Regulatory Light Chain Phosphorylation Research Articles

Elastase alters contractility and promotes an inflammatory synthetic phenotype in airway smooth muscle tissues.

Neutrophil elastase is secreted by inflammatory cells during airway inflammation and can elicit airway hyperreactivity in vivo. Elastase can degrade multiple components of the extracellular matrix. We hypothesized that elastase might disrupt the connections between airway smooth muscle (ASM) cells and the extracellular matrix and that this might have direct effects on ASM tissue responsiveness and inflammation. The effect of elastase treatment on ASM contractility was assessed in vitro in isolated strips of canine tracheal smooth muscle by stimulation of tissues with cumulatively increasing concentrations of acetylcholine (ACh) and measurement of contractile force. Elastase treatment potentiated contractile responses to ACh at low concentrations but suppressed the maximal contractile force generated by the tissues without affecting the phosphorylation of myosin regulatory light chain (RLC). Elastase also promoted the secretion of eotaxin and the activation of Akt in ASM tissues and decreased expression of smooth muscle myosin heavy chain, consistent with promotion of a synthetic inflammatory phenotype. As the degradation of matrix proteins can alter integrin engagement, we evaluated the effect of elastase on the assembly and activation of integrin-associated adhesion junction complexes in ASM tissues. Elastase led to talin cleavage, reduced talin binding to vinculin, and suppressed activation of the adhesome proteins paxillin, focal adhesion kinase, and vinculin, indicating that elastase causes the disassembly of adhesion junction complexes and the inactivation of adhesome signaling proteins. We conclude that elastase promotes an inflammatory phenotype and increased sensitivity to ACh in ASM tissues by disrupting signaling pathways mediated by integrin-associated adhesion complexes.

Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone-treated smooth muscle tissues for phos-tag SDS-PAGE.

Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.

Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

Metabolic Stress-Induced Activation of AMPK and Inhibition of Constitutive Phosphoproteins Controlling Smooth Muscle Contraction: Evidence for Smooth Muscle Fatigue?

Metabolic stress diminishes smooth muscle contractile strength by a poorly defined mechanism. To test the hypothesis that metabolic stress activates a compensatory cell signaling program to reversibly downregulate contraction, arterial rings and bladder muscle strips in vitro were deprived of O2 and glucose for 30 and 60 min (“starvation”) to induce metabolic stress, and the phosphorylation status of proteins involved in regulation of contraction and metabolic stress were assessed in tissues under basal and stimulated conditions. A 15–30 min recovery period (O2 and glucose repletion) tested whether changes induced by starvation were reversible. Starvation decreased basal phosphorylation of myosin regulatory light chain (MLC-pS19) and of the rho kinase (ROCK) downstream substrates cofilin (cofilin-pS3) and myosin phosphatase targeting subunit MYPT1 (MYPT1-pT696 and MYPT1-pT853), and abolished the ability of contractile stimuli to cause a strong, sustained contraction. Starvation increased basal phosphorylation of AMPK (AMPK-pT172) and 3 downstream AMPK substrates, acetyl-CoA carboxylase (ACC-pS79), rhoA (rhoA-pS188), and phospholamban (PLB-pS16). Increases in rhoA-pS188 and PLB-pS16 would be expected to inhibit contraction. Recovery restored basal AMPK-pT172 and MLC-pS19 to control levels, and restored contraction. In AMPKα2 deficient mice (AMPK), the basal level of AMPK-pT172 was reduced by 50%, and MLC-pS19 was elevated by 50%, but AMPK did not prevent starvation-induced contraction inhibition nor enhance recovery from starvation. These results indicate that constitutive AMPK activity participates in constitutive regulation of contractile proteins, and suggest that AMPK activation is necessary, but may not be sufficient, to cause smooth muscle contraction inhibition during metabolic stress.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca2+-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.

Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Ca2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+ , MYPT1SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.

Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn’s disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function.

MYBPC3 mutations are associated with a reduced super-relaxed state in patients with hypertrophic cardiomyopathy.

The “super-relaxed state” (SRX) of myosin represents a ‘reserve’ of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein–C (cMyBP-C). Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3) using samples from human patients with hypertrophic cardiomyopathy (HCM). Left ventricular (LV) samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut) or negative (HCMsmn) and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01) between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations.

Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development

Leiomyosarcoma is a rare malignant smooth muscle tumor which can be very unpredictable. Myosin II is involved in many functions, including cell contraction, migration, and adhesion. The phosphorylation of myosin regulatory light chain (MLC) by myosin light chain kinase (MLCK) determines the activity of Myosin II. However, it is still unclear whether MLC phosphorylation is involved in cell proliferation in leiomyosarcoma. In this study, we aimed to explore the role of MLCK-dependent MLC phosphorylation in leiomyosarcoma development. We found that the expression of MLCK, phosphorylated MLC, and Ki67 in leiomyosarcoma was significantly higher than in leiomyoma and adjacent normal smooth muscle cells. MLCK expression was significantly correlated with phosphorylated MLC level. Kaplan-Meier survival analysis revealed that patients with high expression of MLCK or phosphorylated MLC had shorter overall survival times compared with the patients with low expression of MLCK or phosphorylated MLC. In vitro studies revealed a causative link between MLC phosphorylation and cellular proliferation as expression of phosphomimetic MLC (T19D, S20D) increased cellular proliferation as assessed by Ki67 staining. In contrast, MLCK specific inhibitor reduced cellular proliferation. We concluded that MLCK, phosphorylated MLC and Ki67 were overexpressed in leiomyosarcoma. MLCK dependent MLC phosphorylation might be responsible for the high proliferative state in leiomyosarcoma. MLCK and phosphorylated MLC are potential prognostic indicators of leiomyosarcoma.

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation.

Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.

Use of Phase Loop Plots to Assess Ca2+ Sensitivity of Force Generation in Airway Smooth Muscle

In excitation‐contraction coupling, a transient elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) triggers a force response. The Ca2+ sensitivity of muscle reflects the relationship between [Ca2+]cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca2+]cyt at which 50% force is generated. In smooth muscle, Ca2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca2+]cyt. However, this measurement overlooks the dynamic nature of Ca2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca2+]cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca2+]cyt/force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected.Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca2+]cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca2+‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca2+‐induced rMLC phosphorylation.As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca2+]cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca2+]cyt. By comparison, calyculin A increased force with no effect on [Ca2+]cyt. Although these results suggest changes in Ca2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca2+ sensitivity. These results do not support our hypothesis that dynamic Ca2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca2+]cyt.Support or Funding InformationSupported by NIH grant HL126451 (GCS)

Myosin phosphorylation‐mediated potentiation does not alter the economy of work performed in fast skeletal muscles

The physiological role of myosin regulatory light chain (RLC) phosphorylation in fast skeletal muscle is unclear, as the resultant potentiation of contractile function comes with an undetermined energetic cost. We conducted in vitro experiments (25°C) to quantify contractile economy (i.e. energy cost/work performed) in potentiated extensor digitorum longus (EDL) muscles from wildtype and myosin light chain kinase knockout (skMLCK−/−) mice. Economy values were calculated as the ratio of total work performed to high‐energy phosphate consumption (HEPC) during a period of repeated isovelocity contractions that followed a potentiating stimulus (PS). The PS enhanced contractile parameters (concentric force and work) in both genotypes, although the effect was significantly greater in wildtype (1.38 ± 0.03 and 1.51 ± 0.03) vs. skMLCK−/− (1.10 ± 0.04 and 1.10 ± 0.05) muscles (P &lt; 0.05, n = 8 for all data). During the experimental period of repetitive stimulation in the potentiated state, wildtype muscles performed ~58% more total work than skMLCK−/− muscles (94.05 ± 3.80 vs. 59.55 ± 4.10 J•kg−1; P &lt; 0.001) without a statistical increase in HEPC (19.03 ± 3.37 vs. 16.02 ± 3.41 μmol~P; P = 0.27) or genotype difference in economy (5.74 ± 0.67 vs. 4.61 ± 0.71 J•kg−1μmol~P−1; P = 0.27). Importantly, when relative genotype differences were analyzed, the quantity of total work performed was ~3 fold greater than the increase in HEPC for wildtype muscles. Our results support the idea that myosin RLC phosphorylation‐mediated potentiation is a mechanism capable of augmenting sustained contractile function without significantly increasing energetic cost or decreasing economy.Support or Funding InformationNatural Sciences &amp; Engineering Research Council of Canada (NSERC): Discovery Grant (R. Vandenboom)

Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice.

The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling. The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts. As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.

