Skip to content


  • Research article
  • Open Access
  • Open Peer Review

Expression profiles of muscle disease-associated genes and their isoforms during differentiation of cultured human skeletal muscle cells

  • 1,
  • 2 and
  • 1, 3Email author
BMC Musculoskeletal Disorders201213:262

  • Received: 10 August 2012
  • Accepted: 21 December 2012
  • Published:
Open Peer Review reports



The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins.


We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development.


Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development.


In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.


  • Myogenesis
  • Sarcomere
  • Myoblast
  • Skeletal muscle


The formation of skeletal muscle cells takes place during embryogenesis, postnatal growth and repair of postnatal skeletal muscle. Postnatal growth of muscle and regeneration of adult skeletal muscle tissue following injury is accomplished by activation of quiescent satellite cells that subsequently proliferate, differentiate and fuse with each other or pre-existing myofibers to regenerate myofibers thereby regenerating the muscle tissue [1]. For these characteristics of self-renewal, cultured satellite cells can provide an invaluable insight into the basic mechanisms of sarcomere expression profile, assembly and myofibrillogenesis. Although previous studies have revealed some distinct genetic requirements for embryonic, fetal, postnatal and adult regenerative myogenesis, the basic mechanisms are likely to be similar [2, 3].

Striated muscle formation is a fundamentally conserved process and requires the highly regulated and ordered expression of sarcomeric proteins and their assembly into the sarcomeric contractile unit [4]. The principle components of striated muscle sarcomeres include parallel arrays of actin-containing thin filaments, which overlap with myosin-containing thick filaments. Striated muscle function depends on the precise alignment of actin and myosin filaments, which is achieved by accessory proteins that link the different components and hold them in register with each other [5]. In the sarcomere many proteins cooperate to convert the molecular interactions of actin and myosin efficiently into mechanical force and movement [4].

A common property of many of the sarcomeric components of striated muscle is that they often exist as families of similar isoforms. These isoforms are developmentally regulated and differentially expressed in a tissue-specific manner in cardiac, slow and fast muscle [6, 7]. The generation of distinct protein isoforms within a family can result predominantly by two mechanisms: the differential expression of multigene families or the production of multiple protein variants from a single gene via alternative splicing. Muscle proteins, which exist as various isoforms in developing and mature skeletal and cardiac muscles are functionally unique and exhibit distinct contractile and physiological properties [810].

Although several studies have described that during myofibrillogenesis, the expression of proteins occurs in an ordered sequence [4], the sequential onset of the individual isoforms within a family of sarcomeric proteins is poorly characterized. Elucidation of the expression patterns of sarcomeric proteins and their distinct isoforms during myogenesis, is important to better understand their function, formation and assembly into sarcomeric structure in normal muscle cells. These data will allow a comparison with muscle cells obtained from patients suffering from one of the increasing number of diseases originating from genetic defects in these proteins. Congenital myopathies, a heterogeneous group of muscle disorders defined by distinctive morphologic abnormalities in skeletal muscle fibers, are often caused by structural defects in sarcomeric or cytoskeletal proteins [11]. In addition, distal arthrogryposis (DA) syndromes, a heterogeneous group of disorders with congenital contractures mainly of hands and feet, have recently been associated with mutations in sarcomeric components including ß-tropomyosin (TPM2) [12, 13], fast troponin T (TNNT3) [14], fast troponin I (TNNI2) [12, 15], embryonic myosin heavy chain (MYH3) [16, 17], prenatal myosin heavy chain (MYH8) [18] and slow myosin binding protein C (MYBPC1) [19].

In the present study, we characterize the expression profile of a panel of muscle-specific sarcomeric components that are associated with congenital diseases by combining molecular and morphological techniques on cultured human adult regenerative myoblasts and differentiated myotubes.


Muscle cell Cultures

Five standardized batches of human myoblasts were provided by MYOSIX through a collaborative program with Association Francaise contre les Myopathies (AFM). Skeletal muscle cells from donors with no clinical signs of muscle disease were enzymatically isolated and cultured as previously described [20, 21]. Isolated satellite cells, frozen in liquid nitrogen in their second passage, were quickly thawed and plated on chamber slides (Lab-Tek™ II - CC2™, Nalge Nunc International, Naperville, USA) using Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom AG, Berlin, Germany), supplemented with 20% fetal bovine serum (Invitrogen, GIBCO, Auckland, New Zealand), 100x GlutaMAX (Invitrogen, GIBCO, Paisley, UK), 10 mg/ml penicillin/streptomycin (Biochrom AG, Berlin, Germany), 10 μm./ml insulin (Biochrom AG, Berlin, Germany) and 10 ng/ml fibroblast growth factor (BD Biosciences, Bedford, MA, USA). The cells were incubated at 37°C in a humidified 5% CO2 atmosphere.

