Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Saiti S Halder ◽  
Lorenzo R Sewanan ◽  
Michael J Rynkiewicz ◽  
Jeffrey R Moore ◽  
William J Lehman ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Calcium Sensitivity ◽  
Heart Tissue ◽  
Missense Mutations ◽  
Wild Type ◽  
Twitch Force ◽  
In Vitro Motility ◽  
Isometric Twitch ◽  
Engineered Heart Tissues

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

scholarly journals A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

2021 ◽  
pp. mbc.E21-02-0082
Author(s):  
Samantha K. Barrick ◽  
Lina Greenberg ◽  
Michael J. Greenberg
Keyword(s):  
Dilated Cardiomyopathy ◽  
Calcium Binding ◽  
Thin Filament ◽  
Troponin T ◽  
Calcium Sensitivity ◽  
Cellular Level ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Fluorescence Measurements

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.

scholarly journals Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

2020 ◽  
Author(s):  
Robyn D. Moir ◽  
Christian Lavados ◽  
JaeHoon Lee ◽  
Ian M. Willis
Keyword(s):  
Functional Characterization ◽  
Rna Polymerase Iii ◽  
State Level ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Wild Type ◽  
Functional Deficits ◽  
Pol Iii ◽  
Transcriptional Output

AbstractMutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast to Pol III leukodystrophy mutations in RPC160 can be overcome by a combinatorial strategy.

scholarly journals Metabolomics hallmarks OPA1 variants correlating with their in vitro phenotype and predicting clinical severity

2020 ◽  
Vol 29 (8) ◽  
pp. 1319-1329
Author(s):  
Juan Manuel Chao de la Barca ◽  
Mario Fogazza ◽  
Michela Rugolo ◽  
Stéphanie Chupin ◽  
Valentina Del Dotto ◽  
...  
Keyword(s):  
Metabolic Diseases ◽  
Methodological Approach ◽  
Clinical Severity ◽  
Resolving Power ◽  
Missense Mutations ◽  
Wild Type ◽  
Targeted Metabolomics ◽  
Embryonic Fibroblasts ◽  
Uncertain Significance

Abstract Interpretation of variants of uncertain significance is an actual major challenge. We addressed this question on a set of OPA1 missense variants responsible for variable severity of neurological impairments. We used targeted metabolomics to explore the different signatures of OPA1 variants expressed in Opa1 deleted mouse embryonic fibroblasts (Opa1−/− MEFs), grown under selective conditions. Multivariate analyses of data discriminated Opa1+/+ from Opa1−/− MEFs metabolic signatures and classified OPA1 variants according to their in vitro severity. Indeed, the mild p.I382M hypomorphic variant was segregating close to the wild-type allele, while the most severe p.R445H variant was close to Opa1−/− MEFs, and the p.D603H and p.G439V alleles, responsible for isolated and syndromic presentations, respectively, were intermediary between the p.I382M and the p.R445H variants. The most discriminant metabolic features were hydroxyproline, the spermine/spermidine ratio, amino acid pool and several phospholipids, emphasizing proteostasis, endoplasmic reticulum (ER) stress and phospholipid remodeling as the main mechanisms ranking OPA1 allele impacts on metabolism. These results demonstrate the high resolving power of metabolomics in hierarchizing OPA1 missense mutations by their in vitro severity, fitting clinical expressivity. This suggests that our methodological approach can be used to discriminate the pathological significance of variants in genes responsible for other rare metabolic diseases and may be instrumental to select possible compounds eligible for supplementation treatment.

Altered auto-phosphorylation of novel TNNI3K variants associated with AV-nodal re-entry tachycardia and conduction disease

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
C Pham ◽  
N Munoz-Martin ◽  
S Podliesna ◽  
A Milano ◽  
L Beekman ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Cardiac Disease ◽  
Genetic Variants ◽  
Hek293 Cells ◽  
Cardiac Conduction ◽  
Functional Assay ◽  
Funding Source ◽  
Wild Type ◽  
Cardiac Phenotype

Abstract Background In the past decade, we and others have reported three families with rare genetic variants in TNNI3K, encoding the cardiac-specific troponin-I interacting kinase (TNNI3K), co-segregating with a mixed, but highly penetrant, cardiac phenotype that features predominant atrial/junctional tachycardia occurring in combination with cardiac conduction disease and dilated cardiomyopathy. We demonstrated that while the p.Thr539Ala and p.Gly526Asp TNNI3K variants had decreased auto-phosphorylation activity the p.Glu768Lys variant, present in 3 independent families, leads to increased auto-phosphorylation levels, in line with the finding that increased levels of Tnni3k expression are associated with slower atrial-ventricular conduction in mice. Objective Identifying new genetic variants in the TNNI3K gene associated with cardiac disease and assessing their impact on TNNI3K auto-phosphorylation levels. Methods Through next generation sequencing of a panel of genes associated with cardiac disease we assessed TNNI3K in patients with cardiac arrhythmias and cardiomyopathies. All variants identified were assessed in vitro for effects on auto-phosphorylation. Briefly, wild-type and mutant TNNI3K constructs were transfected into HEK293 cells, protein was extracted after 48 hours and analyzed with anti-flag and anti-phospho-tyrosine antibodies on Western blot. Results We identified 7 novel and rare variants in TNNI3K in 11 additional probands, with predominantly cardiac conduction disease, with or without dilated cardiomyopathy, and atrial-ventricular-re-entry-tachycardia (AVNRT). Of these, multiple variants were found to have aberrant auto-phosphorylation including almost absent auto-phosphorylation capacity for one (TNNI3K-p.Val510Leu). All three-independent wild type TNNI3K transfected HEK293 cell lysates showed similar phosphorylated TNNI3K levels and the kinase-dead negative control demonstrated no phosphorylation activity. Conclusion We here present 7 novel genetic variants in TNNI3K in patients with a remarkable overlap in cardiac phenotype consisting mainly of AVNRT and cardiac conduction disease. We further show that some of these variants alter the auto-phosphorylation of TNNI3K. These results indicate a more prevalent role of variants in TNNI3K in human cardiac disease and a possible in vitro functional assay to assess the pathogenicity of such variants. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme VIDI-91718361)

Abstract 257: Determining the Potential Role of cTnT Isoform Switching in the Development of Early Childhood Tropomyosin-linked DCM

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Melissa Lynn ◽  
Teryn Holeman ◽  
Lauren Tal-Grinspan ◽  
J.P. Jin ◽  
Jil C Tardiff
Keyword(s):  
Troponin T ◽  
Systolic Function ◽  
Phenotypic Variability ◽  
Calcium Sensitivity ◽  
Bimodal Distribution ◽  
Heart Tissue ◽  
Regulatory Function ◽  
General Mechanism ◽  
Endogenous Expression

An oft noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. Recently two unrelated multigenerational families were identified with the tropomyosin (Tm) mutation Asp230Asn (D230N), exhibiting a striking “bimodal” distribution of severity. In these families, many children (<1 year) with D230N Tm presented with a severe form of DCM that led to sudden, often fatal CHF, while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. To explain this improvement, despite the persistence of D230N Tm, we hypothesized that the phenotype is modified by other thin filament (TF) isoforms. Thus we propose the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). We have shown that D230N Tm leads to a more stable (rigid) filament primarily at the C-terminus of Tm near the Tm overlap, a crucial region for TF regulatory function that cTnT modulates. Myofilaments from D230N Tm mice exhibited a small decrease in calcium sensitivity of force development that was significantly reduced in the presence of cTnT1, supportive of a modulatory role. We assessed cardiac performance in our novel D230N Tm x cTnT1 double transgenic (DTg) mice, % FS was similarly reduced for D230N Tm and DTg mice at 2 months likely due to persistent endogenous expression of cTnT1. Divergent cardiac remodeling occurred at 4 months at which point DTg mice had significantly reduced % FS compared to D230N, indicating that additive exposure to cTnT1 is detrimental to the function of D230N hearts. Additionally, In vitro studies on non-failing and failing human heart tissue found that RNA levels of cTnT1 are significantly higher in failing hearts. Thus modulation by cTnT1 could be a more general mechanism for the progressive remodeling seen in heart failure.

High Content Screening Identifies Synthetic Lethality Of Retinoid Receptor Agonists In IKZF1-Mutated BCR-ABL1 positive Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 172-172 ◽  
Author(s):  
Michelle Churchman ◽  
Jonathan Low ◽  
Debbie Payne-Turner ◽  
Shann-Ching Chen ◽  
Jing Ma ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Lymphoblastic Leukemia ◽  
Cell Aggregation ◽  
Missense Mutations ◽  
Wild Type ◽  
Retinoid Receptor ◽  
Self Renewal ◽  
Receptor Agonists

Abstract Expression of BCR-ABL1 is a hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). While CML is typically responsive to tyrosine kinase inhibitor monotherapy, BCR-ABL1-positive ALL is associated with a high frequency of treatment failure and relapse, even in the era of TKI therapy. Alterations of the lymphoid transcription factor gene IKZF1 (IKAROS) are frequent in BCR-ABL1 ALL and in CML at lymphoid blast crisis. Moreover, IKZF1 alterations are associated with poor outcome in BCR-ABL1-positive and -negative B-ALL. IKZF1 alterations are usually heterozygous and include loss-of-function deletions (∼60% of cases), focal deletion resulting in expression of a dominant negative isoform lacking the N-terminal DNA-binding zinc fingers, IK6 (∼30%) and missense mutations (∼10%). In addition, over half of BCR-ABL1-positive ALL cases harbor deletions of CDKN2A/B (INK4/ARF). We used two murine models of BCR-ABL1 leukemia to examine the role of IKZF1 alterations (deletions and IK6) and ARF loss in disease lineage and treatment responsiveness. In the first model, lineage negative Arf-/-, Ikzf1+/- or wild type (WT) bone marrow was transduced with p185 BCR-ABL1-expressing retrovirus and either an IK6-expressing retrovirus or empty vector followed by transplantation into irradiated wild type mice. p185-transduced WT marrow invariably induced a CML-like leukemia, whereas Arf loss and Ikzf1 alterations synergistically drove an aggressive pre-B ALL. In a complementary model, unmanipulated Arf-/-, Ikzf1+/-, or WT marrow was transduced with p185 BCR-ABL and transplanted immediately or after in vitro derivation of pre-B cell cultures. In this model, Arf loss and Ikzf1 haploinsuffiency or expression of IK6 reduced the latency of ALL and reduced responsiveness to dasatinib in vitro and in vivo. Strikingly, perturbation of Ikzf1 activity by haploinsufficiency, expression of IK6, zinc finger missense mutations, or shRNA mediated knockdown was associated with increased expression of the stem cell marker CD90 (Thy1) and the growth of cells in large aggregates in liquid cultures. Ikzf1 alteration was also associated with induction of a hematopoietic stem cell-like gene expression program, and increased self-renewal manifested as enhanced serial colony forming potential. This aberrant growth and self-renewal phenotype was abrogated by neutralizing anti-Thy1 antibodies. Thy1 is a known IKZF1 target, and these findings suggest that IKZF1 alterations derepress Thy1 expression resulting in enhanced stemness and treatment resistance. To identify agents that may enhance TKI responsiveness, we screened the activity of 384 FDA approved compounds in Arf-/- BCR-ABL1+ IK6+ pre-B. We assessed cell viability using CellTiter Glo, and measured the number and size of cell aggregates by high throughput microscopy to identify drugs that selectively inhibit cell aggregation. This identified the retinoid X receptor agonist bexarotene as a potent inhibitor of cell aggregation. A follow-up screen of 128 nuclear hormone receptor effector compounds identified potent anti-aggregation activity of 9-cis retinoic acid, 13-cis retinoic acid, all-trans retinoic acid and carbacyclin. In contrast, N-oleoylethanolamine, an antagonist of perioxisome proliferator-activated receptors that heterodimerize with RXRs, markedly increased cell aggregation. All retinoid receptor agonists induced cell cycle arrest and reduced proliferation without inducing apoptosis, and attenuated Thy1 expression, abrogated colony formation and enhanced the efficacy of dasatinib in vitro and in mice transplanted with Arf-/- p185 IK6 cells. Notably, Ikzf1is a predicted retinoid receptor target, and bexarotene treatment upregulated expression and nuclear localization of wild type IKZF1 in Arf-/- p185 pre-B cells, with and without IK6. Thus, IKZF1 alterations drive lymphoid lineage, and induce self-renewal and drug resistance in BCR-ABL1 ALL. IKZF1 alterations are associated with overexpression of Thy1, a target of IKZF1 transcriptional repression. This phenotype is reversed by retinoid receptor agonists that induce expression of IKZF1, attenuate Thy1 expression and increase sensitivity to TKI therapy. These data provide important new insights into the role of IKZF1 alterations in leukemogenesis, and identify a new therapeutic option in high risk B-ALL. Disclosures: No relevant conflicts of interest to declare.

Two missense mutations identified in venous thrombosis patients impair the inhibitory function of the protein Z dependent protease inhibitor

2012 ◽  
Vol 107 (05) ◽  
pp. 854-863 ◽  
Author(s):  
Nigel P. Birch ◽  
Peter J. Browett ◽  
Paul B. Coughlin ◽  
Anita J. Horvath ◽  
Neil S. Van de Water ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Venous Thrombosis ◽  
Nonsense Mutation ◽  
Healthy Controls ◽  
Missense Mutations ◽  
Wild Type ◽  
Protein Z ◽  
Inhibitory Function

SummaryProtein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 2.0, 1.05–3.7, p=0.044). However, in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.

scholarly journals Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

2016 ◽  
Vol 113 (44) ◽  
pp. 12592-12597 ◽  
Author(s):  
Uros Kuzmanov ◽  
Hongbo Guo ◽  
Diana Buchsbaum ◽  
Jake Cosme ◽  
Cynthia Abbasi ◽  
...  
Keyword(s):  
Mouse Model ◽  
Dilated Cardiomyopathy ◽  
Enrichment Analysis ◽  
Immunoblot Analysis ◽  
Heart Tissue ◽  
Specific Protein ◽  
Wild Type ◽  
Notch 1 ◽  
Affinity Capture ◽  
Confocal Immunofluorescence Microscopy

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.

scholarly journals Rab8 Regulates the Actin-based Movement of Melanosomes

2005 ◽  
Vol 16 (4) ◽  
pp. 1640-1650 ◽  
Author(s):  
Marion L. Chabrillat ◽  
Claire Wilhelm ◽  
Christina Wasmeier ◽  
Elena V. Sviderskaya ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Rab Proteins ◽  
Rab Gtpases ◽  
Wild Type ◽  
In Vitro Motility Assay ◽  
Actin Bundles ◽  
In Vitro Motility ◽  
Rab Protein

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes.

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