This work was supported by grants from the National Natural Science Foundation of China (No. 81100159, Dongwu Lai; 81401147, Juhong Zhang), the Medical and Health Science Program of Zhejiang Province (No. 201646246, Dongwu Lai), and the Health Science and Technology Plan of Hangzhou City (No. 2013A28, Ding Lin).

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
<ol>
	<li>Prepare all solutions as described in Table 1.</li>
</ol>
2. Isolation of Left Ventricular (LV) Cardiomyocytes
NOTE: LV cardiomyocytes are isolated enzymatically using a Langendroff perfusion system as previously described7,8,9,10,11.
<ol>
	<li>Set up the Langendroff perfusion system. Fill the perfusion system with Normal Tyrode (NT) solution, set the temperature at 36.5 &#176;C, and eliminate any air bubbles in the tube.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Gently pipette the pieces with a filtered plastic dropper, and re-suspend in 10 mL KB solution.</li>
	<li>Filter the cells with 150 &#181;m 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 &#176;C in solutions gassed with 100% O2.</li>
</ol>
3. Calcium Reintroduction
<ol>
	<li>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.</li>
	<li>Repeat the above procedure with calcium reintroduction solution B (0.3 mM Ca2+, 3 mL Ca2+-free Tyrode solution, 3 mL NT solution).</li>
	<li>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.</li>
</ol>
4. Set Up of Perfusion System
<ol>
	<li>Connect the inflow tube with the NT solution for chamber perfusion (Figure 1A).</li>
	<li>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).</li>
	<li>Evacuate any air bubbles in the tubes to avoid air blockage.</li>
	<li>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.</li>
</ol>
5. Measurements of Intracellular Ca2+ Transient and Sarcomere Shortening 7,8,9
<ol>
	<li>Pipette 10 &#181;L 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.</li>
	<li>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 &#181;M. Keep in the dark for 20 min at room temperature.</li>
	<li>Centrifuge at 150 g for 30 s, and re-suspend the cardiomyocytes with NT solution 2 times.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Frame the single myocyte by adjusting the cell framing adapter. Ensure that the background is clear.</li>
	<li>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.</li>
	<li>Record fluorescence by a dual-excitation fluorescence photomultiplier system. Run the recording program for the calcium imaging system, click &#34;File/new File&#34; to create a new recording file, and click the &#34;start&#34; button to synchronously record fluorescence and sarcomere length.</li>
</ol>
6. Assessment of SR Ca2+ Reserve and Diastolic Ca2+ Removal Function 7,8,9
<ol>
	<li>Interlink the &#34;Aux Out&#34; port in the stimulator with the &#34;Analog In&#34; port on the valve control system (e.g., valve commander, see Table of Materials) by a BNC cable (to synchronize the TTL signal).</li>
	<li>Preset a program for switching the perfusion valves automatically as previously described8,9; Detailed steps for operation are listed as following.
	<ol>
		<li>Preset the parameters in the valve control software as per Table 2, and click the &#34;Download&#34; button to download the sequence loaded in the program. Click the &#34;Trigger&#34; button to enable the trigger function. 
		NOTE: The valve control system can execute the sequence after receiving TTL signal.</li>
		<li>Preset the stimulator to sequence mode, and set the parameters as per Table 3.</li>
	</ol>
	</li>
	<li>Set the stimulator at &#34;S1 step&#34; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After 60 s basic pacing, roll the stimulator cursor to the &#34;D2 step&#34;. 
	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.</li>
	<li>At the end of the recording, detect the background fluorescence for individual myocytes. Click the &#34;pause&#34; button to pause the file recording, move the microscopic view to a nearby blank area. Click the &#34;record&#34; button to resume recording for seconds, and record numerical 340 and 380 nm intensity values for background correction.</li>
	<li>Open the &#34;Operation/Constant&#34; dialog box and fill the values into the &#34;background&#34; form, respectively; the software can correct Fura 2 ratio values by subtracting the background.</li>
</ol>
7. Data Analysis 9
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Measure the amplitude of the caffeine-induced calcium pulse (Ca-caffeine, an index of SRCa2+ reserve).</li>
	<li>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).</li>
</ol>

<ol>
	<li>Capel, R. A., Terrar, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+Ca+(2+)-dependent+mechanisms+for+the+initiation+of+the+heartbeat.">The importance of Ca (2+)-dependent mechanisms for the initiation of the heartbeat.</a> Front Physiol. 6, 80 (2015).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+cycling+and+signaling+in+cardiac+myocytes.">Calcium cycling and signaling in cardiac myocytes.</a> Annu Rev Physiol. 70, 23-49 (2008).</li><li>Zhao, S. M., Wang, Y. L., Guo, C. Y., Chen, J. L., Wu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+decay+of+Ca2++homeostasis+in+the+development+of+diabetic+cardiomyopathy.">Progressive decay of Ca2+ homeostasis in the development of diabetic cardiomyopathy.</a> Cardiovasc Diabetol. 13, 75 (2014).</li><li>Pereira, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+diabetic+cardiomyocytes.">Calcium signaling in diabetic cardiomyocytes.</a> Cell calcium. 56 (5), 372-380 (2014).</li><li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+sodium+current+inhibition+reverses+electromechanical+dysfunction+in+human+hypertrophic+cardiomyopathy.">Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.</a> Circulation. 127 (5), 575-584 (2013).</li><li>Ferrantini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R4496C+RyR2+mutation+impairs+atrial+and+ventricular+contractility.">R4496C RyR2 mutation impairs atrial and ventricular contractility.</a> J Gen Physiol. 147 (1), 39-52 (2016).</li><li>Li, L., Chu, G., Kranias, E. G., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+myocyte+calcium+transport+in+phospholamban+knockout+mouse+relaxation+and+endogenous+CaMKII+effects.">Cardiac myocyte calcium transport in phospholamban knockout mouse relaxation and endogenous CaMKII effects.</a> Am J Physiol. 274, H1335-H1347 (1998).</li><li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+inhibition+in+heart+failure,+beneficial,+harmful,+or+both.">CaMKII inhibition in heart failure, beneficial, harmful, or both.</a> Am J Physiol Heart Circ Physiol. 302, H1454-H1465 (2012).</li><li>Lai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rho+kinase+inhibitor,+fasudil,+ameliorates+diabetes-induced+cardiac+dysfunction+by+improving+calcium+clearance+and+actin+remodeling.">The Rho kinase inhibitor, fasudil, ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling.</a> J Mol Med (Berl). 95 (2), 155-165 (2017).</li><li>Lai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+Current-Induced+Abnormal+Impulses+in+CaMKII&#948;+Knockout+Mouse+Ventricular+Myocytes.">Stretch Current-Induced Abnormal Impulses in CaMKII&#948; Knockout Mouse Ventricular Myocytes.</a> J Cardiovasc Electrophysiol. 24 (4), 457-463 (2013).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+L-type+calcium+current+and+cardiac+function+in+CaMKII{delta}+knockout+mice.">Alterations of L-type calcium current and cardiac function in CaMKII{delta} knockout mice.</a> Circ Res. 107 (3), 398-407 (2010).</li><li>Roussel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoyl-carnitine+increases+RyR2+oxidation+and+sarcoplasmic+reticulum+Ca2++leak+in+cardiomyocytes:+Role+of+adenine+nucleotide+translocase.">Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: Role of adenine nucleotide translocase.</a> Biochim Biophys Acta. 1852 (5), 749-758 (2015).</li><li>Yaras, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+diabetes+on+ryanodine+receptor+Ca2++release+channel+(RyR2)+and+Ca2++homeostasis+in+rat+heart.">Effects of diabetes on ryanodine receptor Ca2+ release channel (RyR2) and Ca2+ homeostasis in rat heart.</a> Diabetes. 54 (11), 3082-3088 (2005).</li><li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Mediated+Overexpression+of+O-GlcNAcase+Improves+Contractile+Function+in+the+Diabetic+Heart.">Adenovirus-Mediated Overexpression of O-GlcNAcase Improves Contractile Function in the Diabetic Heart.</a> Circ Res. 96 (9), 1006-1013 (2005).</li></ol>

The authors have nothing to disclose.

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 diastole12,1...