To induce differentiation, cells were replenished at 85%-90% confluence with DMEM medium, supplemented with 5% horse serum (Invitrogen, GIBCO, Auckland, New Zealand) and further incubated in this medium for 6 days. The medium was changed three times a week.

RNA isolation, cDNA synthesis, polymerase chain reaction (PCR) and sequence analysis

Total RNA was isolated from proliferating myoblasts and differentiated cell cultures and treated with DNase I using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The total RNA concentration was determined by A260 and A280 (A260/A280=1.7-2.0) measurements using a NanoDrop ND 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA). Synthesis of first-strand complementary DNA (cDNA) was performed from 400 ng total RNA using the iScript cDNA Synthesis kit (BioRad Laboratories, 2000 Alfred Nobel Drive Hercules, CA), according to the manufacturer’s instructions.

The expression of transcripts of a panel of genes encoding skeletal muscle sarcomeric components desmin (DES), titin (TTN), MyHC isoforms (MYH1, MYH2, MYH3, MYH4, MYH7 and MYH8), α-skeletal actin (ACTA1) and α-cardiac actin (ACTC1), tropomyosin isoforms (TPM1, TPM2 and TPM3), troponin T isoforms (TNNT1, TNNT2 and TNNT3), troponin I isoforms (TNNI1, TNNI2 and TNNI3), skeletal muscle myosin-binding protein C isoforms (MYBPC1, MYBPC2) and cardiac myosin-binding protein C (MYBPC3) was analyzed by reverse transcriptase polymerase chain reaction (PCR). In addition, the expression of transcripts of myogenic regulatory factors (MRFs) myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1) and myogenin (MYOG) was analyzed.

The PCR analysis was performed on cDNA in a master mixture (ReddyMix PCR Master Mix, ABgene House, Blenheim Road, UK) containing 20 pmol of each primer. The PCR primers were directed to cDNA sequence flanking exons in order to exclude genomic DNA amplification, although the RNA samples were treated with DNase I. Primer sequences are available on request. PCR amplifications consisted of an initial preheating step for 5 min at 94°C, followed by a touchdown (TD)-PCR. TD-PCR analysis was performed by denaturation at 94°C for 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 1 min with a 1°C temperature decrement per cycle during the first 10 cycles. The subsequent cycles (40 cycles) each consisted of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. The same PCR amplification reaction was performed for all genes, except for the amplification of myosin heavy chain isoforms that consisted of an initial preheating step for 3 min at 94°C, followed by denaturation at 94°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min for 35 cycles. Samples were run in triplicate. The nucleotide sequence determination was carried out by cycle sequencing using an ABI 3730xl DNA sequencer (GATC Biotech AG, Konstanz, Germany).

Immunocytochemistry and immunofluorescence

Immunocytochemical analysis was performed on proliferating myoblasts and myotubes after 6 days of differentiation, grown on chamber slides using LSAB kit (DAKO, Glostrup, Denmark) according to manufacturer’s instructions. The cells were incubated with primary antibodies (Table 1) followed by incubation with EnVision™Flex/HRP. The immunoreactivity was visualized by the indirect peroxidase-antiperoxidase complex method (DAKO, Glostrup, Denmark) using diaminobenzidine as a chromogen. Nuclei were counterstained with hematoxylin (Novocastra, Leica Microsystems Newcastle Ltd). Immunoreactivity was analyzed by light microscopy using the Zeiss Axio Observer microscope (Carl Zeiss AG, Germany) equipped with 10×, 20× and 40× objectives.
Table 1

