Description

The Cardiac Myocyte Core is a component of the Myocyte Biology and Heart Failure program unit of the Penn CVI. This core is designed as a central resource for assisting CVI investigators with experiments requiring isolated cardiac myocytes. Services offered include provision of neonatal rodent myocytes for cell culture experiments, isolation of adult myocytes from a variety of mammalian species, isolated myocyte morphometric analyses and in vitro physiological studies examining cell shortening and responses to agonists. Upon request, interested investigators can receive training that will allow them to perform cell isolations or analyses independently. Contact Ken Margulies at ken.margulies@uphs.upenn.edu to inquire further about these services.

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Contact

Xiaoyin Shan, Ph.D., Director
Email: xiaoyins@mail.med.upenn.edu

George Bratinov, M.D. bratinov@mail.med.upenn.edu
Hongmei Wang, Ph.D. hongmeiann@hotmail.com
Christine Malloy (Administrative Contact) christine.malloy@uphs.upenn.edu

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Instructions for Requesting Services

Adult Cardiac Myocyte Isolation

The core provides interested investigators with assistance in obtaining adult cardiomyocytes from mouse, rat, feline, rabbits, sheep and human hearts. High yield preparations require perfusion-based collagenase isolation performed immediately after cardiac arrest (preferably with high-potassium cold cardioplegia). Isolated cells can be used for:

• Primary cell culture experiments
• In vitro physiological experiments
• Morphology analyses

How to Request Service

Please complete a service order form and email it to Dr. George Bratinov at bratinov@mail.med.upenn.edu
Phone number: (215) 573-2901
Fax Number: (215) 898-3473

Pre-Submission Considerations

• Requests for isolations should be made at least one week in advance to prevent scheduling conflicts.
• Except by special arrangement, the requestor is responsible for procuring heart for the myocyte isolation.
• Subsequent myocyte culture requires aseptic procurement and isolation techniques
• For best results please call or email George Bratinov for instructions on how to handle the heart.

[ Adult Cardiac Myocyte Isolation Request Form ]

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Neonatal Rodent Cardiac Myocyte Isolation

The core provides interested investigators with assistance in obtaining cultures of spontaneously beating, enriched cultures of primary neonatal left-ventricular cardiac myocytes from either mouse or rat hearts. Isolated cells can be cultured in vitro for up to a week. The preparations can be used for:

• Transient transfections, viral infections or pharmacological manipulations
• Co-culture experiments
• In vitro or physiological experiments
• Morphological analyses

How to Request Service

Please complete a service order form and email it to Christine Malloy at christine.malloy@uphs.upenn.edu
Phone number: (215) 573-2999
Fax Number: (215) 898-3473

Pre-Submission Considerations

• Requests for isolations should be made at least one week in advance to prevent scheduling conflicts.
• The neonatal myocytes can be prepared from wild-type animals procured through a central Core IACUC protocol (#801311) or from animals (e.g. transgenic mice) procured by the investigator via an alternative IACUC protocol.
• For investigator-procured animals, live rat or mouse pups (1-4 days old max.) must be delivered to Rm 707 Stellar Chance on morning of scheduled isolation. It is preferred but not essential that 2-day old pups be utilized for isolation experiments.
• For investigator-procured animals, the pups should be accompanied with their mother in a cage and it is the requestors responsibility to collect the mother before the end of the day once the pups have been sacrificed.

[ Neonatal Cardiac Myocyte Isolation Request Form ]

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Cardiac Myocyte Morphologic Analysis

In intact myocardium, myocyte size and shape are heterogeneous and the orientation of myocytes varies considerably in any given sectioning plane. Accordingly, there are many advantages of examining cardiac myocyte morphology in a populating of myocytes derived from a single heart or chamber. Our core uses well-validated techniques [Gerdes 1986] to exploit the advantages of isolated myocytes for morphologic assessment of cell populations. Median cell volume and a size histogram (when needed) are derived from >10,000 iso-osmotically fixed myocytes using a Beckman Z2 Coulter Analyzer. Myocyte length and average width are derived via image analysis of at least 40 isolated myocytes. Other metrics, including average cell thickness and aspect ratios can be derived from these primary measurements.

