Publications
198 results found
Luther PK, Marston SB, 2024, Complex architecture of cardiac muscle thick filaments revealed, Trends in Pharmacological Sciences, Vol: 45, Pages: 191-192, ISSN: 0165-6147
Muscle contraction is orchestrated by the well-understood thin filaments and the markedly complex thick filaments. Studies by Dutta et al. and Tamborrini et al., discussed here, have unravelled the structure of the mammalian heart thick filament in exquisite near-atomic detail and pave the way for understanding physiological modulation pathways and mutation-induced dysfunction and for designing potential drugs to modify defects.
Yang Z, Marston SB, Gould IR, 2023, Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations, JOURNAL OF PHYSICAL CHEMISTRY B, Vol: 127, Pages: 8736-8748, ISSN: 1520-6106
Marston S, 2023, Recent studies of the molecular mechanism of lusitropy due to phosphorylation of cardiac troponin I by protein kinase A, JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, Vol: 44, Pages: 201-208, ISSN: 0142-4319
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Marston S, Pinto J, 2023, Suppression of lusitropy as a disease mechanism in cardiomyopathies, Frontiers in Cardiovascular Medicine, Vol: 9, Pages: 1-12, ISSN: 2297-055X
In cardiac muscle the action of adrenaline on 1 receptors of heart muscle cells is essential to adjust cardiac output to the body’s needs. Adrenergic activation leads to enhanced contractility (inotropy), faster heart rate (chronotropy) and faster relaxation (lusitropy), mainly through activation of protein kinase A (PKA). Efficient enhancement of heart output under stress requires all of these responses to work together. Lusitropy is essential for shortening the heartbeat when heart rate increases. It therefore follows that, if the lusitropic response is not present, heart function under stress will be compromised. Current literature suggests that lusitropy is primarily achieved due to PKA phosphorylation of troponin I (TnI) and phospholamban (PLB). It has been well documented that PKA-induced phosphorylation of TnI releases Ca2+ from troponin C faster and increases the rate of cardiac muscle relaxation, while phosphorylation of PLB increases SERCA activity, speeding up Ca2+ removal from the cytoplasm. In this review we consider the current scientific evidences for the connection between suppression of lusitropy and cardiac dysfunction in the context of mutations in phospholamban and thin filament proteins that are associated with cardiomyopathies. We will discuss what advances have been made into understanding the physiological mechanism of lusitropy due to TnI and PLB phosphorylation and its suppression by mutations and we will evaluate the evidence whether lack of lusitropy is sufficient to cause cardiomyopathy, and under what circumstances, and consider the range of pathologies associated with loss of lusitropy. Finally, we will discuss whether suppressed lusitropy due to mutations in thin filament proteins can be therapeutically restored.
Pavadai E, Rynkiewicz MJ, Yang Z, et al., 2022, Modulation of cardiac thin filament structure by phosphorylated troponin-I analyzed by protein-protein docking and molecular dynamics simulation., Archives of Biochemistry and Biophysics, Vol: 725, Pages: 109282-109282, ISSN: 0003-9861
Tropomyosin, controlled by troponin-linked Ca2+-binding, regulates muscle contraction by a macromolecular scale steric-mechanism that governs myosin-crossbridge-actin interactions. At low-Ca2+, C-terminal domains of troponin-I (TnI) trap tropomyosin in a position on thin filaments that interferes with myosin-binding, thus causing muscle relaxation. Steric inhibition is reversed at high-Ca2+ when TnI releases from F-actin-tropomyosin as Ca2+ and the TnI switch-peptide bind to the N-lobe of troponin-C (TnC). The opposite end of cardiac TnI contains a phosphorylation-sensitive ∼30 residue-long N-terminal peptide that is absent in skeletal muscle, and likely modifies these interactions in hearts. Here, PKA-dependent phosphorylation of serine 23 and 24 modulates Ca2+ and possibly switch-peptide binding to TnC, causing faster relaxation during the cardiac-cycle (lusitropy). The cardiac-specific N-terminal TnI domain is not captured in crystal structures of troponin or in cryo-EM reconstructions of thin filaments; thus, its global impact on thin filament structure and function is uncertain. Here, we used protein-protein docking and molecular dynamics simulation-based protocols to build a troponin model that was guided by and hence consistent with the recent seminal Yamada structure of Ca2+-activated thin filaments. We find that when present on thin filaments, phosphorylated Ser23/24 along with adjacent polar TnI residues interact closely with both tropomyosin and the N-lobe of TnC during our simulations. These interactions would likely bias tropomyosin to an off-state positioning on actin. In situ, such enhanced relaxation kinetics would promote cardiac lusitropy.
Marston S, 2022, Force measurements from myofibril to filament, Frontiers in Physiology, Vol: 12, Pages: 1-10, ISSN: 1664-042X
Contractility, the generation of force and movement by molecular motors, is the hallmark of all muscles, including striated muscle. Contractility can be studied at every level of organization from a whole animal to single molecules. Measurements at sub-cellular level are particularly useful since, in the absence of the excitation-contraction coupling system, the properties of the contractile proteins can be directly investigated; revealing mechanistic details not accessible in intact muscle. Moreover, the conditions can bemanipulated with ease, for instance changes in activator Ca2+, small molecule effector concentration or phosphorylation levels and introducing mutations. Subcellular methods can be successfully applied to frozen materials and generally require the smallest amount of tissue, thus greatly increasing the range of possible experiments compared with the study of intact muscle and cells. Whilst measurement of movement at the subcellular level is relatively simple, measurement of force is more challenging. This mini review will describe current methods for measuring force production at the subcellular level including single myofibril and single myofilament techniques.
Vikhorev P, Vikhoreva N, Yeung W, et al., 2022, Titin-truncating mutations associated with dilated cardiomyopathy alter length-dependent activation and its modulation via phosphorylation, Cardiovascular Research, Vol: 118, Pages: 241-253, ISSN: 0008-6363
Aims Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants.Methods and results We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants.Conclusion Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation.
Marston SB, 2021, Richard Tregear, co-founder of the Journal of Muscle Research and Cell Motility, Journal of Muscle Research and Cell Motility, Vol: 42, Pages: 129-130, ISSN: 0142-4319
Marston S, Alsulami K, 2020, Small molecules acting on myofilaments as treatments for heart and skeletal muscle diseases, International Journal of Molecular Sciences, Vol: 21, Pages: 1-30, ISSN: 1422-0067
Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are themost prevalent forms of the chronic and progressive pathological condition known ascardiomyopathy. These diseases have different aetiologies; however, they share the feature ofhaemodynamic abnormalities which is mainly due to dysfunction in the contractile proteins thatmake up the contractile unit known as the sarcomere. To date, pharmacological treatment optionsare not disease-specific and rather focus on managing the symptoms without addressing the diseasemechanism. Earliest attempts at improving cardiac contractility by modulating the sarcomereindirectly (inotropes) resulting in unwanted effects. In contrast, targeting the sarcomere directly,aided by high throughput screening systems, could identify small molecules with a superiortherapeutic value in cardiac muscle disorders. Here in, an extensive literature review of 21 smallmolecules directed to five different targets was conducted. A simple scoring system was created toassess the suitability of small molecules for therapy by evaluating them in 8 different criteria. Mostof the compounds failed due to lack of target specificity or poor physicochemical properties. Sixcompounds stood out showing a potential therapeutic value in HCM, DCM or HF. OmecamtivMecarbil and Danicamtiv (myosin activators), Mavacamten, CK-274 and MYK-581 (myosininhibitors) and AMG 594 (Ca2+-sensitiser) all are small molecules that allosterically modulatetroponin or myosin. Omecamtiv Mecarbil is currently under phase III trials for heart failure, whileresults from phase III EXPLORER-HCM trial were recently published indicating that Mavacamtenreduced left ventricular outflow tract (LVOT) obstruction and diastolic dysfunction and improvedthe health status of patients with HCM. A novel category of small molecules known as ‘Recouplers’was reported to target a phenomenon termed uncoupling commonly found in familialcardiomyopathies but has not progressed beyond pr
Tucholski T, Cai W, Gregorich ZR, et al., 2020, Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics, Proceedings of the National Academy of Sciences, Vol: 117, Pages: 24691-24700, ISSN: 0027-8424
Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry–based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.
Marston S, Jacques A, Bayliss C, et al., 2020, Donor hearts in the Sydney Heart Bank: reliable control but is it 'normal' heart?, Biophys Rev, Vol: 12, Pages: 799-803, ISSN: 1867-2450
Human heart samples from the Sydney Heart Bank have become a de facto standard against which others can be measured. Crucially, the heart bank contains a lot of donor heart material: for most researchers this is the hardest to obtain and yet is necessary since we can only study the pathological human heart in comparison with a control, preferably a normal heart sample. It is not generally realised how important the control is for human heart studies. We review our studies on donor heart samples. We report the results obtained with 17 different donor samples collected from 1994 to 2011 and measured from 2005 to 2015 by our standard methodology for in vitro motility and troponin I phosphorylation measurements. The donor heart sample parameters are consistent between the hearts, over time and with different operators indicating that Sydney Heart Bank donor hearts are a valid baseline control for comparison with pathological heart samples. We also discuss to what extent donor heart samples are representative of the normal heart.
Wright P, Tsui S, Francis A, et al., 2020, Approaches to high-throughput analysis of cardiomyocyte contractility, Frontiers in Physiology, Vol: 11, ISSN: 1664-042X
The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiologyand pathophysiology of the myocardium. However, the isolation of cardiomyocytes introducesvarious technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low throughput experimental studies, in which single cells are studied over the course of a pharmacologicalor physiologicalintervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-19HTS).Wecan assess the shortening of sarcomeres, cell length, Ca2+handling and cellular morphology of almost 4 cells perminute. Thisincrease in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficientnumbers of cells in each preparation can be assessed for multiple conditions reducingthese batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods thatcompensate for the hierarchical effects of clustering withinheart preparations anddemonstrate asignificant false positive rate which is potentially present in conventional studies. We demonstrate a more stringent way toperform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulnessof the CC-HTSin suchstudies. We specifically focus on the effects of agents thatdirectly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g. isolated muscl
Copeland O, Prasad S, Jabbour A, et al., 2020, Pressure overload is associated with low levels of troponin I and myosin binding protein C phosphorylation in the hearts of patients with aortic stenosis, Frontiers in Physiology, Vol: 11, ISSN: 1664-042X
In previous studies of septal heart muscle from HCM patients with hypertrophic obstructive cardiomyopathy (HOCM, LVOT gradient 50–120 mmHg) we found that the level of phosphorylation of troponin I (TnI) and myosin binding protein C (MyBP-C) was extremely low yet samples from hearts with HCM or DCM mutations that did not have pressure overload were similar to donor heart controls. We therefore investigated heart muscle samples taken from patients undergoing valve replacement for aortic stenosis, since they have pressure overload that is unrelated to inherited cardiomyopathy. Thirteen muscle samples from septum and from free wall were analyzed (LVOT gradients 30–100 mmHg) The levels of TnI and MyBP-C phosphorylation were determined in muscle myofibrils by separating phosphospecies using phosphate affinity SDS-PAGE and detecting with TnI and MyBP-C specific antibodies. TnI was predominantly monophosphorylated and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI. This phosphorylation level was significantly different (p < 0.0001) from both donor heart TnI (1.6 ± 0.06 molsPi/mol TnI) and HOCM heart TnI (0.19 ± 0.04 molsPi/mol TnI). MyBP-C is phosphorylated at up to four sites. In donor heart the 4P and 3P species predominate but in the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated. Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 8) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart. We conclude that pressure overload may be associated with substantial dephosphorylation of troponin I and MyBP-C.
Marston S, Zamora JE, 2020, Troponin structure and function: a view of recent progress, JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, Vol: 41, Pages: 71-89, ISSN: 0142-4319
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- Citations: 46
Marston S, 2019, Small molecule studies: the fourth wave of muscle research, Journal of Muscle Research and Cell Motility, Vol: 40, Pages: 69-76, ISSN: 0142-4319
The study of muscle and contractility is an unusual scientific endeavour since it has from the start been focussed on one problem-What makes muscle work?-and yet has needed a vast range of different approaches and techniques to study it. Its uniqueness lies in the fundamental fascination of a large scale molecular machine that converts chemical energy into mechanical energy at ambient temperature and with high efficiency that is also controlled by an exquisitely intricate yet utterly reliable regulatory system and is an essential component of animal life. The investigation of muscle is as innovative as any other field of research. As soon as one approach appears to be played out another comes along. It is instructive to consider this as a series of waves of novel and heightened activity starting in the 1950s. The thesis of this article is that we are approaching the fourth wave with the recent rise of interest in small molecules as research tools and possible therapies for muscle diseases.
Ehler E, Marston SB, 2019, The European Muscle Conference 2019 Special Issue, JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, Vol: 40, Pages: 67-67, ISSN: 0142-4319
Piroddi N, Witjas-Paalberends ER, Ferrara C, et al., 2019, The homozygous K280N troponin T mutation alters cross-bridge kinetics and energetics in human HCM, Journal of General Physiology, Vol: 151, Pages: 18-29, ISSN: 0022-1295
Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (kACT) and the rate constant of isometric relaxation (slow kREL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces kACT, slow kREL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases kACT, slow kREL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.
Smith JGW, Owen T, Bhagwan JR, et al., 2018, Isogenic pairs of hiPSC-CMs with hypertrophic cardiomyopathy/LVNC-associated ACTC1 E99K mutation unveil differential functional deficits, Stem Cell Reports, Vol: 11, Pages: 1226-1243, ISSN: 2213-6711
Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations.
Cai W, Hite ZL, Lyu B, et al., 2018, Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics, Journal of Molecular and Cellular Cardiology, Vol: 122, Pages: 11-22, ISSN: 0022-2828
Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4 °C or 22 °C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4 °C or 22 °C. Furthermore, significant protein degradation was also observed after incubation at 4 °C or 22 °C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples.
Marston SB, 2018, The molecular mechanisms of mutations in actin and myosin that cause inherited myopathy, International Journal of Molecular Sciences, Vol: 19, ISSN: 1661-6596
The discovery that mutations in myosin and actin genes, together with mutations in theother components of the muscle sarcomere, are responsible for a range of inherited muscle diseases(myopathies) has revolutionized the study of muscle, converting it from a subject of basic science to arelevant subject for clinical study and has been responsible for a great increase of interest in musclestudies. Myopathies are linked to mutations in five of the myosin heavy chain genes, three of themyosin light chain genes, and three of the actin genes. This review aims to determine to what extentwe can explain disease phenotype from the mutant genotype. To optimise our chances of finding theright mechanism we must study a myopathy where there are a large number of different mutationsthat cause a common phenotype and so are likely to have a common mechanism: a corollary tothis criterion is that if any mutation causes the disease phenotype but does not correspond to theproposed mechanism, then the whole mechanism is suspect. Using these criteria, we consider twocases where plausible genotype-phenotype mechanisms have been proposed: the actin “A-triad” andthe myosin “mesa/IHD” models.
Sheehan A, Messer A, Papadaki M, et al., 2018, Molecular defects in cardiac myofilament Ca2+- regulation due to cardiomyopathy-linked mutations can be reversed by small molecules binding to troponin, Frontiers in Physiology, Vol: 9, ISSN: 1664-042X
The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+regulation viatroponin. HCM is usually linked to higher myofilament Ca2+-sensitivitywhilst in both HCM and DCM mutant tissue there is oftenan uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heartto failure under stress. Atpresenttherearenocompoundsorinterventionsthatcanpreventortreatsarcomericcardiomyopathies.Thereisaneedfornoveltherapiesthatactatamorefundamentalleveltoaffectthediseaseprocess.Wedemonstratedthatepigallocatechin-3gallate(EGCG)wasfoundtobecapableofrestoringthecoupledrelationshipbetweenCa2+-sensitivityandTnIphosphorylationinmutantthinfilamentstonormalinvitro,independentofthemutation(15mutationstested).Wehavelabelledthisproperty“re-coupling”.TheactionofEGCGinvitrotoreversetheabnormalitycausedbymyopathicmutationswouldappeartobeanidealpharmaceuticalprofilefortreatmentofinheritedHCMandDCMbutEGCGisknowntobepromiscuousinvivoandisthusunsuitableastherapeuticdrug.Wethereforeinvestigatedwhetherotherstructurallyrelatedcompoundscanre-couplemyofilamentswithouttheseoff-targeteffects.We used the quantitative in vitromotility assay to screen 40 compounds,related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnIphosphorylation was restored to 2.2±0.01 –fold (n=19) compared to 2.0±.24 fold (n=7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitisers,indicating these properties are independent; moreover,re-
Rynkiewicz MJ, Prum T, Hollenberg S, et al., 2017, Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function., Biophysical Journal, Vol: 113, Pages: 2444-2451, ISSN: 0006-3495
Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.
Vikhorev, Smoktunowicz N, Munster A, et al., 2017, Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes., Scientific Reports, Vol: 7, ISSN: 2045-2322
Dilated cardiomyopathy (DCM) is an important cause of heart failure. Single gene mutations in at least 50 genes have been proposed to account for 25–50% of DCM cases and up to 25% of inherited DCM has been attributed to truncating mutations in the sarcomeric structural protein titin (TTNtv). Whilst the primary molecular mechanism of some DCM-associated mutations in the contractile apparatus has been studied in vitro and in transgenic mice, the contractile defect in human heart muscle has not been studied. In this study we isolated cardiac myofibrils from 3 TTNtv mutants, and 3 with contractile protein mutations (TNNI3 K36Q, TNNC1 G159D and MYH7 E1426K) and measured their contractility and passive stiffness in comparison with donor heart muscle as a control. We found that the three contractile protein mutations but not the TTNtv mutations had faster relaxation kinetics. Passive stiffness was reduced about 38% in all the DCM mutant samples. However, there was no change in maximum force or the titin N2BA/N2B isoform ratio and there was no titin haploinsufficiency. The decrease in myofibril passive stiffness was a common feature in all hearts with DCM-associated mutations and may be causative of DCM.
Rowlands C, Owen T, Lawal S, et al., 2017, Age and strain related aberrant Ca2+ release is associated with sudden cardiac death in the ACTC E99K mouse model of hypertrophic cardiomyopathy, American Journal of Physiology: Heart and Circulatory Physiology, Vol: 313, Pages: H1213-H1226, ISSN: 1522-1539
Patients with hypertrophic cardiomyopathy, particularly young adults, can die from arrhythmia, but the mechanism underlying abnormal rhythm formation remains unknown. C57Bl6 × CBA/Ca mice carrying a cardiac actin (ACTC) E99K (Glu99Lys) mutation reproduce many aspects of human hypertrophic cardiomyopathy, including increased myofilament Ca2+ sensitivity and sudden death in a proportion (up to 40%) of young (28−40 day old) animals. We studied the hearts of transgenic (TG; ACTC E99K) mice and their non-TG (NTG) littermates when they were in their vulnerable period (28–40 days old) and when they were adult (8–12 wk old). Ventricular myocytes were isolated from the hearts of TG and NTG mice at these two time points. We also examined the hearts of mice that died suddenly (SCD). SCD animals had approximately four times more collagen compared with age-matched NTG mice, yet myocyte cell size was normal. Young TG mice had double the collagen content of NTG mice. Contraction and Ca2+ transients were greater in cells from young TG mice compared with their NTG littermates but not in cells from adult mice (TG or NTG). Cells from young TG mice had a greater propensity for Ca2+ waves than NTG littermates, and, despite similar sarcoplasmic reticulum Ca2+ content, a proportion of these cells had larger Ca2+ spark mass. We found that the probability of SCD in young TG mice was increased when the mutation was expressed in animals with a CBA/Ca2+ background and almost eliminated in mice bred on a C57Bl6 background. The latter TG mice had normal cellular Ca2+ homeostasis.
Dos Remedios CG, Lal SP, Li A, et al., 2017, The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease., Biophys Rev, Vol: 9, Pages: 431-441, ISSN: 1867-2450
The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly reques
Messer AE, Chan WS, Daley A, et al., 2017, Investigations into the sarcomeric protein and Ca2+-regulation abnormalities underlying hypertrophic cardiomyopathy in cats (felix catus), Frontiers in Physiology, Vol: 8, ISSN: 1664-042X
Hypertrophic cardiomyopathy (HCM) is the most common single gene inherited cardiomyopathy. In cats (Felix catus) HCM is even more prevalent and affects 16% of the outbred population and up to 26% in pedigree breeds such as Maine Coon and Ragdoll. Homozygous MYBPC3 mutations have been identified in these breeds but the mutations in other cats are unknown. At the clinical and physiological level feline HCM is closely analogous to human HCM but little is known about the primary causative mechanism. Most identified HCM causing mutations are in the genes coding for proteins of the sarcomere. We therefore investigated contractile and regulatory proteins in left ventricular tissue from 25 cats, 18 diagnosed with HCM, including a Ragdoll cat with a homozygous MYBPC3 R820W, and 7 non-HCM cats in comparison with human HCM (from septal myectomy) and donor heart tissue. Myofibrillar protein expression was normal except that we observed 20–44% MyBP-C haploinsufficiency in 5 of the HCM cats. Troponin extracted from 8 HCM and 5 non-HCM cat hearts was incorporated into thin filaments and studied by in vitro motility assay. All HCM cat hearts had a higher (2.06 ± 0.13 fold) Ca2+-sensitivity than non-HCM cats and, in all the HCM cats, Ca2+-sensitivity was not modulated by troponin I phosphorylation. We were able to restore modulation of Ca2+-sensitivity by replacing troponin T with wild-type protein or by adding 100 μM Epigallocatechin 3-gallate (EGCG). These fundamental regulatory characteristics closely mimic those seen in human HCM indicating a common molecular mechanism that is independent of the causative mutation. Thus, the HCM cat is a potentially useful large animal model.
Marston S, 2017, Obscurin variants and inherited cardiomyopathies, Biophysical Reviews, Vol: 9, Pages: 239-243, ISSN: 1867-2450
The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and left ventricular non-compaction (LVNC), have been frequently associated with mutations in sarcomeric proteins. In recent years, advances in DNA sequencing technology has allowed the study of the giant proteins of the sarcomere, such as titin and nebulin. Obscurin has been somewhat neglected in these studies, largely because its functional role is far from clear, although there was an isolated report in 2007 of obscurin mutations associated with HCM. Recently, whole exome sequencing methodology (WES) has been used to address mutations in OBSCN, the gene for obscurin, and OBSCN variants were found to be relatively common in inherited cardiomyopathies. In different studies, 5 OBSCN unique variants have been found in a group of 30 end-stage failing hearts, 6 OBSCN unique variants in 74 HCM cases and 3 OBSCN unique variants in 10 LVNC patients. As yet, the number of known potentially disease-causing OBSCN variants is quite small. The reason for this is that mutations in the OBSCN gene have not been recognised as potentially disease-causing until recently, and were not included in large-scale genetic surveys. OBSCN mutations may be causative of HCM, DCM and LVNC and other cardiomyopathies, or they may work in concert with other variants in the same or other genes to initiate the pathology. Currently, the function of obscurin is not well understood, but we anticipate that many more OBSCN variants linked to cardiomyopathy will be found when the large cohorts of patient sequences available are tested. It is to be hoped that the establishment of the importance of obscurin in pathology will stimulate a thorough investigation of obscurin function.
Papakadi M, Marston SB, 2016, The Importance of Intrinsically Disordered Segments of Cardiac Troponin in Modulating Function by Phosphorylation and Disease-Causing Mutations, Frontiers in Physiology, Vol: 7, ISSN: 1664-042X
Troponin plays a central role in regulation of muscle contraction. It is the Ca2+ switch of striated muscles including the heart and in the cardiac muscle is physiologically modulated by PKA-dependent phosphorylation at Ser22 and 23. Many cardiomyopathy-related mutations affect Ca2+ regulation and/or disrupt the relationship between Ca2+ binding and phosphorylation. Unlike the mechanism of heart activation, the modulation of Ca2+-sensitivity by phosphorylation of the cardiac specific N-terminal segment of TnI (1-30) is structurally subtle and has proven hard to investigate. The crystal structure of cardiac troponin describes only the relatively stable core of the molecule and the crucial mobile parts of the molecule are missing including TnI C terminal region, TnI (1-30), TnI (134-149) (‘inhibitory’ peptide) and the C-terminal 28 amino acids of TnT that are intrinsically disordered.Recent studies over the years have been performed to answer this matter by building structural models of cardiac troponin in phosphorylated and dephosphorylated states based on peptide NMR studies. Now these have been updated by more recent concepts derived from molecular dynamic simulations treating troponin as a dynamic structure. The emerging model confirms the stable core structure of troponin and the mobile structure of the intrinsically disordered segments. We will discuss how we can describe these segments in terms of dynamic transitions between a small number of states with the probability distributions being altered by phosphorylation and by HCM or DCM-related mutations that can explain how Ca2+-sensitivity is modulated by phosphorylation and the effects of mutations.
