Intracellular O-linked glycosylation directly regulates cardiomyocyte L-type Ca²⁺ channel activity and excitation–contraction coupling

Cardiomyocyte L-type Ca²⁺ channels (Ca_vs) are targets of signaling pathways that modulate channel activity in response to physiologic stimuli. Ca_v regulation is typically transient and beneficial but chronic stimulation can become pathologic; therefore, gaining a more complete understanding of Ca_v 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_v 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_v 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_v current (I_Ca), but with increased efficacy of adrenergic stimulation, and Ca_v steady-state gating and window current were significantly depolarized. Consistently, OGTKO cardiomyocyte intracellular Ca²⁺ release and contractility were diminished and demonstrated greater beat-to-beat variability. Additionally, we show that the Ca_v α1 and β2 subunits are O-GlcNAcylated while α2δ1 is not. Echocardiographic analyses indicated that the reductions in OGTKO cardiomyocyte Ca²⁺ 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_Ca achieved through altered Ca_v expression, gating, and response to adrenergic stimulation; these mechanisms have significant implications for understanding how EC coupling is regulated in health and disease.