How to Request Service

Please complete a service order form and email it to Christine Malloy at christine.malloy@uphs.upenn.edu
Phone number: (215) 573-2999
Fax Number: (215) 898-3473

Pre-Submission Considerations

• Requests for isolations should be made at least one week in advance to prevent scheduling conflicts.
• Immediately after cell isolation, a concentrated suspension of filtered myocytes should be fixed in an iso-osmotic 1.5% Gluteraldehyde solution that preserves cell morphology for months to years.

Recipe for 1.5% Gluteraldehyde solution
23.5 ml 0.06 M Phosphate Buffer
1.5 ml gluteraldehyde (25% in water solution)
Each sample for analysis should be submitted as a cell suspension in glutaraldehyde

[ Cardiac Myocyte Morphology Request Form ]

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Cardiac Myocyte Functional Analysis

The core provides interested investigators with assistance in performing functional assessments of isolated cardiac myocytes from mammalian hearts. Functional assessments include isolated myocyte shortening and relengthening during field stimulation, intracellular calcium [Ca2+]i transients and assessments of responses to changes in stimulation frequency or selected agonists/antagonists (e.g. isoproterenol for assessment of β1-adrenergic responses). A variety of metrics can be generated from basic myocyte shortening studies including: fractional shortening, time to peak shortening, peak +dL/dt, peak dL/dt, time to 50% relaxation and time to 90% relaxation. Analogous metrics can be derived by analysis [Ca2+]i transients. Typically, these experiments and analyses are performed by experienced core personnel, but interested investigators can be trained to perform either experiments or their analyses by special arrangement.

How to Request Service

Please complete a service order form and email it to Christine Malloy at christine.malloy@uphs.upenn.edu
Phone number: (215) 573-2999
Fax Number: (215) 898-3473

Pre-Submission Considerations

• Requests for cell physiology experiments should be submitted at least two weeks in advance to prevent scheduling conflicts.
• If results of requested experiments are being compared with previous experiments, it is essential to specify the in vitro experimental conditions including: temperature, bath [Ca2+], bath pH, stimulation frequency and [Ca2+]i indicator used (if applicable).
• Physiological experiments involving freshly isolated adult myocytes should be completed within 8 hours of cell isolation.
• Physiological experiments involving cultured myocytes require plating on specially designed cover slips and must include appropriate controls due to physiological adaptations to cell culture.
• Cell shortening experiments are generally not feasible in cultured neonatal myocytes.

[ Cardiac Myocyte Physiology Request Form ]

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Protocols

Human Cardiac Myocyte Isolation Protocol

Prepare Solutions:
Start with 1000 ml Ca free KHB (see recipe below) and supplement with
1,252 grams taurine (to make 10 mM)
Make a Stock 0.5 M CaCl₂ solution each week (555mg CaCl₂/10 ml DI water)

Use the above to make the following three solutions:
Early Rinse solution (500 ml KHB/taurine with 1.01 g butandione monoxime (BDM)
Collagenase solution (200 ml KHB/taurine)
Add 404 mg BDM and
Add 20 L of 0.5 CaCl₂ stock (to achieve 50 M Ca2+)
Add 120 mg Worthington Collagenase II (299 U/mg)
(**added during the perfusion rinse (to make 180 U/ml)

2nd Rinse solution (200 ml KHB/taurine)
Add 404 mg BDM and
Add 80 L of 0.5 CaCl₂ stock (to achieve 200 M Ca2+)

Resuspension solution (100ml) with 1 gram bovine albumin (to make 1% w/v)
Add 40 L of 0.5 CaCl₂ stock (to achieve 200 M Ca2+)

Procedure (perfusion at 13 ml/min for human):

1. Equilibrate rinse and collagenase solutions with 95% 02, 5%C02, warm solutions to 37C, and prime perfusion system with warmed KHB before beginning experiment.
2. Prime all tubing, cannulate aorta, and initiate a non-recirculating rinse with the rinse solution making sure all RBCs are cleared (15-30 min. for human). Clip all superficial runoff from cut vessels. During rinse, add collagenase to collagenase solution.
3. Switch to collagenase solution (re-circulating) and perfuse until myocardium is soft/swollen (30 min. for human)
4. Cut off RV, place LV and septum (no atria) into petri dish, and add some collagenase solution to dish.
5. Carefully dissociate myocytes by first cutting and then using light suction from a cut off transfer pipette.
6. Filter cells through sieve (400 m stainless-steel filter) and repeat with any non-dissociated tissue.
7. Resuspend cells in 15 ml resuspension solution
8. Further increment [Ca2+] gradually as needed to match the conditions for in vitro experiments or culture protocol. Using 0.5M CaC12 stock, every 15 μl will increase a 15 ml volume by 0.5 mM.
   

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Sheep Cardiac Myocyte Isolation Protocol

Prepare Solutions:

Start with 2000 ml Ca free solution (see recipe below) and supplement with
10 mM BDM 2.02 (g)
0.5 mg/ml BSA 1.00 (g)

Make a Stock 0.5 M CaCl₂ solution each week (555mg CaCl₂/10 ml DI water)
Use the above to make the following solutions:
Calcium free solution: use as is
Collagenase solution (200 ml calcium free solution/collagen)
Add 120 mg Worthington Collagenase II (299 U/mg)
(**added during the perfusion rinse (to make 180 U/ml)
2nd Rinse solution (500 ml taurine solution) (see recipe below)
10 mM BDM 0.505 (g)
0.5 mg/ml BSA 0.25 (g)
50 mM Taurine 3.13 (g)
0.1 mM CaCl₂ 100 l of a 0.5 M CaCl₂ stock

Procedure:

1. Make solutions fresh, and pH it to 7.45
2. Keep 0.8L of solution cold in the 4°C refrigerator for tracker to take over to the OR
3. Warm up the solutions for the perfusion up to 37°C
4. After the heart is cannulated, start perfusion with the Calcium Free Solution for 10 min at 25ml/min.
5. Add collagenase (180 units/ml) to 200 ml of the Calcium Free Solution. Recirculate the solution for 10 minutes.
6. Start rinsing the tissue with the Taurine containing solution for 20 min with 0.5L of solution
7. Separate heart between Periinfarct and Distal zones and proceed to mince the tissue
8. Right after the isolation place the myocytes in 1.8mM Ca containing Tyrodes solution (sse recipe below)
9. Proceed to study myocytes

Detailed recipes for Solutions

Calcium Free Solution

	1L	8L	10L
1.134 mM NaCl	7.83	62.65	78.31
2. 11 mM Glucose	1.98	15.85	19.82
3. 10 mM Hepes	2.60	20.82	26.03
4. 4 mM KCl	0.30	2.39	3.00
5. 1.2 mM MgSO4	0.15	1.16	1.45
6. 1.2 mM Na2HPO4	0.17	1.36	1.70
pH solution to 7.45 @ 25°C and 7.34 at 37°C.
When ready to do the isolation add the following ingredients:
	2L
7. 10 mM BDM	2.02
8. 0.5 mg/ml BSA	1.00
Check pH again

Taurine Solutions with Calcium (0.1 mM)

	1L	8L	10L
1. 113 mM NaCl	6.60	52.83	66.04
2. 11 mM Glucose	1.98	15.84	19.80
3. 10 mM Hepes	2.60	20.82	26.00
4. 4 mM KCl	0.30	2.39	2.98
5. 1.2 mM MgSO4	0.14	1.16	1.45
6. 1.2 mM Na2HPO4	0.17	1.36	1.70
pH solution to 7.45 @ 25°C and 7.34 at 37°C.
When ready to do the isolation add the following ingredients:
	0.5 L
7. 10 mM BDM	0.505
8. 0.5 mg/ml BSA	0.25
9. 50 mM Taurine	3.13
10. 0.1 mM CaCl2	0.0055 or 100 l of a 0.5 M CaCl2 stock

Tyrodes

	1L	10L
1. 140 mM NaCl	8.18	81.82
2. 10 mM Glucose	1.80	18.02
3. 10 mM Hepes	2.60	26.03
4. 4 mM KCl	0.30	3.00
5. 1 mM MgCl2	0.20	2.03
pH solution to 7.45 @ 25°C and 7.34 at 37°C.
When ready to do the isolation add the following ingredient:
	0.5 L
6. 1.8 mM CaCl2	1800 l of 0.5 M stock CaCl2 stock
0.5 M CaCl2 stock
- 0.7 g in 12.5 ml of ddH2O

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Feline Cardiac Myocyte Isolation Protocol

1. NON-RECIRCULATING rinse (Ca free)1000 mL KHB, Add 1252 mg taurine

2.  ~20 min RECIRCULATING Digestion (33.333 µM CaCl2)180 mL of Solution 1 (180 mL KHB, 225 mg taurine), Add 180 units/mL collagenase - amount depends on collagenase lot, Add 13 µL 0.5 M CaCl2 stock solution

3. Resuspension Solution (200µM CaCl2)250 mL of Solution 1 (250 mL KHB, 313 mg taurine), Add 2.5g bovine serum albumin (BSA), Add 100 µL 0.5 M CaCl2 stock solution

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Adult Rat Cardiac Myocyte Isolation Protocol

Rats (2-3 months) are injected with sodium pentobarbital and heparin IP before hearts are removed and placed in ice-cold Hepes buffer containing (in mM): 137 NaCl, 4.9 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 15 Glucose, 20 Hepes, 10mM Taurine and 5mM Creatine. Hearts are immediately followed by a 8-14 min perfusion with low-calcium Hepes buffer containing 1mg/ml collagenase and 0.04mg/ml protease. After perfusion, ventricles are collected, minced into small chunks and shaken for 10 min to dissociate the cells. Myocytes are subjected to sequential Hepes buffer supplemented with increasing concentration of calcium before they are cultured in M199 medium on laminin-coated plates. After 30-60 min incubation at 37° C, dead cells are removed by changing the media.

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Mouse Cardiac Myocyte Isolation Protocol

Under development

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Neonatal Rodent Cardiac Myocyte Isolation Protocol

Hearts from 0-1 day old pups are harvested and immediately placed into cold Hanks Balanced Salt Solution (HBSS). Once hearts have been collected, excess tissue, blood vessels and atria are removed, and the remaining right and left ventricular tissue is minced into small pieces and washed several times with HBSS. The cleaned and chopped pieces of ventricular heart muscle are subjected to sequential digests with a Trypsin/HBSS solution at 37 C. The supernatants from the digests are pooled and the cells pelleted. The cells are gently re-suspended and pre-plated for 1-3 hours in nutrient media containing 10% fetal bovine serum and 5% horse serum to remove nonmyocytes. Viable myocytes are counted using trypan blue, seeded onto fibronectin-coated plates and allowed to attach overnight in growth media containing 10% fetal bovine serum, 5% horse serum and penicillin-streptomycin. 5-Bromo-2'-deoxyuridine can be optionally used in culture for the first three days to inhibit proliferation of potential fibroblasts.

Plating of Neonatal Cardiac Myocytes

Upon request, isolated Neonatal cardiac myocytes can be seeded into 35, 60 or 100mm dishes at a confluence of approximately 75%. The number of dishes will be dependent upon the total number of isolated neonatal cardiac myocytes. You will be notified either on the day or next morning to come and collect your samples.

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Cardiac Myocyte Morphological Analysis Protocols

Cell Volume Measurements

Fixed cardiac myocytes are counted and measured as they passed through the aperture tube of a particle size analyzer (Model Z2 Beckman-Coulter, Inc). The median cell volume, MCV, is then taken from the volume measured in at least 10,000 cardiac myocytes. Analysis and shape factor adjustment are used as previously derived and validated by other investigators such as Gerdes1, Hurley2 and Nash3, to derive the final median myocyte volume for each isolated cell preparation.

Light Microscopic Morphometry

An aliquot from the isolated cardiac myocytes is stained with 4% trypan blue. Then, images of randomly selected rod-shaped myocytes with normal striations and no membrane blebs or granularity are obtained with a CCD camera, and Nikon Eclipse 80i microscope (n=40 myocytes per heart) (Zafeiridis et al. 1998). Previous studies have demonstrated that this sample size adequately represents the population at the 95% CI (Gerdes, et al. 1986). The length and profile surface area of myocytes are measured with the Image Pro Plus software, version 5.1. Myocyte length is measured as the longitudinal axis of the best-fitting ellipse. The average width of each myocyte was calculated by the ratio of the profile surface area to the length of the cell.

References

1. Gerdes AM, Moore JA, Hines JM, Kirkland PA, Bishop SP. Regional differences in myocyte size in normal rat heart. Anat Rec. 1986;215:420426.

2. Hurley J. Sizing particles with Coulter counter. Biophys J. 1970;10:7479.

3. Nash GB, Tatham PER, Powell T, Twist TV, Speller RD, Loverock LT. Size measurements on isolated rat heart cells using Coulter analysis and light scatter flow cytometry. Biochim Biophys Acta. 1979;587:99111.

4. Zafeiridis A, Jeevanandam V, Houser SR, and Margulies KB. Regression of cellular hypertrophy after left ventricular assist device support. Circulation 98: 656662, 1998.

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Cardiac Myocyte Functional Analysis Protocols

Measurement of Sarcomere Length Transient during Field Stimulation

A) Freshly isolated cardiac myocytes are resuspended and equilibrated in the Tyrodes buffer containing 1 mM CaCl₂.

B) The cells are loaded into a 37 C perfusion chamber and superfused with Tyrodes buffer containing 1 mM CaCl₂. The cells are then paced at a voltage that is 20% above the threshold. Pacing frequencies can be varied from 0.2 to 2.0 Hz.

C) After a three minute stabilization period, the sarcomere length signal derived from the power spectrum of the video image is recorded using the IonWizard data acquisition software.

D) As an alternative, cell length can be assessed via edge-detection, but edge detection tends to be less stable than the sarcomere length approach

Measurement of Intracellular Calcium Transients during Field Stimulation

A) Freshly isolated cardiomyocytes are incubated with different concentrations (4uM, 6uM, 8uM and 10uM) of Fluo-3AM at room temperature for 15 minutes.

B) The unabsorbed dye is removed by washing with Tyrodes buffer containing 1mM Ca2+. A further 30 minutes incubation is followed to allow complete de-esterification of intracellular Fluo-3 AM esters.

C) The cells are subsequently loaded into a 37 C perfusion chamber and superfused with Tyrodes buffer containing 1 mM CaCl₂. The cells are then paced at a voltage that is 20% above the threshold. Pacing frequencies can be varied from 0.2 to 2.0 Hz.

D) Control experiments comparing cell shortening Fluo-3 loaded and unloaded cells is used to detect excessive buffering induced by the calcium indicator and allow selection of the most appropriate indicator concentration.

E) The myocytes incubated with the highest Fluo-3 concentration without significant buffering are placed in the chamber and allowed to settle. After a three minute stabilization period, the calcium transient signals are recorded using the IonWizard data acquisition software.

 

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