Full Duration At Half Maximum Research Articles

Image-derived mean velocity measurement for prediction of coronary flow reserve in a canonical stenosis phantom using magnetic particle imaging.

IntroductionAim of this study is to evaluate whether magnetic particle imaging (MPI) is capable of measuring velocities occurring in the coronary arteries and to compute coronary flow reserve (CFR) in a canonical phantom as a preliminary study.MethodsFor basic velocity measurements, a circulation phantom was designed containing replaceable glass tubes with three varying inner diameters, matching coronary-vessel diameters. Standardised boluses of superparamagnetic-iron-oxide-nanoparticles were injected and visualised by MPI. Two image-based techniques were competitively applied to calibrate the respective glass tube and to compute the mean velocity: full-duration-at-half-maximum (FDHM) and tracer dilution (TD) method. For CFR-calculation, four necessary settings of the circulation model of a virtual vessel with an inner diameter of 4 mm were generated using differently sized glass tubes and a stenosis model. The respective velocities in stenotic glass tubes were computed without recalibration.ResultsOn velocity level, comparison showed a good agreement (rFDHM = 0.869, rTD = 0.796) between techniques, preferably better for 4 mm and 6 mm inner diameter glass tubes. On CFR level MPI-derived CFR-prediction performed considerably inferior with a relative error of 20–44%.ConclusionsMPI has the ability to reliably measure coronary blood velocities at rest as well as under hyperaemia and therefore may be suitable for CFR calculation. Calibration-associated accuracy of CFR-measurements has to be improved substantially in further studies.

ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression.

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1G93A (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1G93A mice were confirmed with fluorescent probes, MitoSOX™ Red and ROS Brite™570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1G93A in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1G93A-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1G93A transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1G93A. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1G93A directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.

Dysregulation of Mitochondrial Permeability Transition Pore (MPTP) is Associated with Enhanced ROS Production in Skeletal Muscle of an ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease. Oxidative stress is implicated in ALS pathophysiology. Using ROS Brite™ 570, a cytosolic ROS detector, we found a significant increase in ROS production of muscle fibers derived from the ALS G93A mice. Mitochondria are the major source of ROS production. In order to detect early changes in ROS-related mitochondrial metabolic function, we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP monitors dynamic changes of ROS-related mitoflash events at the single mitochondrion level. Remarkably, G93A muscle cells show early and disease stage-dependent changes in mitoflash events. The mitoflash activity at the age of 2 months (before ALS symptom onset) is marked by an increased flashing area in G93A muscle fibers, while their kinetics properties remain unchanged. After ALS onset (3-month old), there are drastic changes in the kinetics of mitoflash signal with prolonged FDHM (full duration at half maximum) of the mitoflash signal. Thus, mitoflash provides a sensitive and quantitative biomarker for evaluating ROS-related mitochondrial dysfunction. It is known that uncontrolled opening of mitochondrial permeability transition pore (mPTP) is a key step to promote mitochondrial ROS production. While the molecular composition of mPTP is still incompletely understood, studies have shown that cyclophilin D (CypD) promotes the opening of mPTP and phosphorylated form of GSK-3β participates in maintaining mPTP in the closed state. We used subcellular fractionation to isolate mitochondria from skeletal muscle of G93A mice, and found an increased CypD expression level in muscle mitochondria, while the phosphorylated form of GSK-3β was significantly reduced in mitochondria of G93A muscle. Thus, both may act as triggering factors for mPTP opening in G93A muscle.

Altered Mitochondrial Superoxide Production in Skeletal Muscle of an ALS Mouse Model during the Disease Progression

