A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart.

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. VIDEO ABSTRACT.

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.

Beta-3 adrenergic receptor overexpression reverses aortic stenosis-induced heart failure and restores balanced mitochondrial dynamics.

Aortic stenosis (AS) is associated with left ventricular (LV) hypertrophy and heart failure (HF). There is a lack of therapies able to prevent/revert AS-induced HF. Beta3 adrenergic receptor (ß3AR) signaling is beneficial in several forms of HF. Here, we studied the potential beneficial effect of ß3AR overexpression on AS-induced HF. Selective ß3AR stimulation had a positive inotropic effect. Transgenic mice constitutively overexpressing human ß3AR in the heart (c-hß3tg) were protected from the development of HF in response to induced AS, and against cardiomyocyte mitochondrial dysfunction (fragmented mitochondria with remodeled cristae and metabolic reprogramming featuring altered substrate use). Similar beneficial effects were observed in wild-type mice inoculated with adeno-associated virus (AAV9) inducing cardiac-specific overexpression of human ß3AR before AS induction. Moreover, AAV9-hß3AR injection into wild-type mice at late disease stages, when cardiac hypertrophy and metabolic reprogramming are already advanced, reversed the HF phenotype and restored balanced mitochondrial dynamics, demonstrating the potential of gene-therapy-mediated ß3AR overexpression in AS. Mice with cardiac specific ablation of Yme1l (cYKO), characterized by fragmented mitochondria, showed an increased mortality upon AS challenge. AAV9-hß3AR injection in these mice before AS induction reverted the fragmented mitochondria phenotype and rescued them from death. In conclusion, our results step out that ß3AR overexpression might have translational potential as a therapeutic strategy in AS-induced HF.

Sustained Elevated Blood Pressure Accelerates Atherosclerosis Development in a Preclinical Model of Disease.

The continuous relationship between blood pressure (BP) and cardiovascular events makes the distinction between elevated BP and hypertension based on arbitrary cut-off values for BP. Even mild BP elevations manifesting as high-normal BP have been associated with cardiovascular risk. We hypothesize that persistent elevated BP increases atherosclerotic plaque development. To evaluate this causal link, we developed a new mouse model of elevated BP based on adeno-associated virus (AAV) gene transfer. We constructed AAV vectors to support transfer of the hRenin and hAngiotensinogen genes. A single injection of AAV-Ren/Ang (1011 total viral particles) induced sustained systolic BP increase (130 ± 20 mmHg, vs. 110 ± 15 mmHg in controls; p = 0.05). In ApoE-/- mice, AAV-induced mild BP elevation caused larger atherosclerotic lesions evaluated by histology (10-fold increase vs. normotensive controls). In this preclinical model, atheroma plaques development was attenuated by BP control with a calcium channel blocker, indicating that a small increase in BP within a physiological range has a substantial impact on plaque development in a preclinical model of atherosclerosis. These data support that non-optimal BP represents a risk for atherosclerosis development. Earlier intervention in elevated BP may prevent or delay morbidity and mortality associated with atherosclerosis.

Pharmacological doses of melatonin impede cognitive decline in tau-related Alzheimer models, once tauopathy is initiated, by restoring the autophagic flux.

