Skeletal muscle myoblasts are capable of proliferation and differentiation in vitro, which imitates early embryonic development and muscle regeneration. In several studies mammalian or avian cultured myocytes have been used as an experimental model to analyze the temporal and spatial expression of myofibrillar proteins during myogenesis in cardiac and skeletal muscles [1, 4, 22–25]. These studies have lead to the understanding that during myofibrillogenesis, the initiation of the expression of the many proteins involved occurs in an ordered sequence, suggesting a specific impact of the individual proteins in the complex mechanisms associated with sarcomere formation . Due to their limited availability and reduced level of differentiation, human cells were only used in few studies. A better knowledge of their expression patterns and contribution to the assembly of cytoskeletal and myofibrillar structures in human cells might provide insights into the pathomechanisms of diseases associated with mutations in genes encoding sarcomeric proteins. In the current study we have used cultured primary human skeletal muscle cells to investigate the expression patterns of a panel of sarcomeric components and their isoforms with a focus on proteins associated with a group of muscle diseases. It is of interest to understand the onset of the expression of such proteins related to the congenital feature of the disease. Our differentiated cultures were predominantly occupied by myotubes, showing a mature sarcomeric cross-striated pattern. We observed a subsequent expression and development stages of myofibril components from an initial unorganized pattern in myoblasts, into a mature cross-striated pattern with clearly distinguishable Z-disks, A/I-junctions, A-bands and M-bands. The appearance of these four well-defined sarcomeric structures, and in particular, the integration of M-band titin, which is a late stage in myofibrillogenesis , indicated the formation of mature sarcomeric structures in vitro. This indicates the high quality of our human tissue culture and confirms its usability as a model system for studying the pathogenesis of muscle diseases caused by defects in sarcomeric or cytoskeletal constituents during skeletal muscle development.
Our results identify α-cardiac actin and α-skeletal actin as the predominant actin isoform in mononucleated myoblasts and in multinucleated differentiated cells, respectively demonstrating that the expression of actin isoforms is developmentally regulated in a temporal, tissue-specific manner. This is in accordance with previous results in chicken and mouse (27–29), that revealed that α-cardiac actin is the main isoform in early skeletal muscle development [27, 28]. Its expression is down-regulated in later development and α-skeletal actin becomes the predominant isoform in adult skeletal muscle fibres .
We also show the predominant expression of the β-TM isoform in proliferating human myoblasts and myotubes during myogenesis in vitro. This indicates an important role for β-TM in early stages of myofibrillogenesis. In addition, our data indicate that myoblasts and early-differentiated myotubes contain predominantly slow TnI, suggesting the importance of this isoform during development. This is further supported by the previous notion that the slow troponin I gene is the major isoform in early fetal heart in vertebrates and it is predominantly expressed during development in fast muscles with a subsequent switch to fast troponin I [30–32]. Similarly, we detected the expression of fast TnI and T solely in differentiated myotubes, indicating their impact in later stages of muscle development. Moreover, in accordance with previous results  the homogeneous expression of cardiac TnI in proliferating myoblasts and early myotubes suggest a role for this protein in the initial stages of myofibrillogenesis.
Association of mutations in slow skeletal muscle MyBPC isoform (MYBPC1) with autosomal dominant DA type 1 has recently been reported . We identified this specific isoform as the major MyBPC variant at the initial phases of myofibrillogenesis in human skeletal muscle myoblasts and early myotubes, indicating that it is the main MyBPC isoform involved in early myofibril development. Accordingly, previous results indicate that slow MyBPC is first expressed in developing skeletal muscle both in mice and humans and fast MyBPC is detected at later developmental stages . Also in accordance with a previous study, which indicated that cardiac MyBPC is not expressed in human skeletal muscles, not even during development , the expression of this isoform was not detected in our cultures. This indicates that the cardiac MyBPC appears not to be essential for human skeletal muscle development. However the cardiac MyBPC transcript was clearly detected in both, proliferating mononucleated myoblasts and myotubes, indicating the ectopic expression of this gene, as previously suggested . In addition, we observed the expression of muscle-specific sarcomeric transcripts in proliferating mononucleated myoblasts, which either suggests the existence of myoblasts that had begun to express these components prior to fusion, or an expression of muscle-specific proteins in mononucleated myoblasts that differentiate prematurely. We found early expression of various MyHC protein isoforms, in a population of proliferating mononucleated myoblasts with an elongated spindle-shaped morphology without fusion. This may indicate the co-existence of early differentiated but non-fused myoblasts within the population of proliferating myoblasts in adult regenerative muscle. In addition, a small number of the proliferating mononucleated myoblasts expressed myogenin, confirming their differentiated state.
The essential roles of sarcomeric proteins have been highlighted by identification of mutations in their genes associated with various diseases. This includes mutations in genes encoding β-TM and γ-TM isoforms (TPM2 and TPM3) in association with congenital myopathies with a range of clinical and morphological phenotypes [12, 13, 35–42]. In addition, mutations in TPM2, TNNI2, MYH3, MYH8 and MYBPC1 have recently been identified to cause the DA syndromes, characterized by congenital contractures [12, 15]. The sequential onset of distinct sarcomeric protein isoforms within a family has not been well-characterized in human, except for the MYH3 and MYH8, which are known to be expressed during fetal development and also during muscle regeneration [43, 44]. The impact of embryonic and fetal MyHC isoforms for normal fetal development was supported by the identification of MYH3 and MYH8 mutations [16, 17, 45][18, 46]. It was suggested that they cause a developmental myopathy resulting in reduced fetal movement and joint contractures [16, 17]. Our results here demonstrated the predominant expression of β-TM, cardiac alpha actin, slow TnI and slow MyBPC isoforms in proliferating human mononucleated myoblasts and myotubes during myogenesis in vitro. This points to a possible role for these protein isoforms in the early stages of myofibrillogenesis. Mutations in such proteins may affect muscle function during early development either through haploinsufficiency with insufficient dosage of a functional protein or a dominant negative effect of the mutated allele by functional or structural alterations.