myofibrils , the workhorses of skeletal muscle , consist of interdigitating thick and thin filaments , and their associated membrane systems .
muscle contraction and relaxation is mediated by the sliding of thick myosin filaments past thin actin filaments , under the strict regulation of ca release and reuptake via the sarcoplasmic reticulum ( sr ) .
in addition to housing the basic thick and thin filaments , the sarcomere also contains several accessory proteins that are involved in the assembly , maintenance , and regulation of contractile activity .
myosin binding protein - c ( mybp - c ) comprises a family of accessory proteins that contributes to the assembly and stabilization of thick filaments , and regulates the formation of cross - bridges between myosin and actin by interacting directly with both filamentous systems ( as reviewed in ) .  
mybp - c was originally identified from mammalian skeletal muscle as an impurity of isolated myosin . using sds - polyacrylamide gel electrophoresis ( sds - page ) , star and offer
were the first to separate a number of unidentified myosin - associated proteins that were consistently found in preparations of purified myosin .
mybp - c , originally termed c - protein for its location on sds - page as band c , was further characterized as a myosin binding protein of ~140  kda using a combination of biochemical methods , ranging from gel filtration , to ammonium sulfate fractionation and single molecule electron microscopy [ 46 ] .
the location of mybp - c at striped intervals within the c - zone of the a - band of skeletal muscle was first observed with x - ray diffraction and immunoelectron microscopy , further supporting its association with the thick myosin filaments .
subsequent studies revealed that mybp - c is arranged along the length of the a - band in 79 transverse stripes that are ~43  nm apart , with ~24 molecules of mybp - c associating with each myosin cross - bridge [ 811 ] .  
the family of mybp - c contains three isoforms : cardiac , slow skeletal , and fast skeletal , which are encoded by separate genes ; in humans , these map to chromosomes 11 , 12 , and 19 , respectively [ 12 , 13 ] .
the different isoforms have been cloned and sequenced from various species , including human , chicken , rabbit and mouse , allowing a thorough comparison of their molecular composition and primary sequence [ 1316 ] .
an ~65% identity is shared among the individual isoforms across species , while an ~50% homology is shared among the human cardiac , slow and fast forms .  
the core structure of mybp - c is composed of seven immunoglobulin ( ig ) domains and three fibronectin type iii ( fn - iii ) repeats , numbered from the nh2-terminus as c1c10 .
the c1 domain is flanked by two unique motifs , one enriched in proline and alanine residues , termed proline / alanine rich motif and a conserved linker , referred to as mybp - c motif .
notably , the cardiac isoform possesses three additional features , which are absent from the skeletal forms of the protein .
these include an ig domain at the extreme nh2-terminus of the molecule , termed c0 , a unique 9-residues long insertion within the mybp - c motif containing phosphorylation sites necessary for the protein 's regulatory role in contraction , and a 28-amino acids long loop in the middle of the c5 domain [ 12 , 16 ] .   in mammals ,
cardiac mybp - c is expressed early in development , along with titin and myosin [ 18 , 19 ] .
the skeletal isoforms of mybp - c , however , are detected later in development , after the expression of titin and myosin , with the expression of slow mybp - c preceding that of fast [ 18 , 20 ] . by contrast , in chicken skeletal muscles the slow and fast isoforms appear concurrently at the end of late embryogenesis . as
development proceeds , though , the amounts of the slow form diminish , while the expression of the fast form persists through adulthood .
the fast and slow skeletal isoforms have been shown to coexpress in the same muscle type and can even coexist in the same sarcomere ; expression of the cardiac isoform , however , is restricted to the developing and mature heart [ 15 , 18 , 2224 ] .
interestingly , a recent study demonstrated that the expression of mybp - c slow is not restricted to skeletal muscle but is also evidenced in the right atrium and interatrial septum of adult mammalian cardiac muscle .  
to date , much of our knowledge on the molecular properties and functional activities of mybp - c originates from the numerous studies that focus on the cardiac isoform .
