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We have determined the effects of myosin binding protein-C (MyBP-C) N-terminal domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C modulates muscle contractility and is capable of interacting with thin filaments. Protein kinase A (PKA) phosphorylates cardiac and slow skeletal MyBP-C, altering myofilament structure and function. We previously used TPA to show that full-length MyBP-C restricts actin torsional dynamics, and effects by cardiac and slow skeletal MyBP-C (cMyBP-C; ssMyBP-C) are relieved by PKA phosphorylation. To determine the effects of MyBP-C N-terminal domains on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin flexibility at saturation of the TPA effect. PKA phosphorylation of ssMyBP-C domains C1-C2 and cMyBP-C domains C0-C2 relieved the restriction of torsional amplitude but also increased the rate of torsional motion, thus increasing actin resilience (rate/amplitude; less brittle). Moreover, effects of cardiac C0-C2 on actin resilience were also PKA-dependent, whereas slow skeletal C1-C2 effects which promote actin resilience persisted independently of phosphorylation. In the case of fast skeletal C1-C2, actin resilience was unaffected and its effect to restrict actin dynamics was unchanged by phosphorylation. The N-terminal domains of cMyBP-C (C0-C2) and skeletal MyBP-C's (C1-C2) had effects on actin dynamics similar to those determined for the full-length MyBP-C isoforms. Effects of phosphomimetic mutations were also investigated. These unique isoform-dependent MyBP-C-induced changes in actin dynamics may play a role in the functional effects of MyBP-C on contraction. This work was supported by NIH grants to DDT (R01 AR032961), to SS (R01 AR067279), and to BC (R00 HL122397).

Myosin Binding Protein-C (MyBP-C) consists of three isoforms: slow-skeletal, fast-skeletal, and cardiac. In the present study, our objective is to elucidate the role of fast-skeletal Myosin Binding Protein-C (fsMyBP-C). Using recombinant MyBP-C N-termini, we have previously demonstrated that the fast-skeletal isoform regulates muscle contraction more similarly to cardiac vs. slow-skeletal MyBP-C. However, the physiological implications of such regulation in skeletal muscle remains unclear. Therefore, we generated homozygous knock-out mice that do not express fsMyBP-C in either alleles, as well as heterozygous mice that express fsMyBP-C in only one allele. Currently, no myopathies are associated with mutations in fsMyBP-C, fsMyBP-C expression is dysregulated in skeletal muscle diseases, such as Duchenne Muscular Dystrophy. Both homozygous and heterozygous mice are viable and do not currently exhibit differences in longevity. However, expression of fsMyBP-C are drastically different: homozygous mice do not express fsMyBP-C, but heterozygous mice exhibit the same pattern of expression in skeletal muscle as wild-type (WT) mice. In WT mice, fsMyBP-C is expressed at levels comparable to slow-skeletal MyBP-C in the extensor digitalis longus (EDL) and tibialis anterior (TA) (fast-type and mixed type skeletal muscles, respectively). Interestingly, small amounts of fsMyBP-C are also expressed in the soleus, a slow-type muscle. To analyze functional changes in EDL and soleus muscles, we used Force-ATPase experiments to analyze steady-state myofilament properties, such as force generation, Ca2+-sensitivity, and tension cost. No significant functional changes were observed in heterozygous mice, nor were they observed in the soleus muscle. However, functional deficits became apparent in EDL muscles of homozygous mice, but could be recovered by the addition of recombinant fsMyBP-C N-termini. We propose that homozygous mice may be a useful model for isolating the effects of fsMyBP-C in health and disease.

Myosin binding protein C (MyBP-C) is a thick filament-associated protein found in striated muscle and may regulate muscle contractility. Separate genes encode the fast and slow skeletal isoforms, and there are potential PKA phosphorylation sites in their functionally important N-terminal regions. Here, we compare mouse N-terminal fast (fC1C2) and slow (sC1C2) skeletal fragments containing the initial ∼50 aa Pro/Ala-rich domain and the C1 and C2 Ig-domains that are linked by the ∼100 aa M-domain. Of the known slow skeletal splice variants, we chose a highly expressed variant lacking the N-terminal 34-59 residue insert. To define the potential mechanisms by which skeletal MyBP-Cs affect contractility and whether these effects are modulated by PKA phosphorylation, we assessed the Ca2+-dependent motility of rabbit skeletal native thin filaments over a surface of rabbit psoas myosin in the presence of C1C2 fragments. While thin filaments were fully regulated, with no motion observed at pCa > 7 in the absence of fragments, the addition of either 0.50 μM fC1C2 or sC1C2 resulted in significant motility. This suggests that skeletal MyBP-C isoforms effectively sensitize the thin filament. Under fully activating conditions (pCa ≤ 5), sC1C2 had little effect on motility whereas fC1C2 inhibited sliding velocities by nearly 50%. Thus, these fragments differ in their modulatory capacities with fC1C2 sensitizing the thin filament to Ca2+ and inhibiting maximal velocities, while the sC1C2 variant exhibits only a single mode of contractile modulation; i.e. thin filament sensitization. Interestingly, PKA phosphorylation in the Pro/Ala (sC1C2) and M-domains (sC1C2 and fC1C2), as confirmed by mass spectrometry, reduced both fragments’ Ca2+ sensitization of the thin filament. Thus, the function of MyBP-C isoforms may be tuned to match the physiological demands of the muscle in which they are expressed.

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser(273), Ser(282), and Ser(302) by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala(273)-Asp(282)-Ala(302)), DAD (Asp(273)-Ala(282)-Asp(302)), and SAS (Ser(273)-Ala(282)-Ser(302)) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (~50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca(2+) sensitivity (pCa50) and cooperativity (nH) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased nH and slightly decreased pCa50. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp(273) and/or Asp(302) (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.

We investigated the influence of cardiac myosin binding protein-C (cMyBP-C) and its constitutively unphosphorylated status on the radial and longitudinal stiffnesses of the myofilament lattice in chemically skinned myocardial strips of the following mouse models: nontransgenic (NTG), effective null for cMyBP-C (t/t), wild-type cMyBP-C expressed into t/t (WT(t/t)), and constitutively unphosphorylated cMyBP-C (AllP-(t/t)). We found that the absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) resulted in a compressible cardiac myofilament lattice induced by rigor not observed in the NTG and WT(t/t). These results suggest that the presence and phosphorylation of the N-terminus of cMyBP-C provides structural support and radial rigidity to the myofilament lattice. Examination of myofilament longitudinal stiffness under rigor conditions demonstrated a significant reduction in cross-bridge-dependent stiffness in the t/t compared with NTG controls, but not in the AllP-(t/t) compared with WT(t/t) controls. The absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) both resulted in a shorter myosin cross-bridge lifetime when myosin isoform was controlled. These data collectively suggest that cMyBP-C provides radial rigidity to the myofilament lattice through the N-terminus, and that disruption of the phosphorylation of cMyBP-C is sufficient to abolish this structural role of the N-terminus and shorten cross-bridge lifetime. Although the presence of cMyBP-C also provides longitudinal rigidity, phosphorylation of the N-terminus is not necessary to maintain longitudinal rigidity of the lattice, in contrast to radial rigidity.

Myosin binding protein-C (MyBP-C) is a thick filament accessory protein of vertebrate striated muscle. It is essential for normal cardiac function, and mutations in MyBP-C cause cardiac and skeletal muscle disease. The 40 nm long molecule is composed of 10 or 11 immunoglobulin- or fibronectin-like domains and is located in up to 9 axial stripes 43 nm apart in each half of the A-band. To understand MyBP-C function it is important to know its structural organization in the sarcomere. Several models have been proposed, in which MyBP-C either wraps around the thick filament, or extends radially from or longitudinally along the filament (or some combination of these). To distinguish between these models, we have used immuno-EM of mouse cardiac myofibrils labeled with antibodies to different domains of MyBP-C to determine the relative axial positions of the domains. Myofibrils were obtained from mouse cardiac muscle by homogenizing chemically skinned hearts under rigor conditions. They were labeled with antibodies specific for the N-terminal domain, the middle of the molecule, and the C-terminal domain, and observed by negative stain EM. Labeled myofibrils showed nine stain-excluding stripes in each half of the A-band. The average distance of each stripe from the middle of the M-line was measured. All antibodies labeled axially within 10 nm of each other, at the same axial positions as the unlabeled stripes, suggesting that the bulk of MyBP-C runs radially or circumferentially. A small axial difference between the C-terminal and the central and N-terminal antigenic sites suggests that a short portion of the C-terminus runs longitudinally. Electron tomography of muscle sections (Luther et al., PNAS 2011) and 3D reconstruction of isolated thick filaments (Zoghbi et al., PNAS 2008) support the radial arrangement.