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 dysfunction2,3,4.
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-clamp5,6, and the caffeine-induced Ca2+ pulse based on Ca2+ fluorescence imaging7,8,9. 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.

<table><tbody><tr><td>NaCl</td><td>Alfa Aesar</td><td>012314</td><td></td></tr><tr><td>MgCl2</td><td>Alfa Aesar</td><td>012315</td><td></td></tr><tr><td>KCl</td><td>Alfa Aesar</td><td>11595</td><td></td></tr><tr><td>HEPEs</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>D-Glucose</td><td>Alfa Aesar</td><td>A16828</td><td></td></tr><tr><td>NaOH</td><td>Alfa Aesar</td><td>A18395</td><td></td></tr><tr><td>KOH</td><td>Alfa Aesar</td><td>A18854</td><td></td></tr><tr><td>KH2PO4</td><td>Alfa Aesar</td><td>011594</td><td></td></tr><tr><td>MgSO4</td><td>Alfa Aesar</td><td>3337</td><td></td></tr><tr><td>L-Glutamic</td><td>Alfa Aesar</td><td>A15031</td><td></td></tr><tr><td>Taurine</td><td>Alfa Aesar</td><td>A12403</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103586001</td><td></td></tr><tr><td>Collagenase Type II</td><td>Worthington</td><td>LS004177</td><td></td></tr><tr><td>BSA</td><td>Roche</td><td>735094</td><td></td></tr><tr><td>caffeine</td><td>Sigma</td><td>C0750</td><td></td></tr><tr><td>Fura-2 AM</td><td>Invitrogen</td><td>F1201</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>Olympus IX 71</td><td></td></tr><tr><td>Langendorff system</td><td>Beijing Syutime Technology Co</td><td>PlexiThermo-S-LANGC</td><td></td></tr><tr><td>Micromanipulator</td><td>Marchauser</td><td>MM33 links</td><td></td></tr><tr><td>Perfusion chamber</td><td>IonOptix</td><td>FHD</td><td></td></tr><tr><td>Valve Controlled Gravity Perfusion System</td><td>ALA</td><td>VC 3-8</td><td></td></tr><tr><td>valve commander software</td><td>ALA</td><td>VC 3 1.0.1.2</td><td></td></tr><tr><td>Precision flow regulator</td><td>Delta Med</td><td>3204315</td><td></td></tr><tr><td>Multi-Barrel Manifold Perfusion Pencil</td><td>AutoMate Scientific</td><td>04-08-[360]</td><td></td></tr><tr><td>Micron Removable Tip</td><td>AutoMate Scientific</td><td>360um i.d.</td><td></td></tr><tr><td>Fluorescence Measurement and Cell Dimensioning Systems</td><td>IonOptix</td><td>Hyperswitch</td><td></td></tr><tr><td>Recording software</td><td>IonOptix</td><td>IonWizard 6.2.59</td><td></td></tr><tr><td>Stimulator</td><td>IonOptix</td><td>MyoPacer EP</td><td></td></tr><tr><td>Sprague-dawley rats</td><td>Beijing Vital River Laboratory Animal Technology Co.</td><td>SCXK 2016-01-007436</td><td></td></tr></tbody></table>

assessment of sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal in isolated ventricular cardiomyocytes

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.

&#160; &#160; &#160; &#160; &#160; &#160; &#160; 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 &#177; 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 &#62; 16.7 mmol/L were considered diabetic. After 8 weeks, the LV myocytes were enzymatically iso...

Watch this Scientific Journal Video about Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes at JoVE.com

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

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.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

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JoVE Journal

Medicine

10.0K Views.  Zhejiang University School of Medicine. 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.

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

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Biology

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Real-Time Analysis of Calcium and Contractility in Isolated Lymph Vessels

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Author Spotlight: Innovative Methods in Lymphedema and Hypertension Research

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...

Immunology and Infection

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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

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.

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

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 ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

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

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Chapters in this video

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca²⁺ Transients and Membrane Currents

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Isolation and Physiological Analysis of Mouse Cardiomyocytes

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

Real-time Evaluation of Absolute, Cytosolic, Free Ca²⁺ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca²⁺ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

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

Analysis of Cardiac Contractile Dysfunction and Ca²⁺ Transients in Rodent Myocytes

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices