Published online before print February 23, 2009, doi: 10.1161/CIRCULATIONAHA.108.798983
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(Circulation. 2009;119:1253-1262.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Cardiac Myosin Binding Protein-C Phosphorylation in a β-Myosin Heavy Chain Background
From the Department of Pediatrics, Cincinnati Children’s Hospital Medical Center (S.S., J.G., R.K., M.S., J.D.M., J.R.), and Department of Molecular and Cellular Physiology, University of Cincinnati (J.N.L.), Cincinnati, Ohio.
Correspondence to Jeffrey Robbins, MLC 7020, 240 Albert Sabin Way, Cincinnati, OH 45229-3039. E-mail jeff.robbins{at}cchmc.org
Received June 12, 2008; accepted December 19, 2008.
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Abstract |
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Background— Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C–null (cMyBP-C(t/t)) hearts, a cMyBP-C phosphomimetic (cMyBP-CAllP+) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly
-myosin heavy chain (-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly β-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a β-MyHC transgenic mouse heart in which >80% of the -MyHC was replaced by β-MyHC, which is the predominant myosin isoform in human cardiac muscle. Methods and Results— To determine the effects of cMyBP-C phosphorylation in a β-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-CWT), nonphosphorylatable cMyBP-C (cMyBP-CAllP–), or cMyBP-CAllP+ were bred into the β-MyHC background (β). These mice were then crossed into the cMyBP-C(t/t) background to ensure the absence of endogenous cMyBP-C. cMyBP-C(t/t)/β and cMyBP-CAllP–:(t/t)/β mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the β-MyHC background. cMyBP-CAllP+:(t/t)/β and cMyBP-CWT:(t/t)/β hearts showed no morbidity and mortality, and cMyBP-CAllP+:(t/t)/β hearts were significantly cardioprotected from ischemia/reperfusion injury.
Conclusions— cMyBP-C phosphorylation is necessary for basal myocardial function in the β-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.
Key Words: cardiovascular diseases • heart failure • molecular biology • myocardial contraction
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Introduction |
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Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is involved in the regulation of myocardial function and cardioprotection,1–4 but its precise functional roles remain obscure. cMyBP-C mutations cause heritable cardiomyopathies, accounting for
15% to 30% of all cardiomyopathic cases.5 A unique feature of cMyBP-C is its multiple phosphorylation sites.6 Three phosphorylated serines are present in cMyBP-C (Ser-273, Ser-282, and Ser-302) that can serve as substrates for protein kinase A (PKA), protein kinase C, and Ca2+-calmodulin–activated kinase II.3,4 Interestingly, the cardiomyopathic Gly278Glu, Gly279Ala, and Arg282Trp mutations are located within the cMyBP-C phosphorylation motif.7
Clinical Perspective p 1262
cMyBP-C phosphorylation is essential for normal cardiac function1 but decreases during the development of human heart failure8 and ischemia/reperfusion (I/R) injury.1 Previously, we explored the role of cMyBP-C phosphorylation in cardiac function by mutating the 3 phosphorylation sites to either nonphosphorylatable alanines (cMyBP-CAllP–)1,9 or to aspartates (cMyBP-CAllP+) to create a phosphomimetic.2 Transgenic (TG) expression of cMyBP-CAllP– in cardiomyocytes resulted in depressed cardiac function and was unable to rescue the cMyBP-C–null (cMyBP-C(t/t)) phenotype, in contrast to expression of wild-type cMyBP-C (cMyBP-CWT) in the cMyBP-C(t/t) background.1 Strikingly, expression of cMyBP-CAllP+ rescued the cMyBP-C(t/t) mice and, in addition, was cardioprotective during I/R injury.2
Rapid and reversible changes in thick filament structure and ordering of myosin heads can be produced in cardiac muscle by changes in cMyBP-C phosphorylation,10 and these changes in structure are accompanied by changes in force production.11,12 The interactions of cMyBP-C with myosin are modulated by cMyBP-C phosphorylation such that when cMyBP-C is dephosphorylated it interacts strongly with the S2 region of myosin, preventing its force-generating interaction with actin. In vitro studies show that when cMyBP-C sites are phosphorylated, myosin S2 interaction is weakened or ablated,2 promoting the interaction of the myosin head with actin, which activates cross-bridge cycling. Because of these interactions, cMyBP-C function is at least partially dependent on the particular myosin isoform with which it interacts.10,13
How applicable are these mouse studies to the human heart? Two distinct myosin isoforms, termed V1 (2 -MyHC plus light chains) and V3 (2 β-MyHC plus light chains), are in the mammalian heart, giving rise to differences in shortening velocity, force, and actomyosin ATPase activity.14 The mouse heart consists of mostly -MyHC, whereas the human heart contains β-MyHC. Phosphorylation of Ser-273, Ser-282, and Ser-302 in cMyBP-C in vitro15 and in vivo2 causes the thick filaments to exhibit a relatively loose structure,2 with changes in myosin orientation, increased contractility, and maximum Ca2+-activated force.15 Weisberg and Winegrad10 examined thick filaments that contained either -MyHC or β-MyHC and studied the effects of cMyBP-C phosphorylation on their structure and contractile parameters. PKA-mediated phosphorylation of cMyBP-C and cMyBP-CAllP+ resulted in cross-bridge extension from the filament backbone, changes in overall orientation, and a decrease in cross-bridge flexibility in the -MyHC–containing filaments.2,10 In contrast, phosphorylation of cMyBP-C in β-MyHC–containing filaments had no effect on cross-bridge extension, the degree of order, or flexibility.10,13 These data call into question the relevance of altered cMyBP-C phosphorylation in modulating contractile parameters in the human heart.
We constructed mice in which the ventricular -MyHC isoform was replaced with >80% β-MyHC (β-TG).14 Although mimicking the MyHC isoform composition of the adult human myocardium, it should be recognized that the Vmax ATPase activity of mouse β-MyHC is substantially larger than that of human β-MyHC in the β-TG mouse heart. We then investigated the impact of cMyBP-C phosphorylation in these mice. The data demonstrate that cMyBP-C phosphorylation can improve myocardial function regardless of the myosin isoform with which it interacts and supports the hypothesis that altering cMyBP-C phosphorylation status in vivo may represent a novel therapeutic target in human heart failure.
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Methods |
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An expanded Methods section is available in the online-only Data Supplement.
TG and Targeted Mouse Models
To determine the efficacy of cMyBP-C phosphorylation in a β-MyHC background, the cMyBP-CWT–(line 21),1 cMyBP-CAllP–– (line 262),1 and cMyBP-CAllP+–(line 34)2 expressing mice were bred to the β-TG mice (line 137)14,16 to generate double TG animals, which were subsequently crossed into the cMyBP-C(t/t) background17 to ensure the absence of endogenous cMyBP-C (cMyBP-CWT:(t/t)/β, cMyBP-CAllP-:(t/t)/β, and cMyBP-CAllP+:(t/t)/β), as described.1 All mice (FVB/N) procedures were in accordance with the Guide for the Use of and Care of Laboratory Animals published by the National Institutes of Health.
Cardiac I/R Injury
To determine the cardioprotective effects of cMyBP-CAllP+ expression, cardiac I/R injury was performed at 8 to 10 weeks.2 Total area at risk, infarcted area, and area not at risk were determined.2 DNA laddering assay and the number of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)–positive nuclei were quantified to assess cardiac apoptosis.2
Statistical Analysis
Results are presented as mean±SE. For comparisons of multiple groups, data were analyzed by a 1-way or 2-way ANOVA. Differences within and between groups were analyzed with 2-way repeated-measures ANOVA, followed by a Tukey post hoc test to compare individual means (SigmaPlot V11.0). P<0.05 was considered significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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The Phosphorylatable Domain of cMyBP-C Interacts With β-MyHC
Myosin is composed of 2 heavy chains and 4 light chains (Figure 1A). The globular head contains the actin binding and ATPase sites. Previously, we showed that cMyBP-C C1–C2 domains interact with
-MyHC at the S2 region.2 This interaction is dynamically regulated by phosphorylation/dephosphorylation of cMyBP-C at serines 273, 282, and 302 (Figure 1A). To confirm that phosphorylation of cMyBP-C also determines β-MyHC interaction, pull-down assays were performed with the use of His-tagged C1–C2 peptides derived from cMyBP-CWT (C1–C2WT) or from C1–C2 peptides in which Ser-273, Ser-282, and Ser-302 were mutated to either alanines (C1–C2AllP–) or aspartates (C1–C2AllP+). The peptide fragments were tested against ventricular protein extracted from mouse predominantly expressing β-MyHC.14 C1–C2WT peptides were also phosphorylated with PKA to determine the relative myosin interaction. Results show that nonphosphorylated C1–C2WT and C1–C2AllP– proteins interact with β-MyHC, whereas PKA-treated C1–C2WT and untreated C1–C2AllP+ do not (Figure 1B). We conclude that dephosphorylated cMyBP-C C1–C2 interacts in vitro with β-MyHC and that phosphorylation ablates the interaction. We were not able to confirm these experiments in vivo because a strong interaction of cMyBP-C exists through its COOH terminus domains with the light meromyosin region of either β- or -MyHC (Figure I in the online-only Data Supplement), such that coimmunoprecipitation occurs irrespective of cMyBP-C phosphorylation when intact proteins are used.
