Skeletal Myosin Binding protein-C Research Articles

Abstract P2047: Fast Skeletal Myosin Binding Protein-C Expression Exacerbates Dysfunction In Heart Failure

Background: During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We have previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences in the functional differences which may affect cardiac function. Also, it is unknown if the fMyBP-C expression in the heart is a pathological or compensatory response. Objectives: We aim to elucidate the consequence of increased fMyBP-C expression in the heart. Methods: To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the absence of cMyBP-C function. Finally, fMyBP-C knockout mice were subjected to transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. Results: We confirmed the upregulation of fMyBP-C in several models of cardiac disease. Genetic over-expression of low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. High expression of fMyBP-C in a heart failure model further exacerbated the cardiac disease, hypertrophy and fibrosis. On the other hand, following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the greater instability of the myosin super-relaxed state compared to wild-type littermates. Conclusions: These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure.

Fast and slow skeletal myosin binding protein-C and aging

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs.

In situ replacement of slow skeletal myosin binding protein-c

Myosin binding protein (MyBP-C) is a modular protein that interacts with both thin and thick filaments and regulates striated muscle contraction. The functional characteristics of MyBP-C have yet to be fully uncovered in all 3 isoforms (cardiac, fast skeletal, and slow skeletal). To manipulate cardiac (c) MyBP-C at its precise position in the sarcomere in situ, we created gene-edited ‘SpyC’ mice that allow us to use a novel ‘cut and paste’ method for cMyBP-C recombination in cardio-myocytes. This ‘cut and paste’ method allows for the in situ removal of endogenous N’-terminal domains from MyBP-C via TEV proteolysis, followed by its replacement with any desired recombinant N’-terminal MyBP-C.

Protein Kinase A Modifies Skeletal Muscle Contractile Function Via Modification Of Mybp-C

Human aging is associated with decreased skeletal muscle (SM) contractile velocity and powerproduction. Interestingly, older muscle exhibits greater tension (force per cross sectional area)compared to younger muscle when velocity is zero (isometric contraction), suggesting age-related contractile deficits are velocity-dependent and distinct from changes in muscle size.Mechanisms explaining this phenomenon are unknown, but evidence in cardiac myocytessuggests that Myosin Binding Protein-C (MyBP-C) has similar, phosphorylation-dependenteffects on maximal velocity (Vmax), power (Pmax), and tension (Tmax). However, this potentialhas not been explored in human SM. Therefore, the PURPOSE of our study is to examine ifphosphorylation of skeletal MyBP-C in older muscle produces contractile outcomes emulatingthose reported in younger muscle. METHODS: Measures of contractile performance, Tmax, Vmax and Pmax, were collected during maximal activation (pCa 4.5) of thirteen SM fibers fromtwo males (41 and 74 yrs.). Measures were acquired before and after incubation with ProteinKinase A (PKA), a known phosphorylating agent of MyBP-C, or relaxing solution without PKA(Control). Phospho-specific changes to MyBP-C were confirmed in parallel experiments on fiberbundles exposed to identical incubation conditions and assessed via SDS-PAGE followed byProQ Diamond and SYPRO Ruby staining. RESULTS: Phospho-specific staining revealed thatPKA selectively modified MyBP-C under these conditions without altering Regulatory LightChain or Troponin I. Following PKA incubation, MHC I fibers produced greater Vmax (0.46 ± 0.29 vs. 0.37 ± 0.25 ML/s, p < 0.05) and Pmax (1.73 ± 0.68 vs. 1.46 ± 0.54 mN/mm2·ML-s,p < 0.05) and lower Tmax (83.72 ± 11.44 vs. 92.19 ± 14.39 mN/mm2, p < 0.05). In contrast, neitherMHC II nor Control MHC I fibers demonstrated significant contractile changes after incubation. CONCLUSION: These exciting pilot data suggest that PKA selectively phosphorylates MyBP-C, resulting in functional differences in older SM fiber contractile performance that mirror theyouthful contractile phenotype.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5–10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5–10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7–10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S–S bonds providing for general stability.

Regulation of Myofilament Force and Loaded Shortening by Skeletal Myosin Binding Protein-C

Myosin binding protein C (MyBP-C) is a 125-140-kD protein located in the C-zone of each half-thick filament. It is thought to be an important regulator of contraction, but its precise role is unclear. Here we investigate mechanisms by which skeletal MyBP-C regulates myofilament function using rat permeabilized skeletal muscle fibers. We mount either slow-twitch or fast-twitch skeletal muscle fibers between a force transducer and motor, use Ca2+ to activate a range of forces, and measure contractile properties including transient force overshoot, rate of force development, and loaded sarcomere shortening. The transient force overshoot is greater in slow-twitch than fast-twitch fibers at all Ca2+ activation levels. In slow-twitch fibers, protein kinase A (PKA) treatment (a) augments phosphorylation of slow skeletal MyBP-C (sMyBP-C), (b) doubles the magnitude of the relative transient force overshoot at low Ca2+ activation levels, and (c) increases force development rates at all Ca2+ activation levels. We also investigate the role that phosphorylated and dephosphorylated sMyBP-C plays in loaded sarcomere shortening. We test the hypothesis that MyBP-C acts as a brake to filament sliding within the myofilament lattice by measuring sarcomere shortening as thin filaments traverse into the C-zone during lightly loaded slow-twitch fiber contractions. Before PKA treatment, shortening velocity decelerates as sarcomeres traverse from ∼3.10 to ∼3.00 µm. After PKA treatment, sarcomeres shorten a greater distance and exhibit less deceleration during similar force clamps. After sMyBP-C dephosphorylation, sarcomere length traces display a brief recoil (i.e., bump) that initiates at ∼3.06 µm during loaded shortening. Interestingly, the timing of the bump shifts with changes in load but manifests at the same sarcomere length. Our results suggest that sMyBP-C and its phosphorylation state regulate sarcomere contraction by a combination of cross-bridge recruitment, modification of cross-bridge cycling kinetics, and alteration of drag forces that originate in the C-zone.

Regulation of myofilament force and loaded shortening by skeletal myosin binding protein C.

Myosin binding protein C (MyBP-C) is a 125-140-kD protein located in the C-zone of each half-thick filament. It is thought to be an important regulator of contraction, but its precise role is unclear. Here we investigate mechanisms by which skeletal MyBP-C regulates myofilament function using rat permeabilized skeletal muscle fibers. We mount either slow-twitch or fast-twitch skeletal muscle fibers between a force transducer and motor, use Ca2+ to activate a range of forces, and measure contractile properties including transient force overshoot, rate of force development, and loaded sarcomere shortening. The transient force overshoot is greater in slow-twitch than fast-twitch fibers at all Ca2+ activation levels. In slow-twitch fibers, protein kinase A (PKA) treatment (a) augments phosphorylation of slow skeletal MyBP-C (sMyBP-C), (b) doubles the magnitude of the relative transient force overshoot at low Ca2+ activation levels, and (c) increases force development rates at all Ca2+ activation levels. We also investigate the role that phosphorylated and dephosphorylated sMyBP-C plays in loaded sarcomere shortening. We test the hypothesis that MyBP-C acts as a brake to filament sliding within the myofilament lattice by measuring sarcomere shortening as thin filaments traverse into the C-zone during lightly loaded slow-twitch fiber contractions. Before PKA treatment, shortening velocity decelerates as sarcomeres traverse from ∼3.10 to ∼3.00 µm. After PKA treatment, sarcomeres shorten a greater distance and exhibit less deceleration during similar force clamps. After sMyBP-C dephosphorylation, sarcomere length traces display a brief recoil (i.e., "bump") that initiates at ∼3.06 µm during loaded shortening. Interestingly, the timing of the bump shifts with changes in load but manifests at the same sarcomere length. Our results suggest that sMyBP-C and its phosphorylation state regulate sarcomere contraction by a combination of cross-bridge recruitment, modification of cross-bridge cycling kinetics, and alteration of drag forces that originate in the C-zone.

