length-dependent effects on cardiac contractile dynamics are different in cardiac muscle containing...

Archives of Biochemistry and Biophysics 535 (2013) 3–13

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Length-dependent effects on cardiac contractile dynamics are different in cardiac muscle containing a- or b-myosin heavy chain

Steven J. Ford ⇑, Murali Chandra Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (VCAPP), Washington State University, 205 Wegner Hall, Pullman, WA 99164, United States

a r t i c l e i n f o a b s t r a c t

Article history:Available online 27 October 2012

Keywords:Papillary muscles Contractile proteins Cardiac myosins

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⇑ Corresponding author. Fax: +1 509 335 4650.E-mail address: [email protected] (S.J. Ford).

1 Abbreviations used: MHC, b-myosin heavy chain; XSL, sarcomere length; LDA, length-dependence of contvirus B-type; PTU, propylthiouracil; HR, high relamonoxime; BES, N,N-bis (2-hydroxyethyl)-2-amino-ethylene glycol tetra-acetic acid; PEP, 10 phosphoenol pML, muscle length; LRD, linear recruit ment-distortion;distortion; Tm, tropomyo sin; TnT, troponin T; TnI, trchain-1; MLC2, myosin light chain-2; LRD, linear recru

Actomyosin crossbridges (XBs) are the fundamental source of force generation and pressure development in the myocardium. Faster kinetics are imparted on XBs comprised of the fast, a-my osin heavy chain (MHC) isoform, whereas slower kinetics are imparted on XBs comprised of the slow, b-MHC isoform.Other factors, such as sarcomere length (SL), influence XB formation, presumably acting through allosteric effects on the kinetics that regulate the XB cycle. We sought to determine whether the slower XBkinetics of b-MHC were more sensitive to such length-dependent effects than those of a-MHC. We studied the SL effects on mechanical properties of demembranated muscle fibers from normal and propylt hi- ouracil-treated mouse hearts, which expressed predominantly a-MHC or b-MHC, respectively.Interestingly, XB detachment kinetics were more length-sensitive in b-MHC fibers, as estimated by tension cost and XB detachment rate constant (c), and as inferred by ktr. The nonlinearity in force responses to various-amplitude step-like changes in muscle length was more pronounced in b-MHC fibers. This phenomenon is attributed to a greater cooperative/allosteric mech anism in b-MHC fibers, as estimated by model parameter c. These data suggest a mechanism whereby greater cooperative/allosteric effects impart an enhanced length-sensitivity of XB cycling kinetics in fibers containing the slower cycling b-MHC.

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Introductio n

Actin–myosin crossbridge (XB)1 formation is the fundamental source of force generati on and contractio n in cardiac muscle. These interaction s are initiated by Ca2+ activatio n of the thin filament,and are modulated by sarcomer e length (SL) [1,2]. Furtherm ore,XBs modulat e thin filament activatio n through cooperat ive mechanisms of activatio n [3–7]. Therefore, these two intrinsi c properties of the sarcomere allow cardiac function to be modulated through structura l changes imposed by either changes in SL or changes incooperative activati on. However, whethe r there is interplay between these mechan isms of cooperative and SL-mediat ed activation isunclear.

ll rights reserved.

Bs, actomyosin crossbridges;ractile activation; FVB, friend xing; BDM, 2,3-butanedioneethane-sulfonic acid; EGTA,yruvate; PK, pyruvate kinase;NRD, nonlinear recruit ment-

oponin I; MLC1, myos in light itment-dist ortion.

SL modulates inotropy by sensitizing the myofilaments to Ca2+

and by increasing the tension developed in cardiac muscle during activation. These phenomenon are thought to occur by two widely-a ccepted mechanisms: (1) an SL-dependent increase inthick and thin myofilament overlap, which coincides with a decrease in myofilament lattice spacing in the intact sarcomere;and (2) an SL-depende nt change in the structure of the myofila-ments, which promotes Ca2+ binding to the troponin complex and acto-myosin interactions. The latter is supported by the find-ings that the structure of thin-filament regulatory proteins is animportant determinan t of the length-d ependence of contractile activation (LDA) [8–11]. Thick-filament myosin binding to actin also plays a modulato ry role in determini ng the SL dependence of Ca2+-activated tension developmen t [12–14]. The structure ofthick-filament proteins, such as myosin regulatory light chain,have been recently linked to LDA [15] and the strain-depe ndence of myosin XB turnover [16]. Furthermor e, titin, which interacts with both thick and thin myofilaments [17], has been linked toLDA (reviewed in [18]). However, whether the SL-dependent structural changes in the thin and thick myofilaments alter XB cycling kinetics, and whether such SL-dependent effects are different ina- or b-MHC XBs, remains poorly understood.

We sought to test our hypothes is that XB cycling kinetics are influenced by SL in a myosin heavy chain (MHC)-dependent

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4 S.J. Ford, M. Chandra / Archives of Biochemistry and Biophysics 535 (2013) 3–13

manner. The rationale for this study is that the different isoforms ofMHC – despite sharing a high degree of homology (e.g. [19,20]) –possess intrinsically different acto-myosin cycling kinetics. For example, cardiac a-MHC possesses faster XB cycling kinetics than does b-MHC, and imparts much faster contractile dynamics inmammalian hearts [21–25]. This implies that the kinetics of the XB cycle are highly-depend ent on the structure of MHC. Therefore,it is conceivable that muscle length may also alter the kinetics ofthe XB cycle via SL-induced changes in myofilament structure.Consequentl y, we hypothes ized that effects of SL may have a different outcome depending on structure of the MHC isoform. This follows that b-MHC XBs possess a longer XB dwell time than a-MHC XBs, and thus the kinetics of b-MHC may have a greater sensitivity of structural changes imposed by changes in SL.

To test our hypothesis, we measured a broad range of contractile parameters in fibers from the ventricle s of normal and PTU- treated mice (expressing a-MHC or b-MHC, respectively) at both SL 2.0 and 2.2 lm. We measured Ca2+-activated maximal tension,ATPase activity, tension cost, and used model-pred icted estimates of force responses to changes in muscle length to characterize contractile dynamic behavior from cardiac muscle fiber preparations.In addition, we studied nonlinear length-depend ent contractile behavior at SL 2.2 lm in a-MHC and b-MHC fibers. Differences innonlinear behavior indicated whether the underlying allosteric mechanism s, whereby length-med iated XB strain affects XBrecruitment dynamics, were different in a-MHC and b-MHC fibers.

Our results provide a novel characterizati on of how SL mediates contractile dynamics, and further how MHC isoform influences the SL effect on contractile dynamics, which is poorly understood . Our findings suggest that the heteroge neity in cardiac MHC isoforms confers unique differences in the SL-dependence of contractile dynamics in a-MHC and b-MHC fibers. We conclude that these differences in SL-depen dent contractil e dynamics may be due to the difference in XB dwell time in a-MHC and b-MHC fibers, such that b-MHC XBs are more sensitive to length-indu ced structural changes in the myofilament that mediate XB cycling dynamics.

Materials and methods

PTU treatment

All mice were housed and treated in accordance with the procedures and guidelines set by the Washington State University Insti- tutional Animal Care and Use Committee. Young adult (3–4 months in age) friend virus B-type (FVB) mice were either un- treated controls or were treated with propylth iouracil (PTU),administere d in drinking water (0.2 g L�1) and in feed for 5 weeks.PTU treatment has been shown to induce a shift ventricular MHC isoform from predomin antly a-MHC to predomin antly b-MHCwithout altering other myofilament proteins [26–28].

Preparation of cardiac muscle samples for protein analysis

Ventricular samples for protein analysis were prepared as described previousl y [29]. After excision, hearts were submerg ed inliquid nitrogen and pulverized using a mortar and pestle. The tissue was then resuspended in 10 lL of a protein extraction buffer per 1 mg of pulverized tissue. The protein extraction buffer contained the following: 2.5% SDS, 10% glycerol, 50 mM tris base (pH6.8 at 4 C), 1 mM DTT, 1 mM PMSF, 4 mM benzamid ine HCl, and contained a fresh cocktail of phosphatas e inhibitors (PhosSTOP,Roche Applied Science) and protease inhibitors (E-64, leupeptin pepstatin, and bestatin). Resuspended tissue was further homoge- nized on ice using a ‘‘Tissue Tearor’’ (Model 985370-395 Biospec

Products , Inc.), sonicated in a water bath at 4 �C, and then centri- fuged at 10K rpm.

Analysis of myofilament protein content and phosphor ylation status incardiac muscle following PTU treatment

Loading amounts were standardi zed between each sample byestimating total protein concentratio n and further optimized based on relative actin concentrations . Equal amounts of protein were loaded and separated on 6.5% SDS gels for analysis of MHC composition or 12.5% gels for analysis of myofilament protein content and phospho rylation status. 12.5% gels were stained with Pro-Q Dia- mond staining solution (Invitrogen P33300, stain; P33310, destain)to determine the levels of phosphorylated protein in ventricul arsamples. Bands correspond ing to phosphorylated proteins were visualized by UV transillumi nation and a BioRad Chemi Doc XRS camera. Following Pro-Q visualization, 12.5% gels were stained with coomass ie brilliant blue staining solution to visualize the isoform expression profiles of myofilament regulatory proteins other than MHC. Relative MHC content was quantified by densitometr icanalysis using ImageJ software (NIH, acquired at http://rsb-web.nih.gov/ij/ ) for coomassie stained gel images.

Preparatio n of detergent -skinned cardiac muscle bundles

Papillary bundles from control and PTU-treated mice were prepared using procedures described previously [22]. Mice were dee- ply anesthet ized using isoflurane and hearts were quickly excised and placed into an ice-cold high relaxing (HR) solution. The HRsolution contained the following (in mM): 20 2,3-butaned ione monoxime (BDM), 50 N,N-bis (2-hydroxyethyl)-2-amino-ethane- sulfonic acid (BES), 30.83 potassium propionate (K-propionate),10 sodium azide, 20 ethylene glycol tetra-acetic acid (EGTA), 6.29 magnesiu m chloride, 6.09 Na2ATP, 4 benzamidin e-HCl, and 1 mMof DTT. pH was carefully adjusted to 7.0 using KOH and the following protease inhibitors are added (in lM): 5 of bestatin, 2 of E-64,10 of leupeptin, 1 of pepstatin and 200 of PMSF. Papillary bundles were extracted from the left ventricle in the HR solution, from which thin muscle fiber bundles (�2–2.5 mm in length and �150–200 lm in width) were dissected . Thin muscle fiber bundles (fibers) were chemically demembran ated overnight at 4 �C in HRsolution containing 1% Triton X-100.

pCa solutions and composition s

Each fiber was bathed in different solutions, with pCa levels ranging from 4.3 to 9.0. Maximal Ca2+ activating solution (pCa4.3) contained the following (in mM): 50 BES, 5 NaN 3, 10 phosphoenol pyruvate (PEP), 10 EGTA, 10.11 CaCl 2, 6.61 MgCl 2, 5.95 Na2ATP and 31 K-propionate, whereas the relaxing solution (pCa9.0) has the following: 50 BES, 5 NaN 3, 10 PEP, 10 EGTA, 0.024 CaCl2, 6.87 MgCl 2, 5.83 Na2ATP and 51.14 K-propio nate. In addition, the maximal activating and relaxing solutions containe d0.5 mg/mL pyruvate kinase (PK), 0.05 mg/mL lactate dehydroge- nase (LDH), with the following cocktail of protease inhibitors (inlM): 10 leupeptin, 1000 pepstatin, 100 PMSF, 20 lM A2P5, 10 lMoligomyci n. The reagent concentratio ns for all intermediate pCa solutions were calculated based on the program by Fabiato (1988) [30] and the pCa solutions were prepared accordin gly.

Simultaneo us measurement of isometric steady-st ate tension and ATPase activity

Simultan eous measureme nts of isometric steady-state tensions and the ATPase activity in various pCa solutions were conducted using methods described previousl y [10,22,31]. Briefly, muscle fi-

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S.J. Ford, M. Chandra / Archives of Biochemistry and Biophysics 535 (2013) 3–13 5

bers were clipped using aluminum clips and the fiber was then at- tached to a servo motor (322C, Aurora Scientific Inc., Ontario,Canada) and a force transducer (AE801, Sensor One Technologies,Sausalito, CA). The sarcomere length (SL) of the fiber was adjusted to 2.0 or 2.2 lm in HR solution using a He–Ne laser diffraction sys- tem. The fiber was then subjected to two cycles of activation and relaxation and the SL of the fiber was readjusted to the respective SL, if necessary. The fiber was then activated with a series of pCa solutions ranging from pCa 4.3 to pCa 9.0. Simultaneous tension and ATPase measureme nts from each fiber in various pCa solutions were used to determine tension cost. Tension values at various pCa were normalized to steady-st ate maximal tension (measured atpCa 4.3) and were used to construct pCa-tension relationship s.Normalized pCa-tension relationship s were fitted using the Hill equation:

TpCa

TMax¼ pCanH

pCanH þ pCanH50

� �ð1Þ

In the equation above, TMax is the maximal isometric tension atpCa 4.3 and TpCa is the tension elicited by a fiber at a given pCa.

The ATPase activity during steady-state isometric tension production was measured using an assay described previously [10,31]. In brief, a near-UV light (340 nm) was projected through the muscle chamber which was split (50:50) for intensity detection at 340 and 400 nm waveleng ths. Light intensity of the beam at340 nm was sensitive to NADH and thus a change in the UV absorbance at 340 nm could be directly correlated to the oxidation ofNADH (and in turn, ATP usage) through enzymati cally-coupled reactions. NADH was added drop-wise to each activating solution to produce a signal substanti ally low at 340 nm to detect changes produced by the following reactions. Pyruvate kinase converts PEP and the ADP generated from the steady-state ATP hydrolysi s ofactomyosin to ATP and pyruvate, which is subsequent ly converted to lactate by LDH in a reaction that is coupled to the oxidation ofNADH to NAD +. Light intensity of the beam at 400 nm was insensi- tive to NADH and therefore served as the reference signal. An ana- log divider and log amplifier produced a signal proportio nal to the amount of ATP consumed (i.e., amount of NADH oxidized) in the activating solution. Changes in the UV absorbance signal for NADH were calibrated by multiple injections of 0.25 nmol of ADP into the activation solution.

Fig. 1. Representative data from mechanical perturbation studies conducted at maximaamplitude (0.5% initial muscle length, ML), sinusoidal length perturbation of increasing fr0.1–4 Hz sweep over a span of 40 s, and a high frequency, 2–40 Hz sweep over a span oused to determine ktr. Force responses (E) to step-like length changes (F) were fitted usibehavior.

Mechano -dynamic studies

We applied sinusoidal muscle length (ML) changes of constant amplitud e (±0.5% of ML), with increasing frequency over time (chirp) on maximally-acti vated muscle fibers (i.e. at pCa 4.3) tomeasure the dynamic force–length relationship as described previously [10,21]. Two chirps, one with frequencies ranging from 0.1 to4 Hz for a time period of 40 s, and the other with frequencies ranging from 1 to 40 Hz for a time period of 5 s, were administ ered toemphasize low and high frequency components of the force response. Representat ive chirp responses and correspond ing length-p erturbation data are shown in Fig. 1A and B, respectively.The overall force response (including both low- and high-frequency components) elicited by the fiber was fitted to a three- component, linear recruitment -distortion (LRD) model which was successfu lly used previously to elicit the dynamic features of constantly-activat ed muscle fibers [22]. In brief, the LRD model pre- dicts a change in muscle force, DF(t), corresponding to a change in ML, DL(t), in a constantly-a ctivated muscle fiber, as follows:

DFðtÞ ¼ E0gðtÞ þ Cx1ðtÞ þ Dx2ðtÞ ð2Þ

In the equation above, g(t) is a variable that describes the dynamic changes in XB recruitment with changes in length, whereas x1(t) and x2(t) represent XB distortion variables, and each possess the units of length. E0, C, and D are scaling coefficients and carry the units of stiffness. E0 is a measure of newly-recruited XBs,whereas C and D are stiffness coefficients proportional to the number of XBs in strongly- or weakly-bou nd states, respectively. The dynamic relationships of g(t), x1(t), and x2(t) with DL(t) are gov- erned by the following different ial equations:

dgðtÞdt¼ �b½gðtÞ � DLðtÞ� ð3Þ

dx1ðtÞdt

¼ �cx1ðtÞ þdLðtÞ

dtð4Þ

dx2ðtÞdt

¼ �dx2ðtÞ þdLðtÞ

dtð5Þ

where b represents the rate constant governin g the XB recruitm ent,and c and d represe nt rate constan ts that govern the distortion dynamic s of strongly- and weakly-bou nd XBs, respective ly.

l activation. The chirp force response (A) is shown corresponding to the constant- equency over time (B). Two frequency sweeps were administered; a low frequency,

f 5 s. Tension redevelopment (C) following a rapid slack, restretch protocol (D) was ng the nonlinear recruitment-distortion model to characterize nonlinear contractile


The LRD model fits were determined by minimizing the least squared errors between the simulated and experimental force responses using nonlinear regression techniques in order to estimate the six model paramete rs, E0, b, C, c, D and d. The LRD model allowed for the unique separation of recruitment dynamics (E0, b)from distortion dynamics (C, c, D and d). The distortion component,Dx2(t) in Eqs. 2, 5 contributed very little to the force response within the frequency range of interest (e.g., �0.1–4 Hz), and therefore,we only report b, E0, c, C paramete rs in this study. A low standard error (<1% of the parameter value) in each of these model parameters indicated that these estimate s were robust and reliable for comparisons in this study.

Rate constant of tension redevelopme nt (ktr)

The rate constant associated with the tension redevelopmen tfollowing rapid slack/res tretch, ktr was estimated based on the protocol described previously [22,32]. In this protocol, the maximally-activa ted fiber (i.e. at pCa 4.3) was rapidly slackened by10% of its preset ML in a step-like fashion. After 25 ms, the motor arm was commanded to swing beyond its original set point, there- by stretching the fiber by 10% past its set point. The muscle fiberwas then rapidly brought back to its set point at which point the muscle fiber was allowed to redevelop force. This method was done in order to break any strongly-bound XBs to minimize residual force from which tension starts to redevelop force. Fig. 1C and D, respectively illustrate a representat ive tension redevelopmen tforce response and the rapid slack-restretch protocol. ktr wasestimated by fitting a mono-expo nential function given byF ¼ ðF0 � FresÞð1� ektr tÞ þ Fres, where Fo is the steady state isometric force and Fres is the residual force from which the fiber starts toredevelop tension. All the fiber records measured during the tension redevelo pment were well fitted by a mono-ex ponential relation with R2 P 0.9.

Determinati on of nonlinear length-depende nt contractile behavior inmuscle

Once the muscle fiber attained steady-state maximal activation (i.e. pCa 4.3), step-like length changes were applied to the fibers, asdescribed previously [33]. In brief, ML was rapidly stretched (by0.5%, 1.0%, 1.5%, and 2.0% of ML) and held at the increased length for 5 s. The motor arm was then commanded to rapidly return (representing release by 0.5%, 1.0%, 1.5%, and 2.0% of ML) to the initial ML, where it was held for another 5 s. Representat ive force responses and correspondi ng step-length perturba tion data are shown in Fig. 1E and F, respectively . A previously-devel oped nonlinear formulation of the recruitment distortion (NRD) model [33] was fitted to the entire family of force responses (Fig. 1E) tocharacterize the XB cycling dynamics in the muscle fiber. The force responses, F(t), to step-like length changes were represented as aproduct of the net stiffness of the fiber, g(t), times the distortion of the fiber, x(t):

FðtÞ ¼ gðtÞxðtÞ ð6Þ

g(t) represents the stiffness of the fiber, and is proportional tothe number of XBs bound at time t. The change in ML, DL(t), due to the step perturbation , results in a change in recruitment of the number of bound XBs, and g(t) changes with time as shown inEq. (7). b is a rate constant that determines the speed at which the recruitment variable g(t) changes and c is a parameter that represents how XB distortion , x(t), influences XB recruitment . x(t)represents the mean distortion of bound XBs, and changes with re- spect to the velocity of ML change, dLðtÞ

dt , as shown in Eq. (8). c is arate constant that determines the speed at which XB distortion dis- sipates, as XBs that were distorted due to dLðtÞ

dt detach and repopu-

late into a non-distorted state. Therefore, c is considered ameasure of the rate of XB detachment and b is considered a measure of the rate of XB recruitment . c is described as a parameter that characterizes how XB strain influences the dynamics of XBrecruitment . Therefore, c may be considered a measure of thin fil-ament-bas ed cooperative/all osteric mechanism s that modulate XB-mediate d effects on other XBs.

dgðtÞdt¼ �b 1þ c

xðtÞ � x0

x0

� �2 !

gðtÞ þ bDLðtÞ ð7Þ

dxðtÞdt¼ �cxðtÞ þ 1

m0

dLðtÞdt

ð8Þ

Eq. (7) contains a variation of the formula tion described in Ford et al. (2010). c was estimated in the model fits with a high degree of confidence, similar to that reported in Ford et al. (2010).

Data analysis

Measurem ents of contractil e function and dynamics from un- treated or PTU-trea ted mouse muscle fibers were compared using two-way ANOVA, with one factor being MHC isoform (a-MHC vs.b-MHC) and the other being SL (2.0 lm vs. 2.2 lm). The interaction effects were evaluated to test the hypothesis that MHC isoform altered the effects of sarcomere length on cardiac function.Subseque nt post hoc multiple pairwise comparisons and made using Fisher’s LSD method. Statistical significance was assumed for p < 0.05.

Results

We sought to understand whether length-depend ent effects oncardiac contractile function and dynamics were different in fiberscontaining a-MHC and b-MHC. To better understand this relationship, we compare d the sarcomere length (SL)-dependence of the relationshi p between tension and ATPase activity at varying levels of Ca2+ activation, and of contractil e dynamic behaviors (i.e. those elicited in chirp responses) in cardiac muscle fibers isolated from normal (a-MHC) or PTU-treated (b-MHC) mice. We also investigated whether cooperative/all osteric mechanisms that underlie nonlinear behavior in step responses differ in a- or b-MHC fibers.Results were interpreted using two-way ANOVA, with significantinteractio n implying that the SL-dependence of a given contractile function parameter is dependent on MHC isoform.

Sarcomer ic protein expression profiles and phosphorylation status innon-treat ed and PTU-treated mouse ventricular myocardiu m

Ventricul ar samples were prepared from non-treated and PTU- treated mice, and protein compositions were separated by SDS PAGE (Fig. 2). MHC isoform expression was shifted from predominantly a-MHC to predomin antly b-MHC following 5 weeks of PTU treatment (Fig. 2A). This shift was quantified using densitometric analysis; non-treated mice expressed a-MHC as �95% of total MHC, whereas PTU-trea ted mice expressed b-MHC as �90% of total MHC. Due to this near-complete shift in MHC expression, we refer to non-treated fibers as a-MHC fibers, and PTU-treated fibers as b-MHC fibers throughout the rest of the manuscript. In agreement with previous studies [34,35], PTU-trea tment did not affect the phospho rylation status of sarcomeric proteins (e.g. cardiac myosin binding protein C (MyBPC), desmin, troponin T (TnT) cardiac a-tropomyos in (a-Tm), troponin I (TnI) myosin light chain-1 (MLC1), or myosin light chain-2 (MLC2); Fig. 2B). Also in agreement with previous studies [23], we found that PTU treatment did not affect the expression profile of other sarcomeri c proteins

Fig. 2. Effect of five weeks of PTU treatment on isoform expression of MHC and other myofilament proteins, and on myofilament protein phosphorylation status in mouse ventricular preparations. Separation of a-MHC and b-MHC isoforms was done on 6.5% SDS polyacrylamide gels, as shown in panel A. Separation of other myofilament proteins was done on 12.5% gels. After separation, the phosphorylation status of myofilament proteins was visualized by UV-transilluminescence following Pro-Q diamond staining of12.5% gels, as shown in panel B. After Pro-Q imaging, gels were stained with coomassie blue to visualize whether the isoform expression profiles of proteins other than MHC were altered by PTU treatment, as shown in panel C.


(e.g. actin, TnT, a-Tm, TnI, MLC1, MLC2 or troponin C (TnC);Fig. 2C).

Effects of SL on tension production and ATPase activity at varying levels of Ca2+ activation in a- vs. b-MHC fibers

The SL-depen dent effects on maximal tension developmen t orATPase activity were not different in a-MHC or b-MHC fibers. For example, the increase in SL from 2.0 to 2.2 lm gave rise to a 14%or 13% increase in maximal tension in a-MHC or b-MHC fibers,respectively (Fig. 3A). Two-way ANOVA revealed that the interaction effect of SL and MHC did not have a significant impact on maximal tension. Therefore, the SL-dependence of maximal tension developmen t was the same for a-MHC or b-MHC fibers. The main effects of an increase in SL (p = 0.0005) or b-MHC expression

Fig. 3. Effect of increase in SL on contractile activation, as estimated by the percent i(DpCa50) (B). Estimates of myofilament Ca2+ sensitivity (pCa50) were determined by fitdeterminants is 10 for each group. ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001, when comparing tension or

(p < 0.0001) significantly increased tension production. Two-way ANOVA also revealed that the interaction effect of SL and MHC on maximal ATPase activity was not significant. Main effects ofb-MHC (p < 0.0001), but not SL, significantly reduced the rate ofATPase activity. Post hoc analysis further revealed that ATPase activity was significantly lower at SL 2.2 lm vs. 2.0 lm in b-MHCfibers (p < 0.05), but not in a-MHC fibers.

We measure d tension production at various levels of Ca2+ acti-vation to construct pCa-tension relationship s in muscle fibers.Hill’s equation was fitted to pCa-tension relationships to get estimates of myofilament calcium sensitivit y (pCa50, Table 1) and cooperati vity of tension development (nH, Table 1). The SL–MHCinteractio n effect was not significant on pCa 50. This suggests that SL-depen dent increase on myofilament Ca2+ sensitivity (DpCa50,Fig. 3B) was not different in a-MHC or b-MHC fibers. As was seen

ncrease in tension development (A), or the increase myofilament Ca2+ sensitivity ting Hill’s model to pCa-tension relationships. Values are mean + SEM. Number ofpCa 50 values at SL 2.0 lm vs. 2.2 lm within a-MHC or b-MHC fibers groups.


with max tension, the main effects of an increase in SL (p = 0.0005)or b-MHC expression (p < 0.0001) significantly increased pCa 50.Along with our finding that SL dependence of tension developmen twas not different , these data suggest that the length-depend ence of myofilament activation was not different in a-MHC or b-MHCfibers. The SL–MHC interactio n effect on nH was significant(p < 0.05); post hoc analysis revealed that the SL-mediated effect on nH was less pronounced in b-MHC fibers than in a-MHC fibers.At SL 2.0 or 2.2 lm, nH was 3.08 ± 0.22 or 2.16 ± 0.09 (DnH = 0.92,p < 0.01) in a-MHC fibers, whereas nH was 2.81 ± 0.07 or2.46 ± 0.06 (DnH = 0.35, p < 0.01), respectivel y.

The slope of the relationship between tension developmen t and ATPase activity at various pCa is reported as tension cost (Fig. 4).The SL–MHC interactio n effect on tension cost was not significant,but the main effects of SL or MHC were both significant; an increase in SL (p = 0.0012) and b-MHC (p < 0.0001) significantly decreased tension cost. Further analysis revealed that the SL- dependent decrease in tension cost was slightly more pronounced

Table 1Maximal tension (mN mm�2) and rate of ATPase activity (pmol mm�3 s�1) estimated in fibpCa-tension relationships to estimate myofilament Ca2+ sensitivity (pCa50) and cooperativieach group.

SL Fibers from normal mice (a-MHC)

2.0 lm 2.2 lm

Max Tension 38.01 ± 0.62 43.75 ± 1.53 *

Max ATPase 319.72 ± 8.99 319.83 ± 18.60pCa50 5.73 ± 0.01 5.78 ± 0.01 *

nH 3.08 ± 0.22 2.16 ± 0.09 *

* p < 0.05 vs. short SL in same (a-MHC or b-MHC) group of fibers.a p < 0.05 vs. a-MHC fibers at same SL.

Fig. 4. Relationship between the rate of ATP hydrolysis and corresponding tension develofibers. Tension cost is determined by the slope of this linear relationship, and the slope vaeach group. ⁄p < 0.05; ⁄⁄p < 0.01.

in b-MHC (�14% decrease, p < 0.01) than in a-MHC (�11% decrease, p < 0.05) fibers (Fig. 4C).

Effects of SL on XB recruitment and distortion dynamics in a- vs. b-MHC fibers

Contractil e dynamic parameters of a-MHC and b-MHC fibers atdifferent SLs were characterized by fitting a linear recruitment -distortion (LRD) model to force responses to chirp length perturbations. Fitted model predictions determined from a-MHC or b-MHC (shown in Fig. 5A and B), illustrate an apparent difference in the contractile dynamics at SL 2.0 and 2.2 lm. Fig. 5C–F are shown to illustrate the SL effects on the XB recruitment or XB distortion model components. SL did not have an apparent influenceon XB recruitment dynamics in either a-MHC (Fig. 5C) or b-MHC(Fig. 5D) fibers. An increase in SL did have an apparent effect ofslowing XB distortion dynamics in a-MHC fibers (Fig. 5E), but this effect seemed more pronounced in b-MHC fibers (Fig. 5F).

ers from normal or PTU-treated mice at SL 2.0 or 2.2 lm. Hill’s equation was fitted toty (nH) parameter s. Values are mean ± SEM. Number of determinants is at least 10 for

Fibers from PTU-treated mice (b-MHC)

2.0 lm 2.2 lm

46.75 ± 0.90 a 52.84 ± 2.37 *,a

163.67 ± 5.15 a 145.90 ± 5.19 a

5.81 ± 0.01 a 5.85 ± 0.01 *,a

2.81 ± 0.07 a 2.46 ± 0.06 *,a

pment at varying levels of Ca2+ activation in normal (A) and PTU-treated (B) mouse lues are represented in C. Values are mean ± SEM. Number of determinants is 10 for


LRD model parameters b and c were used to quantify these SL- dependent effects on XB recruitment or XB distortion dynamics,respectively . The effect of SL on b was not different in a-MHC orb-MHC fibers (Fig. 6A). The SL–MHC interaction effect did not have a significant effect on b, suggestin g that the SL-mediated effect onXB recruitment dynamics was not different in a-MHC or b-MHC fi-bers. b-MHC expression, but not SL, had a significant effect (p < 0.0001) on slowing b.

Interestingl y, the effect of SL on c was different in a-MHC or b-MHC fibers (Fig. 6B). Although the SL–MHC interaction effect was not significant, post hoc analysis revealed differenc es in the SL- dependence of c in a-MHC and b-MHC fibers. At SL 2.0 or2.2 lm, c decrease d from 54 to 47 s�1 (�17% slower, not signifi-cant) in a-MHC fibers, whereas c decreased from 26 to 19 s�1

(�25% slower, p < 0.01) in b-MHC fibers. Since the rate of XBdetachment is considered to be a major determini ng factor for c[21,22], these data suggest that XB detachment rate is sensitive to SL in b-MHC, but not a-MHC fibers.

Fig. 5. Linear recruitment-distortion (LRD) model-predicted force responses to sinusoidaSL 2.0 or 2.2 lm. Model components are shown in panels C–F to illustrate the SL-based effcomponents are shown for fibers from a-MHC (C) or b-MHC (D) fibers. High-frequency mmodel predictions are determined based on average model fits from each group of chirp

Effects of SL on the rate constant of tension redevelopment (ktr) in a-vs. b-MHC fibers

The rate of tension redevelopmen t, following rapid slack and restretch was approximat ed by the monoexponen tial rate constant , ktr. We examined the effects of SL on the rate oftension development by estimating ktr at both SL 2.0 and 2.2 lm. Tension redevelopmen t traces measured from a-MHCor b-MHC fibers, at both SL, are shown in Fig. 7A and B,respectively . The SL–MHC interaction did not have a signifi-cant effect on ktr, however post hoc analysis suggested differences in the SL-depende nce of ktr in a-MHC and b-MHCfibers. When comparing ktr at SL 2.0 vs. 2.2 lm, ktr was not different in a-MHC fibers, but an increase in SL caused ktr

to significantly decrease (�13% decrease, p < 0.01) in b-MHCfibers (Fig. 7C). These findings further suggest that contractile dynamics are more depende nt on SL in b-MHC fibers than ina-MHC fibers.

l length perturbations of increasing frequency in a-MHC (A) and b-MHC (B) fibers atects on XB recruitment (C, D) or XB distortion (E, F) dynamics. Low-frequency model odel components are shown for fibers from a-MHC (E) or b-MHC (F) fibers. Plotted data.

Fig. 6. Model-predicted estimates of the rate constant of length-mediated XB recruitment, b (A), and the rate constant associated with XB distortion, c (B). b approximates the rate of XB recruitment in response to length change. c approximates XB detachment rate and correlates well with tension cost. Parameter values are mean + SEM. Number ofdeterminants is 10 for each group. ⁄⁄p < 0.01.


Nonlinear XB strain-depende nt effect on XB recruitmen t dynamics ina- vs. b-MHC fibers

Nonlinear contractile behavior was characterized by fitting the nonlinear recruitment-di stortion (NRD) model to a family of force responses to various amplitude step-length changes. The NRD model is formulated with a parameter, c, to reproduce the nonlinearity observed in the family of force responses to various amplitude quick stretches and releases [33]. Physiological ly, cinterprets how XB strain negatively impacts the dynamics of XBrecruitment . Because such XB–XB interactions are indirect, c isthought to interpret allosteric/cooper ative mechanism s in the myofilaments. The force responses to step perturbation s elicited a nonlinear behavior that was more pronounced in b-MHC fibersthan was in a-MHC fibers (Fig. 8A). This gave rise to a significant(p < 0.0001) increase in the NRD model parameter c in b-MHC fi-bers (Fig. 8B). This suggests that the allosteric mechanisms,through which XB strain influences XB recruitment, are more prominent in b-MHC fibers than in a-MHC fibers.

Fig. 7. Tension redevelopment following rapid slack and restretch protocol in a-MHC (length change, and are shown as ensemble averages of at least 10 data records. Tension rillustrate any length-dependence of the rate of tension redevelopment. The rate of tensiovalues are shown as mean + SEM. Number of determinants is 10 for each group. ⁄⁄p < 0.

Discussion

We sought to determine whether the length-depe ndence ofcontractil e function and XB cycling dynamics would be different in cardiac fibers containing a-MHC or b-MHC. The rationale for this study was that: (1) a-MHC and b-MHC impart very different contractile dynamics in cardiac muscle [21,22,24,25]; and (2) a-MHCand b-MHC are expresse d different ly across different species [36,37], during developmen t and aging [37–42], and in cardiac dis- ease [43–47], where contractile function is widely varied. The mechanis ms behind these correlations, while unclear, may belinked to differences in XB cycling rates. The slower cycling, and thus, longer dwell time of b-MHC XBs, may allow for allosteric length-se nsing mechanisms of the myofilaments to impart differences in contractile function. We hypothesized that the length- sensing mechanism s that regulate contractile function were different in a-MHC or b-MHC fibers. To test our hypothesis, we approx- imated contractile dynamics in fibers from normal (a-MHC) orPTU-trea ted (b-MHC) mice at SL 2.0 or 2.2 lm. Interestingly, we

A) or b-MHC (B) fibers. Data are normalized to isometric tension attained prior toedevelopment traces at long SL (solid lines) and short SL (dashed lines) are shown ton redevelopment was determined by the monoexponential rate constant, ktr (C). ktr

01.

Fig. 8. Force responses to large-amplitude (2% muscle length) step-like stretch or release (A) in a-MHC (solid lines) or b-MHC (dashed lines) fibers. Data are normalized toisometric tension (FIso) attained prior to length change, and are shown as ensemble averages of at least 10 data records. Nonlinearity in the responses to stretch or release isseen in both a-MHC and b-MHC fibers; the difference in the shape of the transient and presence of a nadir in the responses to large amplitude stretch, but not release,illustrates this nonlinearity. This nonlinearity is ascribed to effects that XB strain has on recruitment of other XB, and is thus considered a measure of allosteric mechanisms ofmyofilament activation. The nadir in the response to stretch was more prominent in b-MHC fibers than in a-MHC fibers, demonstrating a more prominent nonlinearity. The nonlinear recruitment-distortion (NRD) model was fitted to the family of force responses to various amplitude stretch or release to quantify this nonlinearity with the parameter c. As illustrated in panel B, c predicted that the nonlinear behavior was significantly higher in b-MHC fibers. c values are shown as mean + SEM. Number ofdeterminants is 10 for each group. ⁄⁄⁄p < 0.001.


found that b-MHC, but not a-MHC, exhibited an apparent length- dependence of XB detachment dynamics. Furthermore, an increased c was suggestive of increased strain-depende nt allosteric/cooperat ive effects in fibers containing b-MHC XBs.

Length dependence of tension productio n and myofilament Ca2+

sensitivity is not affected by MHC isoform

Length-depend ent activation (LDA) is a well-documen ted phenomenon whereby an increase in SL results in an increase in myofilament tension production and Ca2+ sensitivity [1,2]. LDA is animportant phenomeno n of healthy heart function, and underlies the Frank-Starling law of the heart. Therefore, understa nding the mechanism s that contribute to LDA is central to understanding healthy heart function, but currently remains unclear. However ,two underlying themes are widely accepted to contribute to LDA:(1) changes in myofilament overlap and lattice spacing in response to changes in SL; and (2) changes in the structure of the thin and thick myofilaments in response to changes in SL. The former theme suggests proximity effects that allow for an increase in probability of XB binding as SL increases, whereas the latter theme suggests anallosteric mechanism that allows the sarcomeres to become more sensitive to activation at longer lengths. The latter is supported by the fact that changes in the thin-filament structure influencesLDA [8–11]. Furthermore, the structure of thick filament proteins appears to play a role in LDA. For example, the presence of XBs influence LDA [12–14], and furthermore , alteration s in myosin regulatory light chain have been linked to modulating LDA [15] andthe strain-de pendence of myosin XB cycling [16]. However,whether a-MHC or b-MHC isoform expression leads to differences in length-depe ndent XB cycling dynamics is not currently understood .

We investigated whether the SL dependence of tension and pCa50 were different in a-MHC or b-MHC fibers. Interestin gly, wefound that maximal tension development and pCa 50 were slightly,but significantly higher in b-MHC fibers than was in a-MHC fibers.These findings are different than results published earlier [23–25,47,48], but these discrepancie s could be attributed to differences in preparati on procedures. For example, results obtained inprevious studies were from samples dissected using mincing –vs. microdissecti on in our study – and included both right- and left-ventricu lar preparations [23–25,48] – vs. only left ventricle preparations in our study. Regardless of this discrepan cy, we ob-

served no differenc e in the SL-depende nce of tension developmen t(Fig. 3A) in a-MHC or b-MHC fibers. Similarly, there was no difference in the SL-depende nce of pCa 50 (Fig. 3B), suggesting that the SL-depen dence of contractile activation was not different between a-MHC and b-MHC fibers. Thus, the inotropic responses of tension and myofilament Ca2+ sensitivity to an increase in SL were not different in a-MHC or b-MHC fibers.

Length dependence of contractile dynamics is more sensitive in b-MHCfibers

It is well-documen ted that b-MHC imparts slower contractile dynamic behaviors in cardiac muscle when compared to a-MHC[21–24]. However , whether contractil e dynamics are sensitive tolength, and whether the length-depe ndence of the contractile dynamics is different in a-MHC and b-MHC cardiac muscle is not well understood . This may hold physiological significance, aschanges in preload, and thus changes in SL may allow the heart to impart different contractile dynamics on a beat-to-bea t basis.Thus, we sought to determine whether changes in SL could influ-ence contractil e dynamics, and further, whether MHC isoform plays a role in the determination of the length-depend ence of contractile dynamics.

LRD model parameters were fitted to force responses to sinusoidal length perturbations (as in [21,22]) to determine effects of SLand MHC isoform on contractil e dynamics . Consistent with previous findings, b-MHC isoform had a major impact on slowing both LRD model predicted rates of XB recruitment , b (Figs. 5C, D and 6A), and XB distortion, c (Figs. 5E, F and 6B). We observed no significant SL dependence on the model-predi cted rate constant ofXB recruitment, b, whether fibers were of a-MHC or b-MHC back- ground (Fig. 6A). This indicates that SL did not alter the speed of XBrecruitment in response to length in either a-MHC or b-MHC fibers.Interestin gly, however , an increase in SL significantly slowed c in b-MHC fibers, but not a-MHC fibers (Fig. 6B). This finding was cor- roborated by our measureme nt of tension cost (Fig. 4C), whereby tension cost decrease d as SL increased, a trend that was more pronounced in b-MHC fibers. Both c and tension cost are limited by the rate constant of XB detachment , and are considered parameters that can infer XB detachment kinetics [21,49]. Therefore, our find-ings suggest that an increase in SL speeds XB detachment kinetics in b-MHC, but not a-MHC fibers.


In a two state XB model, ktr estimated at maximal activation can be approximat ed as the sum of XB cycling forward, f, plus detachment, g, rate constants [32]. At maximal activation, ktr was not different in a-MHC fibers, but was significantly lower at the longer SLin b-MHC fibers (Fig. 7C). These findings correlate well with those reported by Korte and McDonald (2007) [50], where a length- dependent decrease of ktr in b-MHC, but not in a-MHC fibers,was also observed. Taken together with findings of decreased cand tension cost – and the finding that b was not affected by SL– ktr findings suggest that an increase in SL decreases the apparent rate of XB detachment, g in b-MHC fibers, without affecting f.

b-MHC exhibits a greater XB strain effect on XB recruitment;implications for cooperative and/or allosteric mechanisms that underlie nonlinear contractile behavior

The NRD model parameter c is a nonlinear interaction term that represents the effect that strained XBs have on the recruitment ofother XBs [33]. The XB strain effect on recruitment of other XBs isassumed to transmit through cooperative and allosteric mechanisms of the thin and thick filaments. For example, the slowing effect of XB strain on XB recruitment may be due, in part, to the compliant realignment of myosin binding sites on actin relative to myosin heads [51]. Therefore, c reflects cooperative and/or allosteric mechanism s operating within sarcomeres . We performed step perturbation analysis at SL 2.2 lm and found that the nonlinearity in the shape of the curves was much more pronounced in b-MHC fibers (Fig. 8A), which gave rise to a significantly higher c(Fig. 8B). Similarly , b and c from the NRD model predictions were slowed by �2-fold in the b-MHC fibers (data not shown), which matched the trends seen in b and c from the chirp LRD model.These trends also correlate well with the MHC-dep endent trends reported for kdf and k1 by Stelzer et al. [25], which are determined by the same phases of the F(t) response to step-length changes that principally determine c and b, respectively .

The significant finding of an increased c indicates that allosteric and/or cooperative activation mechanism s within the myofila-ments are more pronounced in b-MHC fibers. This change may be due to XB feedback effects on the regulator y units (RU), whose activation is tightly coupled to both Ca2+- and XB-induced changes in the thin filament (for review, see [52]). Because XBs themselves affect the balance between RU ‘on’ and ‘off’ states through cooperative activation, b-MHC may have a more prominent strain-dependent effect on XB recruitment dynamics due to its slower cycling.This suggests that the higher duty time of b-MHC XBs imparts agreater effect of XB-strain feedback on RU on/off dynamics, shifting the equilibrium more so toward the RU off state when XBs are strained.

In conclusion, our data suggest that cardiac MHC isoforms dif- ferently influence the outcome of how SL influences contractile dynamics. We report an SL-depen dent decrease in the rate of XBdetachment and ktr in fibers containing b-MHC, but not a-MHC.Furthermore, the nonlinear effect that XB strain has on XB recruitment was greater in b-MHC fibers, as approximat ed by c. These findings are suggestive of a more prominent cooperative mechanism involved in mediating XB cycling dynamics in b-MHC fibers.We attribute this increased cooperative effect to the slower cycling, and thus higher duty time of XBs in b-MHC fibers. b-MHCXBs may be more sensitive to SL-based effects because of their longer attachment time, allowing more time for length-depend ent allosteric effects to impact XB cycling dynamics. Collectiv ely, these findings imply that kinetics of the b-MHC XB cycle are more dependent on allosteric length-sens ing mechanism s in cardiac muscle than the a-MHC XB cycle. These results further suggest that larger mammals may possess a greater SL-depende nce of contractile

dynamics than smaller mammals that express relatively higher a-MHC content. Therefore, our data suggests that animals expressing predominantly b-MHC would be a better model for understanding the Frank-Starling law of the human heart.

Acknowled gments

This work was supported by the National Heart, Lung, and Blood Institute grant R01-HL75643 (to M.C.), and the American Heart Association 10PRE34800 45 and ARCS fellowship s (to S.J.F.).

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