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Regulation of muscle force in the absence of actin-myosin-based cross-bridge

interaction

T. R. Leonard and W. Herzog

Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada

Submitted 16 February 2010; accepted in final form 30 March 2010

Leonard TR, Herzog W. Regulation of muscle force in theabsence of actin-myosin-based cross-bridge interaction. Am J PhysiolCell Physiol 299: C14C20, 2010. First published March 31, 2010;doi:10.1152/ajpcell.00049.2010.For the past half century, the slid-ing filament-based cross-bridge theory has been the cornerstone of ourunderstanding of how muscles contract. According to this theory,active force can only occur if there is overlap between the contractilefilaments, actin and myosin. Otherwise, forces are thought to becaused by passive structural elements and are assumed to vary solelybecause of the length of the muscle. We observed increases in muscleforce by a factor of 3 to 4 above the purely passive forces for activatedand stretched myofibrils in the absence of actin-myosin overlap. We

show that this dramatic increase in force is crucially dependent on thepresence of the structural protein titin, cannot be explained withcalcium activation, and is regulated by actin-myosin-based cross-bridge forces before stretching. We conclude from these observationsthat titin is a strong regulator of muscle force and propose that thisregulation is based on cross-bridge force-dependent titin-actin inter-actions. These results suggest a mechanism for stability of sarcomereson the inherently unstable descending limb of the force-lengthrelationship, and they further provide an explanation for the protectionof muscles against stretch-induced muscle injuries.

myofibrils; titin; myofilament overlap; force production

THE SLIDING FILAMENT-BASED cross-bridge theory has been theparadigm of choice for muscle contraction and force produc-tion for the past half century (1517, 19, 20). According to thistheory, contraction occurs through the interaction of myosin-based cross bridges that attach cyclically to actin and tend topull actin past the myosin filaments toward the center ofsarcomeres (15, 17, 19). This produces muscle contraction andforce. When a muscle is stretched, sarcomeres become longerand actin-myosin filament overlap decreases (Fig. 1), thusdecreasing the number of possible cross-bridge interactionsand active force while passive forces increase (8, 15, 16, 18,31). At sarcomere lengths where actin-myosin filament overlapceases to exist, only passive forces are possible, and these arethought to be essentially invariant at a given muscle or sarco-mere length when due account is given to transient viscous

effects (Fig. 2). However, recent pilot results suggest thatpassive forces in isolated myofibrils might be modulated sub-stantially by active stretching (26). In myofibrils, passiveforces are known to primarily originate from the structuralprotein titin (9, 14, 30, 35), the largest protein currently knownin the natural world, and thus, we hypothesized that titin, inaddition to actin-myosin, might be a strong regulator of forcein actively stretched muscles. Therefore, the purpose of thisstudy was to investigate the forces produced in myofibrils that

were stretched to lengths too great to allow for actin-myosininteractions and to elucidate the role of titin in producing theseforces. Although titin has been associated with force regulationthrough phosphorylation (37) and calcium binding (25), theseeffects were assumed much too small to explain our pilotresults (26).

METHODS AND MATERIALS

Sample preparation.Strips of rabbit psoas muscle were taken fromeuthanized animals using Dumont 3 forceps and tied to wooden sticksto preserve the in situ sarcomere length. These strips of muscle werethen placed in a rigor-glycerol solution with protease inhibitors(Complete, Roche Diagnostics, Montreal, QB, Canada) and stored at20C for 10 to 14 days (32). For experimentation, strips of musclewere placed in a 4C rigor solution, homogenized, and placed in theexperimental chamber (20C). Solutions used are published elsewhere(22, 34).

Ethics approval was granted from the institutional Animal EthicsCommittee.

Testing protocol. Fifty-nine single myofibrils (with an aggregatetotal of 312 sarcomeres) from rabbit psoas were tested in theseexperiments and divided into eight testing groups. Myofibrils withthree to eight sarcomeres in series were used (mean of 5.3) in theseexperiments because of the high magnification of the microscope(100 oil objective with a 2.5 Optovar) and the large magnitude ofthe stretches employed.

Group 1myofibrils (n 12) were nonactivated and stretched in asolution containing ATP.Group 2myofibrils (n 12) were activatedin a calcium ATP solution and then stretched. Group 3 myofibrils(n 6) were treated with a mild trypsin solution (for titin deletion) (7,10, 11) and then kept nonactivated and stretched in a solutioncontaining ATP. Group 4 samples (n 8) were also treated withtrypsin but were then placed in the activating calcium ATP solutionand then stretched. Group 5 myofibrils (n 10) were placed in anactivating (calcium ATP) solution with 20 mM 2,3 butanedionemonoxime (BDM), a cross-bridge inhibitor (34), and then stretched.Group 6(n 5) myofibrils were nonactivated and lengthened to 3.4m and then activated in a calcium ATP solution and then furtherlengthened. Group 7 myofibrils (n 2) were nonactivated andlengthened to5 m, then activated and further lengthened to a meansarcomere length of 6 m. Group 8 myofibrils (n 4) were

activated at optimal length (2.2 m), lengthened to a mean sarcomerelength of5 m, and then deactivated by placement in a relaxingsolution.

Tests for all myofibrils were performed starting at sarcomerelengths between 2.0 and 2.4 m (exceptgroup 6) and then lengthenedat a speed of 0.1 m per sarcomere per second (which is 5% of theinitial sarcomere length per second).

Sarcomere length and force measurements. All tests were con-ducted using an inverted microscope (Zeiss Axiovert 200M) (22)equipped with a 100 oil immersion objective (numerical aperture1.3) and a 2.5 Optovar. Individual sarcomere lengths were measuredusing an ultra-high-resolution linear diode line scan camera (modelSK10680 DJR, Schafter and Kirschoff) with a resolution of 6.7nm/pixel (22). Sarcomere lengths were calculated from Z line to Z line

Address for reprint requests and other correspondence: W. Herzog, Kinesi-ology, Univ. of Calgary, 2500 Univ. Drive, Calgary, AB, Canada T2N 1N4(e-mail: [email protected]).

Am J Physiol Cell Physiol299: C14C20, 2010.First published March 31, 2010; doi:10.1152/ajpcell.00049.2010.

0363-6143/10 Copyright 2010 the American Physiological Society http://www.ajpcell.orgC14


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of adjacent sarcomeres, and only when this was not possible becausethe striation pattern tended to disappear with excessive stretching,average sarcomere length was determined by dividing the specimenlength by the number of sarcomeres. A custom-built piezo-tube motor

with a drawn glass pipette was used to manipulate the length of the

specimen with nanometer resolution. LabView software (National

Instruments, Austin, TX) controlled the motor and data acquisition.

Myofibril forces were determined using custom-built nanofabricated

silicon nitride cantilevers (6) with a stiffness of 22 pn/nm (for passive

and titin-deleted experiments) or 178 pn/nm (for all active experi-

ments). Displacement of one lever attached to the myofibril relative to

a reference lever was measured, and forces were calculated from the

measured displacement and the known lever stiffness (22). Myofibrils

were glued (Dow Corning 3145)(28) to one of the levers and wrapped

around the lever to help prevent detachment. Forces were normalized

to the cross-sectional area by measuring myofibril diameter (28) and

were expressed in units of stress (nN/m2).

RESULTS

Activated and stretched myofibrils show much greater force

within the actin-myosin filament overlap zone (sarcomere

lengths 4.0 m) than nonactivated and stretched myofibrils,

as one would expect (Fig. 3A); however, completely unex-

pected, forces in the activated and stretched myofibrils remain

much higher, and increase more rapidly, than those of myofi-

brils that were lengthened while not activated, even at sarco-

mere lengths beyond 4.0 m (Fig. 3A).

To elucidate the possible role of titin in the increased force

of actively stretched myofibrils, we repeated the active and

Fig. 1. Active force is produced by cyclic interac-tions of myosin-based cross bridges with actin. Top:a myofibril showing sarcomeres arranged in series.The dark (A band) and light (I band) striation patternindicates the myosin and actin filament regions.Second from top: an isolated sarcomere with Z line,M line, and A and I bands. Third from top: sche-matic illustration of a sarcomere with titin, myosin,and actin filaments. The overlap area in the A band(indicated by the dark green-shaded region) is thearea in which myosin cross bridges can attach toactin and so produce sarcomere shortening and ac-tive force. Bottom: the sarcomere is shown in astretched position. The actin-myosin overlap area isnear zero; thus active actin-myosin-based force pro-duction is also near zero while titin is stretched and

provides passive force at this length. Titin spans thehalf-sarcomere from the M line to the Z line. It issymmetrically arranged in both halves of a sarco-mere, and since it is attached to myosin filaments, itcenters these filaments in the middle of the sarco-mere. The I-band region contains the extensibleportion of titin that is thought to act as a molecularspring.

Fig. 2. Representation of the force-length relationship showing active force(red) and passive force (green) contributions with the total force (blackdashed). Up to sarcomere lengths of 3.6 m, active forces dominate;between 3.6 and 4.0 m, passive forces dominate; and beyond 4.0 m, thepassive forces become the only source of force for rabbit psoas myofibrils.[From Gordon et al. (8).]

C15REGULATION OF MUSCLE FORCE

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nonactivated stretch experiments following depletion of titin.In the absence of titin, forces during activated and nonactivatedstretching were essentially zero and did not increase system-atically with increasing sarcomere lengths (blue and yellowtriangles, Fig. 3B), suggesting that titin is crucial for nonacti-vated force production, as suggested by others (9, 14, 30, 35)and that titin is essential for the dramatic increase in force

observed here beyond myofilament overlap in the activated and

stretched myofibrils (Fig. 3A).

Activation of myofibrils has been associated with calcium

binding to titin and an associated increase in titins spring

stiffness and force upon stretch (23, 25). These effects, how-

ever, have been thought to be minor but have only been studied

within the region of actin-myosin filament overlap. Thus, to

elucidate the role of calcium on titins force and stiffnessregulation in the absence of actin-myosin-based cross-bridge

forces, we performed stretch experiments with calcium-acti-

vated myofibrils while inhibiting cross-bridge force through

BDM (2, 34). Stretching activated myofibrils in BDM con-

ditions was successful in abolishing all actin-myosin-based

forces (orange squares, Fig. 3B) and produced passive forces

that were essentially the same as those produced for purely

nonactivated (passive) myofibril stretching (green circles, Fig.

3B). This result suggests that calcium activation, and calcium

binding to titin, has a negligible effect on myofibril force

regulation and cannot explain the results observed in Fig. 3A.

Thus, we hypothesized that actin-myosin-based cross-bridge

forces before stretching are essential to produce the observedforce increase of actively compared with nonactivated and

stretched myofibrils.

To test this hypothesis, we performed a further set of

experiments in which myofibrils were fully activated at differ-

ent sarcomere lengths (2.4 m and 3.4 m/sarcomere) and

then lengthened. The active forces for myofibrils activated at

3.4 m (purple diamonds, Fig. 3A) were about half of those

activated at 2.4 m (red squares, Fig. 3A), and when stretched

beyond actin-myosin overlap, the forces remained smaller than

those of the myofibrils activated and stretched from sarcomere

lengths of 2.4 m, but were greater than those of the nonac-

tivated and stretched myofibrils (green circles, Fig. 3A). These

results suggest that the force regulation observed here atsarcomere lengths beyond actin-myosin filament overlap de-

pends on the active forces before stretching.

To test whether actin-myosin-based cross-bridge forces

could be produced at sarcomere lengths 4.0 m, we

stretched nonactivated myofibrils from sarcomere lengths of2.2 m to a mean sarcomere length of approximately 4.5

to 5 m, then activated them and stretched them further to

sarcomere lengths of6 m (Fig. 4). Activating myofibrils

at sarcomere lengths 4.0 m did not alter the force-time

curves, indicating that cross-bridge interactions did not

occur at these long lengths.

To confirm that the increased passive forces of actively

compared with nonactivated and stretched myofibrils werenot caused by remnant cross-bridge attachments due to

myosin filaments being elongated at their ends as suggested

by previous work (36) or torn from the A-band region due to

the large stretch imposed in our testing, we stretched fully

activated myofibrils from a mean sarcomere length of 2.4m to a mean sarcomere length of5 m and then replaced

the activating with a relaxing solution (Fig. 5). We observed

no effect on force when the solutions were exchanged,

thereby confirming that the increase in force in the absence

of actin-myosin overlap was not due to rogue myosin

filaments attaching to actin even at very long sarcomere

lengths.

Fig. 3.A: force as a function of sarcomere length: mean SE of force versusaverage sarcomere length for myofibrils stretched in a high-calcium (activa-tion) solution from an initial average sarcomere length of 2.4 m (red squares)and 3.4 m (purple diamonds) and for myofibrils stretched in a low-calcium(relaxation) solution (green circles). The force-sarcomere length relationshipcurves for both active myofibrils are significantly higher ( 0.05) than thatfor the nonactivated myofibrils within the myofilament overlap zone as one

would expect (sarcomere length 4 m). However, completely unexpected isthe 34 (2.4 m) and 1.52 times (3.4 m) higher force of the activelycompared with the nonactively stretched myofibrils beyond the myofilamentoverlap zone (sarcomere length 4.0 m, P 0.05) where actin-myosin-based cross-bridge forces are zero and passive forces should be the same(area to the right of the vertical dashed blue line). Forces are normalizedrelative to the myofibrillar cross-sectional area. B: the force-sarcomere lengthrelationships for nonactively stretched myofibrils (green circles), for myofibrilsstretched in a high-calcium activation solution with an added cross-bridgeinhibitor [20 mM 2,3 butanedione monoxime (BDM)] (orange squares), aftertitin deletion in a low-calcium relaxation solution (yellow triangles), and aftertitin deletion in a high-calcium activation solution (blue triangles). Valuesshown are means SE. There is no isometric force within the optimalmyofilament overlap region (2.26 m to 2.43 m) for the active BDMmyofibrils, indicating that BDM alleviated all cross-bridge-related active force.Active BDM-treated myofibrils produced the same force as the nonactivelystretched myofibrils, indicating that calcium activation in the absence ofactin-myosin-based cross-bridge forces had no effect on passive force duringmyofibril stretching. Titin deletion virtually abolished all active or passiveforces, indicating that titin is essential for passive force production and theincreased non-cross-bridge-based forces observed in this experiment.

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DISCUSSION

The main result of this study is that forces in the absence ofactin-myosin-based cross-bridge forces can be modulated sig-nificantly in skeletal muscles, thereby suggesting that theremust be significant mechanisms of force production that are notexplained by the cross-bridge theory and the traditional expec-tation of viscoelastic passive forces. The force regulation

observed here is significantly greater in magnitude than themaximal actin-myosin-based cross-bridge forces at optimalsarcomere length (Fig. 3A).

Specifically, we show increases in force of up to four timesin actively (compared to nonactivated) stretched rabbit psoasmyofibrils at sarcomere lengths beyond actin-myosin filamentoverlap where active, cross-bridge-based forces do not exist(Figs. 4 and 5). When eliminating titin, forces in the activatedand nonactivated and stretched myofibrils remain nearly zeroeven at very long sarcomere lengths, suggesting that titin mustbe present for this force regulation to take place. Furthermore,since titin is well known to be the primary passive forceproducer in rabbit psoas myofibrils (3, 13), it is safe to assume

that titin plays a crucial role in the force regulation observedhere for the first time.

It has been known for some time now that calcium binds totitin upon activation and increases titins resistance to stretch(22, 25). However, this calcium-induced stiffening of titin,although well accepted, has been thought to be of smallmagnitude and thus functionally irrelevant. Nevertheless, to

test whether calcium binding to titin might play a role in thedramatic upregulation of force during active elongation, westretched myofibrils in the activated (activation solution withhigh-calcium concentration) state but inhibited cross-bridgeattachment with BDM (34). In these experiments, the forcesmeasured in the myofibrils were essentially identical to thosemeasured for the purely passive stretches (in relaxing solution),thereby strongly suggesting that calcium activation of titin wasnot responsible for the increased forces during activated myo-fibril stretching observed in this study (Fig. 3B; orange squaresand green circles, respectively). On the basis of these results,we speculate that either active (actin-myosin based) force orcross-bridge attachment to actin is required to produce the

Fig. 5. Individual sarcomere length-time curves and force-time curve for anactively lengthened single myofibril composed of 5 sarcomeres in series fromthe group 8 tests. There are distinct sarcomere length nonuniformities beforeactivation, and upon activation all sarcomeres shorten. The myofibril is thenlengthened to a mean sarcomere length of 5 m. Sarcomere dispersionincreases somewhat at very long sarcomere lengths, but all sarcomeres arestretched beyond 4 m at the final test length. Removing the high-calciumactivating solution and replacing it with a relaxing solution (area to therightof the vertical dashed blue line) did not alter the force observed, suggesting thatthere were no remnant actin-myosin cross-bridge interactions at these verylong sarcomere lengths.

Fig. 4. Individual sarcomere length-time curves and force-time curve for onenonactivated and lengthened myofibril (passive) from the group 7 tests. Allindividual sarcomere lengths could be measured throughout the test, with allsarcomeres 4.0 m in length before the introduction of the activatingsolution; no change in force is observed upon activation (at vertical dashedline), indicating that cross-bridge interactions are not present at these sarco-mere lengths. Further lengthening from a mean sarcomere length of 4.5 m to6 m in a high-calcium activating solution produces force that is essentiallythe same as that observed in purely passively lengthened myofibrils at thecorresponding sarcomere length.




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increased (non-actin-myosin based) forces observed with acti-vated myofibril stretching.

If active force or cross-bridge attachment was required forthis phenomenon to occur, one would expect that the increasein non-actin-myosin-based forces was directly dependent onthe magnitude of the cross-bridge-based forces. To test thishypothesis, we performed another set of experiments where

stretching of the activated myofibrils was not started at optimal(2.4 m) sarcomere length, but at a longer (3.4 m) averagesarcomere length where active forces would be reduced to40% of those at optimal length (Fig. 3A). Stretching myofi-brils with reduced active force also produced forces in thenonoverlap zone (sarcomere lengths 4.0 m) that weresmaller than those obtained for the myofibrils for which activeforce was greater, but produced forces that were greater thanthose of nonactivated and stretched myofibrils (Fig. 3A). Thisresult provides strong evidence that the increase in non-actin-myosin-based forces with active stretching is directly linked toactive force or cross-bridge attachments to actin.

Before attempting to find explanations for a possible titin-based force regulation, we need to make sure that moretraditional explanations might not be possible. Probably themost frequently used explanation for force enhancement fol-lowing muscle stretching is the so-called sarcomere lengthinstability and nonuniformity theory (24, 29). According to thistheory, sarcomeres are unstable on the descending part of theforce-length relationship, and short sarcomeres remain shortupon stretching, while long sarcomeres are pulled beyondoverlap where they are rescued by passive forces (12). How-ever, the sarcomere length nonuniformity theory is an unlikelyexplanation for our results for two reasons: first, if sarcomerelength nonuniformity produced the dramatic increase in thenon-actin-myosin-based cross-bridge forces, then the highestforces measured in our experiments should never exceed the

active forces at optimal sarcomere length, as that would be thelimiting force of the short (active force producing) sarcomeres.

However, this result was not found, because the forces of theactivated and stretched myofibrils were approximately fivetimes those measured at optimal sarcomere length (Fig. 3A).Even if we assume that the short sarcomeres were stretched,and further assume a maximal stretch force enhancement of100%, we would still not be near the forces required to explainour measurements.

Second, if sarcomere length nonuniformity was responsiblefor the observed results, then the long (overstretched) sarco-meres should follow the passive force-extension curve oncethey are pulled beyond actin-myosin filament overlap. How-ever, our experiments observed that nonactivated and stretchedmyofibrils failed mechanically at sarcomere lengths of6.3m and at a force level (263 nN/m2) that was substantiallyless than the forces obtained in the activated and stretchedmyofibrils, thus sarcomere length nonuniformity seems anunlikely explanation for the results observed here.

Nevertheless, to make absolutely sure that sarcomere lengthnonuniformity could not explain the current results, we mea-sured the individual sarcomere lengths of the stretched myofi-brils. Myofibrils are fairly fragile preparations, and one mightexpect that activation alone might produce a vast increase insarcomere length nonuniformities, but this was not observed.Furthermore, and in contrast to the expectations of the sarco-mere length nonuniformity and instability theory (12), weobserved continuous elongation of all sarcomeres when myo-fibrils were stretched. Sarcomere lengths were not uniform,neither for nonactivated and stretched nor for activated andstretched myofibrils, but on the basis of the structural assump-tions underpinning the force-length relationship, we assumed(without verification by a direct observational method likeelectron microscopy) that all sarcomeres were always pulledbeyond actin-myosin filament overlap in all cases where suchmeasurements were made (e.g., Figs. 4, 5, and 6). Furthermore,

activation of myofibrils with all sarcomeres at lengths 4.0m did not produce an increase in force (Fig. 4), nor did

Fig. 6. Two myofibrils lengthened from 2.4 m to amean sarcomere length of5 m, one actively (A)and one nonactively (B). The sarcomere length isshown for all sarcomeres in the two myofibrils, and

although nonuniformities are present initially and con-tinue to be present throughout lengthening, all sarco-meres are eventually beyond myofilament overlap(demarcated by the vertical dashed line), and theforces of the actively stretched myofibril remain muchgreater than that for the nonactivated and stretchedmyofibril at corresponding sarcomere lengths.




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deactivation in this same situation produce a decrease in force(Fig. 5), strongly suggesting that there were no actin-myosin-based cross-bridge forces that affected (or caused) the observedincreases in force in the activated and stretched myofibrils atsarcomere lengths greater than actin-myosin overlap. From allthese observations, we may safely conclude that whatever thecause for the dramatic increase in non-cross-bridge-based

forces during active muscle stretching, it cannot be explainedwith sarcomere instability and the associated development ofsarcomere length nonuniformity. Classic studies by others (5,27) have proposed the transverse cytoskeleton as a regulator offorce during stretch. The transverse cytoskeleton produceslateral force transfer between myofibrils in single fibers (andpresumably in bundles of myofibrils) but because our prepa-ration is based on a single myofibril, this possibility of forceincrease during stretch of active myofibrils is precluded.

In the absence of an obvious traditional explanation for theobserved force increases during active stretching beyond actin-myosin filament overlap (such as the cross-bridge or thesarcomere length nonuniformity theory), we must search forother possibilities. Reviewing the experimental evidence, itappears that titin must be present for the observed forceincreases, but calcium activation of titin does not do the trick.However, it is well known that titin can also change its springstiffness (and thus resistance to stretch) by changing its freespring length in the I-band region by attaching selectively toactin. Here, we would like to tentatively propose that titinbinding to actin might be the cause for the dramatic increase innon-cross-bridge-based forces when muscles are stretched ac-tively compared with when they are stretched in the nonacti-vated state.