Abstract 303: Fast Skeletal Myosin Binding Protein-c Expression in Heart Failure

Background: Heart failure is a devastating disease affecting nearly 5 million Americans. We recently identified the upregulation of fast skeletal myosin binding protein-C (fsMyBP-C) in models of heart failure. fsMyBP-C is a homologue of cardiac MyBP-C, which is normally expressed in the heart. This family of proteins regulate contractility by modulating thick and thin filament interactions. However, the functional role(s) of fsMyBP-C expression in the heart is unknown. Methods: To understand the role of fsMyBP-C expression in the heart, we generated cardiac specific transgenic mice expressing fsMyBP-C under the control of the alpha myosin heavy chain promoter. Mice underwent serial echocardiography to monitor cardiac function. Western blotting and qRT-PCR were performed to determine transgene expression levels. Additionally, fsMyBP-C localization in cardiomyocytes was established by immunofluorescence. Furthermore, to understand the temporal expression profiles of the MyBP-C homologues, we performed qRT-PCR on 9 muscle groups at different developmental stages in healthy mice. Results: qRT-PCR demonstrated an upregulation of the slow MyBP-C transcript in the developing heart, while fsMyBP-C transcript levels increased steadily following birth in WT mice. At 3 months of age, transgenic mice displayed a significant increase in ventricular dimensions compared to NTG littermates by echocardiography (LVID d : 3.72 ± 0.07 vs. 4.17 ± 0.09 mm, p&lt;0.01; LVID s : 2.44 ± 0.09 vs. 2.81 ± 0.09, p&lt;0.05), with no changes in wall thickness. This was accompanied by a 25% increase in stroke volume (p&lt;0.01) and a 30% increase in cardiac output (p&lt;0.05). fsMyBP-C correctly localized to the C-zone of the sarcomere. Furthermore, protein levels showed mild expression of fsMyBP-C, in line with the small upregulation seen in diseased hearts, while qRT-PCR confirmed overexpression of fsMyBP-C in the ventricles. Conclusion: Mice expressing fsMyBP-C in the heart display a mild hypertrophic phenotype at 3 months, with preserved function, possibly indicating compensated hypertrophy. Ongoing studies investigate cardiac function at later stages and cardiac specific conditional knockout of fsMyBP-C to determine the long-term effect of fsMyBP-C regulation in the heart.

Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner

Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.

MYBP-C Phosphorylation Accelerates Sarcomere Shortening as Thin Filaments Slide through the C-Zone

Myosin Binding Protein-C (MyBP-C) is a 140-150 kDa thick filament protein located in the middle third of each sarcomere, i.e., the C-zone, and is likely a key regulator of contraction. Phosphorylation of MyBP-C occurs at 4 residues within its N-terminus and PKA phosphorylation of MyBP-C in hearts is associated with faster cardiac muscle twitch dynamics (Tong et. al. Circ. Res. 2008). Here we investigated potential mechanisms by which MyBP-C phosphorylation regulates myofilament function. We utilized slow-twitch skeletal muscle fibers since PKA phosphorylates slow skeletal (ss)MyBP-C but not (ss)troponin I. Permeabilized rat slow-twitch fibers were mounted between a force transducer and motor, and calcium activated to elicit ∼30% maximal force. We measured rates of force development and transient force overshoots following a slack re-stretch maneuver and monitored sarcomere shortening during load-clamps. Following PKA, force development rates increased ∼40% and there was a near-doubling in the magnitude of transient force overshoot. We also observed faster shortening velocities at all loads following PKA treatment; this may arise from (i) faster cross-bridge cycling rates, (ii) greater number of cross-bridges during load clamps, and/or (iii) attenuation of internal drag imposed by binding of MyBP-C to actin. To address these mechanisms, we quantified sarcomere length shortening as thin filaments traversed into the C-zone. Before PKA, there tended to be a slowdown as sarcomeres shortened from ∼3.05 to 2.90 µm. After PKA, sarcomeres shortened a considerably greater distance during similar load clamps and, at times, shortening even accelerated as thin filaments slid through the C-zone. Overall these results suggest MyBP-C phosphorylation speeds sarcomere shortening and power output by a combination of faster cross-bridge cycling kinetics, recruitment of cross-bridges, and reduction in drag forces as thin filaments slide through the C-zone.

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

Knockdown of fast skeletal myosin-binding protein C in zebrafish results in a severe skeletal myopathy.

