Abstract
Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling.Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human.Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections.Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial “super-hub” Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.
Highlights
As one billion individuals 65 years or older are expected by the year 2030, aging populations will be affected by a sharp increase in chronic diseases (Moslehi et al, 2012)
We took advantage of a recent strategy to synthesize photostable, far-red emitting fluorescent cholesterol analogs (Chol-PEG-KK114; see methods) for live cell imaging based on STimulated Emission Depletion (STED) nanoscopy (Kolmakov et al, 2010; Sezgin et al, 2016)
While few transverse tubule (TT) were visible at the peripheral surface membrane, numerous prominent axial tubule (AT) components were observed deep inside atrial myocyte (AM) (Figure 1A, magnifications)
Summary
As one billion individuals 65 years or older are expected by the year 2030, aging populations will be affected by a sharp increase in chronic diseases (Moslehi et al, 2012). Most frequent in elderly people, electrical and contractile dysfunction of the atria contributes to stroke, heart failure, and atrial fibrillation the latter alone predicted to increase threefold in prevalence by 2050 (Yiin et al, 2014). While atrial fibrillation is often considered a proximal cause of thrombembolic stroke, recent clinical studies question a direct relationship (Brambatti et al, 2014; Martin et al, 2015). An emerging atrial Ca2+ nanodomain model, extending significantly beyond the canonical role of transverse tubule (TT) invaginations in ventricular myocytes (VMs), was proposed: super-hub Ca2+ signaling based on axial tubule (AT) junctions that rapidly activate Ca2+ release and atrial contraction through cell-specific molecular nanodomain mechanisms (Brandenburg et al, 2016b)
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