When NCX switches sides: Experimental and computational insights into Ca²⁺ 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.