Familial hypertrophic cardiomyopathy (FHC) is characterized by a pathological thickening of the muscle surrounding the heart and is the leading cause for sudden cardiac death in young people. There are numerous mutations in sarcomeric proteins that have been implicated in causing FHC. Myosin, the molecular motor that powers cardiac muscle contraction, consists of a globular domain and an elongated α-helical neck region, which is thought to undergo large conformational changes during muscle contraction. The myosin regulatory light chain (RLC) functions to support the neck region, therefore it is not surprising that several single amino acid substitutions in the RLC have been implicated in FHC. Here we studied D166V, an RLC point mutation that is associated with increased left ventricular wall thickness, abnormal electrocardiogram, and decreased isometric force and ATPase in skinned fibers from transgenic mouse hearts. We have expressed porcine cardiac β myosin (the same isoform found in humans) and replaced the endogenous RLC with a human RLC. The exchanged RLC contained either wild type (WT), D166V, S15A, or a double mutant D166V/S15A, or D166V/S15D. The S15D mutation served as a phosphorylation mimic. We performed frictional loading assays using a modified in vitro motility assay and determined the average force produced by a bed of monomeric myosin. While maximal unloaded velocity was unchanged for all of the mutants studied, the force of D166V and D166V/S15A was significantly reduced compared to WT, with an additive decrease in the double mutant. D166V/S15D resulted in a significant increase in force compared to D166V/S15A and was restored to near WT levels. These results suggest that D166V causes a reduction in myosin isometric force production and phosphorylation may act to recover it.

The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. In this work, we studied the mechanical and structural interaction of actin with basic calponin, a differentiation marker in smooth muscle cells, on a single filament level. We imaged fluorescently labeled thermally fluctuating actin filaments and found that at moderate calponin binding densities, actin filaments were more flexible, evident as a reduction in persistence length from 8.0 to 5.8 μm. When calponin-decorated actin filaments were subjected to shear, we observed a marked reduction of filament lengths after decoration with calponin, which we argue was due to shear-induced filament rupture rather than depolymerization. This increased shear susceptibility was exacerbated with calponin concentration. Cryo-electron microscopy results confirmed previously published negative stain electron microscopy results and suggested alterations in actin involving actin subdomain 2. A weakening of F-actin intermolecular association is discussed as the underlying cause of the observed mechanical perturbations.

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.

Abstract Abstract 3097 HGAL is a recently identified germinal center (GC)–specific gene whose expression by tumor cells correlates with a favorable prognosis in patients with diffuse large B-cell and classical Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and decreasing lymphoma cell migration and dissemination. The latter effect may contribute to the less aggressive clinical behavior of HGAL-expressing lymphomas. Actin filaments, usually in association with myosin, are responsible for many types of cell movements. The interaction between myosin and actin is responsible for muscle contraction, migration of nonmuscle cells and also plays a role in cell division. The movement of cells appears to be driven directly by actin polymerization and by actin-myosin interactions. In our previous work (Lu et al, Blood 2007), we demonstrated by co-immunoprecipitation and co-localization studies that HGAL interacts with myosin II and actin. However, the mechanistic consequences of this interaction are unknown. Because the interaction of actin with myosin is the key factor in cell motility and HGAL negatively regulates lymphocyte migration, we hypothesized that HGAL may decelerate cell migration by affecting the binding of myosin to actin. To confirm the direct and not interdependent interaction between myosin-HGAL and actin-HGAL, we performed in vitro co-sedimentation experiments with recombinant HGAL protein and purified actin and myosin, respectively. These studies demonstrated direct and independent binding of HGAL to both myosin and actin. We next demonstrated that the N terminal portion of the HGAL protein (aa 1–118) can bind the head region of myosin II (S1) that contains the actin and ATP binding sites, and to the rod part of the myosin molecule that is responsible for filament formation. While no effect of the HGAL on the actomyosin ATPase activity was observed, it reduced the rate of actin polymerization from G-actin to F-actin. Further binding studies using pyrene-labeled F-actin showed that HGAL increases the binding of myosin to F-actin. Taken together, our results indicate that HGAL is a cellular regulator of actin assembly that can also regulate the actin-myosin interaction. This in turn may explain the role of HGAL as a favorable prognosis marker for patients with diffuse large B-cell and classical Hodgkin lymphomas. Disclosures: No relevant conflicts of interest to declare.

Cardiac muscle myosin is comprised of two heavy chains (MHCs), two essential light chains, and two regulatory light chains (RLCs). The MHC contains both the ATPase and actin binding domains. It has been shown that the contractile properties of myosin can be tuned by the MHC isoform and that the MHC isoform distribution in the human heart changes during heart failure from predominantly beta isoform to all beta. One cause of heart failure is familial hypertrophic cardiomyopathy (FHC) which is triggered by mutation of sarcomeric proteins including the RLC. Although the RLC is spatially separated from the myosin active site, it appears to have a role in tuning myosin kinetics. In order to examine how two RLC mutations implicated in FHC, N47K and R58Q, affect the kinetic and mechanical properties of beta isoform myosin, we exchanged porcine cardiac RLC with recombinant mutant RLC. We examined the contractile properties of these mutants using the in vitro motility assay and compared these results to our earlier results with mutant RLCs on the alpha -MHC background. Regardless of MHC isoform, the mutations cause reductions in force and power output. However, on the alpha MHC backbone, R58Q shows differences in calcium handling and an elevated ATPase rate which is not seen on the beta backbone. Also, both mutants show increases in duty cycle on the alpha MHC but not the beta. These data suggest that small changes in the myosin structure, far from the active site, can disrupt the contractile properties of the motor depending on the MHC isoform.

Muscle thick filaments are primarily composed of the hexameric protein myosin, which is responsible for the generation of force and motion. Myosin consists of an N-terminal globular domain which, among other functions, binds to the thin filament and an α-helical domain, which acts as a lever arm to transmit force and motion. Familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death among young people, is a pathological thickening of the ventricular walls of the heart that has been caused by single point mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Based on our recent work with mutations in the N-terminus of the RLC and the RLC location on the lever arm region, we hypothesized that the RLC-FHC mutations in the C-terminus (P95A, K104E and D166V) may disrupt force generation and contractility. To determine the effects of the FHC-RLC mutations we generated beta isoform myosin bearing mutant light chains by exchanging the porcine cardiac native light chain with recombinant mutant light chains and examined the effects of the mutations with the in vitro motility assay. Contrary to our hypothesis, none of the mutants exhibited changes in force production using the frictional loading motility assay, and maximal filament velocity (Vmax) also remained unchanged. Next, we measured regulated actin filament velocity as a function of calcium concentration. The velocity-pCa dependence showed the expected sigmoidal characteristic for all tested mutant myosins, however, the calcium sensitivity (pCa50) increased for K104E and D166V mutants. In addition, D166V and P95A exhibited higher cooperativity. Data for D166V is consistent with previous studies performed in skinned mouse papillary muscle fibers showing an increase in the calcium sensitivity of force and ATPase (Kerrick et al., 2009, FASEB. J. 23:855-65).

The myosin regulatory light chain (RLC) wraps around the alpha-helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis, with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K show reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation.

Phosphorylation of the myosin regulatory light chain (RLC) in skeletal muscle has been proposed to act as a molecular memory of recent activation by increasing the rate of force development, ATPase activity, and isometric force at submaximal activation in fibers. It has been proposed that these effects stem from phosphorylation-induced movement of myosin heads away from the thick filament backbone. In this study, we examined the molecular effects of skeletal muscle myosin RLC phosphorylation using in vitro motility assays. We showed that, independently of the thick filament backbone, the velocity of skeletal muscle myosin is decreased upon phosphorylation due to an increase in the myosin duty cycle. Furthermore, we did not observe a phosphorylation-dependent shift in calcium sensitivity in the absence of the myosin thick filament. These data suggest that phosphorylation-induced movement of myosin heads away from the thick filament backbone explains only part of the observed phosphorylation-induced changes in myosin mechanics. Last, we showed that the duty cycle of skeletal muscle myosin is strain dependent, consistent with the notion that strain slows the rate of ADP release in striated muscle.

The myosin neck, which is supported by the interactions between light chains and the underlying alpha-helical heavy chain, is thought to act as a lever arm to amplify movements originating in the globular motor domain. Here, we studied the role of the cardiac myosin regulatory light chains (RLCs) in the capacity of myosin to produce force using a novel optical-trap-based isometric force in vitro motility assay. We measured the isometric force and actin filament velocity for native porcine cardiac (PC) myosin, RLC-depleted PC (PC(depl)) myosin, and PC myosin reconstituted with recombinant bacterially expressed human cardiac RLC (PC(recon)). RLC depletion reduced unloaded actin filament velocity by 58% and enhanced the myosin-based isometric force approximately 2-fold. No significant change between PC and PC(depl) preparations was observed in the maximal rate of actin-activated myosin ATPase activity. Reconstitution of PC(depl) myosin with human RLC partially restored the velocity and force levels to near untreated values. The reduction in unloaded velocity after RLC extraction is consistent with the myosin neck acting as a lever, while the enhancement in isometric force can be directly related to enhancement of unitary force. The force data are consistent with a model in which the neck region behaves as a cantilevered beam.

The glutamic acid to lysine mutation at the 22nd amino acid residue (E22K) in the human cardiac myosin regulatory light chain (RLC) gene causes familial hypertrophic cardiomyopathy (FHC) with a phenotype of midventricular obstruction and septal hypertrophy. Our recent histopathology results have shown that the hearts of transgenic E22K mice (Tg-E22K) resemble those of human patients, demonstrating enlarged interventricular septa and papillary muscles. In this study, we show no effect of the E22K mutation on the kinetics of mutated myosin in its ATP-powered interaction with fluorescently labeled single actin filaments compared to nontransgenic or transgenic wild-type (Tg-WT) control mice. Likewise, no change in cross-bridge dissociation rates (g(app)) was observed in freshly skinned papillary muscle fibers. In contrast, maximal force and ATPase were decreased approximately 20% in Tg-E22K skinned papillary muscle fibers and intracellular [Ca2+] and force transients were significantly decreased in intact papillary muscle fibers from Tg-E22K compared to Tg-WT mice. Moreover, energy metabolism measured in isolated working Tg-E22K mouse hearts perfused under conditions of physiologically relevant levels of metabolic demand was similar in Tg-E22K and control hearts before and after 20 min of no-flow ischemia. Our results suggest that the pathological response observed in the E22K myocardium might be triggered by mutation induced changes in the properties of the RLC Ca2+-Mg2+ site, the state of the Ca2+/Mg2+ occupancy and consequently the Ca2+ buffering ability of the RLC. By decreasing the affinity of the RLC for Ca2+, the E22K mutation most likely promotes a Mg2+-saturated RLC producing less force and ATPase than the Ca2+-saturated RLC of WT fibers. Decreased Ca2+ binding may also lead to faster Ca2+ dissociation kinetics in Tg-E22K intact fibers resulting in decreased duration and amplitude of [Ca2+] and force transients. These changes when placed in vivo would result in higher workloads and consequently cardiac hypertrophy.