Scn1b deletion leads to increased tetrodotoxin-sensitive sodium current altered intracellular calcium homeostasis and arrhythmias in murine hearts

摘要

Na⁺ current (INa) is determined not only by the properties of the pore-forming voltage-gated Na⁺ channel (VGSC) α subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive INa but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive NaV1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca²⁺ transients and frequent spontaneous Ca²⁺ release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca²⁺ homeostasis were prevented by 100 nm tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na⁺ channel α subunit, can be partly consequent to disrupted intracellular Ca²⁺ homeostasis in ventricular myocytes.Key points class="unordered" style="list-style-type:disc"> Na⁺ current (INa) results from the integrated function of a molecular aggregate (the voltage-gated Na⁺ channel complex) that includes the β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin-sensitive INa, delayed after-depolarizations, triggered beats, delayed Ca²⁺ transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca²⁺ homeostasis were prevented by 100 nm tetrodotoxin. We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na⁺ channel α subunit, can be partly consequent to disrupted intracellular Ca²⁺ homeostasis. class="head no_bottom_margin" id="__sec2title">IntroductionThe rapid entry of Na⁺ into ventricular myocytes is a key determinant of action potential propagation. This Na⁺ current (INa) is determined not only by the properties of the pore-forming voltage-gated Na⁺ channel (VGSC) α subunit (mostly NaV1.5 in the adult ventricle), but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC β subunit family. Mutations or rare variants in SCN1B (encoding the β1 and β1B subunits) have been associated with some cases of long QT syndrome and of Brugada syndrome (Hu et al. ; Riuro et al. ). Patino et al. () were first to demonstrate that inheriting two loss-of-function SCN1B alleles causes Dravet syndrome (DS), a devastating paediatric epileptic encephalopathy in humans. Patients with DS exhibit developmental delay and/or regression during early childhood and frequent pharmacoresistant seizures (Guerrini & Aicardi, ; Dravet et al. ; Dravet, ; Genton et al. ; Oakley et al. ). A high percentage of patients with DS die during early childhood or adolescence, the majority due to SUDEP (Guerrini & Aicardi, ; Dravet et al. ; Dravet, ; Genton et al. ; Oakley et al. ; Sakauchi et al. ), defined as sudden, unexpected, witnessed or unwitnessed, non-traumatic and non-drowning death in patients with epilepsy (Hirsch et al. ), excluding cases of documented status epilepticus. In the most widely used definition, death may occur with or without evidence of a seizure, and post-mortem examination does not reveal a toxicological or anatomical cause of death. SUDEP accounts for 7.5–17% of all deaths in epilepsy (Schuele et al. ; Shorvon & Tomson, href="#b56" rid="b56" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699179">2011). Indirect evidence variably links SUDEP to seizure-induced apnoea, pulmonary oedema, dysregulation of cerebral circulation, autonomic dysfunction and cardiac arrhythmias (Schuele et al. href="#b53" rid="b53" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699112">2007a,bhref="#b54" rid="b54" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699165">2007b; Surges et al. href="#b58" rid="b58" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699180">2009, href="#b57" rid="b57" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699182">2010; Shorvon & Tomson, href="#b56" rid="b56" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699197">2011). Arrhythmias may be primary or may occur secondary to hormonal or metabolic changes, or autonomic discharges (Schuele et al. href="#b53" rid="b53" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699118">2007a,bhref="#b54" rid="b54" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699203">2007b; Goldman et al. href="#b19" rid="b19" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_471311270">2009; Surges et al. href="#b58" rid="b58" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699193">2009, href="#b57" rid="b57" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699124">2010).Over the past decade, the Scn1b null mouse model has been used to understand the molecular substrate underlying DS, and its possible relation to SUDEP (Chen et al. href="#b9" rid="b9" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699208">2004; Brackenbury et al. href="#b4" rid="b4" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699205">2013). These mice developed severe seizures of multiple aetiologies at approximately postnatal day (P)10, and died by ∼P21. Similar to heterozygous SCN1A-R1407X knockin mice, expressing a human DS mutation (Auerbach et al. href="#b2" rid="b2" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699177">2013), Scn1b null ventricular cardiac myocytes (CMs) had a 1.6-fold increase in transient and persistent INa density, as well as action potential prolongation. Electrocardiograms of Scn1b null mice showed increased QTc intervals (Lopez-Santiago et al. href="#b31" rid="b31" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699123">2007). In spite of the observation that ³H-saxitoxin binding in Scn1b null cardiac membrane preparations was increased approximately two-fold, suggesting an increase in tetrodotoxin (TTX)-sensitive (TTX-S) VGSC expression (Lopez-Santiago et al. href="#b31" rid="b31" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699190">2007), the molecular identity and subcellular location of the VGSC α subunits responsible for increased INa density, and the likelihood of arrhythmia events in these hearts were not defined. Furthermore, due to SUDEP in these mice (Chen et al. href="#b9" rid="b9" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_438699185">2004), these studies were limited to juvenile animals and the phenotype was confounded by the concurrent occurrence of severe seizures consequent to Scn1b deletion in the brain.Here, we have expanded previous studies on juvenile animals and circumvented the limitations consequent to early mortality by developing a new genetically engineered murine model with cardiac-specific Scn1b deletion. Using a combination of macropatch and scanning ion conductance microscopy (SICM) we show that loss of Scn1b in juvenile null animals results in an increase in the TTX-S INa but only in the midsection of the cell, even before full T-tubule formation; the latter occurred concurrent with an increase in message abundance for the neuronal Scn3a mRNA, suggesting an increased abundance of TTX-S NaV1.3 protein and yet its preferential exclusion from the region of the intercalated disc (ID). Ventricular myocytes from cardiac-specific adult Scn1b null animals showed a trend toward increased TTX-S INa specifically at the T-tubules. Interestingly, this was associated with increased Scn3a message, prolonged action potential repolarization, the presence of delayed after-depolarizations (DADs), disruption of intracellular Ca²⁺ homeostasis, and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Overall, our results indicate that loss of Scn1b in the heart leads to increased expression of Scn3a and increased TTX-S INa, as well as to altered Ca²⁺ regulation and a proarrhythmic substrate. We postulate that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na⁺ channel α subunit, can be partly consequent to disrupted intracellular Ca²⁺ homeostasis in ventricular myocytes.