The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Kathleen M Ruppel - One of the best experts on this subject based on the ideXlab platform.
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controlling load dependent kinetics of β Cardiac Myosin at the single molecule level
Nature Structural & Molecular Biology, 2018Co-Authors: Chao Liu, Masataka Kawana, Kathleen M Ruppel, Dan Song, James A SpudichAbstract:Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate of Myosin from actin, which determines the time that Myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which Myosin works. Here we measure the detachment rate of single molecules of human β-Cardiac Myosin and its load dependence. We find that both can be modulated by both small-molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating versus inhibitory perturbations of Cardiac Myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and suggest that Cardiac contractility can be controlled by tuning the load-dependent kinetics of single Myosin molecules.
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mavacamten stabilizes a folded back sequestered super relaxed state of β Cardiac Myosin
bioRxiv, 2018Co-Authors: Robert L Anderson, Saswata S Sarkar, Darshan V Trivedi, Marcus Henze, Henry Gong, Christopher S Rogers, Fiona Wong, Makenna M Morck, J G Seidman, Kathleen M RuppelAbstract:Mutations in β-Cardiac Myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. Previous studies suggest that Myosin function can be regulated by entering a super-relaxed state (SRX) with very slow ATP hydrolysis, but the structural determinants of this state are uncertain. Using a combination of biochemical approaches with electron microscopy and X-ray fiber diffraction, we show that the SRX corresponds to a folded-back state of Myosin with increased ordering of heads around the thick filament backbone. The small molecule mavacamten induces this conformation, while mutations causing HCM destabilize it. These findings provide a structural basis for an important mode of regulation of Cardiac Myosin with implications for the pathogenesis of cardiomyopathy and potential therapeutic development.
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biophysical properties of human β Cardiac Myosin with converter mutations that cause hypertrophic cardiomyopathy
Science Advances, 2017Co-Authors: Masataka Kawana, Shirley Sutton, Kathleen M Ruppel, Saswata S Sarkar, James A SpudichAbstract:Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the β-Cardiac Myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of Myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on Myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of Myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-Cardiac Myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant Myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G Myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human β-Cardiac Myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of Myosin heads participating in force production.
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early onset hypertrophic cardiomyopathy mutations significantly increase the velocity force and actin activated atpase activity of human β Cardiac Myosin
Cell Reports, 2016Co-Authors: Arjun S Adhikari, James A Spudich, Saswata S Sarkar, Chao Liu, Kristina B Kooiker, Daniel Bernstein, Kathleen M RuppelAbstract:Summary Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-Cardiac Myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-Cardiac Myosin. We observed significant increases (20%–90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type Myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of Myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-Cardiac Myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.
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mutations in the catalytic domain of human β Cardiac Myosin that cause early onset hypertrophic cardiomyopathy significantly increase the fundamental parameters that determine ensemble force and velocity
bioRxiv, 2016Co-Authors: Arjun S Adhikari, James A Spudich, Saswata S Sarkar, Chao Liu, Kristina B Kooiker, Daniel Bernstein, Kathleen M RuppelAbstract:Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden Cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in β-Cardiac Myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human β-Cardiac Myosin at the molecular level. We observe extremely large increases (25% - 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant Myosin motors compared to wild type Myosin. In contrast to previous studies of HCM-causing mutations in human β-Cardiac Myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human β-Cardiac Myosin.
James A Spudich - One of the best experts on this subject based on the ideXlab platform.
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controlling load dependent kinetics of β Cardiac Myosin at the single molecule level
Nature Structural & Molecular Biology, 2018Co-Authors: Chao Liu, Masataka Kawana, Kathleen M Ruppel, Dan Song, James A SpudichAbstract:Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate of Myosin from actin, which determines the time that Myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which Myosin works. Here we measure the detachment rate of single molecules of human β-Cardiac Myosin and its load dependence. We find that both can be modulated by both small-molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating versus inhibitory perturbations of Cardiac Myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and suggest that Cardiac contractility can be controlled by tuning the load-dependent kinetics of single Myosin molecules.
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biophysical properties of human β Cardiac Myosin with converter mutations that cause hypertrophic cardiomyopathy
Science Advances, 2017Co-Authors: Masataka Kawana, Shirley Sutton, Kathleen M Ruppel, Saswata S Sarkar, James A SpudichAbstract:Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the β-Cardiac Myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of Myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on Myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of Myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-Cardiac Myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant Myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G Myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human β-Cardiac Myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of Myosin heads participating in force production.
