This work was supported by RO1- AG028614 to JM, RO1- AR063084to NLW, and RO1-AR057404 to JZ.

1. Setting up Osmotic-stress Perfusion System
Figure 1 is a schematic protocol of calcium sparks assessment in intact skeletal muscle fibers.
<ol>
	<li>Set up a three-axis (xyz) micromanipulator capable of positioning the outlet tip of the perfusion system containing a minimum of two channels. This could be made with disposable Luer-syringe barrel with attached three-way Luer-Lok&#160;stopcock to switch on and/or off of the flow of perfusion solutions. These perfusion channels should be capable of delivering &#62;1 ml/min of solution through a single perfusion tip with a &#62;0.2 mm diameter.</li>
	<li>Load two channels of the perfusion system, one with Isotonic Tyrode Solution and the other with Hypotonic Tyrode Solution.</li>
</ol>
2. Preparing Intact Single Flexor Digitorum Brevis (FDB) Muscle Fibers from Mouse
<ol>
	<li>Remove the foot from a mouse euthanized following NIH and IACUC guidelines using a pair of heavy dissecting scissors by cutting through the leg above the ankle joint.</li>
	<li>Pin the foot onto a dissection chamber filled with Minimal Ca2+ Tyrode Solution with the plantar surface facing up.</li>
	<li>Dissect FDB by cutting the tendon and gently pulling up on the tendon to separate the muscle from the surrounding tissue.</li>
	<li>Finish dissection by cutting the distal tendon where it branches into individual digits in the deep layers of the muscle.</li>
	<li>Transfer the FDB muscle by picking up with forceps via its tendons to avoid additional damage to the muscle fibers and place into a tube with a thawed aliquot of Collagenase Digestion Solution prewarmed to 37 &#176;C.</li>
	<li>Incubate the tube containing FDB upright on an orbital shaker at 37 &#176;C for 60-90 min with a speed of 160 rpm. This muscle bundles dissociation protocol needs to be experimentally examined for different strains of animals, especially for those disease/mutant fibers vulnerable to membrane damage.</li>
	<li>Transfer the digested FDB into tube with 700 &#956;l of Isotonic Tyrode Solution and gently triturate to release the fibers by drawing the muscle several times through a series of 200 ml-micropipette tips of gradually decreasing diameter via cutting with a clean razor blade to remove the tips of 200 ml&#160;micropipette. To avoid damaging fibers in particularly weak muscles from disease, make sure the tip is just large enough to allow the fiber bundle to pass through without sticking. Do not force the bundle through too small an opening.</li>
	<li>Plate out the FDB fibers by gently tapping and resuspending FDB fibers in the tube and then drawing out 70 ml (1/10 of total fibers) with a cut 200 ml&#160;micropipette into the center of a 35&#160;mm Delta TPG dish containing 1 ml of Isotonic Tyrode Solution to reduce diffusion across the dish.</li>
	<li>Determine the number of intact single fibers in the dish using a dissection microscope. Add additional aliquots of FDB fibers to obtain 3-4 intact fibers on the dish.</li>
	<li>Store additional isolated muscle fibers at 4 &#176;C and use within 6 hr.</li>
</ol>
3. Fluo-4 AM Dye Loading and Ca2+ Imaging (Sparks Measurement)
<ol>
	<li>Transfer 500 ml of Isotonic Tyrode Solution from dish with plated FDB fibers into a tube with 10 ml Fluo-4 AM stock (1 mM), mix and add back to the dish to a final Fluo-4 AM concentration of 10 mM. Alternatively, pluronic acid can be used to increase dye loading efficiency.</li>
	<li>Load for 60 min at room temperature in the dark.</li>
	<li>Wash the fibers by carefully drawing out 500 ml of Fluo-4 AM-containing Isotonic Tyrode Solution from the dish with an uncut 200 ml-plastic micropipette tip and replace with equal volume of fresh Isotonic Tyrode Solution.</li>
	<li>Repeat this process 3 more times.</li>
	<li>To visualize mitochondrial function, transfer 500 ml of Isotonic Tyrode Solution from dish with FDB fibers into a tube with 5 ml TMRE stock (1 mM), mix, and add back to the dish for a final concentration of 50 nM.</li>
	<li>Incubate at room temperature for 10 min.</li>
	<li>Wash the fibers by carefully drawing out 500 ml of TMRE-containing Isotonic Tyrode Solution from the dish with an uncut 200 ml&#160;plastic micropipette tip and replace with equal volume of fresh Isotonic Tyrode Solution.</li>
	<li>Repeat this process 3 more times.</li>
	<li>Transfer the dish to the confocal microscope and select an intact fiber with clear striations and a smooth sarcolemmal membrane for experimentation.</li>
	<li>Position the tip of the perfusion system 400-500 &#181;m away from the target muscle fiber and off center using micromanipulator controls.</li>
	<li>Begin flow of the Isotonic Tyrode Solution to ensure the FDB fiber stays in place.</li>
	<li>Record the fluorescence signal from Fluo-4 AM with a 40X oil immersion objective and excite Fluo-4 AM with an argon laser (excitation wavelength 488 nm) and record emission signals (wavelength 510-580 nm). The intensity of the fluorescent signal is proportional to the relative level of intracellular Ca2+ concentration ([Ca2+]i).</li>
	<li>Induce Ca2+ transients by changing the osmotic pressure of the extracellular solution in order to produce osmotic stress on fiber. Initially perfuse the fibers with Isotonic Tyrode solution (290 mOsM) (baseline collection for 60 sec) and then with Hypotonic Tyrode solution (170 mOsM) for 100 sec to induce swelling. Perfuse the FDB fibers back with Isotonic Tyrode solution. Generally, only one osmotic shock is applied to a single intact fiber in one delta TPG dish and the spark events last 10 min for data acquisition.</li>
	<li>Record spatial localization of Ca2+ sparks (Figure 2) using a time-series of xy two-dimensional images with scan speed of 166 lps (line per second, and each line comprised of 512 pixels resulting in 3.08 sec/frame ) for each condition of isotonic (1 min), hypotonic stress (100 sec) and post-hypotonic stress (5 min). Store signals for offline analysis to evaluate the distribution and frequency of Ca2+ sparks following completion of the experiment.</li>
	<li>Find a new fiber on a new dish and follow the same osmotic shock protocol to induce Ca2+ transients. Use a confocal line scan (xt) mode (512 pixels in length, sample rate of 2 msec/line scan to record Ca2+ transients for one minute (i.e. 30,000 lines) with the confocal scan line placed directly underneath sarcolemmal membrane (Figure 3). Change the line scan to different focal planes to record three sequential series (at 1-min intervals) of linescans for analysis of the magnitude and kinetics of individual Ca2+ sparks.</li>
</ol>
4. Data Analysis
<ol>
	<li>Collect a large number of xt line scan traces to evaluate the morphology and kinetics of individual Ca2+ sparks parameters, i.e.&#160;the amplitude (F/F0; where F0 is the resting Ca2+ fluorescence), rise time, time to the peak, duration (FDHM, full duration at half magnitude), and spatial width (FWHM, full width at half magnitude) of the recorded sparks. These parameters represent the basic gating properties of RYR Ca2+ channels such as the Ca2+ flux (amplitude), and gating kinetics of RYRs (duration).</li>
	<li>Analyze digital image data with a custom-developed, semi-automatic algorithm which was created with image-processing language IDL (Interactive Data Language), as described previously11,16. Because the unique kinetics of Ca2+ release in the case of Ca2+ sparks induced in FDB fibers by osmotic stress, it is better to combine the manual and automated features of Ca2+ spark events identification and processing with these custom-build image analysis programs11. This algorithm is very useful when it's necessary to manually identify ROI (region of interest) in some muscle disease models. Once a ROI is defined, the algorithm could automatically derive the above mentioned spark parameters which could then be exported to Excel file. Alternatively, the public available image processing software, e.g. SparkMaster in ImageJ, can be used to define the kinetic properties of Ca2+ sparks and their spatial distribution in skeletal muscle17.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sparks+as+a+plastic+signal+for+skeletal+muscle+health,+aging,+and+dystrophy.">Ca2+ sparks as a plastic signal for skeletal muscle health, aging, and dystrophy.</a> Acta. 27 (7), 791-798 (2006).</li><li>Weisleder, N., Zhou, J., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+calcium+sparks+in+intact+and+permeabilized+skeletal+muscle+fibers.">Detection of calcium sparks in intact and permeabilized skeletal muscle fibers.</a> Methods Mol. 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No conflicts of interest declared.

