Predictors of high school dropout, anxiety, and depression in genetic generalized epilepsy.

Affective disorders are overrepresented in epilepsy, and people with epilepsy may be at risk of dropping out from school. The aim of the present study was to assess factors influencing high school dropout, anxiety, and depression in genetic generalized epilepsy (GGE). One hundred and ten people with GGE aged 19-40 years underwent a clinical interview, including the Hospital Anxiety and Depression Scale (HADS) questionnaire. Potential predictors of high school dropout were analyzed with logistic regression, and factors influencing total HADS score were analyzed with linear regression. Having felt excluded because of epilepsy was significantly associated with high school dropout (odds ratio 7.80, P = .009), as was total HADS score (odds ratio 1.22, P = .005). If a participant was currently employed or undergoing education, previous high school dropout was less likely (odds ratio 0.07, P = .005). High school dropout was associated with increased current anxiety and depression (ß = 0.32, P = .005). Epilepsy severity (current drug resistance, current polytherapy, and active generalized tonic-clonic seizures) was not associated with high school dropout, nor with total HADS score. The issue of stigma in epilepsy must be thoroughly addressed in comprehensive care and may be as important as seizure control when it comes to education and quality of life.

Trait impulsivity correlates with active myoclonic seizures in genetic generalized epilepsy.

BACKGROUND: Juvenile myoclonic epilepsy (JME) is a common subtype of genetic generalized epilepsy (GGE) arising in adolescence and is often associated with executive function (EF) deficits. Some EF components like response inhibition have been extensively evaluated in JME, but few studies have focused upon trait impulsivity or compared between GGE subtypes. The aim of the present study was to compare the association of trait impulsivity in JME with other GGE subtypes. METHODS: Patients with GGE aged between 14 and 40 years (n = 137) were divided into those with JME (n = 92) and those with other GGEs (n = 45: 8 childhood absence epilepsy (CAE), 22 juvenile absence epilepsy (JAE), and 15 epilepsy with generalized tonic-clonic seizures only (EGTCS)). The study participants were recruited through medical records of the general population of Buskerud County and the neighboring municipalities, covering 477,000 people or 9.1% of Norway's total population. All participants underwent a clinical interview including the Barratt Impulsiveness Scale (BIS), an established measure of trait impulsivity. We controlled for other potential predictors of BIS score using analysis of covariance (ANCOVA). RESULTS: There were no differences between JME and other types of GGE for BIS scores, but the presence of myoclonic seizures within the last year, irrespective of GGE subtype, was independently associated with significantly increased behavioral impulsivity. CONCLUSIONS: This study demonstrates that trait impulsivity in GGE is most strongly related to the recent occurrence of myoclonic seizures rather than GGE subtype.

Treatment and challenges with antiepileptic drugs in patients with juvenile myoclonic epilepsy.

BACKGROUND: Patients with juvenile myoclonic epilepsy (JME) may have uncontrolled seizures. The purpose of this study was to investigate the use and challenges with antiepileptic drugs (AEDs) and the patients' view of these challenges. METHOD: A questionnaire about the use of AEDs, adherence to therapy, and quality of life was given to patients with JME recruited from Drammen Hospital. Data regarding AEDs were confirmed from medical records at Drammen Hospital, Norway (2007-2018). Additional clinical interviews were performed, and a mixed method approach was applied. RESULTS: Ninety patients with defined JME diagnosis, 54/36 women/men aged 14-39 (mean: 25) years, were included. Only 29 (33%) were seizure-free. Within the last year, 21% experienced generalized tonic-clonic seizures (GTCS), and 68% had myoclonic jerks. Seventy-six (84%) used AEDs, 78% in monotherapy. A total of 10 AEDs were used;: most commonly valproate (n = 33), lamotrigine (n = 27), and levetiracetam (n = 21). Two-thirds of valproate users were men while all other AEDs were used more in females than in men. Valproate and levetiracetam displayed better efficacy against GTCS than lamotrigine. One-third often/sometimes forgot their medication nonintentionally while 14% had intentional poor adherence. The majority reported good quality of life (76%). No significant correlations between the use of AEDs, use of valproate, poor adherence, quality of life score, and seizure freedom were demonstrated. Half of the patients had serum concentrations measured every year, and two-thirds thought this was important. Qualitative interviews elucidated treatment challenges in JME;, adverse effect burden, adherence, and activities of daily life. CONCLUSION: Despite the use of AEDs in the majority of patients, only one-third were seizure-free. Other challenges included polypharmacy, the use of valproate in women, and variable adherence. This points to a need for closer follow-up in patients with JME.

Long-term follow-up with therapeutic drug monitoring of antiepileptic drugs in patients with juvenile myoclonic epilepsy.

BACKGROUND AND PURPOSE: Patients with juvenile myoclonus epilepsy (JME) may experience uncontrolled seizures and challenges regarding adherence. Implementation of therapeutic drug monitoring (TDM) may contribute to individualization of the therapy with antiepileptic drugs (AEDs). The purpose of this study was to investigate how the treatment of patients with JME is monitored and to demonstrate pharmacokinetic variability within and between patients with a long-term TDM approach. METHOD: Retrospective data from patients with JME from the TDM-database at Drammen Hospital and the National Center for Epilepsy in Norway (2007-2018) were included. RESULTS: Data from 80 of 90 patients with JME using AEDs with TDM measurements was included (88%, 49/31 women/men aged 14-39). One third (27, 33%) was seizure free, 19 (24%) had generalized tonic-clonic seizures, and 53 (66%) myoclonic seizures during the last year. The most common AEDs measured included lamotrigine, valproate, and levetiracetam. Long-term TDM demonstrated variability over time expressed as intra-patient median values and inter-patient ranges of 19% (7-47) for valproate, 43% (10-83) for lamotrigine and 35% (6-111) for levetiracetam. Fifteen pecent (83/563) of serum concentrations were below the reference ranges and clould be due to variable adherence. Comedication with valproate for lamotrigine and pregnancy contributed to variability. The applicability is illustrated in a case of 10 years' follow-up in a young woman. CONCLUSION: There was extensive pharmacokinetic variability of AEDs in and between patients with JME. A long-term TDM approach may contribute to closer monitoring of patients with JME and be used as a practical tool during clinical consultations.

Antiepileptic drug withdrawal in juvenile myoclonic epilepsy.

OBJECTIVES: Withdrawal of antiepileptic drugs (AEDs) has been discouraged in juvenile myoclonic epilepsy (JME). However, impulsivity as a consequence of executive dysfunction in JME may influence treatment adherence. The aim of the present study was to assess how common withdrawal of AEDs is in a large and representative JME group. MATERIALS AND METHODS: Patients with genetic generalized epilepsy (GGE) were identified through a retrospective search of medical records at Drammen Hospital, Norway, and invited to a clinical interview. Information related to AED withdrawal was analyzed in those classified as JME. RESULTS: A total of 132 patients with GGE were interviewed (87 JME). Thirty-five patients with JME (40%) discontinued AEDs, of which 74% did so without consulting a doctor. The rate of self-withdrawal was significantly higher in JME than in other types of GGE. Having a parent with psychosocial difficulties was significantly over-represented in the JME self-withdrawal group. Twelve of those who discontinued AEDs (34%) were free from generalized tonic-clonic seizures (GTCS) and without antiepileptic drugs >1 year. All but one of them withdrew AEDs without consulting a doctor. Age at first motor seizure was significantly higher in those with a favorable outcome of AED withdrawal. CONCLUSIONS: Self-withdrawal of AEDs is common in JME, especially in those with troublesome conditions at home. However, about 1/3 may remain free from GTCS without AEDs. The findings indicate a need for a stronger follow-up with appropriate information about the prognosis of the disorder.

Psychosocial complications in juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) constitutes about 10% of all epilepsies. Because of executive dysfunction, people with JME may be prone to impulsivity and risk-taking behavior. Our aim was to investigate whether psychosocial issues associated with impulsivity are more prominent in people with JME than in those with other types of genetic generalized epilepsy (GGE). Patients with GGE were recruited retrospectively through the Drammen Hospital records in Buskerud County, Norway, 1999-2013. They were invited to a semi-structured interview, either at the hospital or at home. Ninety-two patients with JME and 45 with other types of GGE were interviewed. Variables were evaluated in terms of their association with JME versus other GGE diagnosis using a logistic regression model. Juvenile myoclonic epilepsy was associated with use of illicit recreational drugs and police charges, although with borderline significance (odds ratio [OR] 3.4, p = 0.087 and OR 4.2, p = 0.095); JME was also associated with being examined for attention-deficit hyperactivity disorder (ADHD) in females (OR 15.5, p = 0.015), a biological parent with challenges like addiction or violent behavior (OR 3.5, p = 0.032), and use of levetiracetam (OR 5.1, p = 0.014). After controlling for group differences, we found psychosocial complications to be associated with JME, potentially influencing the lives of the individuals and their families to a greater extent than the seizures per se. Thus, JME should be considered a disorder of the brain in a broader sense than a condition with seizures only.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman.

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after Serca2 knockout.

Cardiomyocyte contraction and relaxation are controlled by Ca(2+) handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca(2+) fluxes. Here we investigate whether altered Ca(2+) fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na(+)/Ca(2+)-exchanger proximal to ryanodine receptors in the SR but did not contain Ca(2+)-channels. Ca(2+) measurements demonstrated a switch from SR-driven to Ca(2+) influx-driven Ca(2+) transients in KO. Still, SR Ca(2+) release constituted 20% of the Ca(2+) transient in KO. Mathematical modeling suggested that Ca(2+) influx via Na(+)/Ca(2+)-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca(2+) release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.

Cardiomyocyte-specific disruption of Serca2 in adult mice causes sarco(endo)plasmic reticulum stress and apoptosis.

Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses.

Cre-loxP DNA recombination is possible with only minimal unspecific transcriptional changes and without cardiomyopathy in Tg(alphaMHC-MerCreMer) mice.

Cre-loxP technology for conditional gene inactivation is a powerful tool in cardiovascular research. Induction of gene inactivation can be carried out by per oral or intraperitoneal tamoxifen administration. Unintended transient cardiomyopathy following tamoxifen administration for gene inactivation has recently been reported. We aimed to develop a protocol for tamoxifen-induced gene inactivation with minimal effects on gene transcription and in vivo cardiac function, allowing studies of acute loss of the targeted gene. In mRNA microarrays, 35% of the 34,760 examined genes were significantly regulated in MCM(+/0) compared with wild type. In MCM(+/0), we found a correlation between tamoxifen dose and degree of gene regulation. Comparing one and four intraperitoneal injections of 40 mg·kg(-1)·day(-1) tamoxifen, regulated genes were reduced to 1/5 in the single injection group. Pronounced alteration in protein abundance and acute cardiomyopathy were observed after the four-injection protocols but not the one-injection protocol. For verification of gene inactivation following one injection of tamoxifen, this protocol was applied to MCM(+/0)/Serca2(fl/fl). Serca2 mRNA levels and protein abundance followed the same pattern of decline with one and four tamoxifen injections. The presence of the MCM transgene induced major alterations of gene expression while administration of tamoxifen induced additional but less gene regulation. Thus nonfloxed MCM(+/0) should be considered as controls for mice that carry both a floxed gene of interest and the MCM transgene. One single tamoxifen injection administered to MCM(+/0)/Serca2(fl/fl) was sufficient for target gene inactivation, without acute cardiomyopathy, allowing acute studies subsequent to gene inactivation.

Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes.

AIMS: To describe the overall role of reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) for Ca(2+) wave development. METHODS AND RESULTS: SERCA2 knockout [Serca2(flox/flox) Tg(alphaMHC-MerCreMer); KO] mice allowing inducible cardiomyocyte-specific disruption of the Serca2 gene in adult mice were compared with Serca(flox/flox) (FF) control mice. Six days after Serca2 gene disruption, SERCA2 protein abundance was reduced by 53% in KO compared with FF, whereas SERCA2 activity in field-stimulated, Fluo-5F AM-loaded cells was reduced by 42%. Baseline Ca(2+) content of the sarcoplasmic reticulum (SR) and Ca(2+) transient amplitude and rate constant of decay measured in whole-cell voltage-clamped cells were decreased in KO to 75, 81, and 69% of FF values. Ca(2+) waves developed in only 31% of KO cardiomyocytes compared with 57% of FF when external Ca(2+) was raised (10 mM), although SR Ca(2+) content needed for waves to develop was 79% of FF values. In addition, waves propagated at a 15% lower velocity in KO cells. Ventricular extrasystoles (VES) occurred with lower frequency in SERCA2 KO mice (KO: 3 +/- 1 VES/h vs. FF: 8 +/- 1 VES/h) (P < 0.05 for all results). CONCLUSION: Reduced SERCA2 abundance resulted in decreased amplitude and decay rate of Ca(2+) transients, reduced SR Ca(2+) content, and decreased propensity for Ca(2+) wave development.

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase alpha2-isoform in heart failure.

AIMS: The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS: NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION: Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.

Serotonin increases L-type Ca2+ current and SR Ca2+ content through 5-HT4 receptors in failing rat ventricular cardiomyocytes.

Rats with congestive heart failure (CHF) develop ventricular inotropic responsiveness to serotonin (5-HT), mediated through 5-HT(2A) and 5-HT(4) receptors. Human ventricle is similarly responsive to 5-HT through 5-HT(4) receptors. We studied isolated ventricular cardiomyocytes to clarify the effects of 5-HT on intracellular Ca(2+) handling. Left-ventricular cardiomyocytes were isolated from male Wistar rats 6 wk after induction of postinfarction CHF. Contractile function and Ca(2+) transients were measured in field-stimulated cardiomyocytes, and L-type Ca(2+) current (I(Ca,L)) and sarcoplasmic reticulum (SR) Ca(2+) content were measured in voltage-clamped cells. Protein phosphorylation was measured by Western blotting or phosphoprotein gel staining. 5-HT(4)- and 5-HT(2A)-receptor stimulation induced a positive inotropic response of 33 and 18% (both P < 0.05) and also increased the Ca(2+) transient (44 and 6%, respectively; both P < 0.05). I(Ca,L) and SR Ca(2+) content increased only after 5-HT(4)-receptor stimulation (57 and 65%; both P < 0.05). Phospholamban serine(16) (PLB-Ser(16)) and troponin I phosphorylation increased by 26 and 13% after 5-HT(4)-receptor stimulation (P < 0.05). 5-HT(2A)-receptor stimulation increased the action potential duration and did not significantly change the phosphorylation of PLB-Ser(16) or troponin I, but it increased myosin light chain 2 (MLC2) phosphorylation. In conclusion, the positive inotropic response to 5-HT(4) stimulation results from increased I(Ca,L) and increased phosphorylation of PLB-Ser(16), which increases the SR Ca(2+) content. 5-HT(4) stimulation is thus, like beta-adrenoceptor stimulation, possibly energetically unfavorable in CHF. 5-HT(2A)-receptor stimulation, previously studied in acute CHF, induces a positive inotropic response also in chronic CHF, probably mediated by MLC2 phosphorylation.

The Na+/K+-ATPase alpha2-isoform regulates cardiac contractility in rat cardiomyocytes.

OBJECTIVE: The presence of both alpha1- and alpha2-isoforms of the Na+/K+-ATPase (NKA) in cardiomyocytes indicates different functions. We hypothesized that preferential localization of the alpha2-isoform to the t-tubules, locally controlling the Na+/Ca2+-exchanger (NCX), underlies a specific role in Ca2+ handling. METHODS: We studied NKA isoform distribution in isolated cardiomyocytes from Wistar rats using immunocytochemistry. NKA pump and NCX currents (I(pump) and I(NCX)) were measured in control and detubulated cardiomyocytes. Intracellular Na+ concentration [Na+]i was assessed with the fluorescent dye SBFI. RESULTS: The alpha2-isoform abundance was higher in the t-tubules than in the surface sarcolemma. We established that 0.3 microM ouabain specifically blocked the alpha2-isoform in isolated rat cardiomyocytes. This low concentration blocked 10.7+/-0.6% of I(pump) in control, but only 6.0+/-0.5% in detubulated cardiomyocytes. Moreover, measured and calculated alpha1-specific and alpha2-specific I(pump) in control (547+/-29 pA and 66 pA, respectively) and in detubulated cells (495+/-30 pA and 31 pA, respectively) showed that 53% of the alpha2-isoform, but only 9.5% of the alpha1-isoform, were localized to the t-tubules. Despite the small abundance of the alpha2-isoform (approximately 11% of total NKA), selective inhibition of this isoform induced a 40% increase in contractility in field stimulated cardiomyocytes, but no increase in global [Na+]i. However, inhibition of the alpha2-isoform increased I(NCX) indicating local subsarcolemmal accumulation of Na+ near NCX. CONCLUSIONS: The alpha2-isoform of the NKA is functionally coupled to the NCX and can regulate Ca2+ handling without changing global [Na+]i.