Regulation Of Cardiac Contraction Research Articles

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca(2+) Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils.

Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

The Regulation of ATP-Binding, PI Release and ADP Dissociation from Myosin II in Native Cardiac Myofibrils by the N-Terminal Extension of Cardiac Troponin T

Cardiac troponin T (cTnT) has an amino-terminal variable region that plays a role in modifying the overall protein conformation and functions in the calcium-dependent regulation of cardiac muscle contraction. Previous studies have shown that the N-terminal domain of cTnT mainly affects the tropomyosin binding site 1 that binds to the head-tail junction of tropomyosins in thin filament, a moiety involved in the cooperativity of the myofilaments upon calcium activation. It has also been shown that in comparison with wild type controls, transgenic mouse hearts over-expressing cTnT lacking the N-terminal variable region (cTnT-ND) had a decreased rate of contraction, which specifically elongates the rapid ejection phase while keeping total ejection time unchanged, suggesting that the altered cTnT molecule affects the actomyosin crossbridge kinetics. In the present study, we employed stopped flow analysis of mant-ATP binding, ADP dissociation and phosphate release in order to investigate which step(s) of the ATPase cycle is regulated by the N-terminal variable region of cTnT. The results showed that cTnT-ND myofibrils exhibited a maximum mant-ATP binding rate constant (k2) significantly lower than that of WT control, while the equilibrium constant K1 for the initial actomyosin complex formation remained unchanged over calcium concentration. ADP dissociation and Pi release from cTnT-ND myofibrils showed no significant change with increasing calcium concentration, however ADP release is slower in cTnT-ND myofibrils when compared to WT control. These results provide novel information regarding the function of the N-terminal variable region in modulating specific kinetic steps and selectively altering systolic but not diastolic function of the heart.

The Physiological Relevance of Interactions between cMyBP-C and Actin Studied in a Transgenic Mouse Model

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein that plays an essential role in the regulation of cardiac contraction. Until recently, cMyBP C was thought to hamper cross bridge cycling trough interaction of its N-terminus with the heads of myosin. However, the N-terminal domains of cMyBP-C can also bind with actin and this interaction could well explain the currently known functions of cMyBP-C. For instance, by forming a connection between the thin and the thick filament cMyBP-C could pose a load on the sarcomere and alter its visco-elastic properties. Additionally, cMyBP-C could alter the activation state of the thin filament by intervening with the position of tropomyosin.To study the physiological relevance of an interaction between cMyBP-C and actin, we created a transgenic mouse with a point mutation (L348P) that increases the binding affinity between cMyBP-C and actin in vitro. We hypothesized that stronger binding between cMyBP-C and actin will cause- through afore mentioned mechanisms, which have been demonstrated in different in vitro studies- stiffer cardiomyocytes which would ultimately lead to diastolic dysfunction in the hearts of L348P-Tg mice.Results show that the left atria of L348P-Tg mice are significantly enlarged. Echocardiograms in 12 week old animals demonstrated misshapen E and A waves, making diastolic function difficult to determine. Interestingly, pulse wave and tissue Doppler images were more defined in 24 weeks old mice. Preliminary data show decreased early blood flow over the mitral valve (E) and mitral valve movement (E’) in L348PTg mice, indicative of a stiffer left ventricle. In correspondence with that, isovolumetric relaxation time was prolonged. At neither age we observed systolic dysfunction. Additional in-depth hemodynamic studies to characterize the mechanisms of cardiac function longitudinally in L348P-Tg mice are ongoing.

