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Atrial standstill (AS) is a rare condition characterized by absence of electrical activity within the atria. Studies to date have been limited.The authors sought to describe the clinical characteristics, genetics, and outcomes of patients with AS.This was a retrospective multicenter study of patients 18 years at AS diagnosis, defined as absence of atrial activity documented during an electrophysiology study, device placement, or noninvasive rhythm tracings and confirmed by echocardiogram. Patients with acquired disorders were excluded. Clinical details and genetic variants were recorded and analyzed.Twenty patients were diagnosed at a median age of 6.6 years (IQR: 2.9-10.8 years). Arrhythmias included 16 (80%) with atrial/supraventricular arrhythmias and 8 (40%) with ventricular tachycardia, including 4 with cardiac arrests. A type 1 Brugada pattern was documented in 4. Pacemakers were implanted in 18 (90%). Although atrial leads were attempted in 15, only 4 achieved pacing at implantation. During a median follow-up of 6.9 years (IQR: 1.2-13.3 years), 7 (35%) had thromboembolic events. Of these, none had atrial pacing, 6 were not on anticoagulation, and 1 was on aspirin. Genetic testing identified SCN5A variants in 13 patients (65%). Analyses suggest SCN5A loss-of-function may be one mechanism driving AS. Ventricular arrhythmias and cardiac arrest were more commonly seen in patients with biallelic SCN5A variants.AS may be associated with loss-of-function SCN5A variants. Patients demonstrate atrial and ventricular arrhythmias, and may present challenges during device placement. Patients without the capacity for atrial pacing are at risk for thromboembolic events and warrant anticoagulation.

Rationale: Loss-of-function of the cardiac sodium channel Na V 1.5 causes conduction slowing and arrhythmias. Na V 1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current ( I Na ) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I Na reduction, but the mechanisms underlying microdomain-specific targeting of Na V 1.5 remain largely unknown. Objective: To investigate the role of the microtubule plus-end tracking proteins EB1 (end-binding protein 1) and CLASP2 (cytoplasmic linker associated protein 2) in mediating Na V 1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human-induced pluripotent stem cell-derived cardiomyocytes resulted in enhanced whole-cell I Na , increased action potential upstroke velocity ( V max ), and enhanced Na V 1.5 localization at the plasma membrane as detected by multicolor stochastic optical reconstruction microscopy. Fluorescence recovery after photobleaching experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na V 1.5 forward trafficking. Knockout of MAPRE1 in human induced pluripotent stem cell-derived cardiomyocytes led to reduced whole-cell I Na , decreased V max , and action potential duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish ( mapre1b ) resulted in decreased ventricular conduction velocity and V max as well as increased APD. Stochastic optical reconstruction microscopy imaging and macropatch I Na measurements showed that subacute treatment (2–3 hours) with SB216763 (SB2), a GSK3β (glycogen synthase kinase 3β) inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na V 1.5 and I Na preferentially at the ID region of wild-type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I Na or Na V 1.5 localization in cardiomyocytes from Clasp2 -deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of Na V 1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the microtubule plus-end tracking protein EB1 on Na V 1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I Na within the ID region of cardiomyocytes.

Background Allergen-specific IL-4+ and IL-13+ CD4+ cells (type 2 cells) are essential for helping B cells to class-switch to IgE and establishing an allergic milieu in the gastrointestinal tract. The role of T cells in established food allergy is less clear. Objective We examined the food allergen–specific T-cell response in participants of 2 food allergen immunotherapy trials to assess the relationship of the T-cell response to clinical phenotypes, including response to immunotherapy. Methods Blood was obtained from 84 participants with peanut allergy and 142 participants with egg allergy who underwent double-blind placebo-controlled food challenges. Peanut- and egg-responsive T cells were identified by CD154 upregulation after stimulation with the respective extract. Intracellular cytokines and chemokine receptors were also detected. The response to peanut epicutaneous immunotherapy (Peanut Epicutaneous Phase II Immunotherapy Clinical Trial [CoFAR6]; 49 participants receiving epicutaneous immunotherapy) and egg oral immunotherapy or a baked egg diet (Baked Egg or Egg Oral Immunotherapy for Children With Egg Allergy [CoFAR7]; 92 participants) was monitored over time. Results Peanut-specific type 2 and CCR6+ T cells were negatively correlated with each other and differently associated with immune parameters, including specific IgE level and basophil activation test result. At baseline, type 2 cells, but not CCR6+ cells, were predictive of clinical parameters, including a successfully consumed dose of peanut and baked egg tolerance. Exposure to peanut or egg immunotherapy was associated with a decrease in type 2 cell frequency. At baseline, high egg-specific type 2 cell frequency was the immune feature most predictive of oral immunotherapy failure. Conclusion Food-specific type 2 T cells at baseline are informative of threshold of reactivity and response to immunotherapy.

