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.

A reduction of tropomyosin limits development of sarcomeric structures in cardiac mutant hearts of the Mexican axolotl.

The cardiac lethal mutation in Mexican axolotl (Ambystoma mexicanum) results in a lack of contractions in the ventricle of mutant embryos. Previous studies have demonstrated that tropomyosin, a component of thin filaments, is greatly reduced in mutant hearts lacking myofibril organization. Confocal microscopy was used to examine the structure and comparative amount of tropomyosin at heartbeat initiation and at a later stage. The formation of functional sarcomeres coincided with contractions in normal hearts at stage 35. A-bands and I-bands were formed at stage 35 and did not change at stage 39. The widening of Z-bodies into z-lines was the main developmental difference between stage 35 and 39 normal hearts. Relative to normal hearts, a reduction of sarcomeric protein levels in mutant hearts at stage 35 was found, and a greater reduction occurred at later stages. The lower level of tropomyosin limited the areas where organized myofibrils formed in the mutant. The areas that had tropomyosin staining also had staining for alpha-actinin and myosin. Early myofibrils formed in these areas but the A-bands and I-bands were shorter than normal. At a later stage in the mutant, A-bands and I-bands remained shorter and importantly the Z-bodies also did not form wider z-lines.

Tropomodulin expression in developing hearts of normal and cardiac mutant Mexican axolotl.

In the axolotl, Ambystoma mexicanum, a simple, recessive cardiac-lethal mutation in gene "c" results in the hearts of c/c homozygous animals being deficient in sarcomeric tropomyosin (TM) and failing to form mature myofibrils. Subsequently, the mutant hearts do not beat. A three-step model of myofibril assembly recently developed in cell culture prompted a reassessment of the myofibril assembly process in mutant hearts using a relatively new late marker for thin filament assembly, tropomodulin (Tmod). This is, to the best of our knowledge, the first report of tropomodulin in an amphibian system. Tropomodulin antibodies were immunolocalized to the ends of the thin filaments. Tropomodulin was also found in discrete punctate spots in normal and mutant hearts, often in linear arrays suggestive of early myofibril formation. The tropomodulin spots assessed in stage 41/42 mutant hearts co-localized with antibodies to other myofibrillar proteins indicative of nascent myofibril formation. This suggests a failure of elongation/maturation of nascent myofibrils, which could be a consequence of decreased TM levels or increased Tmod/ TM ratio. Unlike tropomyosin, there is no apparent decrease in the level of Tmod expression in mutant hearts.

Cardiac myofibril formation is not affected by modification of both N- and C-termini of sarcomeric tropomyosin.

Although the role of tropomyosin is well-defined in striated muscle, the precise mechanism of how tropomyosin functions is still unclear. It has been shown that extension of either N- or C-terminal ends of sarcomeric tropomyosin do not affect cardiac myofibrillogenesis, but it is not known whether simultaneous extension of both ends affects the process. For studying structural/functional relationships of sarcomeric tropomyosin, we have chosen the Ambystoma mexicanum because cardiac mutant hearts are deficient in sarcomeric tropomyosin. In this study, we have made an expression construct, pEGFP.TPM4alpha.E-L-FLAG, that, on transfection into normal and mutant axolotl hearts in organ culture, expresses GFP.TPM4alpha.E-L-FLAG fusion protein in which both the N- and C-termini of TPM4alpha are being extended. TPM4alpha is one of the three tropomyosins expressed in normal axolotl hearts. Both confocal and electron microscopic analyses show that this modified sarcomeric tropomyosin can form organized myofibrils in axolotl hearts.

Diminished myofibril organization in mutant axolotl hearts transfected with site-directed mutants of sarcomeric tropomyosins.

We used a model lacking endogenous sarcomeric tropomyosin, the cardiac mutant of the Mexican axolotl, to examine the effect of mutant tropomyosins on sarcomeric myofibril formation. Previous studies have introduced wild-type mouse alpha-tropomyosin into mutant hearts in organ culture with subsequent for-mation of organized myofibrils. This study examines the predominant embry-onic axolotl TPM-4 type tropomyosin (TPM4alpha), containing a conservative re- placement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4alpha) promoted formation of organized myofibrils in hearts without endogenous tropomyosin. Site-directed mutagenesis of 175 glutamic acid with 175 glutamine or 175 lysine was toxic to the formation of organized myofibrils in mutant hearts in the absence of endogenous tropo-myosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4alpha cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4alpha cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4alpha type or TPM1alpha (striated muscle isoform of the TPM1 gene) was sufficient to alter the protein such that organized myofibrils would not form in ventricles of mutant hearts without endogenous tropomyosin.

A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture.

Striated muscle tropomyosin is classically described as consisting of 10 exons, 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, in both skeletal and cardiac muscle. A novel isoform found in embryonic axolotl heart maintains exon 9a/b of striated muscle but also has a smooth muscle exon 2a instead of exon 2b. Translation and subsequent incorporation into organized myofibrils, with both isoforms, was demonstrated with green fluorescent protein fusion protein construct. Mutant axolotl hearts lack sufficient tropomyosin in the ventricle and this smooth/striated chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.

A point mutation in bioactive RNA results in the failure of mutant heart correction in Mexican axolotls.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. The defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or endoderm/mesoderm-conditioned medium. By screening a cDNA library made of total conditioned medium RNA from normal axolotl embryonic endoderm, we isolated a single clone (MIR), the synthetic RNA from which corrects the mutant heart defect by promoting myofibrillogenesis and thus was named MIR (myofibrillogenesis inducing RNA). In the present study, we have examined MIR gene expression in mutant axolotl hearts at early pre-heart-beat developmental stages and found its quantitative expression, as detected by RT-PCR, to be the same as in normal hearts. However, careful analysis of sequence data revealed a G-->U point mutation in the mutant MIR RNA. Further computational analyses, using GENEBEE software to compare normal and mutant MIR RNAs show a significant alteration in RNA secondary structure of the point-mutated MIR RNA. The results from bioassay and confocal microscopy immunofluorescent studies demonstrate that, unlike MIR RNA derived from normal embryos, the mutated MIR RNA does not promote myofibrillogenesis in mutant embryonic hearts and fails to rescue/correct the mutant heart defect.

Expression of tropomyosin in relation to myofibrillogenesis in axolotl hearts.

The anatomy, function and embryonic development of the heart have been of interest to clinicians and researchers alike for centuries. A beating heart is one of the key criteria in defining life or death in humans. An understanding of the multitude of genetic and functional elements that interplay to form such a complex organ is slowly evolving with new genetic, molecular and experimental techniques. Despite the need for ever more complex molecular techniques some of our biggest leaps in knowledge come from nature itself through observations of mutations that create natural defects in function. Such a natural mutation is found in the Mexican axolotl, Ambystoma mexicanum. It is a facultative neotenous salamander well studied for its ability to regenerate severed limbs and tail. Interestingly it also well suited to studying segmental heart development and differential sarcomere protein expression due to a naturally occurring mendelian recessive mutation in cardiac mutant gene "c". The resultant mutants are identified by their failure to beat and can be studied for extended periods before they finally die due to lack of circulation. Studies have shown a differential expression of tropomyosin between the conus and the ventricle indicating two different cardiac segments. Tropomyosin protein, but not its transcript have been found to be deficient in mutant ventricles and sarcomere formation can be rescued by the addition of TM protein or cDNA. Although once thought to be due to endoderm induction our findings indicate a translational regulatory mechanism that may ultimately control the level of tropomyosin protein in axolotl hearts.

Differential expression of tropomyosin during segmental heart development in Mexican axolotl.

The Mexican axolotl, Ambystoma mexicanum, serves as an intriguing model to investigate myofibril organization and heart development in vertebrates. The axolotl has a homozygous recessive cardiac lethal gene "c" which causes a failure of ventricular myofibril formation and contraction. However, the conus of the heart beats, and has organized myofibrils. Tropomyosin (TM), an essential component of the thin filament, has three known striated muscle isoforms (TPM1alpha, TPM1kappa, and TPM4alpha) in axolotl hearts. However, it is not known whether there are differential expression patterns of these tropomyosin isoforms in various segments of the heart. Also, it is not understood whether these isoforms contribute to myofibril formation in a segment-specific manner. In this study, we have utilized anti-sense oligonucleotides to separately knockdown post-transcriptional expression of TPM1alpha and TPM4alpha. We then evaluated the organization of myofibrils in the conus and ventricle of normal and cardiac mutant hearts using immunohistochemical techniques. We determined that the TPM1alpha isoform, a product of the TPM1 gene, was essential for myofibrillogenesis in the conus, whereas TPM4alpha, the striated muscle isoform of the TPM4 gene, was essential for myofibrillogenesis in the ventricle. Our results support the segmental theory of vertebrate heart development.

Anti-sense-mediated inhibition of expression of the novel striated tropomyosin isoform TPM1kappa disrupts myofibril organization in embryonic axolotl hearts.

Striated muscle tropomyosin (TM) is described as containing ten exons; 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. Exon 9a/b has critical troponin binding domains and is found in striated muscle isoforms. We have recently discovered a smooth (exon 2a)/striated (exons 9a/b) isoform expressed in amphibian, avian, and mammalian hearts, designated as an isoform of the TPM1 gene (TPM1kappa). TPM1kappa expression was blocked in whole embryonic axolotl heart by transfection of exon-specific anti-sense oligonucleotide. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed lower transcript expression of TPM1kappa and in vitro analysis confirmed the specificity of the TPM1kappa anti-sense oligonucleotide. Altered expression of the novel TM isoform disrupted myofibril structure and function in embryonic hearts.

Expression of a novel cardiac-specific tropomyosin isoform in humans.

Tropomyosins are a family of actin binding proteins encoded by a group of highly conserved genes. Humans have four tropomyosin-encoding genes: TPM1, TPM2, TPM3, and TPM4, each of which is known to generate multiple isoforms by alternative splicing, promoters, and 3' end processing. TPM1 is the most versatile and encodes a variety of tissue specific isoforms. The TPM1 isoform specific to striated muscle, designated TPM1alpha, consists of 10 exons: 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. In this study, using RT-PCR with adult and fetal human RNAs, we present evidence for the expression of a novel isoform of the TPM1 gene that is specifically expressed in cardiac tissues. The new isoform is designated TPM1kappa and contains exon 2a instead of 2b. Ectopic expression of human GFP.TPM1kappa fusion protein can promote myofibrillogenesis in cardiac mutant axolotl hearts that are lacking in tropomyosin.

Identification, characterization, and expression of a novel alpha-tropomyosin isoform in cardiac tissues in developing chicken.

Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non-muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM-4 type. This novel alpha-Tm tropomyosin isoform, designated as alpha-Tm-2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (alpha-Tm-1) was also expressed in embryonic hearts along with the striated muscle isoform of TM-4. In adult heart, TM-4 was expressed, however, expression of both alpha-Tm-1 and alpha-Tm-2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of alpha-Tm-2 in embryonic and adult skeletal muscle, however, the alpha-Tm-1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the alpha-TM gene, i.e., alpha-smooth muscle tropomyosin (alpha-Sm), alpha-Fibroblast-1 (alpha-F1), and alpha-Fibroblast-2 (alpha-F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of alpha-Tm-1 and alpha-Tm-2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the alpha-TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.

Characterization of a TM-4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl.

A striated muscle isoform of a Tropomyosin (TM-4) gene was characterized and found to be necessary for contractile function in embryonic heart. The full-length clone of this isoform was isolated from the Mexican axolotl (Ambystoma mexicanum) and named Axolotl Tropomyosin Cardiac-3 (ATmC-3). The gene encoded a cardiac-specific tropomyosin protein with 284 amino acid residues that demonstrated high homology to the Xenopus cardiac TM-4 type tropomyosin. Northern blot analysis indicates a transcript of approximately 1.25 kb in size. RT-PCR and in situ hybridization demonstrated that this isoform is predominantly in cardiac tissue. Our laboratory uses an animal model that carries a cardiac lethal mutation (gene c), this mutation results in a greatly diminished level of tropomyosin protein in the ventricle. Transfection of ATmC-3 DNA into mutant hearts increased tropomyosin levels and promoted myofibrillogenesis. ATmC-3 expression was blocked in normal hearts by transfection of exon-specific anti-sense oligonucleotide (AS-ODN). RT-PCR confirmed lower transcript expression of ATmC-3 and in vitro analysis confirmed the specificity of the ATmC-3 exon 2 anti-sense oligonucleotide. These AS-ODN treated hearts also had a disruption of myofibril organization and disruption of synchronous contractions. These results demonstrated that a striated muscle isoform of the TM-4 gene was expressed embryonically and was necessary for normal structure and function of the ventricle.