Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Summary

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca²⁺ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Abstract

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca²⁺ reservoir for contraction, which reuptakes intracellular Ca²⁺ during relaxation. The SR Ca²⁺ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca²⁺ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca²⁺ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa²⁺ reserve and diastolic Ca²⁺ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca²⁺ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca²⁺ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca²⁺-ATPase (SERCA) and Na⁺-Ca²⁺ exchanger (NCX) to diastolic calcium removal was evaluated.

Introduction

Intracellular calcium ([Ca²⁺]_i) recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes¹. As we know, the calcium-induced Ca²⁺ release initiates the excitation-contraction coupling, which translate the electrical signal to contraction. Membrane depolarization activates the sarcolemmal L-type Ca²⁺ channels, which induce Ca²⁺ release from SR into the cytoplasm via ryanodine receptors 2 (RyR2). The transient elevated cytoplasmic Ca²⁺ initiates contraction of myofibrils. During the diastole, cytoplasmic Ca²⁺ is reuptaken into the SR by means of the SR Ca²⁺-ATPase 2 (SERCA2) and pumped out of the cardiomyocyte via the Na⁺-Ca²⁺ exchanger (NCX)². This process leads to contraction-relaxation recycling in the cardiomyocyte.

The cardiac SR is an intracellular membrane network that surrounds the contractile machinery. It serves as a Ca²⁺ reservoir for contraction, and it reabsorbs intracellular Ca²⁺ during relaxation. The SR Ca²⁺ reserve available for beats is determinate for cardiac contractility. Meanwhile, the removal of intracellular Ca²⁺ is critical for cardiac diastole. Under some pathophysiological conditions, such as diabetes and heart failure, impaired Ca²⁺ clearance and depressed SR Ca²⁺ store in cardiomyocytes may be involved in the process of cardiac dysfunction^2,3,4.

For measuring SR Ca²⁺ release and diastolic Ca²⁺ removal in cardiomyocytes, there are two widely used approaches: the integrity of the NCX current based on patch-clamp^5,6, and the caffeine-induced Ca²⁺ pulse based on Ca²⁺ fluorescence imaging^7,8,9. The former approach depends on the fact that the Ca²⁺ released from the SR is largely pumped out of the cell by NCX. However, this approach is limited by its requirement of advanced equipment and skillful operation. In the present study, we describe a convenient approach to assess SR Ca²⁺ reserve and Ca²⁺ removal in myocytes by measuring a caffeine-induced Ca²⁺ pulse based on a Ca²⁺ fluorescence imaging system. Briefly, intracellular Ca²⁺ fluorescence is indicated by Fura-2. By interlinking the stimulation system and perfusion system, we present a program for switching the perfusion and pacing system automatically. 10 mM caffeine was employed to rapidly induce total Ca²⁺ release in the SR. The exponential decay time constants (Tau) of calcium transients and caffeine-induced calcium pulses were obtained from mono-exponential curve fitting, which reflect the contribution of SERCA and NCX to diastolic Ca²⁺ removal accordingly.

Protocol

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Experimental Research Center, China Academy of Chinese Medical Sciences and Zhejiang University.

1. Solution Preparation

1. Prepare all solutions as described in Table 1.

2. Isolation of Left Ventricular (LV) Cardiomyocytes

NOTE: LV cardiomyocytes are isolated enzymatically using a Langendroff perfusion system as previously described^7,8,9,10,11.

1. Set up the Langendroff perfusion system. Fill the perfusion system with Normal Tyrode (NT) solution, set the temperature at 36.5 °C, and eliminate any air bubbles in the tube.
2. Weigh and anesthetize a Sprague-Dawley rat by intraperitoneal injection (ip) of chloral hydrate (400 mg/kg). Confirm the anesthetic status by evaluating tail or toe pinch response after 5 min.
3. Open the abdominal cavity under the xiphoid process with a surgical scissor, lift the xiphoid process, and open the chest with the scissor. Remove the pericardium, slightly lift the heart with curved forceps, identify the aortic arch, and cut off the heart by scissors from the root of the aorta.
4. Transfer the heart to a 60-mm dish and wash it with NT solution. Hold the aorta with two micro-dissecting forceps, and mount it onto the Langendorff perfusion cannula, and then firmly ligate the aorta onto the cannula using surgical sutures.
   NOTE: A skilled operator can finish this process within 15 s.
5. Perfuse the heart with 30 mL NT solution to recover circulation of the coronary arteries. Then, switch the perfusate to 30 mL Ca²⁺-free Tyrode solution (10 mM Taurine, 1 mg/mL BSA) to stop the heartbeat. Next, switch the perfusate to Collagenase A isolation solution (0.6 mg/mL) for enzyme digestion.
   NOTE: Use Collagenase A for experiments with diabetic rats, or Collagenase II for experiments without diabetic rats.
6. After digestion for 20-25 min, quickly change the perfusion solution to the Ca²⁺-free Tyrode solution to stop further digestion. Then, hold the heart with forceps, cut it off from the cannula, and place the heart in a 35-mm dish containing KB solution (see Table 1).
7. Dissect the LV wall with scissors and forceps. Cut off the atrium, right ventricle and atrioventricular junction area. The remaining tissue is the LV; transfer it into a new 35-mm dish with KB solution. Mince the LV tissue into small pieces.
8. Gently pipette the pieces with a filtered plastic dropper, and re-suspend in 10 mL KB solution.
9. Filter the cells with 150 µm aperture stainless steel filter, and transfer to a 15 mL centrifuge tube. Centrifuge at 150 x g for 30 s and discard the supernatant. Re-suspend the myocytes in 10 mL KB solution, free settle for 6 min, discard the supernatant, and re-suspend the pellet in 10 mL KB solution.
   NOTE: All steps were performed at 36 °C in solutions gassed with 100% O₂.

3. Calcium Reintroduction

1. After settling for 20 min in KB solution, discard the supernatant, and re-suspend the myocytes with calcium reintroduction solution A (0.3 mM Ca²⁺, 4.5 mL Ca²⁺-free Tyrode solution, 1.5 mL NT solution) for 10 min.
2. Repeat the above procedure with calcium reintroduction solution B (0.3 mM Ca²⁺, 3 mL Ca²⁺-free Tyrode solution, 3 mL NT solution).
3. Repeat the above procedure with NT solution (1.2 mM Ca²⁺) to purify the available myocytes. Store the isolated LV myocytes in this solution and study them within 4-6 h.

4. Set Up of Perfusion System

1. Connect the inflow tube with the NT solution for chamber perfusion (Figure 1A).
2. For needle perfusion of the target myocyte, connect the multi-barrel manifold tip (e.g., perfusion pencil, referred to as pencil henceforth), fixed in the micromanipulator, to the valve controlled perfusion system. Add NT solution and 10 mM caffeine solution to each column of the pencil (Figure 1A).
3. Evacuate any air bubbles in the tubes to avoid air blockage.
4. Count the drip number from the micron tip of the pencil for 10 s, and manually adjust the flow regulator to keep the flow speed at an approximate velocity of 3 drip/10 s.

5. Measurements of Intracellular Ca²⁺ Transient and Sarcomere Shortening ^7,8,9

1. Pipette 10 µL cell suspension on the slide, count cell numbers by a cell counter under the microscope, and calculate the density. Dilute myocytes to an approximate concentration of 50,000 cell/mL.
2. Add the Fura-2 acetoxymethyl (AM) stock (a calcium sensitive dye) into a 1 mL suspension of myocytes to bring the final concentration to 2 µM. Keep in the dark for 20 min at room temperature.
3. Centrifuge at 150 g for 30 s, and re-suspend the cardiomyocytes with NT solution 2 times.
4. Turn off the light and keep the cells in the dark. Place the myocytes in the perfusion chamber for 15 min. Then start the chamber perfusion (1.5 mL/min) with NT solution. Pace the myocytes with 1 Hz field stimulation using a stimulator (wave duration 4.0 ms, pulse amplitude 8.0 V) for 5 min.
5. Select a myocyte with good shape (rod shape, sharp edge, and clear cross striations) and stable stimulated twitch (no spontaneous contraction) under the 10x microscopic objective lens. Next, change the microscopic magnification to 40x, and rotate the CCD camera orientation to keep the myocyte in a horizontal position.
6. Frame the single myocyte by adjusting the cell framing adapter. Ensure that the background is clear.
7. Expose the myocytes to the light emitted by a Xenon lamp, with 340 or 380 nm wavelength excitation filters, and image the myocytes through a 40x objective. Detect fluorescence signal at 510 nm. In the meantime, note the sarcomere length changes of the myocytes and record using the soft-edge module simultaneously.
8. Record fluorescence by a dual-excitation fluorescence photomultiplier system. Run the recording program for the calcium imaging system, click "File/new File" to create a new recording file, and click the "start" button to synchronously record fluorescence and sarcomere length.

6. Assessment of SR Ca²⁺ Reserve and Diastolic Ca²⁺ Removal Function ^7,8,9

1. Interlink the "Aux Out" port in the stimulator with the "Analog In" port on the valve control system (e.g., valve commander, see Table of Materials) by a BNC cable (to synchronize the TTL signal).
2. Preset a program for switching the perfusion valves automatically as previously described^8,9; Detailed steps for operation are listed as following.
   1. Preset the parameters in the valve control software as per Table 2, and click the "Download" button to download the sequence loaded in the program. Click the "Trigger" button to enable the trigger function.
      NOTE: The valve control system can execute the sequence after receiving TTL signal.
   2. Preset the stimulator to sequence mode, and set the parameters as per Table 3.
3. Set the stimulator at "S1 step" to pace the LV myocytes at 1 Hz. Start the needle perfusion at the speed of 1.5 mL/min with NT solution.
   NOTE: Because the speed of the needle stream is faster than chamber perfusion, the light refraction of the needle stream is different from the light refraction of chamber perfusion. The difference of light refraction indicates the range of effective needle perfusion, which surrounds the target myocyte.
4. Select a target myocyte under the low power microscopic view (in the sequence of downstream to upstream), and make sure it can be reached by the micro tip of the pencil. Change the microscope magnification to 400x. Rotate the CCD camera orientation to keep the myocyte in the horizontal position. Adjust the rectangular aperture under the cell framing adapter to a suitable window that fills with the myocytes. Ensure that there is minimal background; do not allow other myocytes or dead cell debris in this window.
5. Adjust the position of the pencil fixed on the micromanipulator, and carefully place the micro tip at the distance of the radius of vision field to the target myocyte under 400x microscope magnification.
6. Adjust the needle stream range to mostly cover the target myocyte and make sure that the myocyte cannot be swept away by the needle flow.
7. After 60 s basic pacing, roll the stimulator cursor to the "D2 step".
   NOTE: The rest of protocol will be executed automatically by the stimulator and valve control system. Based on the above setting, the protocol could be executed automatically as in Figure 2A, 1 Hz basic pacing with NT solution for 60 s. Then pause pacing and delay for 2 s, rapidly switch to 10 mM caffeine perfusion for 15 s (to functionally release Ca²⁺ storage in the SR), and then switch back to NT solution.
8. At the end of the recording, detect the background fluorescence for individual myocytes. Click the "pause" button to pause the file recording, move the microscopic view to a nearby blank area. Click the "record" button to resume recording for seconds, and record numerical 340 and 380 nm intensity values for background correction.
9. Open the "Operation/Constant" dialog box and fill the values into the "background" form, respectively; the software can correct Fura 2 ratio values by subtracting the background.

7. Data Analysis ⁹

1. For measurements of calcium transients and sarcomere shortening at the basic pacing stage, average the twitch pulses, and then analyze dynamic parameters, such as calcium transients, the time constant of calcium transient decay (Tau-1 Hz), using the software automatically.
   NOTE: If the software does not fit the decay segment well, export the decline trace for manual mono-exponential curve fitting.
2. For caffeine-induced calcium pulses, select only the pulse with a steep wave for analysis of calcium removal function. Exclude cells with signal disturbance, abnormal pulse, or those that flowed away midway.
3. Measure the amplitude of the caffeine-induced calcium pulse (Ca-caffeine, an index of SRCa²⁺ reserve).
4. Obtain the decay time constant of caffeine-induced calcium pulse (Tau-caffeine) by mono-exponential curve fitting (10 s duration) manually from software (Figure 2C).

Results

              Here, we illustrate streptozotocin (STZ)-induced diabetic rats (DM) and age-matched Sprague-Dawley (SD) rats for example. 8-week-old male SD rats (200 ± 20 g) received a single intraperitoneal injection of STZ (70 mg/kg, ip) for the DM group or citrate buffer for the control group. One week after STZ administration, rats with blood glucose > 16.7 mmol/L were considered diabetic. After 8 weeks, the LV myocytes were enzymatically iso...

Discussion

Calcium flux released from the SR is the major Ca²⁺ source for systole in the heart. To some extent, the amplitude of SR Ca²⁺ content and the fractional Ca²⁺ released from the SR reflect the SR Ca²⁺ reserve available for cardiac contraction. On the other hand, the Ca²⁺ reserve of SR depends on the ability of SR Ca²⁺ reuptake, Ca²⁺ leak of SR, and their balance across the SR during diastole^12,1...

Materials

Name	Company	Catalog Number	Comments
NaCl	Alfa Aesar	E31K43
MgCl2	Alfa Aesar	I02T070
KCl	Alfa Aesar	G22u018
HEPEs	Sigma	SLBM 7880V
D-Glucose	Alfa Aesar	10189341
NaOH	Alfa Aesar	10154048
KOH	Alfa Aesar	10144B17
KH2PO4	Alfa Aesar	F21S033
MgSO4	Alfa Aesar	C31U038
L-Glutamic	Alfa Aesar	10149849
Taurine	Alfa Aesar	J5407a
EGTA	Sigma	SLBM6826V
Collagenase A	Roche	10103586001
Collagenase Type II	Worthington	45k16005
BSA	Roche	735094
caffeine	Sigma	C0750
Fura-2 AM	Invitrogen	F1201
Microscope	Olympus	Olympus IX 71
Langendorff system	Beijing Syutime Technology Co	PlexiThermo-S-LANGC
Micromanipulator	Marchauser	MM33 links
Perfusion chamber	IonOptix	FHD
Valve Controlled Gravity Perfusion System	ALA	VC 3-8
valve commander software	ALA	VC 3 1.0.1.2
Precision flow regulator	Delta Med	3204315
Multi-Barrel Manifold Perfusion Pencil	AutoMate Scientific	04-08-[360]
Micron Removable Tip	AutoMate Scientific	360um i.d.
Fluorescence Measurement and Cell Dimensioning Systems	IonOptix	Hyperswitch
Recording software	IonOptix	IonWizard 6.2.59
Stimulator	IonOptix	MyoPacer EP
Sprague-dawley rats	Beijing Vital River Laboratory Animal Technology Co.	SCXK 2016-01-007436