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Phosphoablation of cMyBP-C Is Deleterious in a β-MyHC Background
To examine the effects of cMyBP-C phosphorylation in a β-MyHC background, cMyBP-CWT, cMyBP-CAllP–, and cMyBP-CAllP+ mice were crossed with β-TG mice14 to generate double TG mice, which were then crossed into the cMyBP-C(t/t) background17 to ensure the absence of endogenous cMyBP-C.1 The gross cardiac anatomy of the 7 groups was analyzed (Figure 2A). Both the nontransgenic (NTG) hearts, in which -MyHC is >95% of the total myosin content, and β-TG hearts, in which -MyHC is replaced with >80% β-MyHC, showed normal anatomy, whereas cMyBP-C(t/t) hearts showed markedly increased ventricular wall thickness, myocyte disarray, and fibrosis.1,17
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Importantly, in the β-MyHC background, the cMyBP-C(t/t) mouse phenotype was severe, with cMyBP-C(t/t)/β mouse hearts exhibiting gross pathology and concentric hypertrophy (Figure 2A to 2C) at 6 weeks of age. Expression of normal cMyBP-C or the phosphomimetic in the β-MyHC– and cMyBP-C–null background (cMyBP-CWT:(t/t)/β and cMyBP-CAllP+:(t/t)/β, respectively) resulted in a rescue of the aberrant cardiac gross anatomy (Figure 2A to 2D). In marked contrast, the phosphoablated cMyBP-CAllP-:(t/t)/β experimental group died by 7 weeks after birth (Figure 2E). We hypothesize that the inability of cMyBP-CAllP– to rescue the null lies in its tendency to strongly bind myosin S2, mimicking chronic complete dephosphorylation, whereas cMyBP-CWT continues to be dynamically and differentially phosphorylated on some of its phosphorylatable residues. Similar to NTG, cMyBP-C(t/t), and β-TG mice, the cMyBP-CWT:(t/t)/β and cMyBP-CAllP+:(t/t)/β mice show essentially no signs of increased morbidity and mortality throughout their life spans, confirming the necessity of cMyBP-C phosphorylation in β-MyHC background for normal cardiac function. Levels of cMyBP-C phosphorylation were similar among NTG, β-TG, and cMyBP-CWT:(t/t)/β hearts as quantified by phospho-site–specific antibodies and 1-dimensional isoelectric focusing (Figures II and III in the online-only Data Supplement).
cMyBP-CAllP+ Effects a Complete Rescue of cMyBP-C(t/t) in the β-MyHC Background
To determine the effects of cMyBP-C phosphorylation on whole organ function in a β-MyHC background, 4 groups—NTG, β-TG, cMyBP-CWT:(t/t)/β, and cMyBP-CAllP+:(t/t)/β—were chosen for further studies at 10 to 12 weeks. SDS-PAGE (Figure 3A) and Western blots with the use of anti-cMyBP-C (Figure 3B) show normal myofilament stoichiometry in the cMyBP-CAllP+:(t/t)/β hearts compared with control hearts. RNA analysis revealed (Figure 3C) that the overexpression of β-MyHC is consistent with protein levels (Figure 2D) in the β-TG, cMyBP-CWT:(t/t)/β, and cMyBP-CAllP+:(t/t)/β mice. Atrial natriuretic peptide expression, a sensitive molecular marker for cardiac stress/hypertrophy, was unchanged among the 4 groups, as were the heart/body weight ratios (Figure 3D). Furthermore, M-mode echocardiography showed that NTG, β-TG, cMyBP-CWT:(t/t)/β, and cMyBP-CAllP+:(t/t)/β mice had normal cardiac function (Figure 3E and Table 1). cMyBP-CAllP+:(t/t)/β hearts had normal levels of cardiac troponin I, myosin essential light chain, and phospholamban phosphorylation (Figure IV in the online-only Data Supplement). Altogether, these data suggest that cMyBP-CAllP+:(t/t) in the β-MyHC background does not cause a discernible pathology at the whole organ, molecular, or cellular level.
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cMyBP-CAllP+ Improves Myocardial Contractility in a β-MyHC Background
Myocardial β-adrenergic receptor function is impaired in human heart failure.8 To determine whether cMyBP-C phosphorylation plays a role in mediating the positive inotropic effect of β-adrenergic receptor stimulation in the intact animal, cardiac hemodynamic rates were measured at baseline and during β-adrenergic receptor agonist infusion in the intact closed chest model.1 The β-TG mouse hearts exhibited decreased systolic and diastolic rates compared with the control NTG mice (Figure 4A and 4B).14 Heart rate and left ventricular pressure were significantly improved in the cMyBP-CAllP+:(t/t)/β mouse hearts compared with β-TG and cMyBP-CWT:(t/t)/β mouse hearts (Table 2). In contrast to our previous data obtained in the normal, -MyHC background,2 cMyBP-CAllP+:(t/t)/β has both significantly improved contraction (Figure 4A) and relaxation (Figure 4B) compared with β-TG and cMyBP-CWT:(t/t)/β cohorts, although these parameters did not reach NTG values, emphasizing the critical role that myosin isoform content plays in determining cardiac hemodynamics.
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). Effects on maximal Ca2+-activated Mg2+-ATPase activities (C) of the cMyBP-C phosphomimetic (AllP+) in a β-MyHC background at pCa 4.0 (n=5). One-way ANOVA showed a significant difference between genotypes (P<0.001). P<0.05 compared with NTG (*) and cMyBP-C AllP+:(t/t)/β (
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Data suggest that cMyBP-C phosphorylation influences actomyosin Mg2+-ATPase activity, the kinetics of cross-bridge cycling, and the rate of relaxation.4 In addition, β-adrenergic receptor stimulation has differential effects on the actomyosin Mg2+-ATPase activity depending on whether - or β-MyHC is present.18 Therefore, to assess the role of cMyBP-CAllP+ in a β-MyHC background, actomyosin Mg2+-ATPase activity was measured in the 4 groups. As expected, actomyosin Mg2+-ATPase activity was depressed in the β-TG hearts compared with the NTG hearts (Figure 4C). However, the cardiac myofibrils of the cMyBP-CAllP+:(t/t)/β showed increased maximal actomyosin Mg2+-ATPase activity compared with cMyBP-CWT:(t/t)/β without changes in the Hill coefficients and Ca2+ sensitivity (Table I in the online-only Data Supplement). Our data suggest that increased actomyosin Mg2+-ATPase contributes to improved cardiac function in the cMyBP-CAllP+:(t/t)/β heart.
cMyBP-CAllP+ Enhances Myosin Kinetics at the Isolated Fiber Level
cMyBP-CAllP+ mimics the effects of PKA-mediated phosphorylation of cMyBP-C. The β-TG mice permit testing of the hypothesis that cMyBP-CAllP+ has consequences on cross-bridge cycling in the presence of the slow myosin isoform. Fiber kinetic studies were performed in which cMyBP-CAllP+:(t/t)/β skinned fibers were compared with control fibers in the absence or presence of PKA treatment. As previously determined, NTG fibers show increased shortening velocity (Figure 5A to 5C), power output (Figure 5D to 5F), and unloaded shortening velocity (Figure 5G through 5I) compared with β-TG fibers.14 PKA treatment of NTG fibers (NTG/PKA) led to increased shortening velocity (Figure 5A), power output (Figure 5D), and unloaded shortening velocity (Figure 5G) as well as decreased Ca2+ sensitivity (Figure 5J and Table 3). In contrast, β-TG fibers exhibited reduced shortening velocity (Figure 5A), power output (Figure 5D), and unloaded shortening velocity (Figure 5G) at baseline relative to NTG fibers. On PKA treatment (β-TG/PKA), the β-TG/PKA fibers showed similar responses (Figure 5J and Table 3). cMyBP-CAllP+:(t/t)/β fibers showed increased shortening velocity (Figure 5B), power output (Figure 5E), and unloaded shortening velocity (Figure 5H) compared with cMyBP-CWT:(t/t)/β and β-TG fibers in the absence of altered myofibrillar Ca2+ sensitivity. Treatment of the cMyBP-CAllP+:(t/t)/β fibers with PKA resulted in decreased maximal Ca2+ sensitivity (Figure 5K) compared with cMyBP-CWT:(t/t)/β. Maximum Ca2+-activated isometric forces (Figure 5L), calcium sensitivity (pCa 5.0), and Hill coefficients (Table 3) were no different in all 3 cases. Taken together, the data show that cMyBP-CAllP+ contributes to faster myosin kinetics in a β-MyHC background and that cMyBP-C phosphorylation plays a significant role in regulating the kinetics in human cardiac muscle.
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length) amplitude vs duration (
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cMyBP-CAllP+ Protects the Heart From I/R Injury in a β-MyHC Background
In the mouse, cMyBP-CAllP+ is cardioprotective during I/R injury but is associated with a reduction in β-MyHC levels.2 To determine whether this phenomenon might be relevant in the human heart, NTG, β-TG, cMyBP-CAllP+:(t/t)/β, and cMyBP-CWT:(t/t)/β hearts were subjected to left ventricular cardiac ischemia for 1 hour followed by 24 hours of reperfusion.2 β-TG mice were treated with isoproterenol as a positive control to protect the heart against ischemic injury. Results show that cMyBP-CAllP+:(t/t)/β hearts had a significantly reduced (22±1%) infarcted area when normalized to the area at risk from I/R-injured NTG (32±2%), β-TG (36±3%), and cMyBP-CWT:(t/t)/β (40±3%) controls (Figure 6A and 6B). The area at risk was not significantly different among the 4 groups (Figure 6B). The degree of cardioprotection is essentially equivalent to that observed in the normal mouse -MyHC background.2 As expected, the preconditioned β-TG hearts showed the largest reduction in infarcted area (9±1%). Cardiac injury was apparent in the cMyBP-CWT:(t/t)/β and β-TG hearts, whereas the cMyBP-CAllP+:(t/t)/β hearts were relatively less affected. NTG hearts with I/R injuries showed a modest β-MyHC induction, but this did not occur in the β-MyHC background (Figure 6C). Cardioprotection in the cMyBP-CAllP+:(t/t)/β hearts was accompanied by decreased TUNEL-positive nuclei (Figure 6D and 6E), DNA fragmentation (Figure 6F), and cMyBP-C degradation (Figure 6G) compared with β-TG and cMyBP-CWT:(t/t)/β hearts. Fractional shortening (Figure 6H) and fractional area change (Figure 6I) were significantly improved in the cMyBP-CAllP+:(t/t)/β mice, whereas these parameters were decreased in β-TG and cMyBP-CWT:(t/t)/β mice at 4 weeks. These results demonstrate that cMyBP-C phosphorylation protects the myocardium from cell injury and death irrespective of myosin background.
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Discussion |
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cMyBP-C phosphorylation has consequences on cross-bridge cycling that differ depending on its phosphorylation state and the myosin isoform present.18,19 The present study was directed at determining the structural and functional aspects of cMyBP-C phosphorylation in a mouse heart whose myosin isoform complement mimics that found in the human heart, establishing a rationale for cMyBP-C phosphorylation modulation after I/R as a cardioprotective strategy. Our data clearly show that expression of a cMyBP-C phosphomimetic in a β-MyHC background has different consequences in vivo. Unlike the case for when cMyBP-CAllP+ was expressed in the normal mouse background of cardiac
-MyHC, expression in the β-MyHC background led to improved contraction and relaxation (Figures 4 and 5
) compared with β-TG and cMyBP-CWT:(t/t)/β cohorts. At the same time, placing the cMyBP-C(t/t) genotype in the β-MyHC background exacerbated morbidity and resulted in a dramatically decreased life expectancy with expression of a nonphosphorylatable cMyBP-C being completely ineffective at rescuing the mouse from death. These results underscore the differences that expression of either normal or mutant cMyBP-C can have on cardiac function in a β-MyHC background and emphasize the importance of cMyBP-C in hearts that contain predominantly β-MyHC.
Interestingly, the positions of the cross-bridges are different in filaments containing - and β-MyHC,14 even though the region to which the C1–C2 domain of cMyBP-C binds is highly homologous between the 2 myosin isoforms.10,20,21 With the use of recombinant C1–C2 peptides, we confirmed that cMyBP-C binds to both myosins in the same region in a phosphorylation-dependent manner. In contrast to when it is placed into the -MyHC background,2 cMyBP-CAllP+ improves myocardial contractility at baseline in the β-TG hearts, demonstrating that the influence of cMyBP-C phosphorylation on cardiac contractility is indeed myosin isoform dependent in the whole organ context and that these experiments are relevant to human cardiac function. In contrast to cMyBP-CWT and cMyBP-CAllP+ in vivo, the constant state of cMyBP-CAllP– binding with the myosin S2 region may be associated with increased inhibition of myosin extension during cross-bridge cycles and dysregulate myosin organization in the β-MyHC background rather than in the -MyHC background for the observed detrimental effect. In conclusion, presence of cMyBP-CAllP– or absence of cMyBP-C (cMyBP-C(t/t)) causes decreased cardiac function and cardiac hypertrophy,1,17 suggesting the necessity of cMyBP-C and its phosphorylation for normal cardiac function.
Does phosphorylation of cMyBP-C play a direct role in regulating contraction? The data are convincing that cMyBP-C influences actomyosin Mg2+-ATPase activity and the kinetics of cross-bridge cycling and that cMyBP-C phosphorylation modifies actomyosin Mg2+-ATPase activity and the rate of relaxation.4,22,23 However, failure of cMyBP-C phosphorylation to alter the Ca2+ sensitivity of actomyosin Mg2+-ATPase activity in reconstituted contractile protein systems has been cited by some as strong evidence against a role for phosphorylation of cMyBP-C in regulating contraction.22,24 Reconstituted thick filament proteins do not, however, reproduce the normal thick-filament structure or the steric arrangement of contractile proteins in a filament lattice. Indeed, it is necessary to study the overall effect of total cMyBP-C phosphorylation in an intact mouse model with different myosin backgrounds, and PKA-mediated phosphorylation has differential effects on actomyosin Mg2+-ATPase activity depending on whether -MyHC or β-MyHC is present.25 Our data show that under normal conditions, cMyBP-C phosphorylation increases actomyosin Mg2+-ATPase activity without altering thin-filament Ca2+ sensitivity in a β-MyHC background, suggesting that cMyBP-C phosphorylation may not directly modulate myofilament Ca2+-sensitivity.22 However, changes in Ca2+ sensitivity due to length-dependent activation have not been excluded,26 and cMyBP-C phosphorylation may play a direct role in the cooperative activation of the filament system, force generation, regulating cross-bridge rates of the myosin-actin motors, as well as functioning as a tether.12,27 Our in vitro data suggest that cMyBP-C phosphorylation releases myosin from a myosin interaction constraint, which ultimately allows myosin to increase cross-bridge formation, resulting in faster cycle kinetics.12 These results are consistent with previous studies with the use of fibers in which cMyBP-C has been completely ablated. Those data documented an increase in the unloaded shortening velocity.28 The data, in conjunction with the Mg2+-ATPase activity, suggest that cMyBP-C phosphorylation can accelerate β-MyHC kinetics and myocardial contractility without affecting Ca2+ sensitivity of the thin filaments in a β-MyHC background.
Myofilament protein phosphorylation represents a point of convergence for complex signaling events that ultimately result in cardioprotection and changes in contractile function. Direct manipulation of the contractile apparatus and end points, bypassing receptor-ligand signaling pathways, could have significant advantages in more precisely targeting a therapeutic action and is now a focus of drug development.29 Widespread myocardial ischemia can cause contractile dysfunction, which often persists even after blood flow has been restored. A major finding of the present work is that cMyBP-CAllP+ is associated with significant protection from myocardial injury with better cardiac function and less cellular damage after I/R injury in the β-TG mouse heart. One possible explanation is that cMyBP-CAllP+ is relatively resistant to calpain cleavage,30 which can be activated during I/R. Our recent data show that cMyBP-C is a substrate of calpain, and phosphorylation of cMyBP-C renders the protein resistant to calpain-mediated proteolysis.31 This is consistent with the observation that phosphorylation of cardiac troponin I by PKA significantly reduced its cleavage by calpain.32 Therefore, the presence of an intact active cMyBP-C is required to regulate the formation of the weakly bound state between actin and myosin that leads to entry into force generation by driving the main molecular components: the thick and thin filaments. Furthermore, improved cardiac function after I/R injury could be mediated by cMyBP-CAllP+ resulting from direct enhancement of the myosin-actin interaction. The absence of cMyBP-CAllP+ interaction with the myosin S2 region, leading to increased thick-filament spacing2 and resistance to calpain-mediated proteolysis, may be associated with cardioprotection, independent of myosin background. These data suggest that improved cardiac function after I/R injury could be mediated by directed phosphorylation of cMyBP-C and that contractile protein-based cardioprotection may represent a therapeutic avenue to improve myocardial contractility in the ischemic and failing heart.
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Acknowledgments |
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Sources of Funding
This research was supported by National Institutes of Health grants P01HL69799, P50HL074728, P50HL077101, P01HL059408, and R01HL087862 and the International Collaboration Research Project with the Japanese Health Science Foundation (Dr Robbins) and by the American Heart Association, Ohio Valley Affiliate (Dr Sadayappan).
Disclosures
None.
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References |
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1. Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dorn GW II, Klevitsky R, Seidman CE, Seidman JG, Robbins J. Cardiac myosin binding protein-C phosphorylation and cardiac function. Circ Res. 2005; 97: 1156–1163.
2. Sadayappan S, Osinska H, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD, Seidman CE, Seidman JG, Robbins J. Cardiac myosin binding protein-C phosphorylation is cardioprotective. Proc Natl Acad Sci U S A. 2006; 103: 16918–16923.
3. Mohamed AS, Dignam JD, Schlender KK. Cardiac myosin-binding protein C (MyBP-C): identification of protein kinase A and protein kinase C phosphorylation sites. Arch Biochem Biophys. 1998; 358: 313–319.[CrossRef][Medline] [Order article via Infotrieve]
4. Gautel M, Zuffardi O, Freiburg A, Labeit S. Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? EMBO J. 1995; 14: 1952–1960.[Medline] [Order article via Infotrieve]
5. Spirito P, Seidman CE, McKenna WJ, Maron BJ. The management of hypertrophic cardiomyopathy. N Engl J Med. 1997; 336: 775–785.
6. Yuan C, Guo Y, Ravi R, Przyklenk K, Shilkofski N, Diez R, Cole RN, Murphy AM. Myosin binding protein C is differentially phosphorylated upon myocardial stunning in canine and rat hearts: evidence for novel phosphorylation sites. Proteomics. 2006; 6: 4176–4186.[CrossRef][Medline] [Order article via Infotrieve]
7. 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.
8. El-Armouche A, Pohlmann L, Schlossarek S, Starbatty J, Yeh YH, Nattel S, Dobrev D, Eschenhagen T, Carrier L. Decreased phosphorylation levels of cardiac myosin-binding protein-C in human and experimental heart failure. J Mol Cell Cardiol. 2007; 43: 223–229.[CrossRef][Medline] [Order article via Infotrieve]
9. Nagayama T, Takimoto E, Sadayappan S, Mudd JO, Seidman JG, Robbins J, Kass DA. Control of in vivo left ventricular contraction/relaxation kinetics by myosin binding protein C: protein kinase A phosphorylation dependent and independent regulation. Circulation. 2007; 116: 2399–2408.
10. 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.
11. Hofmann PA, Greaser ML, Moss RL. C-protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2+ activation. J Physiol. 1991; 439: 701–715.
12. Lecarpentier Y, Vignier N, Oliviero P, Guellich A, Carrier L, Coirault C. Cardiac myosin-binding protein C modulates the tuning of the molecular motor in the heart. Biophys J. 2008; 95: 720–728.[CrossRef][Medline] [Order article via Infotrieve]
13. Winegrad S. Myosin binding protein C, a potential regulator of cardiac contractility. Circ Res. 2000; 86: 6–7.
14. Krenz M, Sanbe A, Bouyer-Dalloz F, Gulick J, Klevitsky R, Hewett TE, Osinska HE, Lorenz JN, Brosseau C, Federico A, Alpert NR, Warshaw DM, Perryman MB, Helmke SM, Robbins J. Analysis of myosin heavy chain functionality in the heart. J Biol Chem. 2003; 278: 17466–17474.
15. 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.
16. Krenz M, Robbins J. Impact of beta-myosin heavy chain expression on cardiac function during stress. J Am Coll Cardiol. 2004; 44: 2390–2397.
17. McConnell BK, Jones KA, Fatkin D, Arroyo LH, Lee RT, Aristizabal O, Turnbull DH, Georgakopoulos D, Kass D, Bond M, Niimura H, Schoen FJ, Conner D, Fischman DA, Seidman CE, Seidman JG. Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice. J Clin Invest. 1999; 104: 1235–1244.[Medline] [Order article via Infotrieve]
18. Weisberg A, Winegrad S. Relation between crossbridge structure and actomyosin ATPase activity in rat heart. Circ Res. 1998; 83: 60–72.
19. Stelzer JE, Brickson SL, Locher MR, Moss RL. Role of myosin heavy chain composition in the stretch activation response of rat myocardium. J Physiol. 2007; 579: 161–173.
20. 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]
21. Gruen M, Prinz H, Gautel M. cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on-off fashion. FEBS Lett. 1999; 453: 254–259.[CrossRef][Medline] [Order article via Infotrieve]
22. Stelzer JE, Patel JR, Walker JW, Moss RL. Differential roles of cardiac myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to protein kinase A phosphorylation. Circ Res. 2007; 101: 503–511.
23. McClellan G, Weisberg A, Winegrad S. cAMP can raise or lower cardiac actomyosin ATPase activity depending on alpha-adrenergic activity. Am J Physiol. 1994; 267: H431–H442.[Medline] [Order article via Infotrieve]
24. Hofmann PA, Lange JH III. Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats. Circ Res. 1994; 74: 718–726.
25. Winegrad S, Weisberg A. Isozyme specific modification of myosin ATPase by cAMP in rat heart. Circ Res. 1987; 60: 384–392.
26. Cazorla O, Szilagyi S, Vignier N, Salazar G, Kramer E, Vassort G, Carrier L, Lacampagne A. Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice. Cardiovasc Res. 2006; 69: 370–380.
27. Stelzer JE, Moss RL. Contributions of stretch activation to length-dependent contraction in murine myocardium. J Gen Physiol. 2006; 128: 461–471.
28. 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.
29. Cleland JG, Coletta AP, Clark AL. Clinical trials update from the Heart Failure Society of America meeting: FIX-CHF-4, selective cardiac myosin activator and OPT-CHF. Eur J Heart Fail. 2006; 8: 764–766.
30. Decker RS, Decker ML, Kulikovskaya I, Nakamura S, Lee DC, Harris K, Klocke FJ, Winegrad S. Myosin-binding protein C phosphorylation, myofibril structure, and contractile function during low-flow ischemia. Circulation. 2005; 111: 906–912.
31. Sadayappan S, Greis KD, Robbins J. Phosphorylation-dependent proteolysis and pathogenesis of cardiac myosin binding protein–C. J Mol Cell Cardiol. 2008; 44: S44.[CrossRef]
32. Di Lisa F, De Tullio R, Salamino F, Barbato R, Melloni E, Siliprandi N, Schiaffino S, Pontremoli S. Specific degradation of troponin T and I by mu-calpain and its modulation by substrate phosphorylation. Biochem J. 1995; 308 (pt 1): 57–61.[Medline] [Order article via Infotrieve]
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.798983/DC1.
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