Although the exact molecular details for such a mechanismneed careful evaluation, we would like to suggest the followingscenario. Upon active force production, titin preferentially

attaches to actin, thereby shortening its spring length andincreasing its resistance to stretch (Fig. 7). To fully explain ourobservations, such titin-actin interactions must be modulated

either by active force or by the number of attached crossbridges. Force could be the modulator of titin-actin attachmentthrough stretching of the actin filament, thereby exposing titinattachment sites on actin, similar to the way stretching of thetalin rod enhances binding of vinculin (4). Alternatively, at-tachment of the cross-bridges to actin, and the associatedmovements of the regulatory proteins, troponin and tropomy-

osin, might free up previously covered titin attachment sites onactin. Whatever the detailed mechanisms, the force modula-tions produced by it must be as great as the actively producedmuscle forces through actin-myosin binding by cross bridges.

The idea about titin-actin binding in actively stretched myo-fibrils can be tested relatively easily. Imagine, for example, thattitin is labeled at specific sites with a fluorescent marker so thatlengths of individual titin segments can be carefully measured.If we now stretch a nonactivated myofibril, we would expectall segments to elongate on the basis of their constitutivestress-strain properties. However, if a myofibril is activated andstretched by the same amount and produces substantially moreforce as observed here, we would expect that some titinsegments (those bound to actin) would not stretch (or stretchsignificantly less than in the passive state), while others (thosenot bound) would stretch more and thereby compensate for thefixed attached segments of titin. This then would producegreater stiffness and increased forces during stretching ofactivated muscle.

The proposed mechanism has the advantage that it is inde-pendent of actin-myosin filament overlap and would continueto be in operation at lengths greater than myofilament overlap(i.e., 4.0 m in rabbit psoas). Therefore, this mechanism mightprovide powerful protection against stretch-induced muscleinjuries, the most common mode of damage of skeletal mus-cles. Also, such a stretch-induced modulation of force wouldprovide an elegant explanation for the observed stability of

sarcomeres on the descending limb of the force-length rela-tionship (21, 33), which has been thought (erroneously) to beunstable for more than half a century (12). Finally, the pro-posed mechanism might also offer a partial explanation for theso-called residual force enhancement of muscles followingactive stretching (1).

Conclusion. We conclude from the results of this study thatthere is a powerful mechanism for force regulation in musclesthat is independent of actin-myosin-based cross bridges. Thisforce modulation depends crucially on the presence of titin andactive force. We tentatively suggest that force (or cross-bridgeattachment)-dependent titin-actin interactions cause the dra-matic increase in force of activated and stretched comparedwith nonactivated and stretched muscles.

ACKNOWLEDGMENTS

The nanofabricated cantilevers used for force measurements were built byus at the NanoScale Facility of Cornell University (Ithaca, NY), which issupported by the National Science Foundation.

GRANTS

We gratefully acknowledge the financial support of the Natural Sciencesand Engineering Research Council of Canada, the Canadian Institutes ofHealth Research, the Canada Foundation for Innovation, and the CanadaResearch Chair program.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Fig. 7. Proposed mechanism of force regulation through titin-actin binding.A: passive myofibril lengthening shown with titin spanning the I-band region.No titin-actin interactions occur, so titin is able to extend over its entire freelength.B: active force is thought to enhance interaction and binding of titin toactin thereby shortening titins natural spring length. Upon stretch, strain intitins free spring element is greater for a given absolute increase in sarcomerelength, thereby increasing titins force contribution. Thus, forces in stretchedmuscle are regulated not only through actin-myosin-based cross-bridge forcesbut also through force-dependent interactions of titin with actin.




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