Name: assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling
Uploaded: 2016-08-01T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

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...

<ol>
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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 ...

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