scholarly journals Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I

2020 ◽  
Vol 295 (14) ◽  
pp. 4398-4410 ◽  
Author(s):  
Ivanka R. Sevrieva ◽  
Birgit Brandmeier ◽  
Saraswathi Ponnam ◽  
Mathias Gautel ◽  
Malcolm Irving ◽  
...  
Keyword(s):  
Light Chain ◽  
Troponin I ◽  
Regulatory Light Chain ◽  
Myosin Regulatory Light Chain ◽  
Cardiac Myosin ◽  
Sarcomeric Proteins ◽  
Ventricular Trabeculae

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

2004 ◽  
Vol 286 (6) ◽  
pp. H2089-H2095 ◽  
Author(s):  
Brian B. Roman ◽  
Paul H. Goldspink ◽  
Elyse Spaite ◽  
Dalia Urboniene ◽  
Ron McKinney ◽  
...  
Keyword(s):  
Troponin I ◽  
Cardiac Contraction ◽  
Phosphorylation Sites ◽  
Developed Pressure ◽  
Contraction And Relaxation ◽  
Intracellular Targets ◽  
Camp Levels

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-ε, may slow the rates of cardiac contraction and relaxation (+dP/d t and −dP/d t). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser43 and Ser45 and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater −dP/d t than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/d t increased by 33% and −dP/d t increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

scholarly journals Mavacamten Decreases Maximal Force and Ca2+-sensitivity in the N47K-Myosin Regulatory Light Chain Mouse Model of Hypertrophic Cardiomyopathy

2020 ◽  
Author(s):  
Peter O Awinda ◽  
Marissa Watanabe ◽  
Yemeserach M. Bishaw ◽  
Anna M Huckabee ◽  
Keinan B Agonias ◽  
...  
Keyword(s):  
Heart Disease ◽  
Hypertrophic Cardiomyopathy ◽  
Light Chain ◽  
Isometric Force ◽  
Regulatory Light Chain ◽  
Isometric Tension ◽  
Myosin Regulatory Light Chain ◽  
Cardiac Myosin ◽  
Cross Bridge ◽  
Obstructive Hypertrophic Cardiomyopathy

Morbidity and mortality associated with heart disease is a growing threat to the global population and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca2+-sensitivity of contraction for both genotypes, but this reduction in pCa50 was nearly twice as large for WT-RLC vs. N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca2+-sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM.

scholarly journals The HCM-Linked Ala13thr Mutation in the Cardiac Myosin Regulatory Light Chain Increases Isometric Force Production

2011 ◽  
Vol 100 (3) ◽  
pp. 111a
Author(s):  
Katarzyna Kazmierczak ◽  
Priya Muthu ◽  
Wenrui Huang ◽  
Ana Rojas ◽  
Michelle Jones ◽  
...  
Keyword(s):  
Light Chain ◽  
Isometric Force ◽  
Force Production ◽  
Regulatory Light Chain ◽  
Myosin Regulatory Light Chain ◽  
Cardiac Myosin ◽  
Isometric Force Production

scholarly journals The direct molecular effects of fatigue and myosin regulatory light chain phosphorylation on the actomyosin contractile apparatus

2010 ◽  
Vol 298 (4) ◽  
pp. R989-R996 ◽  
Author(s):  
Michael J. Greenberg ◽  
Tanya R. Mealy ◽  
Michelle Jones ◽  
Danuta Szczesna-Cordary ◽  
Jeffrey R. Moore
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Molecular Species ◽  
Regulatory Light Chain ◽  
Myosin Regulatory Light Chain ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
Individual Molecular Species ◽  
Molecular Effects

