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

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
Published 8/01/2016
0 Comments
  CITE THIS  SHARE 
Biology

You must be subscribed to JoVE to access this content.

Fill out the form below to receive a free trial:

Welcome!

Enter your email below to get your free 10 minute trial to JoVE!





By clicking "Submit," you agree to our policies.

 

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.

Cite this Article

Copy Citation

Zhao, Z. H., Jin, C. L., Jang, J. H., Wu, Y. N., Kim, S. J., Jin, H. H., et al. Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling. J. Vis. Exp. (114), e54057, doi:10.3791/54057 (2016).

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 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 (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+ 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 "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 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 "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.

Subscription Required. Please recommend JoVE to your librarian.

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% Ovia 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 - 24o 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.

Subscription Required. Please recommend JoVE to your librarian.

Representative 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 recording field and minimal background area is included. In the recording field, adjust the dimensions of the purple box by clicking and dragging this box on the computer screen (through the recording program). When the average sarcomere length shows one sharp red peak, start recording (Figure 2A, lower image).

Both sarcomere length and intracellular Ca2+ transients (Fura-2 ratios) are recorded simultaneously from the same myocytes (Figure 3A). The average traces of sarcomere length and Ca2+ transients are shown in Figure 3B. Individually, diastolic/systolic sarcomere lengths, time to peak (PT), and sarcomere shortening are measured to investigate the amplitude and dynamics of myocyte contractility. Time to 50% relaxation (TR50) is analyzed to assess the relaxation of the myocyte. Similarly, the diastolic and systolic Fura-2 ratios (Ca2+ transients), time to peak (PT) of Ca2+ transients and time constant of Ca2+ transient decay (tau) are analyzed to assess myocyte contraction and relaxation (Table 1). In this example, the amplitudes of Ca2+ transients are moderately increased in LV myocytes from a hypertensive rat and contraction is moderately reduced (Figure 3B and Table 1).

Furthermore, the relationships between the Fura - 2 ratio and sarcomere length (indicating the myofilament Ca2+ sensitivity of LV myocytes) are plotted in both sham and hypertensive rats (Figure 3C). The Fura 2 - sarcomere length trajectory during the relaxation phase of the myocyte defines a quasi-equilibrium of cytosol Ca2+, myofilament Ca2+ binding, and sarcomere length14; therefore, the relaxation phase is compared between the two groups. The rightward shift of the trajectory in myocytes from the hypertensive group indicates a reduced myofilament response to Ca2+ (Figure 3C). Accordingly, the intracellular Ca2+ concentration required for half relaxation (EC50) is increased (Figure 3C), referring to myofilament Ca2+-desensitization in hypertension.

