Analysis of Titin in Red and White Muscles: Crucial Role...

Research ArticleAnalysis of Titin in Red and White Muscles Crucial Role onMuscle Contractions Using a Fish Model

Ming-PingWu12 Nen-Chung Chang13 Chi-Li Chung4 Wan-Chun Chiu 5

Cheng-Chen Hsu6 Hui-Min Chen6 Joen-Rong Sheu 1 Thanasekaran Jayakumar1

Duen-Suey Chou 1 and Tsorng-Harn Fong 6

1Department of Pharmacology School of Medicine College of Medicine Taipei Medical University No 250 Wuxing StreetTaipei 11031 Taiwan

2Department of Obstetrics and Gynecology Chi-Mei Medical Center No 901 Zhonghua Road Yongkang DistrictTainan 71004 Taiwan

3Division of Cardiology Department of Internal Medicine Taipei Medical University Hospital No 252 Wuxing StreetTaipei 11031 Taiwan

4Division of Pulmonary Medicine Department of Internal Medical Taipei Medical University Hospital No 252 Wuxing StreetTaipei 11031 Taiwan

5School of Nutrition and Health Sciences College of Nutrition Taipei Medical University No 250Wuxing Street Taipei 11031 Taiwan6Department of Anatomy and Cell Biology School of Medicine College of Medicine Taipei Medical University No 250Wuxing Street Taipei 11031 Taiwan

Correspondence should be addressed to Duen-Suey Chou firdtmuedutw and Tsorng-Harn Fong thfongtmuedutw

Received 20 July 2018 Revised 18 October 2018 Accepted 31 October 2018 Published 18 November 2018

Academic Editor Shoichiro Ono

Copyright copy 2018 Ming-PingWu et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Several studies have compared molecular components between red and white skeletal muscles in mammals However mammalianskeletal muscles are composed of mixed types of muscle fibers In the current study we analyzed and compared the distributions oftitin lipid phosphate ions and fatty acid levels in red and whitemuscles using a fishmodel (Tilapia) which is rich in red and whitemuscles and these are well separated Oil-red O staining showed that red muscle hadmore-abundant lipids than did white muscleA time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmiticacid and oleic acid but whitemuscle containedmore phosphate ions Moreover elastica-vanGieson (EVG) andMito-Tracker greenFM staining showed that collagen and elastic fibers were highly respectively distributed in connective tissues and mitochondria inredmuscleAn electronmicrographic analysis indicated that redmuscle had a relatively higher number ofmitochondria and longersarcomere lengths and Z-line widths while myofibril diameters were thicker in white muscle Myofibrillar proteins separated bySDS-PAGE showed that the major giant protein titin was highly expressed in whitemuscle than in redmuscle Furthermore ratiosof titin to myosin heavy chain (MHC) (titinMHC) were about 13 times higher in white muscle than red muscle We postulatedthat white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres whereasred muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres

1 Introduction

Skeletal muscles are highly preserved tissues in animals arepresent all over the body and form a combined networkwith a notable skeletal system via tendons to resist gravityand facilitate mobility There are two major categories ofmuscle fibers in vertebrates red fibers and white fibers [1]Morphological studies showed that red fibers (slow twitch

oxidative fibers) are small in diameter and have a red colordue to their greater content of myoglobin and rich supply ofcapillaries They have numerous large mitochondria beneaththe sarcolemma and between the myofibrils Lipid dropletsare common in the sarcoplasm of these fibers In contrastwhite fibers (fast-twitch fibers) are larger in diameter Theirmitochondria and lipid droplets are smaller and less numer-ous than those of red fibers [2] The properties of red muscle

HindawiBioMed Research InternationalVolume 2018 Article ID 5816875 11 pageshttpsdoiorg10115520185816875

2 BioMed Research International

fibers make them very effective in postural maintenancewhile white muscle fibers are suited for bursts of intensemuscle activity [3]

In addition to morphological observations physiologistshave reported that red muscle fibers contract more slowlyand are more resistant to fatigue than are white muscle fibersdue to their ability to oxidatively regenerate ATP In contrastATP generation in white muscle fibers depends on anaerobicglycolysis [4] It is accepted that hydrolysis of ATP by ATPaseis directly involved as the energy supply in the process ofmuscle contraction Biochemical studies revealed that theATPase activity of white muscle fibers is 3 times higher thanthat of red muscle fibers [5] However in a resting statewhether ATP is stored for use in muscle fibers and whetherthe stored ATP content in red muscle fibers is more or lessthan that in white muscle fibers are still unclear

Titin with a molecular mass of 3sim4 MDa is a muscle-specific elastic protein that spans from the Z-line to the M-line in the half-sarcomere it is the largest protein knownto date [6 7] The passive tension elasticity and stiffness ofmuscles are correlated with their titin content [8] Previousexperiments on single myofibrils prepared from the rabbitpsoas muscle revealed another powerful mechanism of activeforce production [9] Recent studies showed that regulation ofskeletal muscle force is based on a three-filament model thatincludes titin rather than a two-filament model consistingonly of actin and myosin filaments [10 11] Titin is the thirdmost abundant protein in muscles after myosin and actinTitin contents are about 10 of total myofibrillar proteins inchicken breast muscle [6] and 16 in rabbit skeletal muscle[12] However titin contents between red and white muscleshave not been investigated in a single species

Biochemical analyses of human muscle usually use biop-sies to obtain muscle samples [13]The optimal sample size iscritical for the availability of tissue for processing A biopsycannot produce a large amount of muscle sample and mayincrease the risk of infection On the other hand musclesof mammals (mice rats rabbits and humans) are mostly ofthe mixed type that is muscles contain both red and whitemuscle fibers [14 15] Relative proportions of the differentfiber types vary among species and in humans they showsignificant variability among individuals [1] Although wewanted to quantify and comparemyofibrillar protein contentsbetween red and white muscles mammalian muscles whichcontain mixed muscle fibers cannot be precisely analyzed

Fish are a good animal model for muscle studies becauseaquatic species have a proportionally larger muscle mass Inaddition red and white muscle types in fish are distributed indifferent anatomical locationsThe lateral triangular zone anda superficial thin layer of the trunk is a red color containingred muscle fibers but large portions of the deep layer ofthe dorsal and ventral zones of the trunk are white andcontain abundant white muscle fibers [16] Moreover whitemuscle fibers are associated with fast swimming behaviors infish such as during predation and escape while red musclefibers are correlated with slow movements such as duringmigration and foraging [17 18]

Zebrafish are commonly used in genetic embryonicdevelopment and muscle research [19] Red and white

muscles are also distributed in different region but zebrafishare too small to separate red muscles fromwhite muscles andobtain a sufficient amount of muscle tissues for biochemicalanalyses Therefore we used Tilapia sp a locally commonfish in Taiwan to analyze morphological and myofibril-lar proteins between red and white muscles in a singlespecies

The purpose of this study was to investigate mor-phological differences by histochemistry time-of-flight sec-ondary ion mass spectrometry (TOF-SIMS) and electronmicroscopy and quantify myofibrillar proteins especiallytitin contents between red and white muscles in fish bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)

2 Materials and Methods

21 Experimental Animals In total 10 apparently healthyadult male fish (Tilapia) (Figure 1(a)) with an averagebody weight of 500 plusmn 5 g were purchased from a localtraditional market in Taipei Taiwan and were transportedto the laboratory live in water Fish were killed by cervicaldislocation and then immediately dissected out Red andwhite muscles were mainly located on the lateral and dorsalsides of the trunk respectively (Figure 1(b)) The lateralpart of the trunk containing a red and white muscle block(about a 1-cm2 area) was excised and frozen with liquidnitrogen for cryostat sectioning All experimental protocolswere approved by the Animal Ethics Committee of TaipeiMedical University (license no LAC-2017-0224)

22 Oil-Red O Staining Oil-red O powder (Sigma-AldrichSt LouisMOUSA)was dissolved in 05 2-propanol (KantoChemical Tokyo Japan) The stock was then diluted to a03 Oil-red O solution with distilled H

2O and filtered

through a 022-120583m filter Frozen sections (20 120583m thick) offish muscle sample were fixed with 2 paraformaldehydeand 2 glutaraldehyde (Sigma-Aldrich) in 01 M phosphatebuffer (pH 74) for 10min at room temperature After fixationsections were washed with phosphate buffer three timesand stained with 03 Oil-red O solution for 10 min atroom temperature Finally stained sections were washedwith phosphate buffer three times mounted with aqueousmounting medium and sealed with nail polish Sections wereexamined with an Olympus BH-2 light microscope (TokyoJapan)

23 TOF-SIMS Analysis The TOF-SIMS analysis was car-ried out on a PHI TRIFT IV instrument (ULVAC-PHIKanagawa Japan) This instrument was equipped with a Biliquid metal ion gun Cryostat sections (20 120583m thick) offish muscle tissues were used for a replicate analysis in thisstudy The Bi

3

+ primary ion beam was operated at 30 keVSurface spectra were taken from an area of 500 times 500 120583min order to get an overview of the sample structure andidentify ion species present at the respective surfaces Fourrandom areas were selected for scanning in each sectionfor which four spectra were separately acquired from each

BioMed Research International 3

5 cm

(a)

2 cm

R

W

W

(b)

W

R

(c)

W

R

(d)

Figure 1 Red muscle (R) contains more lipids than white muscle (W) (a) The dotted line shows a cross-section near the cloaca of Tilapia(b) Cross-section red muscle is located on the lateral sides of the trunk (arrows) while white muscle is located on the back and ventral partsof the fish (c) Oil-red O staining of a frozen section red muscle contains more lipids than does white muscle (d) More intracellular lipiddroplets were seen in red muscle

sample Subsequently negative secondary ions flying througha reflectron mass spectrometer were detected with a micro-channel plate assembly operating at 10 keV after accelerationThe high mass resolution and high mass accuracy allowedassignation of sum formulas to peaks even in the highmass range The spectra obtained in the bunched modewere only used to identify and select peaks for imagingEach image was normalized to the intensity of the brightestpixel Data were collected from several image fields on eachsection

24 Elastica-Van Gieson (EVG) Staining We used the EVGstaining method to examine elastic and collagen fibers inconnective tissues Frozen sections (20 120583m thick) of fishmuscle samples were fixed with 4 paraformaldehyde in 01M phosphate buffer (pH 74) for 10 min at room temperatureSubsequently sections were rinsed three times with phos-phate buffer for 5min each and then sequentially stained withresorcin-fuchsin iron hematoxylin and van Gieson solution(purchased from Electron Microscopy Sciences Hatfield PAUSA) according to the manufacturerrsquos instructions After


that sections were dehydrated in an ethanol series Afterbeing treated with xylene sections were mounted withhistological mounting medium Sections were examined withan Olympus BH-2 light microscope

25 Mito-Tracker Green FM Staining Frozen sections (20120583m thick) of fish muscle samples were incubated with 200nM Mito-Tracker green FM (Molecular Probes EugeneOR USA) for 30 min at 37∘C After washing with 01 Mphosphate buffer (pH 74) sections were fixed with 4paraformaldehyde for 10min at room temperature Once sec-tions were rinsed with phosphate buffer they were mountedwith aqueous mounting medium sealed with nail polishand then examined with a Nikon fluorescence microscope(Tokyo Japan)

26 Electron Microscopy (EM) To identify ultrastructuralcharacteristics of red and white muscles we observed theultrastructural morphology using EMA piece of red or whitemuscle block was cut and fixed in a mixed aldehyde solutioncomposed of 2 paraformaldehyde and 2 glutaraldehydein 01 M phosphate buffer (pH 74) Subsequently sampleswere cut longitudinally with the knife-edge parallel to themuscle fiber and postfixed using 1 osmium tetroxide in 01M cacodylate buffer (pH 72) Samples were dehydrated inan ethanol series and embedded in Epon 812 using standardprocedures Ultrathin sections were cut and double-stainedwith uranyl acetate and lead citrate and then examined witha Hitachi H-600 EM (Tokyo Japan)

