Fluorescent Ca Indicator Research Articles

Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Abstract P1101: Mitochondrial Ca Uniporter Complex, ROS And CaT Alternans

Background: Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternation in CaT amplitude and is causally linked to atrial fibrillation (AF). Inhibition of mitochondrial Ca uptake via the mitochondrial Ca uniporter complex (MCUC) facilitates CaT alternans and can lead to an increase in mitochondrial reactive oxygen species(ROS) production that compromises the cellular antioxidative capacity. The objective was to determine the mechanisms through which mitochondrial Ca uptake and ROS production determine the susceptibility of atrial cells to develop CaT alternans. Methods: CaT alternans was measured in single rabbit left atrial myocytes with fluorescence microscopy using the fluorescent Ca indicator Cal-520/AM, and for simultaneous measurements of ROS and alternans, cells were loaded with CM-DCFDA and Cal-590/AM. CaT alternans was induced by increasing the pacing frequency until stable CaT alternans was observed. The degree of alternans was quantified as the alternans ratio (AR = 1 – S/L, where S/L is the ratio of the small to the large amplitude of a pair of alternating CaTs). Results: Exposure of LA myocytes to the specific MCUC inhibitor Ru360 increased the AR by 86%. However, the subsequent stimulation of mitochondrial Ca uptake with the MCUC activator spermine (Spm) decreased AR by 79% (p=0.028). Quercetin (a O2 .- scavenger) and Tempol (a superoxide dismutase mimetic) lowered CaT AR by 96% (p=0.0007) and 79% (p=0.0029), respectively. An oxidative challenge with tert-butylhydroperoxide (tBHP) increased AR by 140% (p=<0.0001), and the reducing agent dithiothreitol (DTT) decreased AR by 39% (p=0.0194). Moreover, DTT treatment rescued CaT alternans previously enhanced by tBHP. During CaT alternans the rate of ROS production increased by 23% (p=0.026), however Spm treatment attenuated the increased ROS production. Conclusion: An oxidizing environment facilitates the development of CaT alternans. During CaT alternans mitochondrial ROS formation is increased and involves MCUC activity.

Reduction of P62/SQSTM1 on Calcium Handling in Stressed Myocytes

Introduction Cytosolic protein, p62/SQSTM1, regulates the protein turnover rate, and it interacts with various stress signaling proteins in cells. Accumulations of p62/SQSTM1 have been reported in hearts with cardiomyopathy. We recently reported that p62/SQSTM1 protein deletions reduced beta-adrenergic response in myocytes and improved chronic ischemia-induced Calcium (Ca) defect. Here, we studied acute and chronic stress-induced Ca handling in myocytes genetically suppressed p62/SQSTM1 protein. Methods Myocytes (n=3 per group) isolated from Wild-type (WT), and p62/SQSTM1+/- (p62 HET) at Base, 5 days, and 10 days after Isoproterenol (ISO; beta-agonist) injections (150 mg/kg). We measured myocytes contractility (Fraction shortening of sarcomeres), systolic Ca release (CaT), SR Ca contents (CaffT), and Fractional Ca release, (CaT/CaffT) in field-stimulated single myocytes. Myocytes loaded with fluorescent Ca indicators (Fura-2AM; cytosolic Ca loading). Heart tissues in each group were processed for western blot (n=3 per group). Results The baseline myocytes contractility and Ca regulation were not different among the groups. P62 HET myocytes significantly increased contractility and fractional Ca release (the function of SR, F/F0; WT base, 0.58±0.07, WT ISO, 0.84±0.01, p62 HET ISO, 0.79±0.04) after acute ISO stimulation (1 uM, p<0.05), similar to WT myocytes. Interestingly, p62 HET myocytes preserved SR Ca contents, whereas WT myocytes increased SR Ca contents. Chronic ischemia-induced p62 HET myocytes improved fractional Ca release; however, both systolic Ca release (F/F0; WT base, 0.24±0.03, WT ISO 10days, 0.29±0.06, p62 HET ISO 10 days, 0.17±0.02) and SR Ca contents (F/F0; WT base, 0.43±0.05, WT ISO, 0.49±0.10, p62 HET ISO, 0.33±0.04) were proportionally reduced (p<0.05). Conclusion Taken together, suppression of p62/SQSTM1 protein restores stress-induced Ca dysregulation in myocytes. p62/SQSTM1 protein may directly contribute to systolic Ca release via beta-adrenergic signaling.

