Heart muscle excitation-contraction (E-C) coupling is governed by Ca 2؉ release units (CRUs) whereby Ca 2؉ influx via L-type Ca 2؉ channels (Cav1.2) triggers Ca 2؉ release from juxtaposed Ca 2؉ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn ؊/؊ ). The structure and protein composition of the cardiac CRU is significantly altered in Trdn ؊/؊ hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn ؊/؊ hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn ؊/؊ myocytes. CRU function is impaired in Trdn ؊/؊ myocytes, with reduced SR Ca 2؉ release and impaired negative feedback of SR Ca 2؉ release on Cav1.2 Ca 2؉ currents (ICa). Uninhibited Ca 2؉ influx via ICa likely contributes to Ca 2؉ overload and results in spontaneous SR Ca 2؉ releases upon ␤-adrenergic receptor stimulation with isoproterenol in Trdn ؊/؊ myocytes, and ventricular arrhythmias in Trdn ؊/؊ mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.cardiac muscle ͉ sarcoplasmic reticulum ͉ calsequestrin ͉ Cav1.2 ͉ RyR2

T he type 2 ryanodine receptor (RYR2) is an integral membrane protein of the cardiomyocyte sarcoplasmic reticulum (SR) that functions as a Ca 2+ -activated Ca 2+ ion channel. Each receptor is a homotetramer, measuring roughly 29×29×12 nm, which can be readily identified in electron micrographs based on its location within the dyadic cleft and on its size and shape. 1,2 Rotary shadowing studies of type 1 ryanodine receptors (RYR1) in skeletal muscle triads 3 and numerous transmission electron micrographs of cardiac muscle 4 left the impression that the tetramers filled the dyadic cleft, forming a defect-free crystalline array, often referred to as a checkerboard. The array's formation is thought to be an intrinsic property of the protein reflecting the homotetramer's 4-fold symmetry whereby adjacent tetramers were noncovalently connected through their adjacent clamp domains.5 This is also thought to provide the structural basis for interprotein allosteric interactions. 6,7 Electron tomography and super-resolution fluorescence microscopy later revealed that the dyad contained subarrays that did not completely fill the cleft, and although neither technique had the resolution to determine the position and orientation of individual tetramers, the super resolution study assumed a regular checkerboard array when fitting their data. 8,9 A single-tilt tomogram with higher resolution indicated that the subarrays were unlikely to be fitted with a simple checkerboard. 10 RYR1 tetramers, purified from skeletal muscle and inserted in artificial bilayers, spontaneously formed 2 different types of array that depended on the free Mg 2+ concentration. Using a nominally Mg 2+ -free buffer, the tetramers formed a checkerboard, but with the addition of 4 mmol/L Mg 2+ , the tetramers were more densely packed in a side-by-side orientation although there was no physical contact between them. 11,12 The organization of the tetramers at the expected intracellular free Mg 2+ concentration of ≈1 mmol/L was not investigated. Whether RYR2 behaves similarly, and if such changes can occur in vivo, is unknown.In this study, we examined dual-tilt tomograms to visualize the position directly of individual RYR2 tetramers in adult rat ventricular myocytes. When fixed in situ, where the Mg Rationale: Single-tilt tomograms of the dyads in rat ventricular myocytes indicated that type 2 ryanodine receptors (RYR2s) were not positioned in a well-ordered array. Furthermore, the orientation and packing strategy of purified type 1 ryanodine receptors in lipid bilayers is determined by the free Mg 2+ concentration. These observations led us to test the hypothesis that RYR2s within the mammalian dyad have multiple and complex arrangements. Objectives:To determine the arrangement of RYR2 tetramers in the dyads of mammalian cardiomyocytes and the effects of physiologically and pathologically relevant factors on this arrangement. Methods and Results:We used dual-tilt electron tomography to produce en-face views of dyads, enabling a direct examination of RYR2 dis...

The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca2+ sparks and dual-tilt electron tomography) and dSTORM imaging of permeabilized Wistar rat ventricular myocytes. Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca2+ spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca2+ spark formation.

Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na(+)/Ca(2+) exchanger, the predominant Na(+) channel isoform rH1, and the L-type calcium channel, Ca(v)1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na(+)/Ca(2+) exchanger or the Na(+) channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors.

These results illustrate that the RYR2 channel plays an essential role in pacing heart rate. Moreover, we find that RYR2 loss-of-function can lead to fatal arrhythmias typically associated with gain-of-function mutations. Given that RYR2 levels can be reduced in pathological conditions, including heart failure and diabetic cardiomyopathy, we predict that RYR2 loss contributes to disease-associated bradycardia, arrhythmia, and sudden death.

Integrin-mediated adhesion to the ECM is essential for normal development of animal tissues. During muscle development, integrins provide the structural stability required to construct such a highly tensile, force generating tissue. Mutations that disrupt integrin-mediated adhesion in skeletal muscles give rise to a myopathy in humans and mice. To determine if this is due to defects in formation or defects in maintenance of muscle tissue, we used an inducible, targeted RNAi based approach to disrupt integrin-mediated adhesion in fully formed adult fly muscles. A decrease in integrin-mediated adhesion in adult muscles led to a progressive loss of muscle function due to a failure to maintain normal sarcomeric cytoarchitecture. This defect was due to a gradual, age dependent disorganization of the sarcomeric actin, Z-line, and M-line. Electron microscopic analysis showed that reduction in integrin-mediated adhesion resulted in detachment of actin filaments from the Z-lines, separation of the Z-lines from the membrane, and eventually to disintegration of the Z-lines. Our results show that integrin-mediated adhesion is essential for maintaining sarcomeric integrity and illustrate that the seemingly stable adhesive contacts underlying sarcomeric architecture are inherently dynamic.

We analyzed the distribution of ryanodine receptor (RyR) and Cav1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Cav1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Cav1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Cav1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Cav1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Cav1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Cav1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Cav1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca2+ regulation and modeling Ca2+ dynamics.

This review highlights recent and ongoing discoveries that are transforming the previously held view of dyad structure and function. New data show that dyads vary greatly in both structure and in their associated molecules. Dyads can contain varying numbers of type 2 ryanodine receptor (RYR2) clusters that range in size from one to hundreds of tetramers and they can adopt numerous orientations other than the expected checkerboard. The association of Ca(v)1.2 with RYR2, which defines the couplon, is not absolute, leading to a number of scenarios such as dyads without couplons and those in which only a fraction of the clusters are in couplons. Different dyads also vary in the transporters and exchangers with which they are associated producing functional differences that amplify their structural diversity. The essential role of proteins, such as junctophilin-2, calsequestrin, triadin, and junctin that maintain both the functional and structural integrity of the dyad have recently been elucidated giving a new mechanistic understanding of heart diseases, such as arrhythmias, hypertension, failure, and sudden cardiac death.