27 Gel Electrophoresis and Densitometric Analysis SDS-PAGE was performed to analyze the myofibrillar proteinsactin myosin heavy chain (MHC) and titin according toChen et al [20] using a 4sim12 step gradient mini-gel withan ambiguous interface A loading range of each sample waselectrophoresed on a ldquocalibration gelrdquo Gel electrophoresiswas performed at a constant 100 V for 2 h at room tempera-ture until the bromophenol blue had reached approximately 5mm above the bottom of the gel After electrophoresis the gelslabs were stainedwithCoomassie brilliant blue R-250 in 50methanol and 10 acetic acid and subsequently destained in a10 methanol and 10 acetic acid solution A densitometricanalysis was performed with a Photo-Print Digital ImagingSystem (IP-008-SD Vilber Lourmat Cedex France) withBio-1D analytic softwareThe optical density integrals (ODIs)of MHC and titin were measured for each loading volumeand the slope of the relationship between theODI and loadingvolume was determined by a linear regression analysis Alsothe ratio of titin to MHC (titinMHC) was calculated as theslope of the titin ODIloading volume divided by the slopeof the MHC ODIloading volume [21] Prestained ProteinLadder PREP1025 (Bioman Scientific Taipei Taiwan) wasused as a molecular weight reference

28 Statistical Analysis Values are expressed as the mean plusmnstandard error of the mean (SEM) Students paired t-testwas used to verify the significance of the differences between

red and white muscles At all times plt005 was consideredsignificant

3 Results

31 Characteristics of Red and White Muscles of Tilapia Ina cross-section of tilapia the red and white muscles wereeasily identified according to the muscle color and locationAs shown in Figure 1(b) white muscle was abundant in thedorsal and ventral parts of the trunk while red muscle wastriangular and located on the lateral side of the trunk Oil-red O staining showed that red muscle contained more lipiddroplets than did white muscle (Figures 1(c) and 1(d))

32 TOF-SIMS Analysis of Red andWhite Muscles Results ofthe TOF-SIMS spectral analysis are shown in Figure 2(a) andthe respective identified peaks of phosphate ions (PO

3

minus mz7996) palmitic acid (180 mz 25524) and oleic acid (181mz 28126) are shown in Figure 2(b) TOF-SIMS also imagedthe distribution of inorganic ions and fatty acids in red andwhitemuscles of tilapiaThis analysis produced an interestingimage in which phosphate ions (PO

3

minus mz 79) were foundto be more enriched in white muscle (Figure 3(a)) than inred muscle In contrast palmitic acid (180 mz 255) andoleic acid (181 mz 281) showed higher levels in red musclecompared to white muscle (Figures 3(b) and 3(c)) Moreoverthe distribution pattern of palmitic acid was similar to that ofoleic acid in red muscle Figure 3(d) shows areas of red andwhite muscles used as standards for the TOF-SIMS analysis

33 Elastic and Collagen Fibers in Connective Tissues of Redand White Muscles We used the EVG staining methodto examine elastic fibers and collagen fibers in connectivetissues Sections showed a high level of connective tissuesbetween red muscle fibers (Figure 4(a)) The size of whitemuscle fibers was obviously larger than red muscle fibers(Figure 4(b)) The EVG staining image clearly indicated thatred muscle contained more connective tissue fibers such ascollagen and elastic fibers than did white muscle (Figures4(a) and 4(b))

34 Mitochondrion Localization of Red and White MuscleFibers Localization of mitochondria in red and white mus-cles was determined using Mito-Tracker green FM stainingIntracellular mitochondria were stained green whereas con-nective tissues between muscle fibers were black (Figures4(c)ndash4(f)) Results showed that abundantmitochondria accu-mulated beneath the sarcolemma of both red and white mus-cle fibers However more mitochondria between myofibrilswere found in red muscle fibers than in white muscle fibers(arrows in Figures 4(e) and 4(f))

35 EM Ultrastructure of Red and White Muscle Fibers Redand white muscle fibers were distinguishable in longitudi-nal EM sections (Figure 5) Red muscle fibers containednumerous large mitochondria and lipid droplets beneath thesarcolemma and between myofibrils (Figure 5(a)) however


140 200180160 220 280260240

30E+625E+620E+615E+610E+650E+5

0

137146

153 163169

281183 199

255

237227

Tota

l Cou

nts

lowast

lowast

0

35E+730E+725E+720E+715E+710E+750E+6

0

1317

26 4263 97

1

71

79To

tal C

ount

s

20 40 60 80 100 120

lowast

(a)

Identified peaks in TOF-SIMS spectrum

Ion mode Ion Lipid molecules Measured mz

Negative PO3-

7896C16H31O2

- Palmitic C 160 25524C18H33O2

-Oleic C 181 28126

(b)

Figure 2 Negative ion TOF-SIMS mass spectrum (a) Partial mass spectrum obtained from a region of interest containing red and whitemuscles in a fish muscle section in the mz range of 0sim300 (b) Values ofmz of identified peak areas are labeled in the TOF-SIMS spectrum

white fibers rarely had mitochondria or lipid droplets (Fig-ure 5(b)) The sarcomere length between two Z-lines waslonger in red muscle fibers (1411 plusmn 0026 120583m) than whitemuscle fibers (1178 plusmn 0081 120583m) (Figures 5(c)ndash5(e)) Thewidth of the Z-line of red muscle fibers (709 plusmn 106 nm)was nearly double that of white muscle fibers (350 plusmn 70 nm)(Figures 5(c) 5(d) and 5(f)) The average diameter of whitemuscle myofibrils was relatively larger (0968 plusmn 0085 120583m)than that of redmusclemyofibrils (0776plusmn 0135120583m) (Figures5(c) 5(d) and 5(g))

36 Myofibrillar Proteins in Red and White Muscles SDS-PAGE was performed to analyze myofibrillar proteins inred and white muscles using 4 sim12 step gradient mini-gels Titin MHC and actin were all visible in Coomassiebrilliant blue-stained step gradient mini-gels (Figure 6(a))Relative proportions of myofibrillar proteins were calculatedby quantitative densitometry A linear regression analysisbetween the ODI and loading volume showed that slopes ofthe ODIloading volume of titin were 21441 plusmn 475 and 40903plusmn 3978 in red and white muscles respectively Thus the ratioof titin in red to white muscle (titin RW) was about 546plusmn74whichwas significantly lower than those ofMHC (MHCRW was 693plusmn 50) and actin (actin RW was 740 plusmn76) (Figure 6(b)) Ratios of titin toMHC (titinMHC) were

206 plusmn 11 and 273 plusmn 05 in red and white musclesrespectively (Figure 6(c)) These results show that the titinprotein was more abundant in white muscle than red muscle

4 Discussion

In this study we used the fish (Tilapia) to analyze the ATPtitin and sarcomere ultrastructure by using novel TOF-SIMSand EM techniques in red and white muscles Results ofOil-red O staining showed that red muscle contained morelipid droplets than did white muscle The spectra and imagesof TOF-SIMS demonstrated that phosphate ions were richin white muscles but more fatty acids (palmitic acid andoleic acid) were found in red muscles In addition collagenand elastic fibers of connective tissue were more enrichedin red muscle than in white muscle Despite both musclescontainingmitochondria beneath the sarcolemma numerouslong and large mitochondria between the myofibrils wereonly seen in red muscle Moreover ultrastructural observa-tions determined that the sarcomere length and Z-line widthwere larger in red muscle but the myofibril diameter wasthicker in white muscle Finally white muscle had moremyofibrillar proteins (titin MHC and actin) than did redmuscleThe ratio of titin to myosin (titinMHC) was lower inredmuscle than in whitemuscle According to these findings


R

W

79 [13Unsaved] (256x256)

(a)

04

06

08

10

0

02

R

W

255 [13Unsaved] (256x256)

(b)

R

W

281 [13Unsaved] (256x256)

(c)

Standard

R

W

(d)

Figure 3 TOF-SIMS images of negative ion and fatty acid compositions of red (R) and white (W)muscles (a) TOF-SIMS ion image showingthe distribution of phosphate (PO3minus) mz 79 (b) TOF-SIMS ion image showing the distribution of palmitic acid mz 255 (c) TOF-SIMS ionimage showing the distribution of oleic acid mz 281 (d) Bright-field standard Bar 100 120583m Image size 500 times 500 120583m (256 times 256 pixels)

we proposed that white muscle has faster and more-powerfulcontractions than red muscle which may result from highlevels of titin and phosphate ions a source of ATPmoleculesbut less connective tissue and a shorter sarcomere length

Previous biochemical histochemical and EM studiesshowed up to a threefold higher lipid content in red musclefibers than in white muscle fibers obtained from a musclebiopsy sample of healthy subjects [22] Those data weresupported by data using Oil-red O and immunofluorescencemicroscopy [23] Characteristics of red muscles in fish arealso a good capillary supply and high levels of mitochondrialipid droplets and glycogen stores [24] Consistent withthose studies the present work also found that red musclecontains higher numbers of mitochondria and lipid dropletsIn addition we found that red muscle contains high amountsof palmitic acid (180 mz 255) and oleic acid (181 mz 281)compared to white muscle TOF-SIMS images showed thatthe distribution pattern of palmitic acid was similar to that ofoleic acid suggesting that lipid droplets in redmuscle contain

both fatty acids These fatty acids in red muscle might be thefuel for the oxidative regeneration of ATP like glycogen inwhite muscle is the source of anaerobic glycolysis for ATPgeneration [25] A previous EM study by Nag [5] showed thatlipid droplets in redmuscle fibers were closely associatedwithmitochondria Red muscle fibers are mainly used for posturemaintenance and sustained energy-efficient exercise Thesestudies suggest that red muscle fibers are resistant to fatiguebecause of their ability to oxidatively regenerate ATP

The level of phosphate ions an actual energy source ofATP was higher in white muscle than red muscle in a restingcondition Available source of ATP at rest can quickly provideenergy for contractile activity in white muscles for burstmovement [26 27] After depleting stored ATP its regen-eration is supported by creatine kinase adenylate kinaseand AMP-activated protein kinase (AMPK) [28] Musclecontractions during exercise were found to increase AMPKactivity and enhance the immediate availability of bothcarbohydrates and fats as fuel for mitochondrial oxidation


R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)

Figure 4 Elastica-van Gieson (EVG) and Mito-Tracker Green FM staining to respectively detect connective tissues and mitochondria (a)and (b) EVG staining labeled connective tissue between muscle fibers Collagen fibers are red and elastic fibers are purple There was moreconnective tissue (arrowheads) between muscle fibers in red muscle (R) than white muscle (W) In addition the size of red muscle fiberswas smaller than that of white muscle fibers (cndashf) Mito-Tracker Green FM staining to label intracellular mitochondria (c) and (e) show themitochondrial distribution images of red muscle (d) and (f) show the mitochondrial distribution images of white muscleThere was a highdensity of green fluorescence surrounding each muscle fiber in both muscles indicating that abundant mitochondria were located beneaththe sarcolemma In addition more green dots or trabecula (arrows) were found in red muscle fibers than in white muscle fibers ((e) and (f))indicating that more-abundant mitochondria were located between myofibrils in red muscle than in white muscle

and increased rates of ATP production [29 30] Anothersource of ATP generation is the glycolytic pathway whichis more effective in white muscle than in red muscle [31]Furthermore it is well known that ATP is regenerated duringthe tricarboxylic acid cycle in mitochondria and this processis more effective in red muscle than in white muscle [32]In contrast white muscle contained few mitochondria andlipid droplets suggesting that white muscle fatigues rapidly

Ultrastructural observations demonstrated that whitemusclehad shorter sarcomere lengths and wider myofibril diametersthan those of red muscle indicating that white muscle couldexhibit stronger and faster contractions than red muscle

