Date of Award
Doctor of Philosophy (PhD)
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.
Yoram Rudy, Stacey Rentschler, Jeanne Nerbonne, Jianmin Cui,