Skeletal muscle, during periods of exertion, experiences several different fatigue-based changes in contractility, including reductions in force, velocity, power output, and energy usage. The fatigue-induced changes in contractility stem from many different factors, including alterations in the levels of metabolites, oxidative damage, and phosphorylation of the myosin regulatory light chain (RLC). Here, we measured the direct molecular effects of fatigue-like conditions on actomyosin's unloaded sliding velocity using the in vitro motility assay. We examined how changes in ATP, ADP, Pi, and pH affect the ability of the myosin to translocate actin and whether the effects of each individual molecular species are additive. We found that the primary causes of the reduction in unloaded sliding velocity are increased [ADP] and lowered pH and that the combined effects of the molecular species are nonadditive. Furthermore, since an increase in RLC phosphorylation is often associated with fatigue, we examined the differential effects of myosin RLC phosphorylation and fatigue on actin filament velocity. We found that phosphorylation of the RLC causes a 22% depression in sliding velocity. On the other hand, RLC phosphorylation ameliorates the slowing of velocity under fatigue-like conditions. We also found that phosphorylation of the myosin RLC increases actomyosin affinity for ADP, suggesting a kinetic role for RLC phosphorylation. Furthermore, we showed that ADP binding to skeletal muscle is load dependent, consistent with the existence of a load-dependent isomerization of the ADP bound state.

scholarly journals Schizosaccharomyces pombe Pak-related protein, Pak1p/Orb2p, phosphorylates myosin regulatory light chain to inhibit cytokinesis

2008 ◽  
Vol 183 (5) ◽  
pp. 785-793 ◽  
Author(s):  
Tsui-Han Loo ◽  
Mohan Balasubramanian
Keyword(s):  
Actin Cytoskeleton ◽  
Schizosaccharomyces Pombe ◽  
Light Chain ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Eukaryotic Cells ◽  
Myosin Regulatory Light Chain ◽  
Related Protein ◽  
Filamentous Actin

p21-activated kinases (Paks) have been identified in a variety of eukaryotic cells as key effectors of the Cdc42 family of guanosine triphosphatases. Pak kinases play important roles in regulating the filamentous actin cytoskeleton. In this study, we describe a function for the Schizosaccharomyces pombe Pak-related protein Pak1p/Orb2p in cytokinesis. Pak1p localizes to the actomyosin ring during mitosis and cytokinesis. Loss of Pak1p function leads to accelerated cytokinesis. Pak1p mediates phosphorylation of myosin II regulatory light chain Rlc1p at serine residues 35 and 36 in vivo. Interestingly, loss of Pak1p function or substitution of serine 35 and serine 36 of Rlc1p with alanines, thereby mimicking a dephosphorylated state of Rlc1p, leads to defective coordination of mitosis and cytokinesis. This study reveals a new mechanism involving Pak1p kinase that helps ensure the fidelity of cytokinesis.

scholarly journals Slow Cardiac Myosin Regulatory Light Chain 2 (MYL2) was Down-Expressed in Chronic Heart Failure Patients

2010 ◽  
Vol 34 (1) ◽  
pp. 30-34 ◽  
Author(s):  
Yuanhong Li ◽  
Gang Wu ◽  
Qizhu Tang ◽  
Congxin Huang ◽  
Hong Jiang ◽  
...  
Keyword(s):  
Heart Failure ◽  
Chronic Heart Failure ◽  
Light Chain ◽  
Regulatory Light Chain ◽  
Myosin Regulatory Light Chain ◽  
Cardiac Myosin ◽  
Heart Failure Patients

scholarly journals Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.

1994 ◽  
Vol 127 (6) ◽  
pp. 1945-1955 ◽  
Author(s):  
B D Ostrow ◽  
P Chen ◽  
R L Chisholm
Keyword(s):  
Atpase Activity ◽  
Light Chain ◽  
Contractile Activity ◽  
Phosphorylation Site ◽  
Regulatory Light Chain ◽  
Site Directed Mutagenesis ◽  
Cellular Functions ◽  
Developmental Defects

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.

scholarly journals Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

2015 ◽  
Vol 112 (30) ◽  
pp. E4138-E4146 ◽  
Author(s):  
Chen-Ching Yuan ◽  
Priya Muthu ◽  
Katarzyna Kazmierczak ◽  
Jingsheng Liang ◽  
Wenrui Huang ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Light Chain ◽  
Heart Function ◽  
Regulatory Light Chain ◽  
Cardiac Myosin ◽  
X Ray Diffraction ◽  
And Function ◽  
Induced Changes ◽  
Constitutive Phosphorylation

Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 →Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca2+ sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.

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