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(Circulation Research. 2006;98:1212.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Ablation of Cardiac Myosin-Binding Protein-C Accelerates Stretch Activation in Murine Skinned Myocardium

Julian E. Stelzer, Sandy B. Dunning, Richard L. Moss

From the Department of Physiology, University of Wisconsin School of Medicine and Public Health, Madison.

Correspondence to Julian E. Stelzer, Department of Physiology, University of Wisconsin Medical School, 601 Science Dr, Madison, WI 53711. E-mail stelzer{at}physiology.wisc.edu

	Abstract

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Cardiac myosin binding protein-C (cMyBP-C) is a thick filament accessory protein that binds tightly to myosin, but despite evidence that mutations in the cMyBP-C gene comprise a frequent cause of hypertrophic cardiomyopathy, relatively little is known about the role(s) of cMyBP-C in myocardium. Based on earlier studies demonstrating the potential importance of stretch activation in cardiac contraction, we examined the effects of cMyBP-C on the stretch activation responses of skinned ventricular preparations from wild-type (WT) and homozygous cMyBP-C knockout mice (cMyBP-C^–/–) previously developed in our laboratory. Sudden stretch of skinned myocardium during maximal or submaximal Ca²⁺ activations resulted in an instantaneous increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (ie, stretch activation) to levels greater than prestretch force. Ablation of cMyBP-C dramatically altered the stretch activation response, ie, the rates of force decay and delayed force transient were accelerated compared with WT myocardium. These results suggest that cMyBP-C normally constrains the spatial position of myosin cross-bridges, which, in turn, limits both the rate and extent of interaction of cross-bridges with actin. We propose that ablation of cMyBP-C removes this constraint, increases the likelihood of cross-bridge binding to actin, and speeds the rate of delayed force development following stretch. Regardless of the specific mechanism, acceleration of cross-bridge cycling in cMyBP-C^–/– myocardium could account for the abbreviation of systolic ejection in this mouse as a direct consequence of premature stretch activation of ventricular myocardium.

Key Words: cross-bridge kinetics • cooperativity • contractile proteins • regulation of contraction

	Introduction

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Familial hypertrophic cardiomyopathy (FHC) in humans is an inherited autosomal dominant disease that, in most cases, is caused by mutations in sarcomeric proteins. Mutations in one sarcomeric protein, cardiac myosin binding protein-C (cMyBP-C), have been reported to be among the most common causes of FHC,^1–3 and yet relatively little is known about its function. To better understand the role of cMyBP-C in myocardial contraction, we previously developed a cMyBP-C–null mouse (cMyBP-C^–/–) specifically lacking cMyBP-C.⁴ cMyBP-C^–/– mice exhibit severe ventricular hypertrophy, systolic dysfunction evident in abbreviation of ejection and reduced stroke volume, and diastolic dysfunction seen as a decreased rate of isovolumic relaxation.^4,5 The present study was undertaken to determine the effects of ablation of cMyBP-C on the dynamic mechanical properties of myocardium to understand the basis for functional deficits in the cMyBP-C^–/– mouse.

During both the isovolumic and ejection phases of systole, the left ventricle undergoes significant torsional deformation as the apex twists counterclockwise (as viewed from the apex) relative to the base,⁶ with the endocardium exhibiting greater torsional strain than the epicardium.⁷ Such a differential in torsional strain across the ventricular wall might be expected to modulate systolic force generation and the timing of force production in different parts of the wall. Because higher force regions would stretch lower force regions during torsional twist, there should be stretch activation (delayed development of force) of subendocardial myocardium, similar to the delayed development of force developed by isolated cardiac muscle following stretch.⁸ Stretch activation may thus play a role in myocardial power generation and systolic ejection.⁹ In this regard, a recent study showed that the rate and amplitude of the stretch activation response in mouse myocardium varies with the level of activation, suggesting that the response to stretch likely is regulated on a beat-to-beat basis.^10,11

Given the systolic dysfunction evident in the cMyBP-C^–/– mouse, we hypothesized that the stretch activation response in cMyBP-C^–/– myocardium would be reduced or absent and thereby account for reduced systolic ejection. Myosin binding protein-C (MyBP-C) is bound to thick filaments in distinct stripes (between 7 to 11) in the C-zone of the A-bands in each half sarcomere.¹² The C terminus of MyBP-C contains a binding domain,¹³ with high affinity for myosin subfragment-2¹⁴ and the myosin molecule contains sites for binding MyBP-C within the S2 and light meromyosin (LMM) fragments.^15,16 Because the periodicity of MyBP-C¹⁷ is very similar to that of myosin (42.9 nm¹⁸) and it binds to myosin in a functionally important region, it is plausible that cMyBP-C plays an important role in the regulation of cross-bridge movement and interaction with actin, as suggested previously.^19–21

To explore whether the reduced ejection fraction observed in cMyBP-C^–/– hearts is attributable to alterations in stretch activation, we compared stretch activation responses in wild-type (WT) and cMyBP-C^–/– myocardium. Contrary to our initial hypothesis that stretch activation in cMyBP-C^–/– myocardium is reduced or absent, we observed no change in the amplitude of stretch activation, which by itself is not consistent with depressed systolic function in the null mouse. However, the rate of rise of force during delayed force development was substantially faster than in WT myocardium, such that the earlier than normal stretch activation response could account for the premature cessation of ejection observed in cMyBP-C^–/– mice.

	Materials and Methods

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Transgenic Mice
Homozygous cardiac MyBP-C–null (cMyBP-C^–/–) mice were generated previously⁴ and maintained in a breeding colony. For experimental measurements, adult mice (3 to 6 months of age) of either sex were used. Aged-matched WT mice of the same background strain as cMyBP-C^–/– mice (SV/129; Taconic Farms, Germantown, NY) were used as controls. All procedures involving animal use were performed according to institutional guidelines approved by the Association for Assessment and Accreditation of Laboratory Care International (AAALAC).

Solutions
Solution compositions were calculated using the computer program developed by Fabiato²² and the stability constants listed by Godt and Lindley²³ corrected to pH 7.0 and 22°C. All solutions contained (in mmol/L) 100 N,N-bis(2 hydroxy-ethyl)-2-aminoethanesulfonic acid (BES), 15 creatine phosphate, 5 dithiothreitol, 1 free Mg²⁺, and 4 MgATP. In addition, pCa 9.0 solution contained (in mmol/L) 7 EGTA and 0.02 CaCl₂, pCa 4.5 contained 7 EGTA and 7.01 CaCl₂, and preactivating solution contained 0.07 EGTA. Ionic strength of all solutions was adjusted to 180 mmol/L with potassium propionate. Solutions containing different amounts of [Ca²⁺]_free were prepared by mixing appropriate volumes of stock solutions of pCa 9.0 and pCa 4.5.

Skinned Myocardial Preparations
Skinned ventricular myocardium for stretch activation experiments was prepared as previously described.²⁴ Briefly, following IP injection of 5000 U heparin/kg body weight, S129 WT mice (3 to 6 months old) were anesthetized with inhaled isoflurane (15% isoflurane in mineral oil) in accordance with institutional animal care guidelines. Their hearts were excised and right and left ventricles were dissected at room temperature in a relaxing solution containing (in mmol/L) 100 KCl, 20 imidazole, 7 MgCl₂, 2 EGTA, and 4 MgATP (pH 7.0) and were then rapidly frozen in liquid nitrogen. To prepare skinned myocardial preparations, the frozen ventricles were thawed and homogenized in relaxing solution for 2 seconds using a Polytron, which yielded multicellular preparations of 100 to 250 µmx600 to 900 µm. The homogenate was centrifuged at 120g for 1 minute and the resulting pellet was washed with fresh relaxing solution and resuspended in relaxing solution containing 250 µg/mL saponin and 1% Triton X-100. After 30 minutes, the skinned preparations were washed with fresh relaxing solution and were dispersed in 50 mL of relaxing solution in a glass Petri dish. The dish was kept on ice at all times, except during the selection of individual preparations for mechanical experiments.

Apparatus and Experimental Protocol
Skinned preparations with well-defined edges and no evident free ends in the middle region were transferred from the Petri dish to a stainless steel experimental chamber²⁵ containing relaxing solution. The ends of a preparation were attached to the arms of a motor (model 312B, Aurora Scientific Inc) and force transducer (model 403; Aurora Scientific Inc), as described earlier.²⁵ The chamber assembly was then placed on the stage of an inverted microscope (Carl Zeiss) fitted with a x40 objective and a closed-circuit television camera (model WV-BL600, Panasonic). Bitmap images of the preparations were acquired using an AGP 4X/2X graphics card and associated software (ATI Technologies Inc) and were used to assess mean sarcomere length during the course of each experiment. Changes in force and motor position were sampled (16-bit resolution, DAP5216a; Microstar Laboratories, Bellevue, Wash) at 2.0 kHz using SLControl software developed in our laboratory²⁶ and saved to computer files for later analysis. Fiber force during the experiments was also recorded on a digital oscilloscope (Nicolet Instrument Corporation, Madison, Wis).

At the start of each experiment, the preparations were stretched to a mean sarcomere length of 2.12 µm, such that resting force was minimal for measurements of steady-state Ca²⁺-activated force and stretch activation. The preparations were initially activated at pCa 4.5 to establish the maximal tension (P_o), and then force was recorded in pCa 9.0 to establish the resting force, which was then subtracted from the total force in activating solutions to yield the Ca²⁺-activated force (P). For stretch activation experiments, fibers were activated in pCa solutions, yielding maximum force (pCa 4.5), 50% of maximum force, and 25% of maximum force. When steady-state tension was reached, a rapid stretch of 1% fiber length (L_o) was imposed and held for 5 seconds before returning the fiber to pCa 9.0.

The stretch activation variables measured are shown in Figure 1. All amplitudes were normalized to prestretch isometric tension to allow comparisons between different levels of activation. Amplitudes were measured as follows: P₁, measured from prestretch steady-state force to the peak of phase 1; P₂, measured from prestretch steady-state force to the minimum force decay; P₃, measured from prestretch steady-state force to the peak value of delayed force; and P_df, difference between P₃ and P₂.

View larger version (17K): [in this window] [in a new window]   Figure 1. Stretch activation response in murine WT myocardium. The force transient shown (bottom) is typical of typical stretch activation responses of WT myocardium following a stretch of 1% of muscle length (top). Once a steady-state isometric force of 50% of maximal was achieved in the presence of Ca2+, the muscle was stretched and then held at the longer length for 5 seconds. The stretch activation response was multiphasic, as reported previously.10 The recorded variables are labeled on the force record and described in the text.

Apparent rate constants were derived for phase 2 (k_rel, sec^–1) from the force decay following the peak of phase 1 and for phase 3 (k_df, sec^–1) from the point of force reuptake following phase 2 to the completion of delayed force development.

Data Analysis
Cross-sectional areas of skinned preparations were calculated by assuming that the preparations were cylindrical and by measuring width of the mounted preparation. Submaximal Ca²⁺-activated force (P) was expressed as a fraction of the maximum Ca²⁺-activated force (P_o) generated at pCa 4.5, ie, P/P_o. Rate constants of force decay were obtained by fitting a single exponential to the time course of decay, ie, y=a(1-exp[–k₁·x]), where a is the amplitude of the single exponential phase and k₁ is the rate constant of decay. Rate constants of delayed force development were obtained by a double exponential fit, y=a·exp(–k₁·x)+b·exp(–k₂·x), where a is the amplitude of the first exponential phase that rises with rate constant k₁ and b is the amplitude of the second exponential phase rising with rate constant of k₂, or were estimated by linear transformation of the half-time of force redevelopment, ie, k₁=–ln0.5x(t_1/2)^–1.¹⁰

All data are reported as means±SEM. Comparisons of stretch activation variables between WT and cMyBP-C^–/– myocardium at different levels of activation were performed using a 1-way ANOVA with a Tukey post hoc test, with statistical significance set at P<0.05.

	Results

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Response of WT and cMyBP-C^–/– Mouse Ventricular Myocardium to Stretch
Figure 1 illustrates a typical response of WT mouse skinned myocardium to a rapid stretch of 1% of initial muscle length during Ca²⁺ activation that yielded a prestretch force of 50% maximal. The initial rapid increase in force (phase 1) is coincident with stretch and is caused by strain of attached cross-bridges²⁷; phase 1 is followed by a rapid decline in force (phase 2) and a delayed force recovery (phase 3) that persists for several seconds before ultimately decaying to the original isometric force. Amplitudes of these phases are indicated in Figure 1 as P₁, P₂, and P₃. Note that P₂ (the minimum at the end of force decay) can fall below prestretch isometric force and can have negative values, whereas P₁ (phase1 amplitude) and P₃ (phase 3 amplitude) are always positive. The values of P₁ in this study were likely underestimated because our data sampling rate was limited, but this variable was not studied in this work.

To allow comparisons of stretch activation responses in WT and cMyBP-C^–/– myocardium at different levels of activation, stretch activation amplitudes were normalized to prestretch isometric force (Figures 2 and 3). The amplitude of phase 1 (P₁) increased as prestretch isometric force increased and did not differ significantly between WT and cMyBP-C^–/– myocardium (Figure 2) within the time resolution of our force transducer. In WT myocardium P₂ amplitude was lowest at maximal activation and highest (most positive) at low levels of activation, whereas P₂ values in cMyBP-C^–/– myocardium were almost always negative and dipped below isometric force at all levels of activation (Figure 3). The dramatic differences in P₂ amplitude at submaximal levels of activation can be clearly seen when the stretch activation responses of WT and cMyBP-C^–/– myocardium are superimposed at activation levels of 50% (Figure 4). The amplitudes of P₁ and P₂ were not corrected for resting force, which in each preparation was less than 5% of the maximum active force at pCa 4.5.

View larger version (19K): [in this window] [in a new window]   Figure 2. Stretch activation responses of WT and cMyBP-C–/– skinned myocardium recorded at various levels of Ca2+ activation. The force responses in response to a stretch of 1% of muscle length were recorded in WT (top) and cMyBP-C–/– (bottom) myocardium. In each case, the force responses were normalized to the prestretch isometric force recorded at the same level of activation, ie, the prestretch force baseline corresponds to P/Po=1.00 in A and D (maximal activation), P/Po=0.52 in B and E (intermediate activation), level activation, and P/Po=0.25 in C and 0.27 in F (low activation).

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of activation on P2 in WT and cMyBP-C–/– myocardium. P2 values normalized to prestretch isometric force were measured as a function of activation from the force responses to stretches of 1% of muscle length in both WT () (n=10) and cMyBP-C–/– () (n=10) myocardium. Data are means±SEM. *Significantly different from WT, P<0.05.

View larger version (14K): [in this window] [in a new window]   Figure 4. Stretch activation responses in WT and cMyBP-C–/– myocardium. Force transients following a stretch of 1% of muscle length were recorded from WT and cMyBP-C–/– myocardium activated with Ca2+ to a prestretch isometric force of 50% maximal. The transients are normalized to prestretch force, which corresponds to the 0 relative force baseline. Apparent rate constants of force decay (krel) and delayed force development (kdf) were 241 sec–1 and 16.5 sec–1 for WT myocardium and 378 sec–1 and 29.6 sec–1 for cMyBP-C–/– myocardium, respectively.

The rapid force decay in phase 2 proceeds with an apparent rate constant k_rel and is thought to represent detachment of cross-bridges that are strained by stretch and their rapid replacement by unstrained cross-bridges.^28,29 The apparent rate constant of force decay (k_rel) was slightly faster as the level of activation decreased for both WT and cMyBP-C^–/– (Figure 5) and was dramatically faster in cMyBP-C^–/– myocardium at submaximal levels of activation (Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 5. Effect of activation on krel in WT and cMyBP-C–/– myocardium. krel values were normalized to prestretch isometric force and are plotted as a function of activation level for WT () (n=10) and cMyBP-C–/– () (n=10) myocardium. The data shown were obtained following stretches of 1% of muscle length. Data are means±SEM. *Significantly different from WT, P<0.05.

The net delayed recruitment of cross-bridges following stretch can be determined from the amplitude of P₃, which has been shown to be highly dependent on prestretch isometric force.^10,30 Normalized values of P₃ decreased as a function of increased activation level such that proportionately fewer cross-bridges were recruited by stretch at higher levels of activation, a phenomenon that did not differ between WT and cMyBP-C^–/– myocardium at any level of activation. However, the negative amplitudes of P₂ in cMyBP-C^–/– myocardium increased the overall amplitude of the phase 3 tension transient compared with WT, as indicated by the large increase in P_df for cMyBP-C^–/– (Figure 6).

View larger version (12K): [in this window] [in a new window]   Figure 6. Effect of activation on Pdf in WT and cMyBP-C–/– myocardium. Pdf values were normalized to prestretch isometric force and are plotted as a function of activation level for WT () (n=10) and cMyBP-C–/– () (n=10) myocardium. The data were obtained following stretches of 1% of muscle length. Data are means±SEM. *Significantly different from WT, P<0.05.

The stretch activation response (phase 3) does not occur as a simple exponential process^10,30 and thus does not fit well with a single exponential equation, especially at low levels of activation. To facilitate comparisons of phase 3 rates of force development (k_df) at different levels of activation, apparent rate constants were derived from the half-times of force development.^10,30 As previously reported,¹⁰ apparent rate constants of force development (k_df) in murine myocardium are activation dependent, increasing with level of prestretch isometric force (Table). Because k_df is generally thought to involve the cooperative recruitment of cross-bridges into strongly bound states,^29,31,32 the acceleration of k_df at high levels of activation can be understood in terms of an increased number of cross-bridges bound to the thin filament, leaving fewer cross-bridges available for recruitment following stretch, thereby reducing the amplitude of stretch activation (as a fraction of prestretch force) and increasing its rate. In the present study, there was no difference in k_df between WT and cMyBP-C^–/– myocardium at maximal activation, but k_df was significantly accelerated at submaximal levels of activation in cMyBP-C^–/– compared with WT myocardium (Figure 4 and the Table), suggesting that cMyBP-C ablation accelerated the recruitment of cross-bridges into force generating states, thereby accelerating the overall rate of the stretch activation response.

View this table: [in this window] [in a new window]   Table 1. Activation Dependence of Phase 3 Delayed-Force Development in WT and cMyBP-C–/– Myocardium

	Discussion

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This study was undertaken to investigate the role of cMyBP-C in cardiac contraction and to specifically determine how ablation of cMyBP-C leads to impairment of cardiac function. Earlier in vivo measurements of left ventricular function showed that cMyBP-C^–/– mouse hearts have reduced ejection, an abbreviated ejection phase, and decreased rates of relaxation compared with WT controls.^4,5 The overall depression of systolic function in null mice occurs despite a normal initial rise in maximal pressure development (dP/dt_max), presumably because the period of ejection is shortened, thereby reducing the volume of blood ejected. Paradoxically, cMyBP-C^–/– myocardium exhibits accelerated rates of force development and increased power at submaximal levels of activation,³³ possibly as a consequence of enhanced levels of thin filament activation. We have previously suggested that ablation of cMyBP-C increases cooperative activation of the thin filament because of increased likelihood of cross-bridge binding.³⁴ Structural models for such a mechanism emerge from consideration of possible interactions of cMyBP-C with myosin because cMyBP-C has been proposed to restrict myosin interaction with actin by tethering the myosin heads to the thick filament¹⁹ or by forming a collar around the backbone of the thick filament.³ Ablation of cMyBP-C would alleviate the constraints on the position of myosin and lead to an increased probability of cross-bridge formation and acceleration of the transition of cross-bridges to force generating states. The resulting upregulation of thin filament activation, or a reduced ability to inactivate the thin filament, can lead to slowed relaxation, which would decrease both the diastolic filling time and the end diastolic volume, ultimately decreasing ejection fraction and depressing overall systolic function.

The stretch activation response is critical to the generation of oscillatory power required for the beating of insect wings³⁵ and is also a prominent process in heart muscle,^8,31,36 where it is thought to play an important role in modulating pump function and power output.^9,37 In this respect, it has been observed that some mutations of sarcomeric proteins in insect muscles³⁸ and mouse myocardium³⁶ alter the stretch activation response and power output. From such studies, it appears that both the rate of force development (k_df) and the amplitude of the additional force recruited by stretch activation (P₃) are associated with modulation of power output, an important determinant of systolic function.

The present study shows that the stretch activation response in myocardium is dramatically altered by ablation of cMyBP-C. When isometrically contracting myocardium is suddenly stretched, there is a corresponding increase in force caused by strain of attached cross-bridges. In turn, strain results in detachment of some cross-bridges and a decrease in force, a process that is quickly reversed by attachment of new cross-bridges and redevelopment of force to greater than prestretch isometric levels. cMyBP-C^–/– myocardium exhibits increases in the apparent rate constant and amplitude of the phase 2 force decay compared with WT. The N terminus of cMyBP-C binds to myosin S2 and/or actin and thus might modulate the interaction between myosin and actin,^3,39–41 so that removal of cMyBP-C could alter rates of cross-bridge attachment or detachment or both. Genetic deletion or acute biochemical extraction of cMyBP-C^–/– from myocardium increases shortening velocity,^19,33 presumably because of increased rates of cross-bridge detachment. Accelerated detachment would explain the observed increase in k_rel and the greater decline in force immediately following stretch of cMyBP-C^–/– myocardium. It is also possible that accelerated transitions to force generating states (k_df) in cMyBP-C^–/– myocardium contribute to increased cross-bridge detachment as a consequence of an increased flux of cross-bridges into and through the force-producing steps preceding detachment.

Changes in the amplitude of P₂ have also been interpreted as indicating reversal of force-producing steps in response to stretch,⁴² such that more negative values of P₂ represent greater reversal of steps such as the phosphate release step.⁴³ A possible adaptive advantage of such reversal is improved efficiency in myocardium subjected to stretch, because cross-bridges could conceivably detach from actin and quickly reattach without consuming ATP.⁴²

Delayed force redevelopment (phase 3) in the response to stretch, ie, stretch activation, is thought to be mediated by recruitment of additional cross-bridges to force generating states.^10,30,31 Here, there was no difference in amplitude of P₃ between WT and cMyBP-C^–/– myocardium, suggesting that the number of cross-bridges recruited by stretch was similar in the 2 cases; however, the rate constant of force redevelopment (k_df) was dramatically accelerated in cMyBP-C^–/– myocardium. Because stretch activation (phase 3) in cardiac muscle involves both cooperative and direct stretch-induced recruitment of cross-bridges,^10,31 the acceleration of k_df in cMyBP-C^–/– myocardium suggests that the rates of force-generating transitions are accelerated and that the contributions resulting from cooperative recruitment of cross-bridges (which tend to slow force generation⁴⁴) are reduced in the absence of cMyBP-C. Thus, the increased power output reported in cMyBP-C^–/– myocytes³² can be explained as a consequence of faster cross-bridge cycling kinetics.

From the stoichiometric ratio of cMyBP-C molecules to myosin heads,⁴⁵ cMyBP-C binds to only a small fraction of myosin heads in WT hearts, and, thus, ablation would directly affect relatively few myosin heads. If cMyBP-C behaves as a tether on myosin, ablation may remove a spatial constraint on these heads^3,4,19,34,46 and thereby increase the probability of binding to actin. Increased binding of small numbers of cross-bridges might not significantly increase overall force (there is no increase in maximum isometric force in cMyBP-C^–/– myocardium) but could still accelerate the rate of force development and cross-bridge cycling kinetics attributable to the sensitivity of the cardiac thin filament to the activating effects of even small numbers of attached cross-bridges (reviewed by Moss et al⁴⁷).

Ablation of cMyBP-C accelerates cross-bridge kinetics (Table and Figure 4) but, seemingly paradoxically, results in diminished stroke volume and cardiac hypertrophy.⁴ However, the apparent acceleration of rate constants of cross-bridge detachment and attachment in cMyBP-C^–/– myocardium can account for depressed pump function. In the context of cardiac contraction in which stronger regions of myocardium stretch weaker parts to induce delayed stretch activation,^9,10 accelerated turnover kinetics would predict a shorter delay in stretch activation, an earlier peak for the cardiac twitch, and earlier completion of the ejection phase. Such a phenomenon might be difficult to appreciate from our recordings because [Ca²⁺] is maintained constant and the force transient is slow to decay. In contrast, in living muscle the transient increase in intracellular Ca²⁺ is over quickly, so that the faster stretch activation response in cMyBP-C^–/– myocardium would result in an earlier early onset of relaxation of force. In effect, the accelerated kinetics of stretch activation are no longer tuned to the twitch kinetics required for a normal ejection period.

Taken together, our results support the idea that cMyBP-C normally constrains the interaction of myosin heads with actin and suggest that the acceleration of cross-bridge cycling kinetics attributable to ablation of cMyBP-C contributes to reduced ejection by abbreviating the period of ejection. Earlier work⁵ has shown that the in vivo, functional effects of cMyBP-C ablation are very similar to those caused by a C-terminal truncation of cMyBP-C,⁵ a common form of cMyBP-C mutations leading to hypertrophic cardiomyopathy in humans. Thus, results from this study may have applicability to the mechanism of contractile dysfunction in cMyBP-C cardiomyopathies, which are among the most common inherited myocardial diseases.^1–3

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute grant HL-47053 (to R.L.M.) and a grant from the American Heart Association (to J.E.S.).

	Footnotes

 
Original received January 3, 2006; revision received March 1, 2006; accepted March 21, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, Benaiche A, Isnard R, Dubourg O, Burban M, Gueffet JP, Millaire A, Desnos M, Schwartz K, Hainque B, Komajda M. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003; 107: 2227–2232.[Abstract/Free Full Text]
2. Flashman E, Redwood C, Moolman-Smook J, Watkins H. Cardiac myosin binding protein C: its role in physiology and disease. Circ Res. 2004; 94: 1279–1289.[Abstract/Free Full Text]
3. Moolman-Smook J, Flashman E, de Lange W, Li Z, Corfield V, Redwood C, Watkins H. Identification of novel interactions between domains of myosin binding protein-C that are modulated by hypertrophic cardiomyopathy missense mutations. Circ Res. 2002; 91: 704–711.[Abstract/Free Full Text]
4. Harris SP, Bartley CR, Hacker TA, McDonald KS, Douglas PS, Greaser ML, Powers PA, Moss RL. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ Res. 2002; 90: 594–601.[Abstract/Free Full Text]
5. Palmer BM, Georgakopoulos D, Janssen PM, Wang Y, Alpert NR, Belardi DF, Harris SP, Moss RL, Burgon PG, Seidman CE, Seidman JG, Maughan DW, Kass DA. Role of cardiac myosin binding protein C in sustaining left ventricular systolic stiffening. Circ Res. 2004; 94: 1249–1255.[Abstract/Free Full Text]
6. Rademakers FE, Rogers WJ, Guier WH, Hutchins GM, Siu CO, Weisfeldt ML, Weiss JL, Shapiro EP. Relation of regional cross-fiber shortening to wall thickening in the intact heart. Three dimensional analysis by NMR tagging. Circulation. 1994; 89: 1174–1182.[Abstract/Free Full Text]
7. Bogaert J, Rademakers FE. Regional nonuniformity of normal adult human left ventricle. Am J Physiol Heart Circ Physiol. 2001; 283: H792–H799.
8. Steiger GJ. Tension transients in extracted rabbit heart muscle preparations. J Mol Cell Cardiol. 1977; 9: 671–685.[Medline] [Order article via Infotrieve]
9. Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, Epstein ND. The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation. Cell. 2001; 107: 631–641.[CrossRef][Medline] [Order article via Infotrieve]
10. Stelzer JE, Larsson L, Fitzsimons DP, Moss RL. Activation dependence of stretch activation in mouse skinned myocardium: implications for ventricular function. J Gen Physiol. 2006; 127: 95–107.[Abstract/Free Full Text]
11. Campbell KB, Chandra M. Functions of stretch activation in heart muscle. J Gen Physiol. 2006; 127: 89–94.[Free Full Text]
12. Bennett P, Craig R, Starr R, Offer G. The ultrastructural location of C-protein, X-protein and H-protein in rabbit muscle. J Muscle Res Cell Motil. 1986; 7: 550–567.[CrossRef][Medline] [Order article via Infotrieve]
13. Gilbert R, Kelly MG, Mikawa T, Fischman DA. The carboxyl terminus of myosin binding protein-C (MyBP-C), C-protein specifies incorporation into the A-band of striated muscle. J Cell Sci. 1996; 109: 101–111.[Abstract]
14. Gruen M, Gautel M. Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C. J Mol Biol. 1999; 286: 933–949.[CrossRef][Medline] [Order article via Infotrieve]
15. Starr R, Offer G. The interaction of C-protein with heavy meromyosin and subfragment-2. Biochem J. 1978; 171: 813–816.[Medline] [Order article via Infotrieve]
16. Moos C, Offer G, Starr R, Bennett P. Interaction of C-protein with myosin, myosin rod and light meromyosin. J Mol Biol. 1975; 97: 1–9.[CrossRef][Medline] [Order article via Infotrieve]
17. Craig R, Offer G. The location of C-protein in rabbit skeletal muscle. Proc R Soc Lond B Biol Sci. 1976; 192: 451–461.[Medline] [Order article via Infotrieve]
18. Huxley HE, Brown W. The low-angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J Mol Biol. 1967; 30: 383–434.[Medline] [Order article via Infotrieve]
19. Hofmann PA, Hartzell HC, Moss RL. Alterations in Ca²⁺ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers. J Gen Physiol. 1991; 97: 1141–1163.[Abstract/Free Full Text]
20. Weisberg A, Winegrad S. Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle. Proc Natl Acad Sci U S A. 1996; 93: 8999–9003.[Abstract/Free Full Text]
21. Weisberg A, Winegrad S. Relation between crossbridge structure and actomyosin ATPase activity in rat heart. Circ Res. 1998; 83: 60–72.[Abstract/Free Full Text]
22. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988; 157: 378–417.[Medline] [Order article via Infotrieve]
23. Godt RE, Lindley BD. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol. 1982; 80: 279–297.[Abstract/Free Full Text]
24. Patel JR, Fitzsimons DP, Buck SH, Muthuchamy M, Wieczorek DF, Moss RL. PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin. Am J Physiol Heart Circ Physiol. 2001; 280: H2732–H2739.[Abstract/Free Full Text]
25. Stelzer JE, Patel JR, Olsson MC, Fitzsimons DP, Leinwand LA, Moss RL. Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium. Am J Physiol Heart Circ Physiol. 2004; 287: H1756–H1761.[Abstract/Free Full Text]
26. Campbell KS, Moss RL. SLControl: PC-based data acquisition and analysis for muscle mechanics. Am J Physiol Heart Circ Physiol. 2003; 285: H2857–H2864.[Abstract/Free Full Text]
27. Huxley AF, Simmons RM. Proposed mechanism of force generation in striated muscle. Nature. 1971; 233: 533–538.[CrossRef][Medline] [Order article via Infotrieve]
28. Davis JS, Rodgers ME. Indirect coupling of phosphate release to de novo tension generation during muscle contraction. Proc Natl Acad Sci U S A. 1995; 92: 10482–10486.[Abstract/Free Full Text]
29. Piazzesi G, Linari M, Reconditi M, Vanzi F, Lombardi V. Cross-bridge detachment and attachment following a step stretch imposed on active single frog muscle fibres. J Physiol. 1997; 498: 3–15.[Medline] [Order article via Infotrieve]
30. Linari M, Reedy MK, Reedy MC, Lombardi V, Piazzesi G. Ca-activation and stretch-activation in insect flight muscle. Biophys J. 2004; 87: 1101–1111.[Abstract/Free Full Text]
31. Campbell KB, Chandra M, Kirkpatrick RD, Slinker BK, Hunter WC. Interpreting cardiac muscle force-length dynamics using a novel functional model. Am J Physiol Heart Circ Physiol. 2004; 286: H1535–H1545.[Abstract/Free Full Text]
32. Lombardi V, Piazzesi G. The contractile response during steady lengthening of stimulated frog muscle fibres. J Physiol. 1990; 431: 141–171.[Abstract/Free Full Text]
33. Korte FS, McDonald KS, Harris SP, Moss RL. Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C. Circ Res. 2003; 93: 752–758.[Abstract/Free Full Text]
34. Stelzer JE, Fitzsimons DP, Moss RL. Ablation of myosin binding protein-C accelerates force development in mouse myocardium. Biophys J. In press.
35. Pringle JW. The Croonian Lecture, 1977. Stretch activation of muscle: function and mechanism. Proc R Soc Lond B Biol Sci. 1978; 201: 107–130.[Medline] [Order article via Infotrieve]
36. Vemuri R, Lankford EB, Poetter K, Hassanzadeh S, Takeda K, Yu ZX, Ferrans VJ, Epstein ND. The stretch-activation response may be critical to the proper functioning of the mammalian heart. Proc Natl Acad Sci U S A. 1999; 96: 1048–1053.[Abstract/Free Full Text]
37. Epstein ND, Davis JS. Sensing stretch is fundamental. Cell. 2003; 112: 147–150.[CrossRef][Medline] [Order article via Infotrieve]
38. Tohtong R, Yamashita H, Graham M, Haeberle J, Simcox A, Maughan D. Impairment of muscle function caused by mutations of phosphorylation sites in myosin regulatory light chain. Nature. 1995; 374: 650–653.[CrossRef][Medline] [Order article via Infotrieve]
39. Squire JM, Luther PK, Knupp C. Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain. J Mol Biol. 2003; 331: 713–724.[CrossRef][Medline] [Order article via Infotrieve]
40. Kunst G, Kress KR, Gruen M, Uttenweiler D, Gautel M, Fink RH. Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2. Circ Res. 2000; 86: 51–58.[Abstract/Free Full Text]
41. Harris SP, Rostkova E, Gautel M, Moss RL. Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism. Circ Res. 2004; 95: 930–936.[Abstract/Free Full Text]
42. Davis JS, Epstein ND. Kinetic effects of fiber type on the two subcomponents of the Huxley-Simmons phase 2 in muscle. Biophys J. 2003; 85: 390–401.[Abstract/Free Full Text]
43. Cooke R. Actomyosin interaction in striated muscle. Physiol Rev. 1997; 77: 671–697.[Abstract/Free Full Text]
44. Campbell K. Rate constant of muscle force redevelopment reflects cooperative activation as well as cross-bridge kinetics. Biophys J. 1997; 72: 254–262.[Abstract/Free Full Text]
45. Martyn DA. Myosin binding protein-C: structural and functional complexity. J Mol Cell Cardiol. 2004; 37: 813–815.[CrossRef][Medline] [Order article via Infotrieve]
46. Oakley CE, Hambly BD, Curmi PMG, Brown LJ. Myosin binding protein C: structural abnormalities in familial hypertrophic cardiomyopathy. Cell Res. 2004; 14: 95–110.[CrossRef][Medline] [Order article via Infotrieve]
47. Moss RL, Razumova M, Fitzsimons DP. Myosin crossbridge activation of cardiac thin filaments: implications for myocardial function in health and disease. Circ Res. 2004; 94: 1290–1300.[Abstract/Free Full Text]

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