T he synchronous rise of the systolic Ca 2+ transient in mammalian ventricular myocytes requires the presence of an extensive and regular transverse (t)-tubular system.1 These t-tubules ensure close apposition of L-type Ca 2+ channels (LTCCs) and sarcoplasmic reticulum (SR) Ca 2+ release channels (ryanodine receptors [RyRs]) forming dyads or couplons where excitation-contraction coupling commences.2,3 The t-tubules are also surrounded by a continuous network of SR, which is thought to assist with amplification of the initial Ca 2+ entry during the action potential and contribute to the synchronous rise of systolic Ca 2+ . 4,5The t-tubule and SR networks are, however, labile with disorganization and loss commonly observed in heart failure (HF). [5][6][7][8][9] In such circumstances the loss of t-tubules leads to dyssynchronous Ca 2+ release patterns, a smaller systolic Ca 2+ transient, and altered β-adrenergic (β-adrenergic receptor) signaling. 6-10 Conversely, recovery from HF is associated with restoration of the t-tubule network along with normalization of β-adrenergic receptor signaling and resynchronization of the systolic Ca 2+ transient. 9,11In This Issue, see p 961More extensive differences in t-tubule organization and density than those occurring in the ventricle during HF are known to exist between the atrium and the ventricle. For example, small mammals (mouse, rat, rabbit, etc) completely lack or possess only a rudimentary, predominantly axially arranged, t-tubule network.2-14 Conversely, some studies have suggested that limited numbers of atrial cells from smaller laboratory species such as the rat have a more ventricular-like t-tubule pattern, 15 although these particular cells may be of different lineage and a feature of the pulmonary vein sleeve region. 16The poorly developed t-tubule network in these atrial myocytes leads to the characteristic early peripheral and delayed central Rationale: Transverse tubules (t-tubules) regulate cardiac excitation-contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear.Objective: To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. Methods and Results:T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 leve...

T ransverse-tubules (t-ts) are invaginations of the plasma membrane, which in cardiac muscle facilitate transmission of the action potential from the exterior to interior of the cell. Dyads or couplons are formed along regions of the t-ts by the close apposition of the junctional portion of the sarcoplasmic reticulum (jSR). It is within the compartment formed by these 2 structures, the dyadic cleft, that the contractile function in the heart is regulated by the interplay between the L-type voltage-gated Ca 2+ channel (LTCC) localized to the t-ts and the ryanodine receptor (RyR2) anchored in the jSR.1 Critical to this regulation is the arrangement of the jSR and t-t membranes, which are held in a precise geometric organization 2 separated by a gap of 12 to 15 nm. Changes to the microanatomy between the 2 membranes, and hence the spatial relationship between the LTCCs and RyR2s, are associated with Ca 2+ handling abnormalities and impaired contractile function, 3 although the effect on systolic Ca 2+ remains equivocal. 4 Our current understanding of the 3-dimensional (3D) organization of t-ts is mainly derived from studies using confocal microscopy. [5][6][7] Elegant experiments have revealed that the t-ts form regular arrays with a periodicity that correlates to the position of the Z-lines. 8 The dimensions and branching patterns are varied and species-dependent. For example, rat myocytes are shown to have a more geometrically complex arrangement compared with the human t-t system, which has a radial distribution within the cell, akin to the spokes of a wheel. 9 Recently, the development of stimulated emission depletion microscopy has pushed the resolution frontier to ≈60 nm in the focal plane, providing ultrastructural details of the t-t system in control and failing rat hearts showing Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation-contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure.Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results:Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collap...

The bacterial pathogen Neisseria meningitidis expresses long, thin, retractile fibers (called type IV pili) from its cell surface and uses these adhesive structures to mediate primary attachment to epithelial cells during host colonization and invasion. PilQ is an outer membrane protein complex that is essential for the translocation of these pili across the outer membrane. Here, we present the structure of the PilQ complex determined by cryoelectron microscopy to 12 Å resolution. The dominant feature of the structure is a large central cavity, formed by four arm features that spiral upwards from a squared ring base and meet to form a prominent cap region. The cavity, running through the center of the complex, is continuous and is effectively sealed at both the top and bottom. Analysis of the complex using selforientation and by examination of two-dimensional crystals indicates a strong C4 rotational symmetry, with a much weaker C12 rotational symmetry, consistent with PilQ possessing true C4 symmetry with C12 quasisymmetry. We therefore suggest that the complex is a homododecamer, formed by association of 12 PilQ polypeptide chains into a tetramer of trimers. The structure of the PilQ complex, with its large and well defined central chamber, suggests that it may not function solely as a passive portal in the outer membrane, but could be actively involved in mediating pilus assembly or modification.

The sinus node is a collection of highly specialised cells constituting the heart’s pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca 2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.