Regulated and aberrant glycosylation modulate cardiac electrical signaling

Abstract

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.

Fig. 1.

Cardiac glycogene expression is regulated. Relative cardiac glycogene expression levels were determined using microarray analysis as described. Bar graphs represent the percentage of the 239 tested glycogenes and the families of glycogenes (glycosyltransferases, glycosidases, and sugar nucleotide synthesis and transporter genes) significantly differentially expressed among myocyte types. (A) Among neonatal and adult atrial and ventricular myocytes (P < 0.01). (B) Developmental and chamber-specific expression differences between two myocyte types. (P < 0.05); NA, neonatal atrium; NV, neonatal ventricle; AA, adult atrium; AV, adult ventricle.

Fig. 2.

N-glycan glycomic profiling indicates marked changes during cardiomyocyte development. MALDI-TOF-MS profiles of the permethylated N-linked glycans of myocytes isolated from neonatal and adult atria and ventricles. (A) Neonatal atrium; (B) neonatal ventricle; (C) adult atrium; (D) adult ventricle. Major peaks are labeled, with the assigned compositions listed in Table S2. (Insets) Expansions of the mass region m/z 2950–3050 (highlighted), showing the regulated change in the relative levels of NeuAc and NeuGc sialic acids during development. Note for the two atrial samples, that the ratio of di-NeuAc to NeuAc/NeuGc to di-NeuGc determined by comparing the relative intensities of these three closely related peaks changes from ≈2.5:1.5:1 in the neonate to 1:1:2 in the adult sample. A similar phenomenon is observed during ventricular development and is repeated throughout the spectra. Each peak was subjected to MSMS analysis to clarify their structure. All molecular ions are present in sodiated form ([M+Na]⁺).

Fig. 3.

Regulated and aberrant expression of ST8sia2 impact AP waveforms. APs were measured from isolated control and ST8sia2^(−/−) neonatal atrial and ventricular myocytes as described. (Top left) Typical AP waveforms measured from control (in black) and ST8sia2^(−/−) (in red) atrial myocytes. (A–C) The mean ± SEM AP waveform parameters (relevant portions of AP marked with arrows). Black, control atrium (n = 7); red, ST8sia2^(−/−) atrium (n = 9); dark blue, control ventricle (n = 7); blue, ST8sia2^(−/−) ventricle (n = 5). (A) Time to AP peak (ms). (B) AP duration at 50% repolarization (APD₅₀, ms). (C) AP duration at 90% repolarization (APD₉₀, ms). Significance tested using a two-tailed t-test and comparing ST8sia2^(−/−) to control parameters. *, significant (P < 0.005); #, not significant (P > 0.1).

Fig. 4.

ST8sia2 expression modulates atrial Na_v gating consistent with changes in AP. ST8sia2^(−/−) and control myocyte Na_v gating. Left panels (A, C, E, G) Atrium, ST8sia2^(−/−) (red, n = 10); control (black, n = 4). Right panels (B, D, F, H) Ventricle, ST8sia2^(−/−) (blue, n = 4); control (dark blue, n = 6). Data are mean ± SEM values. (A and B) Steady state activation. Data are peak normalized conductance at a membrane potential. Lines are fits of data to single Boltzmann relationships. (Insets) Half-activation voltage. (C and D) Steady state channel availability. Data are the fraction of channels available at a constant, fully-activating depolarization. Lines are fits of data to single Boltzmann relationships. (Dashed line) ST8sia2^(−/−) data shifted along voltage axis by −7.3 mV (to mimic measured shift in half-activation voltage). (Insets) Half-inactivation voltage. (E and F) Fast inactivation time constants. Lines are nontheoretical, point-to-point. (Dashed line) The ST8sia2^(−/−) data shifted along voltage axis by −7.3 mV. (Insets) Typical whole cell Na⁺ current traces elicited by stepping to a −40 mV test potential. (G and H) Time constants of recovery from fast inactivation at a −130 mV recovery potential. Significance tested using a two-tailed t-test and comparing ST8sia2^(−/−) to control parameters. *, significant (P ≤ 0.005); #, not significant (P > 0.1).