Abstract 12088: cMyBP-C’S Interaction With Actin is Critical to Maintain Diastolic Function in vivo

Background: cMyBPC’s interaction with actin is critical to maintain diastolic function in vivo Rationale: Extensive studies demonstrate that cardiac myosin binding protein C (cMyBP-C) is a critical mediator of diastolic function through its interaction with actin, myosin and titin. The precise molecular mechanisms underlying the protein’s function remain elusive as cMyBP-C’s structural domains responsible for actin and/or myosin interaction are ill-defined and the physiological consequences of these filament interactions are unknown. Objective: In the present study, we made transgenic mice that lacked cMyBP-C’s actin and/or the head region of the myosin heavy chain (S2-MyHC) sites in order to better understand the molecular mechanisms of cMyBPC-mediated regulation of cardiac contraction, particularly diastolic function. Methods and Results: Using a combination of genetics and functional assays, we defined cMyBP-C’s N-terminal structural domains as well as the critical residues necessary or sufficient to mediate interactions with actin and/or S2-MyHC. Using genetic experiments, we first confirmed that mutation of three lysines to alanines in the C1 domain abolished actin binding (cMyBP-C Act- ). Similarly, mutation of three arginines to alanines in the “m” domain abolished S2-MyHC binding (cMyBP-C S2- ). To determine the functional roles of these sites in vivo, we generated transgenic lines in which normal cMyBP-C was replaced by either cMyBP-C Act-,S2- or wild type (cMyBP-C WT ), transgenically-encoded cMyBP-C. Mutant protein was completely substituted for endogenous cMyBP-C by breeding the transgenes into a cMyBP-C null background. The cMyBP-C Act-,S2- mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis. Echocardiographic Doppler measurements showed that cMyBP-C Act-,S2- mice develop both diastolic and systolic dysfunction. In contrast, the cMyBP-C WT Tg line showed normal cardiac structure and function. Conclusions: These results suggest that the cMyBP-C’s interactions with both actin/S2-MyHC are structurally important to preserve normal cardiac contractility and particularly, cMyBP-C’s interaction with actin is indispensable for normal diastolic function.

Regulatory domain of troponin moves dynamically during activation of cardiac muscle

Heart muscle is activated by Ca2+ to generate force and shortening, and the signaling pathway involves allosteric mechanisms in the thin filament. Knowledge about the structure-function relationship among proteins in the thin filament is critical in understanding the physiology and pathology of the cardiac function, but remains obscure. We investigate the conformation of the cardiac troponin (Tn) on the thin filament and its response to Ca2+ activation and propose a molecular mechanism for the regulation of cardiac muscle contraction by Tn based uniquely on information from in situ protein domain orientation. Polarized fluorescence from bifunctional rhodamine is used to determine the orientation of the major component of Tn core domain on the thin filaments of cardiac muscle. We show that the C-terminal lobe of TnC (CTnC) does not move during activation, suggesting that CTnC, together with the coiled coil formed by the TnI and TnT chains (IT arm), acts as a scaffold that holds N-terminal lobe of TnC (NTnC) and the actin binding regions of troponin I. The NTnC, on the other hand, exhibits multiple orientations during both diastole and systole. By combining the in situ orientation data with published in vitro measurements of intermolecular distances, we construct a model for the in situ structure of the thin filament. The conformational dynamics of NTnC plays an important role in the regulation of cardiac muscle contraction by moving the C-terminal region of TnI from its actin-binding inhibitory location and enhancing the movement of tropomyosin away from its inhibitory position.

Abstract 93: A Gain-of-Function Mutation in Cardiac Myosin Binding Protein-C Increases Viscoelastic Load and Slows Shortening Velocity in Myocytes from Transgenic Mice

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Effects of cMyBP-C on contraction are thought to be mediated in part by limiting the interactions of actin and myosin to slow myocyte shortening velocity and power output. Although interactions with myosin S2 on the thick filament have been proposed as a way in which cMyBP-C could limit shortening velocity (e.g., by creating a drag force on myosin heads), interactions of cMyBP-C with actin could also account for slowed shortening velocity. For instance, cMyBP-C could create a drag that opposes filament sliding by transiently linking thick and thin filaments together. To explore this possibility we created transgenic mice that express a mutant cMyBP-C with a point mutation, L348P (human L352P), located in a conserved sequence within the regulatory M-domain that increases cMyBP-C binding to actin in vitro. We reasoned that if the mutation also enhanced binding to actin in sarcomeres then shortening velocity would be slowed in myocytes from L348P mice. Results show that transgenic mice expressing the L348P mutation are viable and that L348P cMyBP-C is expressed in sarcomeres. Permeabilized myocytes from transgenic mice showed altered force production including reduced maximal force and enhanced calcium sensitivity of tension. Shortening velocity and power output were significantly reduced whereas passive stiffness and myocyte visco-elasticity were significantly increased. Together these data are consistent with the idea that cMyBP-C creates an internal load in the sarcomere by binding to actin.

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation.

A Gain-Of-Function Mutation in Cardiac Myosin Binding Protein-C Increases Viscoelastic Load and Slows Shortening Velocity in Myocytes from Transgenic Mice

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Effects of cMyBP-C on contraction are thought to be mediated in part by limiting the interactions of actin and myosin to slow myocyte shortening velocity and power output. Although interactions with myosin S2 on the thick filament have been proposed as a way in which cMyBP-C could limit shortening velocity (e.g., by creating a drag force on myosin heads), interactions of cMyBP-C with actin could also account for slowed shortening velocity. For instance, cMyBP-C could create a drag that opposes filament sliding by transiently linking thick and thin filaments together. To explore this possibility we created transgenic mice that express a mutant cMyBP-C with a point mutation, L348P (human L352P), located in a conserved sequence within the regulatory M-domain that increases cMyBP-C binding to actin in vitro. We reasoned that if the mutation also enhanced binding to actin in sarcomeres then shortening velocity would be slowed in myocytes from L348P mice. Results show that transgenic mice expressing the L348P mutation are viable and that L348P cMyBP-C is expressed in sarcomeres. Permeabilized myocytes from transgenic mice showed altered force production including reduced maximal force and enhanced calcium sensitivity of tension. Shortening velocity and power output were significantly reduced whereas passive stiffness and myocyte visco-elasticity were significantly increased. Together these data are consistent with the idea that cMyBP-C creates an internal load in the sarcomere by binding to actin.

Molecular Mechanism for the Regulation of Cardiac Muscle Contraction by Troponin

Contraction of cardiac muscle is regulated by Ca2+ ions binding to troponin (Tn) in the actin-containing thin filament, leading to a movement of tropomyosin around the filament that allows myosin heads to bind to actin and generate force. However the molecular structural basis of this Tn-mediated signalling pathway has remained obscure. We investigated the conformation of the cardiac Tn on the thin filament and its response to binding of Ca2+ to elucidate the molecular mechanism of the regulation of contraction in cardiac muscle.Polarized fluorescence from bifunctional rhodamine was used to determine the orientation of the major component of Tn core domain on the thin filaments of cardiac muscle. We showed that the C-terminus of TnC (CTnC), together with the coiled coil formed by the TnI and TnT chains, did not move during activation and acted as a scaffold that holds N-terminus of TnC (NTnC) and the actin binding regions of troponin I. The NTnC, on the other hand, exhibited multiple orientations during both diastole and systole. By combining the in situ orientation data with published in vitro measurements of intermolecular distances, we constructed an atomic model for the in situ structure of the thin filament that suggests a plausible molecular mechanism for the regulation of heart muscle.

Molecular Dynamics-Based Predictions of the Structural and Functional Effects of Disease-Causing Cardiac Troponin Mutations

Cardiac troponin C (cTnC), the regulatory calcium-binding component of the troponin complex, is responsible for the regulation of cardiac muscle contraction in response to varying cytosolic calcium levels. When calcium is bound to cTnC, a conformational change is initiated, which leads to muscle contraction through changes in the interaction with the remainder of the troponin complex and the interaction of the thin filament with myosin. Mutations that alter the ability of cTnC to bind calcium are hypothesized to induce hypertrophic or dilated cardiomyopathies depending on whether calcium affinity is increased or decreased respectively.Several mutations in cTnC have been selected that are associated with these cardiomyopathies. These mutations include A8V, L29Q, L48Q, C84Y, Q122A, E134D, D145E, which are associated with hypertrophic cardiomyopathy. The structural effects of these mutations have been modeled through equilibrium molecular dynamics and their functional impact assessed using free energy calculations.In each mutant that was analyzed, the equilibrated structures were very similar to those of the wild-type according to the following criteria: backbone superposition, site II calcium coordination residues, the size of calcium-cTnC interface, volume and surface area of the cTnC molecule, and the overall energy score of the structure. The most salient difference between the mutants was the relative orientation of the N & C terminal domains of the proteins. Though the variation across mutants was comparable to the variation between repeated simulations of a given mutation. Despite this very limited structural variation, a measurable change in the free energy of calcium binding was calculated between the mutant and wild-type structures.

Human cardiac troponin complex. Structure and functions

Troponin complex is a component of skeletal and cardiac muscle thin filaments. It consists of three subunits - troponin I, T, and C, and it plays a crucial role in muscle activity, connecting changes in intracellular Ca2+ concentration with generation of contraction. In spite of more than 40 years of studies, many aspects of troponin functioning are still not completely understood, and several models describing the mechanism of muscle contraction exist. Being a key factor in the regulation of cardiac muscle contraction, troponin complex is utilized in medicine as a target for some cardiotonic drugs used in the treatment of heart failure. A number of mutations in troponin subunits are associated with development of different types of cardiomyopathy. Moreover, for the last 25 years cardiac isoforms of troponin I and T have been widely used for immunochemical diagnostics of pathologies associated with cardiomyocyte death (myocardial infarction, myocardial trauma, and others). This review summarizes the existing evidence on the structure and function of troponin complex subunits, their role in the regulation of cardiac muscle contraction, and their clinical applications.

Helix Three (H3) of the M-Domain of Cardiac Myosin Binding Protein-C is not Essential for Functional Effects in Permeabilized Rat Trabeculae

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Cardiac specific phosphorylation sites in the M-domain regulate binding of cMyBP-C to myosin and actin. Immediately downstream of the phosphorylation sites is a bundle of three α-helixes that are well conserved across all species and isoforms of MyBP-C, however the functional significance of these helices is unknown. Helix 3 (H3) bears high homology to the inhibitory peptide of troponin I, suggesting it as a potential binding site for interactions with actin. To understand the functional significance of H3, we produced a recombinant N-terminal protein containing domains C1, M and C2 (C1C2) with the H3 sequence deleted (H3KO). We then assessed effects of the mutant H3KO protein on contractile forces in permeabilized rat trabeculae and compared them to effects of the wild-type C1C2. Results showed that 5µM C1C2 significantly increased Ca2+-sensitivity of force (ΔpCa50=0.29±0.03), whereas 5µM H3KO had no effect (ΔpCa50=0.02±0.01) and 10µM H3KO only modestly increased Ca2+-sensitivity (ΔpCa50=0.11±0.02,). Similarly, whereas 10µM C1C2 activated force in the absence of Ca2+ (pCa 9.0), H3KO activated force in the absence of Ca2+ only at concentrations >20µM. Together, these results establish that H3 is not absolutely required for the functional effects of the N-terminal domains of cMyBP-C to either increase Ca2+-sensitivity of force or to activate tension in sarcomeres, but that H3 contributes to the overall efficacy of the N-terminus in mediating these effects. Potentially, H3 could dock the N-terminus of cMyBP-C with actin or extend/stabilize cMyBP-C interactions with actin or other regulatory proteins. This work was supported by NIH HL-080367 (SPH), NDSEG and AHA graduate fellowships (KLB), and AHA undergraduate fellowship (JKK).

Characterization of a Cardiac Troponin C-Troponin I Chimera Protein

Association of the regulatory N-domain of cardiac troponin C (cNTnC) with the switch region of cardiac troponin I (cTnI147-163) is a major step in Ca2+ regulation of myocardial contraction by troponin as it triggers a series of downstream events that leads to contraction. Investigation of this interaction in the presence and absence of ligands, including Ca2+ and cardiotonic drugs, is important to understanding the regulation of cardiac contraction. This is commonly studied in vitro using isolated cNTnC and cTnI147-163; however, this does not accurately reflect the in situ environment since cNTnC and cTnI147-163 are spatially confined in the myofibril by being attached to their respective structural scaffold domains, the C-domain of cTnC (cCTnC) and the troponin T binding region of cTnI (cTnI80-136). We have designed, expressed and characterized a cardiac chimera (cChimera) protein containing cNTnC linked to cTnI144-173 through a linker containing a tobacco etch virus or thrombin protease cleavage site (similar to skeletal chimera of Tiroli et al, 2005). Our results show that Ca2+ binding to cChimera (KD 0.15 ± 0.05 μm) is tighter than to cNTnC (KD 2.6 ± 0.1 μm) and to that observed in skinned muscle fibers (KD 1.2 μm). We assessed binding of the calcium sensitizer dfbp-o, and desensitizer W7, and show that cChimera is particularly useful for studying and searching interactions of cNTnC-cTnI144-173 with potential agonists. Our cChimera also facilitates the production of the switch cTnI peptide using bacterial cultures by enzyme specific proteolysis after expression along with cNTnC. The dynamic properties of cChimera, along with Ca2+ and drug binding properties, make cChimera a convenient system for the investigation of regulation of troponin by small molecules that target the cTnC-cTnI interface.

Familial hypertrophic cardiomyopathy related E180G mutation increases flexibility of human cardiac α-tropomyosin

α-Tropomyosin (αTm) is central to Ca2+-regulation of cardiac muscle contraction. The familial hypertrophic cardiomyopathy mutation αTm E180G enhances Ca2+-sensitivity in functional assays. To investigate the molecular basis, we imaged single molecules of human cardiac αTm E180G by direct probe atomic force microscopy. Analyses of tangent angles along molecular contours yielded persistence length corresponding to ∼35% increase in flexibility compared to wild-type. Increased flexibility of the mutant was confirmed by fitting end-to-end length distributions to the worm-like chain model. This marked increase in flexibility can significantly impact systolic and possibly diastolic phases of cardiac contraction, ultimately leading to hypertrophy.

Cardiomyopathies: Classification, Clinical Characterization, and Functional Phenotypes

Cardiomyopathies: Classification, Clinical Characterization, and Functional Phenotypes

Tropomyosin Flexural Rigidity and Single Ca2+ Regulatory Unit Dynamics: Implications for Cooperative Regulation of Cardiac Muscle Contraction and Cardiomyocyte Hypertrophy

Striated muscle contraction is regulated by dynamic and cooperative interactions among Ca2+, troponin, and tropomyosin on the thin filament. While Ca2+ regulation has been extensively studied, little is known about the dynamics of individual regulatory units and structural changes of individual tropomyosin molecules in relation to their mechanical properties, and how these factors are altered by cardiomyopathy mutations in the Ca2+ regulatory proteins. In this hypothesis paper, we explore how various experimental and analytical approaches could broaden our understanding of the cooperative regulation of cardiac contraction in health and disease.

PI3Ks Maintain the Structural Integrity of T-Tubules in Cardiac Myocytes

BackgroundPhosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.Methods and ResultsGenetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca2+ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca2+ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.ConclusionsPI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca2+-induced Ca2+ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.

Detection, properties, and frequency of local calcium release from the sarcoplasmic reticulum in teleost cardiomyocytes.

Calcium release from the sarcoplasmic reticulum (SR) plays a central role in the regulation of cardiac contraction and rhythm in mammals and humans but its role is controversial in teleosts. Since the zebrafish is an emerging model for studies of cardiovascular function and regeneration we here sought to determine if basic features of SR calcium release are phylogenetically conserved. Confocal calcium imaging was used to detect spontaneous calcium release (calcium sparks and waves) from the SR. Calcium sparks were detected in 16 of 38 trout atrial myocytes and 6 of 15 ventricular cells. The spark amplitude was 1.45±0.03 times the baseline fluorescence and the time to half maximal decay of sparks was 27±3 ms. Spark frequency was 0.88 sparks µm−1 min−1 while calcium waves were 8.5 times less frequent. Inhibition of SR calcium uptake reduced the calcium transient (F/F0) from 1.77±0.17 to 1.12±0.18 (p = 0.002) and abolished calcium sparks and waves. Moreover, elevation of extracellular calcium from 2 to 10 mM promoted early and delayed afterdepolarizations (from 0.6±0.3 min−1 to 8.1±2.0 min−1, p = 0.001), demonstrating the ability of SR calcium release to induce afterdepolarizations in the trout heart. Calcium sparks of similar width and duration were also observed in zebrafish ventricular myocytes. In conclusion, this is the first study to consistently report calcium sparks in teleosts and demonstrate that the basic features of calcium release through the ryanodine receptor are conserved, suggesting that teleost cardiac myocytes is a relevant model to study the functional impact of abnormal SR function.