On the other hand the generally accepted mechanismof active force production in a sarcomere is based on theactin and myosin filament sliding model the so-called cross-bridge theory [33] Briefly myosin heads attach to actin


R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)

Figure 5 Electronmicroscopic (EM) ultrastructural images of red (R) and white (W)muscle fibers (a) Severalmitochondria are located nearthe sarcolemma Lipid droplets can be seen between the myofibrils in red muscle fibers (b) EM image indicating less cytoplasm and fewermitochondria between myofibrils in white muscle fibers (c) A longitudinal section displayed the well-organized sarcomere in red musclefibers (d) Well-organized sarcomeres were also observed in white muscle fibersThe sarcomere length (double-headed arrow) is the distancebetween two Z-lines (e) The sarcomere length was shorter in white muscle fibers (f) The Z-line width was thinner in white muscle fibers(g)The myofibril width was larger in white muscle fibers lowast p lt 0001 L lipid droplet M mitochondria SL sarcomere length Z Z-line Bar05 120583m

filaments and pull the actin filaments towards the M-line inthe center of the sarcomere Thereby the sarcomere shortensand produces an active forceThediscovery of the giant elasticprotein titin dramatically expanded our understanding ofmuscle structure and function [6 8 34] In addition titin alsoplays an important role as a molecular scaffold for thick andpresumably thin filament formation during myofibrillogene-sis [35] It is because of this supportive role that titin damageresults in abnormal sarcomeric organization and myofibrildisassembly [36 37] Considering these important functionsof titin we characterized muscle proteins in tilapia and foundthat titin was more highly expressed in white muscle than redmuscle Titin contents were about 10 of total myofibrillarproteins in chicken breast muscle [6] 16 in rabbit skeletalmuscle and 13 in fish muscle [12] Ratios of titin relative tomyosin were estimated to be 195 for rabbit cardiac muscleand 142 for rabbit skeletal muscle [38] In this study ratiosof titin to myosin were estimated to be 0206 (148) for redmuscle and 0273 (137) for white muscle White muscle

containing a higher titinMHC ratio represents amore-elasticprotein in sarcomere units Since titin is the main passivetension source of muscles white muscle is relatively morerigid than red muscle In addition skeletal muscle forceregulationhighly depends on titin filaments rather than actinand myosin filaments [10 11] White muscle contracts fasterandmore efficiently than redmuscle whichmight result froma higher level of titin and more-organized sarcomeres

Studies have demonstrated that titin stiffness increases inactivated muscle prior to development of force [39] and alsodemonstrated a role for titin in residual force enhancement[40] Another study suggested that titin binds calcium uponactivation thereby increasing its spring stiffness and thatsome proximal part of titin may bind to actin therebypotentially decreasing titinrsquos free spring length in the I-bandregion thus possibly increasing titinrsquos stiffness and its force[9] Given the weight of these evidences it is now moreungenerous to presume that not only cross-bridges but alsotitin contributes to dynamic force production


2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90

titin RW MHC RW actin RW

Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)

Figure 6 SDS-PAGE protein analysis of red (R) and white (W) muscles (a) After loading equal concentrations (2sim10 120583g) of total proteinswhite muscle displayed more myofibrillar proteins than did red muscle Titin myosin heavy chain (MHC) and actin were the three majormyofibrillar proteins (b) Ratios of titin MHC and actin in red muscle to white muscle (c) Mean ratios of titinMHC in red and whitemuscles p lt 001 lowast p lt 0001

5 Conclusion

Thepresent study was carried out to discover the biochemicalcharacteristics which are important in muscle contractionin red and white muscles of fish (Tilapia) Our resultssuggest that white muscle contains higher levels of titin andphosphate ions and shorter sarcomere lengths which may beinvolved in faster and more-powerful muscle contractionsIn addition red muscle was enriched in well-balanced lipiddroplets fatty acids and mitochondria and had longersarcomere lengths for slow long-term contractions Takentogether the present results of titin and energetic parametersin red and white muscles provide evidence that white muscleis more suitable for short and powerful contractile perfor-mance than red muscle

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

The authors declare that no conflicts of interest exist

Authorsrsquo Contributions

Ming-Ping Wu and Nen-Chung Chang contributed equallyto this work


Acknowledgments

This work was supported by Taipei Medical University-TaipeiMedical University Hospital (104TMU-TMUH-20) and Chi-Mei Medical Center-Taipei Medical University (104CM-TMU-10) The authors are grateful to the Core Facility ofTaipei Medical University for technical support

References

[1] S Schiaffino and C Reggiani ldquoFiber types in Mammalianskeletal musclesrdquo Physiological Reviews vol 91 no 4 pp 1447ndash1531 2011

[2] T Ogata ldquoStructure of Motor Endplates in the Different FiberTypes of Vertebrate Skeletal Musclesrdquo Archives of Histology andCytology vol 51 no 5 pp 385ndash424 1988

[3] C Franzini-Armstrong and L D Peachey ldquoStriated muscle-contractile and control mechanismsrdquo 13e Journal of Cell Biol-ogy vol 91 no 3 pp 166sndash186s 1981

[4] Y-C Huang R G Dennis and K Baar ldquoCultured slow vsfast skeletal muscle cells differ in physiology and responsivenessto stimulationrdquo American Journal of Physiology-Cell Physiologyvol 291 no 1 pp C11ndashC17 2006

[5] A C Nag ldquoUltrastructure and adenosine triphosphatase activ-ity of red and white muscle fibers of the caudal region of a fishsalmo gairdnerirdquo 13e Journal of Cell Biology vol 55 no 1 pp42ndash57 1972

[6] K Wang J McClure and A Tu ldquoTitin major myofibrillarcomponents of striated musclerdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 76 no8 pp 3698ndash3702 1979

[7] H Granzier and S Labeit ldquoStructure-function relations of thegiant elastic protein titin in striated and smooth muscle cellsrdquoMuscle amp Nerve vol 36 no 6 pp 740ndash755 2007

