When NCX switches sides: Experimental and computational insights into Ca 2+ regulation in the heart

The Na+/Ca2+ exchanger (NCX) transports Ca2+ and Na+ through the plasma membrane of cardiomyocytes. NCX dysregulation has been related to diastolic dysfunction. NCX inhibition has been identified as a potential therapeutic approach. It can accelerate the decay of the cytosolic Ca2+ concentration (Ca2+i) and improve impaired cardiomyocyte relaxation. We hypothesized that this counterintuitive effect is explained by the subcellular arrangement of NCX and local ion gradients within the intracellular Ca2+ release units. In a parallel model-based and experimental approach, we re-evaluated the location of NCX with regard to the dyadic cleft and its role in modulating Ca2+i. Stimulated emission depletion imaging revealed NCX in close proximity to junctophilin (the marker for the dyadic cleft). We simulated Ca2+ dynamics in the dyadic cleft considering Ca2+ channels, NCX molecules and local concentration gradients. Positioning NCX inside the dyadic cleft in our computational model matched its action on spark rate. In forward mode (Ca2+ out, Na+ in) NCX decreased spontaneous Ca2+ release events (spark rate) in simulations and imaging experiments, while in reverse mode it increased them. In paced cardiomyocytes, NCX inhibition consistently increased diastolic Ca2+. The effects of NCX inhibition on transient amplitude and peak, however, depended on extracellular Ca2+o suggesting a role of reverse-mode NCX activity at high Ca2+o. NCX inhibition prolonged the early rise of Ca2+, corroborating that reverse-mode NCX facilitates the rapid initial increase of Ca2+i during excitation. Our combined imaging, modelling and functional data support the hypothesis that NCX resides in the dyadic cleft where it bidirectionally shapes Ca2+ transients and spark activity. KEY POINTS: Our data suggest that Na+/Ca2+ exchanger (NCX) is localized inside the dyadic cleft in cardiomyocytes, close to junctophilin, as shown by stimulated emission depletion imaging. Computational modelling confirmed NCX's dyadic positioning. It is critical for its effects on local Ca2+ signalling. NCX bidirectionally modulates Ca2+ dynamics: forward mode reduces, reverse mode increases spontaneous Ca2+ sparks. NCX inhibition raises diastolic cytosolic Ca2+ and alters Ca2+ transient amplitude depending on extracellular Ca2+ levels. Reverse-mode NCX facilitates rapid initial Ca2+ rise during excitation, highlighting its role in cardiac Ca2+ regulation and potential therapy.