Ectopic expression and dynamics of TPM1alpha and TPM1kappa in myofibrils of avian myotubes

From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1kappa, containing exon 2a instead of exon 2b contained in TPM1alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1alpha and chicken TPM1kappa and compared their localizations in premyofibrils and mature myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1alpha and TPM1kappa in myotubes. TPM1alpha and TPM1kappa incorporated into premyofibrils, nascent myofibrils, and mature myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in premyofibrils than in mature myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (premyofibrils, nascent myofibrils, mature myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial premyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (premyofibrils to nascent myofibrils to mature myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or myofibrils.     

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.     

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/straited chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.     

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle

TPM1κ is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function. J. Cell. Biochem. 110: 875–881, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm

A strain of axolotl, Ambystoma mexicanum, that carries the cardiac lethal or c gene presents an excellent model system in which to study inductive interactions during heart development. Embryos homozygous for gene c contain hearts that fail to beat and do not form sarcomeric myofibrils even though muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos inevitably die due to lack of circulation. Embryonic axolotl hearts can be maintained easily in organ culture using only Holtfreter's solution as a culture medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing sarcomeric myofibrils by coculturing the mutant heart tube with anterior endoderm from a normal embryo. The induction of muscle differentiation can also be mediated through organ culture of mutant heart tubes in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability of endoderm-conditioned medium to induce cardiac muscle differentiation. The addition of RNA extracted from normal early embryonic anterior endoderm to organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic liver or neural tube did not induce either muscular contraction or myofibrillogenesis. Thus, RNA from anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile activity in mutant hearts, thereby correcting the genetic defect.     

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

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.     

The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes

Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin-containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s-alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha-actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.     

Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.     

Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle

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.     

Myofibril-Inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts

The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c) embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR), is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR) are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.     

Disruption of muscle architecture and myocardial degeneration in mice lacking desmin

Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils and abnormal mitochondrial organization. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. The present data demonstrate the essential role of desmin in the maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity, and show that the absence of desmin leads to muscle degeneration.