Figure 1
Figure 1. Procedures for isolating LV myocytes from rat heart. (A) The Langendorff perfusion system used to perfuse the isolation solution into the heart cannulated via the aorta (inset, magnified image of the mounted heart on the perfusion system). (B) i - iii: The heart after digestion with collagenase solution 1 and dissected LV tissue in a dish and flask. (B) iv - vi: Myocyte suspension after addition of collagenase solution 2, myocyte pellet after centrifugation, and re-suspended myocyte pellet in storage solution. (B) vii: Incubation, incubate LV myocytes in Fura 2AM - containing isolation solution (2 µM Fura 2 AM, 250 µM Ca2+ and with 2 µl poloxamer 407); washout, wash LV myocytes with isolation solution with 500 µM Ca2+; storage, keep Fura-2 AM - loaded LV myocytes in fresh isolation solution containing 500 µM Ca2+. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Measurement of sarcomere shortening and the Fura-2 ratio (indicative of the intracellular Ca2+ level). (A) A diagram of sarcomere, an image of a Fura-2 -loaded LV myocyte and the averaged sarcomere length (the red peak) are displayed on the computer. In the lower panel, the black line is the average of each horizontal pixel line within the purple region of interest. The blue line is the same data zeroed at each end. The red line is the fast Fourier transform (FFT) power spectrum, which represents the number of signals the FFT has calculated. One sharp peak means a clean sarcomere recording. (B) Simultaneous recordings of sarcomere length and the Fura -2 ratio in response to field stimulation (2Hz). (C) Phase-plane plot of the Fura 2 ratio vs. sarcomere length of the same LV myocyte (note that both the actual length/Fura 2 ratio and delta changes of these parameters are analyzed). EC50 (Fura 2 ratio at 50% relaxation, circle indicated by arrow) is the qualitative comparison of myofilament Ca2+ sensitivity between the groups. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Representative results of the analysis of LV myocyte contraction in sham and hypertensive rats. (A) Raw traces of sarcomere shortening and Fura - 2 ratio measurement in LV myocytes from sham and hypertensive rats. (B) Average traces of sarcomere length and Fura - 2 signals. Parameters analyzed in the averaged traces are shown in Table 1. (C) Phase-plane plots of the Fura - 2 ratio vs. sarcomere length (both the actual length and Fura - 2 ratio and delta changes of these parameters) in the two groups. The trajectory loop is shifted to the right and EC50 tends to be higher in hypertension, suggesting myofilament Ca2+ desensitization. PT, time to peak (sec); Tau, time constant of Ca2+ transient decay (sec) (obtained by fitting the decline phase of the Fura - 2 ratio with an exponential function).TR50: time to 50% relaxation (sec). Figure 3 Please click here to view a larger version of this figure.

Parameters Sham Hypertension
Intracellular Ca2+ Diastolic Ca2+ 1.189 1.124
Systolic Ca2+ 1.71 1.691
Amplitude (Δ ratio) 0.521 0.567
Time to peak (PT, s) 0.021 0.031
Tau (s) 0.079 0.076
Sarcomere Diastolic 1.758 1.78
sarcomere length (μm)
Sarcomere 0.122 0.115
Shortening (Δ length,mm)
Time to peak (PT, s) 0.064 0.055
Time to 50% 0.032 0.03
Relaxation (TR50,s)
EC50 [Ca2+]i (Fura-2 ratio) for 50% sarcomere relengthening  0.2382 0.3224

Table 1. Analysis of the Fura - 2 ratio (intracellular Ca2+) and sarcomere length measurements.

Subscription Required. Please recommend JoVE to your librarian.

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 Ca2+ profile individually1, the present method examines both parameters simultaneously in the same cardiac myocytes.

A number of methods are generally used to assess the myofilament Ca2+ sensitivity of muscles or myocytes15,16,17, e.g., examination of the interactions between exogenous Ca2+ and sarcomere/cell length/tension in skinned myocytes (treated with detergents such as saponin or β-esin to permeabilize the membrane). Lengths are measured with photo-diode and laser beam diffraction techniques, and tension with force transducers or carbon fibers). These techniques are widely used in muscle studies because they enable quantitative assessments of myofilament Ca2+ sensitivity. However, endogenous ion channel activities in the plasma membrane and intracellular Ca2+ handling, those are required to initiate the mechanics of myofilament, are neglected in these techniques. In addition, the muscle strips or myocytes under study are pre-stretched to a certain diastolic length, and under these conditions, the initial sarcomere length can differ from the actual diastolic length of the myocytes (especially in disease conditions and with interventions). This highlights the advantage of the current measurement where cellular organelles remain relatively unperturbed, thereby, enabling the comprehensive evaluation of myocyte contractile function.

Understandably, quality of the myocytes (Ca2+-tolerating) and the optimal loading of Fura - 2AM are two key elements for successful assessment of myofilament Ca2+ sensitivity. Accordingly, several modifications are applied to the protocol in order to obtain viable rod-shaped myocytes (60-80% of total cells, as described previously18,19,20). First, a low dose of Ca2+ is added to the solutions during the digestion processes (e.g., the Ca2+ concentration is 50 µM in the collagenase solutions and 200 µM in the storage solution). Second, during the digestion process, BSA is added to the collagenase solutions to enhance membrane stability. Third, most of the solutions are oxygenated during isolation, which enhances the percentage of functional myocytes and the duration of the experiments (6-8 hr). Fourth, isolated myocytes are kept in storage solution containing 200 µM Ca2+.

