This project received funding from the Israel Science Foundation under grant agreement No. 1379/18; the Jabotinsky Scholarship of the Israeli Ministry of Science and Technology for Applied and Engineering Sciences for Direct PhD Students No. 3-15892 for D.S.; and the European Union&#39;s Horizon 2020 research and innovation program under grant agreement No. 858149 (AlternativesToGd).

The joint ethics committee (IACUC) of the Hebrew University and Hadassah Medical Center approved the study protocol for animal welfare (MD-19-15827-1).
1. Krebs-Henseleit buffer preparation
<ol>
	<li>A day before the experiment, prepare a modified version of the Krebs-Henseleit buffer (KHB)26. Initially, dissolve 118 mM NaCl, 4.7 mM KCl, 0.5 mM pyruvate, 1.2 mM MgSO4, 25 mM NaHCO3, and 1.2 mM KH2PO4&#160;in double-distilled H2O.</li>
	<li>Bubble this mixture with 95%/5% O2/CO2&#160;for 20 min, and then add 1.2 mM CaCl2.</li>
	<li>Adjust the pH of the buffer to 7.4 with HCl or NaOH.</li>
	<li>On the day of the experiment, add 10 mM glucose and 72 U/L insulin to the KHB prepared in step 1.2. 
	NOTE: Insulin is added to the perfusion buffer as described in the work of Kolwicz et al.26&#160;and in agreement with previous studies reporting that insulin increases the contractile function27&#160;and the intensity of the hyperpolarized [13C]bicarbonate signal28.</li>
</ol>
2. Perfusion system preparation
<ol>
	<li>Keep a reservoir of 200 mL of KHB in a water bath at 40 &#176;C, and bubble with 95%/5% O2/CO2&#160;at a flow rate of 4 L/min for 1 h prior to cardiac perfusion. Keep the buffer continuously bubbling with this gas mixture throughout the experiment.
	<ol>
		<li>First, set the water bath to 40 &#176;C. Insert the KHB reservoir. Use a peristaltic pump (see Table of Materials) and medical-grade extension tubes to recirculate the KHB between the buffer reservoir and the 10 mm NMR tube at a constant flow rate of 7.5 mL/min.</li>
		<li>Connect three platinum-cured silicone tubes (3 mm i.d.) to the pump (one inflow tube and two outflow tubes for the KH buffer). Insert the outflow and inflow lines into the heated KH buffer. Then, insert the oxygen line into the heated KH buffer.</li>
		<li>Use thin polyether ether ketone (PEEK, see Table of Materials) lines for the buffer and hyperpolarized agent to flow to and from the NMR tube within the spectrometer&#39;s bore.</li>
	</ol>
	</li>
	<li>Ensure that the temperature is maintained at 37-37.5 &#176;C. Follow the steps below.</li>
	<li>Wrap the inflow line (from the buffer reservoir to the NMR tube) with a heating tape set to 42 &#176;C.</li>
	<li>Heat the NMR tube inside the spectrometer with a warm airflow that is regulated by the spectrometer.</li>
	<li>Use an NMR-compatible temperature sensor (see Table of Materials) to measure the temperature inside the NMR tube. The temperature is adjusted to 37-37.5 &#176;C.</li>
</ol>
3. Calibration and preparation of the NMR spectrometer for acquisition
<ol>
	<li>On the day of the experiment, insert a 13C standard sample that contains 1,4-dioxane (Table of Materials) into the spectrometer, and tune and match the NMR probe for 13C. Then, obtain a spectrum showing the thermal equilibrium signal of 1,4-dioxane with a nutation angle of 90&#176;.</li>
	<li>Now exchange the 13C standard sample with a 31P standard sample (Table of Materials), that contains 105 mM of ATP in D2O. Tune and match the NMR probe for 31P. 
	NOTE: A spectrum showing the thermal equilibrium phosphate signals is obtained with a nutation angle of 50&#176;.</li>
	<li>Insert the inflow line, the outflow line, and the temperature probe into a 10 mm NMR tube, and then insert the tube into the magnetic bore. Adjust the heating tape to 42 &#176;C.</li>
	<li>Acquire a&#160;31P NMR spectrum of the circulating KH buffer to be used in that experiment for 30 min, with a nutation angle of 50&#176; and a TR of 1.1 s (1,640 acquisitions).</li>
</ol>
4. Animal preparation, surgical procedure, and perfusion of the heart in the NMR tube
<ol>
	<li>Anesthetize a male HSD:ICR (CD-1) mouse with 3.3% isoflurane in room air (Table of Materials) at 340 mL/min for 5 min using a gas anesthesia system (Table of Materials) in an induction chamber.</li>
	<li>Use nasal anesthesia for the maintenance of general anesthesia with 2.9% isoflurane. 
	NOTE: Care is taken to minimize pain and discomfort to the animal.</li>
	<li>Secure the limbs of the animal with tape, ensure a negative pedal pain reflex, and then inject 300 IU of sodium heparin intraperitoneally.</li>
	<li>Wet the mouse&#39;s chest wall and abdomen thoroughly with 70% alcohol to ensure cleanliness and avoid hair contamination or obstruction during the surgical procedure.</li>
	<li>At 1 min after the heparin injection, cut the skin and muscle of the abdominal cavity with small scissors.</li>
	<li>Place the small curved-jaw hemostat-locking mosquito clamp between the xiphoid process and the chest skin to lift the chest and expose the diaphragm. Puncture and cut the right lobe of the diaphragm.</li>
	<li>Cut the chest across the midline, retract to the sides, and then remove.</li>
	<li>Inject the left ventricle of the heart with 200 IU of sodium heparin to prevent blood coagulation. Then, inject 0.1 mL of ice-cold 0.5 mol/L KCl to achieve cardiac arrest, as previously described25. Cardiac arrest is essential to be able to cannulate the heart.</li>
	<li>Identify the thymus, and remove it using scissors to expose the aorta. Remove residual rib cage tissue.</li>
	<li>Identify the aortic arch, and use curved forceps to place a loose knot with a 3-0 silk suture (Table of Materials) around the ascending aorta. Inject 3 mL of KHB into the left ventricle to remove blood clots from the aorta.</li>
	<li>Use curved forceps to retract the heart inferiorly for better visualization of the ascending aorta.</li>
	<li>Perform cannulation in situ with a 22 G intravenous catheter (Table of Materials). Apply cyanoacrylate adhesive in the cannulated region, and then perform double suture tying. Inject additional KH buffer into the heart, and verify that it flows through the cannulation tube.</li>
	<li>Remove the curved forceps. Disconnect the heart from the surrounding viscera, and perfuse it retrogradely with ice-cold KHB (4 &#176;C) through the intravenous catheter.</li>
	<li>Connect the heart to the inflow line of the perfusion system via the intravenous catheter. Upon the initiation of perfusion with warm buffer (37-37.5 &#176;C) at 7.5 mL/min, the heart begins beating spontaneously.</li>
	<li>Fix the NMR tube with the beating heart, outflow lines, and temperature probe, and insert into the bore of the spectrometer, making sure the heart is at the center of the NMR probe.</li>
</ol>
5. Acquiring data for cardiac energetics and pH
<ol>
	<li>Acquire&#160;31P spectra for about 1 h with a flip angle of 50 &#176; and a TR of 1.1 s.</li>
</ol>
6. DNP spin polarization and dissolution
<ol>
	<li>Prepare a 28.5 mg formulation of [1-13C]pyruvate. This formulation consists of 11.1 mM to 14.0 mM OX063 radical in the neat acid.</li>
	<li>Prepare 4 mL of dissolution medium. The dissolution medium consists of TRIS-phosphate buffer, which contains 11.2 mM NaH2PO4, 38.8 mM Na2HPO4, 33 mM TRIS, and 2 mM HCl. This medium composition is adjusted such that upon the addition of 28.5 mg of [1-13C]pyruvic acid formulation to 4 mL of this buffer (in the dissolution phase), the pH of the resulting solution will be 7.4.</li>
	<li>Perform spin polarization and fast dissolution in a dissolution-DNP (dDNP) spin polarization device according to the manufacturer&#39;s instructions (Table of Materials). Apply microwave irradiation at a frequency of 94.110 GHz for the polarization of the [1-13C]pyruvic acid formulation at 1.45 K to 1.55 K for about 1.5 h.</li>
	<li>Quickly mix the 4 mL of hyperpolarized medium from the dDNP device with a well-oxygenated solution that complements the hyperpolarized dissolution medium to obtain a composition closely matching the perfusion medium. 
