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(Circulation Research. 2007;101:928.)
© 2007 American Heart Association, Inc.

Cellular Biology

Cardiac Myosin-Binding Protein C Is Required for Complete Relaxation in Intact Myocytes

Lutz Pohlmann, Irena Kröger, Nicolas Vignier, Saskia Schlossarek, Elisabeth Krämer, Catherine Coirault, Karim R. Sultan, Ali El-Armouche, Saul Winegrad, Thomas Eschenhagen, Lucie Carrier

From the Institute of Experimental and Clinical Pharmacology and Toxicology (L.P., I.K., S.S., E.K., K.R.S., A.E.A., T.E., L.C.), University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Inserm, U582 (N.V., L.C.), Institut de Myologie, Paris, France; the University Pierre et Marie Curie-Paris6 (N.V., L.C.), UMR S582, IFR14, Paris, France; Inserm, U689 (C.C.), Cardiovascular Research Center, Paris, France; and the Department of Physiology (S.W.), University of Pennsylvania School of Medicine, Philadelphia.

Correspondence to Lucie Carrier, PhD, Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany. E-mail l.carrier{at}uke.uni-hamburg.de

	Abstract

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The role of cardiac myosin-binding protein C (cMyBP-C) in cardiac contraction is still not fully resolved. Experimental ablation of cMyBP-C by various means resulted in inconsistent changes in Ca²⁺ sensitivity and increased velocity of force of skinned preparations. To evaluate how these effects are integrated in an intact, living myocyte context, we investigated consequences of cMyBP-C ablation in ventricular myocytes and left atria from cMyBP-C knock-out (KO) mice compared with wild-type (WT). At 6 weeks, KO myocytes exhibited mild hypertrophy that became more pronounced by 30 weeks. Isolated cells from KO exhibited markedly lower diastolic sarcomere length (SL) without change in diastolic Ca²⁺. The lower SL in KO was partly abolished by the actin-myosin ATPase inhibitors 2,3-butanedione monoxime or blebbistatin, indicating residual actin-myosin interaction in diastole. The relationship between cytosolic Ca²⁺ and SL showed that KO cells started to contract at lower Ca²⁺ without reaching a higher maximum, yielding a smaller area of the phase-plane diagram. Both sarcomere shortening and Ca²⁺ transient were prolonged in KO. Isolated KO left atria exhibited a marked increase in sensitivity to external Ca²⁺ and, in contrast to WT, continued to develop twitch force at low micromolar Ca²⁺. Taken together, the main consequence of cMyBP-C ablation was a defect in diastolic relaxation and a smaller dynamic range of cell shortening, both of which likely result from the increased myofilament Ca²⁺ sensitivity. Our findings indicate that cMyBP-C functions as a restraint on myosin-actin interaction at low Ca²⁺ and short SL to allow complete relaxation during diastole.

Key Words: cardiac myocytes • contraction • familial hypertrophic cardiomyopathy • hypertrophy • transgenic mice

	Introduction

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Cardiac myosin-binding protein C (cMyBP-C) is located in the A-band of the cardiac sarcomere, where it interacts with myosin, actin, and titin.^1–5 It consists of 11 modules labeled C0 to C10 from the N to the C terminus. It is assumed to play important structural and functional roles in health and disease (for reviews, see^6–10). Interest in cMyBP-C has intensified since the discovery that mutations in the human gene are frequently involved in familial hypertrophic cardiomyopathy (FHC).^11–14 Most of them should produce C-terminal truncated cMyBP-Cs^14,15 that are unstable in cardiac myocytes and undetectable in myocardial tissue of patients.^16–18 A recent study suggests that they are rapidly and quantitatively degraded by the ubiquitin-proteasome system.¹⁹

cMyBP-C can be phosphorylated at 3 different sites by cAMP-regulated protein kinase and a Ca²⁺-calmodulin kinase bound to the thick filament (for reviews, see^6–10). Lower amount of phosphorylated cMyBP-C has been found in human failing heart and atrial fibrillation,^20,21 and during low-flow ischemia.²² Recent data suggest that cMyBP-C phosphorylation is cardioprotective.²³

The regulatory role of cMyBP-C on contraction is still controversial, although there is a general agreement that cMyBP-C acts as an internal load. Removal of cMyBP-C can increase the velocity of shortening, force output, and force redevelopment in skinned preparations.^5,24–27 Addition of N-terminal fragments of cMyBP-C in skinned myocytes activated force production in the absence of Ca²⁺.²⁸ In 2 different cMyBP-C knock-out (KO) mice, fractional shortening was reduced,^29,30 whereas no change in dP/dt_max was found.³⁰ The maximum Ca²⁺-activated force (F_max) was unaltered in skinned myocytes partially depleted in cMyBP-C²⁴ or isolated from KO mice.^29,31 The results of ablation or partial extraction of cMyBP-C on Ca²⁺ sensitivity of skinned myocytes or cardiac preparations varied among different studies.^29,31–34 The aim of the present study was to evaluate how these alterations are integrated in an intact, living myocyte context, in which at least some of the compensatory changes for the absence of cMyBP-C remain and can be quantified. For this purpose, we have used intact, freely-suspended adult ventricular myocytes isolated from cMyBP-C WT and KO mice.³⁰ Both sarcomere shortening and Ca²⁺ transients were measured. In addition, the relationship between external Ca²⁺ and force was measured in intact loaded left atrial muscles. The amounts of major myofilament and Ca²⁺-handling proteins were determined to detect potential compensatory changes.

	Materials and Methods

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For an extended description see supplement data (available online at http://circres.ahajournals.org).

Animals
The cMyBP-C KO mice were generated in a blackswiss background as previously described.³⁰

Sarcomere Shortening and Ca²⁺ Transients Measurements in Intact Ventricular Myocytes
Ventricular myocytes were isolated from KO and WT hearts as previously described.²⁰ Cells were incubated in the IonOptix solution (in mmol/L: 135 NaCl, 4.7 KCl, 0.6 KH₂PO₄, 0.6 Na₂HPO₄, 1.2 MgSO₄, 1.25 CaCl₂, 20 glucose, 10 Hepes, pH 7.46) containing 1 µmol/L Fura-2-AM for 20 minutes. Sarcomere shortening and Ca²⁺ transients of intact myocytes were simultaneously assessed on field stimulation (1 Hz with 4 ms duration, 10 V) using a video-based sarcomere length (SL) detection system (IonOptix Corporation) at room temperature. Cells were alternatively excited at 340 and 380 nm with 510 nm emissions using the hyper-switch dual excitation light source. The F340/F380 ratio was used as an index of cytosolic Ca²⁺ concentration.

Response of Isolated Left Atrial Muscles to External Ca²⁺, Determination of Myosin-Heavy Chain Content, Western Blot, and Immunofluorescence Analyses
Methods are detailed in the online data supplement.

	Results

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Contribution of Myocyte Hypertrophy to Ventricular Hypertrophy in cMyBP-C KO Mice
The absence of cMyBP-C in KO mice was verified by immunofluorescence in isolated myocytes (Figure 1A). Ventricular-to-body-weight ratio was 29% and 44% greater in KO than in WT at 6 and 30 weeks, respectively (Figure 1B). Cell length and width of ventricular myocytes were measured in the presence of 10 mmol/L 2,3-butanedione monoxime (BDM). KO myocytes were 18% and 13% longer than WT myocytes at 6 and 30 weeks, respectively, and wider only at 30 weeks by 22% (Figure 1C). Cell area, calculated as cell lengthxwidth, was significantly greater at 6 and 30 weeks (+13% and +28%, respectively), indicating that ventricular hypertrophy in the KO mice was mainly the result of myocyte hypertrophy.

View larger version (32K): [in this window] [in a new window]   Figure 1. Phenotype of mouse ventricles and ventricular myocytes. A, Immunofluorescence analysis of ventricular myocytes isolated from cMyBP-C KO and WT mice and stained with -actinin (green) and cMyBP-C (red) antibodies; inset, x10 magnification. B, Ventricular-to-body-weight-ratio (VW/BW) of 6- and 30-week-old KO and WT mice (WT6, KO6, WT30, and KO30). Number of animals is indicated in the bars. C, Dimensions of ventricular myocytes isolated from 6- and 30-week-old KO and WT mice. Number of cells is indicated in the bars. Values are mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 vs WT, Student t test. D, Representative sarcomere shortening in ventricular myocytes isolated from 6-week-old cMyBP-C KO and WT mice. Traces represent the average of 40 contractions obtained in 1.25 mmol/L external Ca2+.

Impairment of Shortening Kinetics in cMyBP-C KO Myocytes
Sarcomere shortenings of intact WT and KO myocytes were measured in 6-week-old (mild hypertrophy) and 30-week-old (larger degree of hypertrophy) mice (Figure 1D; Table). Diastolic SL was markedly lower in KO than in WT myocytes at both ages. In fact, SL in KO in diastole was identical to SL in WT in systole, ie, sarcomere shortening in KO started where it ended in WT. Whereas maximal sarcomere shortening did not change between KO and WT, the kinetics of both sarcomere shortening and relengthening were slower in KO. This worsened with age. Thus, shortening velocity in KO was 34% and 56% lower and relengthening velocity 33% and 49% lower at 6 and 30 weeks, respectively.

View this table: [in this window] [in a new window]   Table 1. Table. Sarcomere Shortening in Intact cMyBP-C KO and WT Cardiac Myocytes

Interestingly, whereas no correlation was found between diastolic SL and sarcomere shortening in WT (Figure 2A), a slight, significant negative correlation was found in KO. Systolic SL showed the expected negative correlation with sarcomere shortening in both groups at both ages (Figure 2B). However, the slopes were significantly lower in KO and lower with age in both groups. A similar negative correlation was found between systolic SL and relengthening velocity with even more pronounced difference in slope between KO and WT (Figure 2C).

View larger version (34K): [in this window] [in a new window]   Figure 2. Correlation between SL (sarcomere length) and maximum shortening or kinetics of relenghtening. A, Relationship between diastolic SL and fractional sarcomere shortening. B, Relationship between systolic SL and fractional sarcomere shortening. C, Relationship between systolic SL and relengthening velocity. Analyses were performed in 6- and 30-week-old cMyBP-C KO (red) and WT (black) mice. Values are mean±SEM, **P<0.01, ***P<0.001 vs WT, linear regression analysis. The Spearman correlation factor, r, and slope values are indicated in each panel.

Compensatory Changes in cMyBP-C KO Mice
The amount of key myofilament and Ca²⁺-handling proteins was determined to investigate potential adaptations to the ablation of cMyBP-C. MHC isoforms were analyzed by long electrophoresis in a polyacrylamide gel containing glycerol (Figure 3A). A significant shift from - to ß-MHC was observed in KO at 6 weeks and to a lesser extent at 30 weeks. Thus, the amount of ß-MHC was 5-fold and 3-fold higher in KO than in WT at 6 and 30 weeks, respectively. This finding was confirmed by Western blot with a ß-MHC-specific antibody (Figure 3B). The amount of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) and phospholamban (PLB) was identical in KO and WT. However, the level of both phosphorylated forms of PLB was 2- to 3-fold higher in KO than in WT at 30 weeks, but not at 6 weeks of age (Figure 3B). Interestingly, the amount of Na⁺/Ca²⁺ exchanger (NCX) was 34% lower in KO than in WT (Figure 3C).

View larger version (25K): [in this window] [in a new window]   Figure 3. Determination of protein levels in mouse ventricles. A, Representative SDS gel and quantitative analysis of - and ß-myosin heavy chains (/ß-MHC) in left ventricles from 6- and 30-week-old cMyBP-C KO and WT mice (WT6, KO6, WT30, KO30). Values are mean±SEM, *P<0.05 vs WT, Student t test. Number of animals is indicated in the bars. B, Typical examples and quantitative analysis of Western blots performed on ventricular proteins. Proteins (20 µg) were subjected to 10% to 15% SDS-PAGE and transferred to nitrocellulose or PVDF membranes. Western blots were stained with the antibody directed against the indicated proteins. Values are normalized to CSQ and are expressed as mean±SEM performed on 6 to 9 different samples, *P<0.05 vs WT, Student t test. ß-MHC indicates ß-myosin heavy chain; CSQ, calsequestrin; SERCA2, sarco-endoplasmic reticulum Ca2+-ATPase; Tot-PLB, total phospholamban; Ser16-PLB, phospholamban phosphorylated at Ser16; Thr17-PLB, phospholamban phosphorylated at Thr17. C, Representative Western blot stained for NCX (Na+/Ca2+ exchanger) and CSQ and quantitative analysis of ventricular NCX level normalized to CSQ in 6- to 9-week-old WT and KO mice. Values are expressed as mean±SEM of 12 different samples, *P<0.05 vs WT, Student t test.

Residual Diastolic Actin-Myosin Interaction in cMyBP-C KO Myocytes
To test whether lower diastolic SL is explained by active cross-bridge cycling in KO, the effect of 10 mmol/L BDM, a chemical phosphatase that inhibits cross-bridge cycling,³⁵ in the presence of low external Ca²⁺ (0.0125 mmol/L) was examined. Whereas BDM did not change diastolic SL in WT, it increased it by 4.7% in KO (Figure 4A). We then investigated whether the BDM effect is also observed in the complete absence of extracellular Ca²⁺ by adding 1 or 10 mmol/L EGTA. Lowering external Ca²⁺ from 1.25 to 0.0125 mmol/L induced cell arrest, but did not change diastolic SL in both groups (Figure 4B and 4C). Of note, cell arrest developed 2-fold slower in KO (Figure 4B and 4D). EGTA 10 mmol/L did not change diastolic SL in both groups. Whereas BDM did not significantly change diastolic SL in WT, it significantly increased it in KO (Figure 4C). Time from BDM injection to plateau tended to be longer in KO (521±66 versus 380±54 s in WT, P>0.05). Whereas 10 or 30 mmol/L BDM did not fully relax KO cells, 100 mmol/L BDM returned the diastolic SL in KO to WT baseline value (supplemental Figure I). Similar effects were obtained in KO with 10 µmol/L blebbistatin, another inhibitor of actin-activated myosin ATPase³⁶ (1.73±0.02 versus 1.69±0.03 µm in EGTA, n=4).

View larger version (16K): [in this window] [in a new window]   Figure 4. Measurement of diastolic sarcomere length (SL) in intact myocytes under 1-Hz electrical stimulation. A, Diastolic SL measured in 1.25 mmol/L Ca2+ or in 0.0125 mmol/L Ca2+ plus 10 mmol/L BDM in intact myocytes isolated from 6-week-old WT and KO mice. Values are mean±SEM, ***P<0.001 vs Ca2+ 1.25 mmol/L, Student t test. B, Representative SL measurements obtained in WT and KO cMyBP-C myocytes in the following conditions (in mmol/L): Ca2+ 1.25, Ca2+ 0.0125, EGTA 10, EGTA 10 plus BDM 30. C, Diastolic SL measurements performed as in B. Conditions are indicated below the bars. Values are mean±SEM, **P<0.01 vs EGTA, Student t test. D, Time from the last normal twitch to cell arrest induced by lowering external Ca2+ concentration. Values are mean±SEM, *P<0.05 vs WT, Student t test. Number of cells is indicated in the bars.

Alteration of Ca²⁺ Transient Kinetics but Not Diastolic Ca²⁺ in cMyBP-C KO Myocytes
We then investigated whether lower diastolic SL and slower sarcomere shortening in KO are associated with changes in diastolic Ca²⁺ and Ca²⁺ transient kinetics, respectively. Sarcomere shortenings and Ca²⁺ transients were simultaneously measured in Fura-2-AM-preincubated cells from 6-week-old mice. Diastolic Ca²⁺ was unaltered in KO (Figure 5A through 5C). Whereas the increase in time-to-peak shortening was approximately matched by a similar increase in time-to-peak Ca²⁺ (+27%), the slowing in relengthening (+34%) corresponded to only a minor increase in time-to-50% Ca²⁺ decay (+11%). In contrast, no alteration of the maximal amplitude of both sarcomere shortening and Ca²⁺ transients was detected in KO (Figure 5A through 5C). We therefore evaluated whether varying external Ca²⁺ concentration reveals defects in KO. Increasing Ca²⁺ from 0.25 to 2 mmol/L increased the amplitude of Ca²⁺ transient similarly in both groups, but increased fractional shortening to a slightly lower extent in KO (supplemental Figure IIA). In contrast, KO cells, which were not preincubated with Fura-2 exhibited a higher sarcomere shortening at 0.5 mmol/L external Ca²⁺ compared with WT (supplemental Figure IIB). This divergence may relate to the 4-fold lower sarcomere shortening at 0.5 mmol/L external Ca²⁺ in the presence of Fura-2 (<0.9% versus >3.7% without Fura-2), suggesting that, under our conditions, Fura-2 acted as a Ca²⁺ buffer masking differences in response to low external Ca²⁺.

View larger version (33K): [in this window] [in a new window]   Figure 5. Simultaneous measurements of sarcomere shortenings and Ca2+ transients in myocytes isolated from 6-week-old cMyBP-C KO and WT mice. After preincubation of the cells with 1 µmol/L Fura-2-AM for 20 minutes, measurements were performed under 1-Hz electrical stimulation. A, Representative recordings of sarcomere shortenings and Ca2+ transients in WT and KO myocytes. B, Averaged sarcomere shortening and Ca2+ transient of 56 and 65 WT and KO cells, respectively. C, Summary data for the main parameters of sarcomere shortening and Ca2+ transient. Values are mean±SEM, **P<0.01 and ***P<0.001 vs WT, Student t test. Number of cells is indicated in the bars. D, Phase-plane diagram of SL and F340/380 ratio in averaged WT and KO cells. E, Phase-plane diagram of normalized sarcomere shortening and F340/380 ratio in averaged WT and KO cells. F, F340/380 ratios giving 5 (F5), 10 (F10) and 20% (F20) of maximum shortening in averaged WT and KO cells.

Altered Relation Between Sarcomere Length and Cytosolic Ca²⁺ in Intact KO Myocytes
To get a more detailed view of the relation between sarcomere shortening and cytosolic Ca²⁺ we assessed the phase-plane diagrams as previously described for rat myocytes.³⁷ This analysis revealed a counter-clockwise loop (shortening proceeded upwards), which showed 3 abnormalities in KO (Figure 5D through 5F): a shift to lower SL, a start of sarcomere shortening at lower Ca²⁺, and a smaller area of the loop. Whereas the shift to lower SL recapitulates the effect of alkalosis or pharmacological Ca²⁺ sensitization,^37,38 no increase in maximal shortening or leftward shift of the relengthening phase was observed in KO.

Altered Response of Isolated KO Left Atria to External Ca²⁺
To investigate whether the changes in KO myocyte shortening translate into altered contraction of intact loaded muscle preparations, isolated, electrically paced (1 Hz) left atria were challenged with different external Ca²⁺ concentrations (Figure 6A and 6B). When a Ca²⁺-free Tyrode’s solution was applied (calculated external Ca²⁺ 0.002 mmol/L), active twitch force quickly ceased in WT, but continued at a reduced steady state for >5 minutes in KO. Stepwise increases in external Ca²⁺ concentrations reactivated twitch force in WT and revealed a significantly increased sensitivity to external Ca²⁺ in KO (EC₅₀ 1.21±0.17 versus 2.55±0.19 mmol/L in WT, P<0.01). Moreover, cMyBP-C ablation was also associated with a trend toward a reduced slope of the external Ca²⁺-force relationship as indicated by a slight, not significant decrease in the Hill coefficient (2.9±0.7 versus 3.7±0.8 in WT, n=4, P>0.05). These data indicate that, under physiological concentrations of external Ca²⁺, KO myocytes contract at more than half-maximal force.

View larger version (9K): [in this window] [in a new window]   Figure 6. Response of isolated intact left atria to external Ca2+. A, Representative original recordings obtained in cMyBP-C WT and KO atria at 37°C and 1-Hz stimulation. After adjustment of muscle length to the half-maximum of its force-length relationship in a modified Tyrode’s solution containing 1.8 mmol/L external Ca2+, a Ca2+-free Tyrode’s solution was applied (calculated Ca2+ 0.002 mmol/L). Ca2+ was then stepwise reintroduced up to a final concentration of 6.4 mmol/L. B, Relation between force and external Ca2+ in left atria. Values are mean±SEM of 4 different muscles, **P<0.01, 2-way ANOVA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The precise role of cMyBP-C remains incompletely understood, but the prevailing evidence suggests that it acts as an internal load on actin-myosin interactions. The present data support this interpretation by showing that cMyBP-C abrogation is associated with a marked decrease in diastolic SL in unloaded myocytes and a pronounced increase in the sensitivity to external Ca²⁺ in isometrically contracting atria. The fact that these changes were already seen in young age and did not go along with changes in intracellular diastolic Ca²⁺ concentrations support the notion that they are primary consequences of cMyBP-C ablation.

The decrease in diastolic SL in KO from 1.8 µm to 1.7 µm could be the consequence of a reduction in forces restoring SL or an increase in forces shortening SL against the restoring force, ie, increased cross-bridge cycling. The latter is more likely for several reasons. First, in skinned KO myocytes passive tension and its regulation by PKA were unchanged.³¹ This argues against relevant changes in titin, which is also the major component of restoring forces in cardiac myocytes. Second, the difference in SL between KO and WT was sensitive to inhibitors of myosin ATPase (BDM or blebbistatin). This indicates that the diastolic SL in KO is, at least to an important part, attributable to residual cross-bridge cycling. A similar conclusion was recently drawn in a study evaluating the consequences of a troponin I mutation associated with restrictive cardiomyopathy.³⁹ Finally, the former evidence that cMyBP-C acts as an internal load on contraction^24–27 also supports the notion that the reduced resting SL in the absence of cMyBP-C is attributable to the removal of a restraint.

Residual crossbridge cycling in diastole in myocytes lacking cMyBP-C in the absence of an increase in diastolic Ca²⁺ could either indicate a Ca²⁺-independent mechanism³⁹ or be the consequence of the increased myofilament Ca²⁺ sensitivity or both. Several arguments favor the second hypothesis. First, a decreased diastolic cell-length has been observed with alkalosis or pharmacological Ca²⁺ sensitizers.^37,38 Second, the phase-plane diagrams revealed that KO myocytes started to shorten at lower cytosolic Ca²⁺ than WT. Third, loaded KO left atria still exhibited active twitch force in nominally 0.002 mmol/L external Ca²⁺ whereas WT did not, and the EC₅₀ for external Ca²⁺ to stimulate force was more than 2-fold lower than in WT. Preliminary results indicate that the amplitude of L-type Ca²⁺ current was similar in KO and WT myocytes suggesting that the higher sensitivity of KO atria to external Ca²⁺ is independent of increased Ca²⁺ influx (L. Pohlmann, M. Kruse, O. Pongs, E. Eschenhagen, L. Carrier, unpublished data, 2007). However, in contrast to classical Ca²⁺ sensitization, no increase in maximal shortening or leftward shift of the relengthening phase was observed in KO (Figure 5). These data suggest that the ablation of cMyBP-C promotes myofilament Ca²⁺ response predominantly at low, diastolic Ca²⁺, without effect at high Ca²⁺.

This interpretation is well compatible with 2 recent studies on skinned myocyte Ca²⁺ sensitivity. Whereas earlier studies had reported mixed effects of extraction or ablation of cMyBP-C on Ca²⁺ sensitivity in skinned preparations (increase,^31,32 no change,⁴⁰ or decrease^25,29), the newer data on skinned KO preparations demonstrated a lower Hill coefficient of the pCa-force relation, indicating a reduced cooperativity of the myofilament activation.^27,31 A potential mechanistic explanation for this finding comes from a recent study describing a radial displacement of cross-bridges away from the thick filament in the absence of Ca²⁺.⁴¹ These results are consistent with a model in which cMyBP-C normally acts to tether myosin cross-bridges nearer to the thick filament backbone, thereby reducing the likelihood of cross-bridge binding to actin under low Ca²⁺ concentrations.

The present results were obtained in a KO model, in which, in contrast to another model,²⁹ the transcription start site of the cMyBP-C gene was eliminated.³⁰ This strategy results in a complete lack of both cMyBP-C mRNA and protein, but does not exclude that our results are influenced by compensatory mechanisms. Indeed, several observations favor this notion. KO exhibited cardiac hypertrophy and a fetal gene expression program (³⁰ and present study), hyperphosphorylation of PLB at older age and downregulation of NCX. One may speculate that the higher amount of the slow ß-MHC partially compensates for the increased propensity of actin-myosin interaction in cMyBP-C KO, and it is likely that it contributes to the slower sarcomere shortening in KO.^42,43 However, whereas myocyte dysfunction worsened in KO with age the ß-MHC fraction decreased more than 2-fold. This argues against the idea that upregulation of ß-MHC is a major cause of myocyte dysfunction in KO. Similarly, the markedly slower relaxation and Ca²⁺ transient decay observed in KO cells was not associated with and can therefore not be explained by a change in the amount of SERCA2 or PLB. In contrast, the higher level of both phosphorylated forms of PLB in 30-week-old KO mice suggests that the mice partially compensate the intrinsic relaxation deficit by activating a pathway that accelerates Ca²⁺ uptake into the SR and therefore hastens relaxation. Preliminary results indicate a higher density of the ß-receptors in myocardium and a stronger inotropic effect of isoprenaline in myocytes from KO mice, and therefore support this interpretation (L. Pohlmann, T. Rau, T. Eschenhagen, L. Carrier, unpublished data, 2007). Another interesting finding was the slower disappearance of twitch force after removal of extracellular Ca²⁺ in both myocytes and left atria from KO (Figures 4 and 6). This observation points to a defect of KO to extrude Ca²⁺ and is likely related to the decreased levels of NCX (Figure 3C), to the higher Ca²⁺ sensitivity of the myofilaments that may retain Ca²⁺,^39,44 or both. In any case, both alterations likely contribute to the slower sarcomere relengthening and Ca²⁺ decay in KO. It is difficult to decide to which extent the lack of cMyBP-C as such participates in the altered kinetics of shortening, but the significantly lower slope of the correlation between systolic SL and relengthening velocity in KO (Figure 2C) suggests that the absence of cMyBP-C also affects the onset and/or the velocity of relaxation. This is compatible with an inhibitory effect of cMyBP-C on cross-bridge cycling and increased Ca²⁺ sensitivity in KO.

Residual cross-bridge cycling in diastole, incomplete relaxation, and increased Ca²⁺-sensitivity of the myofilaments are expected to translate into diastolic dysfunction, basal hypercontractility, and increased energy expenditure, all of which are typical features of FHC.^45–47 Indeed, the hemodynamic measurements in the same KO mice have revealed decreased dP/dt_min and increased Tau, as well as an increased LV end-systolic meridional wall stress in the basal state.³⁰ On the other hand, cMyBP-C ablation was associated with a reduced absolute response to increasing external Ca²⁺ (from 1.8 to 6.4 mmol/L) in intact atria (Figure 6), a reduced length-dependent activation in skinned myocytes,³¹ and a blunted response to dobutamine in vivo.³⁰ Taken together, these data indicate that the physiological role of cMyBP-C is to increase the dynamic range of myofilament responses mainly by allowing complete relaxation under diastolic conditions. A problem with this model is that, as recently pointed out by de Tombe,⁸ the substantial contractile activation in diastole should be incompatible with life. Whereas this is obviously not the case in mice, the same does not necessarily apply to human in which cardiac function depends much more on regulation of contractile force. Indeed, a homozygous Q76ter mutation, which is expected to produce a functional cMyBP-C KO, has been shown to be associated with massive hypertrophy and sudden death by 9 months of age in a FHC patient.¹⁴

Taken together, our results suggest that cMyBP-C acts as an internal load that tethers myosin heads to the thick-filament backbone and prevents force generation in diastole. Its absence causes cross-bridge cycling at low diastolic Ca²⁺, higher myofilament Ca²⁺ sensitivity with a reduced cooperativity, hypercontractility in the basal state, and reduced dynamic range of contraction. These changes are well compatible with the phenotype of human FHC and, in human, are likely to occur even when the amount of cMyBP-C is discretely reduced as may result from haploinsufficiency.

	Acknowledgments

 
Sources of Funding

This work was supported by the sixth Framework Program of the European Union (Marie Curie EXT-014051), the Deutsche Forschungsgemeinschaft (FOR-604), the Institut National de la Santé et de la Recherche Médicale (PNRMC-A04048DS), the Centre National de la Recherche Scientifique, and the Association Francaise contre les Myopathies (AFM-9471).

Disclosures

None.

	Footnotes

 
Original received December 28, 2006, first submission received March 2, 2007; second resubmission received June 28, 2007; revised second resubmission received August 22, 2007, accepted August 29, 2007.

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