N. Bursac, M. Papadaki, R. J. Cohen, F. J. Schoen, S. R. Eisenberg, R. Carrier, G.

Vunjak-Novakovic and L. E. Freed

Am J Physiol Heart Circ Physiol 277:433-444, 1999.

You might find this additional information useful...

This article cites 41 articles, 18 of which you can access free at:
http://ajpheart.physiology.org/cgi/content/full/277/2/H433#BIBL

This article has been cited by 13 other HighWire hosted articles, the first 5 are:
Region of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers
J. W. Lin, L. Garber, Y. R. Qi, M. G. Chang, J. Cysyk and L. Tung
Am J Physiol Heart Circ Physiol, January 1, 2008; 294 (1): H58-H65.
[Abstract] [Full Text] [PDF]

Biomimetic approach to cardiac tissue engineering

M Radisic, H Park, S Gerecht, C Cannizzaro, R Langer and G Vunjak-Novakovic
Phil Trans R Soc B, August 29, 2007; 362 (1484): 1357-1368.
[Abstract] [Full Text] [PDF]

In vitro tissue engineering of a cardiac graft using a degradable scaffold with an extracellular
matrix-like topography

Downloaded from ajpheart.physiology.org on May 24, 2010

O. Ishii, M. Shin, T. Sueda and J. P. Vacanti
J. Thorac. Cardiovasc. Surg., November 1, 2005; 130 (5): 1358-1363.
[Abstract] [Full Text] [PDF]

Mathematical model of oxygen distribution in engineered cardiac tissue with parallel channel
array perfused with culture medium containing oxygen carriers
M. Radisic, W. Deen, R. Langer and G. Vunjak-Novakovic
Am J Physiol Heart Circ Physiol, March 1, 2005; 288 (3): H1278-H1289.
[Abstract] [Full Text] [PDF]

Medium perfusion enables engineering of compact and contractile cardiac tissue

M. Radisic, L. Yang, J. Boublik, R. J. Cohen, R. Langer, L. E. Freed and G. Vunjak-Novakovic
Am J Physiol Heart Circ Physiol, February 1, 2004; 286 (2): H507-H516.
[Abstract] [Full Text] [PDF]

Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl

on the following topics:
Physiology .. Cardiac Muscle
Physiology .. Heart Muscle
Engineering .. Biomedical Engineering
Physiology .. Rats
Updated information and services including high-resolution figures, can be found at:
http://ajpheart.physiology.org/cgi/content/full/277/2/H433

Additional material and information about AJP - Heart and Circulatory Physiology can be found at:
http://www.the-aps.org/publications/ajpheart

This information is current as of May 24, 2010 .

AJP - Heart and Circulatory Physiology publishes original investigations on the physiology of the heart, blood vessels, and
lymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the
intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the American Physiological
Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ISSN: 0363-6135,
ESSN: 1522-1539. Visit our website at http://www.the-aps.org/.
Cardiac muscle tissue engineering: toward an
in vitro model for electrophysiological studies
N. BURSAC,1,2 M. PAPADAKI,1 R. J. COHEN,1 F. J. SCHOEN,3 S. R. EISENBERG,2
R. CARRIER,1 G. VUNJAK-NOVAKOVIC,1 AND L. E. FREED1
1Division of Health Sciences and Technology, Massachusetts Institute of Technology,
Cambridge 02139; 2Department of Biomedical Engineering, Boston University, Boston 02215; and
3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115
Bursac, N., M. Papadaki, R. J. Cohen, F. J. Schoen,
S. R. Eisenberg, R. Carrier, G. Vunjak-Novakovic, and
L. E. Freed. Cardiac muscle tissue engineering: toward an in
vitro model for electrophysiological studies. Am. J. Physiol.
277 (Heart Circ. Physiol. 46): H433–H444, 1999.—The objec-
tive of this study was to establish a three-dimensional (3-D)
in vitro model system of cardiac muscle for electrophysiologi-
cal studies. Primary neonatal rat ventricular cells containing
lower or higher fractions of cardiac myocytes were cultured on
polymeric scaffolds in bioreactors to form regular or enriched
cardiac muscle constructs, respectively. After 1 wk, