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+ 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.
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)
2. Preparation for the Isolation of Left Ventricular (LV) Myocytes
3. Isolation of LV Myocytes
4. Simultaneous Measurements of Intracellular Ca2+ Transients and Myocyte Contraction
5. Assessment of Myofilament Ca2+ Sensitivity
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. 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. 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. 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). 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.
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.
The authors have nothing to disclose.
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).
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 |