Line-scanning Confocal Microscopy Research Articles

An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging

Local Ca2+ transients such as puffs and sparks form the building blocks of cellular Ca2+ signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500framess−1) imaging of subcellular Ca2+ signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1Gbmin−1) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca2+ events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca2+ release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events.

BK Channel-Mediated Relaxation of Urinary Bladder Smooth Muscle: A Novel Paradigm for Phosphodiesterase Type 4 Regulation of Bladder Function

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

Atrial endothelin-1 signalling in hypertensive rats

Purpose: Hypertension induces cardiac remodelling at the structural and functional level. Endothelin-1 (ET-1) may contribute to cardiac remodelling. It is not known, however, whether atrial ET-1 signalling is altered in hypertension. Here we tested the hypothesis that ET-1 signalling is augmented in the atria of spontaneously hypertensive rats (SHR) and that this may contribute to hypertension-induced atrial arrhythmias. Methods: SHR and control Wistar-Kyoto (WKY) rats were studied at 6-8 months of age. Hearts were removed for morphometric analysis. Atria were snap frozen and analyzed for mRNA and protein expression by qPCR and Western blotting. Isolated atrial myocytes were field stimulated in the presence of 50 nM ET-1, and Ca transients (CaTs) were characterized at the global or subcellular level by epifluorescence (Fura-2/AM) and line scan confocal microscopy (Fluo-4/AM). To mimic atrial tachycardia, atrial tissue slices were stimulated at 5 Hz for up to 20 h and used for Western blotting. Results: At 6-8 months of age, SHR exhibited compensated LV hypertrophy, but left atrial hypotrophy, despite increased atrial mRNA levels of BNP (2-fold) and β-MHC (3.6-fold), while α-MHC was unaffected. There were no differences in global CaTs between atrial myocytes from WKY and SHR. Exposure of atrial myocytes to 50 nM ET-1 caused a 35% increase in systolic Ca in SHR (n=14), but only a 16% increase in WKY (n=21; P<0.05). Moreover, in SHR atrial myocytes, ET-1 induced a larger fractional SR Ca release (P<0.05 vs WKY). The ET-1-induced increase in CaTs occurred in the subsarcolemmal as well as in the central cytoplasm. Notably, ET-1 also induced arrhythmogenic Ca waves both in SHR and WKY atrial myocytes. Further analysis at the molecular level indicated that the atrial mRNA expression of ET-1 was increased (2-fold) in SHR. ETA receptor expression was unaltered at the mRNA level but upregulated (4-fold) at the protein level in the left atria. Gαq and PLCβ (isoforms 1 and 4) mRNA expression were also upregulated in SHR. Simulated atrial tachycardia in atrial tissue slices caused a 2-fold upregulation of ETA receptor expression in both SHR and WKY. Conclusions: At 6-8 months, SHR exhibit increased atrial expression of ET-1, ETA receptors, Gαq and PLCβ. Atrial tachycardia further increases ETA receptor expression. At the functional level, atrial myocytes from SHR respond to ET-1 with increases in systolic Ca and the development of arrhythmogenic Ca waves. Thus, atrial ET-1 signalling is enhanced already at early stages of hypertension and may contribute to the initiation of atrial tachycardia in hypertension.

Calcium spike variability in cardiac myocytes results from activation of small cohorts of ryanodine receptor 2 channels

In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.

A line scanning confocal fluorescent microscope using a CMOS rolling shutter as an adjustable aperture

Traditional confocal microscopy uses a physical aperture barrier to prevent out-of-focus light from reaching the detector. The physical nature of a conventional aperture limits control over the system confocality. We describe a new line scanning confocal microscope that eliminates a need for a physical aperture by employing a software-controllable rolling shutter on a CMOS camera. A confocal image is obtained by synchronizing motion of the rolling shutter and the laser line scanning over a sample. Confocal resolution of this microscope is adjustable in real time and independently established for each fluorescence channel by changing the rolling shutter width. This technology has been implemented in the IN Cell Analyzer 6000 system by GE Healthcare.

Exercise training corrects control of spontaneous calcium waves in hearts from myocardial infarction heart failure rats

Impaired cardiac control of intracellular diastolic Ca(2+) gives rise to arrhythmias. Whereas exercise training corrects abnormal cyclic Ca(2+) handling in heart failure, the effect on diastolic Ca(2+) remains unstudied. Here, we studied the effect of exercise training on the generation and propagation of spontaneous diastolic Ca(2+) waves in failing cardiomyocytes. Post-myocardial infarction heart failure was induced in Sprague-Dawley rats by coronary artery ligation. Echocardiography confirmed left ventricular infarctions of 40 ± 5%, whereas heart failure was indicated by increased left ventricular end-diastolic pressures, decreased contraction-relaxation rates, and pathological hypertrophy. Spontaneous Ca(2+) waves were imaged by laser linescanning confocal microscopy (488 nm excitation/505-530 nm emission) in 2 µM Fluo-3-loaded cardiomyocytes at 37°C and extracellular Ca(2+) of 1.2 and 5.0 mM. These studies showed that spontaneous Ca(2+) wave frequency was higher at 5.0 mM than 1.2 mM extracellular Ca(2+) in all rats, but failing cardiomyocytes generated 50% (P < 0.01) more waves compared to sham-operated controls at Ca(2+) 1.2 and 5.0 mM. Exercise training reduced the frequency of spontaneous waves at both 1.2 and 5.0 mM Ca(2+) (P < 0.05), although complete normalization was not achieved. Exercise training also increased the aborted/completed ratio of waves at 1.2 mM Ca(2+) (P < 0.01), but not 5.0 mM. Finally, we repeated these studies after inhibiting the nitric oxide synthase with L-NAME. No differential effects were found; thus, mediation did not involve the nitric oxide synthase. In conclusion, exercise training improved the cardiomyocyte control of diastolic Ca(2+) by reducing the Ca(2+) wave frequency and by improving the ability to abort spontaneous Ca(2+) waves after their generation, but before cell-wide propagation.

Parallel Signal Readout for Roll-Type Optical Advanced Memory

We demonstrated one-dimensional parallel readout of a roll-type optical advanced memory. Using a line shape focused beam, multiple signals can be detected simultaneously. To demonstrate one-dimensional parallel readout, we fabricated groove-structured roll-type optical advanced memory. The groove-structures were imprinted by a digital versatile disc stamper. We could detect multiple signals from each groove-structured layer without crosstalk by using line-scanning confocal microscopy. We demonstrated the simultaneous detection of eleven signals.

Optimization of pupil design for point-scanning and line-scanning confocal microscopy

Both point-scanning and line-scanning confocal microscopes provide resolution and optical sectioning to observe nuclear and cellular detail in human tissues, and are being translated for clinical applications. While traditional point-scanning is truly confocal and offers the best possible optical sectioning and resolution, line-scanning is partially confocal but may offer a relatively simpler and lower-cost alternative for more widespread dissemination into clinical settings. The loss of sectioning and loss of contrast due to scattering in tissue is more rapid and more severe with a line-scan than with a point-scan. However, the sectioning and contrast may be recovered with the use of a divided-pupil. Thus, as part of our efforts to translate confocal microscopy for detection of skin cancer, and to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. We report a Fourier-analysis-based computational model of confocal microscopy for six configurations. The six configurations are point-scanning and line-scanning, with full-pupil, half-pupil and divided-pupils. The performance, in terms of on-axis irradiance (signal), resolution and sectioning capabilities, is quantified and compared among these six configurations.

In Vivo Fluorescence Imaging of Blood Flow in Mouse Pancreatic Islets

Diabetes is a disease resulting from changes in pancreatic islets, which are insulin secreting micro-organs within the pancreas. With increased blood glucose, insulin is secreted from beta cells in the islets in a coordinated pulsatile manner. At the same time alpha cell glucagon secretion is inhibited. Mechanisms controlling these processes at the intercellular and at the inter-islet level remain unclear. We suggest that the three-dimensional organization of islet cells and the dynamics of islet blood flow have a role in regulating insulin and glucagon secretion. This is suggested by observation that the density of blood vessels within islets is much greater than in surrounding pancreatic tissue, and that most individual islet cells are adjacent to a blood vessel. As an initial test of our hypothesis, we have developed a high-speed in vivo fluorescence imaging method to track pancreatic blood flow in a living mouse. We are also developing methods necessary to analyze the large amounts of data generated. Using high speed line scan confocal microscopy the method has full frame sub-micron spatial and less than 10 ms temporal resolution. Islets are located within the pancreas by using mice with GFP-labeled beta cells. Blood plasma is labeled with a fluorescent dextran, allowing mapping of vascular dimensions and pathway. Individual blood cells are fluorescently labeled by osmotic shock loading with an Alexa dye, which allows tracking of the blood flow. We present current results for blood flow under different levels of blood glucose in clamping experiments. Our previous qualitative results have suggested that there are differences in blood flow parameters at different glucose levels. Here, a more quantitative analysis of blood flow velocity, any observed changes in vessel dimensions, and changes in blood flow coverage inside and outside islets is presented.

Cross structured illumination for high speed high resolution line scanning confocal microscopy

Previous research presented the structured illumination confocal scanning microscope (SICSM) so as to improve the lateral resolution of the confocal microscope. However, the image acquisition speed of the SICSM is very slow and also an alignment error due to the mechanical rotation of a grating and a slit can easily occur. As a theoretical study, in this paper we propose a new SI method, the cross SI method, which improves lateral resolution and image acquisition speed. Performances of the conventional SI and the proposed SI methods are compared by analysis of the modulation transfer function. The proposed SI method shows similar lateral resolution and can shorten the image acquisition time compared to the conventional SI method. The cross structured illumination confocal microscope (CSICM) is combined with the cross SI pattern optics and the line scanning confocal microscope. We have introduced a 2-D diffractive grating, four linear polarizers and four cylindrical lenses in order to create the cross SI pattern. The effects of the cross SI pattern, intensity and visibility, on the system performance are analyzed. The CSICM has double the lateral resolution of the conventional microscope, an optical sectioning ability and a fast image acquisition speed.

Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

Pancreatic islets are highly vascularized and arranged so that regions containing beta-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic ( approximately 300 mg/dl or 16.8 mM) and hypoglycemic ( approximately 50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the beta-cells or yellow fluorescent protein in the alpha-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of beta-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

Glucocorticoids Improve Calcium Cycling in Cardiac Myocytes After Cardiopulmonary Bypass

Glucocorticoids can reduce myocardial dysfunction associated with ischemia and reperfusion injury following cardiopulmonary bypass (CPB) and circulatory arrest. The hypothesis was that maintenance of cardiac function after CPB with methylprednisolone therapy results, in part, from preservation of myocyte calcium cycling. Piglets (5-7 kg) underwent CPB and 120 min of hypothermic circulatory arrest with (CPB-GC) or without (CPB) methylprednisolone (30 mgkg(-1)) administered 6h before and at CPB. Controls (No-CPB) did not undergo CPB or receive glucocorticoids (n=6 per treatment). Myocardial function was monitored in vivo for 120 min after CPB. Calcium cycling was analyzed using rapid line-scan confocal microscopy in isolated, fluo-3-AM-loaded cardiac myocytes. Phospholamban phosphorylation and sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a) protein levels were determined by immunoblotting of myocardium collected 120 min after CPB. Calpain activation in myocardium was measured by fluorometric assay. Preload recruitable stroke work in vivo 120 min after reperfusion decreased from baseline in CPB (47.4±12 versus 26.4±8.3 slope of the regression line, P<0.05), but was not different in CPB-GC (41±8.1 versus 37.6±2.2, P=0.7). In myocytes isolated from piglets, total calcium transient time remained unaltered in CPB-GC (368±52.5 ms) compared with controls (434.5±35.3 ms; P=0.07), but was prolonged in CPB myocytes (632±83.4 ms; P<0.01). Calcium transient amplitude was blunted in myocytes from CPB (757±168 nM) compared with controls (1127±126 nM, P<0.05) but was maintained in CPB-GC (1021±155 nM, P>0.05). Activation of calpain after CPB was reduced with glucocorticoids. Phospholamban phosphorylation and SERCA2a protein levels in myocardium were decreased in CPB compared with No-CPB and CPB-GC (P<0.05). The glucocorticoid-mediated improvement in myocardial function after CPB might be due, in part, to prevention of calpain activation and maintenance of cardiac myocyte calcium cycling.

