A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Junctophilin-2 expression rescues atrial dysfunction through polyadic junctional membrane complex biogenesis.

Atrial dysfunction is highly prevalent and associated with increased severity of heart failure. While rapid excitation-contraction coupling depends on axial junctions in atrial myocytes, the molecular basis of atrial loss of function remains unclear. We identified approximately 5-fold lower junctophilin-2 levels in atrial compared with ventricular tissue in mouse and human hearts. In atrial myocytes, this resulted in subcellular expression of large junctophilin-2 clusters at axial junctions, together with highly phosphorylated ryanodine receptor (RyR2) channels. To investigate the contribution of junctophilin-2 to atrial pathology in adult hearts, we developed a cardiomyocyte-selective junctophilin-2-knockdown model with 0 mortality. Junctophilin-2 knockdown in mice disrupted atrial RyR2 clustering and contractility without hypertrophy or interstitial fibrosis. In contrast, aortic pressure overload resulted in left atrial hypertrophy with decreased junctophilin-2 and RyR2 expression, disrupted axial junctions, and atrial fibrosis. Whereas pressure overload accrued atrial dysfunction and heart failure with 40% mortality, additional junctophilin-2 knockdown greatly exacerbated atrial dysfunction with 100% mortality. Strikingly, transgenic junctophilin-2 overexpression restored atrial contractility and survival through de novo biogenesis of polyadic junctional membrane complexes maintained after pressure overload. Our data show a central role of junctophilin-2 cluster disruption in atrial hypertrophy and identify transgenic augmentation of junctophilin-2 as a disease-mitigating rationale to improve atrial dysfunction and prevent heart failure deterioration.

Axial Tubule Junctions Activate Atrial Ca2+ Release Across Species.

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.