Axial Membrane Tubules in Atrial Cardiomyocytes Confine Ultrarapid Intracellular Calcium Signals through a New Super-Hub Mechanism

Abstract

Endocytic membrane structures called transverse tubules (TTs) are thought to represent the main mechanism for contacts with the junctional sarcoplasmic reticulum (jSR) and intracellular Ca2+ release in ventricular cardiomyocytes (VCM). In contrast, in atrial cardiomyocytes (ACM) we identify previously not described large axial membrane tubules (ATs) at the cell core. Using optimized ACM isolation and live cell superresolution microscopy, we show that AT membrane structures represent both the major (ie. >90% of endocytic tubule structures) and largest tubule component (mean AT circumference 921±24 nm). Due to the unusually large AT membrane surface with a positive curvature exposed to the cytoplasm, we hypothesized that the endocytic AT membrane surface functions as central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined imaging of AT membranes with fast intracellular Ca2+ imaging. Indeed, high-amplitude Ca2+ transients originated in a highly localized fashion rapidly from the AT-hubs. Importantly, Ca2+ transients originating from AT-hubs occured significantly earlier compared to cellular surface locations (p<0.05). This suggests a new EC-coupling model where AT-hubs control rapid centrifugal Ca2+ signals. Sarcomere shortening confirmed significantly faster ACM versus VCM EC-coupling (p<0.05). Furthermore, in situ phospho-epitope imaging correlated ryanodine receptor (RyR2) channel phosphorylation with AT-hub function. Only junctional RyR2 clusters at AT-hubs showed increased phosphorylation compared to non-junctional clusters. Genetic ablation of RyR2-specific phosphoepitopes by Ala-knockin impaired ACM contractility, consistent with loss-of-function and disruption of AT-hub-dependent signaling. Hence, our data support a fundamentally new ACM model of EC-coupling, where AT-membranes function as signaling super-hubs that control high-amplitude ultrarapid Ca2+ signals at the cell core. Genetic ablation of RyR2 cluster phosphorylation further suggests that the super-hub model is relevant to explain atrial function in health and decreased contractility typical for atrial disease forms.