Antibodies used for the immunocytochemical and immunofluorescence analyses







Mouse monoclonal to desmin




1:1000 Anti-mouse Dylight 549


Mouse monoclonal [F5D] to myogenin





Abcam plc

Mouse monoclonal to troponin T, fast






Mouse monoclonal to titin






Mouse monoclonal to sarcomeric actin


Alpha-skeletal actin


1:1000 Anti-mouse Dylight 549


Mouse monoclonal cardiac actin


Alpha-cardiac actin


1:1000 Anti-mouse Dylight 549

Nordic Biolabs Bioreagents

Mouse monoclonal to myosin heavy chain, (developmental)




1:1000 Anti-mouse Dylight 549


Mouse monoclonal to myosin heavy chain, (neonatal)




1:1000 Anti-mouse Dylight 549


Mouse monoclonal to myosin heavy chain, (slow)






Mouse monoclonal to myosin heavy chain, (fast)






Mouse monoclonal to myosin heavy chain, (fast IIa and slow)


MyHC-fast IIa+slow


1:1000 Anti-mouse Dylight 549

Santa Cruz Biotechnology

Mouse monoclonal to sarcomeric tropomyosin






Rabbit polyclonal to beta tropomyosin





Aviva Systems Biology

Rabbit polyclonal to myosin binding protein C, (slow)






Rabbit polyclonal to myosin binding protein C, (fast)






Mouse monoclonal [12F10] to skeletal muscle troponin I, (slow)





Abcam plc

Mouse monoclonal [2F12A11] to skeletal muscle troponin I, (fast)


TNNI- fast



Abcam plc

Mouse monoclonal [284(19C7)] to cardiac troponin I


TNNI- cardiac



Abcam plc

Mouse monoclonal T12 to Z-disk titin




Polyclonal Anti-Mouse Immunoglobulins/FITC 1:1000

Fürst et al., 1988

Mouse monoclonal T3 to A/I junction titin


A/I junction


Polyclonal Anti-Mouse Immunoglobulins/FITC 1:1000

Fürst et al., 1988

Mouse monoclonal T30 to A-band titin




Polyclonal Anti-Mouse Immunoglobulins/FITC 1:1000

Fürst et al., 1989

Mouse monoclonal T51 to M-band titin




Polyclonal Anti-Mouse Immunoglobulins/FITC 1:1000

Obermann et al., 1996

For immunofluorescence assays, slides were washed three times in 0.01 M phosphate-buffered saline (PBS) and fixed in 4% formaldehyde (methanol-free 16% formaldehyde solution, Thermo scientific) for 10 min. Free aldehyde groups were blocked with 50 mM NH4Cl for 10 min and cells were permeabilized in 0,01 M PBS containing 0,1% Triton X-100 for 4 min. Cells were incubated with primary antibodies for 1 h in a humidified chamber at 37°C, followed by incubation with secondary antibodies for 1 h in the dark and three washes with 0.01 M PBS. Finally, the slides were mounted with a coverslip using Prolong® Gold antifade reagent with DAPI (Invitrogen, GIBCO, Auckland, New Zealand) to highlight cell nuclei. The slides were left overnight in the dark at room temperature before examinations.


The experiments were conducted in triplicate with similar results.

RNA expression and sequence analysis

The appearance of the myogenic phenotype in the investigated cell cultures was verified by analyzing the expression of the genes encoding a family of transcription factors known as MRFs including MYOD1, MYF5 and MYOG that are responsible for controlling muscle-specific gene expression. Expression of all MRFs was readily detectable in both proliferating mononucleated myoblasts and cells after 6 days of differentiation (D6) (Figure 1A and B).
Figure 1
Figure 1

RT-PCR analysis of myogenic regulatory factors (MRFs) and a panel of striated muscle sarcomeric genes in myoblasts and cells differentiated for 6 days. RNA isolated from proliferating mononucleated myoblasts (A, C, E and G) and cultures after 6 days of differentiation (B, D, F and H) was analyzed by RT-PCR and the products were separated on agarose gels. The expression of MRF genes (MYOD1, MYF5 and MYOG), TM isoforms, ACTA1, ACTC1, different MyHC isoforms, DES, TTN and MyBPC isoforms, and troponin I and T isoforms was readily detectable in myoblasts and differentiated cells.

