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Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca(2+) required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca(2+)-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca(2+) handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO(+/−)) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO(+/−) myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca(2+) spark frequency and amplitude; (3) slower rate of Ca(2+) release and mild but significant desynchronization of the Ca(2+) transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca(2+) release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

Visuospatial working memory (VSWM) deficits have been demonstrated to occur during the development of type-1-diabetes (T1D). Despite confirming the early appearance of distinct task-related brain activation patterns in T1D patients compared to healthy controls, the effect of VSWM load on functional brain connectivity during task performance is still unknown. Using electroencephalographic methods, the present study evaluated this topic in clinically well-controlled T1D young patients and healthy individuals, while they performed a VSWM task with different memory load levels during two main VSWM processing phases: encoding and maintenance. The results showed a significantly lower number of correct responses and longer reaction times in T1D while performing the task. Besides, higher and progressively increasing functional connectivity indices were found for T1D patients in response to cumulative degrees of VSWM load, from the beginning of the VSWM encoding phase, without notably affecting the VSWM maintenance phase. In contrast, healthy controls managed to solve the task, showing lower functional brain connectivity during the initial VSWM processing steps with more gradual task-related adjustments. Present results suggest that T1D patients anticipate high VSWM load demands by early recruiting supplementary processing resources as the probable expression of a more inefficient, though paradoxically better adjusted to task demands cognitive strategy.

Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca 2+ transients, increased Ca 2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca 2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca 2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca 2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca 2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca 2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca 2+ , increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca 2+ ] i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca 2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca 2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca 2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Patient: Female, 55 Final Diagnosis: Gallbladder adenocarcinoma Symptoms: Abdominal pain Medication: — Clinical Procedure: Cholecystectomy • pancreatic necrosectomy Specialty: Surgery Objective: Rare co-existance of disease or pathology Background: Gallstones are a common cause of acute pancreatitis. The proposed mechanism by which choledocholithiasis induces pancreatitis is mechanical obstruction of the ampulla leading to the reflux of bile into the pancreatic duct or edema resulting from a gallstone’s passage. To our knowledge, there are no previously reported cases of gallbladder adenocarcinoma as a potential cause of acute pancreatitis. Herein, we describe a patient who presented with acute necrotizing pancreatitis, without other associated risk factors, who was found to have a fragmented friable polypoid gallbladder adenocarcinoma. Case Report: A 55-year old Hispanic female with prediabetes presented to the Emergency Department with severe epigastric abdominal pain radiating to her back. The patient’s clinical presentation, laboratory tests and computed tomography imaging were suggestive of acute necrotizing pancreatitis and a gallbladder lesion concerning for neoplasm. After clinical resolution of her pancreatitis, the patient was brought to the operating room for a cholecystectomy. Final pathology revealed a stage T1aN0 gallbladder adenocarcinoma. Conclusions: We have presented a patient with acute necrotizing pancreatitis in the absence of alcohol abuse, gallstones, biliary sludge, hypertriglyceridemia, hypercalcemia, or hereditary predisposition. Without evidence of other etiologies, we hypothesize that the friable tumor fragments of the gallbladder adenocarcinoma might be the underlying cause of pancreatitis in this patient.

BACKGROUND -: Arrhythmia syndromes associated with KCNJ2 mutations have been described clinically, however little is known of the underling arrhythmia mechanism. We create the first patient inspired KCNJ2 transgenic mouse and study effects of this mutation on cardiac function, I(K1), and Ca(2+) handling, to determine the underlying cellular arrhythmic pathogenesis. METHODS -: A cardiac-specific KCNJ2-R67Q mouse was generated and bred for heterozygosity (R67Q(+/−)). Echocardiography was performed at rest, under anesthesia. In vivo electrocardiogram (ECG) recording and whole heart optical mapping of intact hearts was performed before and after adrenergic stimulation in wild-type (WT) littermate controls and R67Q(+/−) mice. I(K1) measurements, action potential (AP) characterization, and intracellular Ca(2+) imaging from isolated ventricular myocytes at baseline and after adrenergic stimulation were performed in WT and R67Q(+/−) mice. RESULTS -: R67Q(+/−) mice (n=17) showed normal cardiac function, structure, and baseline electrical activity compared to WT (n=10). Following epinephrine and caffeine, only the R67Q(+/−) mice had bidirectional ventricular tachycardia (BiVT), ventricular tachycardia, frequent ventricular ectopy and/or bigeminy and optical mapping demonstrated high prevalence of spontaneous and sustained ventricular arrhythmia. Both R67Q(+/−) (n=8) and WT myocytes (n=9) demonstrated typical n-shaped I(K1) IV relationship; however, following isoproterenol, max outward I(K1) increased by ~20% in WT but decreased by ~24% in R67Q(+/−) (p

Background: Arrhythmia syndromes associated with KCNJ2 mutations have been described clinically; however, little is known of the underlying arrhythmia mechanism. We create the first patient inspired KCNJ2 transgenic mouse and study effects of this mutation on cardiac function, I K1 , and Ca 2+ handling, to determine the underlying cellular arrhythmic pathogenesis. Methods: A cardiac-specific KCNJ2 -R67Q mouse was generated and bred for heterozygosity (R67Q +/− ). Echocardiography was performed at rest, under anesthesia. In vivo ECG recording and whole heart optical mapping of intact hearts was performed before and after adrenergic stimulation in wild-type (WT) littermate controls and R67Q +/− mice. I K1 measurements, action potential characterization, and intracellular Ca 2+ imaging from isolated ventricular myocytes at baseline and after adrenergic stimulation were performed in WT and R67Q +/− mice. Results: R67Q +/− mice (n=17) showed normal cardiac function, structure, and baseline electrical activity compared with WT (n=10). Following epinephrine and caffeine, only the R67Q +/− mice had bidirectional ventricular tachycardia, ventricular tachycardia, frequent ventricular ectopy, and/or bigeminy and optical mapping demonstrated high prevalence of spontaneous and sustained ventricular arrhythmia. Both R67Q +/− (n=8) and WT myocytes (n=9) demonstrated typical n-shaped I K1 IV relationship; however, following isoproterenol, max outward I K1 increased by ≈20% in WT but decreased by ≈24% in R67Q +/− ( P +/− myocytes (n=5) demonstrated prolonged action potential duration at 90% repolarization and after 10 nmol/L isoproterenol compared with WT (n=7; P 2+ transient amplitude, 50% decay rate, and sarcoplasmic reticulum Ca 2+ content were not different between WT (n=18) and R67Q +/− (n=16) myocytes. R67Q +/− myocytes (n=10) under adrenergic stimulation showed frequent spontaneous development of early afterdepolarizations that occurred at phase 3 of action potential repolarization. Conclusions: KCNJ2 mutation R67Q +/− causes adrenergic-dependent loss of I K1 during terminal repolarization and vulnerability to phase 3 early afterdepolarizations. This model clarifies a heretofore unknown arrhythmia mechanism and extends our understanding of treatment implications for patients with KCNJ2 mutation.

Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that trigger AF, are linked to discrete atrial regions that experience the highest remodeling and clinically used for AF ablation; however, mechanisms of their arrhythmogenic propensity remain elusive. We employed in vivo ECG telemetry, in vitro optical mapping and confocal imaging of Ca 2+ transients (CaT) from myocytes isolated from the right atrial appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10), caveolin-3 knockout (KO, n=6) and 8-weeks post-myocardial infarction HF (n=8) mice. HF and KO mice showed an increased susceptibility to pacing-induced AF and enhanced ectopy originated exclusively from ICR. Optical mapping in isolated atria showed prolongation of CaT rise up time (CaT-RT) in HF ICR, which suggested a remodeled coupling between L-type Ca 2+ channels (LTCCs) and ryanodine receptors (RyRs) in this specific region. In WT mice, RAA consists of structured myocytes with a prominent transverse-axial tubular system (TATS) while ICR myocytes don’t have TATS. In RAA, CaT-RT depends on LTCCs in TATS triggering RyR, while in ICR, all the LTCCs are localized in surface caveolae where they can activate subsarcolemmal RyRs and lead to a slow diffusion of Ca 2+ inside the cell interior. Downregulation of caveolae was observed specifically in HF ICR. To mimic this, we used cav3-KO mice. Triggered activities were observed in myocytes isolated from HF and KO ICR, which presumably underlie the ectopic activities in tissue level. These myocytes presented significantly unsynchronized sarcoplasmic reticulum (SR) Ca 2+ releases (synchronization index: 10.8±0.9 in WT vs 38.3±4.1 in HF vs 21.5±2.1 in KO, p p 2+ sparks which were linked to CaMKII activity and associated phosphorylation of RyR. Our findings demonstrate that in HF, caveolar disruption creates “hot spots” for arrhythmogenic ectopic activity emanated from discrete vulnerable regions of the right atrium which are associated with desynchronized SR Ca 2+ release and elevated fibrosis.

Challenges in early oral language acquisition in profoundly deaf individuals have an impact on cognitive neurodevelopment. This has led to the exploration of alternative sound perception methods involving training of vibrotactile discrimination of sounds within the language spectrum. In particular, stimulus duration plays an important role in linguistic categorical perception. We comparatively evaluated vibrotactile temporal discrimination of sound and how specific training can modify the underlying electrical brain activity. Fifteen profoundly deaf (PD) and 15 normal-hearing (NH) subjects performed a vibrotactile oddball task with simultaneous EEG recording, before and after a short training period (5 one-hour sessions; in 2.5–3 weeks). The stimuli consisted of 700 Hz pure-tones with different duration (target: long 500 ms; non-target: short 250 ms). The sound-wave stimuli were delivered by a small device worn on the right index finger. A similar behavioral training effect was observed in both groups showing significant improvement in sound-duration discrimination. However, quantitative EEG measurements reveal distinct neurophysiological patterns characterized by higher and more diffuse delta band magnitudes in the PD group, together with a generalized decrement in absolute power in both groups that might reflect a facilitating process associated to learning. Furthermore, training-related changes were found in the beta-band in NH. Findings suggest PD have different cognitive adaptive mechanisms which are not a mere amplification effect due to greater cortical excitability.

Objective Type-1 diabetes (T1D) is a disruptive metabolic disease that has an impact on neurodevelopment through its effects on the structure and function of the brain. One of the cognitive domains affected by T1D is sustained attention. The aim of this study was to analyze this process in children with T1D and compare their results to those of healthy controls. Methods Seventeen T1D children attending regular primary school and a similar group of healthy children matched by gender, age, handedness, and educational level were evaluated while identifying happy faces in a Go-NoGo task presented visually with simultaneous electrophysiological recording. Results Behavioral performance in the two groups was similar but, the T1D children showed greater prefrontal and frontoparietal spectral power in the theta and alpha bands, compared to controls. Distinct patterns of theta lateralization between groups were also observed, with a negative correlation between frontal power magnitudes in delta and theta and glycated hemoglobin levels. Conclusions These results seem to reflect the early deleterious effects of T1D on neurodevelopment, which affects mainly attention allocation processes and the neurofunctional substrates that underlie them. Significance This phenomenon emphasizes the need for studies on neural-specific targets in which T1D affects neurodevelopment.

Rationale: In cardiomyocytes, Na V 1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective: The objective of this study was to test whether Na V 1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1 Δ314-315 or Kir2.1 R44A/R46A with wild-type (WT) Na V 1.5 WT in heterologous cells reduced inward sodium current compared with Na V 1.5 WT alone or coexpressed with Kir2.1 WT . In cell surface biotinylation experiments, expression of Kir2.1 Δ314-315 reduced Na V 1.5 channel surface expression. Glycosylation analysis suggested that Na V 1.5 WT and Kir2.1 WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na V 1.5 WT , and vice versa, whereas coexpression with Kir2.1 Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1 Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na V 1.5 WT and Kir2.1 WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1 WT , Na V 1.5 WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na V 1.5 WT channels interact with AP1 through the Na V 1.5 Y1810 residue, suggesting that, like for Kir2.1 WT , AP1 can mark Na V 1.5 channels for incorporation into clathrin-coated vesicles at the trans -Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions: The Na V 1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na V 1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Sorcin, a penta-EF hand Ca2+-binding protein expressed in cardiomyocytes, is known to interact with ryanodine receptors and other Ca2+ regulatory proteins. To investigate sorcin’s influence on cardiac excitation-contraction coupling and its role in the development of cardiac malfunctions, we generated a sorcin knockout (KO) mouse model. Sorcin KO mice presented ventricular arrhythmia and sudden death when challenged by acute stress induced by isoproterenol plus caffeine. Chronic stress, which was induced by transverse aortic constriction, significantly decreased the survival rate of sorcin KO mice. Under isoproterenol stimulation, spontaneous Ca2+ release events were frequently observed in sorcin KO cardiomyocytes. Sorcin KO hearts of adult, but not young mice developed overexpression of L-type Ca2+ channel and Na+-Ca2+ exchanger, which enhanced ICa and INCX. Consequently, spontaneous Ca2+ release events in sorcin KO cardiomyocytes were more likely to induce arrhythmogenic delayed afterdepolarizations. Our study demonstrates sorcin deficiency may trigger cardiac ventricular arrhythmias due to Ca2+ disturbances, and evidences the critical role of sorcin in maintaining Ca2+ homeostasis, especially during the adrenergic response of the heart.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell–cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease., It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Background The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We described that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin-protein kinase (CaMKII) activity, ryanodine receptors (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. Methods and Results We generated a pre-diabetic model in mice fed with FRD. FRD-mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycemia significantly enhanced [3H]Ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycemia. CaMKII-inhibition prevented hyperglycemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the ROS scavenger Tempol prevented FRD-induced apoptosis in WT-mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodeling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. Conclusions CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favor Ca2+ transit between both organelles. This article is protected by copyright. All rights reserved

With the completion of the human genome in 2003, it became evident what had been suspected for a time through isolated bits of data: a surprisingly small proportion of the genome—only ≈2%!—was actually translated into proteins (DNA→RNA→protein). The vast majority of the DNA had no protein correlate and was considered sterile, receiving the somewhat derisive term junk DNA. However, modern technology, such as next-generation sequencing, has revealed that almost the entire human genome is transcribed (DNA→RNA), meaning therefore that the majority of its nucleotides are associated with at least 1 transcript that does not code for a protein.1 What function do these noncoding RNAs (ncRNAs) serve, then? Work from several fields has painstakingly assembled an emergent picture for ncRNAs in epigenetic processes. Generally speaking, ncRNAs are main actors in the regulation of gene expression, both at the translational and post-translational level. Noncoding RNAs, which are arbitrarily divided into short ncRNAs ( 200 nucleotides), play a critical role in heterochromatin formation, histone modification, DNA methylation, and gene splicing and silencing; dysregulation of their interplay with DNA and its transcription machinery may lead to diseases, such as cancer, autism, and Alzheimer.2 Thus, junk DNA, from which ncRNAs stem from, has turned out to be anything but junk, revealing amazing complexity and fruitful design. Article, see p 1354 Although their expression levels are generally low, the number of known ncRNAs is already in the tens of thousands, evidently greater in magnitude than protein-coding mRNA. To be exact, a few lncRNAs have been wrongly annotated and do code for proteins, but differing from the polyvalent role of their parental lncRNAs, these proteins are known to date to circumscribe their function to modulation of other proteins. A good example is myoreguling,3 a 46-amino acid micropeptide that regulates …

Visualization and kinematic analysis of gait with computer programs and quantitative metrics are nowadays essential tools for the characterization of normal and pathological patterns, as well as for the diagnosis and rehabilitation in gait alterations. However, most of the software that is used for the acquisition systems in the gait labs, estimates a predetermined and restricted number of parameters, conditioning and limiting the work of the medical specialists, either in follow-up studies of individual cases or in the gait description between populations. The objective in this work, is to implement a computational tool that accesses the records of the databases generated by gait analysis equipment, for the visualization and obtaining of quantitative metrics from these kinematic studies, facilitating in this way, the identification of normal and pathological gait patterns, as well as facilitate the statistical estimation inter- and intra-patient in the development of comparative studies.

BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (“PKP2cKO”) 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular (PKP2cKO-LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (PKP2cKO-RV) myocytes showed increased amplitude and duration of Ca(2+) transients, increased [Ca(2+)] in the cytoplasm and sarcoplasmic reticulum (SR), increased frequency of spontaneous Ca(2+) release events (sparks) even at comparable SR load, and dynamic Ca(2+) accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, RyR2 in PKP2cKO-RV cells presented enhanced Ca(2+) sensitivity and preferential phosphorylation in a domain known to modulate Ca(2+) gating. RNAseq at 14 days post-TAM showed no relevant difference in transcript abundance between RV and LV, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca(2+) entry into the cell. Cx43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca(2+), increased frequency of sparks and early stages of RV dysfunction. Cx43 hemichannel block with GAP19 normalized [Ca(2+)](i) homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca(2+) dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Cx43-dependent membrane conduit and repressed by PKC inhibitors. Given that asymmetric Ca(2+) dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca(2+) handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Summary Background Type 1 diabetes (T1D) is a metabolic disorder characterized by recurrent hypo- and hyperglycemic episodes, whose clinical development has been associated with cognitive and working memory (WM) deficits. Objective To contrast quantitative electroencephalography (qEEG) measures between young patients with T1D and healthy controls while performing a visuospatial WM task with two memory load levels and facial emotional stimuli. Methods Four or five neutral or happy faces were sequentially and pseudo-randomly presented in different spatial locations, followed by subsequent sequences displaying the reversed spatial order or any other. Participants were instructed to discriminate between these two alternatives during EEG recording. Results A significant increase in the absolute power of the delta and theta bands, distributed mainly over the frontal region was found during task execution, with a slight decrease of alpha band power in both groups but mainly in control individuals. However, these changes were more pronounced in the T1D patients, and reached their maximum level during the WM encoding phase, even on trials with the lower memory load. In contrast, changes seemed to occur more gradually in controls and results differed significantly only on the trials with the higher WM load. Conclusions These results reflect adaptive WM-processing mechanisms in which cognitive strategies have evolved in T1D patients in order to meet task demands.

Hypertrophic cardiomyopathy (HCM) is triggered mainly by mutations in genes encoding sarcomeric proteins, but a significant proportion of patients lack a genetic diagnosis. We identified a potentially novel mutation in ryanodine receptor 2, RyR2-P1124L, in a patient from a genotype-negative HCM cohort. The aim of this study was to determine whether RyR2-P1124L triggers functional and structural alterations in isolated RyR2 channels and whole hearts. We found that P1124L induces significant conformational changes in the SPRY2 domain of RyR2. Recombinant RyR2-P1124L channels displayed a cytosolic loss-of-function phenotype, which contrasted with a higher sensitivity to luminal [Ca(2+)], indicating a luminal gain of function. Homozygous mice for RyR2-P1124L showed mild cardiac hypertrophy, similar to the human patient. This phenotype, evident at 1 year of age, was accompanied by an increase in the expression of calmodulin (CaM). P1124L mice also showed higher susceptibility to arrhythmia at 8 months of age, before the onset of hypertrophy. RyR2-P1124L has a distinct cytosolic loss-of-function and a luminal gain-of-function phenotype. This bifunctionally divergent behavior triggers arrhythmias and structural cardiac remodeling, and it involves overexpression of CaM as a potential hypertrophic mediator. This study is relevant to continue elucidating the possible causes of genotype-negative HCM and the role of RyR2 in cardiac hypertrophy.

Background: Surgical aortic valve replacement may be complicated by significant perioperative bleeding. TAVR is a less invasive procedure, however bleeding may still occur related to periprocedural complications and anti-thrombotic therapy. We sought to compare baseline characteristics, hemodynamic parameters and clinical outcomes following TAVR between Jehovah’s witness (JW) patients who decline the use of blood products, and non-JW patients. Methods: Demographic, echocardiographic, and hemodynamic data of 245 consecutive patients with symptomatic severe aortic stenosis who underwent TAVR at our institution between January 2013 and December 2015 were analyzed at baseline and at 30 day follow-up. Results: 10 patients were identified as JW refusing blood transfusion. None of the JW patients received blood products. JW and non-JW patients did not differ in respect to hemodynamic or echocardiographic parameters at baseline or 30 days post TAVR, including: blood pressure, heart rate, valve area, gradient, valvuloarterial impedance, cardiac output, stroke volume and ejection fraction. Baseline hemoglobin was higher in the JW versus non-JW groups, respectively (12.76 vs. 11.62 mg/dL p=0.045) and this was sustained at follow-up. Clinical outcomes were similar at 30 days between JW and non-JW patients, notably major bleed (10% vs 8.97%, respectively; p=0.91, defined as a clinical bleeding event with hemoglobin decrease > 2g/dL or transfusion of > 2 units of whole blood/red cells ) and 30 day mortality (0% vs 6.41%, respectively; p=0.19). Conclusion: JW patients were found to have similar hemodynamic and clinical outcomes up to 30 days. TAVR may be considered a reasonable treatment option for patients with symptomatic severe aortic stenosis who refuse transfusion of blood products.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk., eLife digest The human heart contains four distinct chambers that work together to pump blood around the body. In individuals with a condition called atrial fibrillation, two of the chambers (known as the atria) beat irregularly and are unable to push all the blood they hold into the other two chambers of the heart. This can cause heart failure and increases the likelihood of blood clots, which may lead to stroke and heart attacks. Small molecules called calcium ions play a crucial role in regulating how and when the atria contract by driving electrical activity in heart cells. To contract the atria, a storage compartment within heart cells known as the sarcoplasmic reticulum releases calcium ions into the main compartment of the cells. Calcium ions also enter the cell from the surrounding tissue. As the atria relax, calcium ions are pumped back into the sarcoplasmic reticulum or out of the cell by specific transport proteins. Individuals with mutations in a gene called Tbx5 are more likely to develop atrial fibrillation than other people, but it was not clear how such gene mutations contribute to the disease. Here, Dai, Laforest et al. used mice with a mutation in the Tbx5 gene to study how defects in Tbx5 affect electrical activity in heart cells. The experiments found that the Tbx5 gene was critical for calcium ions to drive normal electrical activity in mouse heart cells. Compared with heart cells from normal mice, the heart cells from the mutant mice had decreased flow of calcium ions into the sarcoplasmic reticulum and increased flow of calcium ions out of the cell. These findings provide a direct link between atrial fibrillation and the flow of calcium ions in heart cells. Together with previous work, these findings indicate that multiple different mechanisms could lead to atrial fibrillation, but that many of these involve changes in the flow of calcium ions. Therefore, personalized medicine, where clinicians uncover the specific mechanisms responsible for atrial fibrillation in individual patients, may play an important role in treating this condition in the future.

espanolIntroduccion: Aunque el abuso sexual en la infancia (ASI) es un factor de riesgo para desarrollar depresion mayor en la vida adulta de las mujeres, la informacion sobre las caracteristicas clinicas asociadas con este evento es escasa. Objetivo: Evaluar la historia de ASI y su asociacion con algunas manifestaciones clinicas en mujeres con depresion mayor. Metodo: Se seleccionaron 71 mujeres adultas con depresion mayor en un centro comunitario de salud mental. Fueron evaluadas con Mini Entrevista Neuropsiquiatrica Internacional, Inventario de Depresion de Beck, Escala de Autoevaluacion de Adaptacion Social, Indice de Maltrato Fisico e Indice de Abuso Sexual en la Infancia. Resultados: 53.5 % habia sufrido alguna forma de ASI. No hubo diferencias significativas en edad, escolaridad, sintomatologia depresiva, adaptacion social o maltrato fisico en la infancia entre las mujeres con y sin historia de ASI, solo mayor frecuencia de comorbilidad con el trastorno por ansiedad social (26 versus 6 %) y mayor proporcion de riesgo suicida (68 versus 45.4 %) distinguio a los grupos. Conclusion: Pocas caracteristicas distinguen a las mujeres con depresion mayor con historia de ASI, sin embargo, el pronostico y las implicaciones terapeuticas del mayor riesgo suicida subrayan la importancia de investigar sistematicamente la historia de ASI entre las mujeres con depresion mayor. EnglishIntroduction: Even when child sexual abuse (CSA) is a risk factor for the development of major depression in adult women, data on the clinical features associated with this traumatic event are scarce. Objective: To assess the history of CSA and its association with some clinical manifestations in women with major depression. Method: Seventy-one adult women with major depression were selected in a community-based mental health center. They were assessed with the Mini International Neuropsychiatric Interview, the Beck Depression Inventory, the Social Adaptation Self-evaluation Scale, and childhood physical maltreatment and child sexual abuse indices. Results: Some form of CSA had been suffered by 53.5%. There were no significant differences in age, level of education, depressive symptoms, social adaptation or physical maltreatment during childhood between the women with or without a history of CSA, only a higher frequency of comorbid social anxiety disorder (26% versus 6%) and a higher proportion of suicide risk (68% versus 45.4%) differentiated the groups. Conclusion: Few characteristics distinguish women with major depression with a history of CSA; however, the prognosis and therapeutic implications of a higher suicide risk underscore the importance of systematically investigating the history of CSA among women with major depression.

The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes.We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis inWT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis inWT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization inWT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR–mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles., Facultad de Ciencias Médicas, Facultad de Ciencias Veterinarias, Centro de Investigaciones Cardiovasculares

The existence of a correlation between heart rate (HR), respiratory rate (RR) and skin conductance response (SCR ) has been reported in the literature, as well as the use of these parameters as a measure of the activation level of the autonomous nervous system. This paper introduces a computational tool (SCRATER) developed with the aim to analyze simultaneous recordings of SCR, and heart and respiratory rates, which were calculated from the electrocardiogram recording (ECG) analysis. In this research, a detailed description of each developed algorithm was made, as well as a description of the interface to be used. In the validation of the algorithms used, 192 ECG records and 231 Electro Dermal Activity (EDA) registers of 40 healthy male participants were analyzed, from which the number of QRS complexes and HR in each ECG record and the number of SCRs of each EDA record are calculated. The data obtained were compared with other tools that analyze SCR and HR separately, obtaining comparable results using correlation coefficients. The noise and artifacts present in the records do not allow a correct estimation of the parameters and affect the results of all the tools used in the development of this work. SCRATER offers three main advantages over other tools: 1) free access, 2) open source and 3) does not use coded or exclusive formats. This work provides a free computational tool that allows simultaneous analysis of SCRs, FC and FR .

Background Phosphorylation of the cardiac ryanodine receptor (RyR2) phospho-site S2808 has been touted by the Marks group as a hallmark of heart failure (HF) and a critical mediator of the physiological fight-or-flight response of the heart. In support of this hypothesis, mice unable to undergo phosphorylation at RyR2-S2808 (S2808A) were significantly protected against HF and displayed a blunted response to adrenergic stimulation. However, the issue remains highly controversial because several groups have been unable to reproduce these findings. An important variable not considered before is the genetic background of the mice used to obtain these divergent results. Methods and results We backcrossed a RyR2-S2808A mouse into a congenic C57Bl/6 strain, the same strain used by the Marks group to conduct their experiments. We then performed several key experiments to confirm or discard the genetic background of the mouse as a relevant variable, including induction of HF by myocardial infarction and tests of integrity of adrenergic response. Congenic C57Bl/6 harboring the S2808A mutation showed similar echocardiographic parameters that indicated identical progression towards HF compared to wild type controls, and had a normal response to adrenergic stimulation in whole animal and cellular experiments. Conclusions The genetic background of the different mouse models is unlikely to be the source of the divergent results obtained by the Marks group in comparison to several other groups. Cardiac adrenergic response and progression towards HF proceed unaltered in mice harboring the RyR2-S2808A mutation. Preventing RyR2-S2808 phosphorylation does not preclude a normal sympathetic response nor mitigates the pathophysiological consequences of MI.

Background: Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. Methods: We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). Results: Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM ( P P =0.01), as was autophosphorylated CaMKII ( P PPP3CB ) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P =0.03). 45 Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype ( P =0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [ 3 H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. Conclusions: Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation–positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation–positive and –negative HCM.

Case 1 A 31-year-old man from Acapulco, Guerrero, in the southwest of Mexico, was seen in March 1994 for evaluation of a 1-month history of intermittent migratory subcutaneous swellings on the left scapula, neck, upper lip, and chin. The edema was painful and nonpruritic, with an oval shape of approximately 5–10 cm in diameter. After 2 days of a course of albendazole, 400 mg three consecutive oral doses, one every 12 h, the patient developed a creeping eruption that started on the left side and ended on the right side of the anterior neck. He practiced the custom of eating a very popular Mexican delicacy, “ceviche,” made with raw fish cut into small pieces and mixed with different sauces and lime juice. On physical examination, there was a erythematous and infiltrated serpiginous linear lesion on the neck, 5 cm by 3 mm in size ( Fig. 1). Laboratory studies of peripheral blood revealed a white blood cell (WBC) count of 10,000/mm3 with 39% eosinophils. A specimen for biopsy was carefully taken, with a surgical knife, from the end of the creeping eruption; the lesion disappeared 1 week later leaving a linear pigmentation. Figure 1. Creeping eruption due to G. spinigerum. The arrow marks the end of the lesion from where the specimen for biopsy was taken (Case 1) Histologic examination of the sections stained with hematoxylin and eosin showed, in the reticular dermis, longitudinal and transverse sections of a worm located within an area of necrosis and surrounded by an intense infiltrate of eosinophils ( Fig. 2a). Cutaneous appendages in the immediate neighbourhood of the necrosis area were seen to be surrounded by a dense inflammatory reaction composed of lymphocytes, histiocytes, and some eosinophils. The worm exhibited three distinctive structures: at the anterior end, the cephalic bulb, in the mid-portion, the esophagus, and at the posterior end, the intestine. The cephalic bulb, over one lateral side, was provided with four cuticular spines. The circumference of the intestine was composed of 21–24 columnar cells with six nuclei (average 3 μm in diameter), and one quadrant of the muscular layer consisted of 11–13 muscle cells ( Fig. 2b). These morphological features, according to Japanese descriptions, are consistent with those of Gnathostoma spinigerum.1 Figure 2. (a) Longitudinal and transverse sections of the advanced third stage larva of G. spinigerum are located within an area of necrosis. The following structures are seen: head (H), esophagus (E), intestine (I) (hematoxylin and eosin ×30). (b) High magnification of transverse section through mid-gut level showing: muscular layer (M) and intestinal columnar cells (I) with several nuclei in each cell (arrow) (hematoxylin and eosin ×400) Case 2 A specimen for biopsy with a clinical diagnosis of gnathostomiasis was mailed from Tepic, Nayarit, in the northwest of Mexico, to our dermatopathology laboratory. The data accompanying the specimen mentioned that the piece of tissue had been taken from an edematous and erythematous sinus cord, over a patch of 6×4 cm in diameter, localized on one buttock of a 25-year-old Mexican woman who practiced the custom of eating “ceviche;” no treatment had been given to the patient. Externally, the tissue presented one dark brown cylindrical “U”-shaped organism measuring 9 mm in length and 1 mm in breadth, localized on the center of the surface of the specimen. The minute organism was dissected and scanned using electron microscopy ( Fig. 3a). Figure 3. (a) Scanning electron microscopy of anterior end of G. spinigerum showing the head bulb of the third stage larva with lips (L), four rows of cuticular spines with serrated tips, and part of the body covered with numerous circumferential rows of backwardly directed minute spines (×260). (b) Cavity left by the removal of the parasite. A sparse, mixed infiltrate and slight necrosis are present in the subjacent dermis at the site previously occupied by the worm (hematoxylin and eosin ×100) Histologic examination of the specimen revealed a superficial cavity left by the removal of the worm. The cavity was bounded on both sides by epidermis and its floor contained slightly necrotic dermis with a diffuse inflammatory infiltrate composed of eosinophils and some neutrophils. Blood vessels were surrounded by edema and a sparse infiltrate of lymphoid cells ( Fig. 3b).

We previously showed that ablation of the cardiac Ca2+ release channel/ryanodine receptor (RyR2) phosphorylation at Ser2808 (RyR2-S2808A) has no functional impact on cardiac performance and/or heart failure progression. In this study, we investigated whether this lack of effect may be due to compensatory mechanisms involving altered phosphorylation of the nearby Ser2814 CaMKII phospho-site. We generated a new mouse model where Ser2808 and Ser2814 were genetically replaced by Alanine (RyR2-S2808A/S2814A). Anesthetized RyR2-S2808A/S2814A mice have normal ejection fraction at baseline compared to wild type (WT) mice (55.1±2.6% vs. 55.9±2.4%, respectively, n=7/group) but exhibit ∼20% increase in heart rate (553±33 vs. 460±28 bpm, p