Marston SB, 2016, WHY IS THERE A LIMIT TO THE CHANGES IN MYOFILAMENT Ca2+-SENSITIVITY ASSOCIATED WITH MYOPATHY CAUSING MUTATIONS?, Frontiers in Physiology, Vol: 7, ISSN: 1664-042X
Mutations in striated muscle contractile proteins have been found to be the cause ofa number of inherited muscle diseases; in most cases the mechanism proposed forcausing the disease is derangement of the thin filament-based Ca2+-regulatory systemofthe muscle. When considering the results of experiments reported over the last 15 years,one feature has been frequently noted, but rarely discussed: the magnitude of changesin myofilament Ca2+-sensitivity due to myopathy-causing mutations in skeletal or heartmuscle seems to be always in the range 1.5–3x EC50. Such consistency suggests itmay be related to a fundamental property of muscle regulation; in this article we willinvestigate whether this observation is true and consider why this should be so. Aliterature search found 71 independentmeasurements of HCMmutation-induced changeof EC50 ranging from 1.15 to 3.8-fold with a mean of 1.87 ± 0.07 (sem). We also found11 independent measurements of increased Ca2+-sensitivity due to mutations in skeletalmuscle proteins ranging from 1.19 to 2.7-fold with a mean of 2.00 ± 0.16. Investigationof dilated cardiomyopathy-related mutations found 42 independent determinations showa range of EC50 wt/mutant from 0.3 to 2.3. In addition we found 14 measurements ofCa2+-sensitivity changes due skeletal muscle myopathy mutations ranging from 0.39 to0.63. Thus, our extensive literature search, although not necessarily complete, found that,indeed, the changes in myofilament Ca2+-sensitivity due to disease-causing mutationshave a bimodal distribution and that the overall changes in Ca2+-sensitivity are quitesmall and do not extend beyond a three-fold increase or decrease in Ca2+-sensitivity.We discuss two mechanism that are not necessarily mutually exclusive. Firstly, it couldbe that the limit is set by the capabilities of the Excitation-contraction machinery thatsupplies activating Ca2+ and that striated muscle cannot work in a way compatible withlife outside these limits; o
Zamora JE, Papadaki M, Messer AE, et al., 2016, Troponin structure: its modulation by Ca(2+) and phosphorylation studied by molecular dynamics simulations., Phys Chem Chem Phys, Vol: 18, Pages: 20691-20707
The only available crystal structure of the human cardiac troponin molecule (cTn) in the Ca(2+) activated state does not include crucial segments, including the N-terminus of the cTn inhibitory subunit (cTnI). We have applied all-atom molecular dynamics (MD) simulations to study the structure and dynamics of cTn, both in the unphosphorylated and bis-phosphorylated states at Ser23/Ser24 of cTnI. We performed multiple microsecond MD simulations of wild type (WT) cTn (6, 5 μs) and bisphosphorylated (SP23/SP24) cTn (9 μs) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-171) and cTnT (212-298), including residues not present in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25 Å are needed to sample the interesting conformational shifts both in the native and bis-phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies, cTnC-cTnI interactions that are responsible for the cTn dynamics are altered. We have also shown that phosphorylation does not increase cTn fluctuations, and its effects on the protein-protein interaction profiles cannot be assessed in a significant way. Finally, we propose that phosphorylation could provoke a loss of Ca(2+) by stabilizing out-of-coordination distances of the cTnC's EF hand II residues, and in particular Ser 69.
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