Alterations in autophagy are increasingly being recognized in the pathogenesis of proteinopathies like Alzheimer's disease (AD). This study was conducted to evaluate whether melatonin treatment could provide beneficial effects in an Alzheimer model related to tauopathy by improving the autophagic flux and, thereby, prevent cognitive decline. The injection of AAV-hTauP301L viral vectors and treatment/injection with okadaic acid were used to achieve mouse and human ex vivo, and in vivo tau-related models. Melatonin (10 µmol/L) impeded oxidative stress, tau hyperphosphorylation, and cell death by restoring autophagy flux in the ex vivo models. In the in vivo studies, intracerebroventricular injection of AAV-hTauP301L increased oxidative stress, neuroinflammation, and tau hyperphosphorylation in the hippocampus 7 days after the injection, without inducing cognitive impairment; however, when animals were maintained for 28 days, cognitive decline was apparent. Interestingly, late melatonin treatment (10 mg/kg), starting once the alterations mentioned above were established (from day 7 to day 28), reduced oxidative stress, neuroinflammation, tau hyperphosphorylation, and caspase-3 activation; these observations correlated with restoration of the autophagy flux and memory improvement. This study highlights the importance of autophagic dysregulation in tauopathy and how administration of pharmacological doses of melatonin, once tauopathy is initiated, can restore the autophagy flux, reduce proteinopathy, and prevent cognitive decline. We therefore propose exogenous melatonin supplementation or the development of melatonin derivatives to improve autophagy flux for the treatment of proteinopathies like AD.

Bloodless reperfusion with the oxygen carrier HBOC-201 in acute myocardial infarction: a novel platform for cardioprotective probes delivery.

Reperfusion, despite being required for myocardial salvage, is associated with additional injury. We hypothesize that infarct size (IS) will be reduced by a period of bloodless reperfusion with hemoglobin-based oxygen carriers (HBOC) before blood-flow restoration. In the pig model, we first characterized the impact of intracoronary perfusion with a fixed volume (600 ml) of a pre-oxygenated acellular HBOC, HBOC-201, on the healthy myocardium. HBOC-201 was administered through the lumen of the angioplasty balloon (i.e., distal to the occlusion site) immediately after onset of coronary occlusion at 1, 0.7, 0.4, or 0.2 ml/kg/min for 12, 17, 30, and 60 min, respectively, followed by blood-flow restoration. Outcome measures were systemic hemodynamics and LV performance assessed by the state-of-the-art cardiac magnetic resonance (CMR) imaging. The best performing HBOC-201 perfusion strategies were then tested for their impact on LV performance during myocardial infarction, in pigs subjected to 45 min mid-left anterior descending (LAD) coronary occlusion. At the end of the ischemia duration, pigs were randomized to regular reperfusion (blood-only reperfusion) vs. bloodless reperfusion (perfusion with pre-oxygenated HBOC-201 distal to the occlusion site), followed by blood-flow restoration. Hemodynamics and CMR-measured LV performance were assessed at 7- and 45-day follow-up. In modifications of the HBOC-201 procedure, glucose and insulin were included to support cardiac metabolism. A total of 66 pigs were included in this study. Twenty healthy pigs (5 per infusion protocol) were used in the study of healthy myocardium. Intracoronary administration of HBOC-201 (600 ml) at varying rates, including a flow of 0.4 ml/kg/min (corresponding to a maximum perfusion time of 30 min), did not damage the healthy myocardium. Slower perfusion (longer infusion time) was associated with permanent LV dysfunction and myocardial necrosis. A total of 46 pigs underwent MI induction. Compared with regular reperfusion, bloodless reperfusion with pre-oxygenated HBOC-201 alone increased IS. This effect was reversed by enrichment of pre-oxygenated HBOC-201 solution with glucose and insulin, resulting in no increase in IS or worsening of long-term ventricular function despite further delaying restoration of blood flow in the LAD. Bloodless reperfusion with a pre-oxygenated HBOC-201 solution supplemented with glucose and insulin is feasible and safe, but did not reduce infarct size. This strategy could be, however, used to deliver agents to the myocardium to treat or prevent ischemia/reperfusion injury before blood-flow restoration.

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum ß-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

BACKGROUND: A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. RESULTS: We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. CONCLUSIONS: We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.

First description of the male and redescription of the female of Ixodes tapirus Kohls, 1956 (Acari: Ixodidae), a parasite of tapirs (Perissodactyla: Tapiridae) from the mountains of Colombia, Costa Rica and Panama.

The male of Ixodes tapirus Kohls, 1956 (Acari: Ixodidae) is described for the first time and the female is redescribed in greater detail. Adults of I. tapirus are similar to those of Ixodes guatemalensis Kohls, 1956, Ixodes lasallei Méndez & Ortiz, 1958, Ixodes montoyanus Cooley, 1944 and Ixodes venezuelensis Kohls, 1953 but can be distinguished by their overall size, the amount of sclerotisation of the conscutum and accessory plates, the shape of the scutum, the number of punctations and their pattern on the conscutum and scutum, the depth of the punctations on the basis capituli dorsally, the shape and size of the porose areas and the size and shape of the auriculae. Adults of I. tapirus were collected from tapirs and vegetation in the mountains of Colombia, Panama and recorded from Costa Rica for the first time.

Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.

OBJECTIVES: Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9(DY)) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). APPROACH AND RESULTS: We constructed an AAV-based vector to support targeted transfer of the PCSK9(DY) gene to liver. After injection with 3.5×10(10) viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9(DY) mice fed a high-fat-diet. Advanced lesions in these high-fat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9(DY) infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9(DY) to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE(-/-) AAV-PCSK9(DY) mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE(-/-) AAV-PCSK9(DY)-transexpressing mice as in ApoE(-/-) AAV-Luc controls without altering serum cholesterol levels. CONCLUSIONS: Single intravenous AAV-PCSK9(DY) injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9(DY) gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses.

ß3 adrenergic receptor selective stimulation during ischemia/reperfusion improves cardiac function in translational models through inhibition of mPTP opening in cardiomyocytes.

Selective stimulation of ß3 adrenergic-receptor (ß3AR) has been shown to reduce infarct size in a mouse model of myocardial ischemia/reperfusion. However, its functional long-term effect and the cardioprotective mechanisms at the level of cardiomyocytes have not been elucidated, and the impact of ß3AR stimulation has not been evaluated in a more translational large animal model. This study aimed at evaluating pre-perfusion administration of BRL37344 both in small and large animal models of myocardial ischemia/reperfusion. Pre-reperfusion administration of the ß3AR agonist BRL37344 (5 µg/kg) reduced infarct size at 2-and 24-h reperfusion in wild-type mice. Long-term (12-weeks) left ventricular (LV) function assessed by echocardiography and cardiac magnetic resonance (CMR) was significantly improved in ß3AR agonist-treated mice. Incubation with ß3AR agonist (BRL37344, 7 µmol/L) significantly reduced cell death in isolated adult mouse cardiomyocytes during hypoxia/reoxygenation and decreased susceptibility to deleterious opening of the mitochondrial permeability transition pore (mPTP), via a mechanism dependent on the Akt-NO signaling pathway. Pre-reperfusion BRL37344 administration had no effect on infarct size in cyclophilin-D KO mice, further implicating mPTP in the mechanism of protection. Large-white pigs underwent percutaneous coronary ischemia/reperfusion and 3-T CMR at 7 and 45 days post-infarction. Pre-perfusion administration of BRL37344 (5 µg/kg) decreased infarct size and improved long-term LV contractile function. A single-dose administration of ß3AR agonist before reperfusion decreased infarct size and resulted in a consistent and long-term improvement in cardiac function, both in small and large animal models of myocardial ischemia/reperfusion. This protection appears to be executed through inhibition of mPTP opening in cardiomyocytes.

HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response.

Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.

Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 (PKP2). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells' contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction-mediated diseases.

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.

Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis.

The gene PTTG1 (encoding the pituitary tumor-transforming 1 protein) is overexpressed in several different tumor types, is tumorigenic in vivo and shows transcriptional activity. The PTTG1 protein is cell-cycle regulated and was identified as the human securin (a category of proteins involved in the regulation of sister-chromatid separation) on the basis of biochemical similarities with the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. To unravel the function of human securin in oncogenesis, we carried out a phage-display screening to identify proteins that interact with securin. Notably, we isolated the p53 tumor suppressor. Pull-down and co-immunoprecipitation assays demonstrated that p53 interacts specifically with securin both in vitro and in vivo. This interaction blocks the specific binding of p53 to DNA and inhibits its transcriptional activity. Securin also inhibits the ability of p53 to induce cell death. Moreover, we observed that transfection of H1299 cells with securin induced an accumulation of G2 cells that compensated for the loss of G2 cells caused by transfection with p53. We demonstrated the physiological relevance of this interaction in PTTG1-deficient human tumor cells (PTTG1(-/-)): both apoptotic and transactivating functions of p53 were potentiated in these cells compared to parental cells. We propose that the oncogenic effect of increased expression of securin may result from modulation of p53 functions.

MYH10 activation rescues contractile defects in arrhythmogenic cardiomyopathy (ACM).

The most prevalent genetic form of inherited arrhythmogenic cardiomyopathy (ACM) is caused by mutations in desmosomal plakophilin-2 (PKP2). By studying pathogenic deletion mutations in the desmosomal protein PKP2, here we identify a general mechanism by which PKP2 delocalization restricts actomyosin network organization and cardiac sarcomeric contraction in this untreatable disease. Computational modeling of PKP2 variants reveals that the carboxy-terminal (CT) domain is required for N-terminal domain stabilization, which determines PKP2 cortical localization and function. In mutant PKP2 cells the expression of the interacting protein MYH10 rescues actomyosin disorganization. Conversely, dominant-negative MYH10 mutant expression mimics the pathogenic CT-deletion PKP2 mutant causing actin network abnormalities and right ventricle systolic dysfunction. A chemical activator of non-muscle myosins, 4-hydroxyacetophenone (4-HAP), also restores normal contractility. Our findings demonstrate that activation of MYH10 corrects the deleterious effect of PKP2 mutant over systolic cardiac contraction, with potential implications for ACM therapy.

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.

Atherosclerosis Induced by Adeno-Associated Virus Encoding Gain-of-Function PCSK9.

Induction of atherosclerosis in mice with one or more genetic alterations (e.g., conditional deletion of a gene of interest) has traditionally required crossbreeding with Apoe or Ldlr deficient mice to achieve sufficient hypercholesterolemia. However, this procedure is time consuming and generates a surplus of mice with genotypes that are irrelevant for experiments. Several alternative methods exist that obviate the need to work in mice with germline-encoded hypercholesterolemia. In this chapter, we detail an efficient and increasingly used method to induce hypercholesterolemia in mice through adeno-associated virus-mediated transfer of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene.

Implication of type 4 NADPH oxidase (NOX4) in tauopathy.

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.

Kir2.1 dysfunction at the sarcolemma and the sarcoplasmic reticulum causes arrhythmias in a mouse model of Andersen-Tawil syndrome type 1.

Andersen-Tawil syndrome type 1 (ATS1) is associated with life-threatening arrhythmias of unknown mechanism. In this study, we generated and characterized a mouse model of ATS1 carrying the trafficking-deficient mutant Kir2.1Δ314-315 channel. The mutant mouse recapitulates the electrophysiological phenotype of ATS1, with QT prolongation exacerbated by flecainide or isoproterenol, drug-induced QRS prolongation, increased vulnerability to reentrant arrhythmias and multifocal discharges resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1Δ314-315 cardiomyocytes display significantly reduced inward rectifier K+ and Na+ currents, depolarized resting membrane potential and prolonged action potentials. We show that, in wild-type mouse cardiomyocytes and skeletal muscle cells, Kir2.1 channels localize to sarcoplasmic reticulum (SR) microdomains, contributing to intracellular Ca2+ homeostasis. Kir2.1Δ314-315 cardiomyocytes exhibit defective SR Kir2.1 localization and function, as intact and permeabilized Kir2.1Δ314-315 cardiomyocytes display abnormal spontaneous Ca2+ release events. Overall, defective Kir2.1 channel function at the sarcolemma and the SR explain the life-threatening arrhythmias in ATS1 and its overlap with CPVT.