spots separated and identified by CH1 antibody after 2D Western blot analyses, confirm the expression of TPM3α/TPM3ξ in heart, but some peptides detected could be either TPM3α or TPM3ν. In heart samples, TPM1 protein was the dominant with varying amount of TPM2 and TPM3, while TPM4 expression was not observed. In skeletal muscles, TPM2 was the majority TPM protein expressed. The biological consequences of these varying expression of individual tropomyosin proteins are yet to be established.

Myofibril assembly and the roles of the ubiquitin proteasome system.

De novo assembly of myofibrils in vertebrate cross-striated muscles progresses in three distinct steps, first from a minisarcomeric alignment of several nonmuscle and muscle proteins in premyofibrils, followed by insertions of additional proteins and increased organization in nascent myofibrils, ending with mature contractile myofibrils. In a search for controls of the process of myofibril assembly, we discovered that the transition from nascent to mature myofibrils could be halted by inhibitors of three distinct functions of the ubiquitin proteasome system (UPS). First, inhibition of pathway to E3 Cullin ligases that ubiquitinate proteins led to an arrest of myofibrillogenesis at the nascent myofibril stage. Second, inhibition of p97 protein extractions of ubiquitinated proteins led to a similar arrest of myofibrillogenesis at the nascent myofibril stage. Third, inhibitors of proteolytic action by proteasomes also blocked nascent myofibrils from transitioning to mature myofibrils. In contrast, inhibitors of autophagy or lysosomes did not affect myofibrillogenesis. To probe for differences in the effects of UPS inhibitors during myofibrillogenesis, we analyzed by fluorescence recovery after photobleaching the exchange rates of two selected sarcomeric proteins (muscle myosin II heavy chains and light chains). In the presence of p97 and proteasomal inhibitors, the dynamics of each of these two myosin proteins decreased in the nascent myofibril stage, but were unaffected in the mature myofibril stage. The increased stability of myofibrils occurring in the transition from nascent to mature myofibril assembly indicates the importance of dynamics and selective destruction in the muscle myosin II proteins for the remodeling of nascent to mature myofibrils.

Cardiac myofibrillogenesis inside intact embryonic hearts.

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 mum filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.

Tracking changes in Z-band organization during myofibrillogenesis with FRET imaging.

There are a large number of proteins associated with Z-bands in myofibrils, but the precise arrangements of most of these proteins in Z-bands are largely unknown. Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells from quail embryos were transfected to express fusions of alpha-actinin, FATZ, myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the Z-band. Telethonin was selected for its reported ability to bind FATZ. Statistical analysis of data from FRET imaging studies yield results that are in agreement with published biochemical data suggesting that FATZ and myotilin bind to alpha-actinin near its C-terminus as well as to each other and that a region near the amino-terminus of FATZ is responsible for its interaction with telethonin. In addition, our analysis has revealed changes in the arrangement of alpha-actinin and FATZ that take place during the transition as the z-bodies of premyofibrils fuse to form the Z-bands of mature myofibrils. There was no evidence for a change in the arrangement of myotilin as z-bodies transformed into Z-bands. Myotilin is one Z-band protein that does not exhibit decreased dynamics as z-bodies fuse to form Z-bands. These FRET results from living cells support a stepwise model for the assembly of myofibrils.

Tropomyosin expression and dynamics in developing avian embryonic muscles.

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.

Nonmuscle myosin II in cardiac and skeletal muscle cells.

De novo assembly of contractile myofibrils begins with the formation of premyofibrils where filaments of non-muscle myosin (NM II), and actin organize in sarcomeric patterns with Z-Bodies containing muscle-specific alpha-actinin. Interactions of muscle specific myosin (MM II) with NM II occur in a nascent myofibril stage that precedes the assembly of mature myofibrils. By the final stage of myofibrillogenesis, the only myosin II present in the mature myofibrils is MM II. In this current study of myofibril assembly, the three vertebrate isoforms of NM II (A, B, and C) and sarcomeric alpha-actinin, ligated to GFP family proteins, were coexpressed in avian embryonic skeletal and cardiac muscle cells. Each isoform of NM II localized only in the mini-A-Bands of premyofibrils and nascent myofibrils. There was no evidence of localization of NM II in Z-Bodies of premyofibrils and nascent myofibrils or in Z-Bands of mature myofibrils. Fluorescence Recovery After Photobleaching (FRAP) experiments indicated similar exchange rates in premyofibrils for NM II isoforms A and B, whereas the IIC isoform was significantly less dynamic. Fluorescence Resonance Energy Transfer (FRET) measurements of colocalized fluorescent pairs of different NM II isoforms yielded signals similar to identical pairs, indicating copolymerization of the different NM II pairs. The role of NM II may reside in establishing the future sarcomere pattern in mature myofibrils by binding to the oppositely polarized actin filaments that extend between pairs of Z-Bodies along premyofibrils prior to their transformation into mature myofibrils.