the reader is referred to excellent reviews discussing the structure of cardiac mybp - c , its key roles in maintaining the normal structure of thick filaments and regulating cross - bridge cycling , and its involvement in the development of hypertrophic cardiomyopathy [ 3 , 26 , 27 ] . here
, we will focus on the slow form(s ) of mybp - c found in skeletal muscles .
we will review past and current literature , discuss its role in the organization and stabilization of thick filaments , and provide evidence that mybp - c slow comprises a subfamily of four alternatively spliced proteins that are expressed in variable amounts in slow and fast twitch skeletal muscles .
total rna was isolated with trizol reagent ( invitrogen , carlsbad , ca ) from p1 rat skeletal myotubes cultured for seven days and from adult rat extensor digitorum longus ( edl ) , flexor digitorum brevis ( fdb ) , tibialis anterior ( ta ) , gastrocnemius ( gastroc ) , quadriceps ( quad ) , and soleus muscles .
aliquots containing ~5  g of rna were reverse transcribed using the superscript first strand synthesis system for rt - pcr ( invitrogen , carlsbad , ca ) following the manufacturer 's instructions .
pcr amplification of mybp - c slow transcripts was performed with primer sets that flanked each of the three insertions ; for the nh2-terminal insert : forward-1 ( f1 ) : 5 ccagaacccactaagaaag 3 and reverse-1 ( r1 ) : 5 gatcctcgaggtgcactt caagatcaa 3 , for the insert within ig7 : forward-2 ( f2 ) : 5 gatcgaattcagccc tcctactctt 3 and reverse-2 ( r2 ) : 5 gatcctcgaggggctcgctggcacca 3 and for the cooh - terminal insert : forward-3 ( f3 ) : 5 cacccatgtttactcaaccct 3 and reverse-3 ( r-3 ) 5 gtgacaatatacattgaa 3. to amplify the cooh - terminal insert from edl and ta muscles , it was necessary to reamplify 0.5  l of the original pcr for an additional 30 cycles for a total of 80 cycles .
pcr products were analyzed by electrophoresis in 1% agarose gels and their authenticity was verified by sequence analysis .
homogenates of p1 myotubes cultured for seven days as well as of edl , fdb , ta , gastrocnemious , quadriceps and soleus muscles of adult sprague - dawley rats ( zivic - miller laboratories , zelienople , pa ) were prepared at rt with a brinkman polytron homogenizer at setting 3 ( vwr , west chester , pa ) in 10  mm napo4 , ph 7.2 , 2  mm edta , 10  mm nan3 , 120  mm nacl , 0.5% deoxycholate , 0.5% np-40 , supplemented with complete protease inhibitors ( roche , indianapolis , in ) .
each sample ( ~60  g ) was solubilized in 4xnupage reducing sample buffer ( invitrogen , carlsbad , ca ) at 90c for 5 minutes , fractionated by 412% sds - page bistris gel using mes running buffer ( invitrogen , carlsbad , ca ) , transferred to nitrocellulose and probed with an antibody that recognizes the slow forms of mybp - c ( 300  ng / ml , abnova ) or with an antibody that specifically recognizes the cooh - terminal insert     .
after incubation with the appropriate secondary antibodies , immunoreactive bands were visualized with a chemiluminescence detection kit ( tropix , bedford , ma ) .
immuno - electron microscopy was performed as previously described with minor modifications     [ 29 , 30 ] .
briefly , adult mouse fdb skeletal muscle was fixed both in situ , via whole animal perfusion - fixation and ex vivo with 2% paraformaldehyde in pbs .
samples were snap - frozen in a slush of liquid n2 , cryosectioned ( ~20  m thick ) , and incubated overnight at 4c with the antibody that specifically recognizes the cooh - terminal insert of mybp - c slow .
samples were incubated with secondary goat antirabbit igg adsorbed on 1  nm gold particles ( nanoprobes incorporated ; yaphank , ny ) and subsequently with fluorescein - conjugated antigoat igg ( jackson immunoresearch laboratories inc . , west grove , pa ) .
samples were enhanced with silver ( hiq silver kit , nanoprobes ) for 5 minutes , fixed overnight at 4c in 2% gluteraldehyde and 5  mg / ml tannic acid in 0.2  m cacodylate buffer and postfixed with 50  mm acetate buffer 1% osmium tetroxide .
they were further stained en bloc for 2 hours with 1% uranyl acetate in 65% ethanol , dehydrated , and embedded in araldite ( electron microscopy sciences , fort washington , pa ) .
ultrathin ( 6090  nm ) sections were prepared with an mt5000 ultramicrotome ( lkb instruments inc . ,
gaithersburg , md ) , mounted on grids , labeled with 1% uranyl acetate followed by reynolds lead citrate , and examined with a philips-201 electron microscope .
to date , four different mybp - c slow transcripts have been identified in human skeletal muscle referred to as variants 14 ( figure 1 ; accession numbers nm_002465 , nm_206819 , nm_206820 , and nm_206821 , respectively ) .
the four variants differ from one another at three regions , due to alternative splicing events that result in inclusion of exons 3 and 4 in the proline / alanine - rich motif , exon 23 in the ig7 domain and exon 31 at the extreme cooh - terminus ( figure 1(a ) ) ; these encode novel sequences of 25 ( figure 1(b ) ) , 18 ( figure 1(c ) ) , and 26 ( figure 1(d ) ) amino acids , respectively .
analysis of the primary sequence of the four mybp - c slow variants indicated that variants 1 and 2 contain the nh2-terminal insertion located in the proline / alanine rich motif , variant 3 carries the insertion within domain ig7 , while variant 1 also contains the unique cooh - terminal region ( figure 1(a ) ) .
notably , variant 3 is the prototypical human isoform of mybp - c slow that was characterized by furst and colleagues in 1992 .   to study the relative expression of the four mybp - c slow transcripts in different rat skeletal muscles , we used rt - pcr analysis to amplify the unique regions described above . to this end , we prepared cdnas from a panel of adult and developing rat skeletal muscles that contained distinct compositions of slow and fast twitch skeletal myofibers . these included extensor digitorum longus ( edl ; ~90  :  10 , fast  :  slow ;     [ 31 , 32 ] ) , flexor digitorum brevis ( fdb ; ~80  :  20 , fast  :  slow ; ) , tibialis anterior ( ta ; ~70  :  30 , fast  :  slow ; ) , gastrocnemius ( gastroc ; ~40  :  60 , fast  :  slow ; ) , quadriceps ( quad ; ~60  :  40 , fast  :  slow ; ) , soleus ( 20  :  80 , fast  :  slow , ) , and hindlimb skeletal myotubes of postnatal day 1 ( p1 ) rat pups ( figure 2 ) .
primer sets were designed to flank each of the three novel insertions ( figures 2(a)2(c ) , cartoons in the upper left corner ) .
amplification of two pcr products with distinct sizes within each reaction indicated the presence of a mixed population of transcripts that contained ( larger size product ) and lacked ( smaller size product ) the respective insertion . on the contrary , amplification of one pcr product indicated the presence of a homogeneous population of transcripts that either included or excluded the corresponding insertion , depending on its size .
accordingly , pcr products that carry the nh2-terminal , ig7 and cooh - terminal inserts would be ~600 , ~310 , and ~350 nucleotides long , respectively , whereas pcr products that lack them would be ~530 , ~260 , and ~290 nucleotides long , respectively .  
all skeletal muscles examined , independent of their fiber type composition , contained sufficient amounts of mybp - c slow transcripts to be amplified by conventional rt - pcr .
figure 2(a ) shows the results following amplification of the nh2-terminal insertion located within the proline / alanine rich motif .
all muscle samples express mybp - c slow transcripts that include the nh2-terminal insert , albeit to varying degrees , as seen by the presence of an ~600  nts amplicon ( figure 2(a ) , upper band ) .
this finding suggested that all muscles tested express variants 1 and/or 2 , with edl , ta , and soleus containing the highest amounts .
notably , all seven muscles also contain different amounts of transcripts that lack the nh2-terminal insert , as shown by the presence of an ~530  nts product , indicating that they also express variants 3 and/or 4 , with fdb showing the highest levels ( figure 2(a ) , lower band ) .
contrary to edl , ta , soleus , and fdb that appear to preferentially contain mybp - c slow variants that either include ( edl , ta and soleus ) or exclude ( fdb ) the nh2-terminal insert , gastroc , quad , and p1 myotubes show similar amounts of both amplification products , suggesting that transcripts possessing and lacking the nh2-terminal insert may exist in similar levels within these muscles ( figure 2(a ) lanes 4 - 5 and 7 ) .  
as before , our primers set was designed to amplify mybp - c slow variants that contained or skipped the ig7 insert . only soleus and p1 myotubes possess transcripts that include the unique insertion within ig7 , as indicated by the presence of an ~310  nts band ( figure 2(b ) , lanes 6 and 7 , upper band ) , corresponding to variant 3 .
as expected , all skeletal muscles tested contained transcripts that lack the ig7 insertion , as shown by the amplification of an ~260  nts product , representing variants 1 , 2 and/or 4 .
last , we generated the appropriate primers to amplify the region flanking the cooh - terminal insert of mybp - c slow . with the exception of quadriceps muscle , all other muscles tested , contained an amplification product of ~350 nucleotides , that includes the novel cooh - terminal insert detected only in variant 1 , with soleus and fdb showing the highest amounts ( figure 2(c ) , lanes 6 and 2 , upper band ) .
a second amplification product of ~290 nucleotides was also detected in all seven muscles , which lacks the unique cooh - terminal insertion , present in variant 1 , but includes the common cooh - terminal region shared by variants 2 , 3 , and 4 ( figure 2(c ) , lower band ) .  
, our results suggest that : ( i ) mrna encoding mybp - c slow is present in all skeletal muscles examined , regardless of fiber type composition or age , and ( ii ) complex alternative shuffling of the single mybp - c slow gene in the various muscles results in the differential expression of the four mybp - c slow isoforms .   to correlate the mrna expression of the transcripts that contain or lack the three novel insertions , as seen by the rt - pcr , to the expression of the proteins that they encode , we used western blot analysis .
homogenates from edl , fdb , ta , gastroc , quad , soleus , and p1 skeletal myotubes were separated on 412% gradient gel , which our laboratory has previously shown to provide optimal separation of the mybp - c slow isoforms ( see section 2 , and ) .
homogenates were probed with a commercial antibody that recognizes a region within domain c5 common to all mybp - c slow variants ( figure 3(a ) ) .
we were able to resolve at least 3 immunoreactive bands based on their distinct electrophoretic mobilities .
the top band may represent variant 1 ( ~132  kda , figure 3(a ) , top panel , marked with a blue dot ) , the middle band may correspond to variants 2 and/or 3 ( ~129 and ~128  kda , respectively , figure 3(a ) , top panel , denoted with a red dot ) , and the bottom band may represent variant 4 ( ~126  kda , figure 3(a ) , top panel , marked with a green dot ) . the cartoon shown in the bottom panel of figure 3(a ) illustrates a representative image of several western blots , analyzed at varying exposure times .
the dotted lines indicate immunoreactive bands that only become evident after periods of long exposure .  
consistent with the rt - pcr data , our immunoblots also demonstrated the presence of at least one form of mybp - c slow in each skeletal muscle tested .
edl and gastrocnemious possess three immunoreactive bands with the most prominent being the middle one ( figure 3(a ) , lanes 1 and 4 ) .
fdb also contains three distinct bands , however , the top and middle bands appear to be of similar intensities and occasionally appear as a broad , single band ( figure 3(a ) , lane 2 ) . similar to fdb
, ta also shows two closely migrating bands , but lacks the bottom one ( figure 3(a ) lane 3 ) .
contrary to the rest of the muscles analyzed , quadriceps appears to contain only the middle band , although , the bottom one becomes evident after long exposure times ( figure 3(a ) lane 5 ) .
