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Papers by Galina Flint

Biophysical Journal, Feb 1, 2023

Additional information Versions of research works Versions of Record If this version is the versi... more Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version.

Journal of Molecular and Cellular Cardiology, Feb 1, 2023

Biophysical Journal, Feb 1, 2019

Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiom... more Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiomyopathies, a serious and sometimes deadly heart disorder. Experimental groups have extensively studied many of these mutations in an attempt to uncover the link between genotype and phenotype, but critical atomic level details cannot be determined by these methods. To address this gap, molecular dynamics (MD) simulations are utilized to model and study the changes in conformation and dynamics imposed by these single point mutations. The goal of this research is to move beyond identifying the effects mutations have on the cardiac thin filament, and to determine a set of guidelines to predict the clinical effects unstudied mutations will have on individuals. A set of twenty different clinically determined amino acid mutations located in cardiac troponin T and tropomyosin were chosen for analysis, and MD simulations were performed using the fully atomistic cardiac thin filament previously created by our group. From the simulations, conformation changes imposed by the mutation, both local and distant to the site of mutation, were computed. Steered molecular dynamics along with Jarzynski's equality were utilized to determine the changes in the free energy barrier of calcium binding and dissociation due to mutation. Coupling the changes in conformation and calcium dynamics data together, subsets of mutations were created to begin the process of determining guidelines for mutational predictions.

Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular pe... more Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2’-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host ...

Circulation Research

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyo... more BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficient. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the on state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv ...

Biophysical Journal

Additional information Versions of research works Versions of Record If this version is the versi... more Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detai... more Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the “on” state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Dan...

Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension gen... more Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension generation, are principal drivers of dilated cardiomyopathy (DCM)– the leading cause of heart failure1,2. Progress towards developing precision therapeutics for and defining the underlying determinants of DCM has been cardiomyocyte centric with negligible attention directed towards fibroblasts despite their role in regulating the best predictor of DCM severity, cardiac fibrosis3,4. Given that failure to reverse fibrosis is a major limitation of both standard of care and first in class precision therapeutics for DCM, this study examined whether cardiac fibroblast-mediated regulation of the heart’s material properties is essential for the DCM phenotype. Here we report in a mouse model of inherited DCM that prior to the onset of fibrosis and dilated myocardial remodeling both the myocardium and extracellular matrix (ECM) stiffen from switches in titin isoform expression, enhanced collagen fibe...

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flex... more Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ~1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to mo...

Cell Stem Cell, 2022

Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response.... more Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response. Because transcriptome maturation by muscleblind-like 1 (MBNL1) promotes differentiated cell states, this study investigated whether tactical control of MBNL1 activity could alter myofibroblast activity and fibrotic outcomes. In healthy mice, cardiac fibroblast-specific overexpression of MBNL1 transitioned the fibroblast transcriptome to that of a myofibroblast and after injury promoted myocyte remodeling and scar maturation. Both fibroblast- and myofibroblast-specific loss of MBNL1 limited scar production and stabilization, which was ascribed to negligible myofibroblast activity. The combination of MBNL1 deletion and injury caused quiescent fibroblasts to expand and adopt features of cardiac mesenchymal stem cells, whereas transgenic MBNL1 expression blocked fibroblast proliferation and drove the population into a mature myofibroblast state. These data suggest MBNL1 is a post-transcriptional switch, controlling fibroblast state plasticity during cardiac wound healing.

bioRxiv, 2021

Dynamic fibroblast state transitions are responsible for the heart’s fibrotic response to injury,... more Dynamic fibroblast state transitions are responsible for the heart’s fibrotic response to injury, raising the possibility that tactical control of these transitions could alter maladaptive fibrotic outcomes. Transcriptome maturation by the RNA binding protein Muscleblind Like 1 (MBNL1) has emerged as a potential driver of differentiated cell states. Here genetic lineage tracing of myofibroblasts in the injured heart demonstrated that gains in MBNL1 function corresponded to profibrotic fibroblast states. Similarly, in mice cardiac fibroblast specific MBNL1 overexpression induced a transcriptional myofibroblast profile in healthy cardiac fibroblasts that prevented the fibroproliferative phase of cardiac wound healing. By contrast loss of MBNL1 reverted cardiac fibroblasts to a pro-proliferative epicardial progenitor state that limited cardiac fibrosis following myocardial infarction. This progenitor state transition was associated with an MBNL1-dependent destabilization of the mesench...

JCI Insight, 2020

Conflict of interest: ADM is a cofounder of and has an equity interest in Insilicomed Inc. and an... more Conflict of interest: ADM is a cofounder of and has an equity interest in Insilicomed Inc. and an equity interest in Vektor Medical Inc. He serves on the scientific advisory board of Insilicomed and as scientific advisor to both companies. Some of his research grants have been identified for conflict-of-interest management based on the overall scope of the project and its potential benefit to these companies. The author is required to disclose this relationship in publications acknowledging the grant support; however, the research subject and findings reported in this study did not involve the companies in any way. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict-ofinterest policies.

Circulation, 2020

Background: Increased fatty acid oxidation (FAO) has long been considered a culprit in the develo... more Background: Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes mellitus–induced cardiomyopathy. However, enhancing cardiac FAO by removing the inhibitory mechanism of long-chain fatty acid transport into mitochondria via deletion of acetyl coenzyme A carboxylase 2 (ACC2) does not cause cardiomyopathy in nonobese mice, suggesting that high FAO is distinct from cardiac lipotoxicity. We hypothesize that cardiac pathology–associated obesity is attributable to the imbalance of fatty acid supply and oxidation. Thus, we here seek to determine whether further increasing FAO by inducing ACC2 deletion prevents obesity-induced cardiomyopathy, and if so, to elucidate the underlying mechanisms. Methods: We induced high FAO in adult mouse hearts by cardiac-specific deletion of ACC2 using a tamoxifen-inducible model (ACC2 iKO). Control and ACC2 iKO mice were subjected to high-fat diet (HFD) feeding for 24 weeks to induce obesity. Cardiac ...

Proceedings of the National Academy of Sciences, 2019

Significance The development of several small-molecule myosin activators to rescue cardiac contra... more Significance The development of several small-molecule myosin activators to rescue cardiac contractility of failing hearts is underway, but understanding the multiscale structure-function consequences of these inotropes remains a major challenge. Using 2′-deoxy-adenosine 5′-triphosphate (dATP; a myosin-binding nucleotide) as a molecular probe, we show that a restructuring of prepowerstroke myosin in the presence of 2-deoxy-adenosine 5′-diphosphate and inorganic phosphate (dADP.Pi) (versus ADP.Pi) increases actin–myosin electrostatic interactions and binding kinetics. X-ray diffraction revealed myosin structure with dATP in relaxed myofilament is similar to the activated state with ATP, with S1 head movement toward actin and stabilization, resulting in an improved probability of strong cross-bridge formation upon activation. Thus, the alterations induced by dATP may provide clues to a more optimal structure of cardiac myosin to be attained by myosin-targeted therapies for heart failure.

Biophysical Journal, 2019

Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiom... more Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiomyopathies, a serious and sometimes deadly heart disorder. Experimental groups have extensively studied many of these mutations in an attempt to uncover the link between genotype and phenotype, but critical atomic level details cannot be determined by these methods. To address this gap, molecular dynamics (MD) simulations are utilized to model and study the changes in conformation and dynamics imposed by these single point mutations. The goal of this research is to move beyond identifying the effects mutations have on the cardiac thin filament, and to determine a set of guidelines to predict the clinical effects unstudied mutations will have on individuals. A set of twenty different clinically determined amino acid mutations located in cardiac troponin T and tropomyosin were chosen for analysis, and MD simulations were performed using the fully atomistic cardiac thin filament previously created by our group. From the simulations, conformation changes imposed by the mutation, both local and distant to the site of mutation, were computed. Steered molecular dynamics along with Jarzynski's equality were utilized to determine the changes in the free energy barrier of calcium binding and dissociation due to mutation. Coupling the changes in conformation and calcium dynamics data together, subsets of mutations were created to begin the process of determining guidelines for mutational predictions.