[8] S Lindstedt and K Nishikawa ldquoHuxleysrsquo Missing FilamentForm and Function of Titin in Vertebrate Striated MusclerdquoAnnual Review of Physiology vol 79 pp 145ndash166 2017

[9] T R Leonard and W Herzog ldquoRegulation of muscle force inthe absence of actin-myosin-based cross-bridge interactionrdquoAmerican Journal of Physiology-Cell Physiology vol 299 no 1pp C14ndashC20 2010

[10] W Herzog T Leonard V Joumaa M DuVall and A Pan-changam ldquoThe three filament model of skeletal muscle stabilityand force productionrdquoMolecular amp Cellular Biomechanics vol9 no 3 pp 175ndash191 2012

[11] G Schappacher-Tilp T Leonard G Desch and W HerzogldquoA novel three-filament model of force generation in eccentriccontraction of skeletal musclesrdquo PLoS ONE vol 10 no 3 2015

[12] N Seki and T Watanabe ldquoConnectin content and its post-mortem changes in fish musclerdquo 13e Journal of Biochemistryvol 95 no 4 pp 1161ndash1167 1984

[13] M M Melendez J A Vosswinkel M J Shapiro et al ldquoWallSuction Applied to Needle Muscle Biopsy-A Novel Techniquefor Increasing Sample Sizerdquo Journal of Surgical Research vol142 no 2 pp 301ndash303 2007

[14] M Ali Khan ldquoHistochemical characteristics of vertebratestriated muscle A reviewrdquo Progress in Histochemistry andCytochemistry vol 8 no 4 pp 1ndash47 1976

[15] G L Tunell and M N Hart ldquoSimultaneous Determination ofSkeletal Muscle Fiber Types I IIA and IIB by HistochemistryrdquoJAMA Neurology vol 34 no 3 pp 171ndash173 1977

[16] J M Wakeling and J A Johnston ldquoWhite strain the carp red towhitemuscle gearing ratios in fishrdquo13e Journal of ExperimentalBiology vol 202 no 5 pp 521ndash528 1999

[17] A SaNger and W Stoiber ldquoMuscle Fiber Diversity and Plas-ticityrdquo in Muscle Development and Growth vol 18 of FishPhysiology pp 187ndash250 Elsevier 2001

[18] M Martin-Perez J Fernandez-Borras A Ibarz et al ldquoNewinsights into fish swimming A proteomic and isotopic approachin gilthead sea breamrdquo Journal of Proteome Research vol 11 no7 pp 3533ndash3547 2012

[19] T Hasumura and S Meguro ldquoExercise quantity-dependentmuscle hypertrophy in adult zebrafish (Danio rerio)rdquo Journalof Comparative Physiology B Biochemical Systemic and Envi-ronmental Physiology vol 186 no 5 pp 603ndash614 2016

[20] S-P Chen J-R Sheu C-Y Lai T-Y Lin G Hsiao and T-HFong ldquoDetection of myofibrillar proteins using a step gradientminigel with an ambiguous interfacerdquo Analytical Biochemistryvol 338 no 2 pp 270ndash277 2005

[21] J-H Wei N-C Chang S-P Chen P Geraldine T Jayakumarand T-H Fong ldquoComparative decline of the protein profilesof nebulin in response to denervation in skeletal musclerdquoBiochemical and Biophysical Research Communications vol466 no 1 pp 95ndash102 2015

[22] B Essen E Jansson J Henriksson A W Taylor and B SaltinldquoMetabolic Characteristics of Fibre Types in Human SkeletalMusclerdquoActa Physiologica Scandinavica vol 95 no 2 pp 153ndash165 1975

[23] L J C van Loon R Koopman J H C H Stegen A J MWagenmakers H A Keizer and W H M Saris ldquoIntramyocel-lular lipids form an important substrate source duringmoderateintensity exercise in endurance-trained males in a fasted staterdquo13e Journal of Physiology vol 553 no 2 pp 611ndash625 2003

[24] A Kiessling K Ruohonen and M Bjoslashrnevik ldquoMuscle fibregrowth and quality in fishrdquo Archiv TierzuchtArchives AnimalBreeding vol 49 pp 137ndash146 2006

[25] B Kiens ldquoSkeletal muscle lipid metabolism in exercise andinsulin resistancerdquo Physiological Reviews vol 86 no 1 pp 205ndash243 2006

[26] C J Barclay J K Constable and C L Gibbs ldquoEnergetics offast- and slow-twitch muscles of the mouserdquo 13e Journal ofPhysiology vol 472 no 1 pp 61ndash80 1993

[27] M L Blei K E Conley and M J Kushmerick ldquoSeparatemeasures of ATP utilization and recovery in human skeletalmusclerdquo13e Journal of Physiology vol 465 no 1 pp 203ndash2221993

[28] W W Winder and D M Thomson ldquoCellular energy sensingand signaling by AMP-activated protein kinaserdquo Cell Biochem-istry and Biophysics vol 47 no 3 pp 332ndash347 2007

[29] D Vavvas A Apazidis A K Saha et al ldquoContraction-inducedchanges in acetyl-CoA carboxylase and 51015840-AMP- activatedkinase in skeletal musclerdquo 13e Journal of Biological Chemistryvol 272 no 20 pp 13255ndash13261 1997

[30] J F P Wojtaszewski C MacDonald J N Nielsen et alldquoRegulation of 51015840-AMP-activated protein kinase activity andsubstrate utilization in exercising human skeletal musclerdquoAmerican Journal of Physiology-Endocrinology and Metabolismvol 284 no 4 pp E813ndashE822 2003

[31] C Spamer and D Pette ldquoActivity patterns of phosphofructok-inase glyceraldehydephosphate dehydrogenase lactate dehy-drogenase and malate dehydrogenase in microdissected fastand slow fibres from rabbit psoas and soleus musclerdquo Journal


of Histochemistry amp Cytochemistry vol 52 no 3 pp 201ndash2061977

[32] S C Leary C N Lyons A G Rosenberger J S BallantyneJ Stillman and C D Moyes ldquoFiber-type differences in mus-cle mitochondrial profilesrdquo American Journal of Physiology-Regulatory Integrative andComparative Physiology vol 285 no4 pp R817ndashR826 2003

