Diastolic Sarcoplasmic Reticulum Research Articles

Structural basis for ryanodine receptor type 2 leak in heart failure and arrhythmogenic disorders

Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca2+ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca2+ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca2+ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.

The inotropic and arrhythmogenic effects of acutely increased late INa are associated with elevated ROS but not oxidation of PKARIα.

Acute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress. We aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα. We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis. ATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment. The inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and β-adrenergic receptor (βAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during βAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

Hyperactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo. We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca2+-spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65±0.73 mF/F0 vs. 1.88±0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+-leak induction in the CaMKIIδC TG, both regarding Ca2+-spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction=12.17±2.05% vs. 45.15±3.46% in WT), cardiac hypertrophy (heart weight/tibia length=24.84±3.00 vs. 9.81±0.50mg/mm in WT), or severe premature mortality (median survival of 12weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

Abstract 9834: Lysosomal Ca 2+ Release Contribute to Arrhythmia After Myocardial Infarction

Introduction: In heart failure (HF), mitochondria Ca 2+ handling is involved in arrhythmogenesis by modulating diastolic sarcoplasmic reticulum (SR) Ca 2+ release. Recently, it has been reported that lysosomal Ca 2+ release can trigger a SR Ca 2+ release. Hypothesis: We investigated whether lysosomal Ca 2+ flux could contribute to ischemic HF arrhythmia in a similar manner to mitochondria. Methods: Ischemic HF was induced in wild type FVB/NJ mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after myocardial infarction (MI). Results: The lysosomal-restricted transient receptor potential mucolipin 1 (TRPML1) Ca 2+ release channel, but not the two-pore channel 2, was significantly upregulated by 84.8% in cardiac border zone and 55.4% in a remote zone after MI, and cardiomyocyte TRPML1 was significantly increased in human patients with ischemic or nonischemic cardiomyopathy. As expected, TRPML1 co-localized with the lysosome marker, LAMP1. The master transcriptional lysosomal regulator, dephosphorylated TFEB (i.e., active TFEB) and LAMP1 labelled lysosomes increased after MI. Lysosomal Ca 2+ release directly as indicated by GCaMP7-TRPML1, a fluorescent Ca 2+ sensor coupled to TRPML1, was upregulated in MI mouse CMs. TRPML1 specific agonist, ML-SA1, could evoke Ca 2+ sparks in the CMs isolated from MI mice, and these sparks were inhibited by a specific antagonist, ML-SI1. Furthermore, thapsigargin or high concentration ryanodine, inhibited Ca 2+ loading and release from the SR respectively and prevented TRPML1-dependent Ca 2+ sparks in the MI CMs. TRPML1-dependent sparks were not affected by S107, a drug which specifically inhibits Ca 2+ leak, however. Blocking TRPML1depressed early and delayed afterdepolarizations (EADs and DADs) in CMs isolated from MI mice while increasing TRPML1 activity enhanced triggered activity. The inhibition of TRPML1 had no effect on the APs recorded from sham mouse CMs, however. ML-SA1 could increase induced ventricular fibrillation episodes in MI mice while ML-SI1 could reduce arrhythmia by optical mapping and pacing. Conclusions: Lysosomes contribute to arrhythmic risk in MI by releasing Ca 2+ during diastole causing SR diastolic Ca 2+ release and triggered activity.

Abstract 12531: Electrophysiological Mechanisms of Tachycardia-Induced Cardiomyopathy in Human Myocardium

Introduction: Tachycardia-induced cardiomyopathy (TCM) is a reversible form of heart failure. However, the effects of persistent tachycardia leading to TCM in the human are less understood limiting appropriate management of patients with this underrecognized condition. This study investigated the electrophysiological mechanisms of TCM. Methods and Results: Tachycardic in vitro electrical stimulation (120 bpm versus 60 bpm control) of human specimen was performed. In human twitching ventricular trabeculae from patients with preexistent heart failure, tachycardia for 8h compromised systolic force, while diastolic tension and relaxation time were increased (n=6/8 patients each). Human iPSC-cardiomyocytes (iPSC-CM) of healthy donors were utilized as human model suitable for extended electrical pacing. After 24h of tachycardia, systolic Ca 2+ -transient amplitude of iPSC-CM was reduced, while diastolic Ca 2+ levels and cytosolic Ca 2+ elimination were not changed (Fura-2, n tachycardia/control=49/44 cells). 7d of tachycardia resulted in a progressive decline of Ca 2+ -transient amplitude together with an impaired Ca 2+ elimination (n=73/66) thereby demonstrating the progression of TCM. Sarcoplasmic reticulum (SR) Ca 2+ load (caffeine application) and SERCA activity (K sys -K caff ) were reduced after 7d of tachycardia (n=13/13). Besides an impaired SERCA activity, we could identify an increased diastolic SR Ca 2+ spark frequency after 7d of tachycardia (n=76/79) as another mechanism of reduced SR Ca 2+ load. In voltage clamp studies, I CaL density was reduced after 7d of tachycardia (n=11/12), which may further contribute to impaired systolic Ca 2+ -handling. Conversely, action potential duration was prolonged after 7d of tachycardia compared to control (n=19/21). As a potential explanation for prolonged action potentials however, late sodium current (I NaL ) was enhanced after 7d of tachycardia (n=26/19). Conclusions: This study demonstrates for the first time in human myocardium that persistent tachycardia induces early adverse electrophysiological remodeling that may underlie the progression of TCM. These findings may help to understand the pathophysiology of an underrated but prevalent disease

The Role of Ca2+ Sparks in Force Frequency Relationships in Guinea Pig Ventricular Myocytes.

Calcium sparks are the elementary Ca2+ release events in excitation-contraction coupling that underlie the Ca2+ transient. The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca2+ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca2+]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca2+]myo transients increase in pacing rates consistent with the experiments. The [Ca2+]myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca2+]sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca2+] transients. However, the local control model also suggests that the reduction of the L-type Ca2+ current, with an increase in pacing frequency due to Ca2+-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca2+ transient in the FFR correlated with the highest numbers of SR Ca2+ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca2+ sparks.

The role of the atrium in the development of the Takotsubo Syndrome – investigation of a patient-specific atrial stem cell model

Abstract Background and aims The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) or the PDE4 activator MR-L2 are suitable for Takotsubo cardiomyopathy in vitro. Methods and results We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC-aCMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated them with catecholamines (Iso) to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs and observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial control cells were significantly decreased in aCMs of the TTS patients. This effect was rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, Iso-induced increase in systolic Ca2+ transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol and by MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal microscope Ca2+ measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca2+ leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca2+ kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM. Subsequent treatment with Metoprolol rescued the Ca2+ kinetic parameters and the increased calcium sparks in all cell lines. Conclusion In conclusion, we were able to draw a comprehensive picture on the role of the atrium in the development of arrhythmias in TTS. We found TTS-patient-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol has proven to be an effective treatment option for TTS and preliminary data of MR-L2 demonstrate promising effects as a new patient-specific therapeutic target for TTS under catecholamine-stress. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German centre for cardiovascular research

Reduced sarcoplasmic reticulum Ca2+ pump activity is antiarrhythmic in ischemic cardiomyopathy

We have described an arrhythmic mechanism seen only in cardiomyopathy that involves increased mitochondrial Ca2+ handling and selective transfer of Ca2+ to the sarcoplasmic reticulum (SR). Modeling suggested that mitochondrial Ca2+ transfer to the SR via type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a crucial element of this arrhythmic mechanism. We tested the role of SERCA2a in arrhythmias during ischemic cardiomyopathy. Myocardial infarction (MI) was induced in wild-type (Wt) and SERCA2a heterozygous knockdown (SERCA+/-) mice. Compared with Wt MI mice, SERCA2a heterozygous knockdown (SERCA+/-) MI mice had a substantially lower mortality after 3 weeks of MI without a significant change in MI area. Aside from a significant delay of the cytoplasmic Ca2+ transient decay existed in SERCA+/- compared with Wt, SERCA+/- did not affect cardiac systolic and diastolic function at the whole organ or single cell levels either before or after MI. After MI, SERCA+/- mice had reduced SERCA2a expression in the MI border zone compared with Wt MI mice. SERCA+/- mice had significantly decreased corrected QT intervals and less ventricular tachycardia compared with Wt MI mice. SERCA+/- cardiomyocytes from MI mice showed a reduced action potential duration and reduced triggered activity compared with Wt MI cardiomyocytes. Reduction in arrhythmic risk was accompanied by reduced diastolic SR Ca2+ sparks, reduced SR Ca2+ content, reduced oxidized ryanodine receptor, and increased calsequestrin 2 in SERCA+/- MI mice. SERCA2a knockdown was antiarrhythmic after MI without affecting overall systolic performance. Possible antiarrhythmic mechanisms included reduced SR free Ca2+ and reduced diastolic SR Ca2+ release.

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

Abstract P2106: Arrhythmia As Cause For The Development Of The Takotsubo Syndrome In A Patient-specific Atrial Stem Cell Model

Background and Aims: The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) are suitable for Takotsubo cardiomyopathy in vitro. Methods and Results: We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC- a CMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated the cells with catecholamines to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs, which are besides β-ARs main players involved in cAMP signaling in CMs. We observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial controls were significantly decreased in aCMs of the TTS patients. This effect was completely rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, isoprenaline-induced increase in systolic Ca 2+ transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol as well as the PDE4 activator MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal Ca 2+ measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca 2+ leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca 2+ kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM .Subsequent treatment with the beta-blocker Metoprolol rescued the Ca 2+ kinetic parameters in all cell lines. Conclusion: In conclusion, we were able to draw a comprehensive picture on the role of the arrhythmia in the development of TTS. We found TTS-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol as well as MR-L2 demonstrate promising effects as patient-specific therapeutic target for TTS.

Doxorubicin induces cardiotoxicity in a pluripotent stem cell model of aggressive B cell lymphoma cancer patients

Cancer therapies with anthracyclines have been shown to induce cardiovascular complications. The aims of this study were to establish an in vitro induced pluripotent stem cell model (iPSC) of anthracycline-induced cardiotoxicity (ACT) from patients with an aggressive form of B-cell lymphoma and to examine whether doxorubicin (DOX)-treated ACT-iPSC cardiomyocytes (CM) can recapitulate the clinical features exhibited by patients, and thus help uncover a DOX-dependent pathomechanism. ACT-iPSC CM generated from individuals with CD20+ B-cell lymphoma who had received high doses of DOX and suffered cardiac dysfunction were studied and compared to control-iPSC CM from cancer survivors without cardiac symptoms. In cellular studies, ACT-iPSC CM were persistently more susceptible to DOX toxicity including augmented disorganized myofilament structure, changed mitochondrial shape, and increased apoptotic events. Consistently, ACT-iPSC CM and cardiac fibroblasts isolated from fibrotic human ACT myocardium exhibited higher DOX-dependent reactive oxygen species. In functional studies, Ca2+ transient amplitude of ACT-iPSC CM was reduced compared to control cells, and diastolic sarcoplasmic reticulum Ca2+ leak was DOX-dependently increased. This could be explained by overactive CaMKIIδ in ACT CM. Together with DOX-dependent augmented proarrhythmic cellular triggers and prolonged action potentials in ACT CM, this suggests a cellular link to arrhythmogenic events and contractile dysfunction especially found in ACT engineered human myocardium. CamKIIδ inhibition prevented proarrhythmic triggers in ACT. In contrast, control CM upregulated SERCA2a expression in a DOX-dependent manner, possibly to avoid heart failure conditions. In conclusion, we developed the first human patient-specific stem cell model of DOX-induced cardiac dysfunction from patients with B-cell lymphoma. Our results suggest that DOX-induced stress resulted in arrhythmogenic events associated with contractile dysfunction and finally in heart failure after persistent stress activation in ACT patients.

Effects of Atrial Fibrillation on the Human Ventricle.

Rationale:Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction.Objective:This study investigates the effects and molecular mechanisms of AF on the human LV.Methods and Results:Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca2+ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca2+ levels and Ca2+ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca2+ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ load likely because of an increased diastolic sarcoplasmic reticulum Ca2+ leak. Moreover, cytosolic Na+ concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na+ current as a potential trigger for the detrimentally altered Ca2+/Na+-interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca2+/calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca2+ handling after AF-simulation.Conclusions:AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.

Interplay between mineral bone disorder and cardiac damage in acute kidney injury: from Ca2+ mishandling and preventive role of Klotho in mice to its potential mortality prediction in human

Biomarkers of mineral bone disorders (MBD) including phosphorus, fibroblast growth factor (FGF)-23 and Klotho are strongly altered in patients with acute kidney injury (AKI) who have high cardiac outcomes and mortality rates. However, the crosslink between MBD and cardiac damage after an AKI episode still remains unclear. We tested MBD and cardiac biomarkers in an experimental AKI model after 24 or 72 hours of folic acid injection and we analyzed structural cardiac remodeling, intracellular calcium (Ca2+) dynamics in cardiomyocytes and cardiac rhythm. AKI mice presented high levels of FGF-23, phosphorus and cardiac troponin T and exhibited a cardiac hypertrophy phenotype accompanied by an increase in systolic Ca2+ release 24 hours after AKI. Ca2+ transients and contractile dysfunction were reduced 72 hours after AKI while diastolic sarcoplasmic reticulum Ca2+ leak, pro-arrhythmogenic Ca2+ events and ventricular arrhythmias were increased. These cardiac events were linked to the activation of the calcium/calmodulin-dependent kinase II pathway through the increased phosphorylation of ryanodine receptors and phospholamban specific sites after AKI. Cardiac hypertrophy and the altered intracellular Ca2+ dynamics were prevented in transgenic mice overexpressing Klotho after AKI induction. In a translational retrospective longitudinal clinical study, we determined that combining FGF-23 and phosphorus with cardiac troponin T levels achieved a better prediction of mortality in AKI patients at hospital admission. Thus, monitoring MBD and cardiac damage biomarkers could be crucial to prevent mortality in AKI patients. In this setting, Klotho might be considered as a new cardioprotective therapeutic tool to prevent deleterious cardiac events in AKI conditions.

Abstract 11558: The Role of Oxidized CaMKII in Early Ischemia-Reperfusion Triggered Ventricular Arrhythmias

Introduction: Arrhythmias can complicate revascularization procedures after transmural infarctions and induce hemodynamic instability, but most cases can be adequately treated when they occur in hospital. However, arrhythmias that occur outside of hospital are potentially fatal. Arrhythmias in the early phase of reperfusion are caused by Ca 2+ overload and spontaneous diastolic release of Ca 2+ from the sarcoplasmic reticulum (SR). Indirect evidence suggests that reactive oxygen species (ROS) are important in this process by affecting the activity of key Ca 2+ handling proteins. Specifically, previous publications propose oxidation of the multifunctional Ca 2+ /calmodulin dependent protein kinase type II (CaMKII) to be a critical factor, and a potential therapeutic target. There is, however, a lack of data providing direct evidence for the importance of oxidized CaMKII (ox-CaMKII) for triggered arrhythmias in the early phase of reperfusion. Hypothesis: We hypothesized that ox-CaMKII increases diastolic SR Ca 2+ release events in the early phase of reperfusion. Methods: We evaluated the propensity for early reperfusion arrhythmias in mice resistant to oxidation of CaMKII in the heart (homozygous CaMKIIδ M281/282V mice) and wild type control mice (WT), by the use of Langendorff-perfused hearts exposed to ischemia-reperfusion, confocal line scan imaging of isolated cardiomyocytes transiently exposed to hypoxia and ischemia-like conditions, and immunoblotting. Results: Langendorff-perfused hearts from CaMKIIδ M281/282V mice showed no significant difference in ventricular arrhythmias compared to WT during the early reperfusion phase, both at baseline and during beta-adrenoceptor stimulation with isoprenaline. Immunoblotting of left ventricular tissue from perfused hearts revealed no significant differences in the abundance or phosphorylation levels of Ca 2+ -handling proteins. Ca 2+ imaging experiments without isoprenaline showed no significant difference in the incidence of spontaneous transients, Ca 2+ waves or Ca 2+ sparks between CaMKIIδ M281/282V and WT mice. Conclusions: Initial data indicate that ablation of CaMKII oxidation does not protect mouse hearts against arrhythmias in the early phase of reperfusion after ischemia.

Role of Reduced Sarco-Endoplasmic Reticulum Ca2+-ATPase Function on Sarcoplasmic Reticulum Ca2+ Alternans in the Intact Rabbit Heart.

Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

Simulating cardiac Ca2+ release units: effects of RyR cluster size and Ca2+ buffers on diastolic Ca2+ leak.

Leak of Ca2+ out of the cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) during diastole is vital to regulate SR Ca2+ levels. This leak can become deleterious when large spontaneous RyR-mediated Ca2+ release events evoke proarrhythmic Ca2+ waves that can lead to delayed after-depolarizations. Here, we model diastolic SR Ca2+ leak at individual SR Ca2+ release sites using computer simulations of RyR arrays like those in the dyadic cleft. The results show that RyR arrays size has a significant effect on SR Ca2+ leak, with bigger arrays producing larger and more frequent Ca2+ release events. Moreover, big RyR arrays are more susceptible to small changes in the levels of dyadic Ca2+ buffers. Such changes in buffering shift Ca2+ leak from small Ca2+ release events (involving few open RyRs) to larger events (with many open RyRs). Moreover, by analyzing a large parameter space of possible buffering and SR Ca2+ loads, we find further evidence for the hypothesis that SR Ca2+ leak by RyR arrays can undergo a sudden phase transition.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship.

We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown. To test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship. We used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts. We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Oxidation of Ryanodine Receptors Promotes Ca2+ Leakage and Contributes to Right Ventricular Dysfunction in Pulmonary Hypertension.

Right ventricular (RV) failure is a major cause of death in patients with pulmonary arterial hypertension, and the mechanism of RV failure remains unclear. While the malfunction of RyR2 (ryanodine receptor type 2) on sarcoplasmic reticulum (SR) and aberrant Ca2+ cycling in cardiomyocytes have been recognized in some cardiovascular diseases, their roles in RV failure secondary to pulmonary arterial hypertension require further investigation. In a monocrotaline-induced rat model of pulmonary arterial hypertension, the RV remodeling process was divided into normal, compensated, and decompensated stages according to the hemodynamic and morphological parameters. In both compensated and decompensated stages, significant diastolic SR Ca2+ leakage was detected along with reduced intracellular Ca2+ transient amplitude and SR Ca2+ contents in RV myocytes. RyR2 protein levels decreased progressively during the process, and the thiol oxidation proportions of RyR2 were higher in compensated and decompensated stages than in normal stage. Inhibition of RyR2 oxidation by dithiothreitol or repairing RyR2 directly by dantrolene could restore Ca2+ homeostasis in RV myocytes. Daily intraperitoneal injection of dantrolene delayed decompensation progression and significantly improved the survival rate of pulmonary hypertension rats in decompensated stage (79.3% versus 55.9%; P=0.026). Our findings suggest that diastolic SR Ca2+ leakage via oxidized RyR2 facilitates the development of RV failure. Dantrolene can inhibit diastolic SR Ca2+ leakage in RV cardiomyocytes, delay right cardiac dysfunction, and improve the survival of rats with pulmonary arterial hypertension.

CaMKII delta interaction with neuronal sodium channel Nav1.8 contributes to arrhythmogenic triggers in failing human and mouse cardiomyocytes

Abstract In heart failure, enhanced persistent current through neuronal sodium channel NaV1.8 (INaL) may induce influx of Na+ into cardiomyocytes. This may cause Ca2+ influx via the Na+/Ca2+ exchanger leading to increased proarrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ leak. This Ca2+ may activate Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) which can induce INaL augmentation by phosphorylating NaV1.5 channels leading to a vicious cycle between INaL and CaMKIIδ. Here, we examined whether CaMKIIδ associates with NaV1.8 in human and mouse cardiomyocytes thereby regulating its function. Interaction and co-localisation of CaMKIIδ and NaV1.8 were confirmed by co-immunoprecipitation and immunocytochemistry. Whole-cell patch clamp showed a potent reduction of INaL after addition of novel specific Nav1.8 blockers, either A-803467 (30 nmol/L) or PF-01247324 (1 μmol/L) in failing mouse cardiomyocytes overexpressing CaMKIIδc (CaMKIIδc+/T: −109.4±10.6 vs A-803467: −56.9±11.7 and PF-01247324:−-69.9±8.6 A*ms*F-1). In failing human cardiomyocytes inhibition of either NaV1.8 or CaMKIIδ using AIP (1 μmol/L) or AIP and PF-01247324 together led to a significant and comparable decrease of INaL (control: −93.7±7.1 vs PF-01247324: −56.8±6.6; AIP: −44.2±6.6; AIP+PF-01247324: −39.8±5.4 A*ms*F-1). Furthermore, to confirm whether observed alterations in INaL after inhibition of NaV1.8 are not due to an overall reduction in peak sodium current (INa) we measured INa properties in mouse cardiomyocytes. Importantly, we observed no difference neither in the peak nor in inactivation between wild type (WT), WT with PF-01247324 and in mice lacking NaV1.8. Using confocal microscopy we investigated whether inhibition of the NaV1.8-mediated INaL could attenuate the increase of proarrhythmogenic SR Ca2+ spark frequency (CaSpF) caused by overexpression of CaMKIIδ in mice. We observed a significant reduction of CaSpF in both NaV1.8 inhibitor groups (PF-01247324: 0.51±0.08 and A-803467: 0.57±0.08 μm–1 s–1) compared to control (1.00±0.13 μm–1 s–1). Incubation of human failing cardiomyocytes with either AIP (0.35±0.06 μm–1 s–1) or PF-01247324 (0.44±0.11 μm–1 s–1), or blocking CaMKIIδ and NaV1.8 together (0.30±0.08 μm–1 s–1) resulted in significant decrease of CaSpF compared to control (0.89±0.13 μm–1 s–1). In conclusion, we show for the first time subcellular localisation of the neuronal sodium channel NaV1.8 and its interaction with CaMKIIδ in both human and mouse ventricular cardiomyocytes. Moreover, pharmacological inhibition of NaV1.8 caused a reduction of the augmented INaL and spontaneous diastolic SR-Ca2+ release in both failing human and mouse cardiomyocytes. NaV1.8 and CaMKIIδ interaction seem to play a relevant role for the generation of arrhythmogenic triggers (INaL &amp; spontaneous diastolic SR-Ca2+ release) in both human and mouse cardiomyocytes from failing hearts. Funding Acknowledgement Type of funding source: None