Biophysical Journal, 2017

can alter the mechanical properties of cMyBP-C leading to reduced braking ability and hypercontra... more can alter the mechanical properties of cMyBP-C leading to reduced braking ability and hypercontractility, a hallmark of HCM. To test this hypothesis, we have produced several variants of the C3 domain of cMyBP-C, a central domain of the protein without known protein interactors. As expected, most mutants retain close-to-wild-type structure and thermodynamic stability. However, pathogenic mutants show diminished mechanical stability, as determined by single-molecule atomic force microscopy experiments in force-clamp mode. Application of Bell's model to our experimental data show that pathogenic mutants unfold up to 2 times faster than wild-type protein at physiological forces. This mechanical alteration would lead to softer cMyBP-C tethers. Hence, our results support the new idea that nanomechanical phenotypes in cMyBP-C can trigger the development of HCM. This mechanical perspective can explain why missense mutations and truncations, which induce full loss of the cMyBP-C tether, converge in similar clinical outcomes.

Biophysical Journal, 2016

smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K 4 (equilibrium constant... more smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K 4 (equilibrium constant of the force generation step) than those of a-MHC. There were no differences in K 2 (equilibrium constant of the cross-bridge detachment step), K 5 (Pi association constant), active tension, or rigor stiffness. Our study also demonstrated that there were no differences in the above mentioned parameters between fibers obtained from left and right ventricles, indicating that there is no difference at the myofilament level in both ventricles.

Biophysical Journal, 2014

Journal of muscle research and cell motility, 2015

Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in t... more Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in three main isoforms in skeletal and cardiac muscles. In the present work we studied the functional role of α- and βTm on force development by modifying the isoform composition of rabbit psoas skeletal muscle myofibrils and of regulated thin filaments for in vitro motility measurements. Skeletal myofibril regulatory proteins were extracted (78 %) and replaced (98 %) with Tm isoforms as homogenous ααTm or ββTm dimers and the functional effects were measured. Maximal Ca(2+) activated force was the same in ααTm versus ββTm myofibrils, but ββTm myofibrils showed a marked slowing of relaxation and an impairment of regulation under resting conditions compared to ααTm and controls. ββTm myofibrils also showed a significantly shorter slack sarcomere length and a marked increase in resting tension. Both these mechanical features were almost completely abolished by 10 mM 2,3-butanedione 2-monoxime, ...

Biophysical Journal, Feb 1, 2023

Additional information Versions of research works Versions of Record If this version is the versi... more Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version.

Journal of Molecular and Cellular Cardiology, Feb 1, 2023

Biophysical Journal, Feb 1, 2019

Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiom... more Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiomyopathies, a serious and sometimes deadly heart disorder. Experimental groups have extensively studied many of these mutations in an attempt to uncover the link between genotype and phenotype, but critical atomic level details cannot be determined by these methods. To address this gap, molecular dynamics (MD) simulations are utilized to model and study the changes in conformation and dynamics imposed by these single point mutations. The goal of this research is to move beyond identifying the effects mutations have on the cardiac thin filament, and to determine a set of guidelines to predict the clinical effects unstudied mutations will have on individuals. A set of twenty different clinically determined amino acid mutations located in cardiac troponin T and tropomyosin were chosen for analysis, and MD simulations were performed using the fully atomistic cardiac thin filament previously created by our group. From the simulations, conformation changes imposed by the mutation, both local and distant to the site of mutation, were computed. Steered molecular dynamics along with Jarzynski's equality were utilized to determine the changes in the free energy barrier of calcium binding and dissociation due to mutation. Coupling the changes in conformation and calcium dynamics data together, subsets of mutations were created to begin the process of determining guidelines for mutational predictions.

Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular pe... more Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2’-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host ...

Circulation Research

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyo... more BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficient. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the on state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv ...

Biophysical Journal

Additional information Versions of research works Versions of Record If this version is the versi... more Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detai... more Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the “on” state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Dan...

Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension gen... more Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension generation, are principal drivers of dilated cardiomyopathy (DCM)– the leading cause of heart failure1,2. Progress towards developing precision therapeutics for and defining the underlying determinants of DCM has been cardiomyocyte centric with negligible attention directed towards fibroblasts despite their role in regulating the best predictor of DCM severity, cardiac fibrosis3,4. Given that failure to reverse fibrosis is a major limitation of both standard of care and first in class precision therapeutics for DCM, this study examined whether cardiac fibroblast-mediated regulation of the heart’s material properties is essential for the DCM phenotype. Here we report in a mouse model of inherited DCM that prior to the onset of fibrosis and dilated myocardial remodeling both the myocardium and extracellular matrix (ECM) stiffen from switches in titin isoform expression, enhanced collagen fibe...

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flex... more Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ~1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to mo...

Cell Stem Cell, 2022

Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response.... more Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response. Because transcriptome maturation by muscleblind-like 1 (MBNL1) promotes differentiated cell states, this study investigated whether tactical control of MBNL1 activity could alter myofibroblast activity and fibrotic outcomes. In healthy mice, cardiac fibroblast-specific overexpression of MBNL1 transitioned the fibroblast transcriptome to that of a myofibroblast and after injury promoted myocyte remodeling and scar maturation. Both fibroblast- and myofibroblast-specific loss of MBNL1 limited scar production and stabilization, which was ascribed to negligible myofibroblast activity. The combination of MBNL1 deletion and injury caused quiescent fibroblasts to expand and adopt features of cardiac mesenchymal stem cells, whereas transgenic MBNL1 expression blocked fibroblast proliferation and drove the population into a mature myofibroblast state. These data suggest MBNL1 is a post-transcriptional switch, controlling fibroblast state plasticity during cardiac wound healing.

bioRxiv, 2021

Dynamic fibroblast state transitions are responsible for the heart’s fibrotic response to injury,... more Dynamic fibroblast state transitions are responsible for the heart’s fibrotic response to injury, raising the possibility that tactical control of these transitions could alter maladaptive fibrotic outcomes. Transcriptome maturation by the RNA binding protein Muscleblind Like 1 (MBNL1) has emerged as a potential driver of differentiated cell states. Here genetic lineage tracing of myofibroblasts in the injured heart demonstrated that gains in MBNL1 function corresponded to profibrotic fibroblast states. Similarly, in mice cardiac fibroblast specific MBNL1 overexpression induced a transcriptional myofibroblast profile in healthy cardiac fibroblasts that prevented the fibroproliferative phase of cardiac wound healing. By contrast loss of MBNL1 reverted cardiac fibroblasts to a pro-proliferative epicardial progenitor state that limited cardiac fibrosis following myocardial infarction. This progenitor state transition was associated with an MBNL1-dependent destabilization of the mesench...

JCI Insight, 2020

Conflict of interest: ADM is a cofounder of and has an equity interest in Insilicomed Inc. and an... more Conflict of interest: ADM is a cofounder of and has an equity interest in Insilicomed Inc. and an equity interest in Vektor Medical Inc. He serves on the scientific advisory board of Insilicomed and as scientific advisor to both companies. Some of his research grants have been identified for conflict-of-interest management based on the overall scope of the project and its potential benefit to these companies. The author is required to disclose this relationship in publications acknowledging the grant support; however, the research subject and findings reported in this study did not involve the companies in any way. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict-ofinterest policies.

Circulation, 2020

Background: Increased fatty acid oxidation (FAO) has long been considered a culprit in the develo... more Background: Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes mellitus–induced cardiomyopathy. However, enhancing cardiac FAO by removing the inhibitory mechanism of long-chain fatty acid transport into mitochondria via deletion of acetyl coenzyme A carboxylase 2 (ACC2) does not cause cardiomyopathy in nonobese mice, suggesting that high FAO is distinct from cardiac lipotoxicity. We hypothesize that cardiac pathology–associated obesity is attributable to the imbalance of fatty acid supply and oxidation. Thus, we here seek to determine whether further increasing FAO by inducing ACC2 deletion prevents obesity-induced cardiomyopathy, and if so, to elucidate the underlying mechanisms. Methods: We induced high FAO in adult mouse hearts by cardiac-specific deletion of ACC2 using a tamoxifen-inducible model (ACC2 iKO). Control and ACC2 iKO mice were subjected to high-fat diet (HFD) feeding for 24 weeks to induce obesity. Cardiac ...

Proceedings of the National Academy of Sciences, 2019

Significance The development of several small-molecule myosin activators to rescue cardiac contra... more Significance The development of several small-molecule myosin activators to rescue cardiac contractility of failing hearts is underway, but understanding the multiscale structure-function consequences of these inotropes remains a major challenge. Using 2′-deoxy-adenosine 5′-triphosphate (dATP; a myosin-binding nucleotide) as a molecular probe, we show that a restructuring of prepowerstroke myosin in the presence of 2-deoxy-adenosine 5′-diphosphate and inorganic phosphate (dADP.Pi) (versus ADP.Pi) increases actin–myosin electrostatic interactions and binding kinetics. X-ray diffraction revealed myosin structure with dATP in relaxed myofilament is similar to the activated state with ATP, with S1 head movement toward actin and stabilization, resulting in an improved probability of strong cross-bridge formation upon activation. Thus, the alterations induced by dATP may provide clues to a more optimal structure of cardiac myosin to be attained by myosin-targeted therapies for heart failure.

Biophysical Journal, 2019

Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiom... more Single amino acid mutations to the cardiac thin filament have been shown to cause genetic cardiomyopathies, a serious and sometimes deadly heart disorder. Experimental groups have extensively studied many of these mutations in an attempt to uncover the link between genotype and phenotype, but critical atomic level details cannot be determined by these methods. To address this gap, molecular dynamics (MD) simulations are utilized to model and study the changes in conformation and dynamics imposed by these single point mutations. The goal of this research is to move beyond identifying the effects mutations have on the cardiac thin filament, and to determine a set of guidelines to predict the clinical effects unstudied mutations will have on individuals. A set of twenty different clinically determined amino acid mutations located in cardiac troponin T and tropomyosin were chosen for analysis, and MD simulations were performed using the fully atomistic cardiac thin filament previously created by our group. From the simulations, conformation changes imposed by the mutation, both local and distant to the site of mutation, were computed. Steered molecular dynamics along with Jarzynski's equality were utilized to determine the changes in the free energy barrier of calcium binding and dissociation due to mutation. Coupling the changes in conformation and calcium dynamics data together, subsets of mutations were created to begin the process of determining guidelines for mutational predictions.

Biophysical Journal, 2017

can alter the mechanical properties of cMyBP-C leading to reduced braking ability and hypercontra... more can alter the mechanical properties of cMyBP-C leading to reduced braking ability and hypercontractility, a hallmark of HCM. To test this hypothesis, we have produced several variants of the C3 domain of cMyBP-C, a central domain of the protein without known protein interactors. As expected, most mutants retain close-to-wild-type structure and thermodynamic stability. However, pathogenic mutants show diminished mechanical stability, as determined by single-molecule atomic force microscopy experiments in force-clamp mode. Application of Bell's model to our experimental data show that pathogenic mutants unfold up to 2 times faster than wild-type protein at physiological forces. This mechanical alteration would lead to softer cMyBP-C tethers. Hence, our results support the new idea that nanomechanical phenotypes in cMyBP-C can trigger the development of HCM. This mechanical perspective can explain why missense mutations and truncations, which induce full loss of the cMyBP-C tether, converge in similar clinical outcomes.

Biophysical Journal, 2016

smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K 4 (equilibrium constant... more smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K 4 (equilibrium constant of the force generation step) than those of a-MHC. There were no differences in K 2 (equilibrium constant of the cross-bridge detachment step), K 5 (Pi association constant), active tension, or rigor stiffness. Our study also demonstrated that there were no differences in the above mentioned parameters between fibers obtained from left and right ventricles, indicating that there is no difference at the myofilament level in both ventricles.

Biophysical Journal, 2014

Journal of muscle research and cell motility, 2015

Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in t... more Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in three main isoforms in skeletal and cardiac muscles. In the present work we studied the functional role of α- and βTm on force development by modifying the isoform composition of rabbit psoas skeletal muscle myofibrils and of regulated thin filaments for in vitro motility measurements. Skeletal myofibril regulatory proteins were extracted (78 %) and replaced (98 %) with Tm isoforms as homogenous ααTm or ββTm dimers and the functional effects were measured. Maximal Ca(2+) activated force was the same in ααTm versus ββTm myofibrils, but ββTm myofibrils showed a marked slowing of relaxation and an impairment of regulation under resting conditions compared to ααTm and controls. ββTm myofibrils also showed a significantly shorter slack sarcomere length and a marked increase in resting tension. Both these mechanical features were almost completely abolished by 10 mM 2,3-butanedione 2-monoxime, ...