scholarly journals Mitochondrial calpain inhibition restores defective SR-mitochondrial crosstalk in CPVT rat myocytes

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Shanna Hamilton ◽  
Radmila Terentyeva ◽  
Roland Veress ◽  
Fruzsina Perger ◽  
Benjamin Y. Martin ◽  
...  
Keyword(s):  
Energy Demand ◽  
Structural Damage ◽  
Polymorphic Ventricular Tachycardia ◽  
Structural Defects ◽  
Calpain Inhibitor ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Structural Protein ◽  
Gain Of Function ◽  
Enhanced Production

Cardiac RYR2-mediated sarcoplasmic Ca2+ (SR) release is essential for matching increased energy demand during fight-or-flight response with mitochondrial metabolic output by delivering Ca2+ into the mitochondrial matrix to activate Ca2+-dependent Krebs cycle dehydrogenases. RYR2 complex gain-of-function mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) have been linked to mitochondrial structural damage and enhanced production of reactive oxygen species (ROS). Despite being critical for arrhythmogenesis in CPVT, the exact causes of these phenomena remain undetermined. Taking advantage of a new rat model of CPVT induced by heterozygous RYR2 gain-of-function mutation S2222L, we tested how RYR2 overactivity alters mitochondrial Ca2+ and ROS handling, and how these changes cause mitochondrial structural defects. Injection of epinephrine (1 mg/kg) and caffeine (120 mg/kg) induced bigamy and bidirectional VT in vivo in 100% of CPVT rats. Simultaneous whole-cell patch clamp and confocal Ca2+-imaging demonstrated that under β-adrenergic stimulation with isoproterenol (50 nM), CPVT ventricular myocytes (VMs) exhibited severe Ca2+ mishandling and high propensity for generation of spontaneous Ca2+ waves (SCWs) that cause arrhythmogenic afterdepolarizations. Diminished Ca2+ transient amplitude in CPVT VMs resulted in a significant reduction in mitochondrial matrix–[Ca2+], and thereby a mito-ROS surge, visualized using matrix-targeted biosensors mtRCaMP1h and MLS-HyPer, respectively. Importantly, using novel Ca2+-biosensors targeted to intermembrane space (IMS-GECO), we uncovered that [Ca2+] in this compartment reaches 1 µM, sufficient for activation of Ca2+-dependent protease μ-calpain. Adenoviral overexpression of IMS-targeted calpastatin, an endogenous calpain inhibitor, reduced mito-ROS, restored cytosolic Ca2+ transient amplitude and SR Ca2+ content, and reduced RYR2-mediated SCWs in CPVT VMs. These changes were paralleled by restored expression levels of OPA1, a mitochondrial structural protein responsible for tight cristae organization. Our data suggest that enhanced mito-ROS due to matrix-[Ca2+] reduction in CPVT VMs and unexpectedly high IMS-[Ca2+] promotes IMS-calpain–mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling.

scholarly journals Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins

2006 ◽  
Vol 174 (3) ◽  
pp. 379-390 ◽  
Author(s):  
Steven M. Claypool ◽  
J. Michael McCaffery ◽  
Carla M. Koehler
Keyword(s):  
Point Mutations ◽  
Mutant Gene ◽  
Barth Syndrome ◽  
Membrane Association ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Mitochondrial Membranes ◽  
Membrane Bilayer ◽  
Conserved Residues ◽  
Tm Domain

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215–232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.

scholarly journals Structural defects in rat liver deoxyribonucleic acid. Endogenous single-strained regions in comparison with damage induced in vivo by a carcinogen

1979 ◽  
Vol 179 (2) ◽  
pp. 341-352 ◽  
Author(s):  
B W Stewart ◽  
P H Huang ◽  
M J Brian
Keyword(s):  
Rat Liver ◽  
Structural Damage ◽  
Deae Cellulose ◽  
Minor Component ◽  
Structural Defects ◽  
Structural Abnormality ◽  
Denaturation Kinetics ◽  
Limited Digestion ◽  
A Minor

Rat liver DNA may be separated into two fractions by stepwise elution from benzoylated-DEAE-cellulose with NaCl and caffeine solutions respectively. Other studies using bacterical and yeast DNA suggested that the first fraction contains native DNA, whereas the second may exhibit some degree of single-stranded character. In the present experiments, chromatography of DNA was monitored by labelling in vivo with [methyl-3H]thymidine in rats previously subjected to partial hepatectomy. In animals killed up to 1 h after thymidine injection, radioactivity eluted in the second fraction was inversely related to the incorporation time, being greatest when animals were killed 10 min after radioisotope injection. However, for most experiments, animals were allowed to survive 2-4 weeks after surgery before use, analysis being made on non-dividing DNA. Under these conditions, the proportion of caffeine-eluted DNA was decreased by subjecting the preparation to shear, before chromatography. A procedure that resulted in 12% of the recovered radioactivity being eluted with caffeine was adopted for experiments involving comparisons of the two DNA fractions. Under these conditions, cross-contamination could be detected by rechromatography, but this did not preclude distinction being made between the two fractions in terms of DNA structure. NaCl-eluted DNA did not bind to nitrocellulose filters. Caffeine-eluted DNA was retained by the filters and released by washing with 3mM-Tris/HCl, pH9.4. The fractions did not differ in terms of isopycnic centrifugation in CsCl. The NaCl-eluted fraction migrated as a single band in polyacrylamide gels, and this pattern was not modified by prior digestion with Neurospora crassa endonuclease. In contrast, caffeine-eluted DNA contained a minor component having a wide molecular-weight distribution and was subject to limited digestion by the endonuclease. The kinetics of denaturation of NaCi-eluted DNA in the presence of formaldehyde, in common with unfractionated DNA, were consistent with double-stranded structure. The same analysis of caffeine-eluted DNA revealed structural abnormality equivalent to two defects per 10000 base-pairs. The data are consistent with the minor fraction of rat liver DNA, separated by using benzoylated-DEAE-cellulose, containing regions of local denaturation. We previously showed that administration of the hepatocarcinogen dimethylnitrosamine is associated with an increase in the proportion of caffeine-eluted DNA. In terms of most analysis, differences between DNA fraction from nitrosamine-treated rats were similar to differences exhibited by preparations from control animals. However, structural analysis using denaturation kinetics indicated defects in both the NaCl- and caffeine-eluted DNA isolated from nitrosamine-treated rats. The two fractions differed from each other in that caffeine-eluted DNA exhibited a degree of structural damage far greater than that detected in any preparation from control animals...

scholarly journals STRUCTURAL CHANGES IN ISOLATED LIVER MITOCHONDRIA OF RATS DURING ESSENTIAL FATTY ACID DEFICIENCY

1964 ◽  
Vol 21 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Janet A. Smith ◽  
Hector F. DeLuca
Keyword(s):  
Atpase Activity ◽  
Structural Damage ◽  
Structural Changes ◽  
Essential Fatty Acids ◽  
Saturated Fat ◽  
Respiratory Control ◽  
Liver Mitochondria ◽  
Structural Defects ◽  
Thin Sections ◽  
Fatty Acid Deficiency

Liver mitochondria isolated in 0.44 M sucrose from rats deficient in essential fatty acids (EFA) oxidized citrate, succinate, α-ketoglutarate, glutamate, and pyruvate at a faster rate than did mitochondria isolated from normal rats; however, the oxidation of malate, caprylate, and ß-hydroxybutyrate was not significantly increased. The mitochondria from deficient rats exhibited an increased ATPase activity and extensive structural damage as revealed by electron microscope examination of thin sections. An increase in citrate oxidation and ATPase activity, together with some structural damage, could be demonstrated as early as the 4th week in rats on a fat-free diet. Saturated fat in the diet did not prevent the change in mitochondrial structure but accelerated its appearance. Both the biochemical and structural defects could be reversed within three weeks after feeding deficient rats a source of EFA. In the presence of a phosphate acceptor the effect of EFA deficiency on substrate oxidation was largely eliminated. A trend toward a reduced efficiency of oxidative phosphorylation was noted in mitochondria from EFA-deficient rats, but significant uncoupling was found only in the case of citrate, ß-hydroxybutyrate, and glutamate in the presence of malonate. Together with the increased ATPase activity, the uncoupling of phosphorylation could account for the poor respiratory control found with the deficient preparation. However, EFA deficiency was without effect on the respiration of liver slices, which supports the belief that the observed changes in oxidation and phosphorylation are an artifact resulting from damage sustained by the deficient mitochondria during their isolation.

Anisotropic diffusion based denoising on concrete images and surface crack segmentation

2019 ◽  
Vol 11 (3) ◽  
pp. 395-409
Author(s):  
Diana Andrushia ◽  
N. Anand ◽  
Prince Arulraj
Keyword(s):  
Health Monitoring ◽  
Anisotropic Diffusion ◽  
Structural Damage ◽  
Performance Metrics ◽  
Research Work ◽  
Structural Similarity ◽  
Structural Defects ◽  
Content Type ◽  
Diffusion Filter ◽  
Anisotropic Diffusion Filter

Purpose Health monitoring of concrete is one of the important tasks in the structural health monitoring. The life of any infrastructure relies on the quality of the concrete. The computer vision-based methods are very useful to identify the structural defects. The identification of minor cracks in the noisy concrete image is complex. The purpose of this paper is to denoise the concrete crack images and also segment the cracks. Design/methodology/approach The novelty of the proposed work lies on the usage of anisotropic diffusion filter in the noisy concrete images. Initially anisotropic diffusion filter is applied to smoothen the concrete images. Adaptive threshold and gray level-based edge stopping constant are used in the diffusion process. The statistical six sigma-based method is utilized to segment the cracks from smoothened concrete images. Findings The proposed method is compared with five state-of-the-art-methods with the performance metrics of mean square error, peak signal to noise ratio and mean structural similarity. The experimental results highlight the advantages of the proposed method. Originality/value The novelty of the proposed work lies on the usage of anisotropic diffusion filter in the noisy concrete images. This research work gives the scope for structural damage evaluation by the automation techniques.

Acoustic impact testing and waveform analysis for damage detection in glued laminated timber

Holzforschung ◽  
2017 ◽  
Vol 71 (10) ◽  
pp. 801-811 ◽  
Author(s):  
Feng Xu ◽  
Xiping Wang ◽  
Marko Teder ◽  
Yunfei Liu
Keyword(s):  
Damage Detection ◽  
Structural Damage ◽  
Wavelet Packet ◽  
Acoustic Signals ◽  
Impact Testing ◽  
Waveform Analysis ◽  
Structural Defects ◽  
Glued Laminated Timber ◽  
Acoustic Impact ◽  
First Moment

AbstractDelamination and decay are common structural defects in old glued laminated timber (glulam) buildings, which, if left undetected, could cause severe structural damage. This paper presents a new damage detection method for glulam inspection based on moment analysis and wavelet transform (WT) of impact acoustic signals. Acoustic signals were collected from a glulam arch section removed from service through impact testing at various locations. The presence and positions of internal defects were preliminarily determined by applying time centroid and frequency centroid of the first moment. Acoustic signals were then decomposed by wavelet packet transform (WPT) and the energy of the sub-bands was calculated as characteristics of the response signals. The sub-bands of 0–375 Hz and 375–750 Hz were identified as the most discriminative features that are associated with decay and delamination and therefore are indicative of the presence of delamination or decay defects. A defect diagnosis algorithm was tested for its ability to identify internal decay and delamination in glulam. The results show that depth of delamination in a glulam member can be determined with reasonable accuracy.

scholarly journals Mutations in type I collagen genes resulting in osteogenesis imperfecta in humans.

2002 ◽  
Vol 49 (2) ◽  
pp. 433-441 ◽  
Author(s):  
Anna Gajko-Galicka
Keyword(s):  
Osteogenesis Imperfecta ◽  
Bone Disease ◽  
Type I Collagen ◽  
Wide Spectrum ◽  
Dominant Negative ◽  
Structural Defects ◽  
Collagen Molecule ◽  
Type I ◽  
Structural Protein ◽  
Collagen Genes

Osteogenesis imperfecta (OI), commonly known as "brittle bone disease", is a dominant autosomal disorder characterized by bone fragility and abnormalities of connective tissue. Biochemical and molecular genetic studies have shown that the vast majority of affected individuals have mutations in either the COL1A1 or COL1A2 genes that encode the chains of type I procollagen. OI is associated with a wide spectrum of phenotypes varying from mild to severe and lethal conditions. The mild forms are usually caused by mutations which inactivate one allele of COL1A1 gene and result in a reduced amount of normal type I collagen, while the severe and lethal forms result from dominant negative mutations in COL1A1 or COL1A2 which produce structural defects in the collagen molecule. The most common mutations are substitutions of glycine residues, which are crucial to formation and function of the collagen triple helix, by larger amino acids. Although type I collagen is the major structural protein of both bone and skin, the mutations in type I collagen genes cause a bone disease. Some reports showed that the mutant collagen can be expressed differently in bone and in skin. Since most mutations identified in OI are dominant negative, the gene therapy requires a fundamentally different approach from that used for genetic-recessive disorders. The antisense therapy, by reducing the expression of mutant genes, is able to change a structural mutation into a null mutation, and thus convert severe forms of the disease into mild OI type I.

