Confocal Line Scanning Research Articles

Abstract P3134: Impaired Calcium Homeostasis Underlies Lamin A/C Mutation R331Q Associated Atrial Fibrillation

Background: Atrial fibrillation (AF) is one of the first cardiac manifestations in patients harboring mutations in nucleoskeletal protein lamin A/C (LMNA). However, the underlying arrhythmogenic mechanisms are unclear. Objective: We hypothesize that impaired intracellular Ca 2+ -homeostasis contribute to LMNA mutation-associated atrial arrhythmogenesis. Methods: LMNA mutation R331Q was introduced in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 and cells were differentiated into atrial iPSC-derived cardiomyocytes (iPSC-CMs). L-type Ca 2+ current and intracellular Ca 2+ concentration were measured using patch-clamp technique and epifluorescence microscopy (Fluo-3), respectively. Results: R331Q iPSC-CMs demonstrated increased L-type Ca 2+ current density followed by increased amplitude of systolic Ca 2+ transient (CaT) compared to isogenic control (Fig. A). Sarcoplasmic reticulum (SR) Ca 2+ content was evaluated from caffeine-induced CaT amplitude and integrated Na + -Ca 2+ exchange current, and found to be greater in R331Q iPSC-CMs (Fig. B). Confocal line scan analysis revealed an increased Ca 2+ spark frequency in R331Q iPSC-CMs (Fig. C). In addition, by applying a stepwise increase in stimulation frequency (1-5 Hz), R331Q iPSC-CMs developed Ca 2+ alternans at lower threshold frequency (Fig. D). Conclusions: LMNA mutation R331Q is associated with disturbed intracellular Ca 2+ cycling, promoting occurrence of diastolic Ca 2+ sparks and arrhythmogenic Ca 2+ alternans. Collectively, our findings suggest that impaired Ca 2+ homeostasis may contribute to atrial arrhythmogenesis in patients harboring LMNA mutations.

Time-domain full-field optical coherence tomography with digital confocal line scanning.

Full-field optical coherence tomography (FF-OCT) is a camera-based interferometric microscopy technique that can image deep in tissue with high spatial resolution. However, the absence of confocal gating leads to suboptimal imaging depth. Here, we implement digital confocal line scanning in time-domain FF-OCT by exploiting the row-by-row detection feature of a rolling-shutter camera. A digital micromirror device (DMD) is used in conjunction with the camera to produce synchronized line illumination. An improvement in the SNR by an order of magnitude is demonstrated on a sample of a US Air Force (USAF) target mounted behind a scattering layer.

Abstract 11558: The Role of Oxidized CaMKII in Early Ischemia-Reperfusion Triggered Ventricular Arrhythmias

Introduction: Arrhythmias can complicate revascularization procedures after transmural infarctions and induce hemodynamic instability, but most cases can be adequately treated when they occur in hospital. However, arrhythmias that occur outside of hospital are potentially fatal. Arrhythmias in the early phase of reperfusion are caused by Ca 2+ overload and spontaneous diastolic release of Ca 2+ from the sarcoplasmic reticulum (SR). Indirect evidence suggests that reactive oxygen species (ROS) are important in this process by affecting the activity of key Ca 2+ handling proteins. Specifically, previous publications propose oxidation of the multifunctional Ca 2+ /calmodulin dependent protein kinase type II (CaMKII) to be a critical factor, and a potential therapeutic target. There is, however, a lack of data providing direct evidence for the importance of oxidized CaMKII (ox-CaMKII) for triggered arrhythmias in the early phase of reperfusion. Hypothesis: We hypothesized that ox-CaMKII increases diastolic SR Ca 2+ release events in the early phase of reperfusion. Methods: We evaluated the propensity for early reperfusion arrhythmias in mice resistant to oxidation of CaMKII in the heart (homozygous CaMKIIδ M281/282V mice) and wild type control mice (WT), by the use of Langendorff-perfused hearts exposed to ischemia-reperfusion, confocal line scan imaging of isolated cardiomyocytes transiently exposed to hypoxia and ischemia-like conditions, and immunoblotting. Results: Langendorff-perfused hearts from CaMKIIδ M281/282V mice showed no significant difference in ventricular arrhythmias compared to WT during the early reperfusion phase, both at baseline and during beta-adrenoceptor stimulation with isoprenaline. Immunoblotting of left ventricular tissue from perfused hearts revealed no significant differences in the abundance or phosphorylation levels of Ca 2+ -handling proteins. Ca 2+ imaging experiments without isoprenaline showed no significant difference in the incidence of spontaneous transients, Ca 2+ waves or Ca 2+ sparks between CaMKIIδ M281/282V and WT mice. Conclusions: Initial data indicate that ablation of CaMKII oxidation does not protect mouse hearts against arrhythmias in the early phase of reperfusion after ischemia.

Effects of atrial fibrillation on ventricular remodeling in the human heart

Abstract Atrial fibrillation (AF) is often found in patients with heart failure (HF). Clinical data indicated that the arrhythmic component of AF alone could contribute to left-ventricular (LV) dysfunction. However, the effects of non-tachycardic AF with arrhythmic excitation of the human LV, are unknown. We investigated human LV myocardium from patients with sinus rhythm (SR) or normofrequent AF (mean EF>50%, matched clinical data, derived from septal resections during AVR). In histological analysis we detected no difference between SR (n=17 patients) and AF patients (n=18) regarding the amount and distribution of fibrosis. We isolated human LV cardiomyocytes (CM) and studied cellular Ca-handling (Fura-2). Systolic Ca-transient amplitude of LV CM was reduced in patients suffering from AF (n=8 AF patients vs. 11 SR), while diastolic Ca-levels and Ca-transient kinetics were not significantly changed. These results were confirmed in LV CM from non-failing donors (NF) with AF (n=4 AF patients vs. 8 SR). For the standardized investigation of a normofrequent arrhythmia, we simulated AF in vitro by using arrhythmic (60 bpm, 40% beat-to-beat variability) or rhythmic (60 bpm) field stimulation. Human LV CM from NF SR patients (n=8) showed an impaired Ca-transient amplitude after 24h arrhythmic culture pacing without changes in diastolic Ca and Ca-transient kinetics. For studying a model suitable for more standardized chronic pacing, we utilized human iPSC cardiomyocytes (iPSC-CM) from healthy donors (n=6). After 7 days, arrhythmically paced iPSC-CM exhibited a reduced systolic Ca-transient amplitude, a trend towards a prolonged Ca-elimination time and a reduced sarcoplasmic reticulum Ca-load. Confocal line-scans of arrhythmically paced cells (Fluo-4 AM) showed an increased diastolic Ca-leak from the sarcoplasmic reticulum, possibly underlying the reduced Ca-load. Coupled with the Ca changes, cytosolic Na was elevated after arrhythmia. We found an increased late INa, which could explain the detrimentally altered Ca/Na-interplay. Accordingly, Patch-clamp experiments revealed a prolonged action potential duration after arrhythmia. We further elucidated the underlying mechanisms of this electrophysiological remodeling by showing that oxidative stress (H2O2, LPO) is increased in the LV of patients suffering from AF (n=6 AF patients vs. 6 SR), which was associated with an enhanced NOX2/-4 activity. Consecutively, Ca2+/calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized (CaMKII-Met281/282) in the LV of AF patients (n=7 AF patients vs. 7 SR) leading to an increased CaMKII activity, which adversely regulated EC-coupling protein phosphorylation including RyR2 hyperphosphorylation. Normofrequent arrhythmia/AF impairs human ventricular EC-coupling via increased oxidative stress and enhanced CaMKII. Thus, this translational study provides the first mechanistic characterization and the potential negative impact of isolated AF on the human LV. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Else Kröner-Fresenius-Stiftung (EKFS) and Deutsche Gesellschaft für Innere Medizin

