Abstract 402: Defining Diverse Disease Pathomechanisms Across Thick And Thin Filament Hypertrophic Cardiomyopathy Variants.

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Christopher Toepfer ◽  
Yiangos Psaras ◽  
Francesca Margara ◽  
Manuel Schmid ◽  
Violetta Steeples ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Thin Filament ◽  
Experimental Studies ◽  
Induced Pluripotent Stem Cell ◽  
Thick Filament ◽  
Cellular Level ◽  
Atpase Inhibitor ◽  
Atp Assay ◽  
Causal Mechanisms ◽  
Parametric Data

Hypertrophic cardiomyopathy (HCM) affects as many as ~1 in 500 individuals, and is often typified by hyperdynamic contraction and poor cellular relaxation. HCM can be caused by mutations in a variety of key contractile proteins of the sarcomere. A large proportion of these variants are found in MYBPC3, MYH7, TNNT2, and TNNI3. These genes encode proteins that control cardiac muscle contraction at the thick (MYBPC3 and MYH7) and thin filaments (TNNT2 and TNNI3) of the sarcomere. In this study we use human induced pluripotent stem cell derived cardiomyocytes to model HCM across all of these genes. We do this to define key mechanistic differences between thick and thin filament HCM. We define sarcomeric contractility (SarcTrack) calcium transients (CalTrack) and myosin states using the mant-ATP assay. We use the parametric data from these experimental studies in iPSC-CMs to model possible disease mechanisms in silico. Our experimental analysis highlights that both thick and thin filament HCM variants cause cellular hypercontractility, with slowed cellular relaxation. We find that thick filament HCM variants drive cellular HCM phenotypes by destabilising the myosin interacting heads motif (IHM), showing a marked reduction in the super relaxed state of myosin. Counterintuitively thin filament based HCM variants show a reduction in DRX myosin. When applying Mavacamten the allosteric myosin ATPase inhibitor to our thin and thick filament HCM variant iPSC-CMs we find a dichotomy of cellular responses. The thick filament variants studied all show a clear resolution of cellular HCM. However, not all cellular phenotypes of thin filament HCM are corrected by Mavacamten treatment, although there is benefit. We conclude that causal mechanisms of thick filament HCM are well corrected at the molecular and cellular level by Mavacamten, but these causal mechanisms in thin filament based HCM are not suitably corrected. We highlight key mechanistic pharmacological targets for thin filament variants that could add cellular benefit to HCM phenotype resolution.

scholarly journals Mavacamten rescues increased myofilament calcium sensitivity and dysregulation of Ca2+ flux caused by thin filament hypertrophic cardiomyopathy mutations

2020 ◽  
Vol 318 (3) ◽  
pp. H715-H722 ◽  
Author(s):  
Alexander J. Sparrow ◽  
Hugh Watkins ◽  
Matthew J. Daniels ◽  
Charles Redwood ◽  
Paul Robinson
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Thick Filament ◽  
Calcium Sensitivity ◽  
Calcium Handling ◽  
Cellular Models

Thin filament hypertrophic cardiomyopathy (HCM) mutations increase myofilament Ca2+ sensitivity and alter Ca2+ handling and buffering. The myosin inhibitor mavacamten reverses the increased contractility caused by HCM thick filament mutations, and we here test its effect on HCM thin filament mutations where the mode of action is not known. Mavacamten (250 nM) partially reversed the increased Ca2+ sensitivity caused by HCM mutations Cardiac troponin T (cTnT) R92Q, and cardiac troponin I (cTnI) R145G in in vitro ATPase assays. The effect of mavacamten was also analyzed in cardiomyocyte models of cTnT R92Q and cTnI R145G containing cytoplasmic and myofilament specific Ca2+ sensors. While mavacamten rescued the hypercontracted basal sarcomere length, the reduced fractional shortening did not improve with mavacamten. Both mutations caused an increase in peak systolic Ca2+ detected at the myofilament, and this was completely rescued by 250 nM mavacamten. Systolic Ca2+ detected by the cytoplasmic sensor was also reduced by mavacamten treatment, although only R145G increased cytoplasmic Ca2+. There was also a reversal of Ca2+ decay time prolongation caused by both mutations at the myofilament but not in the cytoplasm. We thus show that mavacamten reverses some of the Ca2+-sensitive molecular and cellular changes caused by the HCM mutations, particularly altered Ca2+ flux at the myofilament. The reduction of peak systolic Ca2+ as a consequence of mavacamten treatment represents a novel mechanism by which the compound is able to reduce contractility, working synergistically with its direct effect on the myosin motor. NEW & NOTEWORTHY Mavacamten, a myosin inhibitor, is currently in phase-3 clinical trials as a pharmacotherapy for hypertrophic cardiomyopathy (HCM). Its efficacy in HCM caused by mutations in thin filament proteins is not known. We show in reductionist and cellular models that mavacamten can rescue the effects of thin filament mutations on calcium sensitivity and calcium handling although it only partially rescues the contractile cellular phenotype and, in some cases, exacerbates the effect of the mutation.

scholarly journals A contraction stress model of hypertrophic cardiomyopathy due to thick filament sarcomere mutations

2018 ◽  
Author(s):  
Rachel Cohn ◽  
Ketan Thakar ◽  
Andre Lowe ◽  
Feria Ladha ◽  
Anthony M. Pettinato ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Induced Pluripotent Stem Cell ◽  
Thick Filament ◽  
3 Dimensional ◽  
Genetic Ablation ◽  
Expression Studies ◽  
Contraction Stress ◽  
P53 Activation ◽  
Induced Pluripotent

Thick filament sarcomere mutations are the most common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered an isogenic panel of four human HCM induced pluripotent stem cell (iPSc) models using CRISPR/Cas9, and studied iPSc-derived cardiomyocytes (iCMs) in 3-dimensional cardiac microtissue (CMT) assays that resemble in vivo cardiac architecture and biomechanics. HCM mutations result in hypercontractility in association with prolonged relaxation kinetics in proportion to mutation pathogenicity but not calcium dysregulation. RNA sequencing and protein expression studies identified that HCM mutations result in p53 activation secondary to increased oxidative stress, which results in increased cytotoxicity that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as an early consequence of thick filament mutations, and the p53 pathway as a molecular marker and candidate therapeutic target for thick filament HCM.

Abstract 4845: Hypertrophic Cardiomyopathy due to Sarcomere Gene Mutations is Associated with More Severe Derangement of Microvascular Function Compared to Myofilament Negative Disease

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Iacopo Olivotto ◽  
Francesca Girolami ◽  
Michael J Ackerman ◽  
Roberto Sciagra ◽  
Stefano Nistri ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Hypertrophic Cardiomyopathy ◽  
Thin Filament ◽  
Troponin I ◽  
Genetic Test ◽  
Troponin T ◽  
Gene Mutations ◽  
Thick Filament ◽  
Microvascular Function ◽  
Coronary Microvascular Dysfunction

Coronary microvascular dysfunction (CMD) is an important primary feature of hypertrophic cardiomyopathy (HCM), contributing to myocardial ischemia and ventricular remodelling, and is predictive of adverse outcome. Whether there is any association between presence/severity of CMD and HCM’s pathogenic substrate remains to be determined. To address this issue, we used positron emission tomography (PET) to assess CMD in an HCM cohort that received comprehensive genetic testing for sarcomeric/myofilament-HCM. We measured maximum (intravenous dipyridamole, 0.56 mg/kg) myocardial blood flow (Dip-MBF), using 13 N-labeled ammonia and PET in 46 HCM patients (age 38±14 years, 32 male). Genetic testing was performed by denaturing high performance liquid chromatography and automatic DNA sequencing of nine myofilament-encoding genes including both thick filament proteins (myosin binding protein C, beta-myosin heavy chain, regulatory and essential light chains); and thin filament proteins (troponin-T, troponin-I, troponin-C, alpha-tropomyosin and alpha-actin). Results . Thirty-four mutations were identified in 30/46 patients (myofilament-HCM; 65%), including 29 with thick filament and 6 with thin filament mutations, as well as 4 with complex genotype. Despite similar age and clinical features, patients with myofilament-HCM showed lower Dip-MBF values than the patients with a negative genetic test (1.6±0.7 versus 2.2±0.9 ml/min/g, respectively; p=0.03). Specifically, 13/30 (43%) patients with myofilament-HCM had a Dip-MBF below the lower tertile for the study group (≤1.38 ml/min/g), compared to 2/16 (12%) patients with a negative genetic test (p=0.034). No difference in Dip-MBF was evident with respect to the particular sarcomeric gene involved (ANOVA p=0.63). HCM due to sarcomere gene mutations is characterized by more severe impairment in microvascular function compared to myofilament negative disease, irrespective of the involved contractile protein. These findings suggest that sarcomeric mutations might be implicated in adverse remodelling of the microcirculation in patients with HCM, and account for the greater prevalence of ventricular dysfunction and failure in this subset.