scholarly journals Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes

2019 ◽  
Vol 30 (9) ◽  
pp. 1587-1603 ◽  
Author(s):  
Onur K. Polat ◽  
Masatoshi Uno ◽  
Terukazu Maruyama ◽  
Ha Nam Tran ◽  
Kayo Imamura ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Structural Damage ◽  
Coiled Coil ◽  
Nonselective Cation Channel ◽  
Gain Of Function ◽  
Function Mechanism ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Trpc6 Channel ◽  
Amplitude Amplification

BackgroundTRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.MethodsWe performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.ResultsBoth lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton.ConclusionsThe gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC’s coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

Abstract 103: MCL-1 Matrix Promotes Mitochondrial Fusion and Protects Against Mitophagy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Leonardo J Leon ◽  
Alexandra G Moyzis ◽  
Asa B Gustafsson
Keyword(s):  
Energy Demand ◽  
Functional Role ◽  
Mitochondrial Morphology ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Matrix ◽  
Functional Importance ◽  
Protein Ratio ◽  
Induced Fission ◽  
Significant Activation

MCL-1 is a less well characterized anti-apoptotic BCL-2 protein which is highly expressed in the myocardium. We recently discovered that loss of MCL-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and development of heart failure. MCL-1 exists both in the outer mitochondrial membrane (MCL-1 OM ) and in the mitochondrial matrix (MCL-1 Matrix ). Because the Mcl-1 -/- knockout mice are missing both MCL-1 Matrix and MCL-1 OM , the functional importance of each form is still unknown. While studies have implicated MCL-1 OM in regulating apoptosis, very little is known about the functional role of MCL-1 Matrix . Here, we examined whether cardiac MCL-1 levels were altered in response to fasting. Our data show that the MCL-1 Matrix /MCL-1 OM protein ratio was significantly increased in mouse hearts after 24 and 48 h of fasting. This was mainly due to a decrease in MCL-1 OM , while MCL-1 Matrix levels were unchanged. We also confirmed that the reduction in MCL-1 OM did not lead to a significant activation of apoptosis. Mitochondria are responsive to alterations in cellular metabolism and culturing cells in “oxidative” media containing galactose or acetoacetate led to a significant increase in the MCL-1 Matrix /MCL-1 OM ratio due to a reduction in MCL-1 OM protein and to elongation of mitochondria. We also confirmed that the reduced MCL-1 OM levels did not result in activation of apoptosis. This suggests that the MCL-1 Matrix /MCL-1 OM ratio is altered in response to changes in metabolism and energy demand without activating apoptosis. We have started dissecting how altering the MCL-1 Matrix /MCL-1 OM ratio affects mitochondrial morphology and turnover. We confirmed that MCL-1 Matrix overexpression led to more fused mitochondria under baseline conditions. Interestingly, we also found that the elongated mitochondria were also more protected against FCCP-induced fission as well as degradation by autophagosomes. Thus, we propose that MCL-1 Matrix protects mitochondria from unnecessary mitophagy to preserve the bioenergetic capacity when nutrients are limited. We are currently evaluating how cardiac restricted overexpression of MCL-1 Matrix (αMHC-MCL-1 Matrix ) affects mitochondrial morphology, mitophagy and bioenergetics during fasting.

scholarly journals Absence of the iron-containing superoxide dismutase in mitochondria from mustard (Brassica campestris)

1981 ◽  
Vol 195 (1) ◽  
pp. 229-233 ◽  
Author(s):  
M L Salin ◽  
S M Bridges
Keyword(s):  
Superoxide Dismutase ◽  
Brassica Campestris ◽  
Polyacrylamide Gel ◽  
Superoxide Dismutases ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Mature Leaves ◽  
Etiolated Seedlings

Mitochondria were isolated from mature leaves as well as etiolated seedlings of Brassica campestris (mustard), a eukaryote previously shown to possess the iron-containing isoenzyme of superoxide dismutase. On the basis of KCN- and H2O2-sensitivity, and on polyacrylamide-gel analysis, only the cuprozinc and mangano superoxide dismutases were found in mitochondria. The iron-containing enzyme was absent. The mangano enzyme was found in the mitochondrial matrix, whereas the cuprozinc enzyme appeared to be localized in the intermembrane space.

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