Axial tubule junctions control rapid calcium signaling in atria
Sören Brandenburg, … , W. Jonathan Lederer, Stephan E. Lehnart
Sören Brandenburg, … , W. Jonathan Lederer, Stephan E. Lehnart
Published September 19, 2016
Citation Information: J Clin Invest. 2016;126(10):3999-4015. https://doi.org/10.1172/JCI88241.
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Research Article Cardiology Cell biology

Axial tubule junctions control rapid calcium signaling in atria

Abstract

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon “super-hubs” thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias.

Authors

Sören Brandenburg, Tobias Kohl, George S.B. Williams, Konstantin Gusev, Eva Wagner, Eva A. Rog-Zielinska, Elke Hebisch, Miroslav Dura, Michael Didié, Michael Gotthardt, Viacheslav O. Nikolaev, Gerd Hasenfuss, Peter Kohl, Christopher W. Ward, W. Jonathan Lederer, Stephan E. Lehnart

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Figure 3

Differential regulation of RyR2 cluster phosphorylation.

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Differential regulation of RyR2 cluster phosphorylation. (A) AMs coimmun...
(A) AMs coimmunostained for RyR2- and PKA-phosphorylated RyR2-pS2808. WT: highphos RyR2 clusters (yellow) aligned in central-axial string-of-pearls, intersecting transverse striations of lowphos RyR2 clusters (green); RyR2-S2808A+/+: zero PKA phosphorylation of RyR2 clusters, confirming phospho-epitope–specific labeling; WT + H89: attenuated highphos RyR2 cluster signals. Scale bars: 10 μm; magnified (×4) regions are indicated by yellow brackets. (B) Histogram showing bimodal frequency distribution of in situ pS2808/RyR2 normalized cluster signals in untreated WT AMs. The main peak indicates abundant lowphos versus fewer highphos RyR2 clusters represented by the shoulder. n = 22 AMs. (C) AMs colabeled for Cav3 and RyR2-pS2808. Highphos RyR2-pS2808 clusters alternated with Cav3 clusters in central AT structures. Scale bar: 10 μm; magnification ×4. (D) Image segmentation confirming highphos RyR2-pS2808 clusters aligned with Cav3-labeled TAT structures. (E) WT, RyR2-S2814A+/+, and WT AMs treated with the CaMK inhibitor AIP were coimmunostained for RyR2 and the CaMK-phosphorylated epitope RyR2-pS2814. Note the central highphos RyR2 clusters (yellow) versus lowphos RyR2 clusters (green). AMs from CaMK phosphorylation–incompetent RyR2-S2814A+/+ mice showed no CaMK phosphorylation in situ. Magnifcation ×4. (F) Frequency histogram of the pS2814/RyR2 cluster distribution indicating abundant lowphos versus few highphos RyR2 clusters in control AMs. n = 19 AMs. (G) AMs coimmunostained for Cav3 and RyR2-pS2814. Highphos RyR2 clusters were associated with Cav3-labeled AT structures as confirmed by image segmentation (H). Scale bar: 10 μm, magnifcation ×4. (I) STED images comparing immunolabeled central RyR2 clusters in VMs versus AMs. Atrial RyR2 clusters identified by red circles show a higher density on Z-lines and a trend toward overall shorter NNDs. Images are representative of 18 VMs and 19 AMs. Scale bars: 500 nm. Box and whisker plot: boxes show lower and upper quartiles; whiskers represent the 5th and 95th percentiles. IF, immunofluorescence. (J) Confocal live AM imaging with di-8-ANEPPS and fluo-4 showing large AT-localized Ca2+ sparks occurring repeatedly. CaT, final 1-Hz pacing evoked Ca2+ transient; red triangles indicate AT-associated Ca2+ sparks; white triangles indicate surface membrane–associated Ca2+ sparks.