B-PO01-008 MULTIPLEX GENOME EDITING FOR THE TREATMENT OF CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia disorder that can cause sudden cardiac death. Variants in the ryanodine receptor type 2 gene (RYR2) cause >60% of CPVT cases. These variants increase RyR2 calcium (Ca2+) leak from the sarcoplasmic reticulum that can trigger ventricular arrythmias. Current therapeutic options are partially effective. Previously, our lab showed that using the CRISPR/Cas9 genome editing system targeting a silent restriction site could disrupt a disease-causing allele preventing VT in a CPVT mouse model with RyR2-R176Q (RQ). To develop a single CRISPR/Cas9 vector that can be used to correct one of several CPVT variants. We designed single guide RNAs (sgRNA) to target the N-terminal RQ and central domain RyR2-R2474S (RS) mutation sites. We cloned vectors with sgRNA targeting RS or RQ, dual guides targeting RS + RQ, and no gRNA as control. We used an in-vitro luciferase assay to determine on and off target editing efficiencies. We generated AAV9 with CRISPR/Cas9 and injected SC p5 mice. 6 weeks after injection, mice underwent ECG stress testing with isoproterenol, caffeine, and programmed electrical stimulation. Isolated ventricular cardiomyocytes loaded with Fluo4 dye were analyzed with confocal line scanning. We found by targeting the RQ site with sgRNA/Cas9, 0/8 mice had pacing-induced VT compared to 6/8 control (p=0.03). Edited cardiomyocytes showed a significant reduction in Ca2+ spark frequency (p<0.01). When targeting the RS site with sgRNA/Cas9, 0/7 mice had inducible VT compared to 6/7 control (p=0.04). The luciferase assay showed similar editing efficiencies with sgRNA and dual gRNA (Δ <10%) with preserved specificity to mutant alleles (>80%). In ongoing experiments, we expect similar efficacies in CPVT mice treated with dual gRNA/Cas9. CRISPR/Cas9 gene editing can treat CPVT by specifically targeting causative mutation sites in multiple channel domains, reducing mutant allele expression and preventing Ca2+ leak. Through successful targeting of the mutation sites, we have promising preclinical data for gene editing as a permanent cure for CPVT.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

Nanoscale Study of Calcium Handling Remodeling in Right Ventricular Cardiomyocytes Following Pulmonary Hypertension.

Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca2+ transients along with a higher local Ca2+-release activity. These Ca2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle.

Atrial fibrillation impairs ventricular function by altering excitation-contraction coupling in the human heart

Abstract Atrial fibrillation (AF) often co-exists in patients with heart failure (HF). Recent clinical evidence suggests that the arrhythmic component of AF alone may contribute to ventricular dysfunction. However, the pathophysiological effects of a non-tachycardic AF on the human ventricle are unknown. To investigate the effects of normofrequent AF on the human ventricle we investigated ventricular myocardium from patients with preserved ejection fraction with sinus rhythm (SR) or AF in the absence of HF (compensated hypertrophy, EF&amp;gt;50%, matched clinical characteristics). In histological analysis we detected no difference between SR (n=9) vs. AF (n=6) regarding the amount and distribution of fibrosis. For functional investigation, Ca-handling was studied (Fura-2 AM). While systolic Ca-transient amplitude was in trend reduced in isolated human ventricular AF cardiomyocytes, we found a significantly prolonged Ca-elimination time (n=17–22 cells/4 pat.). Using caffeine application, a decreased SR Ca-load in AF was detected, which may be explained by a significant decrease in SERCA2a activity (ksys-kCaff, n=10–12/4 pat.). Patch-clamp experiments revealed a prolonged action potential duration in AF cardiomyocytes (n=5/15 cells). For the standardized evaluation of the mechanisms of persistent normofrequent arrhythmia, we simulated AF in vitro by using arrhythmic (1 Hz, 40% R-R-variability) or rhythmic (1 Hz) field stimulation. We performed contractility experiments using in-toto isolated human ventricular trabeculae from explanted human hearts. After 8h of pacing, arrhythmically stimulated human trabeculae showed a significantly reduced systolic force, an increase in diastolic tension and a prolonged relaxation (n=11–12 trabeculae/11 pat.). For studying the cellular effects of persistent normofrequent arrhythmia in a model suitable for chronic pacing (up to 7 days), we utilized human iPSC cardiomyocytes (iPSC-CM) from healthy donors (n=6). After 7 days, arrhythmic paced iPSC-CM showed a significantly reduced systolic Ca-transient amplitude, a prolonged Ca-elimination time (n=35/45 cells) as well as a reduced SR Ca-load and a trend towards a lower SERCA2a activity compared to control (n=11 cells). Confocal line-scans (Fluo-4 AM) showed an increased diastolic SR Ca-release, which might also explain the reduced SR Ca-content (n=45/35 cells). Moreover, in irregularly paced iPSC-CM we found significant increased levels of cytosolic Na (n=69 cells each) and in patch-clamp experiments a significantly prolonged action potential duration (n=14/11 cells/3 diff.). This study demonstrates that a normofrequent arrhythmic ventricular excitation as it occurs in AF impairs human ventricular myocardial function by altering cardiomyocyte excitation-contraction coupling. Thus, this study provides the first translational mechanistic characterization and the potential negative impact of isolated AF in the absence of tachycardia on the human ventricle. Funding Acknowledgement Type of funding source: None

Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport

Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A′, that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG. We hypothesized that these effects are linked and might be explained by a cisternal-specific delay in cargo transport. In pulse chase experiments using a deconvolved, confocal line scanning approach to score the distribution of the tsO45 mutant of VSVG protein in Rab6 depleted cells, we found that anterograde transport at 32 °C, permissive conditions, through the Golgi apparatus was locally delayed, almost tenfold, between medial and trans Golgi cisterna. Cis to medial transport was nearly normal as was trans Golgi to TGN transport. TGN exit was unaffected by Rab6 depletion. These effects were the same with either of two siRNAs. Similar intra-Golgi transport delays were seen at 37 °C with RUSH VSVG or a RUSH GPI-anchored construct using a biotin pulse to release the marker proteins from the ER. Using 3D-SIM, a super resolution approach, we found that RUSH VSVG transport was delayed pre-trans Golgi. These visual approaches suggest a selective slowing of anterograde transport relative to 3 different marker proteins downstream of the trans Golgi. Using a biochemical approach, we found that the onset of VSVG endoglycosidase H resistance in Rab6 depleted cells was delayed. Depletion of neither Rab6A or Rab6A′ isoforms alone had any effect on anterograde transport through the Golgi suggesting that Rab6A and Rab6A′ act coordinately. Delayed cargo transport conditions correlate strongly with a proliferation of Golgi cisternae observed in earlier electron microscopy. Our results strongly indicate that Rab6 is selectively required for rapid anterograde transport from the medial to trans Golgi. We suggest that the observed correlation with localized cisternal proliferation fits best with a cisternal progression model of Golgi function.

Line scanning mechanical streak camera for phosphorescence lifetime imaging.

Phosphorescence lifetime measurement holds great importance in life sciences and material sciences. Due to the long lifetime of phosphorescence emission, conventional approaches based on point scanning time-domain recording suffer from long recording time and low signal-to-noise ratio (SNR). To overcome these difficulties, we developed a line scanning mechanical streak camera for parallel and high SNR imaging. This design offers three key advantages. First, hundreds to thousands of pixels can be recorded simultaneously at high throughput. Second, hundreds of excitation can be accumulated on a single camera frame and read out at once with high quantum efficiency (QE) and low read noise. Third, the system is very simple, only requiring a camera and a scanner. Using a confocal line scanning configuration, we imaged samples of various lifetime ranging from tens of nanoseconds to hundreds of microseconds, which demonstrated the versatility and advantages of this method.

Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young’s modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 μN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 μN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

P6279Mitochondria targeted catalase overexpression protects against beta-adrenergic receptor mediated cardiac remodeling in mice

Abstract Background Beta-adrenergic receptor signaling is widely recognized as a fundamental component in the pathogenesis of chronic heart failure. However, the mechanisms behind beta-adrenergic receptor-mediated remodeling in cardiomyocytes and the myocardium are not fully understood. Oxidative stress has been proposed as a central pathophysiological mediator in cardiovascular disease and heart failure. The triggers and sources of oxidative stress in heart failure remain unclear. In this study we use mice with mitochondria-targeted overexpression of the antioxidant enzyme catalase (mCAT) to link beta-adrenergic receptor-mediated stress to mitochondrial reactive oxidative species (ROS) and the progression of heart failure. Hypothesis Mitochondrial ROS, induced by beta-adrenergic receptor-activation, is a mediator in the progression of the heart failure phenotype. Methods mCAT and wild type mice (n=10) were administered the non-selective beta-adrenergic agonist Isoprotenerol (Iso; 50mg/kg/day) through subcutaneous osmotic pumps for 3 weeks. Hearts were taken for biochemistry (western blotting, qPCR). Cardiomyocytes were isolated and loaded with Fluo-3 AM to study intracellular Ca2+ transients and fractional shortening using confocal line scan microscopy. All experiments were performed in accordance with the Stockholm ethical committee for animal research. Results and conclusions The WT mice displayed an increased heart/body weight ratio following chronic Iso administration. In contrast, mCAT mice displayed resistance to Iso-induced cardiac hypertrophy (p&lt;0.05). Furthermore, chronic Iso exposure in WT mice induced increased ROS-dependent post-translational protein modifications, impaired cardiomyocyte Ca2+ handling and reduced contractility in isolated cardiomyocytes (p&lt;0.05). Cardiomyocytes from mCAT mice did not display the deleterious effects of chronic Iso exposure on cardiomyocyte Ca2+ and contractility. Our study demonstrates that beta-adrenergic receptor stimulation-induced remodeling of the heart, which is similar to what is seen in heart failure, can be prevented by overexpressing catalase in the mitochondria. This indicates an important role of mitochondrial ROS in the link between adrenergic signaling and the development of cardiomyopathy and heart failure. Acknowledgement/Funding Hjärtlungfonden

Late Ca2+ Sparks and Ripples During the Systolic Ca2+ Transient in Heart Muscle Cells.

The development of a refractory period for Ca2+ spark initiation after Ca2+ release in cardiac myocytes should inhibit further Ca2+ release during the action potential plateau. However, Ca2+ release sites that did not initially activate or which have prematurely recovered from refractoriness might release Ca2+ later during the action potential and alter the cell-wide Ca2+ transient. To investigate the possibility of late Ca2+ spark (LCS) activity in intact isolated cardiac myocytes using fast confocal line scanning with improved confocality and signal to noise. We recorded Ca2+ transients from cardiac ventricular myocytes isolated from rabbit hearts. Action potentials were produced by electric stimulation, and rapid solution changes were used to modify the L-type Ca2+ current. After the upstroke of the Ca2+ transient, LCSs were detected which had increased amplitude compared with diastolic Ca2+ sparks. LCS are triggered by both L-type Ca2+ channel activity during the action potential plateau, as well as by the increase of cytosolic Ca2+ associated with the Ca2+ transient itself. Importantly, a mismatch between sarcoplasmic reticulum load and L-type Ca2+ trigger can increase the number of LCS. The likelihood of triggering an LCS also depends on recovery from refractoriness that appears after prior activation. Consequences of LCS include a reduced rate of decline of the Ca2+ transient and, if frequent, formation of microscopic propagating Ca2+ release events (Ca2+ ripples). Ca2+ ripples resemble Ca2+ waves in terms of local propagation velocity but spread for only a short distance because of limited regeneration. These new types of Ca2+ signaling behavior extend our understanding of Ca2+-mediated signaling. LCS may provide an arrhythmogenic substrate by slowing the Ca2+ transient decline, as well as by amplifying maintained Ca2+ current effects on intracellular Ca2+ and consequently Na+/Ca2+ exchange current.

线扫描共聚焦成像技术在生物医学成像中的应用

线扫描共聚焦成像技术在生物医学成像中的应用

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation-contraction coupling, and inefficient Ca-induced Ca release remain major limitations. Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation-contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation-contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation-contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Design of high-resolution wide field of view confocal line scanning laser microscopy

Commercial confocal microscopy usually utilizes two-dimensional galvanometer to scan specimen, the frame rate is limited within 30 f/s, and commercial confocal microscopy is 10 f/s or lower. In order to improve the imaging speed of confocal system, and meet the needs of real-time observation of in vivo imaging, in the high-speed confocal line scanning laser microscopy, the sample is illuminated by one-dimensional scanning laser beam. The imaging speed is greatly improved. At the same time, a slit is placed before the linear array CCD to filter out to the non-focused plane stray light and improve the image quality. Experiments show that the optical magnification is 55, horizontal resolution is higher than 2.2 μm, and the frame rate is up to 50 frame per second when the linear CCD scans with 28 kHz horizontal frequency and 512 pixels×2048 pixels image resolution, image experiment of plant and animal cells prove that this imaging system could be used in ex vivo or in vivo cell image.

Light-sheet microscopy by confocal line scanning of dual-Bessel beams.

We have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.

Dyssynchronous calcium removal in heart failure-induced atrial remodeling.

We tested the hypothesis that in atrial myocytes from a rabbit left ventricular heart failure (HF) model, spatial inhomogeneity and temporal dyssynchrony of Ca removal during excitation-contraction coupling together with increased Na/Ca exchange (NCX) activity generate a substrate for proarrhythmic Ca release. Ca removal occurs via Ca reuptake into the sarcoplasmic reticulum and extrusion via NCX exclusively in the cell periphery since rabbit atrial myocytes lack transverse tubules. Ca removal kinetics were assessed by the time constant τ of decay of local peripheral subsarcolemmal (SS) and central (CT) action potential (AP)-induced Ca transients (CaTs) recorded in confocal line scan mode (using Fluo-4). Spatial and temporal dyssynchrony of Ca removal was quantified by CV TAU, defined as the standard deviation of local τ along the transverse cell axis divided by mean τ. In normal cells CT CaT decline was slower compared with the SS domain, while in HF cells decline was accelerated, became equal in SS and CT regions, and a significant increase of CV TAU indicated an increased Ca removal dyssynchrony. In HF atrial cells NCX upregulation was accompanied by an overall higher incidence of spontaneous Ca waves and a higher propensity of arrhythmogenic Ca waves, defined as waves that triggered APs due to NCX-mediated membrane depolarization. NCX inhibition normalized CV TAU in HF atrial cells and decreased the propensity of Ca waves. In summary, HF atrial myocytes show accelerated but dyssynchronous diastolic Ca removal and altered sarcoplasmic reticulum Ca-ATPase (SERCA) and NCX activity that result in increased susceptibility to arrhythmia.

Hypertensive retinopathy and cerebral small vessel disease in Amerindians living in rural Ecuador: The Atahualpa Project

BackgroundDiagnosis of cerebral small vessel disease (SVD) is a challenge in remote areas where MRI is not available. Hypertensive retinopathy (HTRP) has shown to correlate with SVD in different ethnic groups, but there is no information from indigenous Latin American people. We assessed the usefulness of retinal photographs to detect cases with SVD among Amerindians living in rural Ecuador. MethodsAtahualpa residents aged ≥60years with arterial hypertension or prehypertension were identified during a door-to-door survey. A confocal line scanning laser ophthalmoscope was used to identify and grade HTRP (according to the Keith–Wagener–Barker classification). MRIs were read with attention to the presence of white matter hyperintensities (WMH) of presumed vascular origin and lacunar infarcts. Using logistic regression models, we evaluated whether HTRP was independently associated with neuroimaging signatures of SVD. ResultsOf 323 eligible candidates, 241 (75%) were enrolled. MRI readings revealed moderate-to-severe WMH in 49 (20%) cases and lacunar infarcts in 29 (12%). HTRP Grade 1 was noticed in 90 (37%) individuals and Grade 2–3 in 42 (17%). After adjusting for demographics and cardiovascular risk factors, multivariate analyses showed a significant association between Grades 2–3 HTRP and moderate-to-severe WMH (OR: 3.87, 95% C.I.: 1.64–9.13) but not with lacunar infarcts (OR: 2.22, 95% C.I.: 0.83–5.92). ConclusionAmerindians with HTRP Grades 2–3 are almost four times more likely to have SVD-related subcortical damage than those with no- or only Grade 1-HTRP. Retinal photographs might allow recognition of people who need further investigation and therapy.

Abstract 16781: Dyssynchronous Ca Removal in Atrial Cardiac Myocytes From Failing Hearts

In heart failure atrial remodeling (AR) leads to impaired contractility and relaxation as well as, atrial fibrillation and contributes to negative clinical outcomes. In myocytes cytosolic Ca removal after systolic release is primarily achieved by SERCA pump and Na/Ca exchange (NCX) activity, allowing atrial relaxation. In atrial myocytes that lack a transverse tubule system, NCX is located exclusively in the cell periphery where it is exposed to high concentrations of Ca during the cardiac cycle. Previously we found that the relative contribution of NCX to Ca removal was increased in AR and was associated with a higher propensity of arrhythmogenic Ca waves, i.e. Ca waves that triggered action potentials (APs). We hypothesize that dyssynchrony of Ca removal globally (whole cell) and locally (peripheral subsarcolemmal (SS) and central (CT) domains) is responsible for impaired relaxation and increased arrhythmogenicity. We used confocal line scan imaging and Ca sensitive dyes (Fluo-4/AM) to investigate Ca removal in remodeled atrial myocytes from a rabbit left ventricular volume-pressure overload systolic heart failure model. In control atrial myocytes Ca removal (assessed by the time constant of decay (TAU) of local SS and CT AP-induced Ca transients) is faster in the cell center (CT region) compared to the SS domain (342±32 vs. 393±31 ms, n=10 cells). In AR myocytes however, CT TAU was not different from SS TAU (196±11 vs. 190+14 ms, n=10 cells). In addition, in AR cells local dyssynchrony (defined as the standard deviation of TAU divided by mean TAU) in the CT domain was significantly increased compared to control (0.11±0.03 vs. 0.04±0.01, n=10 AR and CTRL cells). However despite the increased dyssynchrony, CT Ca removal was 50% and SS Ca removal 56% faster in AR myocytes as compared to control cells (n=10 AR and CTRL cells). In summary, AR myocytes show accelerated but dyssynchronous diastolic Ca removal that may result in impaired relaxation and increased susceptibility to rhythm disorders.