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that involves severe skeletal muscle atrophy. Mitochondria are critically affected in ALS muscle. To better understand the role of mitochondria in ALS onset and progression, we generated a double transgenic (dTG) mouse model overexpressing an ALS mutant (SOD1G93A) and a mitochondrial superoxide biosensor (mt-cpYFP) that reports dynamic production of superoxide inside mitochondria termed as (Wang 2008; Fang 2011). We found that flash signal in muscle changed significantly during the disease progression. Before disease onset, 2-month old dTG mice (ALS) showed an increase in mitochondrial superoxide production, which was manifested by a marked increase in the ratio of flashing area to cell area (ALS: 2.3±0.29, n=80, 5 mice; WT: 1.2±0.18, n=74, 4 mice; P=0.008), while there was no significant change in the kinetic parameters of the flash signal at this stage. At end stage (ESALS, 4.5-month old, n=36, 4 mice), there was a drastic decrease in mitochondrial superoxide production characterized by a decrease in the ratio of flashing area to cell area compared to WT (0.69±0.11, P<0.05). Remarkably, the kinetic parameters of the flash signal were significantly slower in ESALS dGT mice. Compared to WT and early stage ALS, there is an increase in the rising time (RT) (WT:15.82±1.38, ALS:14.63±1.2 vs ESALS:23.62±2.97, P=0.008 and 0.001), the half decay time (T50) (WT:9.81±1.13, ALS:11.93±1.43 vs ESALS:20.56±3.65, P<0.001 and 0.013), and the full duration at half maximum (FDHM) (WT:16.76±1.72, ALS:18.28±2.07 vs ESALS:33.37±5.33, P<0.001 and 0.003). Our data demonstrate drastic changes in muscle mitochondrial metabolic function in the course of ALS. Ongoing studies aim to explore the cause and the role of this abnormal mitochondrial function at different ALS stages. Supported by MDA and NIAMS/NIH.

Identification and characterization of calcium sparks in cardiomyocytes derived from human induced pluripotent stem cells.

IntroductionCa2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs) may display unique structural and functional properties than mature adult cardiomyocytes.Methods and resultsCa2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR) Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM) and full duration at half maximum (FDHM) of spontaneous Ca2+ sparks.ConclusionsFor the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

A Method to Characterize Calcium Activity in Stimulated Cultures of Cardiac Myocytes

Cardiac function and disease constitutes a complex physiological process involving several biophysical scales, from the stochastic dynamics of a single calcium release channel to the calcium signal of a single myocyte and the activation of contraction across a cardiac tissue. Integration of signals from several of these scales requires specific tools to study their interdependence.We present a method for automatic detection of calcium signals in cultured cardiomyocytes subjected to external field stimulation. The method is applied to a sequence of confocal fluorescence images, and provides information on both the calcium activity in individual myocytes, the global calcium signal of the imaged field, as well as the propagation of the calcium signal across the cell culture.The approach first segments each cell in the culture and computes its average calcium activity. An automatic classification method then identifies the response of each cell among six different dynamical regimes a) uniform response, b) alternating response, c) irregular response, d) calcium waves, e) phase-lock (conduction block) or f) inactive. The system computes the area, the full duration at half maximum (FDHM), the resting value and the peak of the calcium transient of each cell. Subsequently, it maps the distribution of cells from each group within the imaged field.Finally, the method generates an isochronal map of the activation of each cell in the culture as the calcium front propagates across the culture, computing the linear and angular velocities as well as the propagation direction.The resulting data can be used to quantify how abnormal calcium regulation at the single-cell level affects the propagation of the calcium signal in a myocyte culture. The method is also suitable for a quantitative comparison of the effects of pharmacological or genetic treatments of cell cultures.

Return of Myoplasmic Calcium (Ca) to Resting Levels following Stimulation of Fast- and Slow-Twitch Mouse Muscle Fibers

To study the processes underlying the relaxation and recovery of skeletal muscle, we have used the high affinity fluorescent Ca indicators fluo-3 and fluo-4 to measure Ca transients in fibers from mouse fast-twitch (EDL) and slow-twitch (soleus) muscles (16 oC). Single fibers on the surface of small bundles of fibers were injected with indicator, and fluorescence changes (ΔF) were recorded for up to 60 s following a single action potential (AP). The full-duration at half-maximum (FDHM) of ΔF and the early decay time constant from 50% peak ΔF were larger in slow-twitch than in fast-twitch fibers (∼220 and ∼320 ms, respectively, vs. ∼50 and ∼70 ms). These findings are consistent with the larger FDHM of the Ca transient in slow-twitch fibers (Baylor and Hollingworth, J. Physiol., 2003). Interestingly, on a time scale of seconds, the decay of ΔF to resting levels was much slower in fast-twitch than slow-twitch fibers (average time constants of ∼12 vs. ∼2 s). This long tail of the Ca transient in fast-twitch fibers, which can last up to 60 s, is consistent with the large concentration of the calcium binding protein parvalbumin in EDL but not soleus fibers. Modeling of myoplasmic Ca movements in fast-twitch fibers indicates that, 1 s after an AP, most (∼0.8) of the released Ca remains in the myoplasm where it is mainly (∼95%) bound to parvalbumin. Ca binding by parvalbumin helps to abbreviate the Ca transient during relaxation. However, because parvalbumin brings free [Ca] close to resting levels, the modeled activity of the Ca pump is low. In consequence, Ca pumping continues for tens of seconds, as Ca is slowly returned to the sarcoplasmic reticulum. Supported by NIH and MDA.

Measurement and simulation of myoplasmic calcium transients in mouse slow‐twitch muscle fibres

Bundles of intact fibres from soleus muscles of adult mice were isolated by dissection and one fibre within a bundle was micro-injected with either furaptra or mag-fluo-4, two low-affinity rapidly responding Ca(2+) indicators. Fibres were activated by action potentials to elicit changes in indicator fluorescence (ΔF), a monitor of the myoplasmic free Ca(2+) transient ([Ca(2+)]), and changes in fibre tension. All injected fibres appeared to be slow-twitch (type I) fibres as inferred from the time course of their tension responses. The full-duration at half-maximum (FDHM) of ΔF was found to be essentially identical with the two indicators; the mean value was 8.4 ± 0.3 ms (±SEM) at 16°C and 5.1 ± 0.3 ms at 22°C. The value at 22°C is about one-third that reported previously in enzyme-dissociated slow-twitch fibres that had been AM-loaded with mag-fluo-4: 12.4 ± 0.8 ms and 17.2 ± 1.7 ms. We attribute the larger FDHM in enzyme-dissociated fibres either to an alteration of fibre properties due to the enzyme treatment or to some error in the measurement of ΔF associated with AM loading. ΔF in intact fibres was simulated with a multi-compartment reaction-diffusion model that permitted estimation of the amount and time course of Ca(2+) release from the sarcoplasmic reticulum (SR), the binding and diffusion of Ca(2+) in the myoplasm, the re-uptake of Ca(2+) by the SR Ca(2+) pump, and Δ[Ca(2+)] itself. In response to one action potential at 16°C, the following estimates were obtained: 107 μm for the amount of Ca(2+) release; 1.7 ms for the FDHM of the release flux; 7.6 μm and 4.9 ms for the peak and FDHM of spatially averaged Δ[Ca(2+)]. With five action potentials at 67 Hz, the estimated amount of Ca(2+) release is 186 μm. Two important unknown model parameters are the on- and off-rate constants of the reaction between Ca(2+) and the regulatory sites on troponin; values of 0.4 × 10(8) m(-1) s(-1) and 26 s(-1), respectively, were found to be consistent with the ΔF measurements.

Measurement of Myoplasmic Calcium Transients in Mouse Slow-Twitch Muscle Fibers

Bundles of intact fibers from soleus muscles of adult mice were isolated by dissection and one fiber within a bundle was micro-injected with either furaptra or mag-fluo-4, two low-affinity rapidly-responding Ca2+ indicators. Fibers were activated by action potentials to elicit changes in indicator fluorescence (dF), a monitor of the myoplasmic free Ca2+ transient, and changes in fiber tension. All injected fibers appeared to be slow-twitch (type I) fibers as inferred from the time course of their twitch tension response, which was markedly slower than that in fast-twitch fibers from extensor digitorum longus muscle. The time of peak and full-duration at half maximum (FDHM) of dF were found to be essentially identical with the two indicators: 4.5 + 0.2 and 8.4 + 0.5 ms, respectively, with furaptra (n = 6) and 4.8 + 0.4 and 8.3 + 0.3 ms with mag-fluo-4 (n = 8) (16 oC). Mag-fluo-4's dF was also measured at 22 oC; its FDHM, 5.1 + 0.3 ms (n = 6), is about one-third that reported previously in enzyme-dissociated slow-twitch fibers that had been AM-loaded with mag-fluo-4: 12.4 + 0.8 ms (n = 20) and 17.2 + 1.7 ms (n = 23). We attribute the larger FDHM in enzyme-dissociated fibers either to an alteration of fiber properties due to the enzyme treatment or to some error in the measurement of dF associated with AM-loading. Supported by NIH (GM-086167) and the Muscular Dystrophy Association.

