A Genomic Link From Heart Failure to Atrial Fibrillation Risk: FOG2 Modulates a TBX5/GATA4-Dependent Atrial Gene Regulatory Network.

BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.

The role of RyR2 oxidation in the blunted frequency-dependent facilitation of Ca2+ transient amplitude in rabbit failing myocytes.

Defective Ca2+ regulation plays a key role in the blunted force-frequency response in heart failure (HF). Since HF is commonly associated with oxidative stress, we studied whether oxidation of ryanodine receptor (RyR2) contributes to this defect. In control ventricular myocytes, oxidative stress induced formation of disulfide bonds between RyR2 subunits: intersubunit cross-linking (XL). Western blot analysis and Ca2+ imaging revealed a strong positive correlation between RyR2 XL and sarcoplasmic reticulum (SR) Ca2+ leak. These results illustrate that RyR2 XL can be used as a sensitive indicator of RyR2 dysfunction during oxidative stress. HF myocytes were in a state of oxidative stress since they exhibited an increase in reactive oxygen species (ROS) level, a decrease in ROS defense and an overall protein oxidation. These myocytes were also characterized by RyR2 XL and increased SR Ca2+ leak. Moreover, the frequency-dependent increase of Ca2+ transient amplitude was suppressed due to the inability of the SR to maintain Ca2+ load at high pacing rates. Because SR Ca2+ load is determined by the balance between SR Ca2+ uptake and leak, the blunted frequency-dependent inotropy in HF can be mediated by ROS-induced SR Ca2+ leak. Preventing RyR2 XL in HF myocytes decreased SR Ca2+ leak and increased Ca2+ transients at high pacing rate. We also studied whether RyR2 oxidation alone can cause the blunted frequency-dependent facilitation of Ca2+ transient amplitude in control myocytes. When RyR2 XL was induced in control myocytes to a similar level seen in HF, an increase of Ca2+ transient amplitude at high pacing rate was significantly suppressed. These results suggest that SR Ca2+ leak induced by RyR2 oxidation can play an important role in the blunted frequency-dependent inotropy of HF.

Functional Impact of Ryanodine Receptor Oxidation on Intracellular Calcium Regulation in the Heart.

Type 2 ryanodine receptor (RyR2) serves as the major intracellular Ca2+ release channel that drives heart contraction. RyR2 is activated by cytosolic Ca2+ via the process of Ca2+-induced Ca2+ release (CICR). To ensure stability of Ca2+ dynamics, the self-reinforcing CICR must be tightly controlled. Defects in this control cause sarcoplasmic reticulum (SR) Ca2+ mishandling, which manifests in a variety of cardiac pathologies that include myocardial infarction and heart failure. These pathologies are also associated with oxidative stress. Given that RyR2 contains a large number of cysteine residues, it is no surprise that RyR2 plays a key role in the cellular response to oxidative stress. RyR's many cysteine residues pose an experimental limitation in defining a specific target or mechanism of action for oxidative stress. As a result, the current understanding of redox-mediated RyR2 dysfunction remains incomplete. Several oxidative modifications, including S-glutathionylation and S-nitrosylation, have been suggested playing an important role in the regulation of RyR2 activity. Moreover, oxidative stress can increase RyR2 activity by forming disulfide bonds between two neighboring subunits (intersubunit cross-linking). Since intersubunit interactions within the RyR2 homotetramer complex dictate the channel gating, such posttranslational modification of RyR2 would have a significant impact on RyR2 function and Ca2+ regulation. This review summarizes recent findings on oxidative modifications of RyR2 and discusses contributions of these RyR2 modifications to SR Ca2+ mishandling during cardiac pathologies.

Oxidation of ryanodine receptor after ischemia-reperfusion increases propensity of Ca2+ waves during ß-adrenergic receptor stimulation.

ß-Adrenergic receptor (ß-AR) activation produces the main positive inotropic response of the heart. During ischemia-reperfusion (I/R), however, ß-AR activation can trigger life-threatening arrhythmias. Because I/R is frequently associated with oxidative stress, we investigated whether ryanodine receptor (RyR) oxidation contributes to proarrythmogenic Ca2+ waves during ß-AR activation. Measurements of contractile and electrical activity from Langendorff-perfused rabbit hearts revealed that I/R produces tachyarrhythmias. Ventricular myocytes isolated from I/R hearts had an increased level of oxidized glutathione (i.e., oxidative stress) and a decreased level of free thiols in RyRs (i.e., RyR oxidation). Furthermore, myocytes from I/R hearts were characterized by increased sarcoplasmic reticulum (SR) Ca2+ leak and enhanced fractional SR Ca2+ release. In myocytes from nonischemic hearts, ß-AR activation with isoproterenol (10 nM) produced only a positive inotropic effect, whereas in myocytes from ischemic hearts, isoproterenol at the same concentration triggered spontaneous Ca2+ waves. ß-AR activation produced a similar effect on RyR phosphorylation in control and I/R myocytes. Treatment of myocytes from I/R hearts with the reducing agent mercaptopropionylglycine (100 µM) attenuated RyR oxidization and decreased Ca2+ wave frequency during ß-AR activation. On the other hand, treatment of myocytes from nonischemic hearts with H2O2 (50 µM) increased SR Ca2+ leak and triggered Ca2+ waves during ß-AR activation. Collectively, these results suggest that RyR oxidation after I/R plays a critical role in the transition from positive inotropic to arrhythmogenic effects during ß-AR stimulation. Prevention of RyR oxidation can be a promising strategy to inhibit arrhythmias and preserve positive inotropic effect of ß-AR activation during myocardial infarction. NEW & NOTEWORTHY Oxidative stress induced by ischemia plays a critical role in triggering arrhythmias during adrenergic stimulation. The combined increase in sarcoplasmic reticulum Ca2+ leak (because of ryanodine receptor oxidation) and sarcoplasmic reticulum Ca2+ load (because of adrenergic stimulation) can trigger proarrythmogenic Ca2+ waves. Restoring normal ryanodine receptor redox status can be a promising strategy to prevent arrhythmias and preserve positive inotropic effect of adrenergic stimulation during myocardial infarction.

Increased Energy Demand during Adrenergic Receptor Stimulation Contributes to Ca(2+) Wave Generation.

While ß-adrenergic receptor (ß-AR) stimulation ensures adequate cardiac output during stress, it can also trigger life-threatening cardiac arrhythmias. We have previously shown that proarrhythmic Ca(2+) waves during ß-AR stimulation temporally coincide with augmentation of reactive oxygen species (ROS) production. In this study, we tested the hypothesis that increased energy demand during ß-AR stimulation plays an important role in mitochondrial ROS production and Ca(2+)-wave generation in rabbit ventricular myocytes. We found that ß-AR stimulation with isoproterenol (0.1 µM) decreased the mitochondrial redox potential and the ratio of reduced to oxidated glutathione. As a result, ß-AR stimulation increased mitochondrial ROS production. These metabolic changes induced by isoproterenol were associated with increased sarcoplasmic reticulum (SR) Ca(2+) leak and frequent diastolic Ca(2+) waves. Inhibition of cell contraction with the myosin ATPase inhibitor blebbistatin attenuated oxidative stress as well as spontaneous SR Ca(2+) release events during ß-AR stimulation. Furthermore, we found that oxidative stress induced by ß-AR stimulation caused the formation of disulfide bonds between two ryanodine receptor (RyR) subunits, referred to as intersubunit cross-linking. Preventing RyR cross-linking with N-ethylmaleimide decreased the propensity of Ca(2+) waves induced by ß-AR stimulation. These data suggest that increased energy demand during sustained ß-AR stimulation weakens mitochondrial antioxidant defense, causing ROS release into the cytosol. By inducing RyR intersubunit cross-linking, ROS can increase SR Ca(2+) leak to the critical level that can trigger proarrhythmic Ca(2+) waves.

Ca handling during excitation-contraction coupling in heart failure.

In the heart, coupling between excitation of the surface membrane and activation of contractile apparatus is mediated by Ca released from the sarcoplasmic reticulum (SR). Several components of Ca machinery are perfectly arranged within the SR network and the T-tubular system to generate a regular Ca cycling and thereby rhythmic beating activity of the heart. Among these components, ryanodine receptor (RyR) and SR Ca ATPase (SERCA) complexes play a particularly important role and their dysfunction largely underlies abnormal Ca homeostasis in diseased hearts such as in heart failure. The abnormalities in Ca regulation occur at practically all main steps of Ca cycling in the failing heart, including activation and termination of SR Ca release, diastolic SR Ca leak, and SR Ca uptake. The contributions of these different mechanisms to depressed contractile function and enhanced arrhythmogenesis may vary in different HF models. This brief review will therefore focus on modifications in RyR and SERCA structure that occur in the failing heart and how these molecular modifications affect SR Ca regulation and excitation-contraction coupling.

Mechanisms of Ca²+ handling in zebrafish ventricular myocytes.

The zebrafish serves as a promising transgenic animal model that can be used to study cardiac Ca(2+) regulation. However, mechanisms of sarcoplasmic reticulum (SR) Ca(2+) handling in the zebrafish heart have not been systematically explored. We found that in zebrafish ventricular myocytes, the action potential-induced Ca(2+) transient is mainly (80 %) mediated by Ca(2+) influx via L-type Ca(2+) channels (LTCC) and only 20 % by Ca(2+) released from the SR. This small contribution of the SR to the Ca(2+) transient was not the result of depleted SR Ca(2+) load. We found that the ryanodine receptor (RyR) expression level in zebrafish myocytes was ∼72 % lower compared to rabbit myocytes. In permeabilized myocytes, increasing cytosolic [Ca(2+)] from 100 to 350 nM did not trigger SR Ca(2+) release. However, an application of a low dose of caffeine activated Ca(2+) sparks. These results show that the zebrafish cardiac RyR has low sensitivity to the mechanism of Ca(2+)-induced Ca(2+) release. Activation of protein kinase A by forskolin increased phosphorylation of the RyR in zebrafish myocardium. In half of the studied cells, an increased Ca(2+) transient by forskolin was entirely mediated by augmentation of LTCC current. In the remaining myocytes, the forskolin action was associated with an increase of both LTCC and SR Ca(2+) release. These results indicate that the mechanism of excitation-contraction coupling in zebrafish myocytes differs from the mammalian one mainly because of the small contribution of SR Ca(2+) release to the Ca(2+) transient. This difference is due to a low sensitivity of RyRs to cytosolic [Ca(2+)].

Regulation of sarcoplasmic reticulum Ca²âº leak by cytosolic Ca²âº in rabbit ventricular myocytes.

Sarcoplasmic reticulum (SR) Ca(2+) leak determines SR Ca(2+) content and, therefore, the amplitude of global Ca(2+) transients in ventricular myocytes. However, it remains unresolved to what extent Ca(2+) leak can be modulated by cytosolic [Ca(2+)] ([Ca(2+)](i)). Here, we studied the effects of [Ca(2+)](i) on SR Ca(2+) leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we monitored SR Ca(2+) leak as the change in [Ca(2+)](SR) (with Fluo-5N) after complete SERCA inhibition with thapsigargin (10 µm). Increasing [Ca(2+)](i) from 150 to 250 nM significantly increased SR Ca(2+) leak over the entire range of [Ca(2+)](SR). This increase was associated with an augmentation of both Ca(2+) spark- and non-spark-mediated Ca(2+) leak. Further increasing [Ca(2+)](i) to 350 nM led to rapid [Ca](2+)](SR) depletion due to the occurrence of Ca(2+) waves. The augmentation of SR Ca(2+) leak by high [Ca(2+)](i) was insensitive to inhibition of Ca(2+)-calmodulin-dependent protein kinase II. In contrast, lowering [Ca(2+)](i) to 50 nM markedly decreased SR Ca(2+) leak rate and nearly abolished Ca(2+) sparks. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 µM), changes in [Ca(2+)](i) between 50 and 350 nM did not produce any significant effect on SR Ca(2+) leak, indicating that [Ca(2+)](i) alters SR Ca(2+) leak solely by regulating RyR activity. In summary, [Ca(2+)](i) in the range of 50-350 nM has a significant effect on SR Ca(2+) leak rate mainly via direct regulation of RyR activity. As RyR activity depends highly on [Ca(2+)](i) and [Ca(2+)](SR), SR Ca(2+) leak remains relatively constant during the declining phase of the Ca(2+) transient when [Ca(2+)](SR) and [Ca(2+)](i) change in opposite directions.

A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.

MicroRNA-130a Regulation of Desmocollin 2 in a Novel Model of Arrhythmogenic Cardiomyopathy.

BACKGROUND: MicroRNAs are small noncoding RNA molecules that play a critical role in regulating physiological and disease processes. Recent studies have now recognized microRNAs as an important player in cardiac arrhythmogenesis. Molecular insight into arrhythmogenic cardiomyopathy (AC) has primarily focused on mutations in desmosome proteins. To our knowledge, models of AC due to microRNA dysregulation have not been reported. Previously, we reported on miR-130a mediated down-regulation of Connexin43. OBJECTIVE: Here, we investigate miR-130a-mediated translational repression of Desmocollin2 (DSC2), as it has a predicted target site for miR-130a. DSC2 is an important protein for cell adhesion, which has been shown to be dysregulated in human AC. METHOD & RESULTS: After induction of miR-130a, transgenic mice demonstrated right ventricular dilation. Surface ECG revealed spontaneous premature ventricular complexes confirming an arrhythmogenic phenotype in αMHC-miR130a mice. Using total protein from whole ventricular lysate, western blot analysis demonstrated an 80% reduction in DSC2 levels in transgenic myocardium. Furthermore, immunofluorescent staining confirmed downregulation of DSC2 in transgenic compared with littermate control myocardium. In transgenic hearts, histologic findings revealed fibrosis and lipid accumulation within both ventricles. To validate DSC2 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts, known to express miR-130a. Using a luciferase reporter fused to the 3UTR of DSC2 compared with a control, we found a 42% reduction in luciferase activity with the DSC2 3UTR. This reduction was reversed upon selective inhibition of miR-130a. CONCLUSION: Overexpression of miR-130a results in a disease phenotype characteristic of AC and therefore, may serve as potential model for microRNA-induced AC.

Transcription-factor-dependent enhancer transcription defines a gene regulatory network for cardiac rhythm.

The noncoding genome is pervasively transcribed. Noncoding RNAs (ncRNAs) generated from enhancers have been proposed as a general facet of enhancer function and some have been shown to be required for enhancer activity. Here we examine the transcription-factor-(TF)-dependence of ncRNA expression to define enhancers and enhancer-associated ncRNAs that are involved in a TF-dependent regulatory network. TBX5, a cardiac TF, regulates a network of cardiac channel genes to maintain cardiac rhythm. We deep sequenced wildtype and Tbx5-mutant mouse atria, identifying ~2600 novel Tbx5-dependent ncRNAs. Tbx5-dependent ncRNAs were enriched for tissue-specific marks of active enhancers genome-wide. Tbx5-dependent ncRNAs emanated from regions that are enriched for TBX5-binding and that demonstrated Tbx5-dependent enhancer activity. Tbx5-dependent ncRNA transcription provided a quantitative metric of Tbx5-dependent enhancer activity, correlating with target gene expression. We identified RACER, a novel Tbx5-dependent long noncoding RNA (lncRNA) required for the expression of the calcium-handling gene Ryr2. We illustrate that TF-dependent enhancer transcription can illuminate components of TF-dependent gene regulatory networks.

Pitx2 modulates a Tbx5-dependent gene regulatory network to maintain atrial rhythm.

Cardiac rhythm is extremely robust, generating 2 billion contraction cycles during the average human life span. Transcriptional control of cardiac rhythm is poorly understood. We found that removal of the transcription factor gene Tbx5 from the adult mouse caused primary spontaneous and sustained atrial fibrillation (AF). Atrial cardiomyocytes from the Tbx5-mutant mice exhibited action potential abnormalities, including spontaneous depolarizations, which were rescued by chelating free calcium. We identified a multitiered transcriptional network that linked seven previously defined AF risk loci: TBX5 directly activated PITX2, and TBX5 and PITX2 antagonistically regulated membrane effector genes Scn5a, Gja1, Ryr2, Dsp, and Atp2a2 In addition, reduced Tbx5 dose by adult-specific haploinsufficiency caused decreased target gene expression, myocardial automaticity, and AF inducibility, which were all rescued by Pitx2 haploinsufficiency in mice. These results defined a transcriptional architecture for atrial rhythm control organized as an incoherent feed-forward loop, driven by TBX5 and modulated by PITX2. TBX5/PITX2 interplay provides tight control of atrial rhythm effector gene expression, and perturbation of the co-regulated network caused AF susceptibility. This work provides a model for the molecular mechanisms underpinning the genetic implication of multiple AF genome-wide association studies loci and will contribute to future efforts to stratify patients for AF risk by genotype.

Cytosolic Ca²âº buffering determines the intra-SR Ca²âº concentration at which cardiac Ca²âº sparks terminate.

Single ryanodine receptor (RyR) Ca(2+) flux amplitude (i(Ca-RyR)) decreases as intra-sarcoplasmic reticulum (SR) Ca(2+) levels fall during a cardiac Ca(2+) spark. Since i(Ca-RyR) drives the inter-RyR Ca(2+)-induced Ca(2+) release (CICR) that underlies the spark, decreasing i(Ca-RyR) may contribute to spark termination because RyRs that spontaneously close may stay closed. To test this possibility, we simultaneously measured local cytosolic and intra-SR ([Ca(2+)]cyto and [Ca(2+)]SR) during Ca(2+) sparks in permeabilized rabbit ventricular myocytes. Local cytosolic or intra-SR Ca(2+) dynamics were manipulated using Ca(2+) buffers. Buffer manipulations applied in cells had no effect on individual RyR channels reconstituted in planar lipid bilayers. Presence of a fast cytosolic Ca(2+) buffer (BAPTA) significantly suppressed Ca(2+) spark activity and sparks terminated earlier at a higher than usual [Ca(2+)]SR level (∼80% vs. ∼62%). When cytosolic Ca(2+) buffer power was reduced (i.e. cytosolic EGTA level decreased), sparks terminated later and at a lower than usual [Ca(2+)]SR level (∼45% vs. ∼62%). When intra-SR Ca(2+) buffer power was increased, sparks also terminated later and at a lower than usual [Ca(2+)]SR (∼48% vs. ∼62%). These results suggest that cytosolic local control of inter-RyR CICR by i(Ca-RyR) plays a substantial role during the spark termination process. Thus, alterations in local cytosolic Ca(2+) handling dynamics in the dyadic cleft (Ca(2+) buffering, extrusion, etc.) likely influence Ca(2+) spark termination.

Regulation of sarcoplasmic reticulum Ca(2+) release by cytosolic glutathione in rabbit ventricular myocytes.

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca(2+)-induced Ca(2+) release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca(2+) release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca(2+) release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca(2+) release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca(2+)]SR, increased SR Ca(2+) fractional release, and increased spark- and non-spark-mediated SR Ca(2+) leak. Single-channel recordings from RyR's incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca(2+) load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca(2+) leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca(2+) handling during periods of oxidative stress.