Abstract 1076: Conditional Fkbp12.6 Overexpression In Mouse Cardiac Myocytes Protects From Triggered Ventricular Arrhythmia Through Specific Alteration In Ec Coupling

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Barnabas Gellen ◽  
Ana-Maria Gomez ◽  
Khai Le Quang ◽  
Francois Briec ◽  
Laurent Vinet ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Antiarrhythmic Effect ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Ec Coupling ◽  
Before And After ◽  
Isolated Cardiac Myocytes ◽  
Pre Treatment ◽  
Specific Alteration ◽  
Ventricular Tachycardias

In cardiac myocytes, Ca2+ release from the sarcoplasmic reticulum (SR) into the cytoplasm via the ryanodine receptor (RyR2) activates cell contraction. During diastole, RyR2 are closed and prevent Ca2+ efflux from the SR. One of the major regulators of RyR2 function is FKBP12.6. Binding of FKBP12.6 stabilizes RyR2 in the closed formation in diastole and contributes to synchronized RyR2 opening in systole. Beta-adrenergic stimulation dissociates FKBP12.6 from RyR2, leading to diastolic Ca2+ leak that can trigger ventricular tachycardias (VT). We tested the hypothesis whether FKBP12.6 overexpression in the myocardium can reduce the susceptibility to VT in stress conditions. We developed a mouse model with conditional cardiac specific overexpression of FKBP12.6 using the Tet-Off system. Transgenic (TG) mice and controls (CT) were examined by echocardiography, PV-catheterization, ECG, and underwent intracardiac stimulation to trigger VT before and after pre-treatment with isoproterenol. In isolated cardiac myocytes, SR Ca2+ load, Ca2+ sparks and Ca2+ transient were measured using confocal microscopy, and L-type Ca2+ current was determined by the patch-clamp technique. Echocardiography, PV-catheterization and ECG recording did not reveal differences between Tg (n=11) and CT (n=13) mice. Burst pacing (figure 1) could induce TV in 4 of 24 controls and in 0 of 14 TG mice (n.s.). Following pre-treatment with isoproterenol, TV could be induced in 10 of 23 controls, but only in 1 of 14 TG animals (figure 2, p<0.05). In isolated myocytes, decreased Ca2+ spark frequency, increased Ca2+ spark size, unchanged SR Ca2+ load and decreased Ca2+ transient were observed in TG cells (n=19) as compared to controls (n=48, p<0.05). L-type Ca2+ channel current was found to be decreased in Tg myocytes (n=29 vs n=32, p<0.01). We conclude that myocardial FKBP12.6 overexpression has a protective effect against VT induced by rapid pacing after pretreatment with catecholamines. This antiarrhythmic effect is probably, at least in part, linked to decreased diastolic SR Ca2+ leak. Our results support the hypothesis that increased FKBP12.6 binding to RyR2 might represent a potential therapeutical target in the prevention and treatment of ventricular arrhythmias.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

scholarly journals inhibitor 2-[(4-methylphenyl)sulfonylcarbamido]-1-(4-nitrobenzyl)pyridinium bromide (2-AP27) is a muscarinic M2 receptor antagonist

Medicina ◽  
2009 ◽  
Vol 45 (7) ◽  
pp. 516
Author(s):  
Vytenis Skeberdis ◽  
Vida Gendvilienė ◽  
Danguolė Zablockaitė ◽  
Irma Martišienė ◽  
Antanas Stankevičius
Keyword(s):  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Inhibitory Effect ◽  
Patch Clamp Technique ◽  
Pyridinium Bromide ◽  
K Current ◽  
Isolated Cardiac Myocytes ◽  
Series Of Experiments ◽  
Muscarinic M2 Receptors ◽  
M2 Muscarinic Receptors