The Role Of P62 On Chronic Oxidative Stress‐Induced Myocytes Ca handling

IntroductionRecent studies have shown that the activity of autophagy impairs, and the accumulation of p62 protein, autophagy substrate, in the heart observes in heart failure patients and mouse models of cardiomyopathy. Accumulations of p62 protein are known to activate the intrinsic cellular stress. Abnormal Ca release is partly responsible for oxidative stress‐induced contractile dysfunction of myocytes in ischemic heart disease. Myocytes Ca release is controlled by a function of sarcoplasmic reticulum (SR) Ca store. However, the role of p62 protein on myocytes Ca regulation in the ischemic heart is not clear.PurposeTo investigate the role of p62 on SR Ca regulation in myocytes isolated from mice with the Isoproterenol‐induced myocardial injury.MethodsTo test our hypothesis, both wild‐type (WT) and p62 knockout (KO) mice were used at 10–12 weeks of age. Myocytes isolated from WT and p62 KO mice (n=3 per group) injected with 200 mg/kg Isoproterenol (ISO; beta agonist, twice per day) for 0 (Base), 5 days, and 10 days. SR Ca handling and contractility measured in field‐stimulated single myocytes loaded with fluorescent Ca indicators (Fura‐2AM; cytosolic Ca loading). Amplitudes of caffeine (10 mM, 5 sec)‐induced Ca transients used as estimates of SR Ca store. Sets of heart from WT and p62 mice were processed for western blot (n=3 per group).ResultsThe p62‐deficient myocytes had no difference of myocytes contractility, and SR Ca handling at the baseline compared to WT myocytes (n=35–45 cells per group). After acute ISO exposure (1μM for 1 min), WT myocytes increased systolic Ca release whereas p62‐deficient myocytes did not. An exposure of a submaximal oxidative stress (1μM; H2O2) in p62‐deficient myocytes, however, increased myocytes contractility and the fraction of Ca released from the SR during each beat (fractional Ca release; systolic Ca release over SR Ca store) after acute ISO stimulation (p&lt;0.05). In WT mice injected with ISO for 5 days and 10 days, myocytes had a biphasic effect on myocytes contractility and fractional Ca release (n=15–45 cells per group, p&lt;0.05). Interestingly, in p62‐deficient myocytes further increased myocytes contractility and fractional Ca release (Figure) on chronic ISO injections from 5 days to 10 days. WT mice hearts with chronic exposure to ISO for 10 days increased p62 protein expressions and suppressed the conversion of LC3 I to LC3 II.ConclusionTaken together, our results suggest that accumulations of p62 protein may directly cause SR Ca defects via beta‐adrenergic signaling in myocytes under chronic myocardial injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction between TRPC1 and STIM1 in calcium sensing receptor mediated calcium influx and nitric oxide production in human umbilical vein endothelial cells

Objective: To investigate the interaction of Ca(2+) protein TRPC1 and STIM1 in extracellular Ca(2+) -sensing receptor (CaR)-induced extracellular Ca(2+) influx and the production of nitric oxide (NO). Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and incubated with CaR agonist spermine (activating store-operates cation channels (SOC) and receptor-operated channels (ROC)), CaR negative allosteric modulator Calhex231 (blocking SOC, activating ROC) and ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967(activate SOC, blocking ROC), respectively. The interaction of TRPC1 and STIM1 was determined using the immunofluorescence methods. The interaction between TRPC1 and STIM1 were examined by Co-immuno precipitation. The HUVECs were divided into: TRPC1 and STIM1 short hairpin RNA group (shTRPC1+ shSTIM1 group), vehicle-TRPC1+ vehicle-STIM1 group and control group. The cells were incubated with four different treatments under the action of above mentioned interventions, intracellular Ca(2+) concentration ([Ca(2+) ](i)) was detected using the fluorescence Ca(2+) indicator Fura-2/AM, the production of NO was determined by DAF-FM. Results: (1) The expression of TRPC1 and STIM1 proteins levels in HUVECs: Under the confocal microscope, TRPC1 and STIM1 protein expression showed masculine gender, both located in cytoplasm in the normal control group. Post incubation with Calhex231+ TPA, Ro31-8220 and Go6967, TRPC1 and STIM1 positioned in cytoplasm was significantly reduced, and the combined TRPC1 and STIM1 was also significantly reduced. (2) The interaction of TRPC1 and STIM1 in HUVECs: The relative ratios of Calhex231+ TPA+ Spermine+ Ca(2+) group, Ro31-8220+ Spermine+ Ca(2+) group and Go6976+ Spermine+ Ca(2+) group STIM1/TRPC1 and TRPC1/STIM1 were as follows: (25.98±2.17)% and (44.10±4.01)%, (20.85±1.01)% and (46.31±3.47)%, (23.88±2.05)% and (39.65±2.91)%, which were significantly lower than those in the control group (100.00±4.66)% and (100.00±6.40)% and in the Spermine+ Ca(2+) group (106.04±2.45)% and (107.78±2.66)% (all P<0.05). (3) The influence of joint TRPC1 and STIM1 transfection to four different drugs treated HUVECs on [Ca(2+) ](i) and NO generation: The changes of two excitation fluorescence intensity ratio and NO net fluorescence intensity values were consistent, [Ca(2+) ](i) and NO net fluorescence intensity values were significantly lower in the experimental group than the control group and the vehicle group (all P<0.05), while which were similar between the vehicle group and control group (all P>0.05). Conclusions: Our results indicate that TRPC1 and STIM1 jointly regulate CaR-mediated Ca(2+) influx and nitric oxide generation in HUVECs in the form of binary complex.

Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrial-specific Na+–Ca2 + exchanger knockout mice

Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic “V”-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the “V”-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na–Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a “V”-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm.

R-Cepia1er as a New Tool to Directly Measure [Ca] in the Sarcoplasmic Reticulum of Ventricular Myocytes

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR) determines, in part, the amplitude of SR Ca release which, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best tool available to directly measure [Ca]SR was a low affinity Ca indicator (Fluo-5N). However this dye presents several difficulties, including species-limitation (it does not work in small rodents), dye extrusion, and non-specific cellular localization. Recently a new genetically encoded family of Ca indicators has been generated, named calcium-measuring organelle-entrapped protein indicators (CEPIA). Here we tested the red emitting fluorescence Ca indicator targeted to the SR, R-CEPIA1er, as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat myocytes with an adenovirus expressing the R-CEPIA1er gene, displayed prominent localization of the indicator in the SR and nuclear envelope. Calibration of R-CEPIA1er in rabbit myocytes resulted in a Kd of 617 microM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR. [Ca]SR dynamics measured with R-CEPIA1er were tested in rabbit myocytes and compared to Fluo5-N signal. We found that the time course of the [Ca]SR depletion and fractional [Ca]SR release (FR) induced by an action potential was similar between R-CEPIA1er and Fluo-5N. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, this Ca sensor allowed us to reliably measure [Ca]SR dynamics in rat myocytes. Measurements of [Ca]SR with R-CEPIA1er can be combined with [Ca]cyt indicators (e.g. Fluo-4) to obtain detailed information regarding Ca homeostasis in cardiac myocytes. Moreover, R-CEPIA1er is an optimal tool that can be used to study [Ca]SR handling in commonly used transgenic animals (such as mice and rats).

Colocalization properties of elementary Ca(2+) release signals with structures specific to the contractile filaments and the tubular system of intact mouse skeletal muscle fibers.

Ca(2+) regulates several important intracellular processes. We combined second harmonic generation (SHG) and two photon excited fluorescence microscopy (2PFM) to simultaneously record the SHG signal of the myosin filaments and localized elementary Ca(2+) release signals (LCSs). We found LCSs associated with Y-shaped structures of the myosin filament pattern (YMs), so called verniers, in intact mouse skeletal muscle fibers under hypertonic treatment. Ion channels crucial for the Ca(2+) regulation are located in the tubular system, a system that is important for Ca(2+) regulation and excitation-contraction coupling. We investigated the tubular system of intact, living mouse skeletal muscle fibers using 2PFM and the fluorescent Ca(2+) indicator Fluo-4 dissolved in the external solution or the membrane dye di-8-ANEPPS. We simultaneously measured the SHG signal from the myosin filaments of the skeletal muscle fibers. We found that at least a subset of the YMs observed in SHG images are closely juxtaposed with Y-shaped structures of the transverse tubules (YTs). The distances of corresponding YMs and YTs yield values between 1.3 μm and 4.1 μm including pixel uncertainty with a mean distance of 2.52±0.10 μm (S.E.M., n=41). Additionally, we observed that some of the linear-shaped areas in the tubular system are colocalized with linear-shaped areas in the SHG images.

Analysis of the Effect of β-Endorphin-Like Peptide Pentarphin on Intracellular Ca²⁺ Concentration in Early Mouse Embryos Using Fluorescent Probes.

At the early stages of mammalian embryo development, Ca(2+) influences the processes of cleavage and differentiation. An important role in these processes is played by peptide growth factors. One of them, pentarphin, is a peptide growth factor similar to the central part of β-endorphin molecule and a fragment 369-373 of IgG heavy chain. We used fluorescent Ca(2+) indicators Fluo-3 AM and Fura Red AM. Pentarphin added to embryos in a concentration of 0.1 μM 1.5-2 fold increased intercellular Ca(2+) level in comparison with the initial level.

A systematic approach for assessing Ca²⁺ handling in cardiac myocytes.

In cardiac myocytes, Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store through the opening of ryanodine receptors (RyRs) is the major source of Ca(2+) for activation of myofilaments and contraction. Over the past 20 years, tools have become available to study this release process in detail, allowing new insights into the regulation of SR Ca(2+) release and RyR function. To assess these processes, we recommend and here review a systematic approach that evaluates the essential transport mechanisms and Ca(2+) fluxes in isolated single cardiac myocytes by using fluorescent Ca(2+) indicators and whole-cell recording of membrane voltage and ionic currents under voltage clamp. The approach includes an assessment of the L-type Ca(2+) current as a trigger for opening of RyRs and release of SR Ca(2+), of the SR Ca(2+) content, of intrinsic properties of RyRs, and of Ca(2+)-removal systems.

Measuring the rate constants of Ca²⁺ indicators.

Fluorescent Ca(2+) indicators are widely used to measure the concentration of free Ca(2+) ([Ca(2+)](free)) in biological processes. To determine the exact kinetics of changes in [Ca(2+)](free) and the processes underlying these changes (e.g., Ca(2+) binding to Ca(2+)-binding proteins), it is necessary to know the exact binding properties of the indicator used. Here, we describe how to determine the binding rate constants (k(on) and k(off)) of Ca(2+) indicators.

Measuring the Steady-State Properties of Ca2+ Indicators with a Set of Calibrated [Ca2+] Solutions

Fluorescent Ca(2+) indicators are widely used to measure the concentration of free Ca(2+) ([Ca(2+)]free) in biological processes. By calibrating the dye under the same experimental conditions as employed during its planned use, the actual [Ca(2+)] can be calculated from the measured fluorescence. When using non ratiometric dyes, such as the Oregon Green BAPTA (OGB) family of dyes or the Fluo dyes, the steady-state affinity (K(d)) and the ratio between the maximal and minimal fluorescence (F(ratio) = F(max)/F(min)) of the particular dye are needed for this conversion. Although these values are usually given by the manufacturer, we consistently find that the actual values can differ between various batches delivered by the companies that make the dyes. In this protocol, we provide the recipe for a series of solutions with a known and tightly buffered [Ca(2+)](free) and describe how to use these mixtures to determine the exact K(d) and F(ratio) of a fluorescent Ca(2+) dye.

Automated analysis of dynamic Ca2+ signals in image sequences.

Intracellular Ca(2+) signals are commonly studied with fluorescent Ca(2+) indicator dyes and microscopy techniques. However, quantitative analysis of Ca(2+) imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca(2+) signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca(2+) signaling.

The effect of Cyclin-dependent kinase 5 on voltage-dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state.

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre-synaptic terminals triggered by Ca(2+) influx into the pre-synaptic cytoplasm through voltage-dependent Ca(2+) channels (VDCCs). It is reported that Cdk5 regulates L-, P/Q-, or N-type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca(2+) -channel property of VDCCs, using PC12 cells expressing endogenous, functional L-, P/Q-, and N-type VDCCs. The Ca(2+) influx, induced by membrane depolarization with high K(+) , was monitored with a fluorescent Ca(2+) indicator protein in both undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells. Overall, Ca(2+) influx was increased by expression of Cdk5-p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5-p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5-p35 regulates L-, P/Q-, or N-type VDCCs in a cellular context-dependent manner. Calcium (Ca(2+) ) influx through voltage-dependent Ca(2+) channels (VDCCs) triggers neurotransmitter release from pre-synaptic terminal of neurons. The channel activity of VDCCs is regulated by Cdk5-p35, a neuronal Ser/Thr kinase. However, there have been debates about the regulation of VDCCs by Cdk5. Using PC12 cells, we show that Cdk5-p35 regulates VDCCs in a type (L, P/Q, and N) and differentiation-dependent manner. NGF=nerve growth factor.

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Intracellular calcium stores drive slow non-ribbon vesicle release from rod photoreceptors

Rods are capable of greater slow release than cones contributing to overall slower release kinetics. Slow release in rods involves Ca2+-induced Ca2+ release (CICR). By impairing release from ribbons, we found that unlike cones where release occurs entirely at ribbon-style active zones, slow release from rods occurs mostly at ectopic, non-ribbon sites. To investigate the role of CICR in ribbon and non-ribbon release from rods, we used total internal reflection fluorescence microscopy as a tool for visualizing terminals of isolated rods loaded with fluorescent Ca2+ indicator dyes and synaptic vesicles loaded with dextran-conjugated pH-sensitive rhodamine. We found that rather than simply facilitating release, activation of CICR by ryanodine triggered release directly in rods, independent of plasma membrane Ca2+ channel activation. Ryanodine-evoked release occurred mostly at non-ribbon sites and release evoked by sustained depolarization at non-ribbon sites was mostly due to CICR. Unlike release at ribbon-style active zones, non-ribbon release did not occur at fixed locations. Fluorescence recovery after photobleaching of endoplasmic reticulum (ER)-tracker dye in rod terminals showed that ER extends continuously from synapse to soma. Release of Ca2+ from terminal ER by lengthy depolarization did not significantly deplete Ca2+ from ER in the perikaryon. Collectively, these results indicate that CICR-triggered release at non-ribbon sites is a major mechanism for maintaining vesicle release from rods and that CICR in terminals may be sustained by diffusion of Ca2+ through ER from other parts of the cell.

Ca(2+) handling in mouse embryonic stem cell-derived cardiomyocytes.

Embryonic stem (ES) cells are alternative cell source for cell replacement therapy for cardiac diseases, thus it is important to verify if the cardiomyocytes derived from ES cells have comparable functional parameters similar to the mature cardiomyocytes. Ca(2+) handling is one of the most important parameters of cardiomyocyte since it is involved in the regulation of several main functions of cardiomyocytes, e.g. the excitation-contraction coupling. By applying membrane-permeable fluorescent Ca(2+) indicator and confocal microscopy detection system, change of intracellular Ca(2+) concentration in ES cell-derived cardiomyocytes can be monitored in real-time manner. In this protocol, we describe a method of isolating mouse ES cell-derived cardiomyocytes and recording their global and local Ca(2+) transients.

Urocortin 2 Protects Against Pacing-Induced Alternans via Phosphorylation of Phospholamban in Cardiac Myocytes from Normal and Failing Hearts

Cardiac alternans is a high risk indicator for cardiac arrhythmias, stroke and sudden cardiac death. The cardioactive peptide Urocortin 2 (Ucn2) exhibits beneficial effects in normal and failing hearts, and elicits PKA-dependent positive inotropic and lusitropic effects in normal myocytes. Thus, we investigated if Ucn2 protects against pacing-induced alternans and elucidated the underlying mechanism.Experiments were performed on single rabbit atrial and ventricular myocytes from normal and failing hearts. Chronic heart failure was induced by combined pressure and volume overload. Ca alternans was induced by incrementally increasing the pacing frequency until stable Ca alternans occurred at room temperature. Global Ca transients were measured with the fluorescent Ca indicators Fluo-4 or Indo-1 and monitored simultaneously with mechanical alternans (sarcomere length). In some experiments, cytosolic Ca alternans and intra-SR Ca alternans were simultaneously recorded with the Ca indicators Rhod-2 and Fluo-5N, respectively.The average alternans ratio (AR = 1-(small-amplitude/large-amplitude)) in atrial myocytes was 0.79, and in normal and failing ventricular myocytes the ARs were 0.69 and 0.64, respectively. Ucn2 (100 nM) completely abolished Ca and mechanical alternans (within 2-3 min) in atrial and ventricular myocytes from normal and failing hearts. An increased sarcoplasmic reticulum (SR) Ca content, together with an enhanced SR Ca release flux, suggested that Ucn2 normalized alternans through effects on SR Ca ATPase (SERCA). Ucn2 increased significantly the level of cyclic adenosine monophosphate (cAMP) in normal cells (∼12-fold), and enhanced phosphorylation of phospholamban (PLB) at Ser16 in normal myocytes (∼10-fold) and to a lesser extent (∼5-fold) in failing myocytes. These data demonstrate that Ucn2 rescues alternans presumably via increased SERCA activity in atrial and ventricular myocytes and thus protects normal and failing hearts from proarrhythmic alternans.

Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells

Calcium (Ca(2+)) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca(2+) concentration ([Ca(2+)]i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca(2+)]i is the use of fluorescent Ca(2+) indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca(2+) indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca(2+)-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca(2+)-free and Ca(2+)-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca(2+)-free and Ca(2+)-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca(2+) sensitivity is reduced. Nevertheless, quantitative [Ca(2+)]i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.

Quercetin induces insulin secretion by direct activation of L‐type calcium channels in pancreatic beta cells

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor. Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique. Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 μmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i. Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.