Myosin-binding protein C (MyBPC) in the muscle sarcomere interacts with several contractile and structural proteins. Mutations in the cardiac isoform (MyBPC-3) in humans, or animal knockout, are associated with cardiomyopathy. Function of the fast skeletal isoform (MyBPC-2) in living muscles is less understood. This question was addressed using zebrafish models, combining gene expression data with functional analysis of contractility and small-angle x-ray diffraction measurements of filament structure. Fast skeletal MyBPC-2B, the major isoform, was knocked down by >50% using morpholino antisense nucleotides. These morphants exhibited a skeletal myopathy with elevated apoptosis and up-regulation of factors associated with muscle protein degradation. Morphant muscles had shorter sarcomeres with a broader length distribution, shorter actin filaments, and a wider interfilament spacing compared with controls, suggesting that fast skeletal MyBPC has a role in sarcomere assembly. Active force was reduced more than expected from the decrease in muscle size, suggesting that MyBPC-2 is required for optimal force generation at the cross-bridge level. The maximal shortening velocity was significantly increased in the MyBPC-2 morphants, but when related to the sarcomere length, the difference was smaller, reflecting that the decrease in MyBPC-2B content and the resulting myopathy were accompanied by only a minor influence on filament shortening kinetics. In the controls, equatorial patterns from small-angle x-ray scattering revealed that comparatively few cross-bridges are attached (as evaluated by the intensity ratio of the 11 and 10 equatorial reflections) during active contraction. X-ray scattering data from relaxed and contracting morphants were not significantly different from those in controls. However, the increase in the 11:10 intensity ratio in rigor was lower compared with that in controls, possibly reflecting effects of MyBPC on the cross-bridge interactions. In conclusion, lack of MyBPC-2 results in a severe skeletal myopathy with structural changes and muscle weakness.

The N-Terminal Domains of MyBPC3 Restrict Actin Dynamics and Increase Resilience in a Phosphorylation-Dependent Manner

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).

Skeletal Myosin Binding Protein-C Isoforms Modulate Actomyosin Contractility and are Regulated by Phosphorylation

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 restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C 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 torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Introducing a series of topical special issues of the Journal of Muscle Research and Cell Motility

The Journal of Muscle Research aims to serve its readers not only by publishing original studies in muscle and motility structure and function but also by informing readers of the state of the art in muscle research and cell motility and to promote debate. This issue of the Journal of Muscle Research and Cell Motility is the first of a series of topical special issues that combine original research with review articles related to a specific subject that we plan to publish once or twice a year. Further topical issues are being planned and we would welcome ideas for the themes to be covered in future issues. In this issue, articles are devoted to cardiac myosinbinding protein-C. When C-protein, or myosin-binding protein-C (MyBP-C) was discovered in skeletal muscle in the early 1970s by Gerald Offer (Offer 1972; Offer et al. 1973), MyBP-C was thought to be merely a structural protein component of the thick filament. Interest in the functions of MyBP-C were galvanised when it was found that the cardiac isoform, encoded by the MYBPC3 gene on chromosome 11, was mutated in cases of hereditary hypertrophic cardiomyopathy (HCM) (Bonne et al. 1995; Watkins et al. 1995). By now, mutations in MYBPC3 are the second-most frequently identified mutations that cause HCM (Schlossarek et al. 2011), with carriers of a specific pathogenic mutation amounting up to 4 % of the population of Indian descent (Dhandapany et al. 2009). It has become clear that MyBP-C must play important roles in both the regulation of contractility and as the assembly of muscle myofibrils. MyBP-C is transcribed from three related genes (MYBPC1-3), which encode the slow, fast and cardiac isoforms. Like many other myofibrilassociated structural proteins, MyBP-C is composed of a series of hundred-residue domains of the intracellular immunoglobulin and fibronectin families—10–11 in the case of MyBP-C (Furst and Gautel 1995; Otey et al. 2009). This molecular architecture is shared with the small MyBPC homologue, myosin-binding protein-H (Vaughan et al. 1993) and other myofibrillar proteins like myopalladin, myotilin, the myomesin isogenes and the giant scaffold and signalling proteins titin and obscurin (KontrogianniKonstantopoulos et al. 2009). Earlier work by the laboratories of Gerald Offer, Carl Moos, Roger Starr, Rick Moss, Polly Hofmann, Criss Hartzell and others had implicated MyBP-C in contraction regulation by interactions with actin or myosin filament components, possibly dynamically modulated by phosphorylation by cAMP and calcium/calmodulin dependent protein kinases in the case of the cardiac isoform (see articles in this issue for a review of the literature). The elucidation of the primary structure of both skeletal and cardiac MyBP-C isoforms pinpointed these phosphorylation sites to the N-terminal region within a sequence distinct from the immunoglobulin or fibronectin-like domains that form the remainder of the protein (Einheber and Fischman 1990; Furst et al. 1992; Gautel et al. 1995). This MyBP-C motif has since been implicated as the major domain involved in contraction regulation by interactions that have been localised to both the actin and myosin S. B. Marston NHLI, Myocardial Function, Imperial College London, Imperial Centre for Translational and Experimental Medicine, London W12 0NN, UK e-mail: S.marston@imperial.ac.uk

