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Invaginated sarcolemmal membrane structures in cardiac myocytes form transverse tubules (TTs). The TTs form electrical and chemical signaling conduits from the myocyte exterior to regions deep in the cell such as the junctional sarcoplasmic reticulum ( jSR) critical in excitation-contraction (EC) coupling. Local control of jSR Ca2þ release in ventricular myocytes (VM) depends critically on this organization. In contrast, atrial myocytes (AM) normally have few if any TTs (although the exact abundance has been controversial). In atrial myocytes, the normally sparse distribution of TTs suggests that a critical role in EC coupling remains unlikely. Here, we identify a structurally distinct and mechanistically novel role for even sparsely set TTs in atrial EC coupling. Using novel membrane-preserving live cell techniques, we identify relatively abundant axial membrane tubules (ATs). These ATs are deep intracellular branches of the relatively rare TTs in 100% of AM investigated (n 1⁄4 29). Using live cell STimulated Emission Depletion (STED) superresolution microscopy, we show that atrial AT structures are characteristically different from TTs. ATs represent both the major component of the cell-wide tubule network and the largest tubules: AT circumference in AM 921 + 24 nm vs. VM 630 + 26 nm; p , 0.001 (physiological live cell conditions). These dimensions indicate unprecedentedly large AT surfaces with a positive membrane curvature at the AM center suggesting that ATs could function as a central-axial Ca2þ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined AT and intracellular Ca2þ transient (CaT) imaging. High-amplitude CaTs originated directly from ATs creating highly localized centralaxial Ca2þ signals. Importantly, local CaT onset measurements showed that atrial CaTs occurred significantly earlier at AT compared to surface membrane locations (p , 0.05). To explain the observed CaT behaviors, we imaged in situ the Ser-2808 phospho-epitope of ryanodine receptor (RyR2) Ca2þ release channels necessary for PKA phosphorylation. RyR2 channel clusters at AT-hubs showed increased PKA phosphorylation in contrast to the low phosphorylation state of non-junctional RyR2 clusters in unstimulated AM. Sarcomere shortening experiments confirmed significantly larger and faster AM compared with VM axial contractions (p , 0.05); this effect was significantly augmented by isoproterenol (1 mm) through non-junctional cluster recruitment. Consistent with the proposed AT super-hub model, genetic ablation of RyR2 PKA phosphorylation in Ser-2808-Ala knockin mice significantly attenuated atrial sarcomere shortening and in vivo atrial but not ventricular contractile function. Our data suggest a fundamentally new atrial EC-coupling model where AT structures in atrial myocytes function as large signaling super-hubs that couple central-axial Ca2þ signaling to rapid contractile activation. This supports a critical role of the transverse-axial tubular system in cell-specific atrial EC-coupling mechanisms (i.e. faster contractions than in ventricles) and questions previous models of atrial EC-coupling based on slow intracellular Ca2þ diffusion. Genetic ablation of RyR2 cluster phosphorylation unmasks a previously unknown role of atrial PKA regulation, which may explain atrial function in health versus decreased contractility occuring in atrial fibrillation and heart failure.

168-03: Axial membrane tubules in atrial myocytes initiate rapid intracellular calcium signals through a new “super-hub” mechanism