Voltage-gated sodium channels (Na v ) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Na v are composed of a pore-forming ␣ subunit and often one to several modulating ␤ subunits. Previous work showed that terminal sialic acid residues attached to ␣ subunits affect channel gating. Here we show that the fully sialylated ␤ 1 subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal ␣ subunit isoforms. Under conditions of reduced sialylation, the ␤ 1 -induced gating effect was eliminated. Consistent with this, mutation of ␤ 1 N-glycosylation sites abolished all effects of ␤ 1 on channel gating. Data also suggest an interaction between the cis effect of ␣ sialic acids and the trans effect of ␤ 1 sialic acids on channel gating. Thus, ␤ 1 sialic acids had no effect on the gating of the heavily glycosylated skeletal muscle ␣ subunit. However, when glycosylation of the skeletal muscle ␣ subunit was reduced through chimeragenesis such that ␣ sialic acids did not impact gating, ␤ 1 sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that ␤ 1 N-linked sialic acids can modulate Na v gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Na v can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.

Millions afflicted with Chagas disease and other disorders of aberrant glycosylation suffer symptoms consistent with altered electrical signaling such as arrhythmias, decreased neuronal conduction velocity, and hyporeflexia. Cardiac, neuronal, and muscle electrical signaling is controlled and modulated by changes in voltage-gated ion channel activity that occur through physiological and pathological processes such as development, epilepsy, and cardiomyopathy. Glycans attached to ion channels alter channel activity through isoform-specific mechanisms. Here we show that regulated and aberrant glycosylation modulate cardiac ion channel activity and electrical signaling through a cell-specific mechanism. Data show that nearly half of 239 glycosylation-associated genes (glycogenes) were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. The N-glycan structures produced among cardiomyocyte types were markedly variable. Thus, the cardiac glycome, defined as the complete set of glycan structures produced in the heart, is remodeled. One glycogene, ST8sia2, a polysialyltransferase, is expressed only in the neonatal atrium. Cardiomyocyte electrical signaling was compared in control and ST8sia2 (؊/؊) neonatal atrial and ventricular myocytes. Action potential waveforms and gating of less sialylated voltage-gated Na ؉ channels were altered consistently in ST8sia2 (؊/؊) atrial myocytes. ST8sia2 expression had no effect on ventricular myocyte excitability. Thus, the regulated (between atrium and ventricle) and aberrant (knockout in the neonatal atrium) expression of a single glycogene was sufficient to modulate cardiomyocyte excitability. A mechanism is described by which cardiac function is controlled and modulated through physiological and pathological processes that involve regulated and aberrant glycosylation. action potentials ͉ cardiomyocyte ͉ glycomics ͉ ion channels ͉ sialic acids

Cell surfaces are replete with complex, biologically important glycan structures responsible for multiple cellular functions including cell adhesion and cellular communication. Proper protein glycosylation is essential for normal development and physiology with the effects of improper glycosylation being severe and even lethal. Gene products involved in glycan formation (glycosylation-associated genes) comprise 1–2% of the genome, resulting in a glycome of thousands of structures; possibly more extensive than the proteome. Using GeneChip microarray analyses, we provide evidence that glycosylation-associated gene expression is highly regulated throughout the developing myocardium. Nearly one-third of the glycosylation-associated genes were significantly differentially expressed in neonatal atria versus neonatal ventricles while 46.8% of these same genes were differentially expressed in neonate versus adult ventricles. Quantitative-PCR of individual genes confirmed the microarray analyses. Such gross glycosylation-associated gene expression remodeling likely modifies the complexity and size of glycosylation structures attached to cell surface proteins. To confirm this, glycan structures were determined and compared among myocyte types using mass spectrometry. The data indicated that the observed regulation in gene expression translated into a change in glycan structure. Further, we report that remodeled glycans are likely responsible for alterations in voltage-gated sodium channel activity and action potential waveforms, suggesting a physiological role for regulation of glycosylation-associated genes in the heart. Specifically, differences in sodium channel gating in neonatal atria versus neonatal ventricles were abolished by knocking out a single polysialyltransferase expressed only in neonatal atria. Atrial action potential duration and the rate of depolarization were altered in the absence of this polysialyltransferase, while ventricle action potential waveforms were unaffected. Together, these data indicate that the glycome is tightly controlled in the heart, and proper glycosylation is apparently essential for normal myocyte functions.

Ventricular fibrillation (VF) is the main cause of sudden cardiac death. We hypothesized that VF induced by large scars in an isolated porcine heart model could aid the understanding of VF in human hearts associated with structural disease. The explanted hearts were perfused with blood and Tyrode solution at 37C, and optically imaged with a voltage-sensitive fluorescence dye (di4-ANEPPS excited at 530nm with 150W halogen lamp). The emitted signal was filtered (610nm) and recorded with high speed cameras (MiCAM02, BrainVison, Jp) at 0.7mm spatial resolution. No optical signals could be recorded from the core of chronic infarcts or RF lesions. A total of 10 hearts were used: 4 controls, 3 with lesions generated via RF ablation and 3 with chronic infarcts. We observed the propagation of the depolarization waves and analyzed the VF waveforms at the border zone (BZ) and normal myocardium. We analyzed the VF waves in the frequency domain by calculating the dominant frequency (DF) on select regions of interest using Matlab (Mathworks, Ca). Our results showed that DF is smaller at the BZ compared to healthy tissue. Referenced to the average DF in the control hearts (10.07þ/-0.54 Hz), the DF was slightly smaller in healthy myocardium of infarct hearts (i.e., 8.9þ/ -0.71Hz) and significantly smaller at the border zone (i.e., 6.03 þ/-0.86Hz). In ablated hearts, mean DF in normal myocardium was 9.16þ/-0.7Hz and 7.24þ/-0.66Hz at BZ, respectively. We suggest that these differences are related to the heterogeneous restitution properties as well as the changes in tissue structure at the BZ. The BZ of chronic scars is comprised of a mixture of viable and necrotic fibers; whereas in the acute settings of RF lesions, inflammation and edema are present at the BZ without alteration of fiber directions. Mutations in KCNH2 gene underlie type 2 of the congenital long-QT syndrome (LQT2), in which rapid component of I K (I Kr ) is malfunctional and startled auditory stimuli are specific symptomatic trigger. The latter suggests fast arrhythmogenic mechanism. Therefore, we investigated acute alpha (1A) -and cAMPrelated beta-adrenergic modulation of I Kr in HL-1 cardiomyocytes, wild type (wt-) and two LQT2-associated mutant Kv11.1 channels (Kv11.1-Y43D and Kv11.1-K595E) reconstituted in Chinese Hamster Ovary (CHO) cell line. I Kr and Kv11.1 currents were recorded through whole-cell patch-clamp technique and confocal microscopy of HL-1 cardiomyocytes transfected with GFP-tagged pleckstrin homology domain of phospholipase C-delta (1) , visualized the fluctuations of membrane PIP 2 content. In HL-1 cardiomyocytes expressing human alpha (1A) -adrenoceptor, superfusion with 30 micromol/l phenylephrine significantly reduced I Kr amplitude, shifted current activation to more positive potentials and accelerated kinetics of deactivation. Confocal images demonstrated decline of PIP 2 concentration during phenylephrine exposure. Stimulation of beta (1) -and beta (2) -adrenoceptor downstream enzyme adenylyl cyclase by 5 micromol/l forskolin shifted I K...