Maurocalcin and its analog MCaE12A facilitate Ca2+ mobilization in cardiomyocytes.

Ryanodine receptors are responsible for the massive release of calcium from the sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the effect of MCa and its analog MCaE12A on isolated cardiac ryanodine receptor (RyR2), and showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ transients, calcium sparks and contraction on cardiomyocytes isolated from adult rats or differentiated from human-induced pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes and promotes the abnormal opening of RyR2. We also investigated the effect of MCaE12A on the pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A improves pacemaker activity of sinus node cells obtained from mice lacking L-type Cav1.3 channel, or following selective pharmacologic inhibition of calcium influx via Cav1.3. Our results identify MCaE12A as a high-affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca2+ homeostasis and dynamic of cardiac cells.

In vitro differentiation of W8B2+ human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity.

BACKGROUND: Human cardiac stem cells expressing the W8B2 marker (W8B2+ CSCs) were recently identified and proposed as a new model of multipotent CSCs capable of differentiating into smooth muscle cells, endothelial cells and immature myocytes. Nevertheless, no characterization of ion channel or calcium activity during the differentiation of these stem cells has been reported. METHODS: The objectives of this study were thus to analyze (using the TaqMan Low-Density Array technique) the gene profile of W8B2+ CSCs pertaining to the regulation of ion channels, transporters and other players involved in the calcium homeostasis of these cells. We also analyzed spontaneous calcium activity (via the GCaMP calcium probe) during the in vitro differentiation of W8B2+ CSCs into cardiac myocytes. RESULTS: Our results show an entirely different electrophysiological genomic profile between W8B2+ CSCs before and after differentiation. Some specific nodal genes, such as Tbx3, HCN, ICaT, L, KV, and NCX, are overexpressed after this differentiation. In addition, we reveal spontaneous calcium activity or a calcium clock whose kinetics change during the differentiation process. A pharmacological study carried out on differentiated W8B2+ CSCs showed that the NCX exchanger and IP3 stores play a fundamental role in the generation of these calcium oscillations. CONCLUSIONS: Taken together, the present results provide important information on ion channel expression and intrinsic calcium dynamics during the differentiation process of stem cells expressing the W8B2 marker.

RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome.

AIMS: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. METHODS AND RESULTS: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. CONCLUSION: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

Functional Impact of BeKm-1, a High-Affinity hERG Blocker, on Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells.

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

Mental stress test: a rapid, simple, and efficient test to unmask long QT syndrome.

Aims: QT prolongation during mental stress test (MST) has been associated with familial idiopathic ventricular fibrillation. In long QT syndrome (LQTS), up to 30% of mutation carriers have normal QT duration. Our aim was to assess the QT response during MST, and its accuracy in the diagnosis of concealed LQTS. Methods and results: All patients who are carrier of a KCNQ1 or KCNH2 mutations without QT prolongation were enrolled. A control group was constituted of patients with negative exercise and epinephrine tests. Electrocardiogram were recorded at rest and at the maximum heart rate during MST and reviewed by two physicians. Among the 70 patients enrolled (median age 41±2.1 years, 46% male), 36 were mutation carrier for LQTS (20 KCNQ1 and 16 KCNH2), and 34 were controls. KCNQ1 and KCNH2 mutation carriers presented a longer QT interval at baseline [405(389; 416) and 421 (394; 434) ms, respectively] compared with the controls [361(338; 375)ms; P < 0.0001]. QT duration during MST varied by 9 (4; 18) ms in KCNQ1, 3 (-6; 16) ms in KCNH2, and by -22 (-29; -17) ms in controls (P < 0.0001). These QT variations were independent of heart rate (P < 0.3751). Receiver operating characteristic curve analysis identified a cut-off value of QT variation superior to -11 ms as best predictor of LQTS. It provided 97% sensitivity and 97% specificity of QT prolongation in the diagnosis of LQTS. Conclusion: We identified a paradoxical response of the QT interval during MST in LQTS. Easy to assess, MST may be efficient to unmask concealed LQTS in patients at risk of this pathology.

HIV-Tat induces a decrease in IKr and IKsvia reduction in phosphatidylinositol-(4,5)-bisphosphate availability.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.

Cooperative and antagonistic roles for Irx3 and Irx5 in cardiac morphogenesis and postnatal physiology.

The Iroquois homeobox (Irx) homeodomain transcription factors are important for several aspects of embryonic development. In the developing heart, individual Irx genes are important for certain postnatal cardiac functions, including cardiac repolarization (Irx5) and rapid ventricular conduction (Irx3). Irx genes are expressed in dynamic and partially overlapping patterns in the developing heart. Here we show in mice that Irx3 and Irx5 have redundant function in the endocardium to regulate atrioventricular canal morphogenesis and outflow tract formation. Our data suggest that direct transcriptional repression of Bmp10 by Irx3 and Irx5 in the endocardium is required for ventricular septation. A postnatal deletion of Irx3 and Irx5 in the myocardium leads to prolongation of atrioventricular conduction, due in part to activation of expression of the Na(+) channel protein Nav1.5. Surprisingly, combined postnatal loss of Irx3 and Irx5 results in a restoration of the repolarization gradient that is altered in Irx5 mutant hearts, suggesting that postnatal Irx3 activity can be repressed by Irx5. Our results have uncovered complex genetic interactions between Irx3 and Irx5 in embryonic cardiac development and postnatal physiology.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His-Purkinje network.

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes containing Parkinson disease-associated alpha-synuclein gene mutations precede central nervous system changes.

Parkinson disease (PD) is a neurodegenerative disease with motor as well as non-motor signs in the gastrointestinal tract that include dysphagia, gastroparesis, prolonged gastrointestinal transit time, constipation and difficulty with defecation. The gastrointestinal dysfunction commonly precedes the motor symptoms by decades. Most PD is sporadic and of unknown etiology, but a fraction is familial. Among familial forms of PD, a small fraction is caused by missense (A53T, A30P and E46K) and copy number mutations in SNCA which encodes alpha-synuclein, a primary protein constituent of Lewy bodies, the pathognomonic protein aggregates found in neurons in PD. We set out to develop transgenic mice expressing mutant alpha-synuclein (either A53T or A30P) from insertions of an entire human SNCA gene as models for the familial disease. Both the A53T and A30P lines show robust abnormalities in enteric nervous system (ENS) function and synuclein-immunoreactive aggregates in ENS ganglia by 3 months of age. The A53T line also has abnormal motor behavior but neither demonstrates cardiac autonomic abnormalities, olfactory dysfunction, dopaminergic neurotransmitter deficits, Lewy body inclusions or neurodegeneration. These animals recapitulate the early gastrointestinal abnormalities seen in human PD. The animals also serve as an in vivo system in which to investigate therapies for reversing the neurological dysfunction that target alpha-synuclein toxicity at its earliest stages.

Generation of human induced pluripotent stem cell lines from two patients affected by catecholamine-induced QT prolongation (CIQTP).

Catecholamine-induced QT prolongation (CIQTP) is an inherited cardiac disease characterized by a normal baseline ECG and a risk of sudden cardiac death by ventricular arrhythmia due to a QT prolongation that only appears during catecholergic stimulation, especially mental stress. Induced pluripotent stem cells (hiPSCs) were generated from peripheral blood mononuclear cells collected from two CIQTP-affected patients from two different families. These two hiPSC lines are a valuable model to study biological alterations due to CIQTP.

Deciphering Transcriptional Networks during Human Cardiac Development.

Human heart development is governed by transcription factor (TF) networks controlling dynamic and temporal gene expression alterations. Therefore, to comprehensively characterize these transcriptional regulations, day-to-day transcriptomic profiles were generated throughout the directed cardiac differentiation, starting from three distinct human- induced pluripotent stem cell lines from healthy donors (32 days). We applied an expression-based correlation score to the chronological expression profiles of the TF genes, and clustered them into 12 sequential gene expression waves. We then identified a regulatory network of more than 23,000 activation and inhibition links between 216 TFs. Within this network, we observed previously unknown inferred transcriptional activations linking IRX3 and IRX5 TFs to three master cardiac TFs: GATA4, NKX2-5 and TBX5. Luciferase and co-immunoprecipitation assays demonstrated that these five TFs could (1) activate each other's expression; (2) interact physically as multiprotein complexes; and (3) together, finely regulate the expression of SCN5A, encoding the major cardiac sodium channel. Altogether, these results unveiled thousands of interactions between TFs, generating multiple robust hypotheses governing human cardiac development.

Generation of human induced pluripotent stem cell lines from three patients affected by Catecholaminergic Polymorphic ventricular tachycardia (CPVT) carrying heterozygous mutations in RYR2 gene.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an exercise and emotional stress-induced life-threatening inherited heart rhythm disorder, characterized by an abnormal cellular calcium homeostasis. Most reported cases have been linked to mutations in the gene encoding the type 2 ryanodine receptor gene, RYR2. We generated induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMC) from three CPVT-affected patients, two of them carrying p.R4959Q mutation and one carrying p.Y2476D mutation. These generated hiPSC lines are a useful model to study pathophysiological consequences of RYR2 dysfunction in humans and the molecular basis of CPVT.

Generation of three human induced pluripotent stem cell lines with IRX5 knockout and knockin genetic editions using CRISPR-Cas9 system.

Studies on animal models have shown that Irx5 is an important regulator of cardiac development and that it regulates ventricular electrical repolarization gradient in the adult heart. Mutations in IRX5 have also been linked in humans to cardiac conduction defects. In order to fully characterize the role of IRX5 during cardiac development and in cardiomyocyte function, we generated three genetically-modified human induced pluripotent stem cell lines: two knockout lines (heterozygous and homozygous) and a knockin HA-tagged line (homozygous).

FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.

This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).

Generation of human induced pluripotent stem cell lines from four unrelated healthy control donors carrying European genetic background.

Four human induced pluripotent stem cell (hiPSC) lines have been generated from healthy control European donors, and validated. This resource represents a useful tool for stem cell-based research, as references for developmental studies and disease modeling linked to any type of human tissue and organ, in an ethnical-, sex- and age-matched context. They providea reliable in-vitro model for single cell- and tissue-based investigations, and are also a valuable tool for genome editing-based studies.

Human model of IRX5 mutations reveals key role for this transcription factor in ventricular conduction.

AIMS: Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. METHODS AND RESULTS: Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. CONCLUSION: Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases.

Ion-channel mRNA-expression profiling: Insights into cardiac remodeling and arrhythmic substrates.

Membrane ion channels and transporters are key determinants of cardiac electrical function. Their expression is affected by cardiac region, hemodynamic properties, heart-rate changes, neurohormones and cardiac disease. One of the important determinants of ion-channel function is the level of ion-channel subunit mRNA expression, which governs the production of ion-channel proteins that traffic to the cell-membrane to form functional ion-channels. Ion-channel mRNA-expression profiling can be performed with cDNA microarrays or high-throughput reverse transcription/polymerase chain reaction (PCR) methods. Expression profiling has been applied to evaluate the dependence of ion-channel expression on cardiac region, revealing the molecular basis of regionally-controlled electrical properties as well as the molecular determinants of specialized electrical functions like pacemaking activity. Ion-channel remodeling occurs with cardiac diseases like heart failure, congenital repolarization abnormalities, and atrial fibrillation, and expression profiling has provided insights into the mechanisms by which these conditions affect cardiac electrical stability. Expression profiling has also shown how hormonal changes, antiarrhythmic drugs, cardiac development and altered heart rate affect ion-channel expression patterns to modify cardiac electrical function and sometimes to produce cardiac rhythm disturbances. This article reviews the information obtained to date with the application of cardiac ion-channel expression profiling. With increasing availability and efficiency of high-throughput PCR methods for ion-channel subunit mRNA-expression characterization, it is likely that the application of ion-channel expression profiling will increase and that it will provide important new insights into the determinants of cardiac electrical function in both physiological and pathological situations.

Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts.

Gender-related differences in ventricular electrophysiology are known to be important determinants of human arrhythmic risk, but the underlying molecular basis is poorly understood. The present work aims to provide the first detailed analysis of gender-related cardiac ion-channel gene-distribution, based on samples from non-diseased human hearts. By using a high-throughput quantitative approach, we investigated at a genome-scale the expression of 79 genes encoding ion-channel and transporter subunits in epicardial and endocardial tissue samples from non-diseased transplant donors (10 males, 10 females). Gender-related expression differences involved key genes implicated in conduction and repolarization. Female hearts showed reduced expression for a variety of K(+)-channel subunits with potentially important roles in cardiac repolarization, including HERG, minK, Kir2.3, Kv1.4, KChIP2, SUR2 and Kir6.2, as well as lower expression of connexin43 and phospholamban. In addition, they demonstrated an isoform switch in Na(+)/K(+)-ATPase, expressing more of the alpha1 and less of the alpha3 subunit than male hearts, along with increased expression of calmodulin-3. Iroquois transcription factors (IRX3, IRX5) were more strongly expressed in female than male epicardium, but the transmural gradient remained. Protein-expression paralleled transcript patterns for all subunits examined: HERG, minK, Kv1.4, KChIP2, IRX5, Nav1.5 and connexin43. Our results indicate that male and female human hearts have significant differences in ion-channel subunit composition, with female hearts showing decreased expression for a number of repolarizing ion-channels. These findings are important for understanding sex-related differences in the susceptibility to ventricular arrhythmias, particularly for conditions associated with repolarization abnormalities like Brugada and Long QT syndrome.