This method of assessing Ca2+ sparks in intact skeletal muscle is a useful tool for muscle physiology and disease research. We showed that the Ca2+&#160;spark response was altered in different conditions, including muscular dystrophy11, aging18,19,&#160;as well as in amyotrophic lateral sclerosis20. Our recent study also revealed functional coupling between IP3 receptor and RyR representing a critical component that contributes to Ca<...

Intracellular free Ca2+ ([Ca2+]i) is a versatile and important secondary messenger that regulates multiple cellular functions in excitable cells such as neurons, cardiac, skeletal and smooth muscles (for review see&#160;Stutzmann&#160;and Mattson1). Regulated Ca2+ mobilization and cross-talk between sarcoplasmic reticulum (SR) and T-tubule (TT) membranes are fundamental regulators of muscle physiology. Furthermore, changes in Ca2+ signaling had been shown to be an underlying mechanism of contractile dysfunction in various muscle diseases.
Ca2+ sparks are localized elementary Ca2+ release events originating from opening of the ryanodine receptor (RyR) channel on the sarcoplasmic reticulum (SR) membrane2. In cardiac muscle, sparks occur spontaneously through opening of the RyR2 channel by a Ca2+-induced Ca2+&#160;release (CICR) mechanism 3-5. In skeletal muscle, RyR1 is strictly controlled by the voltage sensor at the TT membrane6,7. Thus Ca2+ sparks are suppressed and rarely detected in resting conditions in intact skeletal muscle fibers. Until recently, the sarcolemmal membrane needed to be disrupted by various chemical or mechanical &#34;skinning&#34; methods to uncouple the suppression of the voltage sensor on RyR1 and allowed for Ca2+ spark events to be detected in skeletal muscle8,9. One method previously described required permeabilization of the membrane of muscle fibers by saponin detergent10.
In 2003, we discovered that either transient hypoosmotic stress or hyperosmotic stress could induce peripheral Ca2+ sparks adjacent to the sarcolemmal membrane in intact muscle fibers11. This method has since been modified to study the molecular mechanism and modulation of Ca2+ release and dynamics12-16. Here we outline the details of our experimental approach for induction, detection and analysis of Ca2+ sparks in intact skeletal muscle. We also present our custom-built spark analysis program that can be used to quantify the elemental properties of individual Ca2+ sparks in skeletal muscle, e.g. spark frequency and amplitude (&#916; F/F0, which reflects the open probability of the RyR channels and the Ca2+ load inside the SR); time to peak (rise time) and duration (FDHM, full duration at half-maximal amplitude) of sparks, as well as the spatial distribution of Ca2+ sparks. In addition, we present evidence that links altered Ca2+ sparks to the various pathophysiological states in skeletal muscle, such as muscular dystrophy and amyotrophic lateral sclerosis.
The advantage of this technique involves the ability to measure Ca2+ in relatively undamaged cells, instead of stripping the muscle fibers, allowing recording of Ca2+ sparks in conditions closer to physiologic. Additionally, our custom-designed program provides more accurate calculations of the properties of the spark in relation to muscle fibers.

<table><tbody><tr><td>Dissecting microscope</td><td></td><td></td><td>Dissect out fibers and check muscle integrity</td></tr><tr><td>Dissection tools -scissors and fine tip forceps</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>DOW CORNING</td><td>184 SIL ELAST KIT</td><td>Make ahead of time for fiber dissection. Mix 5 ml liquid Sylgard components and pour into a 60 mm glass Petri dish and waiting 48 hr to let the Sylgard compound become a clear, colorless solid substrate.&amp;nbsp;</td></tr><tr><td>60 mm glass Petri dish&amp;nbsp;</td><td>Pyrex</td><td>3160</td><td>Fiber dissection</td></tr><tr><td>Isotonic Tyrode Solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Minimal Ca&lt;sup&gt;2+&lt;/sup&gt; Tyrode Solution</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Hypotonic Tyrode Solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Collagenase type I&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C-5138</td><td>Fiber digestion&amp;nbsp;</td></tr><tr><td>Fluo-4 AM&amp;nbsp;</td><td>Invitrogen</td><td>F14201</td><td>Fluorescent Ca&lt;sup&gt;2+&lt;/sup&gt; imaging dyes&amp;nbsp;</td></tr><tr><td>TMRE</td><td>Invitrogen</td><td>T669</td><td>mitochondrial membrane potential fluorescent dyes</td></tr><tr><td>Delta TPG dishes&amp;nbsp;</td><td>Bioptechs</td><td>0420041500C</td><td>35 mm glass bottom dish for imaging</td></tr><tr><td>Spark Fit software</td><td></td><td></td><td>data analysis software</td></tr><tr><td>Radiance 2100 laser scanning confocal microscope or equivalent microscope</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>3 Axis Micromanipulator</td><td>Narishige International</td><td>MHW-3</td><td></td></tr><tr><td>Temperature controllable orbital shaker</td><td>New Brunswick scientific</td><td></td><td>Fiber dissociation</td></tr></tbody></table>

assessment of calcium sparks in intact skeletal muscle fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Earlier studies showed that transient hypoosmotic stress induced peripheral Ca2+ sparks adjacent to the sarcolemmal membrane in intact muscle fibers11. Figure 1 shows the images of intact single muscle fibers with smooth sarcolemmal membrane and characteristic clear striations. Figure 2 shows typical Ca2+ sparks (as xy images) were induced by transient (100 sec) treatment with a hypoosmotic solution that swells the FDB muscle fibers from young, h...

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Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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15.1K Views.  The Ohio State University Wexner Medical Center.  Hier beschreven is een methode om direct te meten calcium vonken, de elementaire eenheden van Ca2 + vrij van sarcoplasmatisch reticulum in intacte spiervezels. Deze methode maakt gebruik van osmotische stress-gemedieerde activering van Ca 2 + vrijlating uit ryanodinereceptor in geïsoleerde spiervezels. De dynamiek en homeostatische aantal intracellulaire Ca2 +-signalen kunnen worden gebruikt om spierfunctie in gezondheid en ziekte te beo...

Video: Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Bioengineering

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Neuroscience

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

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