Simulation of Myoplasmic Calcium Transients in Mouse Slow-Twitch Muscle Fibers

ABSTRACTFiber bundles were dissected from soleus muscles of adult mice and individual slow-twitch fibers were micro-injected with furaptra, a low-affinity rapidly-responding fluorescent Ca indicator. Fiber activity was elicited by action potential stimulation at 16 oC and dfCaD, the fraction of the indicator in the Ca-bound form, was measured. The dfCaD waveform was simulated with a multi-compartment reaction-diffusion model that provides estimates of the amount and time course of Ca release from the sarcoplasmic reticulum (SR), the binding and diffusion of Ca in the myoplasm, the re-uptake of Ca by the SR Ca pump, and the myoplasmic free Ca transient itself (d[Ca]). In response to one action potential (AP), the following spatially-averaged estimates were obtained (concentration units are referred to the myoplasmic water volume): 107 micro-molar for the amount of Ca release; 57 micro-molar/ms and 1.7 ms for the peak and full-duration at half maximum (FDHM) of the release flux; 7.6 micro-molar and 4.9 ms for the peak and FDHM of d[Ca]. In response to five APs at 67 Hz, d[Ca] summated somewhat in response to later action potentials in the train, while the second and subsequent releases declined progressively, from 0.3 to 0.1 times that of the first release. Two important parameters of the model are the on- and off-rate constants of the reaction between Ca and the regulatory sites on troponin. Values of 0.4x108 M−1 s−1 and 26 s−1, respectively, were found to be consistent with the measurements of dfCaD. The peak troponin occupancy is estimated to be 79% and 93% in response to one and five APs, respectively.

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

Comparison of the myoplasmic calcium transient elicited by an action potential in intact fibres of mdx and normal mice

The myoplasmic free [Ca2+] transient elicited by an action potential (Delta[Ca2+]) was compared in fast-twitch fibres of mdx (dystrophin null) and normal mice. Methods were used that maximized the likelihood that any detected differences apply in vivo. Small bundles of fibres were manually dissected from extensor digitorum longus muscles of 7- to 14-week-old mice. One fibre within a bundle was microinjected with furaptra, a low-affinity rapidly responding fluorescent calcium indicator. A fibre was accepted for study if it gave a stable, all-or-nothing fluorescence response to an external shock. In 18 normal fibres, the peak amplitude and the full-duration at half-maximum (FDHM) of Delta[Ca2+] were 18.4 +/- 0.5 microm and 4.9 +/- 0.2 ms, respectively (mean +/- s.e.m.; 16 degrees C). In 13 mdx fibres, the corresponding values were 14.5 +/- 0.6 microm and 4.7 +/- 0.2 ms. The difference in amplitude is statistically highly significant (P = 0.0001; two-tailed t test), whereas the difference in FDHM is not (P = 0.3). A multi-compartment computer model was used to estimate the amplitude and time course of the sarcoplasmic reticulum (SR) calcium release flux underlying Delta[Ca2+]. Estimates were made based on several differing assumptions: (i) that the resting myoplasmic free Ca2+ concentration ([Ca2+]R) and the total concentration of parvalbumin ([Parv(T)]) are the same in mdx and normal fibres, (ii) that [Ca2+](R) is larger in mdx fibres, (iii) that [Parv(T)] is smaller in mdx fibres, and (iv) that [Ca2+]R is larger and [Parv(T)] is smaller in mdx fibres. According to the simulations, the 21% smaller amplitude of Delta[Ca2+] in mdx fibres in combination with the unchanged FDHM of Delta[Ca2+] is consistent with mdx fibres having a approximately 25% smaller flux amplitude, a 6-23% larger FDHM of the flux, and a 9-20% smaller total amount of released Ca2+ than normal fibres. The changes in flux are probably due to a change in the gating of the SR Ca2+-release channels and/or in their single channel flux. The link between these changes and the absence of dystrophin remains to be elucidated.

Effects of Tetracaine on Voltage-activated Calcium Sparks in Frog Intact Skeletal Muscle Fibers

The properties of Ca2+ sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K+] Ringer's are well described by a computational model with a Ca2+ source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 °C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca2+ current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1–5 RyR Ca2+ release channels open during a voltage-activated Ca2+ spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K+] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75–100 μM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca2+ channels per spark, and (b) the single-channel Ca2+ current of an RyR channel is ≤1.2 pA under physiological conditions. The results support the conclusion that some normal voltage-activated sparks (i.e., in the absence of tetracaine) are produced by two or more active RyR Ca2+ channels. The question of how the activation of multiple RyRs is coordinated is discussed.

Altered Ca 2+ sparks and gating properties of ryanodine receptors in aging cardiomyocytes

To investigate the cellular mechanisms for altered cardiac function in senescence, we measured Ca 2+ transients and Ca 2+ sparks in ventricular cardiomyocytes from 6- to 24-month-old Fisher 344 (F344) rat hearts. The single channel properties of ryanodine receptors from adult and senescent hearts were also studied. In senescent myocytes, we observed a decreased peak [Ca 2+] i amplitude and an increased time constant for decay ( τ), both of which correlated with a reduced Ca 2+ content of the sarcoplasmic reticulum (SR). Our studies also revealed that senescent cardiomyocytes had an increased frequency of Ca 2+ sparks and a slight but statistically significant decrease in average amplitude, full-width-at-half-maximum (FWHM) and full-duration-at-half-maximum (FDHM). Single channel recordings of ryanodine receptors (RyR2) demonstrated that in aging hearts, the open probability ( P o) of RyR2 was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca 2+ sparks and decreased size of sparks, respectively. Thus, modifications of normal RyR2 gating properties may play a role in the altered Ca 2+ homeostasis observed in senescent myocytes.

Identification and Spatiotemporal Characterization of Spontaneous Ca2+Sparks and Global Ca2+Oscillations in Retinal Arteriolar Smooth Muscle Cells

To identify spontaneous Ca(2+) sparks and global Ca(2+) oscillations in microvascular smooth muscle (MVSM) cells within intact retinal arterioles and to characterize their spatiotemporal properties and physiological functions. Retinal arterioles were mechanically dispersed from freshly isolated rat retinas and loaded with Fluo-4, a Ca(2+)-sensitive dye. Changes in [Ca(2+)](i) were imaged in MVSM cells in situ by confocal scanning laser microscopy in x-y mode or line-scan mode. The x-y scans revealed discretely localized, spontaneous Ca(2+) events resembling Ca(2+) sparks and more global and prolonged Ca(2+) transients, which sometimes led to cell contraction. In line scans, Ca(2+) sparks were similar to those previously described in other types of smooth muscle, with an amplitude (DeltaF/F(0)) of 0.81 +/- 0.04 (mean +/- SE), full duration at half maximum (FDHM) of 23.62 +/- 1.15 ms, full width at half maximum (FWHM) of 1.25 +/- 0.05 mum, and frequency of 0.56 +/- 0.06 seconds(-1). Approximately 35% of sparks had a prolonged tail (>80 ms), similar to the Ca(2+)"embers" described in skeletal muscle. Sparks often summated to generate global and prolonged Ca(2+) elevations on which Ca(2+) sparks were superimposed. These sparks occurred more frequently (2.86 +/- 025 seconds(-1)) and spread farther across the cell (FWHM = 1.67 +/- 0.08 microm), but were smaller (DeltaF/F(0) = 0.69 +/- 0.04). Retinal arterioles generate Ca(2+) sparks with characteristics that vary during different phases of the spontaneous Ca(2+)-signaling cycle. Sparks summate to produce sustained Ca(2+) transients associated with contraction and thus may play an important excitatory role in initiating vessel constriction. This deserves further study, not least because Ca(2+) sparks appear to inhibit contraction in many other smooth muscle cells.

Calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.