To understand the functional significance of phosphorylation that takes place in the M domain of cMyBP-C, chemically skinned papillary muscle fibers of transgenic mice were studied by sinusoidal length alterations and concomitant tension transients. Muscle fibers were maximally activated at pCa 4.55 in the solution that mimic physiological conditions (5 mM MgATP, 8 mM Pi, 200 mM total ionic strength with K-acetate) in myocytes. WT mice possess phosphorylation sites S273, S282, and S302. SAS is a single mutant S282A, and ADA and DAD are triple mutants S273A/S282D/S302A and S273D/S282A/S302D, respectively. D models for phosphorylation (phosphomimetic), and A models for non-phosphorylation (phosphor-ablation). Isometric tension and stiffness of DAD were respectively ∼0.5x of those of WT, but tension and stiffness of t/t (cMyBP-C null), ADA, and SAS were respectively similar to WT. The fast rate constant 2πc of DAD and t/t was ∼0.6x of WT, but that of ADA and SAS was similar to WT. The intermediate rate constant 2πb of DAD and SAS was ∼1.3x of WT, but that of ADA and t/t was similar to WT. These results demonstrate that cMyBP-C M domain phosphorylation affects the cross-bridge kinetics at ATP binding and phosphate release steps, indicating that phosphorylation affects myosin structure and its interaction with actin. However, pCa-tension and pCa-stiffness studies demonstrated that pCa50 (Ca2+ sensitivity) and nH (cooperativity) were respectively not different among mutants and WT groups, indicating that phosphorylation of cMyBP-C has a minimal effect on the regulatory system. The decreased amount of isometric tension only in DAD indicates that phosphorylation of S273 and S302 are most significant and they diminish the force generation capability, presumably owing to the extra electrostatic interaction of the M domain of cMyBP-C with actin thin filament, which may serve as a break.

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric thick filament assembly protein with regulatory functions in the heart. The cMyBP-C protein differs from the skeletal isoform in that it has a small insertion near the carboxyl terminus that contains 3 phosphorylatable serines, Ser-273, −282 and −302. While the precise functional correlates of cMyBP-C phosphorylation remain obscure, we do know that cMyBP-C is targeted by multiple kinases, such as PKA, PKC, RSK, PKD, CaMKII and PKG, suggesting that it plays a vital role in cardiac signaling. We previously reported that cMyBP-C phosphorylation is essential for normal heart function and that Ser-282 phosphorylation is critical for the subsequent phosphorylation of Ser-302 and normal cardiac function. However, the role of Ser-282 cMyBP-C phosphorylation in cardiac function, particularly as it affects contractile properties and sarcomere organization, is unclear. Therefore, to better understand the mechanisms and significance of cMyBP-C phosphorylation, we established several transgenic mouse models to determine the necessity and sufficiency of Ser-282 phosphorylation for normal cardiac function. Our findings suggested that cMyBP-C phosphorylation at Ser-282 is essential for normal cardiac function and that dephosphorylation at this site accelerates cMyBP-C degradation and cleavage of a 40 kDa fragment. During MI, we showed that cMyBP-C is extensively fragmented when dephosphorylated and that such fragmentation correlates well with contractile dysfunction and heart failure. Meanwhile, we also established that the release of cMyBP-C in the blood post-MI could be a potential diagnostic biomarker for MI. Overall, these studies show that Ser-282 phosphorylation is a critical determinant of Ser-302 phosphorylation and that cMyBP-C dephosphorylation accelerates its degradation and release into the circulation. In conclusion, we provide strong evidence that cMyBP-C phosphorylation directly affects the heart's contractile properties, sarcomere organization and cardioprotection.

Phosphorylation sites in Cardiac MyBP-C have been predicted at Ser273,282 and 302 but studies in intact tissue have identified 5 phosphorylted sites and suggested up to 4.6molsPi/mol MyBP-C is present in human heart.We analysed MyBP-C phosphospecies in human heart myofibrils by phosphate affinity SDS-PAGE using a non-specific antibody raised against the MyBP-C peptide 2-14. We observed six bands corresponding to 0, 1P, 2P, 3P, 4P, 5P phospho-species. Control experiments with pure MyBP-C indicated that the antibody labelled all phosphospecies equally. The assigned phosphorylation levels were confirmed by staining western blots with PhosTools phosphoprotein stain. This separation permits direct quantitative determination of MyBP-C phosphospecies without need for calibration.In donor heart myofibrils the highly phosphorylated species predominated: 0, 7±3%: 1P, 1±1%: 2P, 23±7%: 3P, 41±2%: 4P, 20±8% (n=4) from which total phosphorylation of MyBP-C was calculated to be 3.4molsPi/mol. In failing heart unphosphorylated MyBP-C predominated (0, 48±4%: 1P, 4±4%: 2P, 27±5%: 1±1%: 3P, 17±4%: 4P, 4±2%, n=4) and calculated total phosphorylation was 1.5 molsPi/mol. Total phosphorylation in failing heart myofibrils was 44% of donor and in myectomy samples from HCM patients it was 29% of donor, compared with 45 and 40% respectively determined in previous assays.We conclude that MyBP-C is highly phosphorylated in vivo with significant phosphorylation of at least 5 sites and that phosphorylation is dynamic, being greatly reduced in pathological muscle. Initial tests using antibodies specific to Ser 273, 282 and 302 show distinct patterns on phosphate affinity SDS-PAGE indicating varying preferences for the highly phosphorylated species of MyBP-C in normal and pathological muscle.

Rationale: Cardiac myosin binding protein-C (cMyBP-C) has three phosphorylatable serines at N-terminus (Ser-273, Ser-282, and Ser-302) and phosphorylation states of these serines may alter the thick filament structure and function. Phosphorylation of these serines in cMyBP-C may also alter sarcomere length-dependent modulation of contractile force development (LDA), which would be expected to modulate the Frank-Starling Law of the Heart.Objective: To analyze length dependent activation in cMyBP-C for nonphosphorylated and phosphomimetic states of serines 273, 282, 302 and to study the contribution of cMyBP-C in regulating cardiac contraction at different sarcomere lengths.Methods & Results: We studied the force-Ca2+ relationship in skinned myocytes isolated from (a) nonphosphorylated alanine transgenic mice (cMyBP-CAAA), (b) phosphomimetic aspartic acid transgenic mice (cMyBP-CDDD), and normal cMyBP-CWT mice at 2.0 and 2.3 μm sarcomere length (SL).At short SL, Ca2+ sensitivity was similar in all groups. However, Ca2+ sensitivity was increased in the cMyBP-CAAA group compared to either the WT or the cMyBP-CDDD group. Similar results were obtained for maximum force development. As a consequence, LDA was blunted (∼40%) in cMyBP-CAAA myocardium. There were no differences in the level of cooperativety as indexed by the Hill coefficient in any group.Conclusion: Phosphorylated cMyBP-C has been shown to contribute to regulation of cardiac sarcomere function via modulation of the cMyBP-C-actin interaction as well as the disposition of the cross-bridges in relation to the thin filament. Moreover removal of cMyBP-C results in blunted LDA, and a cardiac dysfunction that can be prevented by cMyBP-CDDD but not cMyBP-CAAA. Our data showed that lack of cMyBP-C phosphorylation results in blunted LDA, similar to that found previously in the absence cMyBP-C. We conclude that cMyBP-C phosphorylation modulates myofilament length dependent activation, possibly via modulation of the cMyBP-C interaction with actin.

Cardiac myosin binding protein-C (cMyBP-C) is a modular protein involved in stabilizing interactions with the thick filament of the sarcomere. The N-terminus of cMyBP-C associates with actin and myosin S2 and the C-terminus interacts with titin and myosin rods. While no high-resolution structure of C-terminal cMyBP-C exists, disruption of this region is proposed to destabilize cMyBP-C and adversely affect cardiac structure and function. In particular, deletion of 25 base pairs (Δ25) in the gene encoding for cMyBP-C results in amino acid substitutions in the C10 domain of cMyBP-C (C10 Δ25) which may be associated with the development of hypertrophic and dilated cardiomyopathies by unknown molecular mechanisms. The prevalence of this mutation is approximately 1% of the world population, underscoring the necessity of determining its role(s) in the pathogenesis of cardiomyopathies. In this study, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies have been used to examine the conformation of wild-type (Wt) and mutant C-terminal domains of cMyBP-C. Comparison of near UV CD spectra revealed alterations in the packing of aromatic residues in C10 Δ25 suggesting it is less stably folded as compared to C10 Wt. C10 Δ25 exhibited less beta-sheet content than C10 Wt as evidenced by the estimation of secondary structure from CD data. NMR analyses of amide proton/nitrogen chemical shifts and line-widths were used to probe the conformation of C10 domains and to map residues of importance in protein-protein association onto cMyBP-C models. Taken together, these data suggest that the Δ25 mutation structurally modulates cMyBP-C sites involved in binding titin and myosin.

Rationale: Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin binding protein-C (cMyBP-C), a cardiac-specific myofilament protein, is proteolyzed post-MI in humans and results in an N-terminal fragment, C0C1f. The presence of C0C1f in cultured adult cardiomyocytes results in decreased Ca2+ transients and cell shortening, in addition to the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear.Objective: To determine how C0C1f causes altered contractility in human cardiac myofilaments in vitro.Methods and Results: We generated recombinant human C0C1f (hC0C1f) and incorporated it into skinned human left ventricular myocytes. Mechanical properties were then studied at sarcomere lengths of 2.0 and 2.3 µm. Our data demonstrate that the presence of hC0C1f in the sarcomere decreased maximal force myofilament Ca2+ sensitivity, increased cooperative activation at short lengths and enhanced length-dependent activation. Furthermore, hC0C1f led to increased cross-bridge cycling kinetics and tension cost at both short and long sarcomere lengths. We further established that the detrimental effects of hC0C1f occur through direct interaction with the thin filament proteins actin and α-tropomyosin (α-TM).Conclusions: Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myofilament preparations. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-TM, suggesting that cleaved C0C1f could act as a poison peptide and disrupt the interaction of native cMyBP-C with the thin filament.Keywords: Cross-bridge cycling kinetics; length-dependent activation; cMyBP-C; C0C1f protein.

Understanding how cellular level mechanical properties change as failing hearts remodel may help identify the protein modifications that produce contractile dysfunction associated with this condition. We therefore procured through wall left ventricular samples from patients undergoing heart transplants at the University of Kentucky and from organ donors who did not have heart failure. Multicellular chemically permeabilized preparations were subsequently obtained from sub epicardial, midwall and sub endocardial regions of these samples by mechanical homogenization and triton treatment. The mechanical properties of the preparations were assessed by attaching them between a force transducer and a motor and subjecting them to a standard series of mechanical assays. There was a 30% decrease in maximum power output (p-value = 0.01) and steady-state force (p-value=0.005) in patients with end-stage heart failure (n=8, total of 72 preparations) as compared to samples from nonfailing organ donors (n=4, total of 36 preparations). The phosphorylation of two cardiac sarcomeric proteins- myosin binding protein-C (cMyBP-C) and troponin-I (cTnI) which have been previously associated with contractile dysfunction were investigated using phospho specific antibodies. Western blot analysis of three major phosphorylated sites of cMyBP-C, PSer273, PSer282 and PSer302 were not significantly different between heart failure and non failing groups but cTnI phosphorylation at PSer22/23 decreased significantly in heart failure (p=0.01). This study suggests that the cellular level mechanical dysfunction seen in heart failure may be in part due to phosphorylation modifications.