[33] A F Huxley and R Niedergerke ldquoStructural changes in muscleduring contraction interference microscopy of living musclefibresrdquo Nature vol 173 no 4412 pp 971ndash973 1954

[34] K Maruyama ldquoConnectin an elastic protein from myofibrilsrdquo13e Journal of Biochemistry vol 80 no 2 pp 405ndash407 1976

[35] C C Gregorio H Granzier H Sorimachi and S LabeitldquoMuscle assembly a titanic achievementrdquo Current Opinion inCell Biology vol 11 no 1 pp 18ndash25 1999

[36] P F M Van Der Ven JW BartschM Gautel H Jockusch andD O Furst ldquoA functional knock-out of titin results in defectivemyofibril assemblyrdquo Journal of Cell Science vol 113 no 8 pp1405ndash1414 2000

[37] J Udaka S Ohmori T Terui et al ldquoDisuse-induced preferentialloss of the giant protein titin depresses muscle performancevia abnormal sarcomeric organizationrdquo 13e Journal of GeneralPhysiology vol 131 no 1 pp 33ndash41 2008

[38] J Suzuki S Kimura and K Maruyama ldquoConnectin contentin rabbit cardiac and skeletal musclerdquo International Journal ofBiochemistry vol 25 no 12 pp 1853ndash1858 1993

[39] A S Cornachione F LeiteM A Bagni and D E Rassier ldquoTheincrease in non-cross-bridge forces after stretch of activatedstriated muscle is related to titin isoformsrdquo American Journalof Physiology-Cell Physiology vol 310 no 1 pp C19ndashC26 2016

[40] W Herzog G Schappacher M DuVall T R Leonard and JA Herzog ldquoResidual force enhancement following eccentriccontractions A new mechanism involving titinrdquo PhysiologyJournal vol 31 no 4 pp 300ndash312 2016

Hindawiwwwhindawicom

International Journal of

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Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



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The Scientific World Journal

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BioinformaticsAdvances in

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MicrobiologyHindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom


fibers make them very effective in postural maintenancewhile white muscle fibers are suited for bursts of intensemuscle activity [3]

In addition to morphological observations physiologistshave reported that red muscle fibers contract more slowlyand are more resistant to fatigue than are white muscle fibersdue to their ability to oxidatively regenerate ATP In contrastATP generation in white muscle fibers depends on anaerobicglycolysis [4] It is accepted that hydrolysis of ATP by ATPaseis directly involved as the energy supply in the process ofmuscle contraction Biochemical studies revealed that theATPase activity of white muscle fibers is 3 times higher thanthat of red muscle fibers [5] However in a resting statewhether ATP is stored for use in muscle fibers and whetherthe stored ATP content in red muscle fibers is more or lessthan that in white muscle fibers are still unclear

Titin with a molecular mass of 3sim4 MDa is a muscle-specific elastic protein that spans from the Z-line to the M-line in the half-sarcomere it is the largest protein knownto date [6 7] The passive tension elasticity and stiffness ofmuscles are correlated with their titin content [8] Previousexperiments on single myofibrils prepared from the rabbitpsoas muscle revealed another powerful mechanism of activeforce production [9] Recent studies showed that regulation ofskeletal muscle force is based on a three-filament model thatincludes titin rather than a two-filament model consistingonly of actin and myosin filaments [10 11] Titin is the thirdmost abundant protein in muscles after myosin and actinTitin contents are about 10 of total myofibrillar proteins inchicken breast muscle [6] and 16 in rabbit skeletal muscle[12] However titin contents between red and white muscleshave not been investigated in a single species

Biochemical analyses of human muscle usually use biop-sies to obtain muscle samples [13]The optimal sample size iscritical for the availability of tissue for processing A biopsycannot produce a large amount of muscle sample and mayincrease the risk of infection On the other hand musclesof mammals (mice rats rabbits and humans) are mostly ofthe mixed type that is muscles contain both red and whitemuscle fibers [14 15] Relative proportions of the differentfiber types vary among species and in humans they showsignificant variability among individuals [1] Although wewanted to quantify and comparemyofibrillar protein contentsbetween red and white muscles mammalian muscles whichcontain mixed muscle fibers cannot be precisely analyzed

Fish are a good animal model for muscle studies becauseaquatic species have a proportionally larger muscle mass Inaddition red and white muscle types in fish are distributed indifferent anatomical locationsThe lateral triangular zone anda superficial thin layer of the trunk is a red color containingred muscle fibers but large portions of the deep layer ofthe dorsal and ventral zones of the trunk are white andcontain abundant white muscle fibers [16] Moreover whitemuscle fibers are associated with fast swimming behaviors infish such as during predation and escape while red musclefibers are correlated with slow movements such as duringmigration and foraging [17 18]

Zebrafish are commonly used in genetic embryonicdevelopment and muscle research [19] Red and white

muscles are also distributed in different region but zebrafishare too small to separate red muscles fromwhite muscles andobtain a sufficient amount of muscle tissues for biochemicalanalyses Therefore we used Tilapia sp a locally commonfish in Taiwan to analyze morphological and myofibril-lar proteins between red and white muscles in a singlespecies

The purpose of this study was to investigate mor-phological differences by histochemistry time-of-flight sec-ondary ion mass spectrometry (TOF-SIMS) and electronmicroscopy and quantify myofibrillar proteins especiallytitin contents between red and white muscles in fish bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)

2 Materials and Methods

21 Experimental Animals In total 10 apparently healthyadult male fish (Tilapia) (Figure 1(a)) with an averagebody weight of 500 plusmn 5 g were purchased from a localtraditional market in Taipei Taiwan and were transportedto the laboratory live in water Fish were killed by cervicaldislocation and then immediately dissected out Red andwhite muscles were mainly located on the lateral and dorsalsides of the trunk respectively (Figure 1(b)) The lateralpart of the trunk containing a red and white muscle block(about a 1-cm2 area) was excised and frozen with liquidnitrogen for cryostat sectioning All experimental protocolswere approved by the Animal Ethics Committee of TaipeiMedical University (license no LAC-2017-0224)

