Brian M. Hagen

Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

STED Live Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Golgi Export of the Kir2.1 Channel Is Driven by a Trafficking Signal Located within Its Tertiary Structure

Mechanisms that are responsible for sorting newly synthesized proteins for traffic to the cell surface from the Golgi are poorly understood. Here, we show that the potassium channel Kir2.1, mutations in which are associated with Andersen-Tawil syndrome, is selected as cargo into Golgi export carriers in an unusual signal-dependent manner. Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, Golgi exit of Kir2.1 is dictated by residues that are embedded within the confluence of two separate domains. This signal patch forms a recognition site for interaction with the AP1 adaptor complex, thereby marking Kir2.1 for incorporation into clathrin-coated vesicles at the trans-Golgi. The identification of a trafficking signal in the tertiary structure of Kir2.1 reveals a quality control step that couples protein conformation to Golgi export and provides molecular insight into how mutations in Kir2.1 arrest the channels at the Golgi.

Proton-sensing Ca2+ Binding Domains Regulate the Cardiac Na+/Ca2+ Exchanger

The cardiac Na+/Ca2+ exchanger (NCX) regulates cellular [Ca2+]i and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C2 regulatory domains, Ca2+ binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, INCX, whereas [H+]i was varied using an ammonium chloride “rebound” method at constant extracellular pH 7.4. At pHi = 7.2 and [Ca2+]i < 120 nm, INCX was less than 4% that of its maximally Ca2+-activated value. INCX increases steeply at [Ca2+]i between 130–150 nm with a Hill coefficient (nH) of 8.0 ± 0.7 and K0.5 = 310 ± 5 nm. At pHi = 6.87, the threshold of Ca2+-dependent activation of INCX was shifted to much higher [Ca2+]i (600–700 nm), and the relationship was similarly steep (nH = 8.0±0.8) with K0.5 = 1042 ± 15 nm. The Vmax of Ca2+-dependent activation of INCX was not significantly altered by low pHi. The Ca2+ affinities for CBD1 (0.39 ± 0.06 μm) and CBD2 (Kd = 18.4 ± 6 μm) were exquisitely sensitive to [H+], decreasing 1.3–2.3-fold as pHi decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C2 regulatory domains CBD1 and CBD2.

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.

Short-Term Mechanical Unloading With Left Ventricular Assist Devices After Acute Myocardial Infarction Conserves Calcium Cycling and Improves Heart Function

OBJECTIVES This study sought to demonstrate that short-term cardiac unloading with a left ventricular (LV) assist device (LVAD) after acute myocardial infarction (MI) can conserve calcium cycling and improve heart function. BACKGROUND Heart failure secondary to MI remains a major source of morbidity and mortality. Alterations in calcium cycling are linked to cardiac dysfunction in the failing heart. METHODS Adult Dorsett hybrid sheep underwent acute MI and were mechanically unloaded with an axial-flow LVAD (Impella 5.0) for 2 weeks (n = 6). Six sheep with MI only and 4 sham sheep were used as controls. All animals were followed for 12 weeks post-MI. Regional strains in the LV were measured by sonomicrometry. Major calcium-handling proteins (CHPs), including sarco-/endoplasmic reticulum calcium ATPase-2α (SERCA-2α), Na(+)-Ca(2+) exchanger-1, and phospholamban, and Ca(2+)-ATPase activity were investigated. The electrophysiological calcium cycling in single isolated cardiomyocytes was measured with the patch-clamp technique. The related ultrastructures were studied with electron microscopy. RESULTS LVAD unloading alleviated LV dilation and improved global cardiac function and regional contractility compared with the MI group. The regional myocardial strain (stretch) was minimized during the unloading period and even attenuated compared with the MI group at 12 weeks. Impaired calcium cycling was evident in the adjacent noninfarcted zone in the MI group, whereas CHP expression was normalized and Ca(2+)-ATPase activity was preserved in the LVAD unloading group. The electrophysiological calcium cycling was also conserved, and the ultrastructural damage was ameliorated in the unloaded animals. CONCLUSIONS Short-term LVAD unloading may conserve calcium cycling and improve heart function during the post-infarct period.

Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology

To avoid spectral interference with common fluorophores in multicolor fluorescence microscopy, a fluid-phase tracer with excitation and emission in the violet end of the visible spectrum is desirable. CM-pyranine is easily synthesized and purified. Its excitation and emission maxima at 401.5 nm and 428.5 nm, respectively, are well suited for excitation by 405-nm diode lasers now commonly available on laser-scanning microscopes. High fluorescence quantum efficiency (Q = 0.96) and strong light absorption (ε405 > 25,000 M-1cm-1) together make CM-pyranine the brightest violet aqueous tracer. The fluorescence spectrum of CM-pyranine is invariant above pH 4, which makes it a good fluid-phase marker in all cellular compartments. CM-pyranine is very photostable, is retained for long periods by cells, does not self-quench, and has negligible excimer emission. The sum of its properties make CM-pyranine an ideal fluorescent tracer. The use of CM-pyranine as a fluid-phase marker is demonstrated by multicolor confocal microscopy of cells that are also labeled with lipid and nuclear markers that have green and red fluorescence emission, respectively.

Superresolution Subspace Signaling

Highly localized activity of ion channels in vascular endothelial cells controls the contraction of surrounding muscle cells and blood flow. Exquisitely local signaling provides robust, precise, and rapid communication in single cells. Nanometer-wide regions constitute the signaling conduit that separates interacting ion channels, organelles, and sensor and effector proteins. Such signaling domains are primary features of many systems, including those responsible for excitation-contraction coupling in the heart (1, 2), smooth muscle tone in the vasculature (3, 4), and Ca2+ signaling in neurons (5). These intimate pathways, also known as “fuzzy spaces” (1), “subspaces” (6), or “nanoscopic spaces,” permit efficient signaling with rapid and reliable information transfer, yet are too small to be seen with a standard optical microscope. Such superresolution domains may become experimentally visible with the implementation of optical superresolution microscopy (7–9). On page 597 in this issue, Sonkusare et al. (10) identify a specific and broadly important local signaling organization in small mesenteric arteries (∼100 µm in diameter) that uses nano meter-wide communication regions to regulate local vascular blood flow.

Nuclear export of misfolded SOD1 mediated by a normally buried NES-like sequence reduces proteotoxicity in the nucleus

Over 170 different mutations in the gene encoding SOD1 all cause amyotrophic lateral sclerosis (ALS). Available studies have been primarily focused on the mechanisms underlying mutant SOD1 cytotoxicity. How cells defend against the cytotoxicity remains largely unknown. Here, we show that misfolding of ALS-linked SOD1 mutants and wild-type (wt) SOD1 exposes a normally buried nuclear export signal (NES)-like sequence. The nuclear export carrier protein CRM1 recognizes this NES-like sequence and exports misfolded SOD1 to the cytoplasm. Antibodies against the NES-like sequence recognize misfolded SOD1, but not native wt SOD1 both in vitro and in vivo. Disruption of the NES consensus sequence relocalizes mutant SOD1 to the nucleus, resulting in higher toxicity in cells, and severer impairments in locomotion, egg-laying, and survival in Caenorhabditis elegans. Our data suggest that SOD1 mutants are removed from the nucleus by CRM1 as a defense mechanism against proteotoxicity of misfolded SOD1 in the nucleus. DOI: http://dx.doi.org/10.7554/eLife.23759.001

Calcium Spark and Wave Behavior in the Intact Rat Heart

Spontaneous Ca2+ release from the sarcoplasmic reticulum and subsequent activation of Ca2+-sensitive conductances have been postulated as mechanisms to develop arrthymogenic ectopic ventricular beats. Previous studies investigating subcellular Ca2+ signaling in the whole-heart have been limited by low temporal and/or spatial resolution. To overcome these obstacles we used an ultra-fast confocal microscope to image the epicardial surface of a Langendorff-perfused heart loaded with the Ca2+ indicator fluo-2. Myocardium displayed normal Ca2+ transients when paced at low frequencies, and sub-cellular alternans at high frequencies (>5Hz). Importantly a pause in the pacing protocol unmasked the development of Ca2+ sparks and spark-triggered Ca2+ waves. The abundance of pause-dependent Ca2+ sparks and waves increased with pacing frequency. Occasionally during the pause after rapid pacing, spiral Ca2+ waves could be observed in a small population of myocytes. Attached figure shows a single myocyte (cell borders indicated by a solid line, top-left) with time; fluorescence depicted on inverted grayscale. The dotted line highlights the spiral Ca2+ wave. This study provides new information on rate-dependent Ca2+ instability in normal rat hearts and lays the foundation for investigations of Ca2+ instabilities due to genetic and acquired heart diseases.View Large Image | View Hi-Res Image | Download PowerPoint Slide