	NOTE: The final volume of the medium perfusing the heart during the hyperpolarized injections with 14 mM hyperpolarized [1-13C]pyruvate is 26 mL. The final composition of the injected medium (after mixing) contains 4.7 mM KCl, 1.2 mM MgSO4, 70 mM NaCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 10 mM glucose, 1.2 mM CaCl2, and 72 U/L insulin.</li>
	<li>Administer the hyperpolarized [1-13C]pyruvate-containing medium to the isolated heart using a continuous flow setup29. 
	NOTE: This is done to ensure that the heart perfusion is not disturbed at any point during the experiment and that the hyperpolarized medium is administered at a known rate and for a known duration.</li>
</ol>
7. Hyperpolarized 13C spectroscopy
<ol>
	<li>Acquire hyperpolarized 13C data using product-selective saturating-excitation pulses15&#160;by applying 2.5 ms cardinal sine (Sinc) pulses, as previously described15,16. Selectively excite [1-13C]lactate and [13C]bicarbonate consecutively at 6 s intervals to obtain a 12 s interval for each metabolite.</li>
	<li>For [1-13C]lactate detection, center the selective Sinc pulse at the [1-13C]pyruvate hydrate frequency (179.4 ppm), which results in signal intensity ratio (lac) of 0.113 for the C1&#160;signals of [1-13C]pyruvate to [1-13C]lactate.</li>
	<li>For [13C]bicarbonate detection, center the selective Sinc pulse at 157.7 ppm, which is 214 Hz down-field of the [13C]bicarbonate signal (161.1 ppm); this results in a signal intensity ratio (bic) of 0.139 for the C1signal of [1-13C]pyruvate to [13C]bicarbonate.</li>
</ol>
8. Determination of the tissue wet weight and volume
<ol>
	<li>At the end of the experiment, detach the heart from the perfusion system, and gently dry it with tissue paper. Subsequently, weigh the heart to obtain the tissue wet weight.</li>
	<li>Determine the volume of the heart using a density factor of 1.05 g/cm3,&#160;as determined previously for the mouse heart30.</li>
</ol>
9. ATP content quantification
<ol>
	<li>Integrate the &#947;-ATP signal of a single acquisition of the ATP standard sample and a 30 min acquisition (TR of 1.1 s and 1,640 acquisitions) of the isolated heart.</li>
	<li>Quantify the ATP content of the heart by comparing the integral of the heart&#39;s &#947;-ATP signal to that of the standard (Table of Materials), where the latter has a known concentration (105 mM), and correct for the number of acquisitions and relaxation effects.</li>
</ol>
10. Resolving the Pi signal of the heart
NOTE: In order to evaluate the tissue pH, it is first necessary to deconvolve the heart&#39;s Pi signal from that of the total Pi signal (Pit). This is done by omitting the signal of the KHB Pi (PiKH) from that of the Pit.
<ol>
	<li>In a 31P spectrum of KHB that shows a single Pi signal (PiKH, Figure 1A), fit the PiKH signal to a Lorentzian function using Excel (Table of Materials).</li>
	<li>The volume visible to the NMR probe (Vp, 1.375 mL), contains more KHB when the sample tube does not contain the heart. To correct for this filling effect, calculate the attenuated buffer signal (Pib) using Eq. 1A. 
	<img alt="Equation 1" src="/files/ftp_upload/63188/63188eq01.jpg" style="margin: auto;" />&#160; &#160;Eq. 1A 
	where Vh is the heart&#39;s volume, as determined in step 8.</li>
	<li>Subtract this signal from the Pit according to Eq. 1B to obtain the Pi signal arising solely from the perfused heart (Pih, Figure 1B). 
	<img alt="Equation 2" src="/files/ftp_upload/63188/63188eq02.jpg" style="margin: auto;" />&#160; &#160;Eq. 1B</li>
</ol>
11. Multi-parametric pH analysis
<ol>
	<li>Perform the conversion of the Pih signal chemical shift distribution to the pH with reference to the chemical shift of PCr using Eq. 231. 
	<img alt="Equation 3" src="/files/ftp_upload/63188/63188eq03.jpg" style="margin: auto;" />&#160; &#160;Eq. 2 
	where &#916;&#948; is the chemical shift difference, pKa is 6.72, &#948; free base is 5.69, and &#948; free acid is 3.27, as previously described31.</li>
	<li>Correct the resulting pH distribution curve for the non-linearity between the Pih chemical shift scale and the pH scale according to Lutz et al.32. A typical pH distribution resulting from this calculation is presented in Figure 1C.</li>
	<li>Analyze the tissue pH distribution with a multi-parametric approach using seven statistical parameters following the work of Lutz et al.32. Four of these parameters are presented here as they appear to be the most descriptive of tissue pH: 1) global maximum pH; 2) weighted mean pH; 3) weighted median pH; and 4) skewness of the pH plot (Figure 1C).</li>
</ol>
12. Calculation of the LDH and PDH activities
NOTE: The production rates of the hyperpolarized metabolites [1-13C]lactate and [13C]bicarbonate are used to calculate the LDH and PDH activities, respectively. In the product selective saturating-excitation approach15, only newly synthesized hyperpolarized metabolites are detected by each selective excitation.
<ol>
	<li>Use the hyperpolarized [1-13C]pyruvate signal as a reference to determine the corresponding metabolite production level.
	<ol>
		<li>During the perfusion with the hyperpolarized medium,&#160;the [1-13C]pyruvate concentration in the NMR tube increases (wash-in), then plateaus (at a maximal concentration of 14 mM), and then decreases (wash-out).</li>
		<li>To identify the time points at which the pyruvate concentration reached a constant level (plateau), correct the [1-13C]pyruvate signal for signal decay resulting from T1&#160;relaxation and RF pulsation using the effective relaxation constant, Teff.</li>
		<li>For each injection, define the Teff&#160;based on its ability to correct the [1-13C]pyruvate decay curve to show this flow dynamics (Eq.3). 
		<img alt="Equation 4" src="/files/ftp_upload/63188/63188eq04.jpg" style="margin: auto;" />&#160; &#160;Eq. 3 
		where <img alt="Equation 5" src="/files/ftp_upload/63188/63188eq05.jpg" style="margin: auto;" /> is the [1-13C]pyruvate signal that was acquired during the [13C]bicarbonate acquisition. The average Teff in the experiments described herein was found to be 35.8 s &#177; 2.3 s (n = 5 hearts).</li>
		<li>Select the data points in which the concentration is within 10% of the maximal corrected [1-13C]pyruvate signal for further analysis.</li>
	</ol>
	</li>
	<li>Use the corresponding data of [1-13C]lactate and [13C]bicarbonate production for the time points selected in step 12.1 for the calculation of the metabolite production rates using Eq. 4A and Eq. 4B, provided that the SNR of the metabolite signal is larger than 2 (threshold for analysis). A typical example of such a selection of time points is shown in Figure 2B (highlighted temporal window).</li>
	<li>Calculate the production rates of each of the selected data points using Eq. 4A and Eq. 4B: 
	<img alt="Equation 17" src="/files/ftp_upload/63188/63188eq17.jpg" style="margin: auto;" />&#160; &#160;Eq. 4A 
	<img alt="Equation 6" src="/files/ftp_upload/63188/63188eq06.jpg" style="margin: auto;" />&#160; &#160;Eq. 4B 
	where <img alt="Equation 7" src="/files/ftp_upload/63188/63188eq07.jpg" style="margin: auto;" /> and <img alt="Equation 8" src="/files/ftp_upload/63188/63188eq08.jpg" style="margin: auto;" /> are the production rates of [1-13C]lactate or [13C]bicarbonate at each time point, respectively. <img alt="Equation 9" src="/files/ftp_upload/63188/63188eq09.jpg" style="margin: auto;" /> and <img alt="Equation 10" src="/files/ftp_upload/63188/63188eq10.jpg" style="margin: auto;" /> are factors that represent the relative excitation of [1-13C]pyruvate and the products [1-13C]lactate or [13C]bicarbonate, respectively. These factors were determined previously to be 0.113 and 0.139, respectively29. Vp is the volume that is detected by the NMR probe (1.375 mL), TR denotes the time interval between two consecutive product selective saturating excitations (12 s for each product), <img alt="Equation 11" src="/files/ftp_upload/63188/63188eq11.jpg" style="margin: auto;" /> and <img alt="Equation 12" src="/files/ftp_upload/63188/63188eq12.jpg" style="margin: auto;" /> are the signals of [1-13C]lactate and [13C]bicarbonate, respectively, and <img alt="Equation 13" src="/files/ftp_upload/63188/63188eq13.jpg" style="margin: auto;" /> and <img alt="Equation 14" src="/files/ftp_upload/63188/63188eq14.jpg" style="margin: auto;" /> are the signals of [1-13C]pyruvate that were acquired during the [1-13C]lactate and [13C]bicarbonate excitations, respectively. [Pyr] is the [1-13C]pyruvate concentration, which was 14 mM during the plateau phase.
