In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine ...receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The role of I(KCa) in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular ...electrophysiologic effects of I(KCa) blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The I(KCa) blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by I(KCa) blockade in all of the groups studied. I(KCa) blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. I(KCa) blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, I(KCa) blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for I(KCa) in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current ...preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.
Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in ...HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic (Cac) and sarcoplasmic reticulum (SR) luminal Ca (CaSR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca leak was significantly enhanced in permeabilized myocytes, resulting in reduced CaSR in HF compared to control cells. Both SR Ca leak and CaSR were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca leak and decreased CaSR in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca and was partially normalized by reducing agents through restoring luminal Ca sensitivity oxidation of control RyR2s enhanced their luminal Ca sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.
RATIONALE:Diastolic spontaneous Ca waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when Ca in sarcoplasmic reticulum (CaSR) reaches a certain ...threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca.
OBJECTIVE:We investigated the mechanisms of DCW generation in myocytes from normal and diseased hearts, using a canine model of post–myocardial infarction ventricular fibrillation (VF).
METHODS AND RESULTS:The frequency of DCWs, recorded during periodic pacing in the presence of a β-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final CaSR, DCWs arose with a distinct time delay after attaining steady CaSR in both experimental groups. Although the rate of CaSR recovery after the SR Ca release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the CaSR at DCW initiation was lower. The restitution of depolarization-induced Ca transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca release in VF myocytes.
CONCLUSIONS:The attainment of a certain threshold CaSR is not sufficient for the generation of DCWs. Postrelease Ca signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca signaling refractoriness due to RyR2 phosphorylation and oxidation is responsible for the increased rate of DCWs observed in VF myocytes and could provide a substrate for synchronization of arrhythmogenic events at the tissue level in hearts prone to VF.
The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) ...required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodelling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.
In heart failure (HF), abnormal myocyte Ca(2+) handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca(2+) ...handling, reduced myocyte contractility, and enhanced arrhythmogenesis during HF progression in a canine model of non-ischaemic HF.
Key Ca(2+) handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) in isolated ventricular myocytes at different stages of HF. The progression of HF was associated with an early and continuous increase in ryanodine receptor (RyR2)-mediated SR Ca(2+) leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca(2+) waves (SCWs) in HF myocytes under conditions of β-adrenergic stimulation. In addition to causing arrhythmogenic-delayed afterdepolarizations, SCWs decreased the amplitude of subsequent electrically evoked Ca(2+) transients by depleting SR Ca(2+). At late stages of HF, Ca(2+) release oscillated essentially independent of electrical pacing. The increased propensity for the generation of SCWs in HF myocytes was attributable to reduced ability of the RyR2 channels to become refractory following Ca(2+) release. The progressive alterations in RyR2 function and Ca(2+) cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation.
These findings suggest that destabilized RyR2 activity due to excessive CaMKII phopshorylation and oxidation resulting in impaired post-release refractoriness is a common mechanism involved in arrhythmogenesis and contractile dysfunction in the failing heart.
Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca
homeostasis, impairing luminal Ca
regulation of the sarcoplasmic reticulum (SR) Ca
release channel, the RyR2 (ryanodine ...receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process.
A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca
and reactive oxygen species imaging in isolated ventricular myocytes (VMs).
The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca
transient amplitude and SR Ca
content, and reduced proarrhythmic spontaneous Ca
waves in TAB VMs under β-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca
-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca
-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44.
A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca
release and Ca
-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.
Atrial fibrillation (AF) may persist due to structural changes in the atria that are promoted by inflammation. C-reactive protein (CRP), a marker of systemic inflammation, predicts cardiovascular ...events and stroke, a common sequela of AF. We hypothesized that CRP is elevated in patients with atrial arrhythmias.
Using a case-control study design, CRP in 131 patients with atrial arrhythmias was compared with CRP in 71 control patients. Among arrhythmia patients, 6 had frequent atrial ectopy or tachycardia, 86 had paroxysmal AF, 39 had persistent AF lasting >30 days, and 70 had lone arrhythmias. CRP was higher in arrhythmia than in control patients (median, 0.21 versus 0.096 mg/dL; P<0.001). Arrhythmia patients in AF within 24 hours before sampling had higher CRP than those in sinus rhythm (0.30 versus 0.15 mg/dL; P<0.001). CRP in controls was not different than in patients with atrial ectopy or tachycardia. Lone arrhythmia patients had a CRP of 0.21 mg/dL, which was not significantly lower than arrhythmia patients with structural heart disease (CRP, 0.23 mg/dL) but higher than controls (P=0.002). Persistent AF patients had a higher CRP (0.34 mg/dL) than paroxysmal AF patients (0.18 mg/dL; P=0.008); both groups had higher CRP levels than controls (P</=0.005).
CRP is elevated in AF patients. This study is the first to document elevated CRP in non-postoperative arrhythmia patients. These findings are reinforced by stepwise CRP elevation with higher AF burden. Although the cause of elevated CRP levels in AF patients remains unknown, elevated CRP may reflect an inflammatory state that promotes the persistence of AF.
Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein ...phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate “global” PP2A function from the activities of multiple “local” holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.
Background: PP2A regulates cardiac excitability and physiology.
Results: PP2A regulation in heart occurs through integrative transcriptional, translational, and post-translational control of three classes of subunits (17 genes) to control holoenzyme synthesis, localization, and maintenance; pathways are mechanistically altered in heart disease.
Conclusion: Multiple mechanisms are present for acute and chronic regulation of specific PP2A populations.
Significance: Results provide molecular insight into cardiac PP2A regulation.
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