SARS-CoV-2 Infection Induces Ferroptosis of Sinoatrial Node Pacemaker Cells.

BACKGROUND: Increasing evidence suggests that cardiac arrhythmias are frequent clinical features of coronavirus disease 2019 (COVID-19). Sinus node damage may lead to bradycardia. However, it is challenging to explore human sinoatrial node (SAN) pathophysiology due to difficulty in isolating and culturing human SAN cells. Embryonic stem cells (ESCs) can be a source to derive human SAN-like pacemaker cells for disease modeling. METHODS: We used both a hamster model and human ESC (hESC)-derived SAN-like pacemaker cells to explore the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the pacemaker cells of the heart. In the hamster model, quantitative real-time polymerase chain reaction and immunostaining were used to detect viral RNA and protein, respectively. We then created a dual knock-in SHOX2:GFP;MYH6:mCherry hESC reporter line to establish a highly efficient strategy to derive functional human SAN-like pacemaker cells, which was further characterized by single-cell RNA sequencing. Following exposure to SARS-CoV-2, quantitative real-time polymerase chain reaction, immunostaining, and RNA sequencing were used to confirm infection and determine the host response of hESC-SAN-like pacemaker cells. Finally, a high content chemical screen was performed to identify drugs that can inhibit SARS-CoV-2 infection, and block SARS-CoV-2-induced ferroptosis. RESULTS: Viral RNA and spike protein were detected in SAN cells in the hearts of infected hamsters. We established an efficient strategy to derive from hESCs functional human SAN-like pacemaker cells, which express pacemaker markers and display SAN-like action potentials. Furthermore, SARS-CoV-2 infection causes dysfunction of human SAN-like pacemaker cells and induces ferroptosis. Two drug candidates, deferoxamine and imatinib, were identified from the high content screen, able to block SARS-CoV-2 infection and infection-associated ferroptosis. CONCLUSIONS: Using a hamster model, we showed that primary pacemaker cells in the heart can be infected by SARS-CoV-2. Infection of hESC-derived functional SAN-like pacemaker cells demonstrates ferroptosis as a potential mechanism for causing cardiac arrhythmias in patients with COVID-19. Finally, we identified candidate drugs that can protect the SAN cells from SARS-CoV-2 infection.

Knockout of the X-linked Fgf13 in the hypothalamic paraventricular nucleus impairs sympathetic output to brown fat and causes obesity.

Fibroblast growth factor (FGF)13, a nonsecreted, X-linked, FGF homologous factor, is differentially expressed in adipocytes in response to diet, yet Fgf13's role in metabolism has not been explored. Heterozygous Fgf13 knockouts fed normal chow and housed at 22°C showed hyperactivity accompanying reduced core temperature and obesity when housed at 30°C. Those heterozygous knockouts showed defects in thermogenesis even at 30°C and an inability to protect core temperature. Surprisingly, we detected trivial FGF13 in adipose of wild-type mice fed normal chow and no obesity in adipose-specific heterozygous knockouts housed at 30°C, and we detected an intact brown fat response through exogenous ß3 agonist stimulation, suggesting a defect in sympathetic drive to brown adipose tissue. In contrast, hypothalamic-specific ablation of Fgf13 recapitulated weight gain at 30°C. Norepinephrine turnover in brown fat was reduced at both housing temperatures. Thus, our data suggest that impaired CNS regulation of sympathetic activation of brown fat underlies obesity and thermogenesis in Fgf13 heterozygous knockouts fed normal chow.-Sinden, D. S., Holman, C. D., Bare, C. J., Sun, X., Gade, A. R., Cohen, D. E., Pitt, G. S. Knockout of the X-linked Fgf13 in the hypothalamic paraventricular nucleus impairs sympathetic output to brown fat and causes obesity.

Enhanced Sensitivity of α3ß4 Nicotinic Receptors in Enteric Neurons after Long-Term Morphine: Implication for Opioid-Induced Constipation.

Opioid-induced constipation is a major side effect that persists with long-term opioid use. Previous studies demonstrated that nicotine-induced contractions are enhanced after long-term morphine exposure in guinea pig ileum. In the present study, we examined whether the increased sensitivity to nicotine could be observed in single enteric neurons after long-term morphine exposure, determined the subunits in mouse enteric neurons, and examined the effect of nicotine in reversing opioid-induced constipation. Nicotine (0.03-1 mM) dose-dependently induced inward currents from a holding potential of -60 mV in isolated single enteric neurons from the mouse ileum. The amplitude of the currents, but not the potency to nicotine, was significantly increased in neurons receiving long-term (16-24 h) but not short-term (10 min) exposure to morphine. Quantitative mRNA analysis showed that nicotinic acetylcholine receptor (nAChR) subunit expression in the mouse ileum was α3 ≥ ß2 > ß4 > α5 > α4 > ß3 > α6. Nicotine-induced currents were obtained in neurons from α7, ß2, α5, and α6 knockout mice. The currents were, however, inhibited by mecamylamine (10 µM) and the α3ß4 blocker α-conotoxin AuIB (3 µM), suggesting that nicotine-induced currents were mediated by the α3ß4 subtype of nAChRs on enteric neurons. Conversely, NS3861, a partial agonist at α3ß4 nAChR, enhanced fecal pellet expulsion in a dose-dependent manner in mice that received long-term, but not short-term, morphine treatment. Overall, our findings suggest that the efficacy of nAChR agonists on enteric neurons is enhanced after long-term morphine exposure, and activation of the α3ß4 subtype of nAChR reverses chronic, but not acute, morphine-induced constipation.

Interneuron FGF13 regulates seizure susceptibility via a sodium channel-independent mechanism.

Developmental and Epileptic Encephalopathies (DEEs), a class of devastating neurological disorders characterized by recurrent seizures and exacerbated by disruptions to excitatory/inhibitory balance in the brain, are commonly caused by mutations in ion channels. Disruption of, or variants in, FGF13 were implicated as causal for a set of DEEs, but the underlying mechanisms were clouded because FGF13 is expressed in both excitatory and inhibitory neurons, FGF13 undergoes extensive alternative splicing producing multiple isoforms with distinct functions, and the overall roles of FGF13 in neurons are incompletely cataloged. To overcome these challenges, we generated a set of novel cell type-specific conditional knockout mice. Interneuron-targeted deletion of Fgf13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while excitatory neuron-targeted deletion of Fgf13 caused no detectable seizures and no survival deficits. While best studied as a voltage-gated sodium channel (Nav) regulator, we observed no effect of Fgf13 ablation in interneurons on Navs but rather a marked reduction in K+ channel currents. Re-expressing different Fgf13 splice isoforms could partially rescue deficits in interneuron excitability and restore K+ channel current amplitude. These results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures and expand our understanding of FGF13 functions in different neuron subsets.

Hydrogen sulfide as an allosteric modulator of ATP-sensitive potassium channels in colonic inflammation.

The ATP-sensitive potassium channel (K(ATP)) in mouse colonic smooth muscle cell is a complex containing a pore-forming subunit (Kir6.1) and a sulfonylurea receptor subunit (SUR2B). These channels contribute to the cellular excitability of smooth muscle cells and hence regulate the motility patterns in the colon. Whole-cell voltage-clamp techniques were used to study the alterations in K(ATP) channels in smooth muscle cells in experimental colitis. Colonic inflammation was induced in BALB/C mice after intracolonic administration of trinitrobenzene sulfonic acid. K(ATP) currents were measured at a holding potential of -60 mV in high K(+) external solution. The concentration response to levcromakalim (LEVC), a K(ATP) channel opener, was significantly shifted to the left in the inflamed smooth-muscle cells. Both the potency and maximal currents induced by LEVC were enhanced in inflammation. The EC(50) values in control were 6259 nM (n = 10) and 422 nM (n = 8) in inflamed colon, and the maximal currents were 9.9 ± 0.71 pA/pF (60 µM) in control and 39.7 ± 8.8 pA/pF (3 µM) after inflammation. As was seen with LEVC, the potency and efficacy of sodium hydrogen sulfide (NaHS) (10-1000 µM) on K(ATP) currents were significantly greater in inflamed colon compared with controls. In control cells, pretreatment with 100 µM NaHS shifted the EC(50) for LEV-induced currents from 2838 (n = 6) to 154 (n = 8) nM. Sulfhydration of sulfonylurea receptor 2B (SUR2B) was induced by NaHS and colonic inflammation. These data suggest that sulfhydration of SUR2B induces allosteric modulation of K(ATP) currents in colonic inflammation.

Opioid-induced hypernociception is associated with hyperexcitability and altered tetrodotoxin-resistant Na+ channel function of dorsal root ganglia.

Opiates are potent analgesics for moderate to severe pain. Paradoxically, patients under chronic opiates have reported hypernociception, the mechanisms of which are unknown. Using standard patch-clamp technique, we examined the excitability, biophysical properties of tetrodotoxin-resistant (TTX-R) Na(+) and transient receptor potential vanilloid 1 (TRPV1) channels of dorsal root ganglia neurons (DRG) (L(5)-S(1)) from mice pelleted with morphine (75 mg) or placebo (7 days). Hypernociception was confirmed by acetic acid-writhing test following 7-day morphine. Chronic morphine enhanced the neuronal excitability, since the rheobase for action potential (AP) firing was significantly (P < 0.01) lower (38 ± 7 vs. 100 ± 15 pA) while the number of APs at 2× rheobase was higher (4.4 ± 0.8 vs. 2 ± 0.5) than placebo (n = 13-20). The potential of half-maximum activation (V(1/2)) of TTX-R Na(+) currents was shifted to more hyperpolarized potential in the chronic morphine group (-37 ± 1 mV) vs. placebo (-28 ± 1 mV) without altering the V(1/2) of inactivation (-41 ± 1 vs. -33 ± 1 mV) (n = 8-11). Recovery rate from inactivation of TTX-R Na(+) channels or the mRNA level of any Na(+) channel subtypes did not change after chronic morphine. Also, chronic morphine significantly (P < 0.05) enhanced the magnitude of TRPV1 currents (-64 ± 11 pA/pF) vs. placebo (-18 ± 6 pA/pF). The increased excitability of sensory neurons by chronic morphine may be due to the shift in the voltage threshold of activation of TTX-R Na(+) currents. Enhanced TRPV1 currents may have a complementary effect, with TTX-R Na(+) currents on opiate-induced hyperexcitability of sensory neurons causing hypernociception. In conclusion, chronic morphine-induced hypernociception is associated with hyperexcitability and functional remodeling of TTX-R Na(+) and TRPV1 channels of sensory neurons.

Chronic Morphine Induces IL-18 in Ileum Myenteric Plexus Neurons Through Mu-opioid Receptor Activation in Cholinergic and VIPergic Neurons.

The gastrointestinal epithelium is critical for maintaining a symbiotic relationship with commensal microbiota. Chronic morphine exposure can compromise the gut epithelial barrier in mice and lead to dysbiosis. Recently, studies have implicated morphine-induced dysbiosis in the mechanism of antinociceptive tolerance and reward, suggesting the presence of a gut-brain axis in the pharmacological effects of morphine. However, the mechanism(s) underlying morphine-induced changes in the gut microbiome remains unclear. The pro-inflammatory cytokine, Interleukin-18 (IL-18), released by enteric neurons can modulate gut barrier function. Therefore, in the present study we investigated the effect of morphine on IL-18 expression in the mouse ileum. We observed that chronic morphine exposure in vivo induces IL-18 expression in the ileum myenteric plexus that is attenuated by naloxone. Given that mu-opioid receptors (MORs) are mainly expressed in enteric neurons, we also characterized morphine effects on the excitability of cholinergic (excitatory) and vasoactive intestinal peptide (VIP)-expressing (inhibitory) myenteric neurons. We found fundamental differences in the electrical properties of cholinergic and VIP neurons such that VIP neurons are more excitable than cholinergic neurons. Furthermore, MORs were primarily expressed in cholinergic neurons, although a subset of VIP neurons also expressed MORs and responded to morphine in electrophysiology experiments. In conclusion, these data show that morphine increases IL-18 in ileum myenteric plexus neurons via activation of MORs in a subset of cholinergic and VIP neurons. Thus, understanding the neurochemistry and electrophysiology of MOR-expressing enteric neurons can help to delineate mechanisms by which morphine perturbs the gut barrier.

An interaction between the III-IV linker and CTD in NaV1.5 confers regulation of inactivation by CaM and FHF.

Voltage gated sodium channel (VGSC) activation drives the action potential upstroke in cardiac myocytes, skeletal muscles, and neurons. After opening, VGSCs rapidly enter a non-conducting, inactivated state. Impaired inactivation causes persistent inward current and underlies cardiac arrhythmias. VGSC auxiliary proteins calmodulin (CaM) and fibroblast growth factor homologous factors (FHFs) bind to the channel's C-terminal domain (CTD) and limit pathogenic persistent currents. The structural details and mechanisms mediating these effects are not clear. Building on recently published cryo-EM structures, we show that CaM and FHF limit persistent currents in the cardiac NaV1.5 VGSC by stabilizing an interaction between the channel's CTD and III-IV linker region. Perturbation of this intramolecular interaction increases persistent current and shifts the voltage dependence of steady-state inactivation. Interestingly, the NaV1.5 residues involved in the interaction are sites mutated in the arrhythmogenic long QT3 syndrome (LQT3). Along with electrophysiological investigations of this interaction, we present structural models that suggest how CaM and FHF stabilize the interaction and thereby limit the persistent current. The critical residues at the interaction site are conserved among VGSC isoforms, and subtle substitutions provide an explanation for differences in inactivation among the isoforms.