Molecular Basis of Class Ib Drug Interactions with the NaV Channel Macromolecular Complex: A Route to Personalized Medicine for Cardiac Arrhythmia

The heart rhythm is precisely controlled by the electrical impulse that propagate in the cardiac tissue. In single cardiomyocytes, the electrical activity generated by action potentials (AP). Cardiac NaV channels (NaV1.5) carry a large influx of Na+ that mediates the initiation and propagation of the AP in both atria and ventricles. Disruption of NaV1.5 function by genetic variants or external factors can result in deadly arrhythmias, such as long QT syndrome and Brugada syndrome. Thus, NaV channels are import therapeutic targets. The class I antiarrhythmics are the modulators of the NaV channels. Although they have been used clinically for over 100 years, detailed mechanisms of their action are not well understood. The NaV channel co-assembles with many regulatory and accessory proteins to form a macromolecular complex that tailor channel function to different cells. The complicated multi-molecular interactions add another level of complexity in dissecting the drug mechanisms. The pore-forming NaV1.5 α-subunit contains four domains (DI-DIV), each with a voltage sensing domain (VSD). The voltage clamp fluorometry (VCF) method probes the conformational changes of each VSD by attaching a fluorophore on it. Here, we utilized VCF to measure how the accessary β-subunits and Class Ib antiarrhythmics affect the conformational dynamics of the NaV1.5. We found that the non-covalently linked β1 and β3 subunits regulate channel gating by altering the DIII and DIV-VSD dynamics. Moreover, results from multiple experiments provided compelling evidence that β1 and β3 bind proximally to the DIII-VSD. The DIII-VSD also plays an important role in channel’s interaction with Class Ib antiarrhythmics, such as lidocaine, ranolazine and mexiletine. Recent clinical studies showed that mexiletine is effective in treating patients with LQT3 syndrome. However, the patient response is variable, depending on the genetic mutation in NaV 1.5. We showed that mexiletine altered the conformation of the DIII-VSD, which is the same VSD that many tested LQT3 mutations affect. Analysis of 15 LQT3 variants showed a strong correlation between the activation of the DIII-VSD and the strength of the inhibition of the channel by mexiletine. Based on this improved molecular-level understanding, we generated a systems-based model that successfully predicted the response of 7 out of 8 patients to mexiletine in a blinded, retrospective clinical trial. The new model can be used to personalize treatment for LQT3 patients, and improving therapeutic decision making. As the non-covalently linked β subunits and the Class Ib antiarrhythmics both interact with the same part of the NaV channel. We further investigated how β expression affects the Class Ib drug effectiveness. We found that β1 differentially modulates lidocaine and ranolazine blockade of NaV1.5. The molecular mechanism underlying this phenomenon is due to altered drug interaction with the DIII-VSD. In human hearts, β1 expresses at levels that are 3-fold higher in the atria compared to ventricles. Thus, this molecular difference can be targeted to develop chamber specific antiarrhythmic therapies. In conclusion, we demonstrated the essential role of the DIII-VSD dynamics in modulating NaV channel response to the Class Ib antiarrhythmics. This molecular interaction is regulated by the accessary β subunits. We hope to apply this mechanistic insight to improve current antiarrhythmic therapeutic approaches

A molecularly detailed Na V 1.5 model reveals a new Class I antiarrhythmic target

Antiarrhythmic treatment strategies remain suboptimal due to our inability to predict how drug interactions with ion channels will affect the ability of the tissues to initiate and sustain an arrhythmia. We built a multiscale molecular model of the N

Modulation of the effects of class Ib antiarrhythmics on cardiac NaV1.5-encoded channels by accessory NaVβ subunits

Native myocardial voltage-gated sodium (NaV) channels function in macromolecular complexes comprising a pore-forming (α) subunit and multiple accessory proteins. Here, we investigated the impact of accessory NaVβ1 and NaVβ3 subunits on the functional effects of 2 well-known class Ib antiarrhythmics, lidocaine and ranolazine, on the predominant NaV channel α subunit, NaV1.5, expressed in the mammalian heart. We showed that both drugs stabilized the activated conformation of the voltage sensor of domain-III (DIII-VSD) in NaV1.5. In the presence of NaVβ1, the effect of lidocaine on the DIII-VSD was enhanced, whereas the effect of ranolazine was abolished. Mutating the main class Ib drug-binding site, F1760, affected but did not abolish the modulation of drug block by NaVβ1/β3. Recordings from adult mouse ventricular myocytes demonstrated that loss of Scn1b (NaVβ1) differentially affected the potencies of lidocaine and ranolazine. In vivo experiments revealed distinct ECG responses to i.p. injection of ranolazine or lidocaine in WT and Scn1b-null animals, suggesting that NaVβ1 modulated drug responses at the whole-heart level. In the human heart, we found that SCN1B transcript expression was 3 times higher in the atria than ventricles, differences that could, in combination with inherited or acquired cardiovascular disease, dramatically affect patient response to class Ib antiarrhythmic therapies

Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.

BACKGROUND -Dysregulation of voltage-gated cardiac Na(+) channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na(+) current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the four (DI-DIV) VSDs to define molecular mechanisms of NaV1.5 modulation. METHOD AND RESULTS -Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry (VCF). Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of two Brugada Syndrome (BrS) mutations (A735V and G752R). A735V shifted DII-VSD voltage-dependence to depolarized potentials, while G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, even though DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate of INa activation and myocyte excitability. CONCLUSIONS -Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate its activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal BrS mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications and anti-arrhythmic drugs alter NaV1.5 at the molecular level

Gating control of the cardiac sodium channel Nav1.5 by its β3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

The auxiliary β3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some β3 mutations predispose individuals to cardiac arrhythmias. The β3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the β3 transmembrane region and its potential for functionally synergizing with the β3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT β3-subunit and in a β3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed β3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT β3-subunit. Removal of the β3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the β3-Glu-176 residue and the β3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation

Enhanced Immune Responses Conferring Cross-Protection by Skin Vaccination With a Tri-Component Influenza Vaccine Using a Microneedle Patch

Skin vaccination using biodegradable microneedle patch (MNP) technology in vaccine delivery is a promising strategy showing significant advantages over conventional flu shots. In this study, we developed an MNP encapsulating a 4M2e-tFliC fusion protein and two types of whole inactivated influenza virus vaccines (H1N1 and H3N2) as a universal vaccine candidate. We demonstrated that mice receiving this tri-component influenza vaccine via MNP acquired improved IgG1 antibody responses with more balanced IgG1/IgG2a antibody responses and enhanced cellular immune responses, including increased populations of IL-4 and IFN-γ producing cells and higher frequencies of antigen-specific plasma cells compared with intramuscular injection. In addition, stronger germinal center reactions, increased numbers of Langerin-positive migratory dendritic cells, and increased cytokine secretion were observed in the skin-draining lymph nodes after immunization with the tri-component influenza MNP vaccine. The MNP-immunized group also possessed enhanced protection against a heterologous reassortant A/Shanghai/2013 H7N9 (rSH) influenza virus infection. Furthermore, the sera collected from 4M2e-tFliC MNP-immunized mice were demonstrated to have antiviral efficacy against reassortant A/Vietnam/1203/2004 H5N1 (rVet) and A/Shanghai/2013 H7N9 (rSH) virus challenges. The immunological advantages of skin vaccination with this tri-component MNP vaccine could offer a promising approach to develop an easily applicable and broadly protective universal influenza vaccine

Over 18% ternary polymer solar cells enabled by a terpolymer as the third component

“Ternary blending” and “random terpolymerization” strategies have both proven effective for enhancing the performance of organic solar cells (OSCs). However, reports on the combination of the two strategies remain rare. Here, a terpolymer PM6-Si30 was constructed by inserting chlorine and alkylsilyl-substituted benzodithiophene (BDT) unit (0.3 equivalent) into the state-of-the-art polymer PM6. The terpolymer exhibitsadeep highest-occupied-molecular-orbital energy and good miscibility with both PM6 and BTP-eC9 (C9) and enables its use as a third component into PM6:PM6-Si30:C9 bulk-heterojunction for OSCs. The resulting cells exhibit maximum power conversion efficiency (PCE) of 18.27%, which is higher than that obtained for the optimized control binary PM6:C9-based OSC (17.38%). The enhanced performance of the PM6:PM6-Si30:C9 cells is attributed to improved charge transport, favorable molecular arrangement, reduced energy loss and suppressed bimolecular recombination. The work demonstrates the potential of random terpolymer as a third component in OSCs and highlights a new strategy for the construction of a ternary system with improved photovoltaic performance

Regulation of Na(+) channel inactivation by the DIII and DIV voltage-sensing domains.

Functional eukaryotic voltage-gated Na(+) (NaV) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery

Towards Trustworthy Artificial Intelligence for Equitable Global Health

Artificial intelligence (AI) can potentially transform global health, but algorithmic bias can exacerbate social inequities and disparity. Trustworthy AI entails the intentional design to ensure equity and mitigate potential biases. To advance trustworthy AI in global health, we convened a workshop on Fairness in Machine Intelligence for Global Health (FairMI4GH). The event brought together a global mix of experts from various disciplines, community health practitioners, policymakers, and more. Topics covered included managing AI bias in socio-technical systems, AI's potential impacts on global health, and balancing data privacy with transparency. Panel discussions examined the cultural, political, and ethical dimensions of AI in global health. FairMI4GH aimed to stimulate dialogue, facilitate knowledge transfer, and spark innovative solutions. Drawing from NIST's AI Risk Management Framework, it provided suggestions for handling AI risks and biases. The need to mitigate data biases from the research design stage, adopt a human-centered approach, and advocate for AI transparency was recognized. Challenges such as updating legal frameworks, managing cross-border data sharing, and motivating developers to reduce bias were acknowledged. The event emphasized the necessity of diverse viewpoints and multi-dimensional dialogue for creating a fair and ethical AI framework for equitable global health.Comment: 7 page

Mechanisms of noncovalent β subunit regulation of NaV channel gating

Voltage-gated Na(+) (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI-DIV), each with a VSD. Our results show that β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability