Samantha Beck Previs

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Significance Myosin-binding protein C (MyBP-C) is a component of myosin filaments, one of the two sets of contractile elements whose relative sliding is the basis of muscle contraction. In the heart, MyBP-C modulates contractility in response to cardiac stimulation; mutations in MyBP-C lead to cardiac disease. The mechanism by which MyBP-C modulates cardiac contraction is not understood. Using electron microscopy and a light microscopic assay for filament sliding, we demonstrate that MyBP-C binds to the other set of filaments, containing actin and the regulatory component, tropomyosin. In so doing, it displaces tropomyosin from its inhibitory position to activate actin filament interaction with myosin, promoting filament sliding. These findings provide insights into the molecular basis of heart function. Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca2+ binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca2+ conditions, cMyBP-C displaces tropomyosin toward its high Ca2+ position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca2+, cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca2+ but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca2+ position on actin, and govern contractile speed by an independent molecular mechanism.

Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling

The localization of MyBP-C within cardiac muscle cells provides efficient mechanochemical excitation-contraction coupling. The beating heart exhibits remarkable contractile fidelity over a lifetime, which reflects the tight coupling of electrical, chemical, and mechanical elements within the sarcomere, the elementary contractile unit. On a beat-to-beat basis, calcium is released from the ends of the sarcomere and must diffuse toward the sarcomere center to fully activate the myosin- and actin-based contractile proteins. The resultant spatial and temporal gradient in free calcium across the sarcomere should lead to nonuniform and inefficient activation of contraction. We show that myosin-binding protein C (MyBP-C), through its positioning on the myosin thick filaments, corrects this nonuniformity in calcium activation by exquisitely sensitizing the contractile apparatus to calcium in a manner that precisely counterbalances the calcium gradient. Thus, the presence and correct localization of MyBP-C within the sarcomere is critically important for normal cardiac function, and any disturbance of MyBP-C localization or function will contribute to the consequent cardiac pathologies.

The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0–C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1–C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0–C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0–C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0–C3 in proportion to the total number phosphomimetic replacements. Under load, C0–C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0–C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0–C3 on F:V was well fitted by a model that assumed C0–C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.

Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

Significance Mutations in cardiac myosin-binding protein C (cMyBP-C) are the leading cause of inherited hypertrophic cardiomyopathy, demonstrating the key role that cMyBP-C plays in the heart’s contractile machinery. Since its discovery >40 years ago, cardiovascular biologists have wondered how cMyBP C’s structure regulates its function. Using atomic force spectroscopy, electron microscopy, in vitro model systems of contractility, and mutant protein expression, we show how phosphorylation affects cMyBP-C’s structure to tune its functions within the contractile apparatus. We also identify a novel molecular mechanism by which calcium ions that trigger each muscle contraction override phosphorylation’s structural and functional impact. This intersection of posttranslational modification and calcium signaling may occur in other biological systems that rely on these pathways to control cellular processes. During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C’s N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain’s extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C’s inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C’s calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C’s phosphorylation state.

Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.

Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin‐actin) or mixed polarity (α‐actinin‐actin) bundles. The stepping dynamics of individual Qdot‐labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVIs preference to stay on track, which may be critical to its role as an intracellular anchor.

Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca(2+)-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.

Diabetes with heart failure increases methylglyoxal modifications in the sarcomere, which inhibit function

Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.

Phosphorylation and Calcium Antagonistically Tune Myosin-Binding Protein C's Molecular Structure and Function

During each heartbeat, calcium activates the sliding of actin-thin filaments towards the centers of myosin-thick filaments to shorten the overall length of cardiac muscle cells. Cardiac myosin binding protein C (cMyBP-C) tunes these interactions. cMyBP-Cs C terminus is strongly bound to the thick filament backbone and its N-terminal domains transiently interact with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocities. Both mechanisms are tunable by phosphorylation of 4 serines within an extensible, intrinsically disordered region of cMyBP-Cs N-terminus, the M-domain. Does phosphorylation impact the structure and molecular mechanics of cMyBP-Cs N terminus? Rotary shadowing electron microscopy showed that M-domain phosphorylation caused N-terminal fragments (domains C0-C3) to shift from an extended, rod-like structure to a compact conformation. Atomic force microscopy studies of N-terminal fragments (C1-C2), showed that phosphorylation reduced the M-domains contour length and extensibility. Taken together, M-domain phosphorylation and its impact on cMyBP-Cs N-terminal structure, suggest a mechanism for tuning cMyBP-Cs function in motility assays. Interestingly, we found that free calcium (0.1 mM) mitigated the structural effects of phosphorylation by shifting the phosphorylated-C0C3 fragments back to the extended, rod-like state, with a freely extensible M-domain, as if unphosphorylated. Functionally, even though phosphorylation reduced cMyBP-Cs ability to inhibit actin filament sliding velocity in the motility assay (24% vs. 40% inhibition), addition of calcium ablated the impact of phosphorylation, fully restoring cMyBP-Cs inhibitory capacity. We conclude that phosphorylation may have its greatest effect on cardiac contractility by tuning cMyBP-Cs ability to sensitize actin-thin filaments to calcium at the low levels present during the onset of contraction. Importantly, calcium levels at the peak contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of the cMyBP-Cs phosphorylation state.

Myosin Va Cargo Transport on Actin Bundles

Myosin Va (myoVa) walks processively while carrying cargo towards the plus end of actin filaments. In cells, parallel actin filament bundles (e.g. stress fibers and filopodia) present a directional challenge to myoVa cargo transport. Therefore, we formed unipolar (fascin) and mixed polarity (alpha-actinin) actin bundles as tracks for expressed myoVa-HMM with a C-terminal biotin tag. In this assay, a single streptavidin-Qdot served as cargo for one or many (∼ 5) myoVa motors. Qdots transported by one or many myoVa molecules traveled in the same direction on unipolar bundles, while moving in either direction on mixed polarity bundles. Qdot speeds were the same regardless of bundle type or number of motors (400nm/s), and similar to that for one or many motors on a single actin filament (Nelson et al., 2009). However, run lengths for single motors were 2-3 times longer on bundles than previously observed on single actin filaments. This suggests that on parallel tracks the leading head has a greater number of actins within its reach, thus decreasing the probability of run termination. Interestingly, on mixed polarity bundles, we observed individual Qdots changing directions in the middle of a run, the frequency of which increases in the multiple motor case. It was not surprising that a Qdot with a single motor can switch directions on a mixed polarity bundle, given myoVas inherent flexibility that allows it to turn up to 150o at actin filament intersections (Ali et al., 2007). These data also suggest that one or many myoVa molecules bound to a single cargo have the ability to jump tracks to neighboring actin filaments. With Qdot-labeling of the individual heads, high spatial resolution studies will confirm this on mixed polarity bundles, and determine whether the motors also wander on unipolar bundles.