Reduced hybrid/complex N-glycosylation disrupts cardiac electrical signaling and calcium handling in a model of dilated cardiomyopathy

Highlights

• MGAT1 encodes the enzyme necessary for initiation of hybrid/complex N-glycosylation.

• Cardiac and cellular excitability is altered with cardiomyocyte MGAT1 deletion.

• MGAT1 deletion directly alters ventricular Na_v N-glycosylation and gating.

• K_v activity in MGAT1 deficient myocytes declines with heart disease progression.

• Myocyte Ca²⁺ handling is impaired by reduced hybrid/complex N-glycosylation.

Abstract

Dilated cardiomyopathy (DCM) is the third most common cause of heart failure, with ~70% of DCM cases considered idiopathic. We showed recently, through genetic ablation of the MGAT1 gene, which encodes an essential glycosyltransferase (GlcNAcT1), that prevention of cardiomyocyte hybrid/complex N-glycosylation was sufficient to cause DCM that led to heart failure and early death. Our findings are consistent with increasing evidence suggesting a link between aberrant glycosylation and heart diseases of acquired and congenital etiologies. However, the mechanisms by which changes in glycosylation contribute to disease onset and progression remain largely unknown. Activity and gating of voltage-gated Na⁺ and K⁺ channels (Na_v and K_v respectively) play pivotal roles in the initiation, shaping and conduction of cardiomyocyte action potentials (APs) and aberrant channel activity was shown to contribute to cardiac disease. We and others showed that glycosylation can impact Na_v and K_v function; therefore, here, we investigated the effects of reduced cardiomyocyte hybrid/complex N-glycosylation on channel activity to investigate whether chronic aberrant channel function can contribute to DCM. Ventricular cardiomyocytes from MGAT1 deficient (MGAT1KO) mice display prolonged APs and pacing-induced aberrant early re-activation that can be attributed to, at least in part, a significant reduction in K_v expression and activity that worsens over time suggesting heart disease-related remodeling. MGAT1KO Na_v demonstrate no change in expression or maximal conductance but show depolarizing shifts in voltage-dependent gating. Together, the changes in MGAT1KO Na_v and K_v function likely contribute to observed anomalous electrocardiograms and Ca²⁺ handling. These findings provide insight into mechanisms by which altered glycosylation contributes to DCM through changes in Na_v and K_v activity that impact conduction, Ca²⁺ handling and contraction. The MGAT1KO can also serve as a useful model to study the effects of aberrant electrical signaling on cardiac function and the remodeling events that can occur with heart disease progression.

Introduction

Dilated cardiomyopathy (DCM), which is characterized by systolic dysfunction and ventricular chamber enlargement, is attributable to a number of congenital and acquired etiologies; however, upwards of 70% of DCM cases are idiopathic [1,2]. Protein glycosylation is a co/posttranslational modification essential for protein functions such as proper folding, targeting to cellular compartments, and modulating receptor/ion channel activities [3,4]. Genome-wide searches identified expression changes in glycosylation-related genes (glycogenes) in idiopathic DCM, including glycosyltransferases and nucleotide sugar transporters [[5], [6], [7]]; proteomic/glycomic studies showed changes in serum N-glycosylation in heart disease models and in humans with DCM risk factors [[8], [9], [10], [11], [12]]. Additionally, models of DCM and heart failure were associated with changes in glycosylation of proteins involved in electromechanical processes [13,14]. The importance of glycosylation in the heart is further highlighted by the observation that ~20% of patients with congenital disorders of glycosylation (CDG), which result in typically modest reductions in protein glycosylation, present with cardiac deficits including idiopathic DCM [15,16]. The data suggest a correlation between changes in extracellular glycosylation and DCM/heart disease. However, for each example, it is not known whether the altered glycosylation is pathogenic or subsequent to disease onset.

Activity and gating of voltage-gated ion channels (VGICs) play a vital role in cardiac excitability and conduction by initiating and shaping cardiomyocyte action potentials (APs) and converting electrical signals into intracellular messages (i.e. Ca²⁺). The voltage-gated Na⁺ channel (Na_v) isoform Na_v1.5 is the primary cardiac isoform while multiple K_v isoforms contribute to repolarization; relevant K_v isoforms vary by species and region of the heart [17]. Aberrant Na_v and K_v activity can lead to inefficient contraction and life-threatening arrhythmias through congenital and acquired etiologies [18,19]. Several Na_v1.5 mutations were associated with DCM and ion channel remodeling was shown to occur in DCM patients and models [[20], [21], [22], [23], [24], [25], [26], [27]]; however, determining the contribution of aberrant VGIC activity to the progression of heart diseases including DCM remains elusive.

Na_vs and K_vs are large transmembrane proteins that are typically modified by protein glycosylation, which can have a significant impact on channel function. Defects in channel surface expression or trafficking associated with mutations that reduce channel glycosylation by removing glycosylation sites were linked to cardiac diseases such as long QT syndrome [[28], [29], [30]]. However, the overwhelming evidence points to a role for sialic acids, carbohydrate residues that bear a negative charge at physiologic pH, in exerting the most significant impact on channel function. We previously showed that cardiac glycosylation is regulated, with changes in sialic acid levels (typically the terminal residue of both N- and O-linked glycan structures) attached to specific VGIC isoforms responsible for the direct modulation of cardiac electrical signaling through electrostatic mechanisms [[31], [32], [33], [34]]. We went on to show that these same changes in protein sialylation cause stress-induced heart failure [35], further suggesting a link among altered VGIC glycosylation, arrhythmias, and heart disease.

The MGAT1 gene product, GlcNAcT1, is responsible and necessary for initiating formation of hybrid/complex branched N-glycans in the medial Golgi by attaching the first N-acetylglucosamine (GlcNAc) residue to trimmed mannose structures (Fig. 1A) [36]. We showed previously that genetic ablation of MGAT1 in cardiomyocytes only (MGAT1KO) resulted in significantly reduced cardiomyocyte hybrid/complex N-glycosylation and was sufficient to cause DCM that deteriorated into heart failure with 100% of MGAT1KO mice dying early [37]. Consistently, MGAT1 expression was shown to be down-regulated in human idiopathic DCM [[5], [6], [7]]. We also showed that MGAT1KO myocytes demonstrated altered excitation-contraction coupling (EC-coupling) consistent with observed changes in voltage-gated Ca²⁺ channel (Ca_v) activity caused by a direct effect on Ca_v α2δ1 subunit N-glycosylation [37]. These data suggest a possible glycosylation-dependent link between excitation and contraction.

Here, we sought to determine the impact of reduced hybrid/complex N-glycosylation on cardiomyocyte electrical excitability and began to elucidate a possible mechanism by which aberrant Na_v and K_v activities contribute to the development of DCM. Our data indicate that cardiomyocyte deletion of MGAT1 has marked effects on cardiac electrical signaling that result in electrocardiogram (ECG) anomalies, likely caused, at least in part, by the significant changes in MGAT1KO cardiomyocyte AP properties. Our data also demonstrate that chronic reductions in complex N-glycosylation impact VGIC gating directly, consistent with an electrostatic mechanism, as well as indirectly through reductions in repolarizing K⁺ current and K_v expression that likely occur through remodeling as systolic function deteriorates in the MGAT1KO heart.