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early onset hypertrophic cardiomyopathy mutations significantly increase the velocity force and actin activated atpase activity of human β Cardiac Myosin
Cell Reports, 2016Co-Authors: Arjun S Adhikari, James A Spudich, Saswata S Sarkar, Chao Liu, Kristina B Kooiker, Daniel Bernstein, Kathleen M RuppelAbstract:Summary Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-Cardiac Myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-Cardiac Myosin. We observed significant increases (20%–90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type Myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of Myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-Cardiac Myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.
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mutations in the catalytic domain of human β Cardiac Myosin that cause early onset hypertrophic cardiomyopathy significantly increase the fundamental parameters that determine ensemble force and velocity
bioRxiv, 2016Co-Authors: Arjun S Adhikari, James A Spudich, Saswata S Sarkar, Chao Liu, Kristina B Kooiker, Daniel Bernstein, Kathleen M RuppelAbstract:Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden Cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in β-Cardiac Myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human β-Cardiac Myosin at the molecular level. We observe extremely large increases (25% - 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant Myosin motors compared to wild type Myosin. In contrast to previous studies of HCM-causing mutations in human β-Cardiac Myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human β-Cardiac Myosin.
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biophysical properties of human β Cardiac Myosin with converter mutations that cause hypertrophic cardiomyopathy
bioRxiv, 2016Co-Authors: Masataka Kawana, Shirley Sutton, Kathleen M Ruppel, Saswata S Sarkar, James A SpudichAbstract:Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hyper-contractility, and therapies are aimed toward controlling the hyperactive physiology. β-Cardiac Myosin comprises ~40 percent of genetic mutations associated with HCM and the converter domain of Myosin is a hot spot for HCM-causing mutations, but the underlying primary effects of these mutations on Myosin9s biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of Myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-Cardiac Myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations R719W, R723G and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant Myosins are decreased compared to wild type, and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15 percent faster for R719W and R723G Myosin, while there is no change in velocity for G741R. ATPase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to wild type. These results indicate that the net biomechanical properties of human β-Cardiac Myosin carrying these converter domain mutations are very similar to wild type or even slightly hypo-contractile, leading us to consider an alternative mechanism for the clinically observed hyper-contractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of Myosin heads participating in force production.
J G Seidman - One of the best experts on this subject based on the ideXlab platform.
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deciphering the super relaxed state of human β Cardiac Myosin and the mode of action of mavacamten from Myosin molecules to muscle fibers
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Robert L Anderson, Saswata S Sarkar, Darshan V Trivedi, Marcus Henze, Henry Gong, Christopher S Rogers, Fiona Wong, Makenna M Morck, Joshua M Gorham, J G SeidmanAbstract:Mutations in β-Cardiac Myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of Myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in Myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of Myosin heads accessible to actin. Here we show that purified human β-Cardiac Myosin exists partly in an SRX and may in part correspond to a folded-back conformation of Myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.
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mavacamten stabilizes a folded back sequestered super relaxed state of β Cardiac Myosin
bioRxiv, 2018Co-Authors: Robert L Anderson, Saswata S Sarkar, Darshan V Trivedi, Marcus Henze, Henry Gong, Christopher S Rogers, Fiona Wong, Makenna M Morck, J G Seidman, Kathleen M RuppelAbstract:Mutations in β-Cardiac Myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. Previous studies suggest that Myosin function can be regulated by entering a super-relaxed state (SRX) with very slow ATP hydrolysis, but the structural determinants of this state are uncertain. Using a combination of biochemical approaches with electron microscopy and X-ray fiber diffraction, we show that the SRX corresponds to a folded-back state of Myosin with increased ordering of heads around the thick filament backbone. The small molecule mavacamten induces this conformation, while mutations causing HCM destabilize it. These findings provide a structural basis for an important mode of regulation of Cardiac Myosin with implications for the pathogenesis of cardiomyopathy and potential therapeutic development.
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hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse α Cardiac Myosin in the laser trap assay
American Journal of Physiology-heart and Circulatory Physiology, 2007Co-Authors: Edward P Debold, Joachim P Schmitt, Christine E. Seidman, J G Seidman, Joseph B Patlak, Samantha Beck, Jeffrey R Moore, David M WarshawAbstract:Point mutations in Cardiac Myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter Myosin's molecular mech...
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Cardiac Myosin binding protein c phosphorylation is cardioprotective
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Sakthivel Sadayappan, Christine E. Seidman, J G Seidman, Hanna Osinska, Raisa Klevitsky, John N Lorenz, Michelle A Sargent, Jeffrey D Molkentin, Jeffrey M RobbinsAbstract:Cardiac Myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with Cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-CAllP+). The allele was bred into a cMyBP-C null background (cMyBP-C(t/t)) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-CAllP+ was incorporated normally into the Cardiac sarcomere and restored normal Cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick–thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the Myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-CAllP+:(t/t) hearts were resistant to ischemia–reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.
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Cardiac Myosin binding protein c phosphorylation and Cardiac function
Circulation Research, 2005Co-Authors: Sakthivel Sadayappan, Christine E. Seidman, J G Seidman, James Gulick, Hanna Osinska, Lisa A Martin, Harvey S Hahn, Gerald W Dorn, Raisa Klevitsky, Jeffrey M RobbinsAbstract:The role of Cardiac Myosin binding protein-C (cMyBP-C) phosphorylation in Cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-CAllP−). A TG line showing &40% replacement with MyBP-CAllP− showed no changes in morbidity or mortality but displayed depressed Cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-CAllP−, the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-CAllP− was incorpo...
Christine E. Seidman - One of the best experts on this subject based on the ideXlab platform.
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hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse α Cardiac Myosin in the laser trap assay
American Journal of Physiology-heart and Circulatory Physiology, 2007Co-Authors: Edward P Debold, Joachim P Schmitt, Christine E. Seidman, J G Seidman, Joseph B Patlak, Samantha Beck, Jeffrey R Moore, David M WarshawAbstract:Point mutations in Cardiac Myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter Myosin's molecular mech...
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Cardiac Myosin binding protein c phosphorylation is cardioprotective
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Sakthivel Sadayappan, Christine E. Seidman, J G Seidman, Hanna Osinska, Raisa Klevitsky, John N Lorenz, Michelle A Sargent, Jeffrey D Molkentin, Jeffrey M RobbinsAbstract:Cardiac Myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with Cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-CAllP+). The allele was bred into a cMyBP-C null background (cMyBP-C(t/t)) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-CAllP+ was incorporated normally into the Cardiac sarcomere and restored normal Cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick–thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the Myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-CAllP+:(t/t) hearts were resistant to ischemia–reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.
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Cardiac Myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Joachim P Schmitt, Ferhaan Ahmad, Andrea Frederico, David A Conner, Martin J Lohse, Ulrike Mende, Amy Armstrong, David M Warshaw, Edward P Debold, Christine E. SeidmanAbstract:Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in Cardiac Myosin, the molecular motor of the heart. The effects of these mutations on Myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the α-Cardiac Myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant Cardiac Myosins exhibited a reduced ability to translocate actin (Vactin) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower Vactin in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in Cardiac Myosin may initiate the events that cause the heart to remodel and become pathologically dilated.
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Cardiac Myosin binding protein c phosphorylation and Cardiac function
Circulation Research, 2005Co-Authors: Sakthivel Sadayappan, Christine E. Seidman, J G Seidman, James Gulick, Hanna Osinska, Lisa A Martin, Harvey S Hahn, Gerald W Dorn, Raisa Klevitsky, Jeffrey M RobbinsAbstract:The role of Cardiac Myosin binding protein-C (cMyBP-C) phosphorylation in Cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-CAllP−). A TG line showing &40% replacement with MyBP-CAllP− showed no changes in morbidity or mortality but displayed depressed Cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-CAllP−, the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-CAllP− was incorpo...
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role of Cardiac Myosin binding protein c in sustaining left ventricular systolic stiffening
Circulation Research, 2004Co-Authors: Bradley M Palmer, Patrick G Burgon, Samantha P Harris, Richard L Moss, Dimitrios Georgakopoulos, Paul M L Janssen, Yuan Wang, Norman R Alpert, Diego F Belardi, Christine E. SeidmanAbstract:Despite advances in the molecular biology of Cardiac Myosin binding protein-C (cMyBP-C), little is understood about its precise role in muscle contraction, particularly in the intact heart. We tested the hypothesis that cMyBP-C is central to the time course and magnitude of left ventricular systolic elastance (chamber stiffening), and assessed mechanisms for this influence in intact hearts, trabeculae, and skinned fibers from wild-type (+/+) and homozygous truncated cMyBP-C (t/t) male mice. cMyBP-C protein was not detected by gel electrophoresis or Western blot in t/t myocardium. cMyBP-C t/t ventricles displayed reduced peak elastance, but more strikingly a marked abbreviation of the systolic elastance time course, which peaked earlier (27.6±2.1 ms) than in +/+ controls (47.8±1.6 ms). Control hearts reached only 42±4% of maximum elastance at the onset of ejection, with substantial further stiffening during ejection. This contrasted to t/t mutants, which reached 77±3% of peak elastance before ejection of peak. These unusual findings were not observed in alternative models involving severe cardiomyopathy, but were recapitulated in a cMyBP-C null mouse. The abbreviated elastance time course and lower peak were consistent with earlier time-to-peak trabecular tension, increased unloaded shortening velocity in t/t skinned muscle strips, and dramatically reduced myofilament stiffness at diastolic calcium concentrations. These results provide novel insights into the role of cMyBP-C in myocardial systolic mechanics. Abnormal sarcomere shortening velocity and abbreviated muscle stiffening may underlie development of Cardiac dysfunction associated with deficient incorporation of cMyBP-C.
Shirley Sutton - One of the best experts on this subject based on the ideXlab platform.
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biophysical properties of human β Cardiac Myosin with converter mutations that cause hypertrophic cardiomyopathy
Science Advances, 2017Co-Authors: Masataka Kawana, Shirley Sutton, Kathleen M Ruppel, Saswata S Sarkar, James A SpudichAbstract:Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the β-Cardiac Myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of Myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on Myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of Myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-Cardiac Myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant Myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G Myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human β-Cardiac Myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of Myosin heads participating in force production.
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biophysical properties of human β Cardiac Myosin with converter mutations that cause hypertrophic cardiomyopathy
bioRxiv, 2016Co-Authors: Masataka Kawana, Shirley Sutton, Kathleen M Ruppel, Saswata S Sarkar, James A SpudichAbstract:Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hyper-contractility, and therapies are aimed toward controlling the hyperactive physiology. β-Cardiac Myosin comprises ~40 percent of genetic mutations associated with HCM and the converter domain of Myosin is a hot spot for HCM-causing mutations, but the underlying primary effects of these mutations on Myosin9s biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of Myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-Cardiac Myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations R719W, R723G and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant Myosins are decreased compared to wild type, and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15 percent faster for R719W and R723G Myosin, while there is no change in velocity for G741R. ATPase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to wild type. These results indicate that the net biomechanical properties of human β-Cardiac Myosin carrying these converter domain mutations are very similar to wild type or even slightly hypo-contractile, leading us to consider an alternative mechanism for the clinically observed hyper-contractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of Myosin heads participating in force production.
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effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β Cardiac Myosin
The Journal of Experimental Biology, 2016Co-Authors: James A Spudich, Masataka Kawana, Tural Aksel, Suman Nag, Shirley Sutton, Ruth F Sommese, Sadie Bartholomew, Saswata S Sarkar, Jongmin Sung, Carol ChoAbstract:Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the Cardiac muscle sarcomere, including human β-Cardiac Myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-Cardiac Myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-Cardiac Myosin. We are using a recombinantly expressed human β-Cardiac Myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant Myosins compared with wild type. Our studies are extending beyond Myosin interactions with pure actin filaments to include the interaction of Myosin with regulated actin filaments containing tropoMyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of Myosin binding protein-C and titin, important regulatory components of both Cardiac and skeletal muscles.
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contractility parameters of human β Cardiac Myosin with the hypertrophic cardiomyopathy mutation r403q show loss of motor function
Science Advances, 2015Co-Authors: Suman Nag, Shirley Sutton, Kathleen M Ruppel, Ruth F Sommese, Zoltan Ujfalusi, Ariana Combs, Stephen J Langer, Leslie A Leinwand, Michael A Geeves, James A SpudichAbstract:Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the Cardiac sarcomere, including human β-Cardiac Myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-Cardiac Myosin, R403Q. Despite numerous studies, most performed with nonhuman or nonCardiac Myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-Cardiac Myosin and new methodologies to characterize in vitro contractility parameters of the R403Q Myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of Myosin with regulated actin filaments containing tropoMyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q Myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q Myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q Myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-Cardiac Myosin contractility as a result of the R403Q mutation.
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harmonic force spectroscopy measures load dependent kinetics of individual human β Cardiac Myosin molecules
Nature Communications, 2015Co-Authors: Jongmin Sung, Suman Nag, Shirley Sutton, Kathleen M Ruppel, Kim I Mortensen, Christian L Vestergaard, Henrik Flyvbjerg, James A SpudichAbstract:Single molecule methods for measuring load dependence are fundamental for molecular motor research. Here, Sung et al. introduce harmonic force spectroscopy, a method that randomly applies varying loads at high frequency, allowing the determination of load dependent parameters of human β-Cardiac Myosin at physiological ATP concentration.