	<ol>
		<li>Determine the rate for each point and then average per hyperpolarized injection.</li>
	</ol>
	</li>
</ol>

There are no disclosures.

We demonstrate an experimental setup that is designed to investigate hyperpolarized [1-13C]pyruvate metabolism, tissue energetics, and pH in an isolated mouse heart model.
The critical steps within the protocol are as follows: 1) ensuring that the pH of the buffer is 7.4; 2) ensuring that all components of the buffer are included; 3) avoiding blood clotting in the cardiac vessels by heparin injections; 4) avoiding ischemic damage to the heart by reducing the metabolic activity (KCl ...

Alterations in cardiac metabolism are associated with a variety of cardiomyopathies and often form the basis of the underlying pathophysiological mechanisms1. However, there are numerous obstacles to studying metabolism in living tissues, as most biochemical assays require the homogenization of the tissue and cell lysis and/or radioactive tracing. Therefore, there is a pressing need for new tools to investigate myocardial metabolism in living tissues. Magnetic resonance (MR) of hyperpolarized 13C-labeled substrates allows for real-time measurements of metabolism in living tissues2, without the use of ionizing radiation, by increasing the MR signal-to-noise (SNR) ratio of the labeled site(s) by several orders of magnitude3. Here, we describe an experimental setup, an acquisition approach, and an analytical approach for studying the rapid metabolism in the isolated mouse heart and, in parallel, present indicators of general tissue energetics and acidity. The cardiac pH is a valuable indicator, as the acid-base balance is disrupted in the early stages of cardiac diseases and conditions such as myocardial ischemia, maladaptive hypertrophy, and heart failure6.
Hyperpolarized [1-13C]lactate and [13C]bicarbonate production from hyperpolarized [1-13C]pyruvate helps in determining the production rates of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH). Most of the previous studies performed using hyperpolarized substrates in the isolated rodent heart either used complex kinetic models to derive the enzymatic activity of LDH and PDH, or reported the signal intensity ratios of the hyperpolarized product to a substrate without calculating the actual enzyme activity rates2,4,5,6,7,8,9,10,11,12,13,14. Here, we used the product selective saturating-excitations approach15, which allows for the monitoring of the enzyme activity in a model-free manner15,16. In this way, the absolute enzymatic rates (i.e., the number of moles of product produced per unit of time) were determined. 31P spectroscopy was utilized to observe the signals of inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate (ATP). A multi-parametric analysis was used to characterize the pH distribution of the heart, as demonstrated by the heterogeneous chemical shift in the Pi signal of the tissue.
The retrogradely perfused mouse heart (Langendorff heart)17,18,19&#160;is an ex vivo model for the intact beating heart. In this model, the heart viability and pH are preserved for at least 80 min20, and it has shown potential for recovery following a prolonged ischemic injury21,22. Nevertheless, inadvertent variability during micro-surgery may lead to variability in the tissue viability across hearts. Previous studies have reported on the deterioration of this heart over time19; for example, a reduction in contractile function of 5%-10% per hour has been observed18. The adenosine triphosphate (ATP) signal has previously been shown to report on the myocardial energetic status and viability23. Here, we noted that the perfused heart may occasionally show unintentional variability in viability levels, as demonstrated by the ATP content, despite the fact that we had an uninterrupted perfusion and oxygen supply. We demonstrate here that normalizing the LDH and PDH rates to the ATP content of the heart reduces the inter-heart variability in these rates.
In the following protocol, we describe the surgical procedure used for heart cannulation, isolation, and consequent perfusion in the NMR spectrometer. Of note, other surgical approaches aimed at isolating and perfusing the mouse heart have been described before24,25.
The methodologies used for acquiring data related to enzymatic rates in the beating heart (using 13C spectroscopy and hyperpolarized [1-13C]pyruvate) and the heart&#39;s viability and acidity (using 31P NMR spectroscopy) are described as well. Finally, the analytical methodologies for determining metabolic enzyme activities and tissue viability and acidity are explained.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HyperSense DNP Polariser</td>
			<td>Oxford Instruments</td>
			<td>52-ZNP91000</td>
			<td>HyperSense, 3.35 T, preclinical dissolution-DNP hyperpolarizer</td>
		</tr>
		<tr>
			<td>NMR spectrometer&#160;</td>
			<td>RS2D</td>
			<td></td>
			<td>NMR Cube, 5.8 T, equiped with a 10 mm broad-band probe</td>
		</tr>
		<tr>
			<td>Peristaltic pump&#160;</td>
			<td>Cole-Parmer</td>
			<td>07554-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature probe</td>
			<td>Osensa</td>
			<td>FTX-100-LUX+</td>
			<td>NMR compatible temprature probe</td>
		</tr>
		<tr>
			<td>Somnosuite low-flow anesthesia system</td>
			<td>Kent Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lines, tubings, suture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum cured silicone tubes</td>
			<td>Cole-Parmer</td>
			<td>HV-96119-16</td>
			<td>L/S 16 I.D. 3.1 mm&#160;</td>
		</tr>
		<tr>
			<td>Thin polyether ether ketone (PEEK) lines</td>
			<td>Upchurch Scientific</td>
			<td></td>
			<td>id. 0.040&#8221;</td>
		</tr>
		<tr>
			<td>Intravenous catheter&#160;</td>
			<td>BD Medical</td>
			<td>381323</td>
			<td>22 G</td>
		</tr>
		<tr>
			<td>Silk suture</td>
			<td>Ethicon</td>
			<td>W577H</td>
			<td>Wire diameter of 3-0</td>
		</tr>
		<tr>
			<td>Chemicals and pharmaceuticals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[1-13C]pyruvic acid</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>CLM-8077-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>21074</td>
			<td>CAS: 10043-52-4</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>CAS: 50-99-77</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Rotexmedica</td>
			<td>HEP5A0130C0160</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid 37%</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td>CAS: 7647-01-0</td>
		</tr>
		<tr>
			<td>Insulin aspart (NovoLog)</td>
			<td>Novo Nordisk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Terrel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>793612</td>
			<td>CAS: 7487-88-9</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td>CAS: 7447-40-7</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td>CAS: 7778-77-0</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Gadot Group</td>
			<td></td>
			<td>CAS: 144-55-8</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td>CAS: 7647-14-5</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>655104</td>
			<td>CAS: 1310-73-2</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S7907</td>
			<td>CAS: 7558-79-4</td>
		</tr>
		<tr>
			<td>Sodium phosphate monbasic dihydrate</td>
			<td>Merck</td>
			<td>6345</td>
			<td>CAS: 13472-35-0</td>
		</tr>
		<tr>
			<td>TRIS (biotechnology grade)</td>
			<td>Amresco</td>
			<td>0826</td>
			<td>CAS: 77-86-1</td>
		</tr>
		<tr>
			<td>Trityl radical OX063</td>
			<td>GE Healthcare AS</td>
			<td>NC100136</td>
			<td>OX063</td>
		</tr>
		<tr>
			<td>NMR standards</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>13C standard sample</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-72A</td>
			<td>40% p-dioxane in benzene-D6</td>
		</tr>
		<tr>
			<td>31P standard sample</td>
			<td>Made in house</td>
			<td></td>
			<td>105 mM ATP and 120 mM phenylphosphonic acid in D2O</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel 2016</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MNova</td>
			<td>Mestrelab Research</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating cardiac metabolism in the isolated perfused mouse heart with hyperpolarized [1-13c]pyruvate and 13c/31p nmr spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ,&#160;in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

The 31P spectra recorded from a mouse heart perfused with KHB and from the buffer alone are shown in Figure 1A. The signals of &#945;-, &#946;-, and &#947;-ATP, PCr, and Pi were observed in the heart. The Pi signal was composed of two main components: in the higher field (left side of the signal), the Pi signal was mostly due to the KHB at a pH of 7.4; in the lower field (right side of the signal), the Pi signal was broader and less homogeneous due to the more acidic environment. ...

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Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH.

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial ...

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1.3K Views.  Hebrew University of Jerusalem. We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH.

Video: Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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 ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...