Investigation of the Molecular Interactions Regulating the Function of Human Cardiac Myosin

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

Triple FRET Imaging of Non-Muscle Myosin II Localization in Migrating Cells

Non-muscle myosin II (NMII) is critical for cell migration because myosin motors drive cell contraction by regulating pulling forces on actin filaments. Myosin regulatory light chain phosphorylation leads to stress fiber assembly and fiber contraction. It is undetermined if NMII phosphorylation increases or decreases during cell migration due to the inherent complexity of myosin regulation. Single metric approaches for illuminating the roles of these proteins have been unsuccessful in determining where the proteins are active simultaneously in the same cell. Our lab has developed two Forster resonance energy transfer (FRET) biosensors to make dynamic measurements of myosin activity. Our NMII phosphorylation sensor together with our myosin light chain phosphatase (MLCP) sensor are able to measure myosin activity in a single cell. Mutations were introduced into both sensors to change the homotransfer and reflect conformational changes that indicate activation. In order to optimize the myosin light chain kinase (MLCK) sensor, KCl was added to determine the range of min/max. During the migration process, changes in NMII phosphorylation, MLCK, and MLCP activity were observed through the use of three separate FRET biosensors transfected together into living cells. We are able to track MLCP, MLCK, and NMII phosphorylation in the same pathway with high accuracy of spatial and temporal resolution. Our data suggests differential regulation of MLCK and MLCP in along a moving membrane. This allows for a more sophisticated understanding of the intermediate and long-term action of myosin phosphorylation during cell migration.

Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial Hypertrophic Cardiomyopathy Phenotype

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and often caused by mutations in genes encoding for major sarcomeric proteins. This study is focused on Arginine to Glutamine substitution (R58Q) in the ventricular myosin regulatory light chain (RLC) resulting in a malignant hypertrophic phenotype. We previously showed a mutation-induced decrease in the endogenous level of cardiac RLC phosphorylation in R58Q mice, resulting in compromised communication between the functional regions of the RLC and affecting the interaction of myosin and actin and cardiac muscle contraction. In this report we explored the therapeutic potential of pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphorylatable Serine-15 (S15D) in the R58Q background. In vitro actin-activated myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal ATPase activity, Vmax (∼30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten constant (in μM) for R58Q (Km=4.04±0.45) was significantly increased compared to WT (Km=1.68±0.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a small but significant increase in Km (2.56±0.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled actin to quantify the dissociation constants (Kd) and stoichiometry of binding (n) between myosin and actin in the absence of nucleotide. Binding of WT-RLC to actin was strong (Kd value =5.5 nM, n=0.89). A large decrease in binding (∼67-fold increase in Kd) was observed for R58Q-RLC. Kd of S15D-R58Q-RLC was only slightly increased (∼3-fold) compared with WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC mutant. Our findings on S15D-R58Q support the idea that myosin phosphorylation may have important translational applications for the treatment of RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 (DSC).

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy.

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca2+ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.

Myosin phosphorylation potentiates steady-state work output without altering contractile economy of mouse fast skeletal muscles.

Skeletal myosin light chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chain (RLC) increases (i.e. potentiates) mechanical work output of fast skeletal muscle. The influence of this event on contractile economy (i.e. energy cost/work performed) remains controversial, however. Our purpose was to quantify contractile economy of potentiated extensor digitorum longus (EDL) muscles from mouse skeletal muscles with (wild-type, WT) and without (skMLCK ablated, skMLCK-/-) the ability to phosphorylate the RLC. Contractile economy was calculated as the ratio of total work performed to high-energy phosphate consumption (HEPC) during a period of repeated isovelocity contractions that followed a potentiating stimulus (PS). Consistent with genotype, the PS increased RLC phosphorylation measured during, before and after isovelocity contractions in WT but not in skMLCK-/- muscles (i.e. 0.65 and 0.05 mol phosphate mol-1 RLC, respectively). In addition, although the PS enhanced work during repeated isovelocity contractions in both genotypes, the increase was significantly greater in WT than in skMLCK-/- muscles (1.51±0.03 versus 1.10±0.05, respectively; all data P<0.05, n=8). Interestingly, the HEPC determined during repeated isovelocity contractions was statistically similar between genotypes at 19.03±3.37 and 16.02±3.41 μmol P; respectively (P<0.27). As a result, despite performing significantly more work, the contractile economy calculated for WT muscles was similar to that calculated for skMLCK-/- muscles (i.e. 5.74±0.67 and 4.61±0.71Jkg-1μmol-1 P, respectively (P<0.27). In conclusion, our results support the notion that myosin RLC phosphorylation enhances dynamic contractile function of mouse fast skeletal muscle but does so without decreasing contractile economy.