BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte.

BACKGROUND: Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. METHODS: Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. RESULTS: We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. CONCLUSIONS: Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.

Development of a Time Projection Chamber Readout with Hybrid Pixel Sensors for Beam Monitoring.

To monitor the position and profile of therapeutic carbon beams in real-time, in this paper, we proposed a system called HiBeam-T. The HiBeam-T is a time projection chamber (TPC) with forty Topmetal-II- CMOS pixel sensors as its readout. Each Topmetal-II- has 72 × 72 pixels with the size of 83 µm × 83 µm. The detector consists of the charge drift region and the charge collection area. The readout electronics comprise three Readout Control Modules and one Clock Synchronization Module. This Hibeam-T has a sensitive area of 20 × 20 cm and can acquire the center of the incident beams. The test with a continuous 80.55 MeV/u 12C6+ beam shows that the measurement resolution to the beam center could reach 6.45 µm for unsaturated beam projections.

Identification of ST1 reveals a selection involving hitchhiking of seed morphology and oil content during soybean domestication.

Seed morphology and quality of cultivated soybean (Glycine max) have changed dramatically during domestication from their wild relatives, but their relationship to selection is poorly understood. Here, we describe a semi-dominant locus, ST1 (Seed Thickness 1), affecting seed thickness and encoding a UDP-D-glucuronate 4-epimerase, which catalyses UDP-galacturonic acid production and promotes pectin biosynthesis. Interestingly, this morphological change concurrently boosted seed oil content, which, along with up-regulation of glycolysis biosynthesis modulated by ST1, enabled soybean to become a staple oil crop. Strikingly, ST1 and an inversion controlling seed coat colour formed part of a single selective sweep. Structural variation analysis of the region surrounding ST1 shows that the critical mutation in ST1 existed in earlier wild relatives of soybean and the region containing ST1 subsequently underwent an inversion, which was followed by successive selection for both traits through hitchhiking during selection for seed coat colour. Together, these results provide direct evidence that simultaneously variation for seed morphology and quality occurred earlier than variation for seed coat colour during soybean domestication. The identification of ST1 thus sheds light on a crucial phase of human empirical selection in soybeans and provides evidence that our ancestors improved soybean based on taste.

Protection of axonal integrity with 48 or 72 h of cerebral hypothermia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia is partially protective for neonatal hypoxic-ischemic encephalopathy (HIE). Damage to the white matter tracts is highly associated with adverse outcomes after HIE, but the effectiveness and optimal duration of hypothermia to attenuate axonal injury are unclear. METHODS: Near-term fetal sheep were randomized to sham control or cerebral ischemia for 30 min with normothermia or cerebral hypothermia from 3 to either 48 or 72 h. Sheep were killed after 7 days. SMI-312-labeled axons and myelin basic protein were quantified in the intragyral white matter of the first and second parasagittal gyri. RESULTS: Ischemia was associated with reduced axonal and myelin area fraction (p < 0.05); loss of axonal and myelin linearity (p < 0.05); and thin, sparse axons, with spheroids, compared to dense, linear morphology in sham controls and associated with induction of microglia in an amoeboid morphology. Both ischemia-48 h hypothermia and ischemia-72 h hypothermia improved axonal area fraction and linearity (p < 0.05), although abnormal morphological features were seen in a subset. Microglial induction was partially suppressed by ischemia-48 h hypothermia, with a ramified morphology. CONCLUSIONS: These data suggest that therapeutic hypothermia can alleviate post-ischemic axonopathy, in part by suppressing secondary inflammation.

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.

KEY POINTS: Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. ABSTRACT: Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z-axis and obscured detection by 2D techniques. Non-overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single 'rogue' RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t-tubules, which enabled quantification of dyadic and non-dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Porcine circovirus type 2 ORF5 protein induces endoplasmic reticulum stress and unfolded protein response in porcine alveolar macrophages.

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.

Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging.

Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.

Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes.

The contractile properties of cardiac myocytes depend on the calcium (Ca(2+)) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~57% of the perimeter of each myofibril. The average size of internal couplons is ~63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥80% of the total RyRs were detected in clusters containing ≥100 receptors. ~56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca(2+)-induced Ca(2+) release (100nm) and were grouped into 'superclusters'. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca(2+) levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2clusters/µm(3)) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca(2+) signalling.

Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes.

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photo-activated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution Photo-Activated Localization Microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and "travelling" events that spread between neighbouring RyR clusters. Travelling sparks exhibited up to 8 distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute ß-adrenergic stimulation augments intra-cluster RyR recruitment to generate larger events. In contrast, RyR "dispersion" during heart failure facilitates the generation of travelling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Prolonged ß-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure.

