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Stem Cell Research & Therapy Jun 2024Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other...
BACKGROUND
Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocyte cells. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart.
METHODS
CMs were derived from hiPSC (hi-PCS-CM) using different concentrations of RA (Control without RA, LRA with 0.05μM and HRA with 0.1 μM) between day 3-6 of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling hiPSC-CM at high cell density in a low collagen hydrogel.
RESULTS
In the HRA group, hiPSC-CMs exhibited highest expression of contractile proteins MYH6, MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are marker genes for left ventricular CMs, was also the highest in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was functionally more similar to the left ventricle. RNAsequencing revealed that the heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA.
CONCLUSION
By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHTs with a phenotype similar to that of the left ventricle or right ventricle.
Topics: Humans; Tretinoin; Myocytes, Cardiac; Cell Differentiation; Induced Pluripotent Stem Cells; Heart Ventricles; Myosin Heavy Chains; Cardiac Myosins; Tissue Engineering; Homeobox Protein Nkx-2.5; T-Box Domain Proteins
PubMed: 38902843
DOI: 10.1186/s13287-024-03741-0 -
Cell Communication and Signaling : CCS Jun 2024African American (AA) women are twice as likely to develop triple-negative breast cancer (TNBC) as women of European descent. Additionally, AA women with TNBC present a...
African American (AA) women are twice as likely to develop triple-negative breast cancer (TNBC) as women of European descent. Additionally, AA women with TNBC present a much more aggressive disease course than their European American (EA) counterparts. Thus, there is an unmet clinical need to identify race-specific biomarkers and improve survival outcomes in AA patients with TNBC. The minus-end directed microtubule motor protein kinesin family member C1 (KIFC1) promotes centrosome clustering and chromosomal instability and is often overexpressed in TNBC. Previous findings suggest that KIFC1 plays a role in cell proliferation and migration in TNBC cells from AAs and that the levels of nuclear KIFC1 (nKIFC1) are particularly high in AA patients with TNBC. The nuclear localization of KIFC1 in interphase may underlie its previously unrecognized race-specific association. In this study, we found that in TNBC cells derived from AAs, nKIFC1 interacted with the tumor suppressor myosin heavy chain 9 (MYH9) over EA cells. Treatment of AA TNBC cells with commercial inhibitors of KIFC1 and MYH9 disrupted the interaction between KIFC1 and MYH9. To characterize the racial differences in the KIFC1-MYH9-MYC axis in TNBC, we established homozygous KIFC1 knockout (KO) TNBC cell lines. KIFC1 KO significantly inhibited proliferation, migration, and invasion in AA TNBC cells but not in EA TNBC cells. RNA sequencing analysis showed significant downregulation of genes involved in cell migration, invasion, and metastasis upon KIFC1 KO in TNBC cell lines from AAs compared to those from EAs. These data indicate that mechanistically, the role of nKIFC1 in driving TNBC progression and metastasis is stronger in AA patients than in EA patients, and that KIFC1 may be a critical therapeutic target for AA patients with TNBC.
Topics: Humans; Triple Negative Breast Neoplasms; Kinesins; Female; Cell Line, Tumor; Myosin Heavy Chains; Cell Proliferation; Cell Movement; Black or African American; White People; Protein Binding
PubMed: 38902769
DOI: 10.1186/s12964-024-01664-0 -
ELife Jun 2024Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with...
Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.
Topics: Melanosomes; Myosin Type V; Animals; Mice; Adaptor Proteins, Signal Transducing; Protein Binding; Humans; Myosin Heavy Chains; Melanocytes
PubMed: 38900147
DOI: 10.7554/eLife.93662 -
Frontiers in Veterinary Science 2024Equid herpesvirus type 8 (EqHV-8) poses a significant threat to equine health, leading to miscarriages and respiratory diseases in horses and donkeys, and results in...
INTRODUCTION
Equid herpesvirus type 8 (EqHV-8) poses a significant threat to equine health, leading to miscarriages and respiratory diseases in horses and donkeys, and results in substantial economic losses in the donkey industry. Currently, there are no effective drugs or vaccines available for EqHV-8 infection control.
METHODS
In this study, we investigated the and antiviral efficacy of Blebbistatin, a myosin II ATPase inhibitor, against EqHV-8.
RESULTS
Our results demonstrated that Blebbistatin significantly inhibited EqHV-8 infection in Rabbit kidney (RK-13) and Madin-Darby Bovine Kidney (MDBK) cells in a concentration-dependent manner. Notably, Blebbistatin was found to disrupt EqHV-8 infection at the entry stage by modulating myosin II ATPase activity. Moreover, experiments revealed that Blebbistatin effectively reduced EqHV-8 replication and mitigated lung pathology in a mouse model.
CONCLUSION
Collectively, these findings suggest that Blebbistatin holds considerable potential as an antiviral agent for the control of EqHV-8 infection, presenting a novel approach to addressing this veterinary challenge.
PubMed: 38898998
DOI: 10.3389/fvets.2024.1390304 -
Nature Communications Jun 2024Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the...
Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct β- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that β- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that β-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.
Topics: rhoA GTP-Binding Protein; Cytokinesis; Formins; Actins; Humans; Myosin Type II; Adaptor Proteins, Signal Transducing; HeLa Cells; Animals; Protein Isoforms
PubMed: 38897998
DOI: 10.1038/s41467-024-49427-2 -
Frontiers in Physiology 2024Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act...
Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act as nucleotide sensors, can substantially determine the kinetic signature of a myosin, ., to either perform fast movement or enable long-range transport and tension generation. Switch-2 essentially contributes to the ATP hydrolysis reaction and determines product release. With few exceptions, class-1 myosin harbor a tyrosine in the switch-2 consensus sequence DIYGFE, at a position where class-2 myosins and a selection of myosins from other classes have a substitution. Here, we addressed the role of the tyrosine in switch-2 of class-1 myosins as potential determinant of the duty ratio. We generated constitutively active motor domain constructs of two class-1 myosins from the social amoeba , namely, Myo1E, a high duty ratio myosin and Myo1B, a low duty ratio myosin. In Myo1E we introduced mutation Y388F and in Myo1B mutation F387Y. The detailed functional characterization by steady-state and transient kinetic experiments, combined with motility and landing assays revealed an almost reciprocal relationship of a number of critical kinetic parameters and equilibrium constants between wild-type and mutants that dictate the lifetime of the strongly actin-attached states of myosin. The Y-to-F mutation increased the duty ratio of Moy1B by almost one order of magnitude, while the introduction of the phenylalanine in switch-2 of Myo1E transformed the myosin into a low duty ratio motor. These data together with structural considerations propose a role of switch-2 in fine-tuning ADP release through a mechanism, where the class-specific tyrosine together with surrounding residues contributes to the coordination of Mg and ADP. Our results highlight the importance of conserved switch-2 residues in class-1 myosins for efficient chemo-mechanical coupling, revealing that switch-2 is important to adjust the duty ratio of the amoeboid class-1 myosins for performing movement, transport or gating functions.
PubMed: 38887318
DOI: 10.3389/fphys.2024.1393952 -
Investigative Ophthalmology & Visual... Jun 2024We investigated the natural history of retinal dystrophy owing to variants in the MYO7A gene.
PURPOSE
We investigated the natural history of retinal dystrophy owing to variants in the MYO7A gene.
METHODS
Fifty-three patients (mean age, 33.6 ± 16.7 years) with Usher syndrome owing to biallelic, mostly pathogenic, variants in MYO7A underwent baseline and two annual follow-up visits. Best-corrected visual acuity (BCVA), semiautomatic kinetic visual field, full-field electroretinogram, color fundus imaging, microperimetry, spectral-domain optical coherence tomography, and fundus autofluorescence were assessed.
RESULTS
At baseline, all patients presented with decreased BCVA (66.4 ± 17.9 Early Treatment Diabetic Retinopathy score and 59.5 ± 21.7 Early Treatment Diabetic Retinopathy score, in the better- and worse-seeing eyes, respectively), restricted semiautomatic kinetic visual field (III4e area, 3365.8 ± 4142.1°2; 4176.4 ± 4400.3°2) and decreased macular sensitivity (9.7 ± 9.9 dB; 9.0 ± 10.2 dB). Spectral-domain optical coherence tomography revealed reduced central macular thickness (259.6 ± 63.0 µm; 250.7 ± 63.3 µm) and narrowed ellipsoid zone band width (2807.5 ± 2374.6 µm; 2615.5 ± 2370.4 µm). Longitudinal analyses (50 patients) showed a significant decrease of BCVA in better-seeing eyes, whereas no changes were observed in worse-seeing eyes for any parameter. BCVA, semiautomatic kinetic visual field (III4e and V4e) and macular sensitivity were related significantly to age at baseline. Hyperautofluorescent foveal patch (16 eyes [31.4%]) and abnormal central hypoautofluorescence (9 eyes [17.6%]) were significantly associated with worse morphological and functional read-outs compared with the hyperautofluorescent ring pattern (22 eyes [43.1%]).