Comparison of the Development to End-Stage Phase in Hypertrophic Cardiomyopathy With Thick Filament Mutations and Thin Filament Mutations

2009 ◽  
Vol 15 (7) ◽  
pp. S174
Author(s):  
Noboru Fujino ◽  
Hidekazu Ino ◽  
Eiichi Masuta ◽  
Akira Funada ◽  
Akihiko Muramoto ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Thin Filament ◽  
Thick Filament ◽  
End Stage

scholarly journals Advancing Drug Discovery for Neurological Disorders Using iPSC-Derived Neural Organoids

2021 ◽  
Vol 22 (5) ◽  
pp. 2659
Author(s):  
Gianluca Costamagna ◽  
Giacomo Pietro Comi ◽  
Stefania Corti
Keyword(s):  
Drug Discovery ◽  
Drug Screening ◽  
Neural Development ◽  
Neurological Disorders ◽  
Large Scale ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Cellular Level ◽  
Complex Data ◽  
Complex Data Sets

In the last decade, different research groups in the academic setting have developed induced pluripotent stem cell-based protocols to generate three-dimensional, multicellular, neural organoids. Their use to model brain biology, early neural development, and human diseases has provided new insights into the pathophysiology of neuropsychiatric and neurological disorders, including microcephaly, autism, Parkinson’s disease, and Alzheimer’s disease. However, the adoption of organoid technology for large-scale drug screening in the industry has been hampered by challenges with reproducibility, scalability, and translatability to human disease. Potential technical solutions to expand their use in drug discovery pipelines include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create isogenic models, single-cell RNA sequencing to characterize the model at a cellular level, and machine learning to analyze complex data sets. In addition, high-content imaging, automated liquid handling, and standardized assays represent other valuable tools toward this goal. Though several open issues still hamper the full implementation of the organoid technology outside academia, rapid progress in this field will help to prompt its translation toward large-scale drug screening for neurological disorders.

scholarly journals Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

2008 ◽  
Vol 41 (5) ◽  
pp. 816-823 ◽  
Author(s):  
R. Adachi ◽  
T. Takaya ◽  
K. Kuriyama ◽  
A. Higashibata ◽  
N. Ishioka ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Thin Filament ◽  
Thick Filament ◽  
Thin Filament Proteins

scholarly journals Cardiomyopathy: Getting Bigger All the Time - Lessons Learned about Heart Disease from Tropomyosin

2021 ◽  
Author(s):  
David F. Wieczorek
Keyword(s):  
Heart Disease ◽  
Hypertrophic Cardiomyopathy ◽  
Genetic Association ◽  
Basic Science ◽  
Thin Filament ◽  
Contractile Proteins ◽  
Lessons Learned ◽  
Genetic Mutations ◽  
Filament Protein ◽  
Science Investigations

In 1990, John and Christine Seidman uncovered the genetic association between mutations in sarcomeric contractile proteins and hypertrophic cardiomyopathy. Since then, the increase in knowledge and understanding of this disease has increased exponentially. Although pathologies associated with the various cardiomyopathies are vastly different, in some cases, the same proteins are causative, but with different genetic mutations. The focus of this article will be on hypertrophic and dilated cardiomyopathies, which are often caused by mutations in sarcomeric contractile proteins. Tropomyosin, a thin filament protein, serves as a paradigm to illustrate how different mutations within the same protein can generate the hypertrophic or dilated cardiomyopathic condition. As such, the significant advances in information derived from basic science investigations has led to the development of novel therapeutics in the treatment of these pathological diseases. This article will illustrate linkages which occur to bridge scientific advances to clinical treatments in cardiomyopathic patients.

Abstract 16380: Disease Specific Induced Pluripotent Stem Cell Derived Cardiomyocytes Represent Pathophysiological Phenotype in Hypertrophic Cardiomyopathy

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Maki Takeda ◽  
Shigeru Miyagawa ◽  
Takuji Kawamura ◽  
Emiko Ito ◽  
Akima Harada ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Induced Pluripotent Stem Cell ◽  
Digital Pcr ◽  
Hereditary Diseases ◽  
Homozygous Mutation ◽  
True Nature ◽  
Established Disease ◽  
Induced Pluripotent ◽  
Cell Motion ◽  
Disease Specific

Although hypertrophic cardiomyopathy (HCM) is a major hereditary heart disease, unknown underlying basic pathology make it difficult for physicians to heal this disease. We hypothesized that the disease specific iPS derived cardiomyocytes from HCM with the myosin binding protein C (MYBPC3) mutation represent the pathophysiological phenotype, and editing the mutation would cancel the diseased phenotype to check the role in the mutation gene. We established iPS cells with heterozygous frameshift mutation (WT/MT-iPS) from patient and performed genome editing to generate cells with repair of mutation (WT/WT-iPS) and homozygous mutation (MT/MT-iPS) using CRISPER-Cas9. Droplet digital PCR revealed that the transcript of the mutant allele was about 30% in WT/MT-iPS derived cardiomyocytes (CMs) compared to the WT allele and also WT/WT-iPSCMs and MT/MT-iPSCMs expressed only the transcript of WT allele and MT allele, respectively. The RNA expression of MYBPC3 was restored in WT/WT-iPSCMs and decreased in MT/MT-iPSCMs assessed by quantitative real-time PCR. Sarcomeric disarray was observed in WT/MT-iPSCMs, and was more prominently expressed in MT/MT-iPSCMs assessed by immunofluorescence staining. Cell motion analyzer revealed that relaxation velocity was significantly decreased and relaxation duration was significantly increased in WT/MT-iPSCMs and MT/MT-iPSCMs compared to WT/WT-iPSCMs. Moreover significant increase in contraction end velocity was observed in MT/MT-iPSCMs compared to WT/MT and WT/WT-iPSCMs. Established disease specific iPS-CMs with MYBPC3 mutation reproduces pathophysiology in HCM, providing important clues that elucidate true nature in patient’s hereditary diseases.

Hypertrophic cardiomyopathy: genetics

ESC CardioMed ◽  
2018 ◽  
pp. 1443-1450
Author(s):  
Mohammed Majid Akhtar ◽  
Luis Rocha Lopes
Keyword(s):  
Genetic Testing ◽  
Hypertrophic Cardiomyopathy ◽  
Mapk Pathway ◽  
Glycogen Storage ◽  
Cellular Level ◽  
Causative Mutation ◽  
Clinical Role ◽  
Genes Encoding ◽  
Associated Proteins

Hypertrophic cardiomyopathy is most commonly transmitted as an autosomal dominant trait, caused by mutations in genes encoding cardiac sarcomere and associated proteins. Knowledge of the genetic pathophysiology of the disease has advanced significantly since the initial identification of a point mutation in the beta-myosin heavy chain (MYH7) gene in 1990. Other genetic causes of the disease include mutations in genes coding for proteins implicated in calcium handling or which form part of the cytoskeleton. The recent emergence of next-generation sequencing allows quicker and less expensive identification of causative mutations. However, a causative mutation is not identified in up to 50% of probands. At present, the primary clinical role of genetic testing in hypertrophic cardiomyopathy is in the context of familial screening, allowing the identification of those at risk of developing the condition. Genetic testing can also be used to exclude genocopies, particularly in the presence of certain diagnostic ‘red flag’ features, where lysosomal, glycogen storage, neuromuscular or Ras-MAPK pathway disorders may be suspected. The role of individual mutations in predicting prognosis is limited at present. However, the higher incidence of sudden cardiac death in the presence of a family history of such, suggests that genetics play a significant role in determining outcome. With an increased understanding of the impact of these mutations on a cellular level and on longer-term clinical outcomes, the aim in future for gene and mutation specific prognosis or potential disease-modifying therapy is closer.

scholarly journals INTRINSIC BIREFRINGENCE OF GLYCERINATED MYOFIBRILS

1971 ◽  
Vol 51 (3) ◽  
pp. 763-771 ◽  
Author(s):  
Richard H. Colby
Keyword(s):  
Thin Filament ◽  
Striated Muscle ◽  
Thick Filament ◽  
Weight Basis ◽  
Macromolecular Structure ◽  
Form Birefringence ◽  
Sliding Filament Theory ◽  
Filament Protein ◽  
Formalin Fixed ◽  
Intrinsic Birefringence

Patterns of intrinsic birefringence were revealed in formalin-fixed, glycerinated myofibrils from rabbit striated muscle, by perfusing them with solvents of refractive index near to that of protein, about 1.570. The patterns differ substantially from those obtained in physiological salt solutions, due to the elimination of edge- and form birefringence. Analysis of myofibrils at various stages of shortening has produced results fully consistent with the sliding filament theory of contraction. On a weight basis, the intrinsic birefringence of thick-filament protein is about 2.4 times that of thin-filament protein. Nonadditivity of thick- and thin-filament birefringence in the overlap regions of A bands may indicate an alteration of macromolecular structure due to interaction between the two types of filaments.

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