Intracellular O-linked Glycosylation Directly Regulates Cardiomyocyte L-type Ca Channel Activity and Excitation-contraction Coupling

Cardiomyocyte L-type Ca channels (Cas) are targets of signaling pathways that modulate channel activity in response to physiologic stimuli. Ca regulation is typically transient and beneficial but chronic stimulation can become pathologic; therefore, gaining a more complete understanding of Ca regulation is of critical importance. Intracellular O-linked glycosylation (O-GlcNAcylation), which is the result of two enzymes that dynamically add and remove single N-acetylglucosamines to and from intracellular serine/threonine residues (OGT and OGA respectively), has proven to be an increasingly important post-translational modification that contributes to the regulation of many physiologic processes. However, there is currently no known role for O-GlcNAcylation in the direct regulation of Ca activity nor is its contribution to cardiac electrical signaling and EC coupling well understood. Here we aimed to delineate the role of O-GlcNAcylation in regulating cardiomyocyte L-type Ca activity and its subsequent effect on EC coupling by utilizing a mouse strain possessing an inducible cardiomyocyte-specific OGT-null-transgene. Ablation of the OGT-gene in adult cardiomyocytes (OGTKO) reduced OGT expression and O-GlcNAcylation by > 90%. Voltage clamp recordings indicated an ~ 40% reduction in OGTKO Ca current (I), but with increased efficacy of adrenergic stimulation, and Ca steady-state gating and window current were significantly depolarized. Consistently, OGTKO cardiomyocyte intracellular Ca release and contractility were diminished and demonstrated greater beat-to-beat variability. Additionally, we show that the Ca α1 and β2 subunits are O-GlcNAcylated while α2δ1 is not. Echocardiographic analyses indicated that the reductions in OGTKO cardiomyocyte Ca handling and contractility were conserved at the whole-heart level as evidenced by significantly reduced left-ventricular contractility in the absence of hypertrophy. The data indicate, for the first time, that O-GlcNAc signaling is a critical and direct regulator of cardiomyocyte I achieved through altered Ca expression, gating, and response to adrenergic stimulation; these mechanisms have significant implications for understanding how EC coupling is regulated in health and disease.