soleus expresses high amounts of the upper and lower bands , but moderate to low amounts of the middle one ( figure 3(a ) lane 6 ) .
finally , developing p1 skeletal myotubes show only one immunoreactive band with the same mobility as the top one , however , following longer exposure , the middle immunoreactive band is also apparent ( figure 3(a ) lane 7 ) .  
we also probed the same muscle homogenates with an antibody that is specific for the cooh - terminal insert and therefore only recognizes mybp - c slow variant 1 ( figure 3(b ) ) .
with the exception of quadriceps , all of the other muscles were immunopositive for variant 1 .
this finding is in agreement with our immunoblot data shown in panel a , and the rt - pcr analysis shown in figure 2(c ) , which revealed that quadriceps was the sole muscle to lack the top immunoreactive band ( i.e. , variant 1 ) and the cooh - terminal insert , respectively .
notably , mybp - c slow variant 1 appears to be expressed more abundantly in soleus muscle and least prominently in developing myotubes , while the remaining muscles tested contained intermediate amounts .
the slight differences in the sizes of the bands detected in the different muscles are likely due to posttranslation modifications , as it has previously been shown that mybp - c slow is capable of phosphorylation [ 37 , 38 ] .   taken together , our rt - pcr and immunoblotting data ( summarized in table 2 ) clearly indicate that all skeletal muscles tested , apart from quadriceps , express variant 1 , albeit to different extents , with soleus and fdb containing the highest amounts .
variants 2 and 3 have complementary expression profiles , with variant 2 being preferentially expressed in muscles that have a higher composition of fast twitch fibers , such as edl , fdb , ta , gastrocnemious and quadriceps , and variant 3 being selectively present in slow twitch muscles , like soleus , and developing myotubes .
finally , variant 4 is detected in all muscles examined , with the exception of ta and developing myotubes .
it therefore appears that different forms of mybp - c slow are present within the same muscle , independently of its fiber type composition , developmental stage , or age .
early studies have confirmed the direct interaction between mybp - c and myosin and identified the sites of binding on both proteins .
the light meromyosin ( lmm ) region of the myosin rod , that forms the backbone of the thick filament , binds the cooh - terminal c10 region of all three mybp - c isoforms [ 3941 ] . positively charged residues on the surface of the c10 domain mediate binding to lmm , however , the interaction is significantly strengthened by the presence of the c8 and c9 regions .
another interaction between the mybp - c motif , located at the nh2-terminus of mybp - c and subfragment 2 ( s2 ) of myosin has been also identified , albeit of weaker affinity .
in addition to binding myosin , mybp - c associates with actin , in a ca dependent manner , through its nh2-terminal proline / alanine rich motif [ 4446 ] .
the ability of mybp - c to directly interact with both myosin and actin facilitates its role as a regulator of cross - bridge formation during contraction .
interestingly , at low ionic strength mybp - c inhibits actomyosin atpase activity , while at higher ionic strength it acts as a mild activator [ 47 , 48 ] .  
in addition to myosin , mybp - c also associates with titin at the a - band [ 14 , 4951 ] .
the cooh - terminal c8-c10 domains of mybp - c directly bind to the 11-domain super - repeat [ ig-(fn - iii)2-ig-(fn - iii)3-ig-(fn - iii)3 ] present in the c - zone portion of titin , and specifically the first ig domain , although flanking motifs further strengthen the interaction [ 52 , 53 ] .
notably , the arrangement of mybp - c in 11 transverse stripes at regular intervals of ~43  nm corresponds to that of the 11-domain super - repeat of titin , to which mybp - c binds .
consequently , it has been suggested that binding to titin 's super - repeats specifies the subsarcomeric distribution of mybp - c in the c - zone of the a - band .  
recent studies have proposed that mybp - c forms a trimeric  collar  around each thick filament in which domains c5c7 of one molecule overlap with domains c8c10 of the neighboring molecule [ 54 , 55 ] . in vitro binding studies using
the respective peptides postulated that this arrangement might apply to the cardiac and fast skeletal isoforms , but not the slow isoform .
interestingly , for these studies flashman and coworkers generated a recombinant protein that contained domains c8c10 , present in all four slow variants , followed by the novel cooh - terminal insertion , present only in variant 1 .
it is , therefore , possible that the presence of the cooh - terminal 26-amino acids may inhibit binding of the c8c10 domains of variant 1 to the c5c7 domains of the neighboring molecule of mybp - slow .
indeed , this is consistent with our recent studies indicating that variant 1 is preferentially localized at the periphery of the m - band .
conversely , an interaction between the respective motifs present in variants 24 is likely , as these contain a short cooh - terminus following domain c10 that , similar to the cardiac and fast isoforms , consists of four amino acids .   through its interactions with myosin and titin
, mybp - c contributes to the stabilization and maintenance of the sarcomeric a - band . in vitro studies
indicate that myosin filaments are capable of forming in the absence of mybp - c , however , its addition results in increased filament length , and improved structure and uniformity across the filament [ 40 , 56 , 57 ] . additionally , in vivo deletion of the myosin and titin binding sites on mybp - c results in disorganized a - bands [ 58 , 59 ] .
consistent with this , the c8c10 domains that harbor the binding sites for myosin and titin are deleted in patients suffering from familial hypertrophic cardiomyopathy [ 3 , 60 , 61 ] .
interestingly , though , normal a - bands are formed in animals deficient of cardiac mybp - c [ 62 , 63 ] , suggesting the presence of a compensatory mechanism that maintains the myosin and titin filaments within the a - band of the sarcomere .
taken together , these studies suggest that mybp - c , through its direct interaction with myosin and titin , and possibly its self - association , may stabilize the sarcomeric a - band .  
in addition to maintaining the structure of thick filaments , mybp - c may also play key roles in regulating contractile function by modulating the rate of cross - bridges formation .
consistent with this , in cardiomyocytes lacking mybp - c , actomyosin filaments exhibited increased power output during contraction and faster rates of force development at half maximal ca activation .
reintroduction of the nh2-terminal region of cardiac mybp - c , containing the myosin s2 binding site ( i.e. , domains c1 and c2 ) , enhanced ca sensitivity and restored the contractile properties of the null cardiocytes to normal levels , suggesting that cardiac mybp - c contributes to the regulation of myofilament tension and their cycling rates .  
contrary to the cardiac isoform that has been directly implicated in the regulation of the contractile properties of cardiomyocytes ( reviewed in ) , studies focusing on the function of the skeletal forms of mybp - c are limited . however , early studies have suggested that the role of mybp - c in skeletal muscle likely parallels that of the cardiac isoform .
consistent with this , the amounts of mybp - c slow were recently found significantly increased in edl muscles from a mouse model in which the kinase domain of titin was deleted .
deficient edl muscles exhibited reduced myofilament ca sensitivity and altered contractile properties , which were restored to normal levels upon extraction of mybp - c slow .
contrary to the slow form of mybp - c , the fast form was significantly downregulated in the same muscles , while the expression levels of other proteins of the a- and m - bands remained unaffected .
it is , therefore , likely that the kinase domain of titin affects the expression of genes involved in the regulation of myofilament ca sensitivity and force production .
experimental evidence has therefore started to emerge suggesting a key role for the skeletal forms of mybp - c in modulating contractility , too .
the presence of mybp - c slow at the c - zones of the a - band has been studied extensively during the last three decades [ 3 , 26 , 68 ] .
recent studies from our laboratory , however , have provided evidence that at least one form of mybp - c slow , specifically variant 1 , has a unique topography in the muscle cell .
detailed immunofluorescence studies combined with confocal microscopy demonstrated that mybp - c slow variant 1 is selectively localized at the periphery of the m - band in adult rat soleus muscle . the unique localization of mybp - c slow variant 1
immunolabeling of ultrathin cryosections prepared from adult mouse fdb muscle with antibodies to the unique cooh - terminus of variant 1 also demonstrated that it preferentially concentrates at the edges of the sarcomeric m - band ( figures 4(a)4(b ) , arrowheads ) .
thus , it appears that the different mybp - c slow isoforms have distinct distributions in skeletal myofibers , with variants 24 localizing at the a - band and variant 1 at the m - band .   at the m - band , mybp - c slow variant
1 interacts with obscurin and four and a half lim protein 1 ( fhl1 ) [ 28 , 69 ] .
the second ig domain of obscurin and the last ig domain of mybp - c slow variant 1 are both necessary and sufficient to support their interaction , however , binding is enhanced significantly in the presence of the novel 26 amino acids at the cooh - terminus of variant 1 .
overexpression of the second ig domain of obscurin in primary cultures of skeletal myotubes inhibited the integration of mybp - c slow variant 1 at the m - band and severely disrupted the formation of m- and a - bands .
these findings suggested that variant 1 might contribute to the assembly and integrity of these structures via its interaction with obscurin and possibly other proteins .
similarly , mcgrath et al . also demonstrated that overexpression or downregulation of fhl1 , which is localized at the a - i junction and the m - band , in adult mouse soleus muscle resulted in impaired thick filament assembly , which was accompanied by reduced sarcomeric incorporation of different forms of mybp - c slow , including variant 1 .
mutations within the cardiac isoform of mybp - c cause familial hypertrophic cardiomyopathy ( reviewed in [ 70 , 71 ] ) . to date
, there are no muscle diseases causally associated with mutations in the transcripts of the skeletal forms of mybp - c .
however , changes in the expression levels of the skeletal isoforms have been reported in hypertrophic and dystrophic skeletal muscles .
the expression levels of mybp - c slow were significantly increased in rat plantaris muscle induced to hypertrophy by surgical ablation of the neighboring soleus and gastrocnemius muscles , whereas the amounts of mybp - c fast were dramatically decreased .
likewise , the protein levels of mybp - c slow were increased in both chicken and mouse dystrophic skeletal muscles [ 20 , 24 ] .
further research is required in order to understand the molecular mechanisms that lead to the differential regulation of the skeletal forms of mybp - c in these models , and their potentially unique roles in regulating the formation and activity of contractile structures .
mybp - c slow comprises a subfamily of at least four isoforms that result from complex alternative splicing of the single mybp - c slow gene .
the presence of the four mybp - c slow variants is not restricted to slow - twitch muscles , as select isoforms are abundantly expressed in fast - twitch muscles , too , where they may coexist with mybp - c fast .
more importantly , the four isoforms of mybp - c slow may be coexpressed in the same muscle , fiber or sarcomere , where they may exhibit unique topographies concentrating either at a - bands ( variants 2 , 3 , and 4 ) or m - bands ( variant 1 ) .
previous and current findings strongly favor a structural role for mybp - c slow at the sarcomeric a- and m - bands , and point to a regulatory role on muscle contraction .
consequently , the detailed characterization of the biochemical and biophysical properties of the four mybp - c slow variants , and the development of the appropriate molecular tools and animal models are imperative in order to study the cell biology and functional properties of this intricate subfamily of proteins .
 myosin binding protein c ( mybp - c ) consists of a family of thick filament associated proteins . 
 three isoforms of mybp - c exist in striated muscles : cardiac , slow skeletal , and fast skeletal . to date , most studies have focused on the cardiac form , due to its direct involvement in the development of hypertrophic cardiomyopathy . here 
 we focus on the slow skeletal form , discuss past and current literature , and present evidence to support that : ( i ) mybp - c slow comprises a subfamily of four proteins , resulting from complex alternative shuffling of the single mybp - c slow gene , ( ii ) the four mybp - c slow isoforms are expressed in variable amounts in different skeletal muscles , ( iii ) at least one mybp - c slow isoform is preferentially found at the periphery of m - bands and ( iv ) the mybp - c slow subfamily may play important roles in the assembly and stabilization of sarcomeric m- and a - bands and regulate the contractile properties of the actomyosin filaments . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Summary and Future Perspectives