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Myoblast differentiation is a complex process whereby the mononuclear muscle precursor cells myoblasts express skeletal-muscle-specific genes and fuse with each other to form multinucleated myotubes. The objective of this study was to identify potentially novel mechanisms that mediate myoblast differentiation. We first compared transcriptomes in C2C12 myoblasts before and 6 days after induction of myogenic differentiation by RNA-seq. This analysis identified 11,046 differentially expressed genes, of which 5615 and 5431 genes were upregulated and downregulated, respectively, from before differentiation to differentiation. Functional enrichment analyses revealed that the upregulated genes were associated with skeletal muscle contraction, autophagy, and sarcomeres while the downregulated genes were associated with ribonucleoprotein complex biogenesis, mRNA processing, ribosomes, and other biological processes or cellular components Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal-muscle-specific genes and the formation of myotubes, confirming a positive role for autophagy in myoblast differentiation and fusion.

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.

Skeletal muscle has high regenerative capacity due to a resident population of adult stem cells (MuSCs). These MuSCs normally exist in a quiescent state but when a muscle is injured, the MuSCs become activated and re-enter the cell cycle, proliferate, differentiate and undergo fusion to form multinucleated myotubes. During myogenesis, dramatic changes occur in cell size, shape, metabolism and motility, which cause cellular stress and alter muscle proteostasis. Heat shock proteins (HSPs) are molecular chaperones with the potential to maintain proteostasis by regulating protein biosynthesis and folding, facilitating transport of polypeptides across intracellular membranes, and preventing stress-induced protein unfolding/aggregation. HSP70 is the most widely studied HSP relevant to skeletal muscle. How HSPs, particularly HSP70, regulate myogenesis and whether manipulation of HSP expression can enhance muscle repair, remain important unanswered questions. In Chapter 4 of this thesis, HSP expression was characterised in C2C12 cells during proliferation and differentiation. Whole cell lysates prepared from proliferating C2C12 cells and C2C12 cells undergoing myogenic differentiation for 1-4 days (D1-D4), were examined for their expression of various HSPs based on SDS-PAGE and western immunoblotting. HSP25 decreased at D4 of differentiation (P < 0.05). HSP40 expression was high in proliferating myoblasts but decreased at the onset of differentiation (P < 0.05). HSP60 decreased between early and late differentiation (P < 0.001). HSP90 and HSP110 were highly expressed in proliferating myoblasts and decreased during early differentiation. Lastly, HSP70 protein expression peaked during early stages of differentiation, just preceding the expression of myogenin (P < 0.05). These findings imply that many of the HSPs are involved in the regulation of myogenesis at different stages, with HSP70 potentially having a role in myoblast fusion. Based on these findings, the studies in Cha

Age-related loss of muscle mass may result from reduced protein synthesis stimulation in response to anabolic stimuli, such as amino acid (AA) supplementation. The exact etiology of anabolic resistance to AA remains unclear. Therefore, the aim of this study was to investigate the anabolic response [cell size, protein synthesis and mechanistic target of rapamycin (mTOR) pathway] to the AA glutamine (a strong anabolic AA highly present in skeletal muscle) in myotubes obtained from 8 young (YW; 21-35 yrs) and 8 older (OW; 65-70 yrs) healthy women. This in vitro model of human primary myogenic cells explores the intrinsic behavior of muscle cells, while excluding potential influences of external factors. We showed that despite lower muscle mass, strength and cardiorespiratory fitness in OW compared to YW, myotube size (myotube diameter and area) and protein synthesis were not altered in OW, and glutamine-induced myotube hypertrophy and protein synthesis were preserved in OW. Apart from a lower glutamine-induced increase in P70S6 kinase phosphorylation in OW, no significant differences in other components of the mTOR pathway were observed between groups. Altogether, our data support the idea that the intrinsic capacity of muscle cells to respond to glutamine stimulation is preserved in healthy older women., Funding Agency:Swedish Research Council for Sport Science

Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals., journal article

[EN] The characterization of the molecular mechanisms that control fibro-adipogenic progenitors (FAPs) differentiation into adipocytes when they are co-cultured with myotubes. FAPs are known to have a dual role in skeletal muscle; on one hand, in healthy conditions, they promote its regenerations, while in pathological conditions they contribute to fatty and fibrous tissue filtration. The molecular mechanism that controls their differentiation into adipocytes is not fully characterized, but cell-cell contact with myotubes seems to play an important role. In this project I tried to characterize the molecular mechanism underlying the inhibition of the adipogenic differentiation.

