Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

The Kir2.1E299V mutation increases atrial fibrillation vulnerability while protecting the ventricles against arrhythmias in a mouse model of short QT syndrome type 3.

AIMS: Short QT syndrome type 3 (SQTS3) is a rare arrhythmogenic disease caused by gain-of-function mutations in KCNJ2, the gene coding the inward rectifier potassium channel Kir2.1. We used a multidisciplinary approach and investigated arrhythmogenic mechanisms in an in-vivo model of de-novo mutation Kir2.1E299V identified in a patient presenting an extremely abbreviated QT interval and paroxysmal atrial fibrillation. METHODS AND RESULTS: We used intravenous adeno-associated virus-mediated gene transfer to generate mouse models, and confirmed cardiac-specific expression of Kir2.1WT or Kir2.1E299V. On ECG, the Kir2.1E299V mouse recapitulated the QT interval shortening and the atrial-specific arrhythmia of the patient. The PR interval was also significantly shorter in Kir2.1E299V mice. Patch-clamping showed extremely abbreviated action potentials in both atrial and ventricular Kir2.1E299V cardiomyocytes due to a lack of inward-going rectification and increased IK1 at voltages positive to -80 mV. Relative to Kir2.1WT, atrial Kir2.1E299V cardiomyocytes had a significantly reduced slope conductance at voltages negative to -80 mV. After confirming a higher proportion of heterotetrameric Kir2.x channels containing Kir2.2 subunits in the atria, in-silico 3D simulations predicted an atrial-specific impairment of polyamine block and reduced pore diameter in the Kir2.1E299V-Kir2.2WT channel. In ventricular cardiomyocytes, the mutation increased excitability by shifting INa activation and inactivation in the hyperpolarizing direction, which protected the ventricle against arrhythmia. Moreover, Purkinje myocytes from Kir2.1E299V mice manifested substantially higher INa density than Kir2.1WT, explaining the abbreviation in the PR interval. CONCLUSION: The first in-vivo mouse model of cardiac-specific SQTS3 recapitulates the electrophysiological phenotype of a patient with the Kir2.1E299V mutation. Kir2.1E299V eliminates rectification in both cardiac chambers but protects against ventricular arrhythmias by increasing excitability in both Purkinje-fiber network and ventricles. Consequently, the predominant arrhythmias are supraventricular likely due to the lack of inward rectification and atrial-specific reduced pore diameter of the Kir2.1E299V-Kir2.2WT heterotetramer.

Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome.

Background: Andersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing the open state of the channel. Methods and Results: We identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch-clamping the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing PIP2 concentrations. Conclusion: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias.

Molecular stratification of arrhythmogenic mechanisms in the Andersen Tawil syndrome.

Andersen-Tawil syndrome (ATS) is a rare inheritable disease associated with loss-of-function mutations in KCNJ2, the gene coding the strong inward rectifier potassium channel Kir2.1, which forms an essential membrane protein controlling cardiac excitability. ATS is usually marked by a triad of periodic paralysis, life-threatening cardiac arrhythmias and dysmorphic features, but its expression is variable and not all patients with a phenotype linked to ATS have a known genetic alteration. The mechanisms underlying this arrhythmogenic syndrome are poorly understood. Knowing such mechanisms would be essential to distinguish ATS from other channelopathies with overlapping phenotypes and to develop individualized therapies. For example, the recently suggested role of Kir2.1 as a countercurrent to sarcoplasmic calcium reuptake might explain the arrhythmogenic mechanisms of ATS and its overlap with catecholaminergic polymorphic ventricular tachycardia. Here we summarize current knowledge on the mechanisms of arrhythmias leading to sudden cardiac death in ATS. We first provide an overview of the syndrome and its pathophysiology, from the patient's bedside to the protein and discuss the role of essential regulators and interactors that could play a role in cases of ATS. The review highlights novel ideas related to some post-translational channel interactions with partner proteins that might help define the molecular bases of the arrhythmia phenotype. We then propose a new all-embracing classification of the currently known ATS loss-of-function mutations according to their position in the Kir2.1 channel structure and their functional implications. We also discuss specific ATS pathogenic variants, their clinical manifestations, and treatment stratification. The goal is to provide a deeper mechanistic understanding of the syndrome toward the development of novel targets and personalized treatment strategies.

Deficiency of T-Cell Intracellular Antigen 1 in Murine Embryonic Fibroblasts Is Associated with Changes in Mitochondrial Morphology and Respiration.

