scholarly journals Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilative Cardiomyopathy (p.R312H and p.E361G)

2021 ◽  
Author(s):  
Constanze Erdmann ◽  
Roua Hassoun ◽  
Sebastian Schmitt ◽  
Setsuko Fujita-Becker ◽  
Antonina J. Mazur ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Functional Characterization ◽  
Biological Properties ◽  
Cell System ◽  
Neonatal Rat ◽  
Actin Binding ◽  
Rat Cardiomyocytes ◽  
Dilative Cardiomyopathy ◽  
Cardiac Actin ◽  
Myosin S1

Abstract The human mutant cardiac α-actins p.A295S or p.R312H (plus p.R312K) and p.E361G correlated with hypertrophic or dilative cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. After purification their biochemical and cell biological properties were analysed and compared to wild type (wt) cardiac actin identically obtained or conventionally isolated from bovine hearts. DNase I inhibition and their polymerization behaviour indicated that all c-α-actins had maintained their native state. Cardiomyopathy type specific differences were observed except for the p.R312K mutant, which behaved like wt c-α-actin. The extent of myosin-S1 ATPase stimulation by the c-actin variants and its Ca2+-sensitivity after decoration with tropomyosin (cTm) and troponin complex (cTn) varied being highest for the HCM p.A295S and lower for both DCM mutants. Similar Ca2+-sensitivity differences were observed by recording the fluorescence increase of pyrene-cTm in the absence or presence of myosin-S1 and/or the actin-binding N-terminal fragment of cardiac myosin binding protein C (N-cMyBP-C). Transfection experiments showed the incorporation of the c-actin variants into existing cytoskeletal elements of non-muscle cells. Wt and p.A295S c-α-actin preferably incorporated into the microfilament system and p.R312H and p.E361G into the submembranous actin network of MDCK cells. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding for HA-tagged c-α-actins showed their incorporation into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferably incorporated at the minus ends. Our data indicate functional differences of the c-α-actins that may be causative for the different cardiomyopathy phenotypes.

scholarly journals Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1082
Author(s):  
Constanze Erdmann ◽  
Roua Hassoun ◽  
Sebastian Schmitt ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Mdck Cells ◽  
Cell System ◽  
Neonatal Rat ◽  
Actin Binding ◽  
Thin Filaments ◽  
Rat Cardiomyocytes ◽  
Actin Organization ◽  
Adaptive Processes ◽  
Microfilament System

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.

Deficiency in Nebulin Repeats of Sarcomeric Nebulette is Detrimental for Cardiomyocyte Tolerance to Exercise and Biomechanical Stress

2021 ◽  
Author(s):  
Ramona M. Vejandla ◽  
Buyan-Ochir Orgil ◽  
Neely R. Alberson ◽  
Ning Li ◽  
Undral Munkhsaikhan ◽  
...  
Keyword(s):  
Systolic Function ◽  
Mechanical Strain ◽  
Focal Adhesions ◽  
Left Ventricular ◽  
Signaling Network ◽  
Treadmill Running ◽  
Neonatal Rat ◽  
Actin Binding ◽  
Rat Cardiomyocytes ◽  
Biomechanical Stress

Background: The actin-binding sarcomeric nebulette (NEBL) protein provides efficient contractile flexibility via interaction with desmin intermediate filaments. NEBL gene mutations affecting the nebulin repeat (NR) domain is known to induce cardiomyopathy. Objective: The study aimed to explore the roles of NEBL in exercise and biomechanical stress response. Methods: We ablated exon3 encoding the first NR of Nebl and created global Nebl3ex-/3ex- knockout mice. Cardiac function, structure and transcriptome was assessed before and after a 4-week treadmill regimen. A Nebl-based exercise signaling network was constructed using systems genetics methods. H9C2 and neonatal rat cardiomyocytes (NRCs) expressing wild-type or mutant NEBL underwent cyclic mechanical strain. Results: Nebl3ex-/3ex- mice demonstrated diastolic dysfunction with preserved systolic function at 6-months of age. After treadmill running, 4-month-old Nebl3ex-/3ex- mice developed concentric cardiac hypertrophy and left ventricular dilation compared to running Nebl+/+ and sedentary Nebl3ex-/3ex- mice. Disturbance of sarcomeric Z-disks and thin filaments architecture, disruption of intercalated disks and mitochondria were found in exercised Nebl3ex-/3ex- mice. A Nebl-based exercise signaling network included Csrp3, Des, Fbox32, Jup, Myh6, and Myh7. Disturbed expression of TM1, DES, JUP, b-catenin, MLP, α-actinin2 and vinculin proteins was demonstrated. In H9C2 cells, NEBL was recruited into focal adhesions at 24-hours post-strain and redistributed along with F-actin at 72-hours post-strain, suggesting time-dependent redistribution of NEBL in response to strain. NEBL mutations cause desmin disorganization in NRCs upon stretch. Conclusions: Upon stretch, NEBL deficiency causes disturbed sarcomere, Z-disks and desmin organization, and prevents NEBL redistribution to focal adhesions in cardiomyocytes, weakening cardiac tolerance to stress.

scholarly journals Prokaryotic virus host predictor: a Gaussian model for host prediction of prokaryotic viruses in metagenomics

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Congyu Lu ◽  
Zheng Zhang ◽  
Zena Cai ◽  
Zhaozhong Zhu ◽  
Ye Qiu ◽  
...  
Keyword(s):  
Prediction Accuracy ◽  
Functional Characterization ◽  
Gaussian Model ◽  
Software Tool ◽  
Biological Properties ◽  
Rapid Identification ◽  
Taxonomic Assignment ◽  
Genus Level ◽  
Alignment Free ◽  
Archaeal Viruses

Abstract Background Viruses are ubiquitous biological entities, estimated to be the largest reservoirs of unexplored genetic diversity on Earth. Full functional characterization and annotation of newly discovered viruses requires tools to enable taxonomic assignment, the range of hosts, and biological properties of the virus. Here we focus on prokaryotic viruses, which include phages and archaeal viruses, and for which identifying the viral host is an essential step in characterizing the virus, as the virus relies on the host for survival. Currently, the method for determining the viral host is either to culture the virus, which is low-throughput, time-consuming, and expensive, or to computationally predict the viral hosts, which needs improvements at both accuracy and usability. Here we develop a Gaussian model to predict hosts for prokaryotic viruses with better performances than previous computational methods. Results We present here Prokaryotic virus Host Predictor (PHP), a software tool using a Gaussian model, to predict hosts for prokaryotic viruses using the differences of k-mer frequencies between viral and host genomic sequences as features. PHP gave a host prediction accuracy of 34% (genus level) on the VirHostMatcher benchmark dataset and a host prediction accuracy of 35% (genus level) on a new dataset containing 671 viruses and 60,105 prokaryotic genomes. The prediction accuracy exceeded that of two alignment-free methods (VirHostMatcher and WIsH, 28–34%, genus level). PHP also outperformed these two alignment-free methods much (24–38% vs 18–20%, genus level) when predicting hosts for prokaryotic viruses which cannot be predicted by the BLAST-based or the CRISPR-spacer-based methods alone. Requiring a minimal score for making predictions (thresholding) and taking the consensus of the top 30 predictions further improved the host prediction accuracy of PHP. Conclusions The Prokaryotic virus Host Predictor software tool provides an intuitive and user-friendly API for the Gaussian model described herein. This work will facilitate the rapid identification of hosts for newly identified prokaryotic viruses in metagenomic studies.

scholarly journals CapZ integrates several signaling pathways in response to mechanical stiffness

2019 ◽  
Vol 151 (5) ◽  
pp. 660-669 ◽  
Author(s):  
Christopher Solís ◽  
Brenda Russell
Keyword(s):  
Mechanical Load ◽  
Phosphorylation Site ◽  
Tight Binding ◽  
Neonatal Rat ◽  
Actin Binding ◽  
Mechanical Stimuli ◽  
Muscle Adaptation ◽  
Capping Protein ◽  
Actin Capping Protein ◽  
Actin Capping

Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the β-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the β-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the β-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.

Necrostatin-1 Analog DIMO Exerts Cardioprotective Effect against Ischemia Reperfusion Injury by Suppressing Necroptosis via Autophagic Pathway in Rats

Pharmacology ◽  
2021 ◽  
Vol 106 (3-4) ◽  
pp. 189-201
Author(s):  
Shigang Qiao ◽  
Wen-jie Zhao ◽  
Huan-qiu Li ◽  
Gui-zhen Ao ◽  
Jian-zhong An ◽  
...  
Keyword(s):  
Cell Death ◽  
Ischemia Reperfusion Injury ◽  
Myocardial Infarct ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Neonatal Rat ◽  
Ligand Docking ◽  
Autophagic Flux ◽  
Myocardial Infarct Size ◽  
Rat Cardiomyocytes

