Andrew R. Ednie

Modulation of voltage-gated ion channels by sialylation.

Control and modulation of electrical signaling is vital to normal physiology, particularly in neurons, cardiac myocytes, and skeletal muscle. The orchestrated activities of variable sets of ion channels and transporters, including voltage-gated ion channels (VGICs), are responsible for initiation, conduction, and termination of the action potential (AP) in excitable cells. Slight changes in VGIC activity can lead to severe pathologies including arrhythmias, epilepsies, and paralyses, while normal excitability depends on the precise tuning of the AP waveform. VGICs are heavily posttranslationally modified, with upward of 30% of the mature channel mass consisting of N- and O-glycans. These glycans are terminated typically by negatively charged sialic acid residues that modulate voltage-dependent channel gating directly. The data indicate that sialic acids alter VGIC activity in isoform-specific manners, dependent in part, on the number/location of channel sialic acids attached to the pore-forming alpha and/or auxiliary subunits that often act through saturating electrostatic mechanisms. Additionally, cell-specific regulation of sialylation can affect VGIC gating distinctly. Thus, channel sialylation is likely regulated through two mechanisms that together contribute to a dynamic spectrum of possible gating motifs: a subunit-specific mechanism and regulated (aberrant) changes in the ability of the cell to glycosylate. Recent studies showed that neuronal and cardiac excitability is modulated through regulated changes in voltage-gated Na(+) channel sialylation, suggesting that both mechanisms of differential VGIC sialylation contribute to electrical signaling in the brain and heart. Together, the data provide insight into an important and novel paradigm involved in the control and modulation of electrical signaling.

Sialic Acids Attached to O-Glycans Modulate Voltage-gated Potassium Channel Gating

Neuronal, cardiac, and skeletal muscle action potentials are produced and conducted through the highly regulated activity of several types of voltage-gated ion channels. Voltage-gated potassium (Kv) channels are responsible for action potential repolarization. Glycans can be attached to glycoproteins through N- and O-linkages. Previous reports described the impact of N-glycans on voltage-gated ion channel function. Here, we show that sialic acids attached through O-linkages modulate gating of Kv2.1, Kv4.2, and Kv4.3. The conductance-voltage (G-V) relationships for each isoform were shifted uniquely by a depolarizing 8–16 mV under conditions of reduced sialylation. The data indicate that sialic acids modulate Kv channel activation through apparent electrostatic mechanisms that promote channel activity. Voltage-dependent steady-state inactivation was unaffected by changes in sialylation. N-Linked sialic acids cannot be responsible for the G-V shifts because Kv4.2 and Kv4.3 cannot be N-glycosylated, and immunoblot analysis confirmed Kv2.1 is not N-glycosylated. Glycosidase gel shift analysis suggested that Kv2.1, Kv4.2, and Kv4.3 were O-glycosylated and sialylated. To confirm this, azide-modified sugar residues involved specifically in O-glycan and sialic acid biosynthesis were shown to incorporate into all three Kv channel isoforms using Cu(I)-catalyzed cycloaddition chemistry. Together, the data indicate that sialic acids attached to O-glycans uniquely modulate gating of three Kv channel isoforms that are not N-glycosylated. These data provide the first evidence that external O-glycans, with core structures distinct from N-glycans in type and number of sugar residues, can modulate Kv channel function and thereby contribute to changes in electrical signaling that result from regulated ion channel expression and/or O-glycosylation.

Expression of the sialyltransferase, ST3Gal4, impacts cardiac voltage-gated sodium channel activity, refractory period and ventricular conduction

