Abstract 5302: Control of Reversible Phosphorylation of Ryanodine Receptor by MiR-1 : Mechanism of Cardiac Arrhythmia

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Dmitry Terentyev ◽  
Andriy Belevych ◽  
Radmila Terentyeva ◽  
Donald E Kuhn ◽  
Geraldine E Malana ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Heart Association ◽  
Regulatory Subunit ◽  
Resting Cells ◽  
Cellular Mechanisms ◽  
Adult Rat ◽  
Disease States ◽  
Intracellular Ca ◽  
Regulate Protein

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1 , have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca cycling in adult rat ventricular myocytes by using methods of electrophysiology, confocal Ca imaging and quantitative immunoblotting. Adenoviral-mediated overexpressions of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca current and flatten cytosolic Ca transients voltage dependency. The frequency of spontaneous Ca sparks recorded in intact resting cells was enhanced, while the sarcoplasmic reticulum Ca content was reduced in miR-1 - overexpressing myocytes as compared with controls. In the presence of beta-adrenergic receptor agonist isoproterenol, rhythmically paced miR-1 -overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca, events that occurred only rarely in control myocytes. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Immunological analysis with phospho-specific antibodies showed that while phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor at Ser-2814 (CaMKII) but not at Ser-2808 (PKA). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 through translational inhibition of this mRNA target, causes CaMKII-dependent hyperphosphorylation of RyR2 via disrupting localization of PP2A activity to this channel, enhances RyR2 activity, and promotes arrhythmogenic SR Ca release. This mechanism could contribute to induction of arrhythmia in disease states accompanied by elevated miR-1 . This research has received full or partial funding support from the American Heart Association, AHA National Center.

scholarly journals cADP ribose and [Ca2+]iregulation in rat cardiac myocytes

2000 ◽  
Vol 279 (4) ◽  
pp. H1482-H1489 ◽  
Author(s):  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Timothy F. Walseth ◽  
Gary C. Sieck
Keyword(s):  
Electrical Stimulation ◽  
Sarcoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Real Time ◽  
Cardiac Myocytes ◽  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Adult Rat ◽  
Intracellular Ca ◽  
Rat Cardiac Myocytes

cADP ribose (cADPR)-induced intracellular Ca2+ concentration ([Ca2+]i) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1–10 μM) induced sustained, concentration-dependent [Ca2+]i responses ranging from 50 to 500 nM, which were completely inhibited by 20 μM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca2+]i waves, increasing concentrations of cADPR increased wave frequency up to ∼250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca2+]i transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca2+]i responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca2+]i responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca2+]i response. These results indicate that in cardiac myocytes, cADPR induces Ca2+ release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.

scholarly journals Arrhythmogenic consequences of intracellular calcium waves

2009 ◽  
Vol 297 (3) ◽  
pp. H997-H1002 ◽  
Author(s):  
Lai-Hua Xie ◽  
James N. Weiss
Keyword(s):  
Ventricular Myocytes ◽  
Polymorphic Ventricular Tachycardia ◽  
Early Afterdepolarizations ◽  
Digitalis Toxicity ◽  
Disease States ◽  
Intracellular Ca ◽  
Free Running ◽  
Rapid Pacing ◽  
Stimulation Current ◽  
A Charge

Intracellular Ca2+ (Cai2+) waves are known to cause delayed afterdepolarizations (DADs), which have been associated with arrhythmias in cardiac disease states such as heart failure, catecholaminergic polymorphic ventricular tachycardia, and digitalis toxicity. Here we show that, in addition to DADs, Cai2+ waves also have other consequences relevant to arrhythmogenesis, including subcellular spatially discordant alternans (SDA, in which the amplitude of the local Cai2+ transient alternates out of phase in different regions of the same cell), sudden repolarization changes promoting the dispersion of refractoriness, and early afterdepolarizations (EADs). Cai2+ was imaged using a charge-coupled device-based system in fluo-4 AM-loaded isolated rabbit ventricular myocytes paced at constant or incrementally increasing rates, using either field stimulation, current clamp, or action potential (AP) clamp. Cai2+ waves were induced by Bay K 8644 (50 nM) + isoproterenol (100 nM), or low temperature. When pacing was initiated during a spontaneous Cai2+ wave, SDA occurred abruptly and persisted during pacing. Similarly, during rapid pacing, SDA typically arose suddenly from spatially concordant alternans, due to an abrupt phase reversal of the subcellular Cai2+ transient in a region of the myocyte. Cai2+ waves could be visualized interspersed with AP-triggered Cai2+ transients, producing a rich variety of subcellular Cai2+ transient patterns. With free-running APs, complex Cai2+ release patterns were associated with DADs, EADs, and sudden changes in AP duration. These findings link Cai2+ waves directly to a variety of arrhythmogenic phenomena relevant to the intact heart.

scholarly journals Mechano-arrhythmogenicity is enhanced during late repolarisation in ischemia and driven by a TRPA1-, calcium-, and reactive oxygen species-dependent mechanism

2021 ◽  
Author(s):  
Breanne Ashleigh Cameron ◽  
T Alexander Quinn
Keyword(s):  
Reactive Oxygen Species ◽  
Ventricular Myocytes ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Cellular Mechanisms ◽  
Sustained Activity ◽  
Intracellular Ca ◽  
Rabbit Ventricular Myocytes ◽  
Induced Changes ◽  
Dependent Mechanism

Background: Cardiac dyskinesis in regional ischemia results in arrhythmias through mechanically-induced changes in electrophysiology ('mechano-arrhythmogenicity') that involve ischemic alterations in voltage-calcium (Ca2+) dynamics, creating a vulnerable period (VP) in late repolarisation. Objective: To determine cellular mechanisms of mechano-arrhythmogenicity in ischemia and define the importance of the VP. Methods and Results: Voltage-Ca2+ dynamics were simultaneously monitored in rabbit ventricular myocytes by dual-fluorescence imaging to assess the VP in control and simulated ischemia (SI). The VP was longer in SI than in control (146±7 vs 54±8 ms; p<0.0001) and was reduced by blocking KATP channels with glibenclamide (109±6 ms; p<0.0001). Cells were rapidly stretched (10-18% increase in sarcomere length over 110-170 ms) with carbon fibres during diastole or the VP. Mechano-arrhythmogenicity, associated with stretch and release in the VP, was greater in SI than control (7 vs 1% of stretches induced arrhythmias; p<0.005) but was similar in diastole. Arrhythmias during the VP were more complex than in diastole (100 vs 69% had sustained activity; p<0.05). In the VP, incidence was reduced with glibenclamide (2%; p<0.05), by chelating intracellular Ca2+ (BAPTA; 2%; p<0.05), blocking mechano-sensitive TRPA1 (HC-030031; 1%; p<0.005), or by scavenging (NAC; 1%; p<0.005) or blocking reactive oxygen species (ROS) production (DPI; 2%; p<0.05). Ratiometric Ca2+ imaging revealed that SI increased diastolic Ca2+ (+9±1%, p<0.0001), which was not prevented by HC-030031 or NAC. Conclusion: In ischemia, mechano-arrhythmogenicity is enhanced specifically during the VP and is mediated by ROS, TRPA1, and Ca2+.

Ca2+-induced Ca2+ release involved in positive inotropic effect mediated by CGRP in ventricular myocytes

1999 ◽  
Vol 276 (1) ◽  
pp. R259-R264 ◽  
Author(s):  
Ming-He Huang ◽  
Paul R. Knight ◽  
Joseph L. Izzo
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Ventricular Myocytes ◽  
Maximum Velocity ◽  
Ventricular Contractility ◽  
Adult Rat ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Pm 10 ◽  
Kinase A

To investigate the effects and mechanisms of calcitonin gene-related peptide (CGRP) on ventricular contractility, ventricular myocytes isolated from adult rat and mouse hearts were exposed to CGRP. Myocyte contractility was assessed by a video edge motion detector, and the intracellular [Ca2+] transients were measured by a spectroflurophotometer in fura 2-loaded myocytes. CGRP exerted a potent concentration-dependent (10 pM–10 nM, EC50 = 44.1 pM) positive inotropism on rat ventricular myocytes. CGRP (1 nM) increased cell shortening during contraction by 140 ± 40% above baselines and increased maximum velocity of contraction and relaxation by 98 and 106%, respectively. CGRP failed to produce any response in the presence of the CGRP1 receptor antagonist. CGRP induced similar inotropic response in mouse ventricular myocytes. CGRP increased the amplitude of [Ca2+] transients of ventricular myocytes by 120 ± 25% above baseline and shortened the time of half-maximum myoplasmic Ca2+ clearance by 30 ± 5%. Increase in intracellular Ca2+mobilization by CGRP was dependent on Ca2+ influx through the activation of the L-type Ca2+ channel, because nifedipine blocked the CGRP-induced increase in [Ca2+] transients. Furthermore, CGRP failed to increase [Ca2+] transients after the inhibition of protein kinase A in ventricular myocytes. These data indicate that stimulation of mammalian ventricular myocardial CGRP1 receptors enhances [Ca2+] transients through the activation of protein kinase A, which in turn activates voltage-dependent L-type Ca2+channels. These events lead to Ca2+-induced intracellular Ca2+ release and enhanced myocyte contraction and facilitated relaxation.

HCO 3 − -dependent alkalinizing transporter in adult rat ventricular myocytes: characterization and modulation

1997 ◽  
Vol 273 (6) ◽  
pp. H2596-H2603 ◽  
Author(s):  
Karine Le Prigent ◽  
Dominique Lagadic-Gossmann ◽  
Emmanuel Mongodin ◽  
Danielle Feuvray
Keyword(s):  
Ventricular Myocytes ◽  
Intracellular Acidification ◽  
Total Acid ◽  
Adult Rats ◽  
Adult Rat ◽  
Ventricular Cells ◽  
Intracellular Ca ◽  
Acid Load ◽  
Decreasing Ph ◽  
Intracellular Acid

The present work was designed to identify the [Formula: see text]-dependent alkalinizing carrier in ventricular myocytes of normal and diabetic adult rats and to determine to what extent this system contributes to acid-equivalent extrusion after an intracellular acidification. We also examined the possible influence of intracellular Ca2+([Formula: see text]) and glycolytic inhibition on the carrier activation. Intracellular pH (pHi) was recorded using seminaphthorhodafluor-1. The [Formula: see text] method was used to induce an intracellular acid load. Evidence is provided for the existence of a Cl−-independent Na+-[Formula: see text]cotransport contributing to pHirecovery from an intracellular acid load in ventricular cells of adult rats. Na+-[Formula: see text]cotransport accounts for 33% of the total acid-equivalent efflux ([Formula: see text]) from normal adult myocytes after intracellular acidification at pHi 6.75 in CO2/[Formula: see text]-buffered solution. In addition, the activity of this carrier, which is not affected either by decreasing [Formula: see text] or by inhibiting Ca2+/calmodulin protein kinase II, is downregulated by inhibition of glycolysis. Under pathophysiological conditions such as diabetes, although total[Formula: see text] was significantly decreased compared with normal myocytes,[Formula: see text] carried by Na+-[Formula: see text]cotransport remained unchanged. However, because of a decrease in Na+/H+exchange, the contribution of this carrier to total[Formula: see text] increased with decreasing pHi (i.e., under conditions that may be associated with an ischemic episode), reaching ∼58% of total[Formula: see text] at pHi 6.75 (vs. ∼33% in normal myocytes).

