This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013068067); by the Brain Korea 21 Graduate Programme of the Korean Ministry of Education, Science and Technology, Seoul National University Hospital, the Korean Society of Hypertension (2013), SK Telecom Research Fund (no. 3420130290) and from the National Natural Science Foundation of China (NSFC&#160;31460265; NSFC 81260035).

The protocol is in accordance with the Guide for the Care and Use of Laboratory Animals published by the UN National Institutes of Health (NIH Publication No. 85-23, revised 1996). It was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (IACUC approval no.: SNU-101213-1).
1. Buffer Preparation (Table Materials and Equipment)
<ol>
	<li>Prepare 300 ml of fresh isolation solution on the day of the experiment (in mM: NaCl, 135; KCl, 5.4; MgCl2, 3.5; glucose, 5; HEPES, 5; Na2HPO4, 0.4; taurine, 20; pH 7.4 with NaOH).</li>
	<li>Prepare 100 ml of fresh storage solution on the day of the experiment (in mM: NaCl 120; KCl 5.4; MgSO4 5; CaCl2 0.2; sodium-pyruvate 5; glucose 5.5; taurine 20; HEPES 10; mannitol 29; pH 7.4 with NaOH).</li>
	<li>Prepare 1L of perfusion solution (in mM: NaCl, 141.4; KCl, 4; NaH2PO4, 0.33; MgCl2, 1; HEPES, 10; glucose, 5.5; CaCl2, 1.8; mannitol, 14.5; pH 7.4 with NaOH).</li>
	<li>Prepare 30 ml of collagenase solution 1 by adding collagenase (1 mg/ml), protease (0.1 mg/ml), bovine serum albumin (BSA, 1.67mg/ml), and Ca2+ (0.05 mM) to 25 ml of isolation solution.</li>
	<li>Prepare 20 ml of collagenase solution 2 by adding collagenase (1 mg/ml), BSA (1.67 mg/ml), and Ca2+ (0.05 mM) to 16.7 ml of isolation solution.</li>
	<li>Prepare 40 ml of BSA solution by adding 0.4 g of BSA to 40 ml of isolation solution. Separate 10 ml and 30 ml into two beakers. Add Ca2+ to 30 ml of BSA solution so that the final Ca2+ concentration in the BSA solution is 1 mM.</li>
</ol>
2. Preparation for the Isolation of Left Ventricular (LV) Myocytes
<ol>
	<li>Transfer 8-12 week-old, male Sprague-Dawley (SD) rats in clean transport cages from the animal facility to the preparation and isolation room.</li>
	<li>Heat two water baths to 37 o C. 
	Note: Use one water bath for the water - jacketed reservoir and the perfusion tubes of the Langendorff perfusion system. Use the other water bath to agitate myocardial tissue trunks in order to separate single myocytes.</li>
	<li>Add 5 ml and 3.3 ml of BSA solution (without Ca2+) to 25 ml of collagenase solution 1 and 16.7 ml collagenase solution 2 to make up the volume to 30 ml and 20 ml, respectively.</li>
	<li>Add isolation solution (100 ml) and collagenase solution 1 (30 ml) to column 1 (Col. 1) and column 2 (Col. 2) in the Langendorff perfusion system, respectively (Figure 1A).</li>
	<li>Oxygenate the isolation solution and collagenase solution 1 in Col. 1 and Col.2 via the oxygen connection tube in the Langendorff perfusion system (Figure 1A). Similarly, oxygenate collagenase solution 2 and the BSA solution in the shaking water bath with 100% O2&#160;via the oxygen connection tube.</li>
</ol>
3. Isolation of LV Myocytes
<ol>
	<li>Anesthetize a SD rat via intraperitoneal injection of pentobarbital sodium (30 mg/kg) and confirm the anesthetic status by toe pinching and lack of withdrawal reflection.</li>
	<li>Move the rat to a dissection tray. In the supine position, secure the four legs to the sides of the body with tape.</li>
	<li>Apply thoracic mid-axial incisions with surgical scissors to open the chest, ensuring not to damage the heart. Use another pair of clean scissors to dissect the heart from the connecting vessels (e.g., superior and inferior vena cava, pulmonary vessels, and aorta) and pericardial membrane13.</li>
	<li>Leave a portion of the aorta of a sufficient length (5 - 8 mm), clamp the aorta with fine forceps, and rapidly mount the cannula of the Langendorff perfusion system within 1 min (Figure 1A). Tie suture thread 4/0 tightly over the aorta.</li>
	<li>Turn on the valve on Col. 1 and perfuse the isolated heart with pre-warmed and oxygenated isolation solution for 10 min (perfusion rate: 12-14 ml/min). Turn off the valve on Col. 1, turn on the valve on Col. 2 and perfuse the heart with collagenase solution 1 for 8 - 10 min.</li>