Axial distribution of myosin binding protein-C is unaffected by mutations in human cardiac and skeletal muscle

Myosin binding protein-C (MyBP-C), a major thick filament associated sarcomeric protein, plays an important functional and structural role in regulating sarcomere assembly and crossbridge formation. Missing or aberrant MyBP-C proteins (both cardiac and skeletal) have been shown to cause both cardiac and skeletal myopathies, thereby emphasising its importance for the normal functioning of the sarcomere. Mutations in cardiac MyBP-C are a major cause of hypertrophic cardiomyopathy (HCM), while mutations in skeletal MyBP-C have been implicated in a disease of skeletal muscle—distal arthrogryposis type 1 (DA-1). Here we report the first detailed electron microscopy studies on human cardiac and skeletal tissues carrying MyBP-C gene mutations, using samples obtained from HCM and DA-1 patients. We have used established image averaging methods to identify and study the axial distribution of MyBP-C on the thick filament by averaging profile plots of the A-band of the sarcomere from electron micrographs of human cardiac and skeletal myopathy specimens. Due to the difficulty of obtaining normal human tissue, we compared the distribution to the A-band structure in normal frog skeletal, rat cardiac muscle and in cardiac muscle of MyBP-C-deficient mice. Very similar overall profile averages were obtained from the C-zones in cardiac HCM samples and skeletal DA-1 samples with MyBP-C gene mutations, suggesting that mutations in MyBP-C do not alter its mean axial distribution along the thick filament.

Effects of Myosin Binding Protein-C Isoform, Phosphorylation, and Domains on the Rotational Dynamics of Actin Filaments

We have determined the effects of Myosin binding protein-C (MyBP-C) and its domains on the microsecond time-scale rotational dynamics of actin, using time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multi-domain thick filament-associated modulator of striated muscle contraction, spanning the interfilament spacing to contact both myosin and actin. Cardiac (c-) and slow skeletal (ss-) MyBP-C are known substrates for Protein kinase-A (PKA), and phosphorylation of cMyBP-C alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin's microsecond structural dynamics, we labeled actin at C374 with erythrosine iodoacetamide and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy (r∞) of the TPA decay in a concentration-dependent manner, indicating restriction of the rotational amplitude of actin dynamics. While PKA phosphorylation had little effect on fast skeletal (fs-) MyBP-C, phosphorylation of cMyBP-C and ssMyBP-C nearly eliminated the effects of these proteins to restrict actin dynamics, despite no change in binding affinity with phosphorylation. Skeletal MyBP-C (C1-C10) affected actin anisotropy at lower concentrations than cMyBP-C (C0-C10), suggesting that skeletal and cardiac N-terminal MyBP-C interactions with actin have distinct properties. The effects of truncated cMyBP-C on actin anisotropy determined that C-terminal domains are important for restricting rotational dynamics, whereas N-terminal domains are important for regulating this effect. These MyBP-C-induced changes in actin dynamics may play a role in the known effects of MyBP-C on the functional actin-myosin interaction. This work was funded by grants from NIH (F32 HL107039-01 to BAC and T32 AR007612 to DDT).

Support for a trimeric collar of myosin binding protein C in cardiac and fast skeletal muscle, but not in slow skeletal muscle

Myosin-binding protein C (MyBPC) is proposed to take on a trimeric collar arrangement around the thick filament backbone in cardiac muscle, based on interactions between cardiac MyBPC domains C5 and C8. We have now determined, using yeast two-hybrid and in vitro binding assays, that the C5:C8 interaction is not dependent on the 28-residue cardiac-specific insert in C5. Furthermore, an interaction of similar affinity occurs between domains C5 and C8 of fast skeletal muscle MyBPC, but not between these domains of the slow skeletal muscle protein. These data have implications for the role and quaternary structure of MyBPC in skeletal muscle.