22 Oil-Red O Staining Oil-red O powder (Sigma-AldrichSt LouisMOUSA)was dissolved in 05 2-propanol (KantoChemical Tokyo Japan) The stock was then diluted to a03 Oil-red O solution with distilled H

2O and filtered

through a 022-120583m filter Frozen sections (20 120583m thick) offish muscle sample were fixed with 2 paraformaldehydeand 2 glutaraldehyde (Sigma-Aldrich) in 01 M phosphatebuffer (pH 74) for 10min at room temperature After fixationsections were washed with phosphate buffer three timesand stained with 03 Oil-red O solution for 10 min atroom temperature Finally stained sections were washedwith phosphate buffer three times mounted with aqueousmounting medium and sealed with nail polish Sections wereexamined with an Olympus BH-2 light microscope (TokyoJapan)

23 TOF-SIMS Analysis The TOF-SIMS analysis was car-ried out on a PHI TRIFT IV instrument (ULVAC-PHIKanagawa Japan) This instrument was equipped with a Biliquid metal ion gun Cryostat sections (20 120583m thick) offish muscle tissues were used for a replicate analysis in thisstudy The Bi

3

+ primary ion beam was operated at 30 keVSurface spectra were taken from an area of 500 times 500 120583min order to get an overview of the sample structure andidentify ion species present at the respective surfaces Fourrandom areas were selected for scanning in each sectionfor which four spectra were separately acquired from each


5 cm

(a)

2 cm

R

W

W

(b)

W

R

(c)

W

R

(d)











3 Results



3


3






140 200180160 220 280260240

30E+625E+620E+615E+610E+650E+5

0

137146

153 163169

281183 199

255

237227

Tota

l Cou

nts

lowast

lowast

0

35E+730E+725E+720E+715E+710E+750E+6

0

1317

26 4263 97

1

71

79To

tal C

ount

s

20 40 60 80 100 120

lowast

(a)



Negative PO3-

7896C16H31O2


-Oleic C 181 28126

(b)





4 Discussion



R

W

79 [13Unsaved] (256x256)

(a)

04

06

08

10

0

02

R

W

255 [13Unsaved] (256x256)

(b)

R

W

281 [13Unsaved] (256x256)

(c)

Standard

R

W

(d)







R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)






R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



5 cm

(a)

2 cm

R

W

W

(b)

W

R

(c)

W

R

(d)











3 Results



3


3






140 200180160 220 280260240

30E+625E+620E+615E+610E+650E+5

0

137146

153 163169

281183 199

255

237227

Tota

l Cou

nts

lowast

lowast

0

35E+730E+725E+720E+715E+710E+750E+6

0

1317

26 4263 97

1

71

79To

tal C

ount

s

20 40 60 80 100 120

lowast

(a)



Negative PO3-

7896C16H31O2


-Oleic C 181 28126

(b)





4 Discussion



R

W

79 [13Unsaved] (256x256)

(a)

04

06

08

10

0

02

R

W

255 [13Unsaved] (256x256)

(b)

R

W

281 [13Unsaved] (256x256)

(c)

Standard

R

W

(d)







R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)






R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









3 Results



3


3






140 200180160 220 280260240

30E+625E+620E+615E+610E+650E+5

0

137146

153 163169

281183 199

255

237227

Tota

l Cou

nts

lowast

lowast

0

35E+730E+725E+720E+715E+710E+750E+6

0

1317

26 4263 97

1

71

79To

tal C

ount

s

20 40 60 80 100 120

lowast

(a)



Negative PO3-

7896C16H31O2


-Oleic C 181 28126

(b)





4 Discussion



R

W

79 [13Unsaved] (256x256)

(a)

04

06

08

10

0

02

R

W

255 [13Unsaved] (256x256)

(b)

R

W

281 [13Unsaved] (256x256)

(c)

Standard

R

W

(d)







R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)






R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



140 200180160 220 280260240

30E+625E+620E+615E+610E+650E+5

0

137146

153 163169

281183 199

255

237227

Tota

l Cou

nts

lowast

lowast

0

35E+730E+725E+720E+715E+710E+750E+6

0

1317

26 4263 97

1

71

79To

tal C

ount

s

20 40 60 80 100 120

lowast

(a)



Negative PO3-

7896C16H31O2


-Oleic C 181 28126

(b)





4 Discussion



R

W

79 [13Unsaved] (256x256)

(a)

04

06

08

10

0

02

R

W

255 [13Unsaved] (256x256)

(b)

R

W

281 [13Unsaved] (256x256)

(c)

Standard

R

W

(d)







R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)






R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



R

W

79 [13Unsaved] (256x256)

(a)

04

06

08

10

0

02

R

W

255 [13Unsaved] (256x256)

(b)

R

W

281 [13Unsaved] (256x256)

(c)

Standard

R

W

(d)







R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)






R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



R

(a)

W

(b)

100 um

R

(c)

100 um

W

(d)

50 um

R

(e)

50 um

W

(f)






R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



R

M

M

M

L

(a)

W

(b) (c)

(d)R W

00

02

04

06

08

10

12

14

16

Sarc

omer

e len

gth

(m

) lowast

(e)R W

0102030405060708090

Z-lin

e wid

th (n

m)

lowast

(f)R W

00

02

04

06

08

10

12

Myo

fibri

l wid

th (

m)

lowast

(g)






2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



2 10864 g

titin

MHC

actin

-170-130-95-72-55-43-34-26

-17-10

_

_

_

R W

2 10864 g

(a)

0

10

20

30

40

50

60

70

80

90


Mea

n ra

tio o

f RW

()

(b)R W

lowast

0

5

10

15

20

25

30M

ean

ratio

of t

itin

MH

C (

)

(c)


5 Conclusion


Data Availability







Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Acknowledgments


References













































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Analysis of Titin in Red and White Muscles: Crucial Role...

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