Ectopic Activity Research Articles

PO-02-145 SPATIAL TRANSCRIPTOMICS IN THE HUMAN PULMONARY VEIN SLEEVE

AF is often treated by ablation to isolate the pulmonary veins where ectopic pacemaker activity or reentry may initiate AF. The relationship of gene expression and histology in the pulmonary vein is unknown. To identify gene expression in pulmonary vein sections and identify regions where gene expression may be indicative of AF pathogenesis. Human pulmonary vein (PV) sleeve tissues form the ostia of the left atrium were obtained from 8 unused lung or heart transplant donors in a protocol approved by the Cleveland Clinic IRB. After tissue sectioning and staining, spatial RNAseq was performed using the 10x Genomics Visium platform. mRNAs from∼1000 to 2500 genes were sequenced in each spatial area. Seurat clustering yielded 15 different clusters. These split into two segregating populations, with one connecting population. Multiple cells and cell types may reside in each 55 μm diameter spatial area. A clear asymmetry of clusters was observed in the PV sections. Cell-type marker genes derived from published human heart single nuclei RNAseq were used to determine the dominant cell types in the 15 different clusters. One group of segregating clusters was enriched in cardiomyocytes, while the other group of segregating clusters was enriched for other cell types including fibroblasts, vascular smooth muscle cells, endothelial cells, and adipocytes. Spatial transcriptomics clearly resolved the venous, cardiomyocyte, and epicardial regions of the PV (Figure, showing histology and spatial expression UMAP clusters for two PV specimens), as well as fibrotic regions. Spatial expression of the AF-associated genes PITX2, SHOX2, and HCN4 was also mapped, confirming higher expression of the cardiac master transcription factor SHOX2 in the PV vs. left atrial appendage (LAA) tissues. Expression of the hyperpolarization-activated ion channel HCN4 was sporadic in both PV and LAA specimens. Spatial transcriptomics identified gene expression profiles in the PV easily identifying the venous vascular smooth muscle, cardiomyocyte, and epicardial layers. The predominant cell type in each spatial region was identified by leveraging heart single nuclei RNAseq data. Spatial transcriptomics may help to discover gene expression and cell types associated with ectopic pacemaker activity in the PV.

Role of localized repolarization and conduction heterogeneities in ventricular arrhythmias and sudden cardiac death

Recent clinical studies have indicated the presence of localized tissue abnormalities in sudden cardiac death (SCD) survivors without apparent structural heart disease, including idiopathic ventricular fibrillation (VF) and J-wave Syndromes. The origin of these abnormalities, showing distinct electrical features on contact mapping, and mechanisms of associated arrhythmias are still a matter of debate. To determine the respective role of localized repolarization and conduction heterogeneities in vulnerability to arrhythmias and SCD, and to characterize their specific electrical signatures. Optical mapping was performed on porcine right ventricular models of either local (i) repolarization shortening, (ii) conduction slowing, or (iii) micro-structural heterogeneity which were created by locally perfusing (i) pinacidil (20 μM n = 13) or (ii) flecainide (2 μM n = 13) via an epicardial catheter (branch of right coronary artery), or (iii) by local epicardial tissue destruction through radiofrequency application (9 micro lesions 30W 1 sec n = 13). Pseudo-ECG were assessed and the number of induced and spontaneous arrhythmias were quantified and mapped in each preparation. An HD grid mapping catheter was used to record bipolar electrograms (n = 5 in each group). Electrograms were normal in model (i), but showed local fragmentation in 40% of preparations in model (ii) with greater effects observed at high pacing frequencies (3 Hz). In model (iii), the structural substrate alone increased the width and number of peaks in the electrograms, and addition of flecainide (2 μM) in this model induced pronounced fragmentation (≥ 3 peaks and ≥ 70ms duration) in 100% of the cases. Arrhythmogenicity was significantly increased in models (i) and (ii) which presented 82% and 45% more spontaneous arrhythmias (P < 0.0001 and 0.05 vs. baseline) and a reduced pacing frequency threshold to induce arrhythmias (−69% and −50%, P < 0.001 vs. baseline). In these models, the frequency of spontaneous activity was increased (+411% P < 0.01 and +181% P < 0.05 vs. baseline) and originated near the heterogeneous area. Microstructural substrates have the most pronounced impact on electrograms, especially when combined with sodium channel blockers, while repolarization heterogeneities do not lead to electrogram fragmentation but are associated with the highest prevalence of spontaneous arrhythmias. Both heterogeneities show increased ectopic activity.