Local is as local does: the unitary nature of SR Ca2+ release in cardiac ventricular myocytes

Local is as local does: the unitary nature of SR Ca²⁺ release in cardiac ventricular myocytes

Real-time, multidimensional in vivo imaging used to investigate blood flow in mouse pancreatic islets

The pancreatic islets of Langerhans are highly vascularized micro-organs that play a key role in the regulation of blood glucose homeostasis. The specific arrangement of endocrine cell types in islets suggests a coupling between morphology and function within the islet. Here, we established a line-scanning confocal microscopy approach to examine the relationship between blood flow and islet cell type arrangement by real-time in vivo imaging of intra-islet blood flow in mice. These data were used to reconstruct the in vivo 3D architecture of the islet and time-resolved blood flow patterns throughout the islet vascular bed. The results revealed 2 predominant blood flow patterns in mouse islets: inner-to-outer, in which blood perfuses the core of beta cells before the islet perimeter of non-beta cells, and top-to-bottom, in which blood perfuses the islet from one side to the other regardless of cell type. Our approach included both millisecond temporal resolution and submicron spatial resolution, allowing for real-time imaging of islet blood flow within the living mouse, which has not to our knowledge been attainable by other methods.

Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode

We describe an optical sectioning microscopy system with no moving parts based on a micro-structured stripe-array light emitting diode (LED). By projecting arbitrary line or grid patterns onto the object, we are able to implement a variety of optical sectioning microscopy techniques such as grid-projection structured illumination and line scanning confocal microscopy, switching from one imaging technique to another without modifying the microscope setup. The micro-structured LED and driver are detailed and depth discrimination capabilities are measured and calculated.

Identification and Functional Characterization of the Hepatic Stellate Cell CD38 Cell Surface Molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

Simulation of a theta line-scanning confocal microscope

We describe a 2-D computational model of the optical propagation of coherent light from a laser diode within human skin to better understand the performance of a confocal reflectance theta microscope. The simulation uses finite-difference time domain (FDTD) computations to solve Maxwell's equations in a synthetic skin model that includes melanin, mitochondria, and nuclei. The theta line-scanning confocal microscope configuration experiences more localized decreases in the signal than the confocal common-path point-scanning microscope. We hypothesize that these decreases result from the bistatic imaging configuration, the imaging geometry, and the inhomogeneity of the index of refraction of the skin. All these factors result in the source path having aberrations different than those of the receiver path. The model predicts signal decreases that are somewhat greater than those seen in experiments. New details on the reflection from a spherical object show that imaging with the theta line scanner leads to somewhat different results than would be seen with a common-path point scanner. The model is used to optimize the design of the theta line-scanning confocal microscope.

Impaired Intracellular Ca2+Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Intracellular Ca2+ Measurements in Live Cells by Rapid Line Scan Confocal Microscopy: Simplified Calibration Methodology

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes.

This study investigated the function of FK506-binding protein (FKBP12.6) using adenoviral-mediated gene transfer to over-express FKBP12.6 (Ad-FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a beta-galactosidase-expressing adenovirus (Ad-LacZ) was used as a control. Peak-systolic intracellular [Ca(2+)] (measured with Fura-2) was higher in the Ad-FKBP12.6 group compared to Ad-LacZ (1 Hz field stimulation at 37 degrees C). The amplitude of caffeine-induced Ca(2+) release was also greater, indicating a higher SR Ca(2+) content in the Ad-FKBP12.6 group. Voltage clamp experiments indicated that FKBP12.6 over-expression did not change L-type Ca(2+) current amplitude or Ca(2+) efflux rates via the Na(+)-Ca(2+) exchanger. Ca(2+) transients comparable to those after Ad-FKBP12.6 transfection could be obtained by enhancing SR Ca(2+) content of Ad-LacZ infected cells with periods of high frequency stimulation. Line-scan confocal microscopy (Fluo-3 fluorescence) of intact cardiomyocytes stimulated at 0.5 Hz (20-21 degrees C) revealed a higher degree of synchronicity of SR Ca(2+) release and fewer non-responsive Ca(2+) release sites in the Ad-FKBP12.6 group compared to control. Ca(2+) spark morphology was measured in beta-escin-permeabilized cardiomyocytes at a free [Ca(2+)](i) of 150 nm. The average values of the spark parameters (amplitude, duration, width and frequency) were reduced in the Ad-FKBP12.6 group. Increasing [Ca(2+)](i) to 400 nm caused coherent propagating Ca(2+) waves in the Ad-FKBP12.6 group but only limited Ca(2+) release events were recorded in the control group. These data indicate that FKBP12.6 over-expression enhances Ca(2+) transient amplitude predominately by increasing SR Ca(2+) content. Moreover, there is also evidence that FKBP12.6 can enhance the coupling between SR Ca(2+) release sites independently of SR content.