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

Summary

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.

Abstract

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.

Introduction

Cardiac excitation-contraction (E-C) coupling is the fundamental scheme for analyzing mechanical properties of the myocardium, i.e., the contractile function of the heart^1,2. 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 (µM) 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 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⁺ ATPase^1,2. 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 relaxation^1-3. 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 heart^1-4.

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⁺ buffers^5,6. 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⁺ buffers^5,6,7. 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 "release" 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 arrhythmias^8,9. 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⁺ sensitivity^8-10. 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 "active" 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 intracellular Ca²⁺ profile, myofilament Ca²⁺ sensitivity and contraction will unearth novel mechanisms underlying myocardial mechanics.

Protocol

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, 3.5; glucose, 5; HEPES, 5; Na₂HPO₄, 0.4; taurine, 20; pH 7.4 with NaOH).
2. 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).
3. 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).
4. 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.
5. 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.
6. 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.

2. Preparation for the Isolation of Left Ventricular (LV) Myocytes

1. Transfer 8-12 week-old, male Sprague-Dawley (SD) rats in clean transport cages from the animal facility to the preparation and isolation room.
2. 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.
3. 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.
4. 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).
5. 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 via the oxygen connection tube.

3. Isolation of LV Myocytes

1. 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.
2. Move the rat to a dissection tray. In the supine position, secure the four legs to the sides of the body with tape.
3. 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.
4. 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.
5. 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.
6. 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).
7. 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.
8. 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.
9. Disperse the myocyte pellets and keep myocytes in 10 ml of pre-oxygenated storage solution at RT (Figure 1Biv-vi).
10. 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.
11. Keep the myocytes in storage solution for 6-8 hr at RT until the end of experiments.

4. Simultaneous Measurements of Intracellular Ca²⁺ Transients and Myocyte Contraction

1. Load LV myocytes with the acetoxymethyl ester Fura-2 AM (2 µM).
   Note: Perform all loading procedures and experiments with loaded myocytes in a dark tube (Figure 1Bvii).
   1. 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 µM, Table Materials and Equipment).
   2. Add Fura-2AM and poloxamer 407 (2 µl), gently disperse the myocyte suspension, and keep the mixture at RT (20 - 24^o C) for 15 min (Figure 1Bvii).
   3. Centrifuge the mixture for 5 sec, discard the supernatant and disperse the myocyte pellet in 1 ml of perfusion solution containing 500 µM Ca²⁺. After 10 min, centrifuge the mixture for 5 sec and discard the supernatant.
   4. Add fresh perfusion solution (500 µM Ca²⁺, 1 ml) and keep the Fura-2AM - loaded myocyte pellet in this solution for recordings.
2. Measurement of LV myocyte contraction and intracellular Ca²⁺
   1. Before recording, fill the perfusion tube that runs through a water jacket with the Tyrode solution, which is pre-warmed to 36 ^o C.
   2. 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).
   3. Press the "start" 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.
      1. Select myocytes that contract stably (not those displaying hyper- or hypo-contraction) for recording.
   4. 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).
   5. Record the changes of sarcomere length in response to field stimulation (Figure 2A and B).
      Note: Use the loaded myocytes within 1 - 2 hr.
   6. 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).

5. Assessment of Myofilament Ca²⁺ Sensitivity

1. 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).
2. In each plot, define Fura -2 ratio at 50% relaxation (EC₅₀, Figure 2C). Compare both the loop and EC₅₀ of each intervention.

Results

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...

Discussion

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

Materials

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
Na2HPO4	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
Na2HPO4	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