Introduction: Mutations in the lamin A/C ( LMNA ) gene have been causally linked to atrial arrhythmias and cardiac conduction disease (CCD) in young adults. However, the mechanism by which the laminopathy leads to perturbed cardiac electrophysiology has not been fully elucidated. Hypothesis: We hypothesize that protein-truncating variants in LMNA will impair the mechano-protection force in the nuclear envelope and will lead to early-onset cardiomyocyte degeneration and CCD. Methods: In a multi-regional registry of early-onset CCD and atrial fibrillation in Japan, we performed whole-exome DNA sequencing of 23 probands. Using CRISPR/Cas9, we generated indels of candidate gene homologues in zebrafish and characterized cardiac physiology using optical mapping technology and immunohistochemistry. Results: Among all probands, our study revealed four rare nonsense variants in the nuclear protein-coding genes. We focused on a LMNA protein-truncating variant, c.339dupT (p.K114X fsX1), and created a variant in the zebrafish lmna that produced a similar truncation. Lmna -/- zebrafish larvae showed shortened atrial action potential duration (APD) (msec.) compared to wild-type controls (162 ± 19 vs 227 ± 79, p lmna -/- embryos exhibited prolonged APD (399 ± 51 vs 322 ± 47, p lmna -/- zebrafish displayed significantly decreased cell numbers and smaller cell size compared to those of controls, which in turn developed into abnormal nuclear structures in adult. Conclusions: These findings suggest that lamin A is a prerequisite for proper atrial cardiomyocyte morphology in embryonic zebrafish, and is indispensable for correct cardiac electrophysiology and -conduction.

Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-alpha factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I-Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on I-Na. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.

Background: Racial inequities for patients with heart failure (HF) have been widely documented. HF patients who receive cardiology care during a hospital admission have better outcomes. It is unknown whether there are differences in admission to a cardiology or general medicine service by race. This study examined the relationship between race and admission service, and its effect on 30-day readmission and mortality Methods: We performed a retrospective cohort study from September 2008 to November 2017 at a single large urban academic referral center of all patients self-referred to the emergency department and admitted to either the cardiology or general medicine service with a principal diagnosis of HF, who self-identified as white, black, or Latinx. We used multivariable generalized estimating equation models to assess the relationship between race and admission to the cardiology service. We used Cox regression to assess the association between race, admission service, and 30-day readmission and mortality. Results: Among 1967 unique patients (66.7% white, 23.6% black, and 9.7% Latinx), black and Latinx patients had lower rates of admission to the cardiology service than white patients (adjusted rate ratio, 0.91; 95% CI, 0.84–0.98, for black; adjusted rate ratio, 0.83; 95% CI, 0.72–0.97 for Latinx). Female sex and age >75 years were also independently associated with lower rates of admission to the cardiology service. Admission to the cardiology service was independently associated with decreased readmission within 30 days, independent of race. Conclusions: Black and Latinx patients were less likely to be admitted to cardiology for HF care. This inequity may, in part, drive racial inequities in HF outcomes.

Background The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood. Objectives Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of subjects with and without peanut allergy (PA). Methods We obtained samples from patients with PA, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (Consortium of Food Allergy Research [CoFAR] 6). Subjects were confirmed as having PA, or if they passed a 1-g peanut challenge, they were termed high-threshold subjects. Healthy control (HC) subjects were also recruited. Peanut-responsive T cells were identified based on CD154 expression after 6 to 18 hours of stimulation with peanut extract. Cells were analyzed by using flow cytometry and single-cell RNA sequencing. Results Patients with PA had tissue- and follicle-homing peanut-responsive CD4 + T cells with a heterogeneous pattern of T H 2 differentiation, whereas control subjects had undetectable T-cell responses to peanut. The PA group had a delayed and IL-2–dependent upregulation of CD154 on cells expressing regulatory T (Treg) cell markers, which was absent in HC or high-threshold subjects. Depletion of Treg cells enhanced cytokine production in HC subjects and patients with PA in vitro , but cytokines associated with highly differentiated T H 2 cells were more resistant to Treg cell suppression in patients with PA. Analysis of gene expression by means of single-cell RNA sequencing identified T cells with highly correlated expression of IL4 , IL5 , IL9 , IL13 , and the IL-25 receptor IL17RB . Conclusions These results demonstrate the presence of highly differentiated T H 2 cells producing T H 2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional T H 2 response was more evident than a Treg cell deficit among peanut-responsive T cells.

