Myofibril Organization Research Articles

SkNAC (skeletal Naca), a muscle‐specific isoform of Naca (nascent polypeptide‐associated complex alpha), is required for myofibril organization

Myofibrillogenesis, the precise assembly of sarcomeric proteins into the highly organized sarcomeres, is essential for muscle cell differentiation and function. Myofibrillogenesis requires proper folding and assembly of newly synthesized sarcomeric proteins. sknac (skeletal naca) is an alternatively spliced isoform of naca, which encodes the nascent polypeptide-associated complex alpha polypeptide that binds to newly synthesized polypeptides emerging from the ribosome. sknac is specifically expressed in skeletal and cardiac muscles. However, little is known about the function of skNAC in muscle development in vivo. To determine skNAC function, we have isolated and characterized the sknac gene from zebrafish. Zebrafish sknac cDNA differs from naca by containing an extra large exon that encodes 815 aa. Knockdown of sknac expression by antisense oligos resulted in zebrafish embryos with skeletal muscle defects. The sknac-knockdown embryos showed a paralyzed phenotype with little muscle contraction. In contrast, injection of a control oligo had no effect. Immunostaining and histological analyses revealed that sknac-knockdown embryos contained disorganized thick and thin filaments. Western blot analysis revealed that myosin protein levels were significantly reduced. Collectively, these results demonstrate that skNAC plays a vital role in myofibril assembly and function during muscle cell differentiation.

Expression of Nkx2.5 in Wild Type, Cardiac Mutant, and Thyroxine-Induced Metamorphosed Hearts of the Mexican Axolotl

Nkx2.5, a homeodomain-containing transcription factor, is known to be necessary for normal heart development in vertebrates. It is one of the earliest lineage-restricted genes expressed in cardiovascular progenitor cells and knowledge of its expression patterns has important therapeutic implications for damaged cardiomyocytes. Mexican axolotl is a unique system to study heart development for two reasons: the presence of a mutant phenotype lacking organized myofibrils due to sarcomeric tropomyosin deficiency and the ability to induce metamorphosis by administration of exogenous thyroid hormone. In this study, we cloned and sequenced the as yet uncharacterized Nkx2.5 cDNA from normal and cardiac mutant axolotl heart RNA. Comparison of cDNA sequences of Nkx2.5 from normal and mutant axolotl hearts did not show differences suggesting that loss of function mutation in Nkx2.5 is not responsible for the mutant phenotype. However, quantitative studies show higher expression of Nkx2.5 in mutant hearts raising the possibility that increased expression of Nkx2.5 may contribute to the mutant phenotype. We also evaluated quantitative changes in expression of Nkx2.5 in axolotl hearts during embryonic and postembryonic heart development induced by exogenous thyroid hormone. There is an apparent increase in Nkx2.5 transcript levels in metamorphosed hearts.

Abstract 5933: Reduced Number of Gap Junctions at the Intercalated Disc in the Ventricles of Boxer Dogs Afflicted with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC)

ARVC is an inherited myocardial disease associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of RV myocardium with fatty or fibro-fatty tissue. Lethal arrhythmias often occur in young patients, before apparent structural damage. It has been hypothesized that loss of Cx43 gap junctions (GJ) may act as a substrate for ventricular arrhythmias in these patients. An inherited form of ARVC is present in the boxer, with clinical symptoms and pathological features similar to those of humans. Here, we used transmission electron microscopy (TEM) to assess the ultrastructure of the intercalated disc (ID) in ARVC-afflicted boxers. Samples from the right and left ventricles (RV and LV) of 1 unafflicted beagle, and 3 ARVC afflicted boxers were examined by TEM. Samples from beagle tissue showed normal morphology, good alignment of the sarcomeres and myofibrils, and attachment of myofibrils to the ID. Samples from the ARVC-afflicted boxer lacked myofibril organization, with few attachments to the ID. Within RV and LV longitudinal sections from one afflicted and one control animal, three parameters were measured: ID length, individual GJ length, and number of GJ per ID. No significant differences were found in the length of IDs or of GJs from RV or LV samples of ARVC vs. control. ID length (μm) was 26.8 +/− 5.4 and 44.9+/− 10.6 in control RV and LV, respectively. ID length in afflicted samples was 31.2+/− 5.0 and 52.3+/−13.5 (pNS). GJ length (μm) was .5+/−.03 and .5+/.05 for RV and LV of the control group, and .4+/−.07 and .6+/−,13 in samples obtained from RV and LV of ARVC afflicted animals (pNS). However, our data indicated a significant (p<.01) decrease in the number of GJ per ID in the ARVC sample by over 80% (5.7+/−.4 RV, 5.8+/− 1.4 LV in control; 1.1+/−.16 RV, 1.5+/−.2 LV in ARVC). Our results suggest that ARVC, both in humans and in boxer dogs, associates with a loss of GJ, which may act as a substrate for lethal arrhythmias. In contrast to human ARVC, no desmosomal protein mutations have been linked to boxer ARVC. This suggests a different genetic basis and yet, a common molecular pathway for ARVC in both species. As such, boxers represent a valuable model to understand the mechanisms leading to malignant ventricular arrhythmias in ARVC patients. This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

Effect of chronic administration of quinine on the myocardium of mice

This study was designed to evaluate the effect of chronic administration of quinine on the myocardium of Swiss albino mice. 20 or 30 mg quinine/kg b.w. were administered for 30 consecutive days, while the control mice received equal amounts of normal saline. The mice were sacrificed 24 hours after the last administration. Some of the hearts were excised and processed for light microscopic study while some were assayed for malondialdehyde (MDA) and catalase activities. The histological findings indicated that the treated sections of the heart were at variance with the control. The myocardium in the control section presents well organized myofibrils with long cylindrical mononucleated cells in contrast to a dose dependent distorted arrangement of myofibrils and scanty cellular components observed in the treated sections. There was a statistically significant decrement in the weight difference and relative percentage weight of the heart in a dose dependent manner just as the activities of MDA and catalase significantly increased in the treated groups. These findings indicate that chronic administration of quinine may have some deleterious effects on the heart of mice and by extension may affect its function.

A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

Heat-shock protein 90α1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos

Heat-shock protein 90alpha (Hsp90alpha) is a member of the molecular chaperone family involved in protein folding and assembly. The role of Hsp90alpha in the developmental process, however, remains unclear. Here we report that zebrafish contains two Hsp90alpha genes, Hsp90alpha1, and Hsp90alpha2. Hsp90alpha1 is specifically expressed in developing somites and skeletal muscles of zebrafish embryos. We have demonstrated that Hsp90alpha1 is essential for myofibril organization in skeletal muscles of zebrafish embryos. Knockdown of Hsp90alpha1 resulted in paralyzed zebrafish embryos with poorly organized myofibrils in skeletal muscles. In contrast, knockdown of Hsp90alpha2 had no effect on muscle contraction and myofibril organization. The filament defects could be rescued in a cell autonomous manner by an ectopic expression of Hsp90alpha1. Biochemical analyses revealed that knockdown of Hsp90alpha1 resulted in significant myosin degradation and up-regulation of unc-45b gene expression. These results indicate that Hsp90alpha1 plays an important role in muscle development, likely through facilitating myosin folding and assembly into organized myofibril filaments.

Structural differentiation of skeletal muscle fibers in the absence of innervation in humans

The relative importance of muscle activity versus neurotrophic factors in the maintenance of muscle differentiation has been greatly debated. Muscle biopsies from spinal cord injury patients, who were trained with an innovative protocol of functional electrical stimulation (FES) for prolonged periods (2.4-9.3 years), offered the unique opportunity of studying the structural recovery of denervated fibers from severe atrophy under the sole influence of muscle activity. FES stimulation induced surprising recovery of muscle structure, mass, and force even in patients whose muscles had been denervated for prolonged periods before the beginning of FES training (up to 2 years) and had almost completely lost muscle-specific internal organization. Ninety percent (or more) of the fibers analyzed by electron microscopy showed a striking recovery of the ultrastructural organization of myofibrils and Ca(2+)-handling membrane systems. This functional/structural restoration follows a pattern that mimics some aspects of normal muscle differentiation. Most importantly, the recovery occurs in the complete absence of motor and sensory innervation and of nerve-derived trophic factors, that is, solely under the influence of muscle activity induced by electrical stimulation.

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.

Myogenic Stage, Sarcomere Length, and Protease Activity Modulate Localization of Muscle-specific Calpain

p94/calpain 3 is a Ca(2+)-binding intracellular protease predominantly expressed in skeletal muscles. p94 binds to the N2A and M-line regions of connectin/titin and localizes in the Z-bands. Genetic evidence showing that compromised p94 proteolytic activity leads to muscular dystrophy (limb-girdle muscular dystrophy type 2A) indicates the importance of p94 function in myofibrils. Here we show that a series of p94 splice variants is expressed immediately after muscle differentiation and differentially change localization during myofibrillogenesis. We found that the endogenous N-terminal (but not C-terminal) domain of p94 was not only localized in the Z-bands but also directly bound to sarcomeric alpha-actinin. These data suggest the incorporation of proteolytic N-terminal fragments of p94 into the Z-bands. In myofibrils localization of exogenously expressed p94 shifted from the M-line to N2A as the sarcomere lengthens beyond approximately 2.6 and 2.8 microm for wild-type and proteaseinactive p94, respectively. These data demonstrate for the first time that p94 proteolytic activity is involved in responses to muscle conditions, which may explain why p94 inactivation causes limb-girdle muscular dystrophy.

The ubiquitin-selective chaperone CDC-48/p97 links myosin assembly to human myopathy

Protein degradation in eukaryotes often requires the ubiquitin-selective chaperone p97 for substrate recruitment and ubiquitin-chain assembly. However, the physiological relevance of p97, and its role in developmental processes, remain unclear. Here, we discover an unanticipated function for CDC-48/p97 in myosin assembly and myofibril organization, both in Caenorhabditis elegans and humans. The developmentally regulated assembly of a CDC-48-UFD-2-CHN-1 complex links turnover of the myosin-directed chaperone UNC-45 to functional muscle formation. Our data suggest a similarly conserved pathway regulating myosin assembly in humans. Remarkably, mutations in human p97, known to cause hereditary inclusion-body myopathy, abrogate UNC-45 degradation and result in severely disorganized myofibrils, detrimental towards sarcomeric function. These results identify a key role for CDC-48/p97 in the process of myofibre differentiation and maintenance, which is abolished during pathological conditions leading to protein aggregation and inclusion-body formation in human skeletal muscle.

Muscle‐specific expression of the smyd1 gene is controlled by its 5.3‐kb promoter and 5′‐flanking sequence in zebrafish embryos

Zebrafish SmyD1 is a SET and MYND domain-containing protein that plays an important role in myofiber maturation and muscle contraction. SmyD1 is required for myofibril organization and sarcomere assembly during myofiber maturation. Whole-mount in situ hybridization revealed that smyd1 mRNAs are specifically expressed in skeletal and cardiac muscles in zebrafish embryos. However, it is unknown if smyd1 is expressed in other striated muscles, such as cranial and fin muscles, and moreover, the regulatory elements required for its muscle-specific expression. We report here the analyses of smyd1 expression using smyd1-gfp transgenic zebrafish. smyd1-gfp transgenic zebrafish were generated using the 5.3-kb smyd1 promoter and its 5'-flanking sequence. GFP expression was found in the skeletal and cardiac muscles of smyd1-gfp transgenic embryos. GFP expression appeared stronger in slow muscles than fast muscles in transgenic zebrafish larvae. In addition, GFP expression was also detected in cranial and fin muscles of smyd1-gfp transgenic zebrafish larvae. In situ hybridization confirmed smyd1 mRNA expression in these tissues, suggesting that the expression of the smyd1-gfp transgene recapitulated that of the endogenous smyd1 gene. Deletion analysis revealed that the 0.5-kb sequence in the proximal promoter of smyd1 was essential for its muscle specificity. Together, these data indicate that smyd1 is specifically expressed in most, if not all, striated muscles, and the muscle specificity is controlled by the 5.3-kb promoter and flanking sequences.

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.

Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

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.

Correction for Tan et al., SmyD1, a histone methyltransferase, is required for myofibril organization and muscle contraction in zebrafish embryos

developmental biology. For the article “SmyD1, a histone methyltransferase, is required for myofibril organization and muscle contraction in zebrafish embryos,” by Xungang Tan, Josep Rotllant, Huiqing Li, Patrick DeDeyne, and Shao Jun Du, which appeared in issue 8, February 21, 2006, of Proc. Natl. Acad. Sci. USA (103, 2713–2718; first published February 13, 2006; 10.1073/pnas.0509503103), the author name Patrick DeDeyne should have appeared as Patrick De Deyne. The online version has been corrected. The corrected author line appears below. Xungang Tan, Josep Rotllant, Huiqing Li, Patrick De Deyne, and Shao Jun Du

Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells

A central question in muscle biology is how costameres are formed and become aligned with underlying myofibrils in mature tissues. Costameres are composed of focal adhesion proteins, including vinculin and paxillin, and anchor myofibril Z-bands to the sarcolemma. In the present study, we investigated the process of costamere formation (“costamerogenesis”) in differentiating primary mouse myoblasts. Using vinculin and paxillin as costameric markers, we found that two additional focal adhesion components, α5β1 integrin and focal adhesion kinase (FAK), are associated with costameres. We have characterized costamerogenesis as occurring in three distinct stages based on the organizational pattern of these costameric proteins. We show that both costamerogenesis and myofibrillogenesis are initiated at sites of membrane contacts with the extracellular matrix and that their maturation is tightly coupled. To test the importance of FAK signaling in these processes, we analyzed cells expressing a dominant negative form of FAK (dnFAK). When cells expressing dnFAK were induced to differentiate, both costamerogenesis and myofibrillogenesis were disrupted although the expression of constituent proteins was not inhibited. Likewise, inhibiting FAK activity by reducing FAK levels using an siRNA approach also resulted in an inhibition of costamerogenesis and myofibrillogenesis. The relationship between costamere and myofibril formation was tested further by treating myotube cultures with potassium or tetrodotoxin to block contraction and disrupt myofibril organization. This also resulted in inhibition of costamere maturation. We present a model of costamerogenesis whereby signaling through FAK is essential for both normal costamerogenesis and normal myofibrillogenesis which are tightly coupled during skeletal myogenesis.

Myocardial Remodeling in Embryonic Heart: An Inductive Interaction Mediated by Periostin

Where mesenchyme in the developing heart contacts adjacent myocardial tissue the myocardium progressively “disappears”. It is replaced by fibrous tissue (e.g. the fibrous annulus or tendinous cords). Periostin, a fasciclin-related protein, is secreted by the mesenchyme which accumulates at boundary interfaces. Similarly, periostin increases in adult hearts undergoing either ischemic or non-ischemic myocardial remodeling that leads to scarring. In periostin null adult hearts, myocardial tissue that normally regresses persists and is frequently accompanied by premature heartbeats of non-sinus origin. Based on cell sorting data, there were more myocardial cells in periostin adult null hearts but 30% of the cells remained undifferentiated. We hypothesized that periostin promoted fibroblast differentiation but inhibited myocardial differentiation or stability of the myocardial phenotype. To test this hypothesis, we used adenoviral vectors expressing full-length periostin protein in culture assays or in vivo. When added to culture media of E.D. 13 explants containing mesenchyme and myocardium, the latter was reduced (~80%) but fibroblastic tissue proliferated. Direct injection of the virus (but not controls) into the pericardial space or ventricular walls resulted in myocardial thinning and loss of compact myocardium. Co-culturing monolayers of mesenchyme with embryonic myocardial cells separated by a barrier resulted in progressive loss of myofibril organization in myocytes. As there was no evidence of augmented apoptosis at boundary interfaces either in vivo or in cultured monolayers, we propose that periostin induces loss of myocardial tissue, possibly through a transdifferentiation mechanism. Supported by HL 33576, HL53228

SmyD1, a histone methyltransferase, is required for myofibril organization and muscle contraction in zebrafish embryos

Histone modification has emerged as a fundamental mechanism for control of gene expression and cell differentiation. Recent studies suggest that SmyD1, a novo SET domain-containing protein, may play a critical role in cardiac muscle differentiation. However, its role in skeletal muscle development and its mechanism of actions remains elusive. Here we report that SmyD1a and SmyD1b, generated by alternative splicing of SmyD1 gene, are histone methyltransferases that play a key role in skeletal and cardiac muscle contraction. SmyD1a and SmyD1b are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. Knockdown of SmyD1a and SmyD1b expression by morpholino antisense oligos resulted in malfunction of skeletal and cardiac muscles. The SmyD1 morphant embryos (embryos injected with morpholino oligos) could not swim and had no heartbeat. Myofibril organization in the morphant embryos was severely disrupted. The affected myofibers appeared as immature fibers with centrally located nuclei. Together, these data indicate that SmyD1a and SmyD1b are histone methyltransferases and play a critical role in myofibril organization during myofiber maturation.

Targeted disruption of N-RAP gene function by RNA interference: A role for N-RAP in myofibril organization

N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.

Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo

Nerve activity is known to be an important regulator of muscle phenotype in the adult, but its contribution to muscle development during embryogenesis remains unresolved. We used the zebrafish embryo and in vivo imaging approaches to address the role of activity-generated signals, acetylcholine and intracellular calcium, in vertebrate slow muscle development. We show that acetylcholine drives initial muscle contraction and embryonic movement via release of intracellular calcium from ryanodine receptors. Inhibition of this activity-dependent pathway at the level of the acetylcholine receptor or ryanodine receptor did not disrupt slow fibre number, elongation or migration but affected myofibril organisation. In mutants lacking functional acetylcholine receptors myofibre length increased and sarcomere length decreased significantly. We propose that calcium is acting via the cytoskeleton to regulate myofibril organisation. Within a myofibre, sarcomere length and number are the key parameters regulating force generation; hence our findings imply a critical role for nerve-mediated calcium signals in the formation of physiologically functional muscle units during development.