Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke

Woody, Michael S.; Greenberg, Michael J.; Barua, Bipasha; Winkelmann, Donald A.; Goldman, Yale E.; Ostap, E. Michael

doi:10.1038/s41467-018-06193-2

Cited by 118 publications

(224 citation statements)

References 50 publications

Supporting

Mentioning

188

Contrasting

Order By: Relevance

“…We propose that a similar mechanism could be relevant in ΔK210 cells, which show reductions in myosin-based contractility and sarcomeric disorganization when patterned on glass. Consistent with this idea, treatment of hPSC-CMs containing a different DCM mutation with Omecamtiv mecarbil, a myosin-based thin filament activator (41, 77, 78), leads to an improvement in sarcomeric organization (76). We speculate that some of the salutary effects of the drug are due to the restoration of tension necessary for proper mechanosensing.…”

Section: Discussionmentioning

confidence: 75%

“…Porcine ventricular cardiac myosin (MYH7) is 97% identical to human, while murine cardiac myosin (MYH6) is only 92% identical. Porcine cardiac myosin has very similar biophysical properties to human cardiac myosin, including the kinetics of the ATPase cycle, step size, and sensitivity to load (38–41), making it an ideal myosin for biophysical studies.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

27Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a 28 major indicator for heart transplant. The disease is frequently caused by mutations of 29 sarcomeric proteins; however, it is not well understood how these molecular mutations 30 lead to alterations in cellular organization and contractility. To address this critical gap in 31 our knowledge, we studied the molecular and cellular consequences of a DCM mutation 32 in troponin-T, DK210. We determined the molecular mechanism of DK210 and used 33 computational modeling to predict that the mutation should reduce the force per 34 sarcomere. In mutant cardiomyocytes, we found that DK210 not only reduces contractility, 35 but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to 36 changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and 37 disease). These results link the molecular and cellular phenotypes and implicate 38 alterations in mechanosensing as an important factor in the development of DCM. 39Results 100 DK210 decreases calcium sensitivity in an in vitro motility assay 101We set out to decipher the molecular mechanism of the DK210 mutation in vitro. 102The molecular effects of cardiomyopathy mutations depend on the myosin isoform (7-9, 103 35-37) and therefore, we used porcine cardiac ventricular myosin (38). Porcine ventricular 104 cardiac myosin (MYH7) is 97% identical to human, while murine cardiac myosin (MYH6) 105 is only 92% identical. Porcine cardiac myosin has very similar biophysical properties to 106 human cardiac myosin, including the kinetics of the ATPase cycle, step size, and 107 sensitivity to load (38-41), making it an ideal myosin for biophysical studies. 108Given the role of troponin-T in thin filament regulation, we first determined whether 109 the DK210 mutation affects calcium-based regulation of myosin binding to thin filaments 110 using an in vitro motility assay (42). Reconstituted thin filaments, consisting of porcine 111 cardiac actin and recombinantly expressed human troponin and tropomyosin, were added 112 to a flow cell coated with porcine cardiac myosin in the presence of ATP. The speed of 113 filament translocation was measured as a function of added calcium. As has been 114 reported previously, the speed of regulated thin filament translocation increased 115 sigmoidally with increasing Ca 2+ concentration (43), ( Figure 1B). Data were fit with the Hill 116 equation to obtain the pCa50 (i.e., the concentration of calcium necessary for half-117 maximal activation). Consistent with previous studies using mouse cardiac, rabbit cardiac, 118 and rabbit skeletal muscle fibers (31, 33, 44), DK210 shows a right-shifted curve (pCa50 119 = 5.7 ± 0.1) compared to the WT (pCa50 = 6.1 ± 0.1; p < 0.0001), meaning more calcium 120 is needed for the same level of activation. This suggests that the mutant could show 121 impaired force production during a calcium transient. 122 7 123 Molecular mechanism of DK210-induced changes in thin filament regulat...

show abstract

Section: Discussionmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The three-state model provides a useful framework to understand the mechanism of perturbations in skeletal and cardiac thin-filament regulation, such as drug treatments (1,6), protein isoform changes (7), disease-causing mutations (8), and post-translational modifications (9,10). The formalism laid out by McKillop and Geeves enables the determination of the equilibrium constants that govern transitions between states, and thus the population of thin-filament regulatory units in each state ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Computational tool to study perturbations in muscle regulation and its application to heart disease

Barrick

Clippinger

Greenberg

2019

Preprint

Self Cite

View full text Add to dashboard Cite

25Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in 26 the sarcomere and generate pulling forces. This process is regulated by calcium, and it 27 can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations 28 (e.g., b-adrenergic stimulation), and pharmacological interventions. Therefore, it is 29 important to have a methodology to robustly determine the mechanism of these 30 perturbations and statistically evaluate their effects. Here, we present an approach to 31 measure the equilibrium constants that govern muscle activation, estimate uncertainty in 32 these parameters, and statistically test the effects of perturbations. We provide a 33 MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials 34 that make this approach accessible. The hypothesis testing and error estimation 35 approaches described here are broadly applicable, and the provided tools work with other 36 types of data, including cellular measurements. To demonstrate the utility of the 37 approach, we apply it to determine the biophysical mechanism of a mutation that causes 38 familial hypertrophic cardiomyopathy. This approach is generally useful for studying the 39 mechanisms of muscle diseases and therapeutic interventions that target muscle 40 contraction. 41 42

show abstract

“…The potential importance of a folded-back sequestered state of cardiac myosin has recently been highlighted in diseases like HCM, and also in the mechanism of action of cardiac inhibitors like mavacamten (15,37,38). The cardiac activator omecamtiv mecarbil has also been shown to stabilize the open state of cardiac myosin while also impacting the kinetics of actin-bound myosin motors (39)(40)(41). Thus, further research into the determinants of the sequestered state of myosin and the role of MyBP-C in holding myosin heads in a reserved state can lead to novel insights into drug discovery for cardiac activators and inhibitors.…”

Section: Discussionmentioning

confidence: 99%

The molecular basis of hypercontractility caused by the hypertrophic cardiomyopathy mutations R403Q and R663H

Sarkar

et al. 2019

Preprint

View full text Add to dashboard Cite

Hypertrophic cardiomyopathy (HCM) mutations in ß-cardiac myosin and myosin binding protein-C (MyBP-C) cause hypercontractility of the heart. We show that hypercontractility caused by the HCM myosin mutation R663H cannot be explained by changes in the fundamental parameters such as actin-activated ATPase, intrinsic force, velocity of pure actin or regulated thin filaments, or the pCa50 of the velocity of regulated thin filaments. The same conclusion was made earlier for the HCM myosin mutation R403Q (Nag et al. 2015). Using enzymatic assays for the number of functionally-available heads in purified human ß-cardiac myosin preparations, we provide evidence that both R403Q and R663H HCM myosin mutations cause hypercontractility by increasing the number of functionally-accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of myosin binding protein-C.

show abstract

Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke

Cited by 118 publications

References 50 publications

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Computational tool to study perturbations in muscle regulation and its application to heart disease

The molecular basis of hypercontractility caused by the hypertrophic cardiomyopathy mutations R403Q and R663H

Contact Info

Product

Resources

About