Transcriptional profiling of ion channel genes in Brugada syndrome and other right ventricular arrhythmogenic diseases.

AIMS: Brugada syndrome is an inherited sudden-death arrhythmia syndrome. Na(+)-current dysfunction is central, but mutations in the SCN5A gene (encoding the cardiac Na(+)-channel Nav1.5) are present in only 20% of probands. This study addressed the possibility that Brugada patients display specific expression patterns for ion-channels regulating cardiac conduction, excitability, and repolarization. METHODS AND RESULTS: Transcriptional profiling was performed on right-ventricular endomyocardial biopsies from 10 unrelated Brugada probands, 11 non-diseased organ-donors, seven heart-transplant recipients, 10 with arrhythmogenic right-ventricular cardiomyopathy, and nine with idiopathic right-ventricular outflow-tract tachycardia. Brugada patients showed distinct clustering differences vs. the two control and two other ventricular-tachyarrhythmia groups, including 14 of 77 genes encoding important ion-channel/ion-transporter subunits. Nav1.5 and K(+)-channels Kv4.3 and Kir3.4 were more weakly expressed, whereas the Na(+)-channel Nav2.1 and the K(+)-channel TWIK1 were more strongly expressed, in Brugada syndrome. Differences were also seen in Ca(2+)-homeostasis transcripts, including stronger expression of RYR2 and NCX1. The molecular profile of Brugada patients with SCN5A mutations did not differ from Brugada patients without SCN5A mutations. CONCLUSION: Brugada patients exhibit a common ion-channel molecular expression signature, irrespective of the culprit gene. This finding has potentially important implications for our understanding of the pathophysiology of Brugada syndrome, with possible therapeutic and diagnostic consequences.

The ß4 subunit of the voltage-gated calcium channel (Cacnb4) regulates the rate of cell proliferation in Chinese Hamster Ovary cells.

The ß subunits of Voltage-Gated Calcium Channel (VGCC) are cytosolic proteins that interact with the VGCC pore -forming subunit and participate in the trafficking of the channel to the cell membrane and in ion influx regulation. ß subunits also exert functions independently of their binding to VGCC by translocation to the cell nucleus including the control of gene expression. Mutations of the neuronal Cacnb4 (ß4) subunit are linked to human neuropsychiatric disorders including epilepsy and intellectual disabilities. It is believed that the pathogenic phenotype induced by these mutations is associated with channel-independent functions of the ß4 subunit. In this report, we investigated the role of ß4 subunit in cell proliferation and cell cycle progression and examined whether these functions could be altered by a pathogenic mutation. To this end, stably transfected Chinese Hamster Ovary (CHO-K1) cells expressing either rat full-length ß4 or the rat C-terminally truncated epileptic mutant variant ß1-481 were generated. The subcellular localization of both proteins differed significantly. Full-length ß4 localizes almost exclusively in the cell nucleus and concentrates into the nucleolar compartment, while the C-terminal-truncated ß1-481 subunit was less concentrated within the nucleus and absent from the nucleoli. Cell proliferation was found to be reduced by the expression of ß4, while it was unaffected by the epileptic mutant. Also, full-length ß4 interfered with cell cycle progression by presumably preventing cells from entering the S-phase via a mechanism that partially involves endogenous B56δ, a regulatory subunit of the phosphatase 2A (PP2A) that binds ß4 but not ß1-481. Analysis of ß4 subcellular distribution during the cell cycle revealed that the protein is highly expressed in the nucleus at the G1/S transition phase and that it is translocated out of the nucleus during chromatin condensation and cell division. These results suggest that nuclear accumulation of ß4 at the G1/S transition phase affects the progression into S-phase resulting in a decrease in the rate of cell proliferation. Regulation of the cell cycle exit is a critical step in determining the number of neuronal precursors and neuronal differentiation suggesting that mutations of the ß4 subunit could affect neural development and formation of the mature central nervous system.