PO-02-229 AUTOMATED LOCALIZATION OF ATRIAL ECTOPIC BEATS USING 12-LEAD ECG MEAN SPATIO-TEMPORAL VECTOR POSITION: IMPORTANCE OF INCORPORATING PATIENT-SPECIFIC ANATOMIC MODELS

Localizing the origin of atrial ectopic activity from the 12-lead ECG can be challenging, particularly in patients with atrial pathology. To identify the origin of paced atrial ectopic beats (simulating atrial ectopy) using software that quantifies the mean spatio-temporal vector position of cardiac depolarization projected onto generic and patient-specific atrial models. 12-lead ECG data were collected during sinus rhythm (SR) and during regular pacing from the high and low right atrium (HRA/LRA), proximal and distal coronary sinus (CS), pulmonary veins (LSPV, LIPV, RSPV, RIPV), and left atrial appendage (LAA). The origin and trajectory of the atrial signals were calculated with software that analyzes P-waves from the 12-lead ECG and automatically constructs a net vector of electrical activity projected onto a standard generic model of human atria (CineECG; Nieuwerbrug, The Netherlands). The model atria were split into 9 segments, and the software accuracy was scored based on localization of the paced atrial signal origin to the correct segment. To see if patient-specific anatomy improved localization, we also incorporated atrial anatomy from cardiac MRIs acquired from a subset of 12 patients and compared localization to the generic model. We enrolled 19 patients (mean age 70±9, 4 female) with paroxysmal (63%) or persistent (37%) AF and left atrial (LA) enlargement (mean LA volume 58 cc/m2) undergoing pulmonary vein isolation for treating AF. Using the generic atrial model, the software correctly localized 105/142 (74%) beats to the correct atrial segment (Figure). Accuracy was better for RA compared to LA signals (90% vs. 62%, p < 0.05). Localization worked best in the coronary sinus (19/19, 100%) and worst in the LPVs (14/33, 42%). Patient-specific models improved localization of PV signals, particularly left-sided ones (generic vs. patient-specific: RPVs 70% vs 85%, p = 0.36, LPVs 42% vs 76%, p = 0.03). Accuracy improved and was similar for RA vs. LA localization when using patient-specific models (90% vs. 82%, p = 0.48). The novel software algorithm can automatically localize paced signals in both the left and right atria despite significant atrial pathology. Patient-specific anatomic models improve localization. This software holds promise for automatically localizing and classifying atrial arrhythmias and guiding therapy, including catheter ablation, based on standard 12-lead ECG data.

Activation of Exchange Proteins directly Activated by cAMP (EPAC) lengthens action potential duration by inhibiting potassium currents in atrial cardiomyocytes

IntroductionAtrial fibrillation (AF) is the most frequent sustained cardiac arrhythmia worldwide. Several mechanisms have been described in AF pathogenesis and sustainability such as dysregulation of calcium cycling and action potential (AP) duration. Effectors involved in these dysregulations are potential relevant therapeutic targets. Among them, the Exchange Proteins directly Activated by cAMP (EPAC) has been shown to increase arrhythmogenic activity in ventricular cardiomyocytes by lengthening of AP duration. However, the electrophysiological impact of EPAC at the atrial level remains to be established. ObjectiveTo evaluate the propensity of EPAC activation to induce atrial arrhythmogenic activity and by which mechanisms. MethodMyocytes from human right atrial appendages and wild type (WT) and EPAC1-/- mice atria were dissociated by enzymatic digestion. AP and potassium currents were recorded using the whole cell configuration of the patch clamp technique. EPAC proteins were pharmacologically activated by acute superfusion of 8-CPTAM (10μM), and the selective role of EPAC1 was evaluated by its selective inhibition by superfusion of AM-001 (20μM). ResultsIn mouse atrial myocytes, activation of EPAC by 8-CPTAM significantly increases AP duration at 90% of repolarization (APD90). Interestingly, this effect is higher in WT myocytes than in EPAC1-/-. Moreover, AM-001, which fails to correct AP lengthening in EPAC1-/- myocytes, reverses partially but significantly the effect of 8-CPTAM in WT myocytes. In human atrial myocytes, 8-CPTAM treatment lengthens APD90 by 32.3%. Additionally, in both WT mouse and human atrial myocytes, the treatment by 8-CPTAM induces an inhibition of both peak and sustained components of the K+ currents. Finally, effect of 8-CPTAM on K+ currents seems to be weaker in EPAC1-/- myocytes than in WT. ConclusionOur results show that EPAC activation lengthens AP duration in mouse and human atrial myocytes. Furthermore, inhibition of K+ currents participates in this AP duration modulation. Thus, these data suggest that EPAC proteins might be involved in atrial arrhythmogenic ectopic activity. Nevertheless, the role of each isoform of EPAC in these modulations and EPAC-induced regulation of other ionic currents remain to be specified.

An ERK-dependent molecular switch antagonizes fibrosis and promotes regeneration in spiny mice (Acomys).

Although most mammals heal injured tissues and organs with scarring, spiny mice (Acomys) naturally regenerate skin and complex musculoskeletal tissues. Now, the core signaling pathways driving mammalian tissue regeneration are poorly characterized. Here, we show that, while immediate extracellular signal-regulated kinase (ERK) activation is a shared feature of scarring (Mus) and regenerating (Acomys) injuries, ERK activity is only sustained at high levels during complex tissue regeneration. Following ERK inhibition, ear punch regeneration in Acomys shifted toward fibrotic repair. Using single-cell RNA sequencing, we identified ERK-responsive cell types. Loss- and gain-of-function experiments prompted us to uncover fibroblast growth factor and ErbB signaling as upstream ERK regulators of regeneration. The ectopic activation of ERK in scar-prone injuries induced a pro-regenerative response, including cell proliferation, extracellular matrix remodeling, and hair follicle neogenesis. Our data detail an important distinction in ERK activity between regenerating and poorly regenerating adult mammals and open avenues to redirect fibrotic repair toward regenerative healing.

RWP-RK Domain 3 (OsRKD3) induces somatic embryogenesis in black rice

BackgroundPlants have the unique capability to form embryos from both gametes and somatic cells, with the latter process known as somatic embryogenesis. Somatic embryogenesis (SE) can be induced by exposing plant tissues to exogenous growth regulators or by the ectopic activation of embryogenic transcription factors. Recent studies have revealed that a discrete group of RWP-RK DOMAIN-CONTAINING PROTEIN (RKD) transcription factors act as key regulators of germ cell differentiation and embryo development in land plants. The ectopic overexpression of reproductive RKDs is associated with increased cellular proliferation and the formation of somatic embryo-like structures that bypass the need for exogenous growth regulators. However, the precise molecular mechanisms implicated in the induction of somatic embryogenesis by RKD transcription factors remains unknown.ResultsIn silico analyses have identified a rice RWP-RK transcription factor, named Oryza sativa RKD3 (OsRKD3), which is closely related to Arabidopsis thaliana RKD4 (AtRKD4) and Marchantia polymorpha RKD (MpRKD) proteins. Our study demonstrates that the ectopic overexpression of OsRKD3, which is expressed preferentially in reproductive tissues, can trigger the formation of somatic embryos in an Indonesian black rice landrace (Cempo Ireng) that is normally resistant to somatic embryogenesis. By analyzing the transcriptome of induced tissue, we identified 5,991 genes that exhibit differential expression in response to OsRKD3 induction. Among these genes, 50% were up-regulated while the other half were down-regulated. Notably, approximately 37.5% of the up-regulated genes contained a sequence motif in their promoter region, which was also observed in RKD targets from Arabidopsis. Furthermore, OsRKD3 was shown to mediate the transcriptional activation of a discrete gene network, which includes several transcription factors such as APETALA 2-like (AP2-like)/ETHYLENE RESPONSE FACTOR (ERF), MYB and CONSTANS-like (COL), and chromatin remodeling factors associated with hormone signal transduction, stress responses and post-embryonic pathways.ConclusionsOur data show that OsRKD3 modulates an extensive gene network and its activation is associated with the initiation of a somatic embryonic program that facilitates genetic transformation in black rice. These findings hold substantial promise for improving crop productivity and advancing agricultural practices in black rice.

Attenuation of stretch-induced arrhythmias following chemical ablation of Purkinje fibres, in isolated rabbit hearts.

Purkinje fibres (PFs) play an important role in some ventricular arrhythmias and acute ventricular stretch can evoke mechanically-induced arrhythmias. We tested whether Purkinje fibres, play a role in these arrhythmias. Pseudo-ECGs were recorded in isolated, Langendorff-perfused, rabbit hearts in which the left ventricular endocardial surface was also irrigated with Tyrode, via an indwelling catheter placed in the left ventricular lumen. The number and period of ectopic activations was measured during left ventricular lumen inflation via an indwelling fluid-filled balloon (500μL added over 2s and maintained for 15s in total). Mechanically-induced arrhythmias occurred in 70% of balloon inflations: they were maximal in the first 5s and ceased within 15s. Brief, (10s) irrigation of the left ventricular lumen with Lugol solution (IK/I2), via the indwelling catheter, reduced inflation-induced ectopics by 98% (p < 0.05). Ablation of endocardial PFs by Lugol was confirmed by Triphenyltetrazolium Chloride staining. Optical mapping revealed the left ventricular epicardial activation patterns of ectopics could have PF-mediated and focal sources. In silico modelling predicted ectopic sources originating in the endocardial region propagate to and through the Purkinje fibres network. Acute distention-induced ectopics are multi-focal, their attenuation by Lugol, their activation patterns and in silico modelling indicate a participation of Purkinje fibres in these arrhythmias.

Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve split-hand/foot malformation type 3

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern induced by AER-specific enhancers of Fgf8. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

Simultaneous activation of Tor and suppression of ribosome biogenesis by TRIM-NHL proteins promotes terminal differentiation

Tissue development and homeostasis depend on the balance between growth and terminal differentiation, but the mechanisms coordinating these processes remain elusive. Accumulating evidence indicates that ribosome biogenesis (RiBi) and protein synthesis, two cellular processes sustaining growth, are tightly regulated and yet can be uncoupled during stem cell differentiation. Using the Drosophila adult female germline stem cell and larval neuroblast systems, we show that Mei-P26 and Brat, two Drosophila TRIM-NHL paralogs, are responsible for uncoupling RiBi and protein synthesis during differentiation. In differentiating cells, Mei-P26 and Brat activate the target of rapamycin (Tor) kinase to promote translation, while concomitantly repressing RiBi. Depletion of Mei-P26 or Brat results in defective terminal differentiation, which canberescued by ectopic activation of Tor together with suppression of RiBi. Our results indicate that uncoupling RiBi and translation activities by TRIM-NHL activity creates the conditions required for terminal differentiation.

Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster

A-kinase anchoring protein (AKAP) comprises a family of scaffold proteins, which decides the subcellular localisation of a combination of signalling molecules. Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. We have earlier reported that Spoon suppresses ribonuclear foci formed by trinucleotide repeat expanded transcripts associated with Spinocerebellar Ataxia 8 neurodegeneration in Drosophila. However, the role of Spoonbill in cellular signalling was unexplored. In this report, we have unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. We have shown here that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that the spoon interacts with components of the JNK pathway, TGF-β activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, we found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. Hence, we propose that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson’s Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis.

Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibility in a canine model of elite exercise.

The health benefits of regular physical exercise are well known. Even so, there is increasing evidence that the exercise regimes of elite athletes can evoke cardiac arrhythmias including ventricular fibrillation and even sudden cardiac death (SCD). The mechanism of exercise-induced arrhythmia and SCD is poorly understood. Here, we show that chronic training in a canine model (12 sedentary and 12 trained dogs) that mimics the regime of elite athletes induces electrophysiological remodeling (measured by ECG, patch-clamp, and immunocytochemical techniques) resulting in increases of both the trigger and the substrate for ventricular arrhythmias. Thus, 4 months sustained training lengthened ventricular repolarization (QTc: 237.1±3.4 ms vs. 213.6±2.8 ms, n=12; APD90: 472.8±29.6 ms vs. 370.1±32.7 ms, n=29 vs. 25), decreased transient outward potassium current (6.4±0.5 pA/pF vs. 8.8±0.9 pA/pF at 50 mV, n=54 vs. 42), and increased the short-term variability of repolarization (29.5±3.8 ms vs. 17.5±4.0 ms, n=27 vs. 18). Left ventricular fibrosis and HCN4 protein expression were also enhanced. These changes were associated with enhanced ectopic activity (number of escape beats from 0/hr to 29.7±20.3/hr) in vivo and arrhythmia susceptibility (elicited ventricular fibrillation: 3 of 10 sedentary dogs vs. 6 of 10 trained dogs). Our findings provide in vivo, cellular electrophysiological and molecular biological evidence for the enhanced susceptibility to ventricular arrhythmia in an experimental large animal model of endurance training.

Moesin is an effector of tau-induced actin overstabilization, cell cycle activation, and neurotoxicity in Alzheimer’s disease

In Alzheimer's disease, neurons acquire phenotypes that are also present in various cancers, including aberrant activation of the cell cycle. Unlike cancer, cell cycle activation in post-mitotic neurons is sufficient to induce cell death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimer's disease and related "tauopathies." Here we combine network analyses of human Alzheimer's disease and mouse models of Alzheimer's diseaseand primary tauopathy with studies in Drosophila to discover that pathogenic forms of tau drive cell cycle activation by disrupting a cellular program involved in cancer and the epithelial-mesenchymal transition (EMT). Moesin, an EMT driver, is elevated in cells harboring disease-associated phosphotau, over-stabilized actin, and ectopic cell cycle activation. We further find that genetic manipulation of Moesin mediates tau-induced neurodegeneration. Taken together, our study identifies novel parallels between tauopathy and cancer.

An FGF timer for zygotic genome activation.

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timing of gene induction during the earliest phases of embryogenesis. Here we used high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate Ciona with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by ERK-mediated derepression of the ERF repressor. Depletion of ERF results in ectopic activation of FGF target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the eight- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also used by vertebrates.

Sleep-related breathing disorders in patients with heart failure with reduced and mildly reduced ejection fraction: main types and their dependence on heart failure etiology

Aim. To identify and study the nature of sleep-related breathing disorders (SBDs) in a cohort of hospitalized patients with heart failure (HF) with reduced and mildly reduced ejection fraction (EF), as well as to clarify the relationship between SBD type, etiology and severity of HF.Material and methods. The study included 117 patients with HF with reduced and mildly reduced ejection fraction hospitalized at the National Medical Research Center for Therapy and Preventive Medicine from 2019 to 2021. All patients underwent clinical and paraclinical examination, including cardiorespiratory sleep study. Patients were divided into three groups according to the type and severity of SBD: no or mild SBD, predominantly with obstructive sleep apnea (OSA) and predominantly with central sleep apnea (CSA). Severity of SBD and clinical data were compared between these groups.Results. A total of 5 patients (4,27%) did not have any SBDs, while 47 (40,17%) were diagnosed with CSA, and 65 (55,56%) — OSA of varying severity. The proportions of patients with moderate and severe CSA and OSA differed insignificantly and amounted to 35,9% (n=42) and 44,4% (n=52), respectively. There were following proportions of diseases related to HF: coronary artery disease (41,88%), nonischemic cardiomyopathy (26,5%), arrhythmogenic cardiomyopathy (15,38%) and other causes (16,24%) (hypertension, myocarditis, heart defects). We found that reduced EF &lt;40%, end-diastolic volume &gt;210 ml, and ventricular ectopy (&gt;300 extrasystoles/day) were associated with CSA, and body mass index &gt;30 kg/m2 was traditionally associated with OSA.Conclusion. More than half of HF patients with reduced and mildly reduced EF have SBDs. Decreased LVEF and ventricular ectopic activity are associated with CSA, while increased body mass index is associated with OSA. Consideration of SBD risk factors may improve patient phenotyping for individualized therapy.

Regional Differences in Ca2+ Signaling and Transverse-Tubules across Left Atrium from Adult Sheep.

Cardiac excitation-contraction coupling can be different between regions of the heart. Little is known at the atria level, specifically in different regions of the left atrium. This is important given the role of cardiac myocytes from the pulmonary vein sleeves, which are responsible for ectopic activity during atrial fibrillation. In this study, we present a new method to isolate atrial cardiac myocytes from four different regions of the left atrium of a large animal model, sheep, highly relevant to humans. Using collagenase/protease we obtained calcium-tolerant atrial cardiac myocytes from the epicardium, endocardium, free wall and pulmonary vein regions. Calcium transients were slower (time to peak and time to decay) in free wall and pulmonary vein myocytes compared to the epicardium and endocardium. This is associated with lower t-tubule density. Overall, these results suggest regional differences in calcium transient and t-tubule density across left atria, which may play a major role in the genesis of atrial fibrillation.

Generation and characterization of a Ddx4-iCre transgenic line for deletion in the germline beginning at genital ridge colonization.

Germline-specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole-body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved Cre (iCre) sequence in-frame at the 3' end of the Ddx4 locus to generate the Ddx4-P2A-iCre allele. Our functional assessment of this new allele, designated Ddx4iCreJoBo , reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5-12.5) germline deletion when compared to the inducible Oct4CreERT2 line. We found the Ddx4iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26RLacZ and R26RtdTomato ) and a floxed gene of interest (Criptoflox ) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for Ddx4iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4iCreJoBo line as useful for germline studies in which early gonadal deletion is required.

Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.

Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor indicates a cell-specific response that promotes CD44 activation.

Hearing loss caused by the death of cochlear hair cells (HCs) might be restored through regeneration from supporting cells (SCs) via dedifferentiation and proliferation, as observed in birds. In a previous report, ERBB2 activation in a subset of cochlear SCs promoted widespread down-regulation of SOX2 in neighboring cells, proliferation, and the differentiation of HC-like cells. Here we analyze single cell transcriptomes from neonatal mouse cochlear SCs with activated ERBB2, with the goal of identifying potential secreted effectors. ERBB2 induction in vivo generated a new population of cells with de novo expression of a gene network. Called small integrin-binding ligand n-linked glycoproteins (SIBLINGs), these ligands and their regulators can alter NOTCH signaling and promote cell survival, proliferation, and differentiation in other systems. We validated mRNA expression of network members, and then extended our analysis to older stages. ERBB2 signaling in young adult SCs also promoted protein expression of gene network members. Furthermore, we found proliferating cochlear cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2 signaling in cochlear SCs can alter the microenvironment, promoting proliferation and cell rearrangements. Together these results suggest a novel mechanism for inducing stem cell-like activity in the adult mammalian cochlea.

Overexpression of Liriodenron WOX5 in Arabidopsis Leads to Ectopic Flower Formation and Altered Root Morphology

Roots are essential for plant growth, and studies on root-related genes, exemplified by WUSCHEL-RELATED HOMEOBOX5 (WOX5), have mainly concentrated on model organisms with less emphasis on the function of these genes in woody plants. Here, we report that overexpression of the WOX5 gene from Liriodendron hybrid (LhWOX5) in Arabidopsis leads to significant morphological changes in both the aerial and subterranean organs. In the Arabidopsis aerial parts, overexpression of LhWOX5 results in the production of ectopic floral meristems and leaves, possibly via the ectopic activation of CLV3 and LFY. In addition, in the Arabidopsis root, overexpression of LhWOX5 alters root apical meristem morphology, leading to a curled and shortened primary root. Importantly, these abnormal phenotypes in the aerial and subterranean organs caused by constitutive ectopic expression of LhWOX5 mimic the observed phenotypes when overexpressing AtWUS and AtWOX5 in Arabidopsis, respectively. Taken together, we propose that the LhWOX5 gene, originating from the Magnoliaceae plant Liriodendron, is a functional homolog of the AtWUS gene from Arabidopsis, while showing the highest degree of sequence similarity with its ortholog, AtWOX5. Our study provides insight into the potential role of LhWOX5 in the development of both the shoot and root.

Endocardial role in arrhythmias induced by acute ventricular stretch and the involvement of Purkinje fibres, in isolated rat hearts

Purkinje fibres (PFs) play an important role in some ventricular arrhythmias and acute ventricular stretch can evoke mechanically-induced arrhythmias. We tested whether PFs and specifically TRPM4 channels, play a role in these mechanically-induced arrhythmias. Pseudo-ECGs and left ventricular (LV) activation, measured by optical mapping, were recorded in isolated, Langendorff-perfused, rat hearts. The LV endocardial surface was irrigated with experimental agents, via an indwelling catheter. The number and period of ectopic activations was measured during LV lumen inflation via an indwelling fluid-filled balloon (100 μL added over 2 s, maintained for 38 s). Mechanically-induced arrhythmias occurred during balloon inflation: they were multifocal, maximal in the first 5 s and ceased within 20 s. Optical mapping revealed activation patterns indicating PF-mediated and ectopic focal sources. Irrigation of the LV lumen with Lugol solution (IK/I2) for 10s reduced ectopics by 93% (n = 16, P < 0.001); with ablation of endocardial PFs confirmed by histology. Five min irrigation of the LV lumen with 50 μM 9-Phenanthrol, a blocker of TRPM4 channels, reduced ectopics by 39% (n = 15, P < 0.01). Immunohistochemistry confirmed that TRPM4 was more abundant in PFs than myocardium. Our results show that the endocardial surface plays an important role in these mechanically-induced ectopic activations. Ectopic activation patterns indicate a participation of PFs in these arrhythmias, with a potential involvement of TRPM4 channels, shown by the reduction of arrhythmias by 9-Phenanthrol.