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged ß-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of ß-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

Nanoscale Organisation of Ryanodine Receptors and Junctophilin-2 in the Failing Human Heart.

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 µm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.

Etiology-Dependent Impairment of Diastolic Cardiomyocyte Calcium Homeostasis in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Whereas heart failure with reduced ejection fraction (HFrEF) is associated with ventricular dilation and markedly reduced systolic function, heart failure with preserved ejection fraction (HFpEF) patients exhibit concentric hypertrophy and diastolic dysfunction. Impaired cardiomyocyte Ca2+ homeostasis in HFrEF has been linked to disruption of membrane invaginations called t-tubules, but it is unknown if such changes occur in HFpEF. OBJECTIVES: This study examined whether distinct cardiomyocyte phenotypes underlie the heart failure entities of HFrEF and HFpEF. METHODS: T-tubule structure was investigated in left ventricular biopsies obtained from HFrEF and HFpEF patients, whereas cardiomyocyte Ca2+ homeostasis was studied in rat models of these conditions. RESULTS: HFpEF patients exhibited increased t-tubule density in comparison with control subjects. Super-resolution imaging revealed that higher t-tubule density resulted from both tubule dilation and proliferation. In contrast, t-tubule density was reduced in patients with HFrEF. Augmented collagen deposition within t-tubules was observed in HFrEF but not HFpEF hearts. A causative link between mechanical stress and t-tubule disruption was supported by markedly elevated ventricular wall stress in HFrEF patients. In HFrEF rats, t-tubule loss was linked to impaired systolic Ca2+ homeostasis, although diastolic Ca2+ removal was also reduced. In contrast, Ca2+ transient magnitude and release kinetics were largely maintained in HFpEF rats. However, diastolic Ca2+ impairments, including reduced sarco/endoplasmic reticulum Ca2+-ATPase activity, were specifically observed in diabetic HFpEF but not in ischemic or hypertensive models. CONCLUSIONS: Although t-tubule disruption and impaired cardiomyocyte Ca2+ release are hallmarks of HFrEF, such changes are not prominent in HFpEF. Impaired diastolic Ca2+ homeostasis occurs in both conditions, but in HFpEF, this mechanism for diastolic dysfunction is etiology-dependent.

A novel PCV2 ORF5-interacting host factor YWHAB inhibits virus replication and alleviates PCV2-induced cellular response.

Porcine circovirus type 2 (PCV2) infection causes porcine circovirus associated diseases (PCVAD) worldwide. Identification of host factors that interact with viral proteins is a fundamental step to understand the pathogenesis of PCV2. Our previous study reported that ORF5, a newly identified PCV2 viral protein supports PCV2 replication and interacts with multiple host factors. Here, we showed that a host factor YWHAB is an ORF5-interacting protein and plays essential roles during PCV2 infection. By using protein-protein interaction assays, we confirmed that YWHAB directly interacts with PCV2-ORF5 protein. We further showed that YWHAB expression was potently induced upon ORF5 overexpression and PCV2 infection. Remarkably, we found that the YWHAB strongly inhibited PCV2 replication, suggesting its role in defending PCV2 infection. By using the ectopic overexpression and gene knockdown approaches, we revealed that YWHAB inhibits PCV2-induced endoplasmic reticulum stress (ERS), autophagy, reactive oxygen species (ROS) production and apoptosis, suggesting its vital role in alleviating PCV2-induced cellular damage. Together, this study demonstrated that an ORF5-interacting host factor YWHAB affects PCV2 infection and PCV2-induced cellular response, which expands the current understanding of YWHAB biological function and might serves as a new therapeutic target to manage PCV2 infection-associated diseases.

Porcine Circovirus Type 2 ORF5 Protein Induces Autophagy to Promote Viral Replication via the PERK-eIF2α-ATF4 and mTOR-ERK1/2-AMPK Signaling Pathways in PK-15 Cells.

Porcine circovirus type 2 (PCV2) is the primary causative agent that causing porcine circovirus-associated disease (PCVAD). The open reading frame 5 (ORF5) protein is a newly discovered non-structural protein in PCV2, which the function in viral pathogenesis remains unknown. The aim of this study was to investigate the mechanism of PCV2 ORF5 protein on autophagy and viral replication. The pEGFP-tagged ORF5 gene was ectopic expressed in PK-15 cells and an ORF5-deficient PCV2 mutant strain (PCV2ΔORF5) were used to infected PK-15 cells. This study demonstrated that the ORF5 is essential for the of PCV2-induced autophagy. The ORF5 protein triggers the phosphorylation of PERK, eIF2α and the expression of downstream transcription factor ATF4. In addition, ORF5 protein activated the AMPK-ERK1/2-mTOR signaling pathways. These findings suggest that ORF5 play essential roles in the induction of autophagy by PCV2. We further revealed that PCV2 ORF5 promotes viral replication through PERK-eIF2α-ATF4 and AMPK-ERK1/2-mTOR pathways. In conclusion, we showed that PCV2 ORF5 induces autophagy to promote virus replication in PK-15 cells.

MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells.

Classical swine fever virus (CSFV) is one of the most important viral pathogens leading worldwide threats to pig industry. MicroRNAs (miRNAs) play important roles in regulating virus replication, but whether miRNAs affect CSFV infection is still poorly understood. In previous study, we identified four miRNAs that were down-regulated by CSFV in swine umbilical vein endothelial cells (SUVEC). In this study, miR-140, one of the most potently down-regulated genes was investigated. We found that the miRNA expression was significantly inhibited by CSFV infection. Subsequent studies revealed that miR-140 mimics significantly inhibited CSFV replication, while the inhibition of endogenous miR-140 enhanced CSFV replication. By using bioinformatics prediction, luciferase reporter system, real-time fluorescence quantitative PCR (RT-qPCR) and Western blot assays, we further demonstrated that miR-140 bind to the 3' UTR of Rab25 mRNA to regulate its expression. We also analyzed the expression pattern of Rab25 in SUVECs after CSFV infection. The results showed that CSFV infection induced Rab25 expression. Finally, Rab25 was found to promote CSFV replication. In conclusion, this study demonstrated that CSFV inhibits miR-140 expression and miR-140 inhibits replication by binding to host factor Rab25.

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a ß-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein.

Porcine circovirus type 2 (PCV2) is the infectious agent of postweaning multisystemic wasting syndrome (PMWS). The recently discovered open reading frame 5 (ORF5) in PCV2 genome encodes a non-structural protein. Previous study revealed that ORF5 protein inhibits cell proliferation and may interact with host transmembrane glycoprotein NMB (GPNMB). However, whether the GPNMB affects PCV2 replication and the underlying molecular mechanisms are still unknown. In this study, the transcriptome maps of PCV2-infected and ORF5-transfected porcine alveolar macrophages 3D4/2 (PAM) cells were profiled. The GPNMB gene was down-regulated in PCV2-infected and ORF5-transfected PAMs. By using glutathione S-transferase (GST) pull-down, co-immunoprecipitation (co-IP) and confocal microscopy approaches, we convincingly showed that PCV2 ORF5 protein interacts with GPNMB. Furthermore, by utilizing lentivirus mediated overexpression or knockdown approach, we showed that the cellular GPNMB significantly inhibits PCV2 replication and ORF5 expression. Moreover, GPNMB overexpressing leads to an increased Cyclin A expression and a reduced S phase, whereas GPNMB knockdown causes a decreased Cyclin A expression and a prolonged S phase. In conclusion, we identified a novel host factor GPNMB that interacts with PCV2 ORF5 protein and restricts PCV2 replication.

Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

Increased collagen within the transverse tubules in human heart failure.

AIMS: In heart failure transverse-tubule (t-tubule) remodelling disrupts calcium release, and contraction. T-tubules in human failing hearts exhibit increased labelling by wheat germ agglutinin (WGA), a lectin that binds to the dystrophin-associated glycoprotein complex. We hypothesized changes in this complex may explain the increased WGA labelling and contribute to t-tubule remodelling in the failing human heart. In this study we sought to identify the molecules responsible for this increased WGA labelling. METHODS AND RESULTS: Confocal and super-resolution fluorescence microscopy and proteomic analyses were used to quantify left ventricle samples from healthy donors and patients with idiopathic dilated cardiomyopathy (IDCM). Confocal microscopy demonstrated both WGA and dystrophin were located at t-tubules. Super-resolution microscopy revealed that WGA labelling of t-tubules is largely located within the lumen while dystrophin was restricted to near the sarcolemma. Western blots probed with WGA reveal a 5.7-fold increase in a 140 kDa band in IDCM. Mass spectrometry identified this band as type VI collagen (Col-VI) comprised of α1(VI), α2(VI), and α3(VI) chains. Pertinently, mutations in Col-VI cause muscular dystrophy. Western blotting identified a 2.4-fold increased expression and 3.2-fold increased WGA binding of Col-VI in IDCM. Confocal images showed that Col-VI is located in the t-tubules and that their diameter increased in the IDCM samples. Super-resolution imaging revealed Col-VI was restricted to the t-tubule lumen where increases were associated with displacement in the sarcolemma as identified from dystrophin labelling. Samples were also labelled for type I, III, and IV collagen. Both confocal and super-resolution imaging identified that these collagens were also present within t-tubule lumen. CONCLUSION: Increased expression and labelling of collagen in IDCM samples indicates fibrosis may contribute to t-tubule remodelling in human heart failure.