Aminopyridines are known to inhibit acetylcholine-activated K+ current (IKACh) in cardiac myocytes. The aim of this study was to examine the effect of 2-aminopyridine sulfonylcarbamide derivative 2-AP27 on isoprenaline-stimulated L-type Ca2+ current (ICaL) and to identify whether 2-AP27 acts via blocking of muscarinic M2-receptors in frog cardiomyocytes. The whole-cell configuration of the patch-clamp technique was used to record ICaL in enzymatically isolated cardiac myocytes. Isoprenaline (0.1 μM), an agonist of β1-β2-adrenoreceptors, stimulated the ICaL up to 475±61% (n=4) (P<0.05) vs. control. Then, in the first series of experiments, carbachol (0.01 μM), an agonist of M2 muscarinic receptors, reduced the stimulatory effect of isoprenaline to 42±15% vs. isoprenaline alone. 2- AP27 (100 μM) alone completely abolished the inhibitory effect of carbachol on isoprenaline-stimulated ICaL, which recovered to 95±5.8% of the effect of isoprenaline. In the second series of experiments, adenosine (1 μM), an agonist of A1-adenosine receptors, reduced the stimulatory effect of isoprenaline on ICaL to 56±10% (n=3) (P<0.05). Then 2-AP27 (100 μM) applied in the presence of adenosine, had no effect on ICaL, which remained at 51±7.9% (n=3) (P<0.05) of the effect of isoprenaline. These results suggest that 2-AP27, a new derivative of 2-AP, containing 4-toluolsulfonylcarbamide instead of amino group and quaternizated nitrogen by 4-nitrobenzylbromide in pyridine ring, is acting as an antagonist of muscarinic M2 receptors in frog ventricular myocytes.

scholarly journals Loss of Mitochondrial Ca 2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy

Circulation ◽  
2021 ◽  
Author(s):  
Edoardo Bertero ◽  
Alexander Nickel ◽  
Michael Kohlhaas ◽  
Mathias Hohl ◽  
Vasco Sequeira ◽  
...  
Keyword(s):  
Ejection Fraction ◽  
Diastolic Dysfunction ◽  
Cardiac Myocytes ◽  
Barth Syndrome ◽  
Integrated Analysis ◽  
Preserved Ejection Fraction ◽  
Adrenergic Stimulation ◽  
Ec Coupling ◽  
Qrs Prolongation

Background: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Since mitochondrial function and ROS formation are regulated by excitation-contraction (EC) coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. Methods: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin ( Taz -KD) compared to wild-type (WT) littermates. Respiratory chain assembly and function, ROS emission, and Ca 2+ uptake were determined in isolated mitochondria. EC coupling was integrated with mitochondrial redox state, ROS, and Ca 2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo . Results: Taz -KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca 2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, partly compensated by accelerated diastolic Ca 2+ decay through preactivated sarcoplasmic reticulum Ca 2+ ATPase (SERCA). Taz deficiency provoked heart-specific loss of mitochondrial Ca 2+ uniporter (MCU) protein that prevented Ca 2+ -induced activation of the Krebs cycle during β-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo , Taz -KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to β-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca 2+ export via the mitochondrial Na + /Ca 2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo . Conclusions: Downregulation of MCU, increased myofilament Ca 2+ affinity, and preactivated SERCA provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca 2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

Alpha-adrenergic regulation of phosphoinositide metabolism and protein kinase C in isolated cardiac myocytes

1991 ◽  
Vol 260 (3) ◽  
pp. C635-C642 ◽  
Author(s):  
T. Kaku ◽  
E. Lakatta ◽  
C. Filburn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Inositol Phosphates ◽  
Total Activity ◽  
Phosphoinositide Metabolism ◽  
Adrenergic Stimulation ◽  
Adrenergic Regulation ◽  
Kinase C ◽  
Isolated Cardiac Myocytes

alpha 1-Adrenergic regulation of phosphoinositide metabolism and protein kinase C translocation was studied in isolated rat cardiac myocytes. Exposure of [3H]inositol-labeled myocytes to norepinephrine in the presence of propranolol caused a dose-dependent increase in [3H]inositol phosphates. Norepinephrine also increased the level of membrane-associated protein kinase C from approximately 10% of total activity to 18%, with a dose response similar to that for generation of inositol phosphates. Depolarization of myocytes with 30 mM KCl had no effect on inositol phosphates or membrane-associated protein kinase C but potentiated the effect of submaximal norepinephrine on both parameters. The potentiation of protein kinase C translocation was amplified when extracellular Ca2+ was increased to 4 mM, resulting in membrane association of one-third of the total cellular activity. These data show that activation of protein kinase C occurs during alpha 1-adrenergic stimulation of cardiac myocytes and that elevation of intracellular Ca2+ amplifies this effect at least in part through increased phosphoinositide metabolism.

Regulation of coronary vascular tone via redox modulation in the α1-adrenergic-angiotensin-endothelin axis of the myocardium

2009 ◽  
Vol 296 (1) ◽  
pp. H226-H232 ◽  
Author(s):  
Osamu Yamaguchi ◽  
Takashi Kaneshiro ◽  
Shu-ichi Saitoh ◽  
Toshiyuki Ishibashi ◽  
Yukio Maruyama ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Receptor Antagonist ◽  
Cardiac Myocytes ◽  
Chain Complex ◽  
Oxidase Inhibitor ◽  
Vascular Cells ◽  
Adrenergic Stimulation ◽  
Transport Chain ◽  
Isolated Cardiac Myocytes ◽  
Coronary Arterioles

We hypothesized that α1-adrenoceptor stimulation of cardiac myocytes results in the production of an endothelin (ET)-releasing factor that stimulates the coronary vasculature to release ET and, by manipulating the redox state of cardiac and vascular cells, may influence the extent of α1-adrenergic-ET-1 vasoconstriction. Dihydroethidium (DHE) and dichlorodihydrofluorescein (DCF) intensities were increased by phenylephrine stimulation in isolated rat cardiac myocytes, which were enhanced by the mitochondrial electron transport chain complex I inhibitor rotenone (DHE: 20.4 ± 1.2-fold and DCF: 25.2 ± 0.9-fold, n = 8, P < 0.01, respectively) but not by the NADPH oxidase inhibitor apocynin. Olmesartan, an angiotensin II type 1 receptor antagonist, and enalaprilate did not change DHE and DCF intensities by phenylephrine. Next, we measured the vasoconstriction of isolated, pressurized rat coronary arterioles (diameter: 74 ± 8 μm) in response to supernatant collected from isolated cardiac myocytes. The addition of supernatant from phenylephrine-stimulated myocytes to a 2-ml vessel bath ( n = 8 each) caused volume-dependent vasoconstriction (500 μl: −14.8 ± 2.2%). Olmesartan and TA0201, an ET type A receptor antagonist, converted vasoconstriction into vasodilation (8.5 ± 1.2% and 10.5 ± 0.5%, P < 0.01, respectively) in response to supernatant from phenylephrine-stimulated myocytes, which was eliminated with catalase. Vasoconstriction was weakened using supernatant from phenylephrine with rotenone-treated myocytes. Treatment of arterioles with apocynin to myocyte supernatant converted vasoconstriction into vasodilation (7.8 ± 0.8%, P < 0.01). These results suggest that α1-adrenergic stimulation in cardiac myocytes produces angiotensin I and H2O2 and that angiotensin releases ET-1 through NADPH oxidase in coronary arterioles. Thus, coronary vasoconstriction via the α-adrenergic-angiotensin-ET axis appears to require redox-mediated signaling in cardiac and vascular cells.

scholarly journals Metabolic Inhibition Induces Transient Increase of L-type Ca2+ Current in Human and Rat Cardiac Myocytes

2019 ◽  
Vol 20 (6) ◽  
pp. 1501 ◽  
Author(s):  
Rimantas Treinys ◽  
Giedrius Kanaporis ◽  
Rodolphe Fischmeister ◽  
Jonas Jurevičius
Keyword(s):  
Cardiac Myocytes ◽  
Early Phase ◽  
Metabolic Inhibition ◽  
Metabolic Inhibitors ◽  
Initial Increase ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Rat Cardiomyocytes ◽  
Wide Range ◽  
Rat Cardiac Myocytes

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-adrenergic receptors. The whole-cell patch–clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.

scholarly journals Effects of catecholamines on protein synthesis in cardiac myocytes and perfused hearts isolated from adult rats. Stimulation of translation is mediated through the α1-adrenoceptor

1990 ◽  
Vol 266 (3) ◽  
pp. 727-736 ◽  
Author(s):  
S J Fuller ◽  
C J Gaitanaki ◽  
P H Sugden
Keyword(s):  
Protein Synthesis ◽  
Cardiac Myocytes ◽  
Adrenergic Stimulation ◽  
Adult Rats ◽  
Kinase C ◽  
Isolated Cardiac Myocytes ◽  
Perfused Hearts ◽  
Intracellular Alkalinization ◽  
Adrenergic Effects ◽  
Stimulation Of

Protein-synthesis rates in freshly isolated cardiac myocytes from adult rats were acutely stimulated by 20-30% by 1 microM-adrenaline, by 1 microM-noradrenaline or by 1 microM-phenylephrine, but were not stimulated by 1 microM-isoprenaline. Stimulation by 1 microM-adrenaline was completely prevented by 100 nM-prazosin. Yohimbine was much less effective in preventing stimulation, and 20 microM-DL-propranolol was completely ineffective. The stimulation of protein synthesis by adrenaline was still observed after inhibition of transcription by actinomycin D. None of these manipulations affected myocyte ATP contents. In anterogradely perfused hearts, protein-synthesis rates were stimulated by 1-2 microM-adrenaline in the presence of 10 microM-DL-propranolol (to decrease the beta-adrenergic effects of adrenaline). ATP contents were not altered, but phosphocreatine contents were increased. These observations lead us to conclude that cardiac protein synthesis can be stimulated acutely at the level of translation by alpha 1-adrenergic stimulation. We discuss possible roles for protein kinase C and intracellular alkalinization in the mediation of this effect.

Cytoprotective Effect of Tocotrienol-Rich Fraction and α-Tocopherol Vitamin E Isoforms Against Glutamate-Induced Cell Death in Neuronal Cells

2014 ◽  
Vol 84 (3-4) ◽  
pp. 0140-0151 ◽  
Author(s):  
Thilaga Rati Selvaraju ◽  
Huzwah Khaza’ai ◽  
Sharmili Vidyadaran ◽  
Mohd Sokhini Abd Mutalib ◽  
Vasudevan Ramachandran ◽  
...  
Keyword(s):  
Vitamin E ◽  
Cell Viability ◽  
Neuronal Cells ◽  
Positive Control ◽  
Post Treatment ◽  
Mitochondrial Activity ◽  
Before And After ◽  
Pre Treatment ◽  
Tocotrienol Rich Fraction ◽  
Major Mediator

Glutamate is the major mediator of excitatory signals in the mammalian central nervous system. Extreme amounts of glutamate in the extracellular spaces can lead to numerous neurodegenerative diseases. We aimed to clarify the potential of the following vitamin E isomers, tocotrienol-rich fraction (TRF) and α-tocopherol (α-TCP), as potent neuroprotective agents against glutamate-induced injury in neuronal SK-N-SH cells. Cells were treated before and after glutamate injury (pre- and post-treatment, respectively) with 100 - 300 ng/ml TRF/α-TCP. Exposure to 120 mM glutamate significantly reduced cell viability to 76 % and 79 % in the pre- and post-treatment studies, respectively; however, pre- and post-treatment with TRF/α-TCP attenuated the cytotoxic effect of glutamate. Compared to the positive control (glutamate-injured cells not treated with TRF/α-TCP), pre-treatment with 100, 200, and 300 ng/ml TRF significantly improved cell viability following glutamate injury to 95.2 %, 95.0 %, and 95.6 %, respectively (p < 0.05).The isomers not only conferred neuroprotection by enhancing mitochondrial activity and depleting free radical production, but also increased cell viability and recovery upon glutamate insult. Our results suggest that vitamin E has potent antioxidant potential for protecting against glutamate injury and recovering glutamate-injured neuronal cells. Our findings also indicate that both TRF and α-TCP could play key roles as anti-apoptotic agents with neuroprotective properties.

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