Inhibition of SIRT1 deacetylase and p53 activation uncouples the anti-inflammatory and chemopreventive actions of NSAIDs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. METHODS: Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. RESULTS: Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. CONCLUSION: Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.

Identification of novel loci for the generation of reporter mice.

Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress.

In vivo imaging of early signs of dopaminergic neuronal death in an animal model of Parkinson's disease.

The Parkinson's disease (PD) evolves over an extended period of time with the onset occurring long before clinical signs begin to manifest. Characterization of the molecular events underlying the PD onset is instrumental for the development of diagnostic markers and preventive treatments, progress in this field is hindered by technical limitations. We applied an imaging approach to demonstrate the activation of Nrf2 transcription factor as a hallmark of neurodegeneration in neurotoxin-driven models of PD. In dopaminergic SK-N-BE neuroblastoma cells, Nrf2 activation was detected in cells committed to die as proven by time lapse microscopy; in the substantia nigra pars compacta area of the mouse brain, the Nrf2 activation preceded dopaminergic neurodegeneration as demonstrated by in vivo and ex vivo optical imaging, a finding confirmed by co-localization experiments carried out by immunohistochemistry. Collectively, our results identify the Nrf2 signaling as an early marker of neurodegeneration, anticipating dopaminergic neurodegeneration and motor deficits.

Design and validation of a reporter mouse to study the dynamic regulation of TFEB and TFE3 activity through in vivo imaging techniques.

TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor's validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism.Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

Sex-Specific Microglial Responses to Glucocerebrosidase Inhibition: Relevance to GBA1-Linked Parkinson's Disease.

Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson's Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5-2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.

Compliance between Food and Feed Safety: Eight-Year Survey (2013-2021) of Aflatoxin M1 in Raw Milk and Aflatoxin B1 in Feed in Northern Italy.

Aflatoxins (AFs) are fungal metabolites that are found in feed and food. When ruminants eat feed contaminated with aflatoxin B1 (AFB1), it is metabolised and aflatoxin M1 (AFM1) is excreted in the milk. Aflatoxins can result in hepatotoxic, carcinogenic, and immunosuppressive effects. The European Union thus set a low threshold limit (50 ng/L) for presence of AFM1 in milk. This was in view of its possible presence also in dairy products and that quantification of these toxins is mandatory for milk suppliers. In the present study, a total of 95,882 samples of whole raw milk, collected in northern Italy between 2013 and 2021, were evaluated for presence of AFM1 using an ELISA (enzyme-linked immunosorbent assay) method. The study also evaluated the relationship between feed materials collected from the same farms in the same area during the same period (2013-2021) and milk contamination. Only 667 milk samples out of 95,882 samples analysed (0.7%) showed AFM1 values higher than the EU threshold limit of 50 ng/L. A total of 390 samples (0.4%) showed values between 40 and 50 ng/L, thus requiring corrective action despite not surpassing the regulatory threshold. Combining feed contamination and milk contamination data, some feedingstuffs seem to be more effective in defying potential carryover of AFs from feed to milk. Combining the results, it can be concluded that a robust monitoring system that covers both feed, with a special focus on high risk/sentinel matrices, and milk is essential to guarantee high quality and safety standards of dairy products.

Molecular imaging of cytochrome P450 activity in mice.

Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs.

An Eight-Year Survey on Aflatoxin B1 Indicates High Feed Safety in Animal Feed and Forages in Northern Italy.

Aflatoxins (AFs) remain the main concern for the agricultural and dairy industries due to their effects on the performances and quality of livestock production. Aflatoxins are always unavoidable and should be monitored. The objective of this paper is to bring to light a significant volume of data on AF contamination in several animal feed ingredients in Northern Italy. The Regional Breeders Association of Lombardy has been conducting a survey program to monitor mycotoxin contamination in animal feeds, and in this paper, we present data relating to AFB1 contamination. In most cases (95%), the concentrations were low enough to ensure compliance with the European Union's (EU's) maximum admitted levels for animal feed ingredients. However, the data show a high variability in AF contamination between different matrices and, within the same matrix, a high variability year over year. High levels of AFs were detected in maize and cotton, especially in the central part of the second decade of this century, i.e., 2015-2018, which has shown a higher risk of AF contamination in feed materials in Northern Italy. Variability due to climate change and the international commodity market affect future prospects to predict the presence of AFs. Supplier monitoring and control and reduced buying of contaminated raw materials, as well as performing analyses of each batch, help reduce AF spread.

Increased Sensitivity of Computed Tomography Scan for Neoplastic Tissues Using the Extracellular Vesicle Formulation of the Contrast Agent Iohexol.

