Background Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite over a decade of investigation. Post-translational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-translational modifications and disease. We recently identified a novel pathway for post-translational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by CaMKII. However, a role for this pathway in cardiac disease has not been evaluated. Methods and Results We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Nav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large animal model of acquired heart disease and in failing human myocardium. Conclusions We identify the mechanism for two human arrhythmia variants that affect Nav1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.

Normal heart rhythm (sinus rhythm) depends on regular activity of the sinoatrial node (SAN), a heterogeneous collection of specialized myocytes in the right atrium. SAN cells, in general, possess a unique electrophysiological profile that promotes spontaneous electrical activity (automaticity). However, while automaticity is required for normal pacemaking, it is not necessarily sufficient. Less appreciated is the importance of the elaborate structure of the SAN complex for proper pacemaker function. Here, we review the important structural features of the SAN with a focus on how these elements help manage a precarious balance between electrical charge generated by the SAN (“source”) and the charge needed to excite the surrounding atrial tissue (“sink”). We also discuss how compromised “source-sink” balance due, for example to fibrosis, may promote SAN dysfunction, characterized by slow and/or asynchronous pacemaker activity and even failure, in the setting of cardiovascular disease (e.g., heart failure, atrial fibrillation). Finally, we discuss implications of the “source-sink” balance in the SAN complex for cell and gene therapies aimed at creating a biological pacemaker as replacement or bridge to conventional electronic pacemakers.

Ankyrin-B is a multifunctional adapter protein responsible for localization and stabilization of select ion channels, transporters, and signaling molecules in excitable cells including cardiomyocytes. Ankyrin-B dysfunction has been linked with highly penetrant sinoatrial node (SAN) dysfunction and increased susceptibility to atrial fibrillation. While previous studies have identified a role for abnormal ion homeostasis in ventricular arrhythmias, the molecular mechanisms responsible for atrial arrhythmias and SAN dysfunction in human patients with ankyrin-B syndrome are unclear. Here, we develop a computational model of ankyrin-B dysfunction in atrial and SAN cells and tissue to determine the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human patients with ankyrin-B syndrome. Our simulations predict that defective membrane targeting of the voltage-gated L-type Ca(2+) channel Cav1.3 leads to action potential shortening that reduces the critical atrial tissue mass needed to sustain reentrant activation. In parallel, increased fibrosis results in conduction slowing that further increases the susceptibility to sustained reentry in the setting of ankyrin-B dysfunction. In SAN cells, loss of Cav1.3 slows spontaneous pacemaking activity, whereas defects in Na(+)/Ca(2+) exchanger and Na(+)/K(+) ATPase increase variability in SAN cell firing. Finally, simulations of the intact SAN reveal a shift in primary pacemaker site, SAN exit block, and even SAN failure in ankyrin-B-deficient tissue. These studies identify the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human disease. Importantly, ankyrin-B dysfunction involves changes at both the cell and tissue levels that favor the common manifestation of atrial arrhythmias and SAN dysfunction.

Normal cardiac excitability depends on the coordinated activity of specific ion channels and transporters within specialized domains at the plasma membrane and sarcoplasmic reticulum. Ion channel dysfunction due to congenital or acquired defects has been linked to human cardiac arrhythmia. More recently, defects in ion channel-associated proteins have been associated with arrhythmia. Ankyrin-B is a multifunctional adapter protein responsible for targeting select ion channels, transporters, cytoskeletal proteins, and signaling molecules in excitable cells, including neurons, pancreatic β-cells, and cardiomyocytes. Ankyrin-B dysfunction has been linked to cardiac arrhythmia in human patients and ankyrin-B heterozygous (ankyrin-B(+/-)) mice with a phenotype characterized by sinus node dysfunction, susceptibility to ventricular arrhythmias, and sudden death ("ankyrin-B syndrome"). At the cellular level, ankyrin-B(+/-) cells have defects in the expression and membrane localization of the Na(+)/Ca(2+) exchanger and Na(+)-K(+)-ATPase, Ca(2+) overload, and frequent afterdepolarizations, which likely serve as triggers for lethal cardiac arrhythmias. Despite knowledge gathered from mouse models and human patients, the molecular mechanism responsible for cardiac arrhythmias in the setting of ankyrin-B dysfunction remains unclear. Here, we use mathematical modeling to provide new insights into the cellular pathways responsible for Ca(2+) overload and afterdepolarizations in ankyrin-B(+/-) cells. We show that the Na(+)/Ca(2+) exchanger and Na(+)-K(+)-ATPase play related, yet distinct, roles in intracellular Ca(2+) accumulation, sarcoplasmic reticulum Ca(2+) overload, and afterdepolarization generation in ankyrin-B(+/-) cells. These findings provide important insights into the molecular mechanisms underlying a human disease and are relevant for acquired human arrhythmia, where ankyrin-B dysfunction has recently been identified.

