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.

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.

Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a "leaky" gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long-term morphine treatment () but not upon direct exposure of glia to morphine (). LPS, a major bacterial product, also increased ATP-induced currents, as well as expression of P2X4, P2X7, IL6, IL-1β mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS-induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine-induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine-related inflammation and constipation.-Bhave, S., Gade, A., Kang, M., Hauser, K. F., Dewey, W. L., Akbarali, H. I. Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

Ross GR, Gade AR, Dewey WL, Akbarali HI. Opioid-induced hypernociception is associated with hyperexcitability and altered tetrodotoxin-resistant Na ϩ channel function of dorsal root ganglia. Am J Physiol Cell Physiol 302: C1152-C1161, 2012. First published December 21, 2011; doi:10.1152/ajpcell.00171.2011Opiates 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 tetrodotoxinresistant (TTX-R) Na ϩ and transient receptor potential vanilloid 1 (TRPV1) channels of dorsal root ganglia neurons (DRG) (L5-S1) 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 Ͻ 0.01) lower (38 Ϯ 7 vs. 100 Ϯ 15 pA) while the number of APs at 2ϫ rheobase was higher (4.4 Ϯ 0.8 vs. 2 Ϯ 0.5) than placebo (n ϭ 13-20). The potential of half-maximum activation (V1/2) of TTX-R Na ϩ currents was shifted to more hyperpolarized potential in the chronic morphine group (Ϫ37 Ϯ 1 mV) vs. placebo (Ϫ28 Ϯ 1 mV) without altering the V1/2 of inactivation (Ϫ41 Ϯ 1 vs. Ϫ33 Ϯ 1 mV) (n ϭ 8 -11). Recovery rate from inactivation of TTX-R Na ϩ channels or the mRNA level of any Na ϩ channel subtypes did not change after chronic morphine. Also, chronic morphine significantly (P Ͻ 0.05) enhanced the magnitude of TRPV1 currents (Ϫ64 Ϯ 11 pA/pF) vs. placebo (Ϫ18 Ϯ 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 ϩ currents. Enhanced TRPV1 currents may have a complementary effect, with TTX-R Na ϩ 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 ϩ and TRPV1 channels of sensory neurons.morphine hyperalgesia pain transient receptor potential vanilloid 1 channels CLINICAL MANAGEMENT OF PAIN proceeds in a stepwise fashion, based on pain intensity and duration as depicted in the analgesic ladder generated by the World Health Organization (60). Typically, narcotic opiate regimens, including morphine, are used to treat moderate to severe pain. Paradoxically, patients undergoing chronic opiate therapy have reported abnormal pain, even in regions away from the initial site of pain (4,26,27,49). In addition to tolerance development to the analgesic effects of opiates being a major impediment to pain therapy, chronic opiate-induced hypernociception has become a critical problem. This phenomenon of opiate-induced hypernociception is also demonstrated in several experimental animal models such as thermal and tactile nociception in mice (56) as well as in a rat model of narcotic bowel syndro...