Background: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. Methods: Optical mapping was performed in zebrafish hearts to determine Ca 2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca 2+ handling. L-type calcium channel (LTCC) current ( I Ca,L ) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. Results: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in I Ca,L and Cavα1c abundance, whereas LITAF knockdown increased I Ca,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD ( neural precursor cell expressed developmentally downregulated protein ) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on I Ca,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of I Ca,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. Conclusions: LITAF acts as an adaptor protein promoting NEDD4-1–mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

In centralized immune monitoring for a multi-center allergen immunotherapy trial, we observed frequent loss of CD4+ T cell integrity following staining of cultured PBMCs with our regulatory T cell flow cytometry panel. Samples were marked by a loss of total cellular events, altered scatter properties, and reduced CD3+CD4+ events. This occurred only in samples that were stained with Foxp3 and were therefore treated with Foxp3 fixation-permeabilization buffer. We identified granulocyte contamination in samples associated with a loss of integrity, and went on to test the impact of granulocyte depletion on day-old blood samples. Granulocyte depletion prevented loss of cell integrity and CD3+CD4+ events, and reduced variability in detection of Foxp3+ cells. Addition of purified neutrophils back to PBMCs altered scatter properties and detection of CD4+ T cells. Implementation of a granulocyte depletion step in our standard operating protocols has reduced assay failure due to loss of sample integrity from 31% to 0%. Routine incorporation of a granulocyte depletion step during PBMC isolation is recommended prior to downstream immune monitoring in blood with next-day processing.

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- Speg fl/fl ) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca 2+ ) and transverse-tubule imaging of ventricular myocytes from MCM- Speg fl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca 2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM- Speg fl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM- Speg fl/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 Ca 2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.

Thirteen is the charm in anaphylaxis Immunoglobulin E (IgE) is a type of antibody associated with allergies and response to parasites such as worms. When high-affinity, allergen-specific IgE binds its target, it can cross-link receptors on mast cells that induce anaphylaxis. It remains unclear, however, how B cells are instructed to generate high-affinity IgE. Gowthaman et al. discovered a subset of T follicular helper cells (T FH 13) that direct B cells to do just that. T FH 13 cells are induced by allergens but not during parasite infection. Transgenic mice lacking these cells show impaired production of high-affinity, anaphylactic IgE. T FH 13 cells, which are elevated in patients with food and aeroallergies, may be targeted in future antianaphylaxis therapies. Science , this issue p. eaaw6433

// Juan Wang 1,2 , Liang Peng 2 , Ruihua Zhang 2 , Zihan Zheng 2,3 , Chun Chen 4,5 , Ka Lung Cheung 6 , Miao Cui 7 , Guanglin Bian 2 , Feihong Xu 2 , David Chiang 2 , Yuan Hu 2 , Ye Chen 1 , Geming Lu 2 , Jianjun Yang 2 , Hui Zhang 2 , Jianfei Yang 8 , Hongfa Zhu 7 , Shu-hsia Chen 2 , Kebin Liu 9 , Ming-Ming Zhou 6 , Andrew G. Sikora 2 , Liwu Li 5 , Bo Jiang 1 and Huabao Xiong 2 1 Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China 2 Department of Medicine, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 3 Department of Biology, College of Arts and Sciences, University of North Carolina at Chapel Hill, NC, USA 4 Amgen Inc., Thousand Oaks, CA, USA 5 Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA 6 Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 7 Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 8 Tempero Pharmaceuticals, GlasxoSmithKline, Cambridge, MA, USA 9 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USA Correspondence to: Huabao Xiong, email: // Bo Jiang, email: // Keywords : 5-FU, TH17, TS, Immunology and Microbiology Section, Immune response, Immunity Received : November 24, 2015 Accepted : March 07, 2016 Published : March 24, 2016 Abstract While it is well established that treatment of cancer patients with 5-Fluorouracil (5-FU) can result in immune suppression, the exact function of 5-FU in the modulation of immune cells has not been fully established. We found that low dose 5-FU selectively suppresses T H 17 and T H 1 cell differentiation without apparent effect on Treg, T H 2, and significantly suppresses thymidylate synthase (TS) expression in T H 17 and T H 1 cells but has a lesser effect in tumor cells and macrophages. Interestingly, the basal expression of TS varies significantly between T helper phenotypes and knockdown of TS significantly impairs T H 17 and T H 1 cell differentiation without affecting the differentiation of either Treg or T H 2 cells. Finally, low dose 5-FU is effective in ameliorating colitis development by suppressing T H 17 and T H 1 cell development in a T cell transfer colitis model. Taken together, the results highlight the importance of the anti-inflammatory functions of low dose 5-FU by selectively suppressing T H 17 and T H 1 immune responses.

Mutations in the lamin A/C ( LMNA ) gene have been causally linked to dilated cardiomyopathy with conduction disease. However, the mechanism by which the laminopathy leads to electrophysiological disorders has not been fully elucidated. In a multi-regional registry of early-onset cardiac conduction

Background: Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca 2+ ) homeostasis and elevated reactive oxygen species. CaMKII (Ca 2+ /calmodulin-dependent protein kinase II) is vital for normal Ca 2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice. Conclusions: Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca 2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy.

Background: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors. Methods: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%–40%), and severe HF (ejection fraction Results: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum. Conclusions: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular “sponge” of PP1c.

The molecular mechanisms underlying the early development of atrial fibrillation (AF) remain poorly understood. Emerging evidence suggests that abnormal epigenetic modulation via microRNAs (miRNAs) might be involved in the pathogenesis of paroxysmal AF (pAF).To identify key molecular changes associated with pAF, we conducted state-of-the-art transcriptomic studies to identify the abnormal miRNA-mRNA interactions potentially driving AF development.High-quality total RNA including miRNA was isolated from atrial biopsies of age-matched and sex-matched pAF patients and control patients in sinus rhythm (SR; n=4 per group) and used for RNA-sequencing and miRNA microarray. Results were analyzed bioinformatically and validated using quantitative real-time (qRT)-PCR and 3'UTR luciferase reporter assays.113 genes and 49 miRNAs were differentially expressed (DE) in pAF versus SR patients. Gene ontology analysis revealed that most of the DE genes were involved in the "gonadotropin releasing hormone receptor pathway" and "p53 pathway". Of these DE genes, bioinformatic analyses identified 23 pairs of putative miRNA-mRNA interactions that were altered in pAF (involving 15 miRNAs and 17 mRNAs). Using qRT-PCR and 3'UTR luciferase reporter assays, the interaction between upregulation of miR-199a-5p and downregulation of FKBP5 was confirmed in samples from pAF patients.Our combined transcriptomic analysis and miRNA microarray study of atrial samples from pAF patients revealed novel pathways and miRNA-mRNA regulations that may be relevant in the development of pAF. Future studies are required to investigate the potential involvement of the gonadotropin releasing hormone receptor and p53 pathways in AF pathogenesis.

Reversible phosphorylation of proteins is a delicate yet dynamic balancing act between kinases and phosphatases, the disturbance of which underlies numerous disease processes. While our understanding of protein kinases has grown tremendously over the past decades, relatively little is known regarding protein phosphatases. This may be because protein kinases are great in number and relatively specific in function, and thereby amenable to be studied in isolation, whereas protein phosphatases are much less abundant and more unspecific in their function. To achieve subcellular localization and substrate specificity, phosphatases depend on partnering with a large number of regulatory subunits, protein scaffolds and/or other interactors. This added layer of complexity presents a significant barrier to their study, but holds the key to unexplored opportunities for novel pharmacologic intervention. In this review we focus on the serine/threonine protein phosphatase type-1 (PP1), which plays an important role in cardiac physiology and pathophysiology. Although much work has been done to investigate the role of PP1 in cardiac diseases including atrial fibrillation and heart failure, most of these studies were limited to examining and manipulating the catalytic subunit(s) of PP1 without adequately considering the PP1 interactors, which give specificity to PP1’s functions. To complement these studies, three unbiased methods have been developed and applied to the mapping of the PP1 interactome: bioinformatics approaches, yeast two-hybrid screens, and affinity-purification mass spectrometry. The application of these complementary methods has the potential to generate a detailed cardiac PP1 interactome, which is an important step in identifying novel and targeted pharmacological interventions.

