Introduction: Adults after repair of Tetralogy of Fallot (ToF) have an increased risk of heart failure, arrhythmias and sudden death.
We hypothesize that electrophysiological remodeling of the right atrium (RA), right ventricle (RV) and right ventricular outflow tract (RVOT) is responsible.
Methods: We collected RA, RV and RVOT biopsies from 10 ToF patients (6 male) at the time of pulmonary valve replacement. Biopsies were also taken from 9 control patients (4 male) with left ventricular outflow tract obstruction without evidence of septal defects, previous arrhythmia or right-sided heart disease. We performed: (i) extracellular matrix (ECM) quantification; (ii) RT-qPCR to quantify mRNA for ion channels, transporters, connexins, inflammatory markers and ECM constituents; (iii) proteomics (liquid chromatography and mass spectrometry (MS)) on 6 patients per group to quantify ~1600 proteins; and (iv) Western Blot for selected proteins.
Results: Mean age at surgery was 32±4 and 22±2 years in the ToF and control groups respectively. There were significant changes in the relative abundance of mRNAs for ion channels, adrenergic receptors, ECM, and heart failure markers (ANP and BNP) in ToF patients compared with controls. MS highlighted over 300 significant differences, specifically changes in proteoglycans and downregulation of mitochondrial respiratory complexes 1 and 3 in ToF patients compared with controls. No significant difference in fibrosis on histological analysis between the two groups was seen. A selection of changes is displayed in Table 1.
Conclusion: The RA and RVOT undergo significant remodeling in ToF. Changes in ion channels, transporters and Ca2+-handling (especially SERCA2A, RYR2, Calsequestrin) as well as gap junction proteins (Cx43) are likely to alter ionic currents, with increased arrhythmia risk. This remodeling is similar to changes in heart failure and ischemia-reperfusion and may contribute to the arrhythmias seen in ToF.
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Summary Listeria monocytogenes is a Gram‐positive intracellular parasite and the causative organism of human listeriosis. In this article we demonstrate that L. monocytogenes encodes a functional member of the CodY family of global regulatory proteins that is responsive to both GTP and branched chain amino acids. By transcript analyses we identified the CodY regulon in L. monocytogenes and demonstrated that it comprises genes involved in amino acid metabolism, nitrogen assimilation as well as genes involved in sugar uptake and incorporation, indicating a role for CodY in L. monocytogenes in both carbon and nitrogen assimilation. A Δ relA mutation reduced expression of the CodY regulon in early stationary phase and introduction of a Δ codY mutation into a Δ relA strain restored virulence. These data indicate that the avirulence of the Δ relA mutant can in part be explained by the continued repression of the CodY regulon . The phenotypes of Δ relA and Δ codY mutants were studied in J774.A1 and Caco‐2 cells and the Δ relA mutation shown to effect intracellular growth. These results provide the first direct evidence that the activity of a CodY‐type protein influences pathogenesis and provides new information on the physiological adaptation of L. monocytogenes to post‐exponential phase growth and virulence.
We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca2+ sparks, and BK channel vasoregulation. Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high-speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional "ceiling" for this pressure --sensitive oxidative pathway. During steady state pressure - induced constriction, any additional Ca2+ sensitive-K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine-induced Ca2+ transient in pressurized arteries from mice with oxidant-resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure-induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.
Abstract Interleukin (IL)-1α is a suggested dual-function cytokine that diverged from IL-1β in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of IL-1α, including a nuclear localisation sequence (NLS) and histone acetyl transferase (HAT)-binding domains. Why evolution modified pro-IL-1α’s subcellular location and protein interactome, and how this shaped IL-1α’s intracellular role, is unknown. TurboID proximity labelling with pro-IL-1α suggested a nuclear role for pro-IL-1α that involved interaction with HATs, including EP300. We also identified and validated inactivating mutations in the pro-IL-1α NLS of multiple mammalian species. However, HAT-binding domains were also conserved in species that had lost pro-IL-1α nuclear localisation. Together, these data suggest that HAT binding and nuclear localisation occurred together, and that while some species lost the NLS in their pro-IL-1α, HAT binding was maintained. The NLS was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the NLS confers some important biological outcome.
The formation of healthy tissue involves continuous remodelling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here we examine how tensin3 contributes to formation of fibrillar adhesions (FBs) and fibronectin fibrillo-genesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for formation of α5β1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.
The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here, we examine how tensin3 contributes to the formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors such as talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for the formation of α5β1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.
Abstract Organisation of the transcription cycle is facilitated by the reversible phosphorylation of the C-terminal domain of RNA Polymerase II (RNAPII-CTD) and its accessory factors. The PNUTS-PP1 protein phosphatase is crucial for mRNA synthesis and processing, yet the complete spectrum of its physiological targets in these processes remain elusive. Here, using quantitative phosphoproteomics, we discover that Cdk11, in addition to various spliceosomal and RNA processing factors, associates with PNUTS, and that disruption of PP1-binding results in hyperphosphorylation of Cdk11 at an evolutionarily conserved Serine residue, seven amino acid residues C-terminal to DFG residues in the activation loop. In vitro experiments reveal a role for Ser DFG+7 in modulating Cdk11 kinase activity towards RNAPII-CTD Ser5. Making use of a novel technique to conditionally disrupt PP1 binding, we show that PNUTS-PP1 normally serves to restrain Cdk11 phosphorylation in vivo . Mutational analysis shows that cdk11 is not only essential for survival but also plays a widespread role in regulating normal mRNA expression and splicing. Notably, we find that a phosphomimetic mutation in cdk11 exhibits distinct biological effects compared to loss of cdk11 function, including defective processing of intronic small nucleolar RNAs, diminished intronic RNA Pol II velocity, and a decrease in intergenic transcription. These findings underscore physiologically significant roles of Cdk11 dephosphorylation by PNUTS-PP1 in the regulation of mRNA transcription and processing.
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