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Biology

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

Published: August 1st, 2016

DOI:

10.3791/54057

1Department of Physiology & Biomedical Sciences, Ischemic/hypoxic Disease Institute, Seoul National University College of Medicine, 2Yan Bian University Hospital, 3Institute of Cardiovascular Sciences, University of Manchester

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 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.

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+ concentr....

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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)

  1. Prepare 300 ml of fresh isolation solution on the day of the experiment (in mM: NaCl, 135; KCl, 5.4; MgCl2.......

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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.......

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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

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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 31460265; NSFC 81260035).

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Name Company Catalog Number Comments
Sprague Dawley rat Koatech 8-12 weeks
Pentobarbital Sodium Hanlim Pharmaceutical (Korea) AHN901 Insurance code:645301220
NaCl Sigma S9625
KCl Sigma P4504
NaH2PO4 Sigma S8282
HEPES Sigma H3375
Glucose Sigma G8270
CaCl2 Biosesang C2002
MgCl2 Biosesang M2001
Mannitol Sigma M4125
MgSO4 Sigma M5921
Sodium Pyruvate Sigma P2256
Taurine Merck 8.08616.1000
Na2HPO Sigma 71649
Bovine Fetal Albumin Sigma A7906
Collagenase Type 2 Worthington LS004177
Protease Sigma P6911
Fura-2 (AM) Molecular Probes F1221
Pluronic F127 20% solution in DMSO Invitrogen P3000MP
Shaking Water Bath Chang Shin Scientific Model: C-108
IonWizard Softwae Suite IonOptix Ltd Experimental Builder Acquisition and Analysis of EC Coupling Data in Myocytes
Myocyte Calcium & Contractility Recording System IonOptix Ltd
Circulating Water Bath BS-Tech BW2-8
Myocyte Fluorescence Microscope Nikon DIATPHOTO 200
MyoCam-S Power IonOptix
Fluorescence & Video Detection IonOptix MyoCam-S
CFA300
PMT400
Fluorescence & System Interface IonOptix FSI700
Excitation Light Source IonOptix mSTEP
High intensity ARC Lamp Power supply Cairn Reseach
Filter wheel controller IonOptix GB/MUS200
Digital Stimulator Medical Systems Corportion S-98 Mutimode
Compositions of Experimental Solutions
Name Company Catalog Number Comments
Isolation Solution (pH: 7.4, NaOH)
NaCl Sigma S9625 Concentration (mmol) 135
KCl Sigma P4504 Concentration (mmol) 5.4
HEPES Sigma H3375 Concentration (mmol) 5
Glucose Sigma G8270 Concentration (mmol) 5
MgCl2 Biosesang M2001 Concentration (mmol) 3.5
Taurine Sigma CB2742654 Concentration (mmol) 20
Na2HPO Sigma 71649 Concentration (mmol) 0.4
Storage Solution (pH: 7.4, NaOH)
NaCl Sigma S9625 Concentration (mmol) 120
KCl Sigma P4504 Concentration (mmol) 5.4
HEPES Sigma H3375 Concentration (mmol) 10
Glucose Sigma G8270 Concentration (mmol) 5.5
CaCl2 Biosesang C2002 Concentration (mmol) 0.2
Mannitol Sigma M4125 Concentration (mmol) 29
MgSO4 Sigma M5921 Concentration (mmol) 5
Sodium Pyruvate Sigma P2256 Concentration (mmol) 5
Taurine Sigma CB2742654 Concentration (mmol) 20
Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)
NaCl Sigma S9625 Concentration (mmol) 141.4
KCl Sigma P4504 Concentration (mmol) 4
NaH2PO4 Sigma S8282 Concentration (mmol) 0.33
HEPES Sigma H3375 Concentration (mmol) 10
Glucose Sigma G8270 Concentration (mmol) 5.5
CaCl2 Biosesang C2002 Concentration (mmol) 1.8      For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM
MgCl2 Biosesang M2001 Concentration (mmol) 1
Mannitol Sigma M4125 Concentration (mmol) 14.5
Collangenase Solution 1
Isolation Solution (30mL)
Bovine Fetal Albumin (BSA solution 5 ml) Concentration (mmol) 1.67 mg/mL
Collagenase Type 2 Worthington LS004177 Concentration (mmol) 1 mg/mL
Protease Sigma P6911 Concentration (mmol) 0.1 mg/mL
CaCl2 Biosesang C2002 Concentration (mmol) 0.05 mM
Collangenase Solution 2
Isolation Solution (20mL)
Bovine Fetal Albumin (BSA solution 3.3 mL) Concentration (mmol) 1.67 mg/mL
Collagenase Type 2 Worthington LS004177 Concentration (mmol) 1 mg/mL
CaCl2 Biosesang C2002 Concentration (mmol) 0.05 mM
BSA solution
Isolation Solution (40mL)
Bovine Fetal Albumin Sigma A7906 Concentration (mmol) 400 mg
CaCl2 Biosesang C2002 Concentration (mmol) 1mM

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