187 results found
Vikhorev P, Tucholski T, Cai W, et al., Differential Regulation of Post-translational Modifications in Human Hypertrophic Cardiomyopathy Revealed by Top-down Proteomics, US HUPO 2020 - 16th Annual conference
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
Marston S, Zamora JE, 2019, Troponin structure and function: a view of recent progress., J Muscle Res Cell Motil
The molecular mechanism by which Ca2+ binding and phosphorylation regulate muscle contraction through Troponin is not yet fully understood. Revealing the differences between the relaxed and active structure of cTn, as well as the conformational changes that follow phosphorylation has remained a challenge for structural biologists over the years. Here we review the current understanding of how Ca2+, phosphorylation and disease-causing mutations affect the structure and dynamics of troponin to regulate the thin filament based on electron microscopy, X-ray diffraction, NMR and molecular dynamics methodologies.
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.
Vikhorev P, Yeung W, Li A, et al., Contractility of myofibrils from patients with dilated cardiomyopathy associated mutations, Alpbach Meeting on Muscle Molecular Motors
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.
Abou Al Saud S, 2016, Investigation of Phenotypic Rescue of Mybpc3 Deficient Mouse
Mutations in the myosin binding protein C gene (MYBPC3) are a frequent cause of hypertrophic cardiomyopathy (HCM) and calcineurin plays a major role in hypertrophic remodelling. However, a functional link between MYBPC3 mutations and calcineurin has not been investigated. Mybpc3 knock out (KO) mice were generated and an increase in the regulator of calcineurin 1 (Rcan1) mRNA expression was detected, indicating an increase in calcineurin activity (Knöll et al. unpublished data). Accordingly, it was hypothesised that calcineurin, particularly its major β-isoform (CnAβ), plays a role in the pathogenesis of these mice. Therefore we investigated Mybpc3/CnAβ double KO (dKO) mice. Our results confirmed that the severe heart failure (HF) phenotype observed in Mybpc3 KO mice is completely rescued by additional ablation of CnAβ as judged by echocardiography, gravimetric analysis, histology and electron microscopy studies. We also have measured muscle contractility in skinned cardiac trabeculae from dKO mice and demonstrated that the rescue was present at the level of the contractile apparatus. Moreover, this rescue was specific to Mybpc3 KO mice as the phenotype of a mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K actin was more severe in ACTC E99K/ CnAβ double transgenic (dTG) mice.Crucially, it was found that ventricular myosin light chain (MLC2v) was hyperphosphorylated in the Mybpc3/CnAβ dKO mice. Furthermore, calcineurin was shown to dephosphorylate MLC2v in vitro. We therefore investigated whether MLC2v hyperphosphorylation per se could rescue the Mybpc3 KO phenotype. Mybpc3 KO mice were injected with AAV9 overexpressing pseudophosphorylated MLC2v (S14/15D; AAV9-pMLC2v), cardiomyocytes were transfected with adv-pMLC2v, and a construct to create a TG mice overexpressing pMLC2v was made.The AAV9-pMLC2v injected mice were studied in detail. A maximal improvement in cardiac function was observed
Toepfer CN, Sikkel MB, Caorsi V, et al., 2016, A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation., American Journal of Physiology - Heart and Circulatory Physiology, Vol: 311, Pages: H465-H475, ISSN: 0363-6135
Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic 'compensation' and congestive 'decompensation'. Nothing is known about the ability of un-infarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drive progression of compensation. We hypothesized that enhanced crossbridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared to non-infarcted controls. Isometric force during submaximal activations was raised >2.4-fold, whilst power was 2-fold greater. EM and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein, and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.
Chan C, Fan J, Messer AE, et al., 2016, Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function, Biochimica et Biophysica Acta - Molecular Basis of Disease, Vol: 1862, Pages: 1453-1458, ISSN: 0006-3002
In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.
Messer A, Bayliss C, El-Mezgueldi M, et al., 2016, Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity by troponin I phosphorylation, Archives of Biochemistry and Biophysics, Vol: 601, Pages: 113-120, ISSN: 1096-0384
We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thinfilaments reconstituted with actin and human cardiac tropomyosin. Weused the quantitative in vitro motility assay to study Ca2+-regulation ofunloaded movement and its modulation by troponin I phosphorylation.Troponin from a patient with the K280N TnT mutation showed nodifference in Ca2+-sensitivity when compared with donor heart troponinand the Ca2+-sensitivity was also independent of the troponin Iphosphorylation level (uncoupled). The recombinant K280N TnT mutationincreased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnTmutation in troponin from transgenic mouse increased Ca2+-sensitivity andwas also completely uncoupled. Five TnT mutations (∆14, ∆28+7, ∆E160,S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutationsin TnT, Ca2+-sensitisation together with uncoupling in vitro is the usualresponse and both factors may contribute to the HCM phenotype. We alsofound that Epigallocatechin-3-gallate (EGCG) can restore coupling to alluncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both theabnormalities found in troponin with a TnT HCM mutation suggesting itmay have therapeutic potential.
Vikhorev P, Marston S, THE EFFECT OF DCM-ASSOCIATED MUTATIONS IN TITIN ON HUMAN CARDIAC MYOFIBRIL ELASTICITY AND CONTRACTILITY., 15th Alpbach Motors Workshop. Myosin & Muscles, and other Motors., Pages: 115-115
Vikhorev P, Marston S, Ferenczi M, 2016, Instrumentation to Study Myofibril Mechanics from Static to Artificial Simulations of Cardiac Cycle, MethodsX, Vol: 3, Pages: 156-170, ISSN: 2215-0161
Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable myofibrils can be obtained from frozen tissue.We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single myofibrils. The control of myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently.This single myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.