Myosin binding protein-C: an essential protein in skeletal and cardiac muscle

Myosin binding protein C (MyBP-C, also known as C-protein), was discovered in skeletal muscle nearly 40 years ago (Offer et al. 1973). It is a major component of the sarcomere that is located on 7–9 stripes of 43 nm spacing in each half of the A-band as shown by immunolabelling and electron microscopy (Bennett et al. 1986). There are three isoforms, slow skeletal, fast skeletal and cardiac muscle, encoded by the genes MYBPC1 (Furst et al. 1992), MYBPC2 (Weber et al. 1993) and MYBPC3 (Gautel et al. 1995), respectively. Two key publications in 1995 reported that mutations in cardiac MyBP-C (cMyBP-C) are a major cause of hypertrophic cardiomyopathy (HCM) (Bonne et al. 1995; Watkins et al. 1995), a disease that we now know affects 1% of the world’s population (Barefield and Sadayappan 2010). This heralded massive research efforts to understand the role of cMyBP-C in the myocardium. The function of cMyBP-C is not completely understood, but it is thought to be involved in filament assembly and in regulation of cardiac contraction (Flashman et al. 2004; Oakley et al. 2004). In comparison, the skeletal isoforms (slow skeletal, ssMyBP-C; fast skeletal, fsMyBPC) have not received the same level of scrutiny, possibly because no associated skeletal myopathies have so far been reported. All that is now set to change with the new study by Gurnett et al. (2010) showing skeletal muscle disorder resulting from mutations in skeletal MyBP-C. All three isoforms of MyBP-C share a conserved domain architecture, composed of seven immunoglobulin (IgI) domains and three fibronectin type III (FnIII) domains depicted as C1-C10 (Fig. 1), with a 105 residue region between C1 and C2 called the MyBP-C motif and a prolineand alanine-rich (PA) region near the N-terminus. MyBP-C binds to LMM and titin via its C-terminal domains C7 to C10 (Okagaki et al. 1993; Freiburg and Gautel 1996). The cardiac isoform differs from the skeletal isoform in three major ways: cMyBP-C has an additional Ig domain, C0, at the N-terminus; it has 4 phosphorylation sites in the MyBP-C motif and the domain C5 has a proline-rich 25 residue insertion (Gautel et al. 1995; Barefield and Sadayappan 2010). cMyBP-C phosphorylation has a major role in regulating myocardial function and is cardioprotective (McClellan et al. 2001; Sadayappan et al. 2006). Mutations causing HCM have been found along the whole length of the cMyBP-C protein. Of the 165 MYBPC3 mutations found so far (Alcalai et al. 2008), 70% are nonsense or frame shift mutations, expected to result in truncated proteins. These truncated proteins have so far been undetectable in myocardial samples (Marston et al. 2009). Studying two familial cases of distal arthrogryposis type 1 (DA1), Gurnett et al. (2010) have now identified the first MyBP-C mutations to affect skeletal muscle, both of them missense mutations in the slow-twitch skeletal MYBPC1 gene. In this rare disease which affects 1 in 10,000, there is congenital contracture of distal limbs resulting from various intrinsic and extrinsic factors that restrict movement of the foetus in the uterus. However no additional anomalies are observed (Gurnett et al. 2010). Interestingly, other protein mutations implicated in the disease, which include Commentary for Journal of Muscle Research and Cell Motility on: Gurnett, C.A., D.M. Desruisseau, K. McCall, R. Choi, Z.I. Meyer, M. Talerico, S.E. Miller, J.S. Ju, A. Pestronk, A.M. Connolly, T.E. Druley, C.C. Weihl, and M.B. Dobbs Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1. Hum Mol Genet. 19:1165–1173.

Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin.

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.