Caffeine-induced Ca2+ sparks in mouse ventricular myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. H666-H669 ◽  
Author(s):  
Michael Ritter ◽  
Zhi Su ◽  
Kenneth W. Spitzer ◽  
Hideyuki Ishida ◽  
William H. Barry
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cell Surface ◽  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Surface Membrane ◽  
Release Channel ◽  
Whole Cell ◽  
Cell Surface Membrane ◽  
Naturally Occurring ◽  
Intracellular Ca

Ca2+ sparks are spatially localized intracellular Ca2+ release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor, RyR) opening induced by Ca2+ influx via L-type Ca2+ channels or by spontaneous RyR openings and have been thought to reflect Ca2+ release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca2+ concentration ([Ca2+]i) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca2+]i transient consists of the summation of sparks and that Ca2+ sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca2+ channel-RyR relation determine spark properties.

Abstract 1508: Stim1-Mediated Store-Operated Calcium (SOC) Influx in Calcineurin-Dependent Cardiac Hypertrophy

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Xiang Luo ◽  
Samvit Tandan ◽  
Nan Jiang ◽  
Beverly A Rothermel ◽  
Joseph A Hill
Keyword(s):  
Ventricular Myocytes ◽  
Cyclopiazonic Acid ◽  
Heart Association ◽  
Cell Types ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Isolated Cells ◽  
Neonatal Cardiomyocytes ◽  
South Central ◽  
Intracellular Ca

Introduction : Calcineurin is activated by Ca 2+ /calmodulin and promotes pathological remodeling of the heart. In many cell types, store-operated calcium (SOC) entry, a process triggered by depletion of ER Ca stores, plays an obligate role in repleting Ca stores, and hence, in subsequent sustained increases in intracellular Ca. Little is known regarding SOC Ca influx in heart or its role in stress responsiveness. Methods and Results : Using RT-PCR, immunofluorescence staining, and Western blot methods, we found that Stim1, a recently identified molecular component of the SOC channel, is expressed in both neonatal rat ventricular myocytes (NRVMs) and adult myocytes. Working with isolated cells, we used Fura-2 to measure SOC Ca influx, finding that this mechanism does, in fact, exist in cardiomyocytes. However, SOC Ca influx was substantially more robust in neonatal myocytes than adult. Given that SOC current was prominent in neonatal cells, we tested ventricular myocytes isolated from calcineurin-transgenic mice, a model where the fetal gene program is prominently activated. Here, we found that Stim1 is up-regulated (1.9±0.4-fold, p<0.05). Treatment of NRVMs with angiotensin II (100nM) elicited similar increases in Stim1 (2.3±0.2-fold, p<0.01). To probe the molecular basis of cardiac SOC current, we expressed mutant (D76A, constitutively active; ΔERM, dominant negative) forms of human Stim1 in NRVMs. D76A markedly increased SOC Ca influx triggered by thapsigargin (2μM) or cyclopiazonic acid (50μM). In contrast, ΔERM blocked SOC Ca influx. Conclusion: SOC Ca entry exists prominently in neonatal cardiomyocytes but is down-regulated in adult cells. Stim1 participates in SOC current and is up-regulated during hypertrophic transformation of the myocardium. We conclude that SOC Ca currents are part of the fetal gene program in heart and may play a significant role in the sustained Ca overload of heart failure. This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).

scholarly journals The cellular force-frequency response in ventricular myocytes from the varanid lizard, Varanus exanthematicus

2010 ◽  
Vol 298 (3) ◽  
pp. R567-R574 ◽  
Author(s):  
Daniel E. Warren ◽  
Gina L. J. Galli ◽  
Simon M. Patrick ◽  
Holly A. Shiels
Keyword(s):  
Ventricular Myocytes ◽  
Fold Increase ◽  
Force Frequency ◽  
Cellular Mechanisms ◽  
Shortening Velocity ◽  
Frequency Dependent ◽  
Intracellular Ca ◽  
Force Frequency Response ◽  
Frequency Relationship

To investigate the cellular mechanisms underlying the negative force-frequency relationship (FFR) in the ventricle of the varanid lizard, Varanus exanthematicus , we measured sarcomere and cell shortening, intracellular Ca2+ ([Ca2+]i), action potentials (APs), and K+ currents in isolated ventricular myocytes. Experiments were conducted between 0.2 and 1.0 Hz, which spans the physiological range of in vivo heart rates at 20–22°C for this species. As stimulation frequency increased, diastolic length, percent change in sarcomere length, and relaxation time all decreased significantly. Shortening velocity was unaffected. These changes corresponded to a faster rate of rise of [Ca2+]i, a decrease in [Ca2+]i transient amplitude, and a seven-fold increase in diastolic [Ca2+]i. The time constant for the decay of the Ca2+ transient (τ) decreased at higher frequencies, indicating a frequency-dependent acceleration of relaxation (FDAR) but then reached a plateau at moderate frequencies and did not change above 0.5 Hz. The rate of rise of the AP was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization tended to decrease, but it was only significant at 1.0 Hz. The decrease in APD was not due to frequency-dependent changes in the delayed inward rectifier ( IKr) or the transient outward ( Ito) current, as neither appeared to be present in varanid ventricular myocytes. Our results suggest that a negative FFR relationship in varanid lizard ventricle is caused by decreased amplitude of the Ca2+ transient coupled with an increase in diastolic Ca2+, which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.

scholarly journals IP3R1 regulates Ca2+ transport and pyroptosis through the NLRP3/Caspase-1 pathway in myocardial ischemia/reperfusion injury

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Guixi Mo ◽  
Xin Liu ◽  
Yiyue Zhong ◽  
Jian Mo ◽  
Zhiyi Li ◽  
...  
Keyword(s):  
Myocardial Ischemia ◽  
Ischemia Reperfusion Injury ◽  
Cell Model ◽  
Ischemia Reperfusion ◽  
Inflammatory Factors ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Caspase 1 ◽  
Intracellular Ca ◽  
Myocardial Ischemia Reperfusion

AbstractIntracellular ion channel inositol 1,4,5-triphosphate receptor (IP3R1) releases Ca2+ from endoplasmic reticulum. The disturbance of IP3R1 is related to several neurodegenerative diseases. This study investigated the mechanism of IP3R1 in myocardial ischemia/reperfusion (MI/R). After MI/R modeling, IP3R1 expression was silenced in myocardium of MI/R rats to explore its role in the concentration of myocardial enzymes, infarct area, Ca2+ level, NLRP3/Caspase-1, and pyroptosis markers and inflammatory factors. The adult rat cardiomyocytes were isolated and cultured to establish hypoxia/reperfusion (H/R) cell model. The expression of IP3R1 was downregulated or ERP44 was overexpressed in H/R-induced cells. Nifedipine D6 was added to H/R-induced cells to block Ca2+ channel or Nigericin was added to activate NLRP3. IP3R1 was highly expressed in myocardium of MI/R rats, and silencing IP3R1 alleviated MI/R injury, reduced Ca2+ overload, inflammation and pyroptosis in MI/R rats, and H/R-induced cells. The binding of ERP44 to IP3R1 inhibited Ca2+ overload, alleviated cardiomyocyte inflammation, and pyroptosis. The increase of intracellular Ca2+ level caused H/R-induced cardiomyocyte pyroptosis through the NLRP3/Caspase-1 pathway. Activation of NLRP3 pathway reversed the protection of IP3R1 inhibition/ERP44 overexpression/Nifedipine D6 on H/R-induced cells. Overall, ERP44 binding to IP3R1 inhibits Ca2+ overload, thus alleviating pyroptosis and MI/R injury.

scholarly journals Cardiac small-conductance calcium-activated potassium channels in health and disease

2021 ◽  
Vol 473 (3) ◽  
pp. 477-489 ◽  
Author(s):  
Xiao-Dong Zhang ◽  
Phung N. Thai ◽  
Deborah K. Lieu ◽  
Nipavan Chiamvimonvat
Keyword(s):  
Ventricular Myocytes ◽  
Pulmonary Veins ◽  
Differential Sensitivity ◽  
Sk Channels ◽  
Sk Channel ◽  
Ventricular Cells ◽  
Intracellular Ca ◽  
Life Threatening ◽  
Health And Disease ◽  
Small Conductance

AbstractSmall-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

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