Figure 4 – Increasing surface/inner CRU NCX expression ratio inhibits SCRs and DADs but does not affect SAPs.

A) Increasing surface/inner CRU NCX expression ratio (i.e., increasing NCX density in surface CRUs without changing whole-cell NCX expression) monotonically decreases the rate threshold of SCRs (i) and DADs (ii), whereas SAP remains absent in all conditions. This effect is greater in cells with dense and intermediate tubular structures vs cells with sparse tubules. B) Effect of altered surface/inner CRU NCX expression ratio on voltage (i) and global cytosolic Ca²⁺ concentration (ii) in cells with sparse (left), intermediate (middle), and dense (right) tubules following pacing at 3 Hz to examine the occurrence of DADs and SCRs. C) Mechanism underlying increasing surface/inner CRU NCX expression ratio inhibiting SCRs and DADs. Biomarkers were determined from the first 100 ms of no-stimulation period and normalized to those of cells with a retained surface/inner CRU NCX expression ratio of 1.0. Higher surface/inner CRU NCX expression ratio is associated with reduced NCX contribution to Ca²⁺ extrusion by inner coupled CRUs, resulting in higher cleft Ca²⁺ concentration in inner CRUs with no visible effect in surface CRUs. Higher inner cleft Ca²⁺ concentration in inner CRUs causes increased RyR P_o and RyR leak, leading to increased SCRs and DADs. Since fewer inner CRUs containing NCX exist in sparsely tubulated cells to begin with, the impact of varying NCX distribution is more pronounced in cells with tubules. As such, the consequence of changing cleft Ca²⁺ concentration is greater in cells with dense and intermediate tubules rather than those with sparse tubules, explaining the observed differences in SCRs and DADs between cells with varying tubule densities.