The sequential glycosylation process typically ends with sialic acid residues added through trans-Golgi sialyltransferase activity. Individuals afflicted with congenital disorders of glycosylation often have reduced glycoprotein sialylation and present with multi-system symptoms including hypotonia, seizures, arrhythmia and cardiomyopathy. Cardiac voltage-gated Na(+) channel (Nav) activity can be influenced by sialic acids likely contributing to an external surface potential causing channels to gate at less depolarized voltages. Here, a possible pathophysiological role for reduced sialylation is investigated by questioning the impact of gene deletion of the uniformly expressed beta-galactoside alpha-2,3-sialyltransferase 4 (ST3Gal4) on cardiac Nav activity, cellular refractory period and ventricular conduction. Whole-cell patch-clamp experiments showed that ventricular Nav from ST3Gal4 deficient mice (ST3Gal4(-/-)) gated at more depolarized potentials, inactivated more slowly and recovered from fast inactivation more rapidly than WT controls. Current-clamp recordings indicated a 20% increase in time to action potential peak and a 30ms decrease in ST3Gal4(-/-) myocyte refractory period, concurrent with increased Nav recovery rate. Nav expression, distribution and maximal Na(+) current levels were unaffected by ST3Gal4 expression, indicating that reduced sialylation does not impact Nav surface expression and distribution. However, enzymatic desialylation suggested that ST3Gal4(-/-) ventricular Nav are less sialylated. Consistent with the shortened myocyte refractory period, epicardial conduction experiments using optical mapping techniques demonstrated a 27% reduction in minimum ventricular refractory period and increased susceptibility to arrhythmias in ST3Gal4(-/-) ventricles. Thus, deletion of a single sialyltransferase significantly impacts ventricular myocyte electrical signaling. These studies offer insight into diseases of glycosylation that are often associated with pathological changes in excitability and highlight the importance of glycosylation in cardiac physiology.

Channel sialic acids limit hERG channel activity during the ventricular action potential.

Activity of human ether‐a‐go‐go‐related gene (hERG) 1 voltage‐gated K+ channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N‐glycosylation modified hERG channel function. Voltage‐dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N‐glycans, and following enzymatic deglycosylation of surface N‐glycans. For each condition of reduced glycosylation, hERG channel steady‐state activation and inactivation relationships were shifted linearly by significant depolarizing ~9 and ~18 mV, respectively. The hERG window current increased significantly by 50‐150%, and the peak shifted by a depolarizing ~10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20–80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50‐70‐ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N‐glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.—Norring, S. A., Ednie, A. R., Schwetz, T. A., Du, D., Yang, H., Bennett, E. S. Channel sialic acids limit hERG channel activity during the ventricular action potential. FASEB J. 27, 622–631 (2013). www.fasebj.org

Statistical Metamodeling and Sequential Design of Computer Experiments to Model Glyco-Altered Gating of Sodium Channels in Cardiac Myocytes

Glycan structures account for up to 35% of the mass of cardiac sodium (Nav) channels. To question whether and how reduced sialylation affects Nav activity and cardiac electrical signaling, we conducted a series of in vitro experiments on ventricular apex myocytes under two different glycosylation conditions, reduced protein sialylation (ST3Gal4-/-) and full glycosylation (control). Although aberrant electrical signaling is observed in reduced sialylation, realizing a better understanding of mechanistic details of pathological variations in INa and AP is difficult without performing in silico studies. However, computer model of Nav channels and cardiac myocytes involves greater levels of complexity, e.g., high-dimensional parameter space, nonlinear and nonconvex equations. Traditional linear and nonlinear optimization methods have encountered many difficulties for model calibration. This paper presents a new statistical metamodeling approach for efficient computer experiments and optimization of Nav models. First, we utilize a fractional factorial design to identify control variables from the large set of model parameters, thereby reducing the dimensionality of parametric space. Further, we develop the Gaussian process model as a surrogate of expensive and time-consuming computer models and then identify the next best design point that yields the maximal probability of improvement. This process iterates until convergence, and the performance is evaluated and validated with real-world experimental data. Experimental results show the proposed algorithm achieves superior performance in modeling the kinetics of Nav channels under a variety of glycosylation conditions. As a result, in silico models provide a better understanding of glyco-altered mechanistic details in state transitions and distributions of Nav channels. Notably, ST3Gal4-/- myocytes are shown to have higher probabilities accumulated in intermediate inactivation during the repolarization and yield a shorter refractory period than WTs. The proposed statistical design of computer experiments is generally extensible to many other disciplines that involve large-scale and computationally expensive models.

Reduced Sialylation Impacts Ventricular Repolarization by Modulating Specific K+ Channel Isoforms Distinctly

Background: Glycosylation results from the coordinated activities of glycogene products that can modulate ion channel function. Results: Gene ablation of the sialyltransferase ST3Gal4 impacts sialylation and gating of specific cardiac voltage-gated K+ channel isoforms and thereby ventricular repolarization. Conclusion: Individual glycogene products serve unique roles in affecting cardiac voltage-gated K+ channel activity. Significance: Protein glycosylation significantly contributes to ventricular electrical signaling. Voltage-gated K+ channels (Kv) are responsible for repolarizing excitable cells and can be heavily glycosylated. Cardiac Kv activity is indispensable where even minimal reductions in function can extend action potential duration, prolong QT intervals, and ultimately contribute to life-threatening arrhythmias. Diseases such as congenital disorders of glycosylation often cause significant cardiac phenotypes that can include arrhythmias. Here we investigated the impact of reduced sialylation on ventricular repolarization through gene deletion of the sialyltransferase ST3Gal4. ST3Gal4-deficient mice (ST3Gal4−/−) had prolonged QT intervals with a concomitant increase in ventricular action potential duration. Ventricular apex myocytes isolated from ST3Gal4−/− mice demonstrated depolarizing shifts in activation gating of the transient outward (Ito) and delayed rectifier (IKslow) components of K+ current with no change in maximum current densities. Consistently, similar protein expression levels of the three Kv isoforms responsible for Ito and IKslow were measured for ST3Gal4−/− versus controls. However, novel non-enzymatic sialic acid labeling indicated a reduction in sialylation of ST3Gal4−/− ventricular Kv4.2 and Kv1.5, which contribute to Ito and IKslow, respectively. Thus, we describe here a novel form of regulating cardiac function through the activities of a specific glycogene product. Namely, reduced ST3Gal4 activity leads to a loss of isoform-specific Kv sialylation and function, thereby limiting Kv activity during the action potential and decreasing repolarization rate, which likely contributes to prolonged ventricular repolarization. These studies elucidate a novel role for individual glycogene products in contributing to a complex network of cardiac regulation under normal and pathologic conditions.

Sialic acids attached to N- and O-glycans within the Nav1.4 D1S5–S6 linker contribute to channel gating

BACKGROUND Voltage-gated Na+ channels (Nav) are responsible for the initiation and conduction of neuronal and muscle action potentials. Nav gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms. METHODS Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Nav1.4 D1S5-S6 linker modulate Nav gating. RESULTS All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data. CONCLUSIONS The data indicate that sialic acids attached to both N- and O-glycans residing within the Nav1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar. GENERAL SIGNIFICANCE Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that environmental, metabolic, and/or congenital changes in glycosylation that impact sugar substrate levels, could lead, potentially, to changes in Nav sialylation and gating that would modulate AP waveforms and conduction.

Reduced myocyte complex N-glycosylation causes dilated cardiomyopathy

Protein glycosylation is an essential posttranslational modification that affects a myriad of physiologic processes. Humans with genetic defects in glycosylation, which result in truncated glycans, often present with significant cardiac deficits. Acquired heart diseases and their associated risk factors were also linked to aberrant glycosylation, highlighting its importance in human cardiac disease. In both cases, the link between causation and corollary remains enigmatic. The glycosyltransferase gene, mannosyl (α‐1,3‐)‐glycoprotein β‐1,2‐N‐acetylglucosaminyltransferase (Mgat1), whose product, N‐acetylglucosaminyltransferase 1 (GlcNAcT1) is necessary for the formation of hybrid and complex N‐glycan structures in the medial Golgi, was shown to be at reduced levels in human end‐stage cardiomyopathy, thus making Mgat1 an attractive target for investigating the role of hybrid/complex N‐glycosylation in cardiac pathogenesis. Here, we created a cardiomyocyte‐specific Mgatl knockout (KO) mouse to establish a model useful in exploring the relationship between hybrid/complex N‐glycosylation and cardiac function and disease. Biochemical and glycomic analyses showed that Mgat1KO cardiomyocytes produce predominately truncated N‐glycan structures. All Mgat1KO mice died significantly younger than control mice and demonstrated chamber dilation and systolic dysfunction resembling human dilated cardiomyopathy (DCM). Data also indicate that a cardiomyocyte L‐type voltage‐gated Ca2+ channel (Cav) subunit (α281) is a GlcNAcT1 target, and Mgat1KO Cav activity is shifted to more‐depolarized membrane potentials. Consistently, Mgat1KO cardiomyocyte Ca2+ handling is altered and contraction is dyssynchronous compared with controls. The data demonstrate that reduced hybrid/complex N‐glycosylation contributes to aberrant cardiac function at whole‐heart and myocyte levels drawing a direct link between altered glycosylation and heart disease. Thus, the Mgat1KO provides a model for investigating the relationship between systemic reductions in glycosylation and cardiac disease, showing that clinically relevant changes in cardiomyocyte hybrid/complex N‐glycosylation are sufficient to cause DCM and early death.—Ednie, A. R., Deng, W., Yip, K.‐P., Bennett, E. S. Reduced myocyte complex N‐glycosylation causes dilated cardiomyopathy. FASEB J. 33, 1248–1261 (2019). www.fasebj.org