[EN] The characterization of the molecular mechanisms that control fibro-adipogenic progenitors (FAPs) differentiation into adipocytes when they are co-cultured with myotubes. FAPs are known to have a dual role in skeletal muscle; on one hand, in healthy conditions, they promote its regenerations, while in pathological conditions they contribute to fatty and fibrous tissue filtration. The molecular mechanism that controls their differentiation into adipocytes is not fully characterized, but cell-cell contact with myotubes seems to play an important role. In this project I tried to characterize the molecular mechanism underlying the inhibition of the adipogenic differentiation.

[EN] The characterization of the molecular mechanisms that control fibro-adipogenic progenitors (FAPs) differentiation into adipocytes when they are co-cultured with myotubes. FAPs are known to have a dual role in skeletal muscle; on one hand, in healthy conditions, they promote its regenerations, while in pathological conditions they contribute to fatty and fibrous tissue filtration. The molecular mechanism that controls their differentiation into adipocytes is not fully characterized, but cell-cell contact with myotubes seems to play an important role. In this project I tried to characterize the molecular mechanism underlying the inhibition of the adipogenic differentiation.

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, -17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the -17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the -17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping.

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, -17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the -17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the -17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping.

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

In mice persistently infected since birth with the prototypic arenavirus lymphocytic choriomeningitis viurs, viral antigen and RNA are readily detected in most organs and cell types but remarkably absent in skeletal muscle. Here we report that mouse C2C12 myoblasts that are readily infected by LCMV, become highly refractory to LCMV infection upon their differentiation into myotubes. Myotube's resistance to LCMV was not due to an intracellular restriction of virus replication but rather an impaired cell entry mediated by the LCMV surface glycoprotein. Our findings provide an explanation for the observation that in LCMV carrier mice myotubes, which are constantly exposed to blood-containing virus, remain free of viral antigen and RNA despite myotubes express high levels of the LCMV receptor alpha dystroglycan and do not pose an intracellular blockade to LCMV multiplication.

Insulin resistance in polycystic ovary syndrome (PCOS) is due to a postbinding defect in signaling that persists in cultured skin fibroblasts and is associated with constitutive serine phosphorylation of the insulin receptor (IR). Cultured skeletal muscle from obese women with PCOS and age- and body mass index-matched control women (n = 10/group) was studied to determine whether signaling defects observed in this tissue in vivo were intrinsic or acquired. Basal and insulin-stimulated glucose transport and GLUT1 abundance were significantly increased in cultured myotubes from women with PCOS. Neither IR beta-subunit abundance and tyrosine autophosphorylation nor insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity differed in the two groups. However, IRS-1 protein abundance was significantly increased in PCOS, resulting in significantly decreased PI 3-kinase activity when normalized for IRS-1. Phosphorylation of IRS-1 on Ser312, a key regulatory site, was significantly increased in PCOS, which may have contributed to this signaling defect. Insulin signaling via IRS-2 was also decreased in myotubes from women with PCOS. In summary, decreased insulin-stimulated glucose uptake in PCOS skeletal muscle in vivo is an acquired defect. Nevertheless, there are intrinsic abnormalities in glucose transport and insulin signaling in myotubes from affected women, including increased phosphorylation of IRS-1 Ser312, that may confer increased susceptibility to insulin resistance-inducing factors in the in vivo environment. These abnormalities differ from those reported in other insulin resistant states consistent with the hypothesis that PCOS is a genetically unique disorder conferring an increased risk for type 2 diabetes. Copyright © 2005 the American Physiological Society.

Insulin resistance in polycystic ovary syndrome (PCOS) is due to a postbinding defect in signaling that persists in cultured skin fibroblasts and is associated with constitutive serine phosphorylation of the insulin receptor (IR). Cultured skeletal muscle from obese women with PCOS and age- and body mass index-matched control women (n = 10/group) was studied to determine whether signaling defects observed in this tissue in vivo were intrinsic or acquired. Basal and insulin-stimulated glucose transport and GLUT1 abundance were significantly increased in cultured myotubes from women with PCOS. Neither IR beta-subunit abundance and tyrosine autophosphorylation nor insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity differed in the two groups. However, IRS-1 protein abundance was significantly increased in PCOS, resulting in significantly decreased PI 3-kinase activity when normalized for IRS-1. Phosphorylation of IRS-1 on Ser312, a key regulatory site, was significantly increased in PCOS, which may have contributed to this signaling defect. Insulin signaling via IRS-2 was also decreased in myotubes from women with PCOS. In summary, decreased insulin-stimulated glucose uptake in PCOS skeletal muscle in vivo is an acquired defect. Nevertheless, there are intrinsic abnormalities in glucose transport and insulin signaling in myotubes from affected women, including increased phosphorylation of IRS-1 Ser312, that may confer increased susceptibility to insulin resistance-inducing factors in the in vivo environment. These abnormalities differ from those reported in other insulin resistant states consistent with the hypothesis that PCOS is a genetically unique disorder conferring an increased risk for type 2 diabetes. Copyright © 2005 the American Physiological Society.

Statins, which are known as cholesterol-lowering drugs, have several additional effects including the enhancement of bone formation and the stimulation of smooth muscle cell proliferation. In this study, we investigated the signal pathway of simvastatin operating in C2C12 myoblast cells. Myotube formation of C2C12 cells was efficiently blocked by 1 muM simvastatin, and mevalonic acid was able to cancel this effect. Geranylgeranyl pyrophosphate restored the myotube formation, whereas farnesyl pyrophosphate did not. These findings demonstrate that the Rho family, such as Rho, Rac and Cdc42, occurring downstream of geranylgeranyl pyrophosphate in the mevalonic acid pathway, was involved in the simvastatin-mediated blockage of myotube formation. An inhibitor of Rho kinase did not influence the myotube formation; whereas an inhibitor of Rac blocked this process. Taken together, we conclude that the differentiation of C2C12 cells into myotubes was blocked by simvastatin through the pathway mediated by Rac, not by Rho., Journal of muscle research and cell motility, 29(2-5), pp.127-134; 2008

This work combines expertise in stem cell biology and bioengineering to define the system for geometric control of proliferation and differentiation of myogenic progenitor cells. We have created an artificial niche of myogenic progenitor cells, namely, modified extracellular matrix (ECM) substrates with spatially embedded growth or differentiation factors (GF, DF) that predictably direct muscle cell fate in a geometric pattern. Embedded GF and DF signal progenitor cells from specifically defined areas on the ECM successfully competed against culture media for myogenic cell fate determination at a clearly defined boundary. Differentiation of myoblasts into myotubes is induced in growth-promoting medium, myotube formation is delayed in differentiation-promoting medium, and myogenic cells, at different stages of proliferation and differentiation, can be induced to coexist adjacently in identical culture media. This method can be used to identify molecular interactions between cells in different stages of myogenic differentiation, which are likely to be important determinants of tissue repair. The designed ECM niches can be further developed into a vehicle for transplantation of myogenic progenitor cells maintaining their regenerative potential. Additionally, this work may also serve as a general model to engineer synthetic cellular niches to harness the regenerative potential of organ stem cells.

Predmet istraživanja ovog rada je humani mišićni protein Ankrd2 koji se eksprimira u sporim mišićnim vlaknima i potencijalni je transkripcioni faktor čija je količina povećana u toku hipertrofije skeletnih mišića. To je protein od 37 kDa koji poseduje signal za lokalizaciju u jedru, četiri ankirinska ponovka i veoma je sličan jedarnom proteinu humanih endotelnih ćelija, C-193, čija je ekspresija indukovana citokinima. Pacovski i mišji ortologoni humanog proteina C-193, CARP odnosno MARP su pretežno eksprimirani u srcu i verovatno su uključeni u kontrolu hipertrofije srca. Pretpostavlja se da Ankrd2 u skeletnim mišićima ima sličnu funkciju kao CARP/MARP/C-193 u srcu. Za utvrdjivanje intraćelijske lokalizacije i ekspresije proteina Ankrd2 u različitim tkivima napravljena su tri mišja poliklonska antitela (na ceo protein, N-terminalni i C-terminalni region) i monoklonsko antitelo (na C-terminalni region proteina Ankrd2). Western blot analizom proteinskih ekstrakata različitih humanih tkiva pokazano je da se protein Ankrd2 eksprimira uglavnom u skeletnim mišićima i u manjoj meri u srcu i bubregu. U toku diferencijacije humanih (CHQ5B) i mišjih (C2C12) mišićnih ćelija dolazi do povećanja količine proteina Ankrd2. U cilju detaljne analize intraćelijske lokalizacije proteina Ankrd2 uradjeni su eksperimenti indirektne imunofluorescencije koristeći humane mioblaste i miotube. Generalno, u toku diferencijacije povećava se i broj ćelija koje fluoresciraju i intenzitet signala. Takodje, u humanim mioblastima, za razliku od miotuba, detektovana je specifična jedarna fluorescencija. Jedarni signal je u obliku tački i eksperimenti kolokalizacije su pokazali da se proteini Ankrd2 i PML nalaze u istim jedarnim strukturama nazvanim PML jedarna tela. U cilju izučavanja proteinsko-proteinskih interakcija pripremljeni su rekombinantni proteini fuzionisani sa GST markerom (F-Ankrd2 (5-333 ak), N-Ankrd2 (5-120 ak) i C-Ankrd2 (279-333 ak)) koji su korišćeni u GST „pull-down” eksperimentima, The object of this study was the new human muscle protein Ankrd2 found preferentially in slow muscle fibers that is possibly a transcription factor up-regulated in hypertrophy. It is a protein of 37 kDa, which has a signal for nuclear targeting, four ankyrin repeat motifs and shows significant similarity to a cytokine inducible nuclear protein C-193 from human endothelial cells. The rat and mouse orthologs of human C-193 called respectively CARP and MARP are mainly expressed in heart and probably involved in the control of cardiac hypertrophy. It is possible that Ankrd2 may play a similar role to CARP/MARP/C-193, but in skeletal muscle rather than heart. For intracellular localization and tissue distribution of Ankrd2 three mouse polyclonal antibodies (raised against the full length, N-terminal and C-terminal regions of the protein) and one monoclonal antibody (raised against C-terminal region of Ankrd2) were made. Western blot analysis of protein extracts isolated from different human tissues showed that Ankrd2 is expressed mainly in human skeletal muscle and to a lesser extent in heart and kidney. During muscle cell differentiation there is an increase of Ankrd2 signal in both mouse (C2C12) and human (CHQ5B) muscle cells that can be detected by Western blot analysis. Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes with the scope of pinpointing the intracellular localization of Ankrd2 protein. In general, during differentiation both the number of fluorescing cells and the intensity of signal increase. Also, in human myoblasts but not in differentiated myotubes very specific nuclear fluorescence can be detected. The nuclear signal is in the form of speckles and co-localization experiments showed that Ankrd2 and PML co-localize in PML nuclear bodies. In order to study protein-protein interactions GST recombinant proteins F-Ankrd2 (5-333 aa), N-Ankrd2 (5-120 aa) and C-Ankrd2 (279-333 aa) were prepared and used for the GST „pull-d

Predmet istraživanja ovog rada je humani mišićni protein Ankrd2 koji se eksprimira u sporim mišićnim vlaknima i potencijalni je transkripcioni faktor čija je količina povećana u toku hipertrofije skeletnih mišića. To je protein od 37 kDa koji poseduje signal za lokalizaciju u jedru, četiri ankirinska ponovka i veoma je sličan jedarnom proteinu humanih endotelnih ćelija, C-193, čija je ekspresija indukovana citokinima. Pacovski i mišji ortologoni humanog proteina C-193, CARP odnosno MARP su pretežno eksprimirani u srcu i verovatno su uključeni u kontrolu hipertrofije srca. Pretpostavlja se da Ankrd2 u skeletnim mišićima ima sličnu funkciju kao CARP/MARP/C-193 u srcu. Za utvrdjivanje intraćelijske lokalizacije i ekspresije proteina Ankrd2 u različitim tkivima napravljena su tri mišja poliklonska antitela (na ceo protein, N-terminalni i C-terminalni region) i monoklonsko antitelo (na C-terminalni region proteina Ankrd2). Western blot analizom proteinskih ekstrakata različitih humanih tkiva pokazano je da se protein Ankrd2 eksprimira uglavnom u skeletnim mišićima i u manjoj meri u srcu i bubregu. U toku diferencijacije humanih (CHQ5B) i mišjih (C2C12) mišićnih ćelija dolazi do povećanja količine proteina Ankrd2. U cilju detaljne analize intraćelijske lokalizacije proteina Ankrd2 uradjeni su eksperimenti indirektne imunofluorescencije koristeći humane mioblaste i miotube. Generalno, u toku diferencijacije povećava se i broj ćelija koje fluoresciraju i intenzitet signala. Takodje, u humanim mioblastima, za razliku od miotuba, detektovana je specifična jedarna fluorescencija. Jedarni signal je u obliku tački i eksperimenti kolokalizacije su pokazali da se proteini Ankrd2 i PML nalaze u istim jedarnim strukturama nazvanim PML jedarna tela. U cilju izučavanja proteinsko-proteinskih interakcija pripremljeni su rekombinantni proteini fuzionisani sa GST markerom (F-Ankrd2 (5-333 ak), N-Ankrd2 (5-120 ak) i C-Ankrd2 (279-333 ak)) koji su korišćeni u GST „pull-down” eksperimentima, The object of this study was the new human muscle protein Ankrd2 found preferentially in slow muscle fibers that is possibly a transcription factor up-regulated in hypertrophy. It is a protein of 37 kDa, which has a signal for nuclear targeting, four ankyrin repeat motifs and shows significant similarity to a cytokine inducible nuclear protein C-193 from human endothelial cells. The rat and mouse orthologs of human C-193 called respectively CARP and MARP are mainly expressed in heart and probably involved in the control of cardiac hypertrophy. It is possible that Ankrd2 may play a similar role to CARP/MARP/C-193, but in skeletal muscle rather than heart. For intracellular localization and tissue distribution of Ankrd2 three mouse polyclonal antibodies (raised against the full length, N-terminal and C-terminal regions of the protein) and one monoclonal antibody (raised against C-terminal region of Ankrd2) were made. Western blot analysis of protein extracts isolated from different human tissues showed that Ankrd2 is expressed mainly in human skeletal muscle and to a lesser extent in heart and kidney. During muscle cell differentiation there is an increase of Ankrd2 signal in both mouse (C2C12) and human (CHQ5B) muscle cells that can be detected by Western blot analysis. Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes with the scope of pinpointing the intracellular localization of Ankrd2 protein. In general, during differentiation both the number of fluorescing cells and the intensity of signal increase. Also, in human myoblasts but not in differentiated myotubes very specific nuclear fluorescence can be detected. The nuclear signal is in the form of speckles and co-localization experiments showed that Ankrd2 and PML co-localize in PML nuclear bodies. In order to study protein-protein interactions GST recombinant proteins F-Ankrd2 (5-333 aa), N-Ankrd2 (5-120 aa) and C-Ankrd2 (279-333 aa) were prepared and used for the GST „pull-d