scholarly journals Suppression of snake-venom cardiotoxin-induced cardiomyocyte degeneration by blockage of Ca2+ influx or inhibition of non-lysosomal proteinases

1988 ◽  
Vol 256 (1) ◽  
pp. 89-95 ◽  
Author(s):  
W F Tzeng ◽  
Y H Chen
Keyword(s):  
Latent Period ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Neonatal Rat ◽  
Cell Degeneration ◽  
Acetoxymethyl Ester ◽  
Calmodulin Antagonist ◽  
Rat Cardiomyocytes ◽  
Fura 2 ◽  
Bath Medium

The incubation of 10(5) single neonatal rat cardiomyocytes with 1 microM-cardiotoxin in a bath medium, Tyrode solution in the presence of 1 mM-Ca2+, at 37 degrees C evoked the following chain of events. Firstly, there appeared a latent period of about 10 min during which the cells behaved normally. Neither lactate dehydrogenase nor ATP leaked from the cells. Cytosolic free Ca2+ increased considerably, as measured by the fluorescence intensity of fura-2-Ca2+ complex. At the same time a large portion of endogenous ATP was depleted. Secondly, after the latent period, the cell beating became irregular and eventually stopped. Thirdly, blebs appeared on the cell surface, leading to cell degeneration. If, before the appearance of blebs, the cells were washed with the bath medium exhaustively or incubated in the presence of the toxin antibody, cytosolic free Ca2+ and endogenous ATP returned to normal levels and cells resumed regular beating. Preincubation of the cells with 3.75 microM-flunarizine or 3.75 microM-diltiazem (both are Ca2+ antagonists), or 1.5 microM-fura-2 acetoxymethyl ester (a chelate for Ca2+), or 200 microM-leupeptin or 50 microM-antipain (both are proteinase inhibitors) considerably suppressed the toxin's ability to degenerate the cells. On the other hand, lysosomal proteinase inhibitor, autophage inhibitor, serine proteinase inhibitor, phospholipase inhibitor and calmodulin antagonist did not inhibit the toxin's activity. The results suggest that the toxin may act on the extracellular surface of intact cardiomyocytes to increase cytosolic free Ca2+. The subsequent cell degeneration may result from the activation of a Ca2+-dependent non-lysosomal proteolytic system.

Endothelin-stimulated glucose uptake: effects of intracellular Ca2+, cAMP and glucosamine

2002 ◽  
Vol 103 (s2002) ◽  
pp. 418S-423S ◽  
Author(s):  
Jinshyun R. WU-WONG ◽  
Cathleen E. BERG ◽  
Brian D. DAYTON
Keyword(s):  
Glucose Uptake ◽  
Biosynthetic Pathway ◽  
Neonatal Rat ◽  
Intracellular Camp ◽  
Acetoxymethyl Ester ◽  
Hexosamine Biosynthetic Pathway ◽  
Rat Cardiomyocytes ◽  
Amino Acid Peptide ◽  
Neonatal Rat Cardiomyocytes ◽  
Camp Levels

Endothelin-1 (ET-1) is a 21-amino-acid peptide that binds to G-protein-coupled receptors to evoke biological responses. Previously we have shown that ET-1 stimulates glucose uptake in 3T3-L1 adipocytes and neonatal rat cardiomyocytes, but the mechanism is not completely understood. ET-1 is known to modulate intracellular Ca2+ and cAMP levels. Depletion of intracellular Ca2+ by treating 3T3-L1 adipocytes with EDTA and 1,2-bis(2-amino-5-methylphenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra-acetoxymethyl ester (MAPTAM) did not have a significant effect on ET-1-induced glucose uptake. Forskolin, a potent stimulator which stimulates adenylate cyclase and increases the intracellular cAMP level, partially inhibited insulin-stimulated glucose uptake in 3T3-L1 cells, but had no significant impact on the effect of ET-1. Forskolin also did not show an effect on the tyrosine phosphorylation of a 75kDa protein induced by ET-1. Glucosamine treatment causes insulin resistance in cells, possibly by entering the hexosamine biosynthetic pathway. In neonatal rat cardiomyocytes, glucosamine treatment blocked both insulin and ET-1-stimulated glucose uptake and also eliminated the translocation of IRAP, an aminopeptidase in GLUT4-containing vesicles, from the cytoplasm to the plasma membrane. These results suggest that ET-1-induced glucose uptake is independent of its effects on modulating intracellular Ca2+ and cAMP levels, but is likely linked to the hexosamine biosynthetic pathway.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

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.

Cross-bridge cycling gives rise to spatiotemporal heterogeneity of dynamic subcellular mechanics in cardiac myocytes probed with atomic force microscopy

2010 ◽  
Vol 298 (3) ◽  
pp. H853-H860 ◽  
Author(s):  
Evren U. Azeloglu ◽  
Kevin D. Costa
Keyword(s):  
Contractile Activity ◽  
Major Axis ◽  
Neonatal Rat ◽  
Pharmacological Treatments ◽  
Rat Cardiomyocytes ◽  
Force Microscopy ◽  
Cross Bridge ◽  
Apparent Modulus ◽  
Atomic Force ◽  
And Function

To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 ± 5.1 kPa and a diastolic value of 7.8 ± 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 ± 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function.

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