	<li>Dismount the digested heart by cutting the aorta and transfer it by holding the aorta with forceps into the flask containing fresh isolation solution (Figure 1Bi). Use fine scissors and forceps to cut most of the LV free wall (including the septum) into smaller pieces (~22 mm, Figure 1Bii).</li>
	<li>Transfer the pieces into a flask containing pre-warmed and oxygenated fresh collagenase solution 2 (Figure 1Biii). Shake for 10 min. Keep delivering oxygen to the myocyte-containing collagenase solution 2.</li>
	<li>Move the myocyte - suspension to a 10 ml centrifuge tube using droppers with a suction bulb and add Ca2+ - containing BSA solution (1:1 in volume). Centrifuge at 30 g for 2 min and discard the supernatant. Re-suspend the myocyte pellet in 5 ml of BSA solution. Centrifuge and discard the supernatant.</li>
	<li>Disperse the myocyte pellets and keep myocytes in 10 ml of pre-oxygenated storage solution at RT (Figure 1Biv-vi).</li>
	<li>Repeat the procedures (steps 3.7 - 3.9) with the remaining LV tissue in the flask. 
	Note: Keep repeating the procedures (steps 3.7 - 3.9) again until most of the LV tissue disappears to obtain a good yield of myocytes.</li>
	<li>Keep the myocytes in storage solution for 6-8 hr at RT until the end of experiments.</li>
</ol>
4. Simultaneous Measurements of Intracellular Ca2+ Transients and Myocyte Contraction
<ol>
	<li>Load LV myocytes with the acetoxymethyl ester Fura-2 AM (2 &#181;M). 
	Note: Perform all loading procedures and experiments with loaded myocytes in a dark tube (Figure 1Bvii).
	<ol>
		<li>Centrifuge the myocyte suspension (1 ml) at 2,000 x g for 10 sec. Discard the supernatant and re-suspend the myocyte pellet in 1 ml of Tyrode solution with a low Ca2+ concentration (250 &#181;M, Table Materials and Equipment).</li>
		<li>Add Fura-2AM and poloxamer 407 (2 &#181;l), gently disperse the myocyte suspension, and keep the mixture at RT (20 - 24o C) for 15 min&#160;(Figure 1Bvii).</li>
		<li>Centrifuge the mixture for 5 sec, discard the supernatant and disperse the myocyte pellet in 1 ml of perfusion solution containing 500 &#181;M Ca2+. After 10 min, centrifuge the mixture for 5 sec and discard the supernatant.</li>
		<li>Add fresh perfusion solution (500 &#181;M Ca2+, 1 ml) and keep the Fura-2AM - loaded myocyte pellet in this solution for recordings.</li>
	</ol>
	</li>
	<li>Measurement of LV myocyte contraction and intracellular Ca2+
	<ol>
		<li>Before recording, fill the perfusion tube that runs through a water jacket with the Tyrode solution, which is pre-warmed to 36 o C.</li>
		<li>Place a few drops of the Fura 2 AM - loaded LV myocyte suspension on the chamber of an inverted fluorescence microscope for 5 - 8 min. Slowly perfuse the Tyrode solution (2.2 ml/min).</li>
		<li>Press the &#34;start&#34; button on the front panel of the digital stimulator to start field stimulation (2 Hz). 
		Note: The output voltage (10 V, 5 msec duration) is applied to the myocytes in the chamber through platinum wires positioned on either side of the chamber.
		<ol>
			<li>Select myocytes that contract stably (not those displaying hyper- or hypo-contraction) for recording.</li>
		</ol>
		</li>
		<li>Adjust the myocyte of choice in the horizontal position of the video-based sarcomere detection system and adjust the focus of the microscope to obtain optimal images of sarcomeres. Position the purple rectangular box on the area in which clear sarcomeres are clearly observed until the average of sarcomere lengths (red peak) displays a singular sharp peak (Figure 2A, lower image).</li>
		<li>Record the changes of sarcomere length in response to field stimulation (Figure 2A and B). 
		Note: Use the loaded myocytes within 1 - 2 hr.</li>
		<li>Adjust the aperture of the camera so that the field in the video-based sarcomere detection system is the size of the myocyte (Figure 2A). Stimulate the myocyte with excitation at 360 nm/380 nm and emission at 510 nm (acquisition frequency 1,000 Hz). Record sarcomere shortening and the Fura2 AM ratio with field stimulation (Figure 2B).</li>
	</ol>
	</li>
</ol>
5. Assessment of Myofilament Ca2+ Sensitivity
<ol>
	<li>Average the sarcomere lengths and Ca2+ transients at steady-state (10 - 20 traces) and plot the phase-plane loop of the Fura-2 ratio vs. sarcomere length of the same myocyte (both with measured values and delta changes; Figure 2C).</li>
	<li>In each plot, define Fura -2 ratio at 50% relaxation (EC50, Figure 2C). Compare both the loop and EC50 of each intervention.</li>
</ol>

<ol>
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The authors declare that they have no competing financial interests.

Here, we describe the protocols to assess changes of myofilament Ca2+ sensitivity in single isolated cardiac myocyte and emphasize the importance of measuring this parameter alongside electrophysiological properties, intracellular Ca2+ transients, and myofilament dynamics. This is because the recordings of one or two of the parameters may not explicate the mechanisms underlying cardiac contraction and relaxation. Unlike conventional methods that measure myocyte contraction and the intracellular Ca<s...

Cardiac excitation-contraction (E-C) coupling is the fundamental scheme for analyzing mechanical properties of the myocardium, i.e., the contractile function of the heart1,2. E-C coupling is initiated by membrane depolarization secondary to the activities of sarcolemmal ion channels (e.g., the voltage-gated Na+ channel, which can be measured via patch-clamp techniques). Subsequent activation of voltage-gated L-type Ca2+ channels (LTCCs) and Ca2+ influx via LTCCs trigger the bulk of Ca2+ release through ryanodine receptors (RyRs), increasing the cytosolic Ca2+ concentration from the nanomolar (nM) to micromolar (&#181;M) level. Such an increase in cytosolic Ca2+ promotes Ca2+ binding to troponin C (TnC) in thin filaments and elicits conformational changes of the filament complex to facilitate the actin-myosin interaction and attains myocardial contraction3. Conversely, the cytosolic Ca2+ is re-uptaken&#160;back into the sarcoplasmic reticulum (SR) through the Ca2+-ATPase in SR (SERCA2a) or is extruded out of the myocyte via the Na+/Ca2+ exchanger and the plasmalemmal Ca2+ ATPase1,2. Consequently, the decline in cytosolic Ca2+ instigates conformational changes of thin filaments back to the original state, resulting in the dissociation of actin-myosin and myocyte relaxation1-3. In this scheme, the activity of SERCA2a is generally considered to determine the speed of myocardial relaxation because it accounts for 70 - 90% of cytosolic Ca2+ removal in most mammalian heart cells1. As such, abnormal Ca2+ handling by LTCC, RyR and SERCA2a, etc. has been considered the primary mechanisms for impaired contractility and relaxation in the diseased heart1-4.
In reality, free cytosolic Ca2+ that functions as the messenger in E-C coupling accounts for around 1% of total intracellular Ca2+ and the majority of Ca2+ is bound to intracellular Ca2+ buffers5,6. This is due to the fact that various Ca2+ buffers are abundant in cardiac myocytes, e.g., membrane phospholipids, ATP, phosphocreatine, calmodulin, parvalbumin, myofibril TnC, myosin, SERCA2a, and calsequestrin in the SR.5,6,7. Among them, SERCA2a and TnC are the predominant Ca2+ buffers5,6,7. Furthermore, Ca2+ binding to its buffers is a dynamic process during twitch (e.g., 30-50% of Ca2+ binds to TnC and dissociate from it during Ca2+ transients7) and the change in Ca2+ binding cause additional &#34;release&#34; of free Ca2+ to the cytosol, results in the alterations of the intracellular Ca2+ concentration. Consequently, perturbation of the intracellular Ca2+ level induces abnormal myofilament movements, which are the precursors of contractile dysfunction and arrhythmias8,9. Many factors (both physiological and pathological) can be the sources of post-transcriptional modifications of myofilament proteins, which influence myofilament Ca2+ buffering and myofilament Ca2+ sensitivity8-10. Recently, it was reported that mutations in myofilament proteins increase the Ca2+ binding affinity and intracellular Ca2+ handling, triggering pause-dependent potentiation of Ca2+ transients, abnormal Ca2+ release, and arrhythmias8. In line with this concept, we have also shown that myofilament Ca2+ desensitization in hypertensive rat hearts secondary to the up-regulation of neuronal nitric oxide synthase is associated with elevated diastolic and systolic Ca2+ levels11, which in turn, increases the vulnerability of the LTCC to Ca2+-dependent inactivation12. Hence, myofilament Ca2+ sensitivity is an &#34;active&#34; regulator of intracellular Ca2+ homeostasis and myocyte contractile function. It has become necessary to analyze interactions between myofilament and Ca2+ handling proteins for thorough investigation of myocyte E-C coupling and cardiac function.
Here, we describe a protocol that assesses the changes of myofilament Ca2+ sensitivity in isolated cardiac myocytes. Comprehensive analysis of&#160;intracellular Ca2+ profile, myofilament Ca2+ sensitivity and contraction will unearth novel mechanisms underlying myocardial mechanics.

<table border="0" cellpadding="0" cellspacing="0" width="1136">
	<tbody>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">Sprague Dawley rat</td>
			<td style="width:131px;">Koatech</td>
			<td style="width:125px;"></td>
			<td style="width:565px;">8-12 weeks</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:315px;">Pentobarbital Sodium</td>
			<td style="width:131px;">Hanlim Pharmaceutical (Korea)</td>
			<td style="width:125px;">AHN901</td>
			<td style="width:565px;">Insurance code:645301220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Taurine</td>
			<td style="width:131px;">Merck</td>
			<td style="width:125px;">8.08616.1000</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fura-2 (AM)</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:125px;">F1221</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Pluronic F127 20% solution in DMSO</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:125px;">P3000MP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking Water Bath</td>
			<td style="width:131px;">Chang Shin Scientific</td>
			<td style="width:125px;">Model: C-108</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:315px;">IonWizard Softwae Suite</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;">Experimental Builder</td>
			<td style="width:565px;">Acquisition and Analysis of EC Coupling Data in Myocytes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:315px;">Myocyte Calcium &#38; Contractility Recording System</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Circulating Water Bath</td>
			<td style="width:131px;">BS-Tech</td>
			<td style="width:125px;">BW2-8</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:315px;">Myocyte Fluorescence Microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:125px;">DIATPHOTO 200</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:315px;">MyoCam-S Power</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="36">
			<td height="119" rowspan="3" style="height:119px;width:315px;">Fluorescence &#38; Video Detection</td>
			<td rowspan="3" style="width:131px;">IonOptix</td>
			<td style="width:125px;">MyoCam-S</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:125px;">CFA300</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:125px;">PMT400</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Fluorescence &#38; System Interface</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">FSI700</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Excitation Light Source</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">mSTEP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">High intensity ARC Lamp Power supply</td>
			<td style="width:131px;">Cairn Reseach</td>
			<td></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Filter wheel controller</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">GB/MUS200</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;">Digital Stimulator</td>
			<td style="width:131px;">Medical Systems Corportion</td>
			<td>S-98 Mutimode</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compositions of Experimental Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name</td>
			<td style="width:131px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:565px;">Comments</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:315px;">Isolation Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 135</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 3.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;">Concentration (mmol) 0.4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Storage Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 120</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.2</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 29</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:315px;">Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 141.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;">Concentration (mmol) 0.33</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td>Concentration (mmol) 1.8&#160;&#160;&#160;&#160;&#160; For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 1</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 14.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 1</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (30mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 5 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;">Concentration (mmol) 0.1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 2</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (20mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 3.3 mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">BSA solution</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (40mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;">Concentration (mmol) 400 mg</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 1mM</td>
		</tr>
	</tbody>
</table>

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.

LV myocytes are isolated from normal and hypertensive rat hearts. Rod-shaped myocytes with clear striations (representing sarcomeres) and stable contractions in response to field stimulation are considered to be the optimal myocytes and are selected for recordings (Figure 2A). In the example shown in Figure 2A, a Fura 2 AM -loaded LV myocyte is positioned horizontally and the aperture of the camera is adjusted so that the myocyte occupies most of the reco...

Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

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.

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 ...

assessment-myofilament-ca2-sensitivity-underlying-cardiac-excitation

Research

JoVE Journal

Biology

9.4K Views.  Seoul National University College of Medicine. 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. 

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

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+ ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ 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 Ca2+ 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 Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...