The expression of tropomyosin isoforms TPM1, TPM2, TPM3, α-skeletal and α-cardiac actin (ACTA1 and ACTC1), MyHC isoforms (MYH1, MYH2, MYH3, MYH4, MYH7, MYH8), DES, TTN, slow, fast and cardiac myosin-binding protein C isoforms (MYBPC1, MYBPC2, MYBPC3), troponin I isoforms (TNNI1, TNNI2, TNNI3), and troponin T isoforms (TNNT1, TNNT2, TNNT3) was detected in both myoblasts and D6 cells (Figure 1A-H).

In order to confirm and verify the expression of each specific transcript in both myoblasts and differentiated cells, a sequence analysis of the amplicons was performed. Using the NCBI BLAST website, the 100% identity to the reference sequences were confirmed for the MRF transcripts (MYOD1, MYF5 and MYOG), as well as the muscle specific alternative splicing variant of the investigated sarcomeric transcripts (Additional files 1, 2, 3, Figure S1–S3).

Immunocytochemistry and immunofluorescence

Mononucleated myoblasts were differentiated into multinucleated cells to induce the development of myotubes containing cross-striated myofibrils. The mature sarcomeric cross-striated pattern of the myofibrils within differentiated cells was confirmed by immunostaining with a panel of titin antibodies: all sarcomeric structures (Z-disk, A/I-junction, A-band and M-band) were clearly detected after only 5 days of differentiation (Figure 2).
Figure 2
Figure 2

Immunofluorescence micrographs of stained myoblasts and 5-day myotube cultures. Staining with monoclonal antibodies recognizing titin epitopes in the Z-disk (A and E), A/I junction (B and F), A-band (C and G) and M-band (D and H) reveal development of mature cross-striated myofibrils. Nuclei were stained with DAPI (blue). The bars represent 10 μm.

To confirm the myogenic identity of the analyzed cells they were stained with an antibody against desmin. The expression of desmin was homogeneous and the antibody strongly stained virtually all cells of both proliferating myoblasts (Figure 3A) and differentiated (D6) cultures (Figure 4A). In order to determine the stage of differentiation of the mononucleated myoblasts and the differentiated cells, an antibody against myogenin was used. Only a small number of the myonuclei of proliferating mononucleated cells were found to express myogenin (Figure 3B), whereas the majority of the nuclei were myogenin positive in the multinucleated differentiated cells confirming their more advanced differentiation level (Figure 4B).
Figure 3
Figure 3

Immunocytochemical analysis of proliferating human mononucleated myoblasts. (A) The strong homogeneous staining for desmin revealed the myogenic potential of virtually all mononucleated cells. (B) Only a small number of the myonuclei of proliferating cells expressed myogenin (arrows). (C) A subset of myoblasts showed immunoreactivity with antibody against titin. (D-E-F-G-H) Demonstrate positive staining of a few cells with antibodies against MyHC-embryonic, MyHC-neonatal, MyHC-slow/beta cardiac, MyHC-fast and MyHC-fast IIa+slow, respectively. Insets demonstrate considerable numbers of mononucleated myoblasts expressing various MyHC isoforms, as indicated. Homogeneous staining of proliferating mononucleated cells with antibodies against slow (I) and fast (J) MyBPC respectively. Uniform and strong staining of proliferating myoblasts with antibody against alpha-cardiac actin (K). The cells were weakly stained with antibody against alpha-skeletal actin (L). The bar represents 10 μm and 100 μm for the insets.

Figure 4
Figure 4

Immunocytochemical analysis of human cells after 6 days of differentiation. (A) shows strong homogeneous staining for desmin of the cells. (B) The differential capacity of the multinucleated cells was demonstrated by myogenin-positive staining of the majority of the nuclei. (C) Strong homogeneous staining of differentiated cells with an antibody against titin. (D) The vast majority of differentiated cells showed strong staining with the antibody against MyHC-embryonic and (E) many myotubes expressed neonatal MyHC. (F-H) expression of fast and slow MyHC was revealed in a subset of fused cells using antibodies against these isoforms. Positive staining of differentiated cells with antibodies against slow (I) and fast (J) MyBPC, respectively. The differentiated cells showed weak positive immunoreactivity with the antibody against alpha-cardiac actin (K) but stronger positive staining with alpha-skeletal actin (L). The bar represents 10 μm.

In order to examine the sequential onset of the expression of sarcomeric proteins and their isoforms, we performed immunocytochemical analyses on mononucleated myoblasts and differentiated cells using a large panel of antibodies (Table 1). While the expression of titin was observed in a subset of myoblasts (Figure 2A-D and 3C), this protein was expressed and organized into a cross-striated pattern in differentiated cells (Figure 2E-H and 4C). The expression analysis of various MyHC isoforms was performed using five different MyHC antibodies. Although MyHC isoforms were predominantly detected in differentiated cells, about 10% of mononucleated myoblasts also expressed embryonic, neonatal, slow and fast MyHC (Figure 3D-H, insets). The vast majority of multinucleated myotubes stained strongly with the antibody against embryonic MyHC (Figure 4D-H). The expression pattern of slow and fast MyBPC was studied using two specific antibodies. While virtually all proliferating mononucleated and differentiated cells appeared to be stained with the antibody against slow MyBPC, these cells were weakly stained with the antibody against fast MyBPC (Figure 3I-J and 4I-J). In addition, no specific staining of proliferating mononucleated myoblasts or multinucleated differentiated cells with cardiac-specific MyBPC antibody was detected (data not shown).

Immunohistochemical analysis with alpha-cardiac and alpha-skeletal actin antibodies revealed uniform staining of proliferating myoblasts (Figure 3K and L). While strong expression of alpha-cardiac actin was detected in the myoblasts (Figure 3K), these cells appeared weakly stained by the alpha-skeletal actin antibody (Figure 3L). In contrast, in multinucleated differentiated cells the intensity of staining for alpha-skeletal actin appeared to be much stronger than for alpha-cardiac actin (Figure 4K and L).

The expression of tropomyosin isoforms was studied in both myoblasts and differentiated cells using two different antibodies. The monoclonal anti-sarcomeric tropomyosin antibody detects all three TM isoforms (α-, β- and γ-TM isofoms), whereas the TPM2 antibody is specific for the β-TM isoform. Although both antibodies produced uniform staining of proliferating mononucleated cells (Figure 5A and B), the intensity of staining of multinucleated cells appeared to be much stronger (Figure 5C and D). While expression of the fast TnT isoform was undetectable in proliferating myoblasts, strong expression was detected in multinucleated cells (Figure 5E and F). To assess the expression of TnI isoforms, specific anti-slow and anti-fast troponin I antibodies were used. Expression of slow TnI isoforms was detected in both mononucleated and multinucleated differentiated cells (Figure 5G and H), whereas the fast TnI isoform was solely expressed in myotubes (Figure 5I and J). Notably, a subset of the multinucleated myotubes formed myofibre-like cells with peripheral nuclei (Figure 5H and J; arrows). Immunocytochemical analyses of cardiac TnI confirmed a uniform staining of proliferating mononucleated myoblasts (Figure 5K) and a vast majority of myotubes (Figure 5L). Immunofluorescence analysis confirmed these results (Additional file 4: Figure S4).
Figure 5
Figure 5

Immunohistochemical analysis of human myoblasts and cells after 6 days of differentiation. Homogeneous staining of cells with (A) antibody against three different sarcomeric TM isoforms and (B) a specific antibody against beta-tropomyosin, β-TM isoform. (C) Strong positive staining of multinucleated cells with an antibody against sarcomeric TM isoforms. (D) Staining of cells with specific antibody against beta-tropomyosin. (E) The mononucleated cells were negative for fast troponin T expression, while positive immunoreactivity was observed in (F) multinucleated cells, as illustrated by the anti-fast TNNT MAb staining. Homogeneous staining of (G) proliferating myoblasts and (H) multinucleated myotubes with antibody against slow isoform of troponin I. No expression of fast troponin I was detected in (I) proliferating myoblasts whereas positive staining was observed in (J) differentiated cells by an antibody against this isoform. Homogeneous immunoreactivity of both (K) proliferating myoblasts and (L) multinucleated cells with antibodies against cardiac isoform of troponin I. A subset of the multinucleated myotubes have a myofibre-like appearance with peripherally located nuclei (H and J; arrows). The bar represents 10 μm.

Supplemental information

Supplemental Appendix includes four figures.


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, 2225]. 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 [4]. 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 [26], 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 [29].

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 [3032]. 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 [33] 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 [19]. 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 [3]. Also in accordance with a previous study, which indicated that cardiac MyBPC is not expressed in human skeletal muscles, not even during development [3], 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 [34]. 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, 3542]. 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.


In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. We show that many proteins involved in muscle diseases are readily detected in the stages of skeletal muscle cell differentiation that can be reached in vitro. The early and uniform expression of these proteins suggests their impact on the developmental mechanisms involved in the initial stages of myofibril assembly, differentiation and formation of muscle. This indicates that myoblasts isolated from patients with a mutation in one of the investigated genes may be an invaluable tool to analyze the effects of these mutations on sarcomere assembly and disassembly or myofibril turnover. It would provide us new insights into development of muscle to indicate whether these diseases are disorders of myofibrillogenesis and muscle development.



The study was supported by a grant from the Swedish Research Council (Project No 073018 to HT), Åke Wiberg’s Foundation to HT. We thank the company Myosix and Association Francaise contre les Myopathies (AFM) for giving free access to the myoblast batches.

Authors’ Affiliations

Department of Pathology, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, SE, 413 45, Sweden
Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, Bonn, 53121, Germany
Department of Clinical and Medical Genetics, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, SE, 413 45, Sweden


  1. Sanger JW, Kang S, Siebrands CC: How to build a myofibril. J Muscle Res Cell Motil. 2005, 26: 343-354.View ArticlePubMedGoogle Scholar
  2. Fürst DO, Osborn M, Weber K: Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J Cell Biol. 1989, 109: 517-527. 10.1083/jcb.109.2.517.View ArticlePubMedGoogle Scholar
  3. Gautel M, Fürst DO, Cocco A, Schiaffino S: Isoform transitions of the myosin binding protein C family in developing human and mouse muscles: lack of isoform transcomplementation in cardiac muscle. Circ Res. 1998, 82: 124-129. 10.1161/01.RES.82.1.124.View ArticlePubMedGoogle Scholar
  4. Ehler E, Gautel M: The sarcomere and sarcomerogenesis. Adv Exp Med Biol. 2008, 642: 1-14. 10.1007/978-0-387-84847-1_1.View ArticlePubMedGoogle Scholar
  5. Clark KA, McElhinny AS, Beckerle MC, Gregorio CC: Striated muscle cytoarchitecture: an intricate web of form and function. Annu Rev Cell Dev Biol. 2002, 18: 637-706. 10.1146/annurev.cellbio.18.012502.105840.View ArticlePubMedGoogle Scholar
  6. Perry SV: Properties of the muscle proteins–a comparative approach. J Exp Biol. 1985, 115: 31-42.PubMedGoogle Scholar
  7. Bandman E: Contractile protein isoforms in muscle development. Dev Biol. 1992, 154: 273-283. 10.1016/0012-1606(92)90067-Q.View ArticlePubMedGoogle Scholar
  8. Lynch GS, Stephenson DG, Williams DA: Analysis of Ca2+ and Sr2+ activation characteristics in skinned muscle fibre preparations with different proportions of myofibrillar isoforms. J Muscle Res Cell Motil. 1995, 16: 65-78. 10.1007/BF00125311.View ArticlePubMedGoogle Scholar
  9. Bottinelli R, Reggiani C: Human skeletal muscle fibres: molecular and functional diversity. Prog Biophys Mol Biol. 2000, 73: 195-262. 10.1016/S0079-6107(00)00006-7.View ArticlePubMedGoogle Scholar
  10. Pette D, Staron RS: Myosin isoforms, muscle fiber types, and transitions. Microsc Res Tech. 2000, 50: 500-509. 10.1002/1097-0029(20000915)50:6<500::AID-JEMT7>3.0.CO;2-7.View ArticlePubMedGoogle Scholar
  11. Goebel HH: Congenital myopathies in the new millennium. J Child Neurol. 2005, 20: 94-101. 10.1177/08830738050200020201.View ArticlePubMedGoogle Scholar
  12. Sung SS, Brassington AM, Grannatt K: Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes. Am J Hum Genet. 2003, 72: 681-690. 10.1086/368294.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Tajsharghi H, Kimber E, Holmgren D, Tulinius M, Oldfors A: Distal arthrogryposis and muscle weakness associated with a beta-tropomyosin mutation. Neurology. 2007, 68: 772-775. 10.1212/01.wnl.0000256339.40667.fb.View ArticlePubMedGoogle Scholar
  14. Sung SS, Brassington AM, Krakowiak PA: Mutations in TNNT3 cause multiple congenital contractures: a second locus for distal arthrogryposis type 2B. Am J Hum Genet. 2003, 73: 212-214. 10.1086/376418.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Kimber E, Tajsharghi H, Kroksmark AK, Oldfors A, Tulinius M: A mutation in the fast skeletal muscle troponin I gene causes myopathy and distal arthrogryposis. Neurology. 2006, 67: 597-601. 10.1212/01.wnl.0000230168.05328.f4.View ArticlePubMedGoogle Scholar
  16. Toydemir RM, Rutherford A, Whitby FG: Mutations in embryonic myosin heavy chain (MYH3) cause Freeman-Sheldon syndrome and Sheldon-Hall syndrome. Nat Genet. 2006, 38: 561-565. 10.1038/ng1775.View ArticlePubMedGoogle Scholar
  17. Tajsharghi H, Kimber E, Kroksmark AK: Embryonic myosin heavy-chain mutations cause distal arthrogryposis and developmental myosin myopathy that persists postnatally. Arch Neurol. 2008, 65: 1083-1090. 10.1001/archneur.65.8.1083.View ArticlePubMedGoogle Scholar
  18. Toydemir RM, Chen H, Proud VK: Trismus-pseudocamptodactyly syndrome is caused by recurrent mutation of MYH8. Am J Med Genet A. 2006, 140: 2387-2393.View ArticlePubMedGoogle Scholar
  19. Gurnett CA, Desruisseau DM, McCall K: Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1. Hum Mol Genet. 2010, 19: 1165-1173. 10.1093/hmg/ddp587.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Vilquin JT, Marolleau JP, Sacconi S: Normal growth and regenerating ability of myoblasts from unaffected muscles of facioscapulohumeral muscular dystrophy patients. Gene Ther. 2005, 12: 1651-1662. 10.1038/ ArticlePubMedGoogle Scholar
  21. Lecourt S, Marolleau JP, Fromigue O: Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro. Exp Cell Res. 2010, 316: 2513-2526. 10.1016/j.yexcr.2010.04.020.View ArticlePubMedGoogle Scholar
  22. van der Ven PF, Schaart G, Jap PH: Differentiation of human skeletal muscle cells in culture: maturation as indicated by titin and desmin striation. Cell Tissue Res. 1992, 270: 189-198. 10.1007/BF00381893.View ArticlePubMedGoogle Scholar
  23. van der Ven PF, Fürst DO: Assembly of titin, myomesin and M-protein into the sarcomeric M band in differentiating human skeletal muscle cells in vitro. Cell Struct Funct. 1997, 22: 163-171. 10.1247/csf.22.163.View ArticlePubMedGoogle Scholar
  24. Sanger JW, Wang J, Fan Y, White J, Sanger JM: Assembly and dynamics of myofibrils. J Biomed Biotechnol. 2010, 2010: 858606-View ArticlePubMedPubMed CentralGoogle Scholar
  25. Sanger JW, Chowrashi P, Shaner NC: Myofibrillogenesis in skeletal muscle cells. Clin Orthop Relat Res. 2002, 403: 153-162.View ArticleGoogle Scholar
  26. van der Loop FT, van der Ven PF, Fürst DO: Integration of titin into the sarcomeres of cultured differentiating human skeletal muscle cells. Eur J Cell Biol. 1996, 69: 301-307.PubMedGoogle Scholar
  27. Hayward LJ, Schwartz RJ: Sequential expression of chicken actin genes during myogenesis. J Cell Biol. 1986, 102: 1485-1493. 10.1083/jcb.102.4.1485.View ArticlePubMedGoogle Scholar
  28. Bains W, Ponte P, Blau H, Kedes L: Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro. Mol Cell Biol. 1984, 4: 1449-1453.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Vandekerckhove J, Bugaisky G, Buckingham M: Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms. J Biol Chem. 1986, 261: 1838-1843.PubMedGoogle Scholar
  30. Saggin L, Gorza L, Ausoni S, Schiaffino S: Troponin I switching in the developing heart. J Biol Chem. 1989, 264: 16299-16302.PubMedGoogle Scholar
  31. Bhavsar PK, Dhoot GK, Cumming DV: Developmental expression of troponin I isoforms in fetal human heart. FEBS Lett. 1991, 292: 5-8. 10.1016/0014-5793(91)80820-S.View ArticlePubMedGoogle Scholar
  32. Sutherland CJ, Esser KA, Elsom VL, Gordon ML, Hardeman EC: Identification of a program of contractile protein gene expression initiated upon skeletal muscle differentiation. Dev Dyn. 1993, 196: 25-36. 10.1002/aja.1001960104.View ArticlePubMedGoogle Scholar
  33. Ferrer-Martinez A, Montell E, Montori-Grau M: Long-term cultured human myotubes decrease contractile gene expression and regulate apoptosis-related genes. Gene. 2006, 384: 145-153.View ArticlePubMedGoogle Scholar
  34. Miller TE, You L, Myerburg RJ, Benke PJ, Bishopric NH: Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases. Genet Med. 2007, 9: 23-33. 10.1097/GIM.0b013e31802d74de.View ArticlePubMedGoogle Scholar
  35. Laing NG, Wilton SD, Akkari PA: A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nat Genet. 1995, 9: 75-79. 10.1038/ng0195-75.View ArticlePubMedGoogle Scholar
  36. Penisson-Besnier I, Monnier N, Toutain A, Dubas F, Laing N: A second pedigree with autosomal dominant nemaline myopathy caused by TPM3 mutation: a clinical and pathological study. Neuromuscul Disord. 2007, 17: 330-337. 10.1016/j.nmd.2007.01.017.View ArticlePubMedGoogle Scholar
  37. Clarke NF, Kolski H, Dye DE: Mutations in TPM3 are a common cause of congenital fiber type disproportion. Ann Neurol. 2008, 63: 329-337. 10.1002/ana.21308.View ArticlePubMedGoogle Scholar
  38. Ohlsson M, Quijano-Roy S, Darin N: New morphologic and genetic findings in cap disease associated with beta-tropomyosin (TPM2) mutations. Neurology. 2008, 71: 1896-1901. 10.1212/01.wnl.0000336654.44814.b8.View ArticlePubMedGoogle Scholar
  39. Donner K, Ollikainen M, Ridanpaa M: Mutations in the beta-tropomyosin (TPM2) gene–a rare cause of nemaline myopathy. Neuromuscul Disord. 2002, 12: 151-158. 10.1016/S0960-8966(01)00252-8.View ArticlePubMedGoogle Scholar
  40. Lehtokari VL: Ceuterick-de Groote C, de Jonghe P, et al.: Cap disease caused by heterozygous deletion of the beta-tropomyosin gene TPM2. Neuromuscul Disord. 2007, 17: 433-442. 10.1016/j.nmd.2007.02.015.View ArticlePubMedGoogle Scholar
  41. Tajsharghi H, Ohlsson M, Lindberg C, Oldfors A: Congenital myopathy with nemaline rods and cap structures caused by a mutation in the beta-tropomyosin gene (TPM2). Arch Neurol. 2007, 64: 1334-1338. 10.1001/archneur.64.9.1334.View ArticlePubMedGoogle Scholar
  42. Monnier N, Lunardi J, Marty I: Absence of beta-tropomyosin is a new cause of Escobar syndrome associated with nemaline myopathy. Neuromuscul Disord. 2009, 19: 118-123. 10.1016/j.nmd.2008.11.009.View ArticlePubMedGoogle Scholar
  43. Feghali R, Leinwand LA: Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain. J Cell Biol. 1989, 108: 1791-1797. 10.1083/jcb.108.5.1791.View ArticlePubMedGoogle Scholar
  44. Karsch-Mizrachi I, Travis M, Blau H, Leinwand LA: Expression and DNA sequence analysis of a human embryonic skeletal muscle myosin heavy chain gene. Nucleic Acids Res. 1989, 17: 6167-6179. 10.1093/nar/17.15.6167.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Alvarado DM, Buchan JG, Gurnett CA, Dobbs MB: Exome sequencing identifies an MYH3 mutation in a family with distal arthrogryposis type 1. J Bone Joint Surg Am. 2011, 93: 1045-1050.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Veugelers M, Bressan M, McDermott DA: Mutation of perinatal myosin heavy chain associated with a Carney complex variant. N Engl J Med. 2004, 351: 460-469. 10.1056/NEJMoa040584.View ArticlePubMedGoogle Scholar
  47. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:


© Abdul-Hussein et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.