BACKGROUND Cardiovascular disease is a leading cause of death worldwide. Arrhythmias are associated with significant morbidity and mortality related to cardiovascular disease. Recent work illustrates that many cardiac arrhythmias are initiated by a pathologic imbalance between kinase and phosphatase activities in excitable cardiomyocytes. OBJECTIVE To test the relationship between myocyte kinase/phosphatase imbalance and cellular and whole animal arrhythmia phenotypes associated with ankyrin-B cardiac syndrome. METHODS By using a combination of biochemical, electrophysiological, and in vivo approaches, we tested the ability of calcium/calmodulin-dependent kinase (CaMKII) inhibition to rescue imbalance in kinase/phosphatase pathways associated with human ankyrin-B-associated cardiac arrhythmia. RESULTS The cardiac ryanodine receptor (RyR 2 ), a validated target of kinase/phosphatase regulation in myocytes, displays abnormal CaMKII-dependent phosphorylation (pS2814 hyperphosphorylation) in ankyrin-B +/− heart. Notably, RyR 2 dysregulation is rescued in myocytes from ankyrin-B +/− mice overexpressing a potent CaMKII-inhibitory peptide (AC3I), and aberrant RyR 2 open probability observed in ankyrin-B +/− hearts is normalized by treatment with the CaMKII inhibitor KN-93. CaMKII inhibition is sufficient to rescue abnormalities in ankyrin-B +/− myocyte electrical dysfunction including cellular afterdepolarizations, and significantly blunts whole animal cardiac arrhythmias and sudden death in response to elevated sympathetic tone. CONCLUSIONS These findings illustrate the complexity of the molecular components involved in human arrhythmia and define regulatory elements of the ankyrin-B pathway in pathophysiology. Furthermore, the findings illustrate the potential impact of CaMKII inhibition in the treatment of a congenital form of human cardiac arrhythmia.

Food allergy has rapidly increased in prevalence, suggesting an important role for environmental factors in disease susceptibility. The immune response of food allergy is characterized by IgE production, and new findings from mouse and human studies indicate an important role of the cytokine IL-9, which is derived from both T cells and mast cells, in disease manifestations. Emerging evidence suggests that route of exposure to food, particularly peanut, is important. Exposure through the skin promotes sensitization while early exposure through the gastrointestinal tract promotes tolerance. Evidence from mouse studies indicate a role of the microbiome in development of food allergy, which is supported by correlative human studies showing a dysbiosis in food allergy. There is no approved treatment for food allergy, but emerging therapies are focused on allergen immunotherapy to provide desensitization, while pre-clinical studies are focused on using adjuvants or novel delivery approaches to improve efficacy and safety of immunotherapy., Highlights • Emerging evidence suggests that route of exposure to food allergens in early life determines sensitization versus tolerance. • The microbiota and dietary factors appear to play a key role in susceptibility to food allergy. • Immunotherapy applied via different routes is currently the most promising form of experimental treatment for food allergy.

The association between systolic cardiac dysfunction and arrhythmia development in patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) is generally assumed but has not been extensively studied. The purpose of this study was to describe arrhythmias in patients with DMD and BMD in the present era and determine whether arrhythmia development is associated with cardiac dysfunction. This is a single-center retrospective review of 237 Holters from 91 patients with DMD (mean = 17 ± 4 years, range 3 to 27 years) and 64 Holters from 21 patients with BMD (mean = 18 ± 7 years, range 4 to 31 years) with corresponding echocardiography. Holters were stratified by age of patient at the time of study and ejection fraction: normal (≥55%), mild (55% and ≥45%), moderate (45% and ≥30%), and severe (30%). Arrhythmias included frequent atrial and ventricular premature complexes (10/hr), couplets, and runs of supraventricular and ventricular tachycardias. Arrhythmias occurred in 44% of DMD and 57% of BMD patients and were significantly associated with decrease in cardiac function. Clinically significant arrhythmias (supraventricular tachycardia and ventricular tachycardia) occurred in 10% of all Holters obtained in patients with DMD and 25% of all Holters obtained in patients with BMD. Subgroup analysis of Holters from patients with DMD demonstrated that arrhythmias increased with decreasing ejection fraction regardless of age, but that age was also a significant predictor of arrhythmia development. In conclusion, among patients with DMD or BMD, arrhythmias increase with development of cardiac dysfunction.

Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson's trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility.

Allergen immunotherapy for the treatment of food allergy has been a subject of intensive study within the last 10 years. After an unsuccessful attempt with subcutaneous immunotherapy for peanut allergy, other routes with varying degrees of safety and efficacy have been tested for peanut, milk, and egg allergies. In this review, we summarize the results to date with oral immunotherapy, sublingual immunotherapy, and epicutaneous immunotherapy for the treatment of food allergy. While results of immunotherapy trials are promising, increases in efficacy are commonly associated with an increased side effect profile. There is a need for additional research beginning at the preclinical level to develop safe and effective treatments for food allergy.

Background— Cardiac device implantation can be complicated by inability to adequately place leads because of significant lead capture issues. This study sought to determine whether there are genetic bases that underlie poor lead capture. Methods and Results— Retrospective review of all patients with structurally normal hearts who underwent new device implantation at Texas Children’s Hospital between 2009 and 2014 was performed. Patients with inability to capture at 10 V or a final capture threshold ≥3 V at 0.4 ms during implant were analyzed. Among a total of 136 patients (median age, 13 years; range, 3 days to 46 years), 11 patients (8.1%) who underwent dual chamber device implantation had elevated thresholds in the atria (4), ventricle (3), or both chambers (4; atrial-lead threshold, 4.7±4.3 versus 0.7±0.3 V; ventricular-lead, 3.0±3.3 versus 0.7±0.3 V). All 11 patients presented with sinus node dysfunction and 10 had atrial arrhythmias. At implant, inability to find atrial capture was seen in 4 patients. Three demonstrated intermittent complete loss of ventricular capture after implantation: 1 has recurrent syncope, 2 eventually died. Genetic testing performed in 10 demonstrated 7 patients with 6 distinct SCN5A mutations, all predicted to be severe loss-of-function mutations by bioinformatic analyses. In the remaining patients, although putative pathogenic mutations were not found, multiple SCN5A polymorphisms were identified in 2 and a desmin mutation in 1. Conclusions— This study suggests that significant capture issues at implant may be because of loss-of-function SCN5A mutations, providing new insights into SCN5A function. Recognition of this association may be critical for planning device implantation strategies and patient follow-up.

Background The molecular mechanisms underlying the early development of atrial fibrillation (AF) remain poorly understood. Emerging evidence suggests that abnormal epigenetic modulation via microRNAs (miRNAs) might be involved in the pathogenesis of paroxysmal AF (pAF).

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 β 2 -adrenergic receptor lysosomal targeting and ubiquitination, which in turn resulted in the elevation of β 2 -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. β 2 -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 β 2 -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.

Background— Enhanced sarcoplasmic reticulum Ca 2+ -leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein is elevated in atria of patients with paroxysmal AF, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RyR2-3′-untranslated region and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated sarcoplasmic reticulum Ca 2+ -leak. Methods and Results— Quantitative real-time polymerase chain reaction showed that the levels of mature miR-106b, miR-93, and miR-25 were lower in atria of patients with paroxysmal AF when compared with patients in sinus rhythm. In vitro assay showed that miR-93 reduced RyR2-3′-untranslated region luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25 −/− mice was increased by 42% when compared with wild-type littermates but still maintained a normal subcellular distribution. Ca 2+ -spark frequency and total sarcoplasmic reticulum Ca 2+ -leak were increased in atrial myocytes of miR-106b-25 −/− mice. Telemetry ECG recordings revealed that miR-106b-25 −/− mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than wild-type littermates. Increased sarcoplasmic reticulum Ca 2+ -release and AF susceptibility in miR-106b-25 −/− mice were abolished by the RyR2 blocker K201. Conclusions— These results suggest that miR-106b-25 cluster–mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in paroxysmal AF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression.

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in the clinical setting [(1)][1]. In the general population, the prevalence of AF increases with age, ranging from 0.5% in patients younger than 40 years to 5% in patients older than 65 years [(2)][2]. Less is known about the

Background Peanut allergy is common, life-threatening, and without therapeutic options. We evaluated peanut epicutaneous immunotherapy (EPIT) by using Viaskin Peanut for peanut allergy treatment. Objective We sought to evaluate the clinical, safety, and immunologic effects of EPIT for the treatment of peanut allergy. Methods In this multicenter, double-blind, randomized, placebo-controlled study, 74 participants with peanut allergy (ages 4-25 years) were treated with placebo (n = 25), Viaskin Peanut 100 μg (VP100; n = 24) or Viaskin Peanut 250 μg (VP250; n = 25; DBV Technologies, Montrouge, France). The primary outcome was treatment success after 52 weeks, which was defined as passing a 5044-mg protein oral food challenge or achieving a 10-fold or greater increase in successfully consumed dose from baseline to week 52. Adverse reactions and mechanistic changes were assessed. Results At week 52, treatment success was achieved in 3 (12%) placebo-treated participants, 11 (46%) VP100 participants, and 12 (48%) VP250 participants ( P = .005 and P = .003, respectively, compared with placebo; VP100 vs VP250, P = .48). Median change in successfully consumed doses were 0, 43, and 130 mg of protein in the placebo, VP100, and VP250 groups, respectively (placebo vs VP100, P = .014; placebo vs VP250, P = .003). Treatment success was higher among younger children ( P = .03; age, 4-11 vs >11 years). Overall, 14.4% of placebo doses and 79.8% of VP100 and VP250 doses resulted in reactions, predominantly local patch-site and mild reactions ( P = .003). Increases in peanut-specific IgG 4 levels and IgG 4 /IgE ratios were observed in peanut EPIT-treated participants, along with trends toward reduced basophil activation and peanut-specific T H 2 cytokines. Conclusions Peanut EPIT administration was safe and associated with a modest treatment response after 52 weeks, with the highest responses among younger children. This, when coupled with a high adherence and retention rate and significant changes in immune pathways, supports further investigation of this novel therapy.

Cardiomyocytes rely on a highly specialized subcellular architecture to maintain normal cardiac function. In a little over a decade, junctophilin-2 (JPH2) has become recognized as a cardiac structural protein critical in forming junctional membrane complexes (JMCs), which are subcellular domains essential for excitation–contraction coupling within the heart. While initial studies described the structure of JPH2 and its role in anchoring junctional sarcoplasmic reticulum and transverse-tubule (T-tubule) membrane invaginations, recent research has an expanded role of JPH2 in JMC structure and function. For example, JPH2 is necessary for the development of postnatal T-tubule in mammals. It is also critical for the maintenance of the complex JMC architecture and stabilization of local ion channels in mature cardiomyocytes. Loss of this function by mutations or down-regulation of protein expression has been linked to hypertrophic cardiomyopathy, arrhythmias, and progression of disease in failing hearts. In this review, we summarize current views on the roles of JPH2 within the heart and how JPH2 dysregulation may contribute to a variety of cardiac diseases.

BackgroundAtrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown.ObjectivesThe purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits.MethodsCardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry–based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit–PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation.ResultsA total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone.ConclusionsSubcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c–R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+ remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.

Aims Altered Ca2+ handling in atrial fibrillation (AF) has been associated with dysregulated protein phosphatase 1 (PP1) and subcellular heterogeneities in protein phosphorylation, but the underlying mechanisms remain unclear. This is due to a lack of investigation into the local , rather than global, regulation of PP1 on different subcellular targets such as ryanodine receptor type 2 (RyR2), especially in AF. Methods and results We tested the hypothesis that impaired local regulation of PP1 causes RyR2 hyperphosphorylation thereby promoting AF susceptibility. To specifically disrupt PP1's local regulation of RyR2, we used the spinophilin knockout (Sp−/−) mice ( Mus musculus ) since PP1 is targeted to RyR2 via spinophilin. Without spinophilin, the interaction between PP1 and RyR2 was reduced by 64%, while RyR2 phosphorylation was increased by 43% at serine (S)2814 but unchanged at S2808. Lipid bilayer experiments revealed that single RyR2 channels isolated from Sp−/− hearts had an increased open probability. Likewise, Ca2+ spark frequency normalized to sarcoplasmic reticulum Ca2+ content was also enhanced in Sp−/− atrial myocytes, but normalized by Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-93 and AIP and also by genetic inhibition of RyR2 S2814 phosphorylation. Finally, Sp−/− mice exhibited increased atrial ectopy and susceptibility to pacing-induced AF, both of which were also prevented by the RyR2 S2814A mutation. Conclusion PP1 regulates RyR2 locally by counteracting CaMKII phosphorylation of RyR2. Decreased local PP1 regulation of RyR2 contributes to RyR2 hyperactivity and promotes AF susceptibility. This represents a novel mechanism for subcellular modulation of calcium channels and may represent a potential drug target of AF.

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.

Background— Electrical, structural, and Ca 2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca 2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca 2+ -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 [Ca 2+ ] i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca 2+ current, and Na + /Ca 2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca 2+ leak and incidence of delayed after-depolarizations in pAF. Ca 2+ -transient amplitude and sarcoplasmic reticulum Ca 2+ load (caffeine-induced Ca 2+ -transient amplitude, integrated Na + /Ca 2+ -exchange current) were larger in pAF. Ca 2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca 2+ 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 Ca 2+ leak and sarcoplasmic reticulum Ca 2+ -release events, causing delayed after-depolarizations/triggered activity in pAF. Conclusions— Increased diastolic sarcoplasmic reticulum Ca 2+ 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 Ca 2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation-contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in car- diomyocytes. Methods and results Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; aMHC- shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by .70% inaMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes impli- cated in TT development remained mostly unchanged. At the same time, intracellular Ca 2+ handling was disrupted in ventricular myocytes fromaMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection frac- tion, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8. Conclusion Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.

Background: Both nuclear receptor subfamily 2 group F member 1 (NR2F1) and microRNAs (miRNAs) have been shown to play critical roles in the developing and functional inner ear. Based on previous studies suggesting interplay between NR2F1 and miRNAs, we investigated the coregulation between NR2F1 and miRNAs to better understand the regulatory mechanisms of inner ear development and functional maturation. Results: Using a bioinformatic approach, we identified 11 potential miRNAs that might coregulate target genes with NR2F1 and analyzed their targets and potential roles in physiology and disease. We selected 6 miRNAs to analyze using quantitative real-time (qRT) -PCR and found that miR-140 is significantly down-regulated by 4.5-fold (P=0.004) in the inner ear of NR2F1 knockout (Nr2f1 –/– ) mice compared to wild-type littermates but is unchanged in the brain. Based on this, we performed chromatin-immunoprecipitation followed by qRT-PCR and confirmed that NR2F1 directly binds and regulates both miR-140 and Klf9 in vivo. Furthermore, we performed luciferase reporter assay and showed that miR-140 mimic directly regulates KLF9-3’UTR, thereby establishing and validating an example coregulatory network involving NR2F1, miR-140, and Klf9. Conclusions: We have described and experimentally validated a novel tissue-dependent coregulatory network for NR2F1, miR-140, and Klf9 in the inner ear and we propose the existence of many such coregulatory networks important for both inner ear development and function. Citation: Chiang DY, Cuthbertson DW, Ruiz FR, Li N, Pereira FA (2013) A Coregulatory Network of NR2F1 and microRNA-140. PLoS ONE 8(12): e83358.

Purpose: Atrial fibrillation (AF) is the most common cardiac arrhythmia with a growing prevalence yet current pharmaceutical treatments are ineffective. Previous studies suggest that protein phosphatase 1 (PP1) is dysregulated globally in AF patients but its local regulation on key Ca2+-handling proteins such as ryanodine receptor type-2 (RyR2) is not known. In this study we hypothesize that decreased PP1 regulation of RyR2 promotes AF through increasing Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2. Methods: To study the local regulation of RyR2 by PP1, we used spinophilin knockout mice (KO), since PP1 targets RyR2 via spinophilin. The interaction between RyR2 and PP1 was measured using co-immunoprecipitation while RyR2 phosphorylation level was quantified using Western blot. RyR2 activity was determined by measuring 1) single RyR2 channel activity in a lipid bilayer system and 2) Ca2+ sparks frequency in atrial myocytes loaded with Fluo4-AM. Baseline cardiac characteristics were determined using echocardiography and histological staining while AF inducibility was assessed by intracardiac electrophysiology study. Results: The interaction of PP1 with RyR2 was reduced by 55% in the KO hearts compared to WT (P

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca(2+)-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) of β cells. This elevation in [Ca(2+)]cyt leads to activation of Ca(2+)/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca(2+)-release channel implicated in Ca(2+)-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca(2+) leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca(2+)]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca(2+) pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.