The peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), were examined using single fluorophore localization-based super-resolution microscopy in rat ventricular myocytes. JPH2 was strongly associated with RyR clusters. Estimates of the colocalizing fraction of JPH labeling with RyR was ∼90% within 30 nm of RyR clusters. This is comparable to fractions estimated from confocal data (∼87%). Similarly, most RyRs were associated with JPH2 labeling in super-resolution images (∼81% within 30 nm of JPH2 clusters). The shape of associated RyR clusters and JPH2 clusters were very similar, but not identical, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked.

1. It is apparent from the literature that there are significant differences in excitation-contraction coupling between species, particularly in the density of calcium transporting proteins in the t-system and sarcoplasmic reticulum (SR) Ca(2+) release channels. Unfortunately, there is a lack of information as to how the principal structures that link electrical excitation to the activation of calcium-induced calcium release (CICR) are different between human and animal models (particularly rat). 2. Comparison of wheat germ agglutinin and caveolin-3 labelling revealed a non-uniform distribution of surface membrane glycosylation in the rat, rabbit and human, and that the rat t-system appeared more complex in geometry than the latter species. Analysis of the t-system skeleton showed that the t-system was highly branched in the rat compared with that of the human (0.8 ± 0.08 and 0.2 ± 0.07 branch points per μm(2), respectively; P < 0.001). 3. We also compared the distribution of contractile machinery, sodium-calcium exchange, SR and ryanodine receptors (RyR) in rat and human. F-Actin and RyR labelling was used to estimate the area of contractile apparatus supplied by each RyR cluster. In the rat, each RyR cluster supplied an average cross-sectional area of contractile machinery of 0.36 ± 0.03μm(2) compared with 0.49 ± 0.04 μm(2) in human (P = 0.048). Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a) labelling showed that the SR formed a tight network of loops surrounding contractile fibrils that were denser than the t-tubule network, but otherwise appeared similar in both species. 4. In general, the results show a higher density in structures involved in CICR in the rat compared with human.

Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk. Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

[This corrects the article on p. e17901 in vol. 6.].

BACKGROUND The cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure. METHODS AND RESULTS The organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearson's correlation coefficient in failing hearts. CONCLUSIONS The results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.

AIMS Therapeutic advances in prevention and treatment of myocardial infarction (MI) have decreased patient mortality and increased concern about efficient repair and scar formation, processes that are necessary to attenuate complications such as adverse remodelling and heart failure. Since the rapid accumulation and activity of cardiac fibroblasts is critical for proper scar formation, we hypothesized that infarct fibroblasts are generated by a cardiac-resident progenitor cell population. METHODS AND RESULTS We found that infarct fibroblasts in C57BL/6 mice are generated by a mesenchymal stem cell (MSC) population that responds robustly to injury by proliferating and accumulating in the infarct. We report that stem cell-derived fibroblasts contribute to the formation of a scar after an infarction by differentiating into matrix-producing fibroblasts closely associated with fibrillar collagen in the infarct. Further characterization of these cells revealed a heterogenous population with expression of both stem cell and canonical cardiac fibroblast markers, suggesting that some have a commitment to the fibroblast phenotype. Our in vitro study of these cells shows that they have extended self-renewal capability and express the primitive marker Nanog. In keeping with these observations, we also report that these cells are multipotent and differentiate readily into fibroblasts as well as other mesenchymal lineages. CONCLUSION Cells with the properties of MSCs participate in wound healing after MI in the adult heart.

Intracellular Ca(2+) dynamics provides excitation-contraction coupling in cardiac myocytes. Under pathological conditions, spontaneous Ca(2+) release events can lead to intracellular Ca(2+) travelling waves, which can break, giving transitory or persistent intracellular re-entrant Ca(2+) scroll waves. Intracellular Ca(2+) waves can trigger cellular delayed after-depolarizations of membrane potential, which if they occur in a cluster of a few hundred neighbouring myocytes may lead to cardiac arrhythmia. Quantitative prediction of the initiation and propagation of intracellular Ca(2+) waves requires the dynamics of Ca(2+)-induced Ca(2+) release, and the intracellular spatial distribution of Ca(2+) release units (CRUs). The spatial distribution of ryanodine receptor clusters within a few sarcomeres was reconstructed directly from confocal imaging measurements. It was then embedded into a three-dimensional ventricular cell model, with a resting membrane potential and simple stochastic Ca(2+)-induced Ca(2+) release dynamics. Isotropic global Ca(2+) wave propagation can be produced within the anisotropic intracellular architecture, by isotropic local Ca(2+) diffusion, and the branching Z-disc structure providing inter Z-disc pathways for Ca(2+) propagation. The branching Z-disc provides a broader spatial distribution of ryanodine receptor clusters across Z-discs, which reduces the likelihood of wave initiation by spontaneous Ca(2+) releases. Intracellular Ca(2+) dynamics during catecholaminergic polymorphic ventricular tachycardia (CPVT) was simulated phenomenologically by increasing the Ca(2+) sensitivity factor of the CRU, which results in an increased rate of Ca(2+) release events. Flecainide has been shown to prevent arrhythmias in a murine model of CPVT and in patients. The modelled actions of flecainide on the time course of Ca(2+) release events prevented the initiation of Ca(2+) waves.

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca(2+) with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca(2+) transient due to reduced ability of the L-type Ca(2+) channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca(2+), with some cellular regions failing to elicit Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca(2+) sparks. Monte-Carlo simulations of spark activation by L-type Ca(2+) current supported the idea that the decreased synchrony of Ca(2+) spark production associated with the loss of the notch could be explained by reduced Ca(2+) influx from open Ca(2+) channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca(2+) release in heart failure, without changes in (for example) SR Ca(2+)-ATPase uptake.

Detection of specific sequences of target DNA is of high importance in many fields, especially in medicinal diagnostics. DNA sensors should exhibit fast response to minute concentrations of the target sequence and have the ability to distinguish single-base mismatches from fully complementary target. This study focuses on the response of an electrochemical, CdTe nanoparticle-modified hairpin DNA sensor. The stem-loop structured probes and the blocking poly(ethylene glycol)(PEG) molecules were self-assembled on the gold electrode through S-Au bonding, to form a mixed monolayer employed as the sensing platform. Water-soluble CdTe nanoparticles were covalently attached to the hairpin probes (HPPs) and impedance spectroscopy was used for investigation of the electron transfer processes at a modified gold electrode before and after hybridization with the target DNA. The sensor showed reliable and sensitive detection of 4.7 fM of target. Although the selectivity of the sensor towards one-base mismatch targets needs to be improved, discrimination of non-complementary targets was achieved.

Localization microscopy techniques based on localizing single fluorophore molecules now routinely achieve accuracies better than 30 nm. Unlike conventional optical microscopies, localization microscopy experiments do not generate an image but a list of discrete coordinates of estimated fluorophore positions. Data display and analysis therefore generally require visualization methods that translate the position data into conventional images. Here we investigate the properties of several widely used visualization techniques and show that a commonly used algorithm based on rendering Gaussians may lead to a 1.44-fold loss of resolution. Existing methods typically do not explicitly take sampling considerations into account and thus may produce spurious structures. We present two additional visualization algorithms, an adaptive histogram method based on quad-trees and a Delaunay triangulation based visualization of point data that address some of these deficiencies. The new visualization methods are designed to suppress erroneous detail in poorly sampled image areas but avoid loss of resolution in well-sampled regions. A number of criteria for scoring visualization methods are developed as a guide for choosing among visualization methods and are used to qualitatively compare various algorithms.