Epilepsy and neurobehavioral abnormalities in mice with a dominant-negative KCNB1 pathogenic variant.

Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay, and often have elevated risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel KV2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons. In addition to its canonical role as a voltage-gated potassium conductance, KV2.1 also serves a highly conserved structural function organizing endoplasmic reticulum-plasma membrane junctions clustered in the soma and proximal dendrites of neurons. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, and atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected KV2.1 clustering at endoplasmic reticulum-plasma membrane junctions, KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-wild-type lowered induction of these structures relative to KV2.1-WT alone, consistent with a dominant negative effect. To model this variant in vivo, we introduced Kcnb1G379R into mice using CRISPR/Cas9 genome editing. We characterized neuronal expression, neurological and neurobehavioral phenotypes of Kcnb1G379R/+ (Kcnb1R/+) and Kcnb1G379R/G379R (Kcnb1R/R) mice. Immunohistochemistry studies on brains from Kcnb1+/+, Kcnb1R/+ and Kcnb1R/R mice revealed genotype-dependent differences in the expression levels of KV2.1 protein, as well as associated KV2.2 and AMIGO-1 proteins. Kcnb1R/+ and Kcnb1R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. Spontaneous seizures were observed in Kcnb1R/R mice, as well as seizures induced by exposure to novel environments and/or handling. Both Kcnb1R/+ and Kcnb1R/R mutants were more susceptible to proconvulsant-induced seizures. In addition, both Kcnb1R/+ and Kcnb1R/R mice exhibited abnormal interictal EEG activity, including isolated spike and slow waves. Overall, the Kcnb1G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1-associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.

Overexpression of Latent TGFß Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFß.

Latent TGFß binding proteins (LTBPs) regulate the extracellular availability of latent TGFß. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFß family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFß and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFß family ligand binding protein with the capacity to modify muscle disease through overexpression.

Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy.

Disruption of the dystrophin complex causes muscle injury, dysfunction, cell death and fibrosis. Excess transforming growth factor (TGF) ß signaling has been described in human muscular dystrophy and animal models, where it is thought to relate to the progressive fibrosis that characterizes dystrophic muscle. We now found that canonical TGFß signaling acutely increases when dystrophic muscle is stimulated to contract. Muscle lacking the dystrophin-associated protein γ-sarcoglycan (Sgcg null) was subjected to a lengthening protocol to produce maximal muscle injury, which produced rapid accumulation of nuclear phosphorylated SMAD2/3. To test whether reducing SMAD signaling improves muscular dystrophy in mice, we introduced a heterozygous mutation of SMAD4 (S4) into Sgcg mice to reduce but not ablate SMAD4. Sgcg/S4 mice had improved body mass compared with Sgcg mice, which normally show a wasting phenotype similar to human muscular dystrophy patients. Sgcg/S4 mice had improved cardiac function as well as improved twitch and tetanic force in skeletal muscle. Functional enhancement in Sgcg/S4 muscle occurred without a reduction in fibrosis, suggesting that intracellular SMAD4 targets may be important. An assessment of genes differentially expressed in Sgcg muscle focused on those encoding calcium-handling proteins and responsive to TGFß since this pathway is a target for mediating improvement in muscular dystrophy. These data demonstrate that excessive TGFß signaling alters cardiac and muscle performance through the intracellular SMAD pathway.

Augmentation of Kv4.2-encoded currents by accessory dipeptidyl peptidase 6 and 10 subunits reflects selective cell surface Kv4.2 protein stabilization.

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

Sex and Gene Influence Arrhythmia Susceptibility in Murine Models of Calmodulinopathy.

BACKGROUND: Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. METHODS: Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. RESULTS: Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. CONCLUSIONS: We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.

Region-specific parasympathetic nerve remodeling in the left atrium contributes to creation of a vulnerable substrate for atrial fibrillation.

Atrial fibrillation (AF) is the most common heart rhythm disorder and a major cause of stroke. Unfortunately, current therapies for AF are suboptimal, largely because the molecular mechanisms underlying AF are poorly understood. Since the autonomic nervous system is thought to increase vulnerability to AF, we used a rapid atrial pacing (RAP) canine model to investigate the anatomic and electrophysiological characteristics of autonomic remodeling in different regions of the left atrium. RAP led to marked hypertrophy of parent nerve bundles in the posterior left atrium (PLA), resulting in a global increase in parasympathetic and sympathetic innervation throughout the left atrium. Parasympathetic fibers were more heterogeneously distributed in the PLA when compared with other left atrial regions; this led to greater fractionation and disorganization of AF electrograms in the PLA. Computational modeling revealed that heterogeneously distributed parasympathetic activity exacerbates sympathetic substrate for wave break and reentry. We further discovered that levels of nerve growth factor (NGF) were greatest in the left atrial appendage (LAA), where AF was most organized. Preferential NGF release by the LAA - likely a direct function of frequency and regularity of atrial stimulation - may have important implications for creation of a vulnerable AF substrate.

Genetic Mosaicism in Calmodulinopathy.

BACKGROUND: CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families. METHODS: CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation. RESULTS: Genetic studies identified 2 novel CaM variants (CALM3-E141K in 2 cases; CALM1-E141V) and one previously reported CaM pathogenic variant (CALM3-D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3-E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3-D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca2+ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca2+-dependent inactivation of L-type Ca2+ channels and prolonged action potential duration. CONCLUSIONS: We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca2+ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.

Inactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase enzymes increases during the meiotic maturation of porcine oocytes.

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11beta-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol-cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111+/-6 vs 2041+/-115 fmol cortisone/oocyte.24 h; P<0.001). Likewise, HSD11B activities were 17+/-1 fold higher in DOs from expanded COCs than in those from compact COCs (P<0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146+/-18 to 1857+/-276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P<0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation.

Novel calmodulin mutations associated with congenital long QT syndrome affect calcium current in human cardiomyocytes.

BACKGROUND: Calmodulin (CaM) mutations are associated with cardiac arrhythmia susceptibility including congenital long QT syndrome (LQTS). OBJECTIVE: The purpose of this study was to determine the clinical, genetic, and functional features of 2 novel CaM mutations in children with life-threatening ventricular arrhythmias. METHODS: The clinical and genetic features of 2 congenital arrhythmia cases associated with 2 novel CaM gene mutations were ascertained. Biochemical and functional investigations were conducted on the 2 mutations. RESULTS: A novel de novo CALM2 mutation (D132H) was discovered by candidate gene screening in a male infant with prenatal bradycardia born to healthy parents. Postnatal course was complicated by profound bradycardia, prolonged corrected QT interval (651 ms), 2:1 atrioventricular block, and cardiogenic shock. He was resuscitated and was treated with a cardiac device. A second novel de novo mutation in CALM1 (D132V) was discovered by clinical exome sequencing in a 3-year-old boy who suffered a witnessed cardiac arrest secondary to ventricular fibrillation. Electrocardiographic recording after successful resuscitation revealed a prolonged corrected QT interval of 574 ms. The Ca(2+) affinity of CaM-D132H and CaM-D132V revealed extremely weak binding to the C-terminal domain, with significant structural perturbations noted for D132H. Voltage-clamp recordings of human induced pluripotent stem cell-derived cardiomyocytes transiently expressing wild-type or mutant CaM demonstrated that both mutations caused impaired Ca(2+)-dependent inactivation of voltage-gated Ca(2+) current. Neither mutant affected voltage-dependent inactivation. CONCLUSION: Our findings implicate impaired Ca(2+)-dependent inactivation in human cardiomyocytes as the plausible mechanism for long QT syndrome associated with 2 novel CaM mutations. The data further expand the spectrum of genotype and phenotype associated with calmodulinopathy.