To obtain optimal loading with Fura 2AM, two different Ca2+ concentrations (250 and 500 µM) are used and poloxamer 407 (2 µl, 20% prepared in dimethyl sulfoxide) is added to enhance the permeability of Fura-2AM through the membrane and its stability in myocytes. It should be noted that all Ca2+ indicator dyes are Ca2+ buffers21. Therefore, researchers should avoid using a high concentration of Fura-2 AM or a longer incubation to avoid excessive buffering and reduced myocyte contractility. It is recommended that myocyte contraction without Fura - 2 loading is routinely checked, which is an essential step to estimate the quality of myocyte and the loading status. Variability in loading is inevitable. Therefore, it is important to check the variability of myocyte contraction, specifically, whether the number of contracting myocytes in the chamber is similar before and after loading. Analysis of hyper- or hypo-contracting myocytes should be avoided because they do not represent the average status of myocytes and may cause biased results and evaluations.

It should be noted that the measured changes in the Fura 2 ratio are reflections of the intracellular Ca2+ level, rather than the actual chemical concentration of Ca2+. Therefore, the method described here evaluates qualitative changes of myofilament Ca2+ sensitivity, rather than the actual sensitivity of myofilaments to Ca2+. Calibration of Fura - 2 signal to the intracellular Ca2+ concentration is the solution to acquire reliable Ca2+ concentrations in individual myocytes. The calibration procedure requires loading myocytes with various concentrations of Ca2+-conjugated Fura - 2 AM (Ca2+ concentration ranging from 0 to 39 µM), and the obtained Fura - 2 ratios are calculated with the following formula: [Ca2+]i = Kd * (R - Rmin) / (Rmax - R) * F380max / F380min21. It is possible to derive the pooled average values of Ca2+ concentrations through such a calibration procedure. However, because the loading of Ca2+ indicators is variable among myocytes and calibration is not performed in an individual cell, Ca2+ concentrations may not be acquired accurately. Furthermore, if F360/F380 is measured rather than F340/F380 (as in the present study), the Fura 2 ratio is less accurate (because F360 is the isobestic wavelength of Fura-2 fluorescence22). Nevertheless, it is still a valid method to assess qualitative changes in myofilament Ca2+ sensitivity in physiological experiments, especially in diseased human hearts, where myofilaments can be concomitantly altered after changes in the intracellular environment. It is recommended that this method is combined with alternative methods (as described previously in the Discussion section) to precisely analyze the true sensitivity of myofilaments to Ca2+.

The other limitation of the method in the study of myocyte contraction is the unloaded condition. It may underestimate or overestimate the changes in myofilament Ca2+ sensitivity. Therefore, to accurately evaluate myofilament Ca2+ sensitivity, analysis should be performed with alternative measurements for both qualitative and quantitative assessment.

The technique is applicable for assessing myocardial function in healthy and diseased hearts, including human cardiac samples, where the cellular redox environment and post-transcriptional modifications are changed, resulting in concomitant alterations in myofilament functions. In particular, ion channels, intracellular ion homeostasis and regulatory proteins in myofilaments are interactive; therefore, all these components function in concert to determine heart performance in vivo (e.g., as measured in the echocardiography).

In conclusion, we describe a method to evaluate the changes of myofilament Ca2+ sensitivity and emphasize the importance of analyzing this parameter in conjunction with cardiac electrophysiology, intracellular Ca2+ handling, and myocyte contraction to obtain a full profile of myocyte function. Myofilament Ca2+ sensitivity should be measured routinely in mechanistic studies using diseased heart models, where changes in these parameters as well as various intracellular signaling pathways are interrelated.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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).

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
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

DOWNLOAD MATERIALS LIST

References

  1. Bers, D. M., et al. Cardiac excitation-contraction coupling. Nature. 415, (6868), 198-205 (2002).
  2. Eisner, D. A., et al. Integrative analysis of calcium cycling in cardiac muscle. Circ Res. 87, (12), 1087-1094 (2000).
  3. Palmiter, K. A., et al. Molecular mechanisms regulating the myofilament response to Ca2+: implications of mutations causal for familial hypertrophic cardiomyopathy. Basic Res Cardiol. 92, (S1), 63-74 (1997).
  4. Missiaen, L., et al. Abnormal intracellular Ca2+ homeostasis and disease. Cell Calcium. 28, (1), 1-21 (2000).
  5. Trafford, A. W., et al. A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes. Pflugers Arch. 437, (3), 501-503 (1999).
  6. Berlin, J. R., et al. Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. Biophys J. 67, (4), 1775-1787 (1994).
  7. Robertson, S. P., et al. The time-course of Ca2+ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca2+. Biophys J. 34, (3), 559-569 (1981).
  8. Schober, T., et al. Myofilament Ca2+ sensitization increases cytosolic Ca2+ binding affinity, alters intracellular Ca2+ homeostasis, and causes pause-dependent Ca2+-triggered arrhythmia. Circ Res. 112, (2), 170-179 (2012).
  9. Briston, S. J., et al. Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during β-adrenergic stimulation in cardiac myocytes. Cardiovasc Res. 104, (2), 347-354 (2014).
  10. Patel, B. G., Wilder, T., John Solaro, R. J., et al. Novel control of cardiac myofilament response to calcium by S-glutathionylation at specific sites of myosin binding protein C. Front Physiol. 4, 336 (2013).
  11. Jin, C. Z., et al. Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart. J Mol Cell Cardiol. 60, 107-115 (2013).
  12. Wang, Y., et al. Modulation of L-type Ca2+ channel activity by neuronal nitric oxide synthase and myofilament Ca2+ sensitivity in cardiac myocytes from hypertensive rat. Cell Calcium. 58, (3), 264-274 (2015).
  13. Louch, W. E., Sheehan, K. A., Wolska, B. M., et al. Methods in cardiomyocyte isolation, culture, and gene transfer. J Mol Cell Cardiol. 51, (3), 288-298 (2011).
  14. Spurgeon, H. A., et al. Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation. J Physiol. 447, 83-102 (1992).
  15. Preston, L. C., et al. Functional effects of the DCM mutant Gly159Asp troponin C in skinned muscle fibres. Pflugers Arch. 453, (6), 771-776 (2007).
  16. Willott, R. H., et al. Mutations in Troponin that cause HCM, DCM AND RCM: what can we learn about thin filament function? J Mol Cell Cardiol. 48, (5), 882-892 (2010).
  17. Yasuda, S. I., et al. A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers. Am J Physiol Heart Circ Physiol. 281, (3), H1442-H1446 (2001).
  18. Sears, C. E., et al. Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling. Circ Res. 92, (5), e52-e59 (2003).
  19. Ashley, E. A., et al. Cardiac nitric oxide synthase 1 regulates basal and beta-adrenergic contractility in murine ventricular myocytes. Circulation. 105, (25), 3011-3016 (2002).
  20. Zhang, Y. H., et al. Reduced phospholamban phosphorylation is associated with impaired relaxation in left ventricular myocytes from neuronal NO synthase-deficient mice. Circ Res. 102, (2), 242-249 (2008).
  21. Roe, M. W., Lemasters, J. J., Herman, B., et al. Assessment of Fura-2 for measurements of cytosolic free calcium. Cell Calcium. 11, (2-3), 63-73 (1990).
  22. Grynkiewicz, G., et al. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 260, (6), 3440-3450 (1985).

Comments

0 Comments


    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Video Stats