higher animals possess distinct muscle classes that are specialised for certain tasks : the vertebrate heart pumps blood life - long without rest , smooth muscles ensheathing the gut propel food without voluntary control , and body muscles move in a precise , consciously controlled manner to enable body movements , body posturing and facial expressions . to optimally fulfill these different tasks
, each muscle class requires distinct contractile , metabolic and electrophysiological properties . the molecular basis for these functional distinctions is generated during development and results in a dramatically different morphology for each of the three muscle classes .
smooth muscles are mononucleated and can be activated by a variety of neuronal , hormonal , autocrine / paracrine signals or changes in load and length .
they are activated through electrical coupling after neuronal firing and show regular striations along their myofibrils .
each muscle fiber contains many , often hundreds , of nuclei and has a defined neuromuscular junction that triggers contractions .
each fiber houses many highly ordered myofibrils that are laterally aligned to form stereotypical cross - striations . in this review , we discuss recent progress on mechanisms of differential transcription and alternative splicing that instruct functional differences between muscle types .
we focus on mammalian skeletal muscle and drosophila body muscle as the best understood model systems .
mammalian skeletal muscle fibers are historically classified as slow ( type 1 , red muscle ) or fast ( type 2 , white muscle ) fibers .
they generally can produce higher forces than slow fibers , are glycolytic and fatigue rather quickly .
in contrast , slow fibers produce lower forces , primarily use oxidative metabolism and are more fatigue - resistant ( reviewed in ) .
each individual human skeletal muscle consists of many , often several hundred , muscle fibers with a characteristic fiber - type composition .
for example , the extensor digitorum longus ( edl ) muscle in the foot is mainly composed of fast fibers , whereas the soleus muscle in the lower leg contains mainly slow fibers . however , the individual fiber composition of each muscle will adapt to exercise regime , such that the soleus muscle of a sprint athlete will incorporate more fast fibers as compared to that of a marathon runner , which will be  slower  .
the different functional properties of skeletal muscle fiber types in mice arise during fetal muscle development and are further modified during postnatal life .
the general myogenic transcription factors myod , myf5 , mrf4 and myogenin are required for the correct development of most , if not all , skeletal muscles early in embryogenesis ( reviewed in ) . subdivision into distinct muscle fiber types arises during late fetal development in mice through initiation of the fetal myogenic program .
it was recently shown that the expression of nuclear factor one x ( nfix ) switches the embryonic to the fetal program by repressing embryonic and activating fetal myogenic genes such as muscle creatine kinase ( mck ) or -enolase .
this enables the next steps of fiber - type specification by the differential expression of additional transcription factors .
the best studied factors are six1 and six4 , which promote the fast fiber fate , together with their cofactor eya1 ( ) . their action is supported by the transcriptional repressor sox6 , which represses slow genes in fast fibers .
together , this complex interplay between general and specific transcription factors establishes the typical fiber - type distribution at the end of murine fetal muscle development .
postnatally , muscle fiber - type distribution is significantly reorganized , coinciding with substantial muscle growth after birth .
neuronal innervation , together with calcium - calcineurin signaling , is a key player at this stage .
increased calcineurin signaling promotes the slow fiber fate , potentially through the downstream cooperation of mef2d with the transcriptional coactivator pgc-1 , which induces the expression of slow fiber genes , such as myoglobin , or genes required for mitochondrial oxidative metabolism .
varying levels of neuronal activity , and thus calcineurin signaling , also promote the differential recruitment of nfat family members to the promoters of activity - dependent genes . an nfatc2/3/4 complex specifies transcription of fast fiber genes , while the nuclear import of nfatc1 driven by slow nerve activity redirects the complex to activate transcription of slow genes . as in embryogenesis , general muscle transcription factors cooperate with fiber type - specific transcription factors to achieve differential expression of fiber type - specific genes during adult muscle differentiation .
the best - studied examples of differentially expressed sarcomeric components in mammalian body muscle are the myosin heavy chain ( myhc ) isoforms .
different fiber types express different myhc isoforms from the various muscle myosin ii genes in mammals . during the embryonic to fetal myogenic switch
is lost and type 2a fast fibers express myhc-2a , while slow fibers express myhcbeta/ slow ( reviewed in ) . myhc expression is at least partially regulated by nfat family members downstream of neuronal activity , as myhc - slow is cooperatively controlled by all four nfat family members , while myhc-2a is controlled by nfatc2/3/4 . while further details of upstream regulation are unclear , the expression of myhc isoforms with different molecular properties , for example variable cross - bridge lengths with actin during contraction , underlies part of the functional differences between fiber types ( reviewed in ) .
additionally , specific myhc isoforms are combined with fiber type - specific isoforms of the troponin  tropomyosin complex to adjust the calcium sensitivity of different fiber types . as a consequence ,
slow fibers already start contracting at low cytoplasmic calcium concentrations ; whereas , fast fibers require higher calcium levels to initiate contraction and show a steeper tension increase upon further calcium influx due to a larger cooperativity in their calcium response .
however , the physiological differences between fast and slow fibers depend on more than just differences in myosin , troponin or tropomyosin isoforms . a recent proteomics analysis in mice compared
the slow soleus muscle with the fast edl muscle and identified 551 proteins that vary significantly between the two muscles .
most notably , these differentially expressed proteins were enriched for proteins involved in contraction , ion homeostasis , glycolysis and oxidation , emphasizing their roles in the different physiologies of these muscles . despite this significant progress , our mechanistic understanding of how a combination of transcription factors and signaling molecules assembles functionally distinct muscle types and regulates such a broad repertoire of cellular genes remains limited .
this is in part due to the high complexity and plasticity of the mammalian system : every muscle consists of many muscle fibers of different fiber types and is patterned by a complex interplay between autonomous and non - autonomous inputs on its transcriptional machinery at various stages during embryonic and adult development .
complementary to vertebrate studies , research in drosophila has added valuable insights into how different body muscle types are generated during development .
tubular muscle fibers in the head , legs , thorax and abdomen are responsible for most movements of the fly .
tubular muscles are very similar to vertebrate skeletal muscles , as they contain laterally aligned myofibrils that lead to a typical cross - striated pattern ( fig .
nerve stimulation results in calcium release , which triggers actomyosin contraction and thus leads to a synchronous stimulation - contraction pattern . in stark contrast ,
drosophila indirect flight muscles ( ifms ) are asynchronous muscles that require stretch - activation in addition to calcium stimulation to induce their contractions .
this stretch - activation mechanism allows very fast ( 200 hz ) ifm oscillations , coupled with high force production ( up to 80 w / kg muscle ) , enabling flight .
calcium does not cycle during contractions , but instead remains at an elevated level during the entire flight period due to continuous low frequency nerve stimulation .
ifms have high mitochondrial content for longlasting oxidative metabolism , little sarcoplasmic reticulum , and a fibrillar stretch - sensitive organization of their myofibrils ( fig .
how the striking morphological and physiological differences between tubular and fibrillar muscle arise during development can be mechanistically investigated with modern drosophila genetics .
the drosophila tool kit includes systematic , tissue - specific loss of function studies with genome - wide rnai approaches using recently established transgenic genome - wide rnai libraries .
the binary gal4-uas system allows tissue - specific gene knockdown at a given developmental time period , e.g. during pupal stages , thereby preventing pleiotropic phenotypes .
a systematic muscle - specific rnai screen identified more than 2000 genes with a putative role in muscle , about 300 of which are required for normal flight behavior and thus likely function in ifms .
detailed morphological analysis of the ifms upon knock - down of these 300 genes identified the conserved zinc - finger transcription factor spalt major ( salm ) as a muscle - type specific selector gene for the fibrillar muscle fate . in salm
knock - down animals , the fibrillar flight muscles are morphologically transformed to tubular , leg - like muscles ( fig .
salm is expressed specifically in fibrillar muscle during development and , if misexpressed , is sufficient to switch tubular muscle to the fibrillar fate ( fig
. 1 g ) , making it a bone - fide muscle - type selector gene .
importantly , this function of spalt is not restricted to drosophila , but is conserved over at least 280 million years of evolution in most flying insects .
interestingly , spalt - like ( sall ) proteins are conserved to mammals and some sall family members are expressed in the mammalian heart .
the heart is a very stiff muscle , whose contraction is also stretch - modulated , a phenomenon described as the frank  starling mechanism , which links cardiac ejection to cardiac filling with stronger ejection upon larger filling .
mutations in sall1 lead to townes - brocks syndrome , a multi - organ syndrome that includes heart abnormalities , suggesting an evolutionarily conserved role of sall family members in stretch - activated muscle .
how does salm execute this switch from tubular to fibrillar muscle development and to what extent does salm control the characteristic properties of stretch - activated ifms ?
salm is the founding member of the conserved family of spalt zinc - finger transcription factors , which play important roles during the development of many organ systems , such as the eyes , wings and trachea in insects . in mammals ,
spalt - like ( sall ) genes are similarly required for the development of various organs including the heart and for specification of the mouse inner cell mass - derived lineages .
sall proteins are localized to the nucleus in mammals and drosophila and have been reported to act as both transcriptional repressors   and activators , depending on the system studied . despite their crucial roles in organ development , specific dna binding motifs for most of the spalt family members , in particular for drosophila salm , remain elusive . in ifms , salm either directly or indirectly activates the expression of fibrillar muscle specific genes such as flightin ( fln ) or troponin c isoform 4 ( tpnc4 ) and at the same time represses tubular muscle - specific genes like troponin c41 ( tpnc41 ) or muscle protein 20 ( mp20 ) .
importantly , it has been shown that all of these proteins directly regulate the contractile properties of the ifms : tpnc4 is critical for stretch - activation of the myofibrils , whereas fln is required for proper assembly of the thick filaments in ifms contributing to their high stiffness , an essential mechanical property for stretch - activation .
in addition , salm up - regulates a wide range of mitochondrial genes specifically in ifms , which mainly rely on oxidative metabolism , thereby adapting them to the high energy demand of flight .
thus , salm not only switches myofiber fate by changing the transcriptional profile of core sarcomeric genes , it also determines most of the physiological differences between tubular and fibrillar muscle fibers .
it is poorly understood how salm achieves ifm - specific target gene expression , especially when considering that salm function is also essential to activate distinct sets of targets in eye , wing or trachea cells .
one possible mechanism is that salm cooperates with or modifies the activity of the essential muscle - specific transcription factor mef2 at particular mef2-dependent enhancers in ifms . since vertebrate mef2 family members are well - known to cooperate with bhlh factors of the myod family , as well as with hdacs and hats , to achieve different regulatory outputs in distinct muscle types , it is plausible that salm similarly cooperates with or modifies the activity of drosophila mef2 at particular mef2-dependent enhancers in ifms .
such cooperative interactions of mef2 to instruct spatio - temporally restricted expression of target genes have also been demonstrated with the fusion competent myoblast - specific transcription factor lame duck ( lmd ) , the cardiogenic transcription factor tinman and the general mesodermal factor twist .
mef2 is also required for ifm differentiation , being essential for proper formation and maturation of contractile filaments . however
, whether cooperative interaction of salm with mef2 at enhancers of fibrillar muscle genes occurs awaits further investigation .
salm may additionally act downstream or cooperatively with the homeodomain proteins homothorax ( hth ) and extradenticle ( exd ) . despite broad expression in adult tubular leg and abdominal muscle as well as fibrillar ifms , adult muscle - specific loss of hth or exd results in a specific transformation of ifms to tubular muscles , and mis - expression of hth or exd in jump muscle , another large tubular muscle in the thorax ,
notably , while ifms express high levels of salm , wild - type jump muscle expresses low levels of salm ; thus , it is attractive to hypothesize that the fibrillar fate results from transcriptional cooperativity of salm with hth and exd , which in wild type only occurs during development of the fibrillar ifms .
these examples suggest that the cooperation of general myogenic transcription factors with muscle - type specific transcriptional regulators constitutes a basic principle for muscle fiber - type specification conserved from drosophila to vertebrates .
interestingly , the fibrillar ifm fate is not only determined by a transcriptional switch , but also by a change in the splicing pattern between fibrillar and tubular muscle .
mf is a muscle - specific thick - filament associated protein conserved in insects that is putatively involved in the assembly of thick filaments .
muscle - specific rnai - mediated knock - down of mf is pupal lethal , supporting an important role in fiber assembly . in drosophila ,
the mf locus generates multiple gene isoforms , including a short isoform that is specifically expressed in ifms and longer isoforms that are expressed in tubular muscles .
mrna sequencing of ifms , salm knock - down ifms as well as tubular leg and jump muscles reveals that the major difference between long and short mf isoforms is a regulated splicing event joining exon 5 with 6 in fibrillar muscles or exon 5 with 7 in tubular muscles ( fig .
the exon 56 junction is preferentially used in fibrillar ifms , whereas this splice event rarely occurs in tubular muscles or salm knock - down ifms ( fig .
although two specific mf splicing events in larval muscles are regulated by muscleblind ( mbl ) , the splicing factor regulating the switch from exon 56 to 57 splicing in fibrillar versus tubular muscle remains to be identified .
in addition to mf , several other structural genes are reported to be alternatively spliced between tubular and fibrillar muscle , including drosophila troponin - t ( upheld , up ) , tropomyosin 1 ( tm1 ) , myosin alkali light chain ( mlc1 ) , myosin heavy chain ( mhc ) ( for review see ) and projectin ( bent , bt ) , a drosophila titin homolog . in salm
knock - down animals , splicing of all these genes is likewise switched to the tubular instead of the fibrillar pattern , consistent with the hypothesis of a functional transition in the alternative splicing landscape to that normally found in tubular instead of fibrillar muscle .
in general , inclusion or exclusion of particular coding sequences due to alternative splicing likely leads to the production of proteins with different regulatory or biophysical properties . while the functional significance of long versus short mf isoforms remains to be determined , aberrant splicing of drosophila troponin t ( tnt , up ) in ifms results in functional impairment ( flightlessness ) caused by defects in the myofibrillar apparatus , with diffuse z - lines and the formation of so - called
these morphological and functional defects are presumably due to changes in tnt activity by differential phosphorylation of c - terminally located alternative exons , which affects tnt function and sarcomere stability .
another target of extensive splicing regulation is the large drosophila titin homolog , projectin ( bt ) .
projectin is one of the largest drosophila proteins composed of 47 annotated exons , including multiple exons encoding a conserved proline - glutamic acid - valine - lysine ( pevk)-rich domain .
interestingly , the pevk domain from vertebrate titin was shown to be elastic in vitro and in vivo , likely due to entropic changes upon stretch , suggesting a spring - like function during sarcomere contraction cycles .
this implies that a difference in pevk domain length between fiber types may modulate the stiffness of vertebrate muscle .
indeed , vertebrate heart contains shorter titin isoforms with shorter pevk - rich domains resulting in an increased passive stiffness of the adult heart compared to skeletal muscle (; reviewed in )
. mice with a deletion of the short pevk domain of the heart - specific isoform display a hypertrophic cardiomyopathy most likely caused by the changed elastic properties of titin .
the drosophila titin - like protein projectin also displays elasticity and is proposed to function as a molecular spring . although titin 's fiber - type specific splice events might not be directly conserved between insects and vertebrates , ifms do possess a high passive resting stiffness that is required to facilitate stretch - activation with minimal sarcomeric displacement .
ifms normally contain the shortest observed pevk domain of only a few amino acids , and presumably a shift to one of the longer 363 or 464 amino acid domains found in tubular muscle would decrease passive resting stiffness and likely disrupt the stretch - activation mechanism powering flight .
a final example of alternative splicing directly affecting sarcomeric function is drosophila myosin heavy chain ( mhc ) , the motor protein that produces contractile force in muscle , which in contrast to vertebrates is encoded by the single mhc gene in drosophila .
alternative splicing generates the wide diversity of mhc protein isoforms expressed in drosophila muscle fibers .
ifms express a different complement of alternatively spliced exons than larval mhc isoforms , resulting in mhc proteins with distinct physiological properties .
in particular , the relay domains encoded by exon 9 variants result in variations in mgatpase activity and actin sliding velocity and affect myofibril assembly and stability , while variants in the converter domain encoded by exon 11 affect caatpase , mgatpase , and actin sliding velocity .
thus , alterations in mhc splicing in ifms would affect myofibril assembly and stability and also change the fine - tuning of the myosin atpase and actin filament motility .
taken together , these three examples strongly suggest that muscle fiber - type selector genes switch not only the transcriptional but also the splicing status of the muscle to ultimately control muscle fiber - type specific morphological and physiological properties .
regulation of splicing in muscle is not limited to insects and is also well documented in vertebrate muscle .
mef2 family members modulate the differentiation of many tissues including muscle , and mef2d produces a unique muscle - specific splice isoform , mef2d2 .
while the broadly expressed mef2d1 isoform inhibits expression of late muscle differentiation genes due to phosphorylation by pka followed by association with corepressors , the muscle - specific isoform mef2d2 escapes pka phosphorylation due to exon switching and thus can recruit the transactivator ash2l to activate transcription of late muscle targets .
vertebrates have three troponin t ( tnt ) genes encoding cardiac , slow and fast tnt isoforms .
the fast tnt gene undergoes extensive muscle fiber - type specific splicing to produce different isoforms that affect its ca sensitivity and thus regulate actomyosin interactions .
these examples strongly suggest that isoform switching is a basic principle in both vertebrate and drosophila muscle to fine - tune muscle fiber - type specific functional properties
. proper control of the splicing machinery is relevant for muscle function in mice and humans , as mis - regulation of splicing can lead to major muscle and heart diseases .
while strong evidence supports the involvement of splicing factors such as muscleblind - like   and rbfox   in muscular dystrophies , the relationship between rna - binding motif protein 20 ( rbm20 ) and titin in heart disease is perhaps the most provocative link to - date between alternative splicing and muscle disease . as discussed above , titin is the elastic component of the myofibril , maintaining the precise structural arrangement of thick and thin filaments and generating passive muscle stiffness .
alternative splicing of titin 's pevk region modifies the  spring  property , with shorter , stiffer versions typically found in the adult vertebrate heart .
mutations affecting titin splicing that reduce passive stiffness are associated with dilated cardiomyopathy ( dcm ) and account for nearly one third of all cases of familial dcm .
titin splicing is at least partially regulated by rna - binding motif protein 20 ( rbm20 ) , which promotes exon skipping by repressing particular splicing events , normally resulting in the shorter titin isoforms expressed in the heart .
mutation of the rbm20 gene results in mis - splicing , notably resulting in the expression of longer titin isoforms ( fig .
2d ) , which results in symptoms of dilated cardiomyopathy including ventricular enlargement , arrhythmia , extensive fibrosis and increased rate of sudden death in both rat and human . moreover , rbm20 regulates conserved splicing events in at least 30 additional genes , including the enigma protein zasp / cypher , that are involved in sarcomere organization and ion transport in the sarcoplasmic reticulum , several of which are also associated with dcm .
this example illustrates that correct alternative splicing is critical to muscle - type physiology and function .
muscle fiber - type specification is a complex process controlled by coordinated regulation of both transcription and alternative splicing .
research in model organisms such as flies provides an important complement to vertebrate studies in understanding the basic principles contributing to the development of different muscle - types . in the case of
the evolutionarily conserved fibrillar selector gene salm from drosophila , mechanisms of transcriptional regulation of not only sarcomeric components but also genes dictating the physiologic status of the muscle , in addition to pervasive regulation of alternative splicing , are likely relevant to the underpinnings of cardiomyopathy in humans .
while recent studies have started to provide insight into how functional and morphological differences are generated during development , many important questions await detailed clarification .
one important challenge is to generate a complete network of both transcriptional and splicing regulators expressed in different muscle types , as factor cooperativity and feed - forward or feed - back mechanisms are important in fine - tuning muscle fiber - type specific physiological properties .
the differences in physiological properties between fiber - types are functionally dictated by the biophysical properties of alternatively spliced forms of signaling and structural proteins .
thus , characterizing first which alternate protein isoforms are present in each fiber - type and second how inclusion or exclusion of particular protein domains affects muscle physiology will be essential to understand muscle fiber - type specific function .
the ultimate goal is to molecularly define how different fiber fates are specified during normal development and how these fiber - types instruct the construction of physiologically different molecular machines .
these insights may be applied for treating disease symptoms such as muscle wasting and cachexia , or to increase muscle fatigue resistance for occupational reasons .
 muscles coordinate body movements throughout the animal kingdom . 
 each skeletal muscle is built of large , multi - nucleated cells , called myofibers , which are classified into several functionally distinct types . 
 the typical fiber - type composition of each muscle arises during development , and in mammals is extensively adjusted in response to postnatal exercise . 
 understanding how functionally distinct muscle fiber - types arise is important for unraveling the molecular basis of diseases from cardiomyopathies to muscular dystrophies . in this review , 
 we focus on recent advances in drosophila and mammals in understanding how muscle fiber - type specification is controlled by the regulation of transcription and alternative splicing . 
 we illustrate the cooperation of general myogenic transcription factors with muscle fiber - type specific transcriptional regulators as a basic principle for fiber - type specification , which is conserved from flies to mammals . 
 we also examine how regulated alternative splicing of sarcomeric proteins in both flies and mammals can directly instruct the physiological and biophysical differences between fiber - types . thus , research in drosophila 
 can provide important mechanistic insight into muscle fiber specification , which is relevant to homologous processes in mammals and to the pathology of muscle diseases . 