T-cell intracellular antigen 1 (TIA1) is a multifunctional RNA-binding protein involved in regulating gene expression and splicing during development and in response to environmental stress, to maintain cell homeostasis and promote survival. Herein, we used TIA1-deficient murine embryonic fibroblasts (MEFs) to study their role in mitochondria homeostasis. We found that the loss of TIA1 was associated with changes in mitochondrial morphology, promoting the appearance of elongated mitochondria with heterogeneous cristae density and size. The proteomic patterns of TIA1-deficient MEFs were consistent with expression changes in molecular components related to mitochondrial dynamics/organization and respiration. Bioenergetics analysis illustrated that TIA1 deficiency enhances mitochondrial respiration. Overall, our findings shed light on the role of TIA1 in mitochondrial dynamics and highlight a point of crosstalk between potential pro-survival and pro-senescence pathways.

A Heterologous Cell Model for Studying the Role of T-Cell Intracellular Antigen 1 in Welander Distal Myopathy.

Welander distal myopathy (WDM) is a muscle dystrophy characterized by adult-onset distal muscle weakness, prevalently impacting the distal long extensors of the hands and feet. WDM is an autosomal dominant disorder caused by a missense mutation (c.1362G>A; p.E384K) in the TIA1 (T-cell intracellular antigen 1) gene, which encodes an RNA-binding protein basically required for the posttranscriptional regulation of RNAs. We have developed a heterologous cell model of WDM to study the molecular and cellular events associated with mutated TIA1 expression. Specifically, we analyzed how this mutation affects three regulatory functions mediated by TIA1: (i) control of alternative SMN2 (survival motor neuron 2) splicing; (ii) formation, assembly, and disassembly of stress granules; and (iii) mitochondrial dynamics and its consequences for mitophagy, autophagy, and apoptosis. Our results show that whereas WDM-associated TIA1 expression had only a mild effect on SMN2 splicing, it led to suboptimal adaptation to environmental stress, with exacerbated stress granule formation that was accompanied by mitochondrial dysfunction and autophagy. Overall, our observations indicate that some aspects of the cell phenotype seen in muscle of patients with WDM can be recapitulated by ectopic expression of WDM-TIA1 in embryonic kidney cells, highlighting the potential of this model to investigate the pathogenesis of this degenerative disease and possible therapeutics.

Transcriptome-wide analysis links the short-term expression of the b isoforms of TIA proteins to protective proteostasis-mediated cell quiescence response.

Control of gene expression depends on genetics and environmental factors. The T-cell intracellular antigens T-cell intracellular antigen 1 (TIA1), TIA1-like/related protein (TIAL1/TIAR) and human antigen R (HuR/ELAVL1) are RNA-binding proteins that play crucial roles in regulating gene expression in both situations. This study used massive sequencing analysis to uncover molecular and functional mechanisms resulting from the short-time expression of the b isoforms of TIA1 and TIAR, and of HuR in HEK293 cells. Our gene profiling analysis identified several hundred differentially expressed genes (DEGs) and tens of alternative splicing events associated with TIA1b, TIARb and HuR overexpression. Gene ontology analysis revealed that the controlled expression of these proteins strongly influences the patterns of DEGs and RNA variants preferentially associated with development, reproduction, cell cycle, metabolism, autophagy and apoptosis. Mechanistically, TIA1b and TIARb isoforms display both common and differential effects on the regulation of gene expression, involving systematic perturbations of cell biosynthetic machineries (splicing and translation). The transcriptome outputs were validated using functional assays of the targeted cellular processes as well as expression analysis for selected genes. Collectively, our observations suggest that early TIA1b and TIARb expression operates to connect the regulatory crossroads to protective proteostasis responses associated with a survival quiescence phenotype.

Ablation of the stress protease OMA1 protects against heart failure in mice.

Heart failure (HF) is a major health and economic burden in developed countries. It has been proposed that the pathogenesis of HF may involve the action of mitochondria. We evaluate three different mouse models of HF: tachycardiomyopathy, HF with preserved left ventricular (LV) ejection fraction (LVEF), and LV myocardial ischemia and hypertrophy. Regardless of whether LVEF is preserved, our results indicate that the three models share common features: an increase in mitochondrial reactive oxygen species followed by ultrastructural alterations in the mitochondrial cristae and loss of mitochondrial integrity that lead to cardiomyocyte death. We show that the ablation of the mitochondrial protease OMA1 averts cardiomyocyte death in all three murine HF models, and thus loss of OMA1 plays a direct role in cardiomyocyte protection. This finding identifies OMA1 as a potential target for preventing the progression of myocardial damage in HF associated with a variety of etiologies.

T-Cell Intracellular Antigens and Hu Antigen R Antagonistically Modulate Mitochondrial Activity and Dynamics by Regulating Optic Atrophy 1 Gene Expression.

Mitochondria undergo frequent morphological changes to control their function. We show here that T-cell intracellular antigens (TIA1b/TIARb) and Hu antigen R (HuR) have antagonistic roles in mitochondrial function by modulating the expression of mitochondrial shaping proteins. Expression of TIA1b/TIARb alters the mitochondrial dynamic network by enhancing fission and clustering, which is accompanied by a decrease in respiration. In contrast, HuR expression promotes fusion and cristae remodeling and increases respiratory activity. Mechanistically, TIA proteins downregulate the expression of optic atrophy 1 (OPA1) protein via switching of the splicing patterns of OPA1 to facilitate the production of OPA1 variant 5 (OPA1v5). Conversely, HuR enhances the expression of OPA1 mRNA isoforms through increasing steady-state levels and targeting translational efficiency at the 3' untranslated region. Knockdown of TIA1/TIAR or HuR partially reversed the expression profile of OPA1, whereas knockdown of OPA1 or overexpression of OPA1v5 provoked mitochondrial clustering. Middle-term expression of TIA1b/TIARb triggers reactive oxygen species production and mitochondrial DNA damage, which is accompanied by mitophagy, autophagy, and apoptosis. In contrast, HuR expression promotes mitochondrion-dependent cell proliferation. Collectively, these results provide molecular insights into the antagonistic functions of TIA1b/TIARb and HuR in mitochondrial activity dynamics and suggest that their balance might contribute to mitochondrial physiopathology.

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

Electrons feed into the mitochondrial electron transport chain (mETC) from NAD- or FAD-dependent enzymes. A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here, we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. Thus, CoQ redox status acts as a metabolic sensor that fine-tunes mETC configuration in order to match the prevailing substrate profile.

Genome-wide profiling reveals a role for T-cell intracellular antigens TIA1 and TIAR in the control of translational specificity in HeLa cells.

TIA (T-cell intracellular antigens)-knockdown HeLa cells show an increase in ribosomes and translational machinery components. This increase correlates with specific changes in translationally up-regulated mRNAs involved in cell-cycle progression and DNA repair, as shown in polysomal profiling analysis. Our data support the hypothesis that a concerted activation of both global and selective translational rates leads to the transition to a more proliferative status in TIA-knockdown HeLa cells.

ROS-triggered phosphorylation of complex II by Fgr kinase regulates cellular adaptation to fuel use.

Electron flux in the mitochondrial electron transport chain is determined by the superassembly of mitochondrial respiratory complexes. Different superassemblies are dedicated to receive electrons derived from NADH or FADH2, allowing cells to adapt to the particular NADH/FADH2 ratio generated from available fuel sources. When several fuels are available, cells adapt to the fuel best suited to their type or functional status (e.g., quiescent versus proliferative). We show that an appropriate proportion of superassemblies can be achieved by increasing CII activity through phosphorylation of the complex II catalytic subunit FpSDH. This phosphorylation is mediated by the tyrosine-kinase Fgr, which is activated by hydrogen peroxide. Ablation of Fgr or mutation of the FpSDH target tyrosine abolishes the capacity of mitochondria to adjust metabolism upon nutrient restriction, hypoxia/reoxygenation, and T cell activation, demonstrating the physiological relevance of this adaptive response.

Long-term reduction of T-cell intracellular antigens leads to increased beta-actin expression.

BACKGROUND: The permanent down-regulated expression of T-cell intracellular antigen (TIA) proteins in HeLa cells improves cytoskeleton-mediated functions such as cell proliferation and tumor growth. METHODS: Making use of human and mouse cells with knocked down/out expression of T-cell intracellular antigen 1 (TIA1) and/or TIA1 related/like (TIAR/TIAL1) proteins and classical RNA (e.g. reverse transcription-quantitative polymerase chain reaction, polysomal profiling analysis using sucrose gradients, immunoblotting, immunoprecipitation, electrophoretic mobility shift assays, ultraviolet light crosslinking and poly (A+) test analysis) and cellular (e.g. immunofluorescence microscopy and quimeric mRNA transfections) biology methods, we have analyzed the regulatory role of TIA proteins in the post-transcriptional modulation of beta-actin (ACTB) mRNA. RESULTS: Our observations show that the acquisition of above cellular capacities is concomitant with increased expression levels of the actin beta subunit (ACTB) protein. Regulating TIA abundance does not modify ACTB mRNA levels, however, an increase of ACTB mRNA translation is observed. This regulatory capacity of TIA proteins is linked to the ACTB mRNA 3'-untranslated region (3'-UTR), where these proteins could function as RNA binding proteins. The expression of GFP from a chimeric reporter containing human ΑCΤΒ 3'-UTR recapitulates the translational control found by the endogenous ACTB mRNA in the absence of TIA proteins. Additionally, murine embryonic fibroblasts (MEF) knocked out for TIA1 rise mouse ACTB protein expression compared to the controls. Once again steady-state levels of mouse ACTB mRNA remained unchanged. CONCLUSIONS: Collectively, these results suggest that TIA proteins can function as long-term regulators of the ACTB mRNA metabolism in mouse and human cells.

Identification of a set of miRNAs differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling.

BACKGROUND: T-cell intracellular antigen (TIA) proteins function as regulators of cell homeostasis. These proteins control gene expression globally at multiple levels in response to dynamic regulatory changes and environmental stresses. Herein we identified a micro(mi)RNA signature associated to transiently TIA-depleted HeLa cells and analyzed the potential role of miRNAs combining genome-wide analysis data on mRNA and miRNA profiles. RESULTS: Using high-throughput miRNA expression profiling, transient depletion of TIA-proteins in HeLa cells was observed to promote significant and reproducible changes affecting to a pool of up-regulated miRNAs involving miR-30b-3p, miR125a-3p, miR-193a-5p, miR-197-3p, miR-203a, miR-210, miR-371-5p, miR-373-5p, miR-483-5p, miR-492, miR-498, miR-503-5p, miR-572, miR-586, miR-612, miR-615-3p, miR-623, miR-625-5p, miR-629-5p, miR-638, miR-658, miR-663a, miR-671-5p, miR-769-3p and miR-744-5p. Some up-regulated and unchanged miRNAs were validated and previous results confirmed by reverse transcription and real time PCR. By target prediction of the miRNAs and combined analysis of the genome-wide expression profiles identified in TIA-depleted HeLa cells, we detected connections between up-regulated miRNAs and potential target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis suggest that target genes are related with biological processes associated to the regulation of DNA-dependent transcription, signal transduction and multicellular organismal development as well as with the enrichment of pathways involved in cancer, focal adhesion, regulation of actin cytoskeleton, endocytosis and MAPK and Wnt signaling pathways, respectively. When the collection of experimentally defined differentially expressed genes in TIA-depleted HeLa cells was intersected with potential target genes only 7 out of 68 (10%) up- and 71 out of 328 (22%) down-regulated genes were shared. GO and KEGG database analyses showed that the enrichment categories of biological processes and cellular pathways were related with innate immune response, signal transduction, response to interleukin-1, glomerular basement membrane development as well as neuroactive ligand-receptor interaction, endocytosis, lysosomes and apoptosis, respectively. CONCLUSION: All this considered, these observations suggest that individual miRNAs could act as potential mediators of the epigenetic switch linking transcriptomic dynamics and cell phenotypes mediated by TIA proteins.

Knockdown of T-cell intracellular antigens triggers cell proliferation, invasion and tumour growth.

TIA (T-cell intracellular antigen) proteins function as DNA/RNA trans-acting regulators to expand transcriptome and proteome diversity in mammals. In the present paper we report that the stable silencing of TIA1 and/or TIAR/TIAL1 (TIA1-related/like protein 1) expression in HeLa cells enhances cell proliferation, anchorage-dependent and -independent growth and invasion. HeLa cells lacking TIA1 and/or TIAR generate larger and faster-growing epithelial tumours with high rates of proliferation and angiogenesis in nude mice xenografts. Protein array analysis of a collection of human tumours shows that TIA1 and TIAR protein expression is down-regulated in a subset of epithelial tumours relative to normal tissues. Our results suggest a link between the epigenetic control exerted by TIA proteins and the transcriptional and post-transcriptional regulation of a subset of specific genes involved in tumour progression. Taken together, these results are consistent with a role for TIA proteins as growth/tumour-suppressor genes.