Faculty Bibliography

BACKGROUND: Mutations in SCN2B, encoding voltage-gated sodium channel beta2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that beta2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. METHODS AND RESULTS: To understand the pathophysiological roles of beta2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of beta2-subunits in the ventricle is to chaperone voltage-gated sodium channel alpha-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. CONCLUSIONS: Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (Vj) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the alpha- and delta-group connexins possess a G2 residue not found in the beta- and gamma-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Delta) affected the Vj gating, increased the single channel conductance (gammaj), and decreased the relative K(+)/Cl(-) permeability (PK/PCl) ratio of the Cx40 gap junction channel. The conserved alpha/beta-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in Vj gating and noisy low gammaj gap junction channels that reduced the gammaj of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all alpha/beta/delta/gamma-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.

Problem: Intrauterine infections activate a proinflammatory cascade involving cytokines and other mediators that lead to preterm labor. Endotoxin tolerance (ET) is a phenomenon in which exposure to a dose of endotoxin renders tissues less responsive to subsequent exposures. The mechanism underlying ET is not fully understood. To our knowledge, no previous studies have elucidated the role of ET in human placenta. Using placental explants, we examined this phenomenon and whether exosomes play part in it. Method of Study: Placental explants from term and second-trimester pregnancies were cultured and exposed to low dose LPS for three days. Media were collected daily, and the explants were re-exposed to LPS. Cytochalasin-D (inhibitor of exosomes release and uptake) was added with LPS in some groups. TNF-aalpha and IL-10 in placental explants media were determined by ELISA. Exosomes were isolated from media by Total Exosome Isolation Kit, and miRNAs inside exosomes were analyzed by RT-PCR. Results: LPS treatment for 24 hours stimulated the secretion of placental pro-inflammatory cytokines. However, repeated treatment of the placental explants with LPS significantly reduced the subsequent pro-inflammatory effect, indicating ET. The anti-inflammatory cytokine, IL-10, was also induced by LPS; however, its levels were not affected on repeated LPS treatments. Cytochalasin-D treatment resulted in the loss of ET; nevertheless, it did not change IL-10 secretion. We observed that LPS increased exosomes secretion from placental explants. Moreover, miR-146a and others, which negatively modulate inflammatory response, were found higher in the LPS treated exosomes. Taking together, these findings suggest that ET is mediated by exosomes. Conclusion: This study illustrates, for the first time, that LPS induces ET in human placenta, and that exosomes mediate this phenomenon. We speculate that dysregulation of placental exosomes production, and thus tolerance to infection, might be linked to the exaggerated inflammatory response that leads to preterm labor

Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 degrees C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 degrees C but are unexcitable at 40 degrees C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.

Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and 'angle view' scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC NaV1.5. We show that the N-cadherin-NaV1.5 association is not random, that NaV1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of NaV1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex.

Background: This randomized, double-blind phase 3 trial examined the efficacy and safety of SU vs PBO in post-nephrectomy patients ( pts) with locoregional RCC at high risk (per modified UISS criteria) of tumor recurrence. Methods: Treatment-naive pts (n = 615) with locoregional RCC (>T3 and/or N1-2) received 50 mg/d SU or PBO in a 4-wks-on/2-wks-off schedule for 1 yr until disease recurrence, occurrence of secondary malignancy, significant toxicity, or consent withdrawal. One dose reduction to 37.5 mg/d was allowed. Baseline imaging was centrally reviewed to exclude pts with suspicion of metastases. Primary endpoint was disease-free survival (DFS) assessed by central review. Secondary endpoints included investigator-assessed DFS, overall survival (OS), safety, and pt-reported outcomes. Results: Baseline characteristics were balanced between the SU (n = 309) and PBO (n = 306) arms. Median (m) number of cycles and relative dose intensity (SU) were 9 and 88.4%, respectively. Fewer DFS events were seen with SU (113; 36.6%) vs PBO (144; 47.1%). The trial met its primary endpoint: DFS by central review was significantly longer for SU vs PBO (HR, 0.761; P = 0.030), and these results were supported by secondary DFS analyses in all randomized pts and a higher-risk subgroup (Table 1). OS data were immature at data cutoff (mOS not reached in either arm). Grade >3 adverse events (AEs) were more frequent with SU (62.1%) vs PBO (21.1%), but serious AE incidence was similar (21.9% vs 17.1%); no death occurred due to treatment toxicity. (Table Presented) Conclusions: SU prolonged DFS in pts with locoregional RCC at high risk for recurrence. Given the increase in DFS and the manageable safety profile, SU represents a potential new treatment option as adjuvant therapy in RCC

KEY POINTS: Na(+) current (INa ) results from the integrated function of a molecular aggregate (the voltage-gated Na(+) channel complex) that includes the beta subunit family. Mutations or rare variants in Scn1b (encoding the beta1 and beta1B 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 Ca(2+) transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) 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 alpha subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis. ABSTRACT: Na(+) current (INa ) is determined not only by the properties of the pore-forming voltage-gated Na(+) channel (VGSC) alpha subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC beta subunit family. Mutations or rare variants in Scn1b (encoding the beta1 and beta1B 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 NaV 1.3 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 Ca(2+) transients and frequent spontaneous Ca(2+) release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) 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 alpha subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis in ventricular myocytes.