2Lavrentyev Institute of Hydrodynamics, Novosibirsk, Russian Federation. The study of osmotic and ion balance is of particular importance for transporting epithelial cells that have to maintain stable intracellular medium and cell volume upon intensive transcellular osmolyte and water fluxes. We propose an approach based on mathematical modeling that allows quantitative estimation of the rate of transmembrane ion transport in rat renal collecting duct principal cells. Our goals were: 1) to estimate membrane permeabilities for sodium, potassium and chloride ions; 2) to estimate rates of passive transmembrane fluxes for sodium, potassium and chloride ions and the flux through Na/K-pump; 3) to assess the activity of KCC and NKCC cotransporters in the cells under examination. The mathematical model describing transmembrane ion fluxes was used to analyze the results of experimental measurements of cell volume and intracellular sodium concentration dynamics. The use of high-performance calculations on computer cluster made it possible to obtain the correspondence between the values of model permeability parameters and experimentally measured cell physiological characteristics. Quantitative estimates of permeability parameters and the rates of transmembrane fluxes were obtained for renal collecting duct principal cells. Also the model made it possible to detect the decrease in sodium and potassium membrane permeability of collecting duct cells from animals kept on high salt diet. This approach sets grounds for interpretation of experimental data to estimate cell physiological characteristics whose accurate experimental measurement is a hardly achievable goal. Sodium coupled betaine transporter BetP is a representative member of the BCCT transporter family. Its major function is to accumulate osmolytes in order to prevent cell death upon change in cytoplasmic osmotic pressure. BetP is one of the key models for understanding how the generalized LeuT-fold transporters function, due to the availability of its crystal structure in multiple conformational states. Recent experimental studies have shown that BetP-G153D can transport choline, instead of betaine, under pH gradient across the cellular membrane. Currently no satisfactory explanation exists for the substrate transport mechanism in the context of this dramatic single-site mutation. Aiming to gain better insight into this phenomenon, we have performed several microseconds of molecular dynamics simulations of the BetP-G153D mutant based on an inward-facing crystal structure. The results indicate that the protonation state of the mutation site Asp153 is closely coupled to large-scale gating motion of the entire transporter. Specifically, changing in the protonation state of Asp153 leads to distinct substrate release kinetics via control over conformation fluctuation in transmembrane helix TM6 and rearrangement of interaction network of charged residues at the intracellular side. These results suggest a novel mechanism for substrate transport control in transporters like...

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Background: Normal cardiac excitability depends upon the coordinated activity of ion channels and transporters. Mutations in genes encoding ion channels affecting their biophysical properties have been known for over twenty years as a root cause of potentially fatal human electrical rhythm disturbance (arrhythmias). More recently, defects in ion channel associated protein (e.g. adapter, regulatory, cytoskeletal proteins) have been shown to cause arrhythmia. Mathematical modeling is ideally suited to integrate large volumes of cellular and in vivo data from human patients and animal disease models with the over goal of determining cellular mechanisms for these atypical human cardiac diseases that involve complex defects in ion channel membrane targeting and/or regulation. Methods and Results: Computational models of ventricular, atrial, and sinoatrial cells were used to determine the mechanism for increased susceptibility to arrhythmias and sudden death in human patients with inherited defects in ankyrin-based targeting pathways. The loss of ankyrin-B was first incorporated into detailed models of the ventricular myocyte to identify the cellular mechanism for arrhythmias in human patients with loss-of-function mutations in ANK2 (encodes ankyrin-B). Mathematical modeling was used to identify the cellular pathway responsible for abnormal Ca 2+ handling and cardiac arrhythmias in ventricular cells. A multi-scalar computational model of ankyrin-B deficiency in atrial and sinoatrial cells and tissue was then developed to determine the mechanism for the increased susceptibility to atrial fibrillation in these human patients. Finally, a state-based Markov model of the voltage-gated Na + channel was incorporated into a ventricular cell model and parameter estimation was performed to determine the mechanism for a new class of human arrhythmia variants that confer susceptibility to arrhythmia by interfering with a regulatory complex comprised of a second member of the ankyrin family, ankyrin-G. Conclusions: Ca 2+ accumulation was observed at baseline in the ankyrin-B deficient ventricular model, with pro-arrhythmic And finally to my family, especially Mom and Dad-words cannot describe my gratitude for your constant love. You have always encouraged me to keep going, even when I didn't think I could. You were my first teachers in life and have been with me every step of the way. All of the sacrifices you have made through the years to provide me with the best education have not gone unnoticed. You were the ones to teach me if I worked for it, I will appreciate it much more than if it was given to me. Although I may not have believed it at the time, I know now just how true it is. For all of the countless phone calls, the headaches I may have caused, the food I stole from home, and the many incidentals along the way, thank you. You have asked me so many times and I'm finally able to say after all these years of school, I'm done.