CONCLUSIONS
Our European multicentric study offers the first prospective longitudinal analysis in one of the largest cohorts of MYO7A patients described to date, confirming the slow disease progression. More important, this study emphasizes the key role of fundus autofluorescence patterns in retinal impairment staging and advocates its adoption as an objective biomarker in patient selection for future gene therapy clinical trials.
Topics: Humans; Male; Female; Adult; Prospective Studies; Tomography, Optical Coherence; Visual Acuity; Middle Aged; Electroretinography; Myosin VIIa; Visual Fields; Young Adult; Adolescent; Usher Syndromes; Genetic Therapy; Child; Visual Field Tests; Europe; Fluorescein Angiography; Follow-Up Studies; Aged; Longitudinal Studies; Disease Progression; Myosins; Retina
PubMed: 38884554
DOI: 10.1167/iovs.65.6.25 -
Proceedings of the National Academy of... Jun 2024Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various...
Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various immune receptors and associated proteins are often dynamically distributed in specific nanodomains on the plasma membrane (PM). However, the exact molecular mechanism and functional relevance of this nanodomain targeting in plant immunity regulation remain largely unknown. By utilizing high spatiotemporal resolution imaging and single-particle tracking analysis, we show that myosin XIK interacts with remorin to recruit and stabilize PM-associated kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) within immune receptor FLAGELLIN SENSING 2 (FLS2)-containing nanodomains. This recruitment facilitates FLS2/BIK1 complex formation, leading to the full activation of BIK1-dependent defense responses upon ligand perception. Collectively, our findings provide compelling evidence that myosin XI functions as a molecular scaffold to enable a spatially confined complex assembly within nanodomains. This ensures the presence of a sufficient quantity of preformed immune receptor complex for efficient signaling transduction from the cell surface.
Topics: Arabidopsis; Arabidopsis Proteins; Plant Immunity; Immunity, Innate; Protein Serine-Threonine Kinases; Myosins; Protein Kinases; Cell Membrane; Signal Transduction; Plant Diseases
PubMed: 38875149
DOI: 10.1073/pnas.2312415121 -
Open Biology Jun 2024Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here,...
Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here, we generated transgenic MYH7 R453C and MYH6 R453C piglets and found both developed typical cardiac hypertrophy. Unexpectedly, we found serious fibrosis and cardiomyocyte loss in the ventricular of MYH7 R453C, not MYH6 R453C piglets, similar to HCM patients. Then, RNA-seq analysis and western blotting identified the activation of ERK1/2 and PI3K-Akt pathways in MYH7 R453C. Moreover, we observed an increased expression of fetal genes and an excess of reactive oxygen species (ROS) in MYH7 R453C piglet models, which was produced by Nox4 and subsequently induced inflammatory response. Additionally, the phosphorylation levels of Smad2/3, ERK1/2 and NF-kB p65 proteins were elevated in cardiomyocytes with the MYH7 R453C mutation. Furthermore, epigallocatechin gallate, a natural bioactive compound, could be used as a drug to reduce cell death by adjusting significant downregulation of the protein expression of Bax and upregulated Bcl-2 levels in the H9C2 models with MYH7 R453C mutation. In conclusion, our study illustrated that TGF-β/Smad2/3, ERK1/2 and Nox4/ROS pathways have synergistic effects on cardiac remodelling and inflammation in MYH7 R453C mutation.
Topics: Animals; Myosin Heavy Chains; Transforming Growth Factor beta; NADPH Oxidase 4; Reactive Oxygen Species; NF-kappa B; Signal Transduction; Swine; Myocytes, Cardiac; Humans; Cardiac Myosins; Disease Models, Animal; MAP Kinase Signaling System; Animals, Genetically Modified; Smad2 Protein; Mutation; Smad3 Protein; Ventricular Remodeling; Cardiomyopathy, Hypertrophic; Rats
PubMed: 38862020
DOI: 10.1098/rsob.230427 -
Journal of the American Heart... Jun 2024
Topics: Humans; Cardiac Myosins; Animals; Myocardial Contraction; Energy Metabolism; Myocardium
PubMed: 38860415
DOI: 10.1161/JAHA.124.035479