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1.  Investigating the role of uncoupling of troponin I phosphorylation from changes in myofibrillar Ca2+-sensitivity in the pathogenesis of cardiomyopathy 
Contraction in the mammalian heart is controlled by the intracellular Ca2+ concentration as it is in all striated muscle, but the heart has an additional signaling system that comes into play to increase heart rate and cardiac output during exercise or stress. β-adrenergic stimulation of heart muscle cells leads to release of cyclic-AMP and the activation of protein kinase A which phosphorylates key proteins in the sarcolemma, sarcoplasmic reticulum and contractile apparatus. Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation and relaxation (lusitropic effect). Recent studies have shown that this relationship between Ca2+-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca2+-sensitizing drugs can promote uncoupling, whilst one Ca2+-desensitizing drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling. We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process.
doi:10.3389/fphys.2014.00315
PMCID: PMC4142463  PMID: 25202278
troponin I; phosphorylation; cardiomyopathies; Ca sensitivity; heart muscle; myofilament
2.  GSK3β Phosphorylates Newly Identified Site in the Pro-Ala Rich Region of Cardiac Myosin Binding Protein C and Alters Cross-Bridge Cycling Kinetics in Human 
Circulation research  2012;112(4):633-639.
Rationale
Cardiac myosin binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and thereby fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However recent work has shown that up to four phosphate groups are present in human cMyBP-C.
Objective
To identify and characterize additional phosphorylation sites in human cMyBP-C.
Methods and results
Cardiac MyBP-C was semi-purified from human heart tissue. Tandem mass-spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine (Pro-Ala) rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared to PKA-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.
Conclusion
GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the Pro-Ala rich region and increases kinetics of force development, suggesting a non-canonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.
doi:10.1161/CIRCRESAHA.112.275602
PMCID: PMC3595322  PMID: 23277198
Cardiac myosin binding protein-C; phosphorylation; GSK3β; heart failure; contractile proteins; hypertrophy/remodeling; myocardial contractility
3.  Point/Counterpoint Troponin phosphorylation and myofilament Ca2+-sensitivity in heart failure: increased or decreased? 
Heart failure is characterised by depressed myocyte contractility and is considered to involve a complex malfunction of adrenergic regulation, Ca2+-handling and the contractile apparatus. Most studies on the contractile apparatus have focused on troponin, the Ca2+-dependent regulator of myofibrillar activity. Importantly, phosphorylation of troponin I secondary to beta-adrenergic receptor activation is known to induce reduced myofilament Ca2+ sensitivity. In muscle samples from explanted failing human hearts, troponin I phosphorylation levels are very low and Ca2+-sensitivity is high. In contrast, some animal models used to study the mechanisms of heart failure give the opposite result- high levels of troponin I phosphorylation and low Ca2+-sensitivity. Which is right?
doi:10.1016/j.yjmcc.2008.07.004
PMCID: PMC2610448  PMID: 18691597
4.  The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy 
Point mutations targeting muscle thin filament proteins are the cause of a number of cardiomyopathies. In many cases, biological effects of the mutations are well-documented, whereas their structural and mechanical impact on filament assembly and regulatory function is lacking. In order to elucidate molecular defects leading to cardiac dysfunction, we have examined the structural mechanics of two tropomyosin mutants, E180G and D175N, which are associated with hypertrophic cardiomyopathy (HCM). Tropomyosin is an α–helical coiled-coil dimer which polymerizes end-to-end to create an elongated superhelix that wraps around F-actin filaments of muscle and non-muscle cells, thus modulating the binding of other actin-binding proteins. Here, we study how flexibility changes in the E180G and D175N mutants might affect tropomyosin binding and regulatory motion on F-actin. Electron microscopy and Molecular Dynamics simulations show that E180G and D175N mutations cause an increase in bending flexibility of tropomyosin both locally and globally. This excess flexibility is likely to increase accessibility of the myosin-binding sites on F-actin, thus destabilizing the low-Ca2+ relaxed-state of cardiac muscle. The resulting imbalance in the on-off switching mechanism of the mutants will shift the regulatory equilibrium towards Ca2+-activation of cardiac muscle, as is observed in affected muscle, accompanied by enhanced systolic activity, diastolic dysfunction, and cardiac compensations associated with HCM and heart failure.
doi:10.1016/j.bbrc.2012.06.141
PMCID: PMC3412897  PMID: 22789852
actin; cardiomyopathy; electron microscopy; Molecular Dynamics; tropomyosin
5.  Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease* 
The Journal of Biological Chemistry  2013;288(19):13446-13454.
Background: Cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle function.
Results: Phosphorylation affects mechanical parameters of cardiac muscle contraction during shortening.
Conclusion: Phosphorylation impacts mechanical function of cardiac muscle and is altered during cardiac disease.
Significance: Understanding RLC regulation by phosphorylation in cardiac muscle contraction is crucial for understanding changes in disease.
Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.
doi:10.1074/jbc.M113.455444
PMCID: PMC3650382  PMID: 23530050
Cardiac Muscle; Muscle; Myosin; Phosphorylation; Physiology
6.  Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy 
Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca2+-activated force was much lower in HCM myocytes (14 ± 1 kN/m2) than in donor myocytes (23 ± 3 kN/m2; P < 0.01), though cross-bridge kinetics (ktr) during maximal Ca2+ activation were 10% faster in HCM myocytes. Myofibrillar Ca2+ sensitivity in HCM myocytes (pCa50 = 6.40 ± 0.05) was higher than for donor myocytes (pCa50 = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca2+ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca2+-activated force and high Ca2+ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.
Research Highlights
► The passive stiffness of skinned HCM cardiac myocytes was similar to that of normal (donor) myocytes. ► Maximum Ca-activated force production was reduced by 40% in HCM vs donor myocytes. ► This loss of force could contribute to systolic dysfunction in HCM hearts. ► Myofibrillar Ca sensitivity was higher in HCM than in donor myocytes. ► The enhanced Ca sensitivity could compensate for the smaller maximum force but would tend to cause diastolic dysfunction. ► These characteristics were common to all HCM patients studied, suggesting the changes were secondary consequence of the underlying genetic variants.
doi:10.1016/j.yjmcc.2010.06.006
PMCID: PMC2954357  PMID: 20615414
Hypertrophic cardiomyopathy; Skinned cardiac myocytes; Viscoelasticity; Ca2+ sensitivity; Cross-bridge kinetics
8.  The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy 
Cardiovascular Research  2008;79(3):481-491.
Aim
The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle.
Methods
Myosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate–polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain.
Results
The fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 ± 0.034 for myectomy myosin vs. 0.305 ± 0.019 µm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 ± 5% compared with 11 ± 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30–45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 ± 0.25 vs. 3.58 ± 0.40%) and reduced relaxation rates compared with donor myocytes (TT50% = 0.32 ± 0.09 vs. 0.17 ± 0.02 s).
Conclusion
Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.
doi:10.1093/cvr/cvn094
PMCID: PMC2492731  PMID: 18411228
Contractile apparatus; Contractile function; Hypertrophy; Protein phosphorylation; Myosin; Myectomy; Hypertrophic cardiomyopathy

Results 1-8 (8)