Marston S, Messer A, Papadaki M, 2016, (De-)sensitisation versus uncoupling: what drives cardiomyopathies in the thin filament? The known unknowns., Cardiovascular Research, Vol: 109, ISSN: 1755-3245
Wilkinson R, Song W, Smoktunowicz N, et al., 2015, A DILATED CARDIOMYOPATHY MUTATION BLUNTS ADRENERGIC RESPONSE AND INDUCES CONTRACTILE DYSFUNCTION UNDER CHRONIC ANGIOTENSIN II STRESS, American Journal of Physiology-Heart and Circulatory Physiology, Vol: 309, Pages: H1936-H1946, ISSN: 1522-1539
We investigated cardiac contractility in the ACTC 361G transgenic mouse model of dilated cardiomyopathy (DCM). No differences in cardiac dimensions or systolic function were observed in young mice whilst young adult mice exhibited only mild diastolic abnormalities. Dobutamine had an inotropic and lusitropic effect on nontransgenic (NTG) mouse heart. At 37°C the effects of dobutamine were most evident at higher frequencies (doubling of (dFmin/dt/)F, t90 halved at 10 Hz ) but in the ACTC E361G mouse (dFmin/dt/)F was not altered and t90 was only reduced 25%. Pressure-volume measurements showed increases in dP/dt and decreases in tau in ACTC E361G mouse that were 1/4-1/3 of the changes in NTG mouse consistent with blunting of the lusitropic response the inotropic effect of dobutamine was also blunted in ACTC E361G mice and the dobutamine-stimulated increase in cardiac output was reduced from 2100 µl/min to 900 µl/min. Mice were treated with high doses of Angiotensin II for 4 weeks. The chronic stress treatment evoked symptoms of systolic heart failure in ACTC E361G mice but not in NTG. There was a significant reduction in rates of pressure increase and decrease as well as reduced end-systolic pressure and increased volume. Ejection fraction and cardiac output were reduced in the ACTC E361G mouse, indicating dilated cardiomyopathy. In vitro DCM-causing mutations uncouple the relationship between Ca2+-sensitivity and troponin I phosphorylation. We conclude that this leads to a reduced response to β1 agonists and reduced cardiac reserve that predisposes the heart to DCM under conditions of chronic stress.
Donkervoort S, Papadaki M, de Winter JM, et al., 2015, TPM3 deletions cause a hypercontractile congenital muscle stiffness phenotype., Annals of Neurology, Vol: 78, Pages: 982-994, ISSN: 1531-8249
OBJECTIVE: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one case. METHODS: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured. We used quantitative in vitro motility assay (IVMA) to measure Ca(2+) -sensitivity of thin filaments reconstituted with recombinant Tpm3.12 ΔE218 and ΔE224. RESULTS: Contractility studies on permeabilized myofibers demonstrated reduced maximal active tension from both patients with increased Ca(2+) sensitivity with altered cross-bridge cycling kinetics in ΔE224 fibers. In vitro motility studies showed a two-fold increase in Ca(2+) -sensitivity of the fraction of filaments motile and the filament sliding velocity concentrations for both mutations. INTERPRETATION: This data indicates that Tpm3.12 deletions ΔE218 and ΔE224 result in increased Ca(2+) sensitivity of the troponin-tropomyosin complex, resulting in abnormally active interaction of actin and myosin complex. Both mutations are located in the charged motifs of the actin-binding residues of tropomyosin 3, thus disrupting the electrostatic interactions that facilitate accurate tropomyosin binding with actin necessary to prevent the on-state. The mutations destabilize the off-state and result in excessively sensitized excitation-contraction coupling of the contractile apparatus. This work expands the phenotypic spectrum of TPM3-related disease and provides insights into the pathophysiological mechanisms of the actin-tropomyosin complex. This article is protected by copyright. All rights reserved.
Marston SB, Montgiraud C, Munster AB, et al., 2015, OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency, PLOS One, Vol: 10, ISSN: 1932-6203
BackgroundStudies of the functional consequences of DCM-causing mutations have been limited to afew cases where patients with known mutations had heart transplants. To increase the numberof potential tissue samples for direct investigation we performed whole exon sequencingof explanted heart muscle samples from 30 patients that had a diagnosis of familial dilatedcardiomyopathy and screened for potentially disease-causing mutations in 58 HCM orDCM-related genes.ResultsWe identified 5 potentially disease-causing OBSCN mutations in 4 samples; one samplehad two OBSCN mutations and one mutation was judged to be not disease-related. Alsoidentified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one eachin TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level ofobscurin mRNA was significantly greater and more variable in healthy donor samples thanthe DCM samples but did not correlate with OBSCN mutations. A single obscurin proteinband was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The threesamples with OBSCN mutations had significantly lower levels of obscurin immunoreactivematerial than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of controllevel).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.ConclusionsOBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency.Mutations in the obscurin gene should be considered as a significant causal factorof DCM, alone or in concert with other mutations.
Yuen M, Cooper ST, Marston SB, et al., 2015, Muscle weakness in TPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres., Human Molecular Genetics, Vol: 24, Pages: 6278-6292, ISSN: 1460-2083
Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterised by generalised muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in twelve TPM3-myopathy patients.We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin.Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres.Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitising drugs may represent a useful treatment for this condition.
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