Aim: It has been reported that necrostatin-1 (Nec-1) is a specific necroptosis inhibitor that could attenuate programmed cell death induced by myocardial ischemia/reperfusion (I/R) injury. This study aimed to observe the effect and mechanism of novel Nec-1 analog (Z)-5-(3,5-dimethoxybenzyl)-2-imine-1-methylimidazolin-4-1 (DIMO) on myocardial I/R injury. Methods: Male SD rats underwent I/R injury with or without different doses of DIMO (1, 2, or 4 mg/kg) treatment. Isolated neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment with or without DIMO (0.1, 1, 10, or 100 μM). Myocardial infarction was measured by TTC staining. Cardiomyocyte injury was assessed by lactate dehydrogenase assay (LDH) and flow cytometry. Receptor-interacting protein 1 kinase (RIP1K) and autophagic markers were detected by co-immunoprecipitation and Western blotting analysis. Molecular docking of DIMO into the ATP binding site of RIP1K was performed using GLIDE. Results: DIMO at doses of 1 or 2 mg/kg improved myocardial infarct size. However, the DIMO 4 mg/kg dose was ineffective. DIMO at the dose of 0.1 μM decreased LDH leakage and the ratio of PI-positive cells followed by OGD/R treatment. I/R or OGD/R increased RIP1K expression and in its interaction with RIP3K, as well as impaired myocardial autophagic flux evidenced by an increase in LC3-II/I ratio, upregulated P62 and Beclin-1, and activated cathepsin B and L. In contrast, DIMO treatment reduced myocardial cell death and reversed the above mentioned changes in RIP1K and autophagic flux caused by I/R and OGD/R. DIMO binds to RIP1K and inhibits RIP1K expression in a homology modeling and ligand docking. Conclusion: DIMO exerts cardioprotection against I/R- or OGD/R-induced injury, and its mechanisms may be associated with the reduction in RIP1K activation and restoration impaired autophagic flux.

scholarly journals Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression

2021 ◽  
Vol 22 (7) ◽  
pp. 3561
Author(s):  
Jumpei Ito ◽  
Tomomi Minemura ◽  
Sébastien Wälchli ◽  
Tomoaki Niimi ◽  
Yoshitaka Fujihara ◽  
...  
Keyword(s):  
Transcriptional Repressor ◽  
Neonatal Rat ◽  
Protective Mechanism ◽  
Pathological State ◽  
Rat Cardiomyocytes ◽  
Adult Mice ◽  
Spontaneous Action ◽  
Sirna Knockdown ◽  
Spontaneous Action Potentials ◽  
Ca2 Channels

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.

Mechanical activity in heart regulates translation of α-myosin heavy chain mRNA but not its localization

1999 ◽  
Vol 276 (6) ◽  
pp. H2013-H2019 ◽  
Author(s):  
Gordana Nikcevic ◽  
Maria C. Heidkamp ◽  
Merja Perhonen ◽  
Brenda Russell
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mrna Translation ◽  
Neonatal Rat ◽  
Mechanical Activity ◽  
Mrna Levels ◽  
Rat Cardiomyocytes ◽  
Significant Difference ◽  
Polysomal Fraction ◽  
Mechanical Transduction

Mechanical inactivity depresses protein expression in cardiac muscle tissue and results in atrophy. We explore the mechanical transduction mechanism in spontaneously beating neonatal rat cardiomyocytes expressing the α-myosin heavy chain (α-MyHC) isoform by interfering with cross-bridge function [2,3-butanedione monoxime (BDM), 7.5 mM] without affecting cell calcium. The polysome content and α-MyHC mRNA levels in fractions from a sucrose gradient were analyzed. BDM treatment blocked translation at initiation (162 ± 12% in the nonpolysomal RNA fraction and 43 ± 6% in the polysomal fraction, relative to control as 100%; P < 0.05). There was an increase in α-MyHC mRNA from the nonpolysomal fraction (120.5 ± 7.7%; P < 0.05 compared with control) with no significant change in the heavy polysomes. In situ hybridization of α-MyHC mRNA was used to estimate message abundance as a function of the distance from the nucleus. The mRNA was dispersed through the cytoplasm in spontaneously beating cells as well as in BDM-treated cells (no significant difference). We conclude that direct inhibition of contractile machinery, but not calcium, regulates initiation of α-MyHC mRNA translation. However, calcium, not pure mechanical signals, appears to be important for message localization.

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