Computed tomography (CT) is a diagnostic medical imaging modality commonly used to detect disease and injury. Contrast agents containing iodine, such as iohexol, are frequently used in CT examinations to more clearly differentiate anatomic structures and to detect and characterize abnormalities, including tumors. However, these contrast agents do not have a specific tropism for cancer cells, so the ability to detect tumors is severely limited by the degree of vascularization of the tumor itself. Identifying delivery systems allowing enrichment of contrast agents at the tumor site would increase the sensitivity of detection of tumors and metastases, potentially in organs that are normally inaccessible to contrast agents, such as the CNS. Recent work from our laboratory has identified cancer patient-derived extracellular vesicles (PDEVs) as effective delivery vehicles for targeting diagnostic drugs to patients' tumors. Based on this premise, we explored the possibility of introducing iohexol into PDEVs for targeted delivery to neoplastic tissue. Here, we provide preclinical proof-of-principle for the tumor-targeting ability of iohexol-loaded PDEVs, which resulted in an impressive accumulation of the contrast agent selectively into the neoplastic tissue, significantly improving the ability of the contrast agent to delineate tumor boundaries.

Increased Ca2+ sensitivity of the ryanodine receptor mutant RyR2R4496C underlies catecholaminergic polymorphic ventricular tachycardia.

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca(2+) handling underlie this disease. Here we analyze the underlying Ca(2+) release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2(R4496C) mutation. RyR2(R4496C-/-) littermates (wild type) were used as controls. [Ca(2+)](i) transients were obtained by field stimulation in fluo-3-loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca(2+)](i) transients and sarcoplasmic reticulum Ca(2+) load were similar in wild-type and RyR2(R4496C) cells. However, paced RyR2(R4496C) ventricular myocytes presented abnormal Ca(2+) release during the diastolic period, viewed as Ca(2+) waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca(2+) release was enhanced at faster stimulation rates and by beta-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca(2+) sparks were more frequent in RyR2(R4496C) myocytes, indicating increased RyR2(R4496C) activity. When permeabilized cells were exposed to different cytosolic [Ca(2+)](i), RyR2(R4496C) showed a dramatic increase in Ca(2+) sensitivity. Isoproterenol increased [Ca(2+)](i) transient amplitude and Ca(2+) spark frequency to the same extent in wild-type and RyR2(R4496C) cells, indicating that the beta-adrenergic sensitivity of RyR2(R4496C) cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2(R4496C) mutation is attributable to the increased Ca(2+) sensitivity of RyR2(R4496C), which induces diastolic Ca(2+) release and lowers the threshold for triggered activity.

Dietary essential amino acids restore liver metabolism in ovariectomized mice via hepatic estrogen receptor α.

In female mammals, the cessation of ovarian functions is associated with significant metabolic alterations, weight gain, and increased susceptibility to a number of pathologies associated with ageing. The molecular mechanisms triggering these systemic events are unknown because most tissues are responsive to lowered circulating sex steroids. As it has been demonstrated that isoform alpha of the estrogen receptor (ERα) may be activated by both estrogens and amino acids, we test the metabolic effects of a diet enriched in specific amino acids in ovariectomized (OVX) mice. This diet is able to block the OVX-induced weight gain and fat deposition in the liver. The use of liver-specific ERα KO mice demonstrates that the hepatic ERα, through the control of liver lipid metabolism, has a key role in the systemic response to OVX. The study suggests that the liver ERα might be a valuable target for dietary treatments for the post-menopause.

Transplantation of autologous extracellular vesicles for cancer-specific targeting.

Nano- and microsized extracellular vesicles (EVs) are naturally occurring cargo-bearing packages of regulatory macromolecules, and recent studies are increasingly showing that EVs are responsible for physiological intercellular communication. Nanoparticles encapsulating anti-tumor theranostics represent an attractive "exosome-interfering" strategy for cancer therapy. Methods: Herein, by labeling plasma-derived EVs with indocyanine green (ICG) and following their biodistribution by in vivo and ex vivo imaging, we demonstrate the existence of nanoparticles with a highly selective cancer tropism in the blood of colorectal cancer (CRC) patients but not in that of healthy volunteers. Results: In CRC patient-derived xenograft (PDX) mouse models, we show that transplanted EVs recognize tumors from the cognate nanoparticle-generating individual, suggesting the theranostic potential of autologous EVs encapsulating tumor-interfering molecules. In large canine breeds bearing spontaneous malignant skin and breast tumors, the same autologous EV transplantation protocol shows comparable safety and efficacy profiles. Conclusions: Our data show the existence of an untapped resource of intercellular communication present in the blood of cancer patients, which represents an efficient and highly biocompatible way to deliver molecules directly to the tumor with great precision. The novel EV-interfering approach proposed by our study may become a new research direction in the complex interplay of modern personalized cancer therapy.

Unexpected structural and functional consequences of the R33Q homozygous mutation in cardiac calsequestrin: a complex arrhythmogenic cascade in a knock in mouse model.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2(R33Q/R33Q) mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2(R33Q/R33Q) mice develop bidirectional VT on exposure to environmental stress whereas CASQ2(R33Q/R33Q) myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2(R33Q/R33Q) hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2(R33Q/R33Q) CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.

Differential Somatic Cell Count as a Marker for Changes of Milk Composition in Cows with Very Low Somatic Cell Count.

The recent availability of a high-throughput milk analyzer performing a partial differential somatic cell count (DSCC) opened new opportunities in investigations on bovine udder health. This analyzer has a potential limitation on the accuracy of measurements when the somatic cell count (SCC) is below 50,000 cells/mL, values characterizing a good proportion of lactating cows in many herds. We obtained data for cows below this threshold, assessed the repeatability of these measurements and investigated the relationship between DSCC and udder health, milk composition and yield. Overall, 3022 cow milk test records performed on a Fossomatic™ 7/DC (Foss A/S, Hillerød, Denmark) were considered; 901 of them had an SCC ≤ 50,000 cells/mL. These latter samples were analyzed by qPCR to identify the presence of bacteria. Overall, 20.75% of the samples (187) were positive. However, the health status did not have any significant association with DSCC. The analysis of the association of DSCC on milk fat, protein and casein showed a significant decrease in their proportions as the DSCC increased, whereas it was not observed for milk yield and lactose. Therefore, DSCC in very low SCC cows may be suggested as a marker to identify early changes in milk composition.

Ryanodine receptor and calsequestrin in arrhythmogenesis: what we have learnt from genetic diseases and transgenic mice.

The year 2001 has been pivotal for the identification of the molecular bases of catecholaminergic polymorphic ventricular tachycardia (CPVT): a life-threatening genetic disease that predisposes young individuals with normal cardiac structure to cardiac arrest. Interestingly CPVT has been linked to mutations in genes encoding the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2): two fundamental proteins involved in regulation of intracellular Ca(2+) in cardiac myocytes. The critical role of the two proteins in the heart has attracted interests of the scientific community so that networks of investigators have embarked in translational studies to characterize in vitro and in vivo the mutant proteins. Overall in the last seven years the field has substantially advanced but considerable controversies still exist on the consequences of RyR2 and CASQ2 mutations and on the modalities by which they precipitate cardiac arrhythmias. With so many questions that need to be elucidated it is expected that in the near future the field will remain innovative and stimulating. In this review we will outline how research has advanced in the understanding of CPVT and we will present how the observations made have disclosed novel arrhythmogenic cascades that are likely to impact acquired heart diseases.

Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2(R4496C+/-)). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2(R4496C+/-) myocytes (P=0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2(R4496C+/-) myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 micromol/L) but not by K201 (1 micromol/L or 10 micromol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2(R4496C+/-) mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6 interaction both in WT and RyR2(R4496C+/-) either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2(R4496C+/-) myocytes and ventricular arrhythmias in RyR2(R4496C+/-) mice; and (3) RyR2-FKBP12.6 interaction in RyR2(R4496C+/-) is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex.

Systemic Administration and Targeted Delivery of Immunogenic Oncolytic Adenovirus Encapsulated in Extracellular Vesicles for Cancer Therapies.

Oncolytic viruses (OV) are engineered to infect, replicate in and kill cancer cells. Currently, the OV therapeutic approach is mainly restricted to neoplasia amenable to direct local administration of viral particles, while the possibility of a systemic delivery of cancer-tropic viruses would extend the OV application to the treatment of metastatic neoplasia. Herein, we applied in vivo/ex vivo imaging to demonstrate that cancer tropism is achieved when OV are encapsulated inside extracellular vesicles (EV) administered intravenously (i.v.), but not when injected intraperitoneally (i.p.). Moreover, we show that the therapeutic procedure adopted does not alter the immunomodulatory properties of the viruses.

Sex-Specific Features of Microglia from Adult Mice.

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases.

Clinical phenotype and functional characterization of CASQ2 mutations associated with catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Four distinct mutations in the human cardiac calsequestrin gene (CASQ2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. METHODS AND RESULTS: We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2(G112+5X)) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2(L167H)) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2(G112+5X) did not bind Ca2+, whereas CASQ2(L167H) had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of I(Ca)-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2(G112+5X). CONCLUSIONS: CASQ2(L167H) and CASQ2(G112+5X) alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca2+ release and calcium content. In addition, CASQ2(G112+5X) displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.

Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock.

Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover, heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts. In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli.