Atrial Myocyte NLRP3/CaMKII Nexus Forms a Substrate for Postoperative Atrial Fibrillation.

RATIONALE: Postoperative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when postoperative triggers act on a preexisting vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown. OBJECTIVE: To identify cellular POAF mechanisms in right atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery. METHODS AND RESULTS: Multicellular action potentials, membrane ion-currents (perforated patch-clamp), or simultaneous membrane-current (ruptured patch-clamp) and [Ca2+]i-recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca2+-transient amplitude was smaller, although spontaneous sarcoplasmic reticulum (SR) Ca2+-release events and L-type Ca2+-current alternans occurred more frequently. CaMKII (Ca2+/calmodulin-dependent protein kinase-II) protein-expression, CaMKII-dependent phosphorylation of the cardiac RyR2 (ryanodine-receptor channel type-2), and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca2+-content was unchanged in POAF despite greater SR Ca2+-leak, with a trend towards increased SR Ca2+-ATPase activity. Patients with POAF also showed stronger expression of activated components of the NLRP3 (NACHT, LRR, and PYD domains-containing protein-3)-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1ß caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in an immortalized mouse atrial cardiomyocyte cell-line (HL-1-cardiomyocytes) and enhanced spontaneous SR Ca2+-release events in both POAF cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2 dysfunction and increased SR Ca2+-uptake are sufficient to reproduce the Ca2+-handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF subjects in response to interleukin-1ß. CONCLUSIONS: Preexisting Ca2+-handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca2+-releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.

SPEG (Striated Muscle Preferentially Expressed Protein Kinase) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity.

RATIONALE: Junctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear. OBJECTIVE: To determine the role of SPEG in healthy and failing adult hearts. METHODS AND RESULTS: Proteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice. CONCLUSIONS: The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes.

Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as 'junctions', are fast communication hubs within excitable cells such as neurones and muscle. Here, we have examined two transgenic murine models probing the role of junctophilin-2, a membrane-tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single-molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR; in this paper, we use RyR to refer to the myocardial isoform RyR2). Although this change was associated with much enlarged RyR clusters that, due to their size, should be more excitable, functionally it caused a mild inhibition in the Ca2+ signalling output of the junctions (Ca2+ sparks). Analysis of the single-molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the Ca2+ signalling properties of the junction and thus the contractile function of the cell.

Genetic deletion of Rnd3/RhoE results in mouse heart calcium leakage through upregulation of protein kinase A signaling.

RATIONALE: Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation. The biological function of Rnd3 in the heart remains unexplored. OBJECTIVE: To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism. METHODS AND RESULTS: By loss-of-function approaches, we discovered that Rnd3 is involved in calcium regulation in cardiomyocytes. Rnd3-null mice died at the embryonic stage with fetal arrhythmias. The deletion of Rnd3 resulted in severe Ca(2+) leakage through destabilized ryanodine receptor type 2 Ca(2+) release channels. We further found that downregulation of Rnd3 attenuated ß2-adrenergic receptor lysosomal targeting and ubiquitination, which in turn resulted in the elevation of ß2-adrenergic receptor protein levels leading to the hyperactivation of protein kinase A (PKA) signaling. The PKA activation destabilized ryanodine receptor type 2 channels. This irregular spontaneous Ca(2+) release can be curtailed by PKA inhibitor treatment. Increases in the PKA activity along with elevated cAMP levels were detected in Rnd3-null embryos, in neonatal rat cardiomyocytes, and noncardiac cell lines with Rnd3 knockdown, suggesting a general mechanism for Rnd3-mediated PKA signaling activation. ß2-Adrenergic receptor blocker treatment reduced arrhythmia and improved cardiac function. CONCLUSIONS: Rnd3 is a novel factor involved in intracellular Ca(2+) homeostasis regulation in the heart. Deficiency of the protein induces ryanodine receptor type 2 dysfunction by a mechanism that attenuates Rnd3-mediated ß2-adrenergic receptor ubiquitination, which leads to the activation of PKA signaling. Increased PKA signaling in turn promotes ryanodine receptor type 2 hyperphosphorylation, which contributes to arrhythmogenesis and heart failure.

Crosstalk between RyR2 oxidation and phosphorylation contributes to cardiac dysfunction in mice with Duchenne muscular dystrophy.

BACKGROUND: Patients with Duchenne muscular dystrophy (DMD) are at risk of developing cardiomyopathy and cardiac arrhythmias. Studies in a mouse model of DMD revealed that enhanced sarcoplasmic reticulum (SR) Ca(2+) leak contributes to the pathogenesis of cardiac dysfunction. In view of recent data suggesting the involvement of altered phosphorylation and oxidation of the cardiac ryanodine receptor (RyR2)/Ca(2+) release channel, we hypothesized that inhibition of RyR2 phosphorylation in a mouse model of DMD can prevent SR Ca(2+) leak by reducing RyR2 oxidation. METHODS AND RESULTS: Confocal Ca(2+) imaging and single RyR2 channel recordings revealed that both inhibition of S2808 or S2814 phosphorylation, and inhibition of oxidation could normalize RyR2 activity in mdx mice. Moreover, Western blotting revealed that genetic inhibition of RyR2 phosphorylation at S2808 or S2814 reduced RyR2 oxidation. Production of reactive oxygen species (ROS) in myocytes from mdx mice was reduced by both inhibition of RyR2 phosphorylation or the ROS scavenger 2-mercaptopropionyl glycine (MPG). Finally, it was shown that ROS production in mdx mice is proportional to the activity of RyR2-mediated SR Ca(2+) leak, and likely generated by Nox2. CONCLUSIONS: Increased ROS production in the hearts of mdx mice drives the progression of cardiac dysfunction. Inhibition of RyR2 phosphorylation can suppress SR Ca(2+) leak in mdx mouse hearts in part by reducing RyR2 oxidation.

Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation.

BACKGROUND: Electrical, structural, and Ca2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+ -release events in paroxysmal AF (pAF) are unknown. METHOD AND RESULTS: Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+ /Ca2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+ -transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+ -transient amplitude, integrated Na+/Ca2+ -exchange current) were larger in pAF. Ca2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+ -release events, causing delayed after-depolarizations/triggered activity in pAF. CONCLUSIONS: Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Ryanodine receptor-mediated calcium leak drives progressive development of an atrial fibrillation substrate in a transgenic mouse model.

BACKGROUND: The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca(2+) leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca(2+) release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms. METHODS AND RESULTS: CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca(2+) release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca(2+)/calmodulin-dependent protein kinase II-mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca(2+) release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca(2+)-dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. CONCLUSIONS: RyR2-mediated SR Ca(2+) leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca(2+) release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.

Oxidative stress and ca(2+) release events in mouse cardiomyocytes.

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca(2+) signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca(2+) through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca(2+) signaling in situ, by analyzing Ca(2+) sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca(2+) spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca(2+) release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca(2+) depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca(2+) release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca(2+) release events.

Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice.

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca(2+) leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca(2+) leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca(2+) release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca(2+) imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca(2+) leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na(+)/Ca(2+)-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd(2+) administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca(2+) leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca(2+). This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca(2+) leak and heart failure development.

Inhibition of CaMKII phosphorylation of RyR2 prevents induction of atrial fibrillation in FKBP12.6 knockout mice.

RATIONALE: Abnormal calcium release from sarcoplasmic reticulum (SR) is considered an important trigger of atrial fibrillation (AF). Whereas increased Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity has been proposed to contribute to SR leak and AF induction, downstream targets of CaMKII remain controversial. OBJECTIVE: To test the hypothesis that inhibition of CaMKII-phosphorylated type-2 ryanodine receptors (RyR2) prevents AF initiation in FKBP12.6-deficient (-/-) mice. METHODS AND RESULTS: Mice lacking RyR2-stabilizing subunit FKBP12.6 had a higher incidence of spontaneous and pacing-induced AF compared with wild-type mice. Atrial myocytes from FKBP12.6-/- mice exhibited spontaneous Ca(2+) waves (SCaWs) leading to Na(+)/Ca(2+)-exchanger activation and delayed afterdepolarizations (DADs). Mutation S2814A in RyR2, which inhibits CaMKII phosphorylation, reduced Ca(2+) spark frequency, SR Ca(2+) leak, and DADs in atrial myocytes from FKBP12.6-/-:S2814A mice compared with FKBP12.6-/- mice. Moreover, FKBP12.6-/-:S2814A mice exhibited a reduced susceptibility to inducible AF, whereas FKBP12.6-/-:S2808A mice were not protected from AF. CONCLUSIONS: FKBP12.6 mice exhibit AF caused by SR Ca(2+) leak, Na(+)/Ca(2+)-exchanger activation, and DADs, which promote triggered activity. Genetic inhibition of RyR2-S2814 phosphorylation prevents AF induction in FKBP12.6-/- mice by suppressing SR Ca(2+) leak and DADs. These results suggest suppression of RyR2-S2814 phosphorylation as a potential anti-AF therapeutic target.

Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation.

BACKGROUND: Delayed afterdepolarizations (DADs) carried by Na(+)-Ca(2+)-exchange current (I(NCX)) in response to sarcoplasmic reticulum (SR) Ca(2+) leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. METHODS AND RESULTS: Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca(2+)](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca(2+)](i) and SR Ca(2+) content (integrated I(NCX) during caffeine-induced Ca(2+) transient) were unchanged, whereas diastolic SR Ca(2+) leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca(2+) leak. Calmodulin expression (60%), Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca(2+) leak, the frequency of spontaneous Ca(2+) release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca(2+) sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca(2+)](i) and I(NCX) density revealed I(NCX) upregulation in cAF. Spontaneous Ca(2+) release events accompanied by inward I(NCX) currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca(2+)](i) (diastolic [Ca(2+)](i)-voltage coupling gain) was higher in cAF compared with control. CONCLUSIONS: Enhanced SR Ca(2+) leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I(NCX) for a given SR Ca(2+) release and increased diastolic [Ca(2+)](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

A novel role of TGFBI in macrophage polarization and macrophage-induced pancreatic cancer growth and therapeutic resistance.

Tumor-associated macrophages (TAMs), as a major and essential component of tumor microenvironment (TME), play a critical role in orchestrating pancreatic cancer (PaC) tumorigenesis from initiation to angiogenesis, growth, and systemic dissemination, as well as immunosuppression and resistance to chemotherapy and immunotherapy; however, the critical intrinsic factors responsible for TAMs reprograming and function remain to be identified. By performing single-cell RNA sequencing, transforming growth factor-beta-induced protein (TGFBI) was identified as TAM-producing factor in murine PaC tumors. TAMs express TGFBI in human PaC and TGFBI expression is positively related with human PaC growth. By inducing TGFBI loss-of-function in macrophage (MΦs) in vitro with siRNA and in vivo with Cre-Lox strategy in our developed TGFBI-floxed mice, we demonstrated disruption of TGFBI not only inhibited MΦ polarization to M2 phenotype and MΦ-mediated stimulation on PaC growth, but also significantly improved anti-tumor immunity, sensitizing PaC to chemotherapy in association with regulation of fibronectin 1, Cxcl10, and Ccl5. Our studies suggest that targeting TGFBI in MΦ can develop an effective therapeutic intervention for highly lethal PaC.

Single-cell RNA sequencing to characterize the response of pancreatic cancer to anti-PD-1 immunotherapy.

Pancreatic cancer (PaC) is resistant to immune checkpoint therapy, but the underlying mechanisms are largely unknown. In this study, we have established four orthotopic PaC murine models with different PaC cell lines by intra-pancreatic inoculation. Therapeutic examinations demonstrate that only tumors induced with Panc02-H7 cells respond to αPD-1 antibody treatment, leading to significantly reduced tumor growth and increased survival in the recipient mice. Transcriptomic profiling at a single-cell resolution characterizes the molecular activity of different cells within tumors. Comparative analysis and validated experiments demonstrate that αPD-1-sensitive and -resistant tumors differently shape the immune landscape in the tumor microenvironment (TME) and markedly altering effector CD8+ T cells and tumor-associated macrophages (TAMs) in their number, frequency, and gene profile. More exhausted effector CD8+ T cells and increased M2-like TAMs with a reduced capacity of antigen presentation are detected in resistant Panc02-formed tumors versus responsive Panc02-H7-formed tumors. Together, our data highlight the correlation of tumor-induced imbalance of macrophages with the fate of tumor-resident effector CD8+ T cells and PaC response to αPD-1 immunotherapy. TAMs as a critical regulator of tumor immunity and immunotherapy contribute to PaC resistance to immune checkpoint blockade.

Ryanodine receptor phosphorylation by calcium/calmodulin-dependent protein kinase II promotes life-threatening ventricular arrhythmias in mice with heart failure.

BACKGROUND: approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. METHODS AND RESULTS: mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. CONCLUSIONS: our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

LMO7 as an Unrecognized Factor Promoting Pancreatic Cancer Progression and Metastasis.

Pancreatic cancer (PC) is one of the most lethal human malignancies without effective treatment. In an effort to discover key genes and molecular pathways underlying PC growth, we have identified LIM domain only 7 (LMO7) as an under-investigated molecule, which highly expresses in primary and metastatic human and mouse PC with the potential of impacting PC tumorigenesis and metastasis. Using genetic methods with siRNA, shRNA, and CRISPR-Cas9, we have successfully generated stable mouse PC cells with LMO7 knockdown or knockout. Using these cells with loss of LMO7 function, we have demonstrated that intrinsic LMO7 defect significantly suppresses PC cell proliferation, anchorage-free colony formation, and mobility in vitro and slows orthotopic PC tumor growth and metastasis in vivo. Mechanistic studies demonstrated that loss of LMO7 function causes PC cell-cycle arrest and apoptosis. These data indicate that LMO7 functions as an independent and unrecognized druggable factor significantly impacting PC growth and metastasis, which could be harnessed for developing a new targeted therapy for PC.

Cardiac-specific ablation of glutaredoxin 3 leads to cardiac hypertrophy and heart failure.

Growing evidence suggests that redox-sensitive proteins including glutaredoxins (Grxs) can protect cardiac muscle cells from oxidative stress-induced damage. Mammalian Grx3 has been shown to be critical in regulating cellular redox states. However, how Grx3 affects cardiac function by modulating reactive oxygen species (ROS) signaling remains unknown. In this study, we found that the expression of Grx3 in the heart is decreased during aging. To assess the physiological role of Grx3 in the heart, we generated mice in which Grx3 was conditionally deleted in cardiomyocytes (Grx3 conditional knockout (CKO) mice). Grx3 CKO mice were viable and grew indistinguishably from their littermates at young age. No difference in cardiac function was found comparing Grx3 CKO mice and littermate controls at this age. However, by the age of 12 months, Grx3 CKO mice exhibited left ventricular hypertrophy with a significant decrease in ejection fraction and fractional shortening along with a significant increase of ROS production in cardiomyocytes compared to controls. Deletion of Grx3 also impaired Ca2+ handling, caused enhanced sarcoplasmic reticulum (SR) calcium (Ca2+ ) leak, and decreased SR Ca2+ uptake. Furthermore, enhanced ROS production and alteration of Ca2+ handling in cardiomyocytes occurred, prior to cardiac dysfunction in young mice. Therefore, our findings demonstrate that Grx3 is an important factor in regulating cardiac hypertrophy and heart failure by modulating both cellular redox homeostasis and Ca2+ handling in the heart.

Structural and functional characterization of ryanodine receptor-natrin toxin interaction.

Cysteine-rich secretory proteins (CRISPs) are widely distributed, and notably occur in the mammalian reproductive tract and in the salivary glands of venomous reptiles. Most CRISPs can inhibit ion channels, such as the cyclic nucleotide-gated ion channel, potassium channel, and calcium channel. Natrin is a CRISP that has been purified from snake venom. Its targets include the calcium-activated potassium channel, the voltage-gated potassium channel, and the calcium release channel/ryanodine receptor (RyR). Immunoprecipitation experiments showed that natrin binds specifically to type 1 RyR (RyR1) from skeletal muscle. Natrin was found to inhibit both the binding of ryanodine to RyR1, and the calcium-channel activity of RyR1. Cryo-electron microscopy and single-particle image reconstruction analysis revealed that natrin binds to the clamp domains of RyR1. Docking of the crystal structure of natrin into our cryo-electron microscopy density map of the RyR1 + natrin complex suggests that natrin inhibits RyR1 by stabilizing a domain-domain interaction, and that the cysteine-rich domain of natrin is crucial for binding. These findings help reveal how natrin toxin inhibits the RyR calcium release channel, and they allow us to posit a generalized mechanism that governs the interaction between CRISPs and ion channels.

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation.

Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular diseases that often co-exist. Animal models have suggested complex AF-promoting atrial structural, electrical, and Ca2+-handling remodeling in the setting of HF, but data in human samples are scarce, particularly regarding Ca2+-handling remodeling. Here, we evaluated atrial remodeling in patients with severe left ventricular (LV) dysfunction (HFrEF), long-standing persistent ('chronic') AF (cAF) or both (HFrEF-cAF), and sinus rhythm controls with normal LV function (Ctl) using western blot in right-atrial tissue, sharp-electrode action potential (AP) measurements in atrial trabeculae and voltage-clamp experiments in isolated right-atrial cardiomyocytes. Compared to Ctl, expression of profibrotic markers (collagen-1a, fibronectin, periostin) was higher in HFrEF and HFrEF-cAF patients, indicative of structural remodeling. Connexin-43 expression was reduced in HFrEF patients, but not HFrEF-cAF patients. AP characteristics were unchanged in HFrEF, but showed classical indices of electrical remodeling in cAF and HFrEF-cAF (prolonged AP duration at 20% and shorter AP duration at 50% and 90% repolarization). L-type Ca2+ current (ICa,L) was significantly reduced in HFrEF, cAF and HFrEF-cAF, without changes in voltage-dependence. Potentially proarrhythmic spontaneous transient-inward currents were significantly more frequent in HFrEF and HFrEF-cAF compared to Ctl, likely resulting from increased sarcoplasmic reticulum (SR) Ca2+ load (integrated caffeine-induced current) in HFrEF and increased ryanodine-receptor (RyR2) single-channel open probability in HFrEF and HFrEF-cAF. Although expression and phosphorylation of the SR Ca2+-ATPase type-2a (SERCA2a) regulator phospholamban were unchanged in HFrEF and HFrEF-cAF patients, protein levels of SERCA2a were increased in HFrEF-cAF and sarcolipin expression was decreased in both HFrEF and HFrEF-cAF, likely increasing SR Ca2+ uptake and load. RyR2 protein levels were decreased in HFrEF and HFrEF-cAF patients, but junctin levels were higher in HFrEF and relative Ser2814-RyR2 phosphorylation levels were increased in HFrEF-cAF, both potentially contributing to the greater RyR2 open probability. These novel insights into the molecular substrate for atrial arrhythmias in HF-patients position Ca2+-handling abnormalities as a likely trigger of AF in HF patients, which subsequently produces electrical remodeling that promotes the maintenance of the arrhythmia. Our new findings may have important implications for the development of novel treatment options for AF in the context of HF.

EL20, a potent antiarrhythmic compound, selectively inhibits calmodulin-deficient ryanodine receptor type 2.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disorder caused by mutations in the cardiac ryanodine receptor RyR2 that increase diastolic calcium cation (Ca2+) leak from the sarcoplasmic reticulum (SR). Calmodulin (CaM) dissociation from RyR2 has been associated with diastolic Ca2+ leak in heart failure. OBJECTIVE: Determine whether the tetracaine-derivative compound EL20 inhibits abnormal Ca2+ release from RyR2 in a CPVT model and investigate the underlying mechanism of inhibition. METHODS: Spontaneous Ca2+ sparks in cardiomyocytes and inducible ventricular tachycardia were assessed in a CPVT mouse model, which is heterozygous for the R176Q mutation in RyR2 (R176Q/+ mice) in the presence of EL20 or vehicle. Single-channel studies using sheep cardiac SR or purified RyR2 reconstituted into proteoliposomes with and without exogenous CaM were used to assess mechanisms of inhibition. RESULTS: EL20 potently inhibits abnormal Ca2+ release in R176Q/+ myocytes (half-maximal inhibitory concentration = 35.4 nM) and diminishes arrhythmia in R176Q/+ mice. EL20 inhibition of single-channel activity of purified RyR2 occurs in a similar range as seen in R176Q/+ myocytes (half-maximal inhibitory concentration = 8.2 nM). Inhibition of single-channel activity for cardiac SR or purified RyR2 supplemented with 100-nM or 1-µM CaM shows a 200- to 1000-fold reduction in potency. CONCLUSION: This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.

Oxidized CaMKII (Ca2+/Calmodulin-Dependent Protein Kinase II) Is Essential for Ventricular Arrhythmia in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca2+) homeostasis and elevated reactive oxygen species. CaMKII (Ca2+/calmodulin-dependent protein kinase II) is vital for normal Ca2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca2+ homeostasis and arrhythmias in cardiomyocytes. Reactive oxygen species induce CaMKII to become autonomously active. We hypothesized that genetic inhibition of CaMKII oxidation (ox-CaMKII) in a mouse model of Duchenne muscular dystrophy can alleviate abnormal Ca2+ homeostasis, thus, preventing ventricular arrhythmia. The objective of this study was to test if selective loss of ox-CaMKII affects ventricular arrhythmias in the mdx mouse model of Duchenne muscular dystrophy. METHODS AND RESULTS: 5-(6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate staining revealed increased reactive oxygen species production in ventricular myocytes isolated from mdx mice, which coincides with elevated ventricular ox-CaMKII demonstrated by Western blotting. Genetic inhibition of ox-CaMKII by knockin replacement of the regulatory domain methionines with valines (MM-VV [CaMKII M281/282V]) prevented ventricular tachycardia in mdx mice. Confocal calcium imaging of ventricular myocytes isolated from mdx:MM-VV mice revealed normalization of intracellular Ca2+ release events compared with cardiomyocytes from mdx mice. Abnormal action potentials assessed by optical mapping in mdx mice were also alleviated by genetic inhibition of ox-CaMKII. Knockout of the NADPH oxidase regulatory subunit p47 phox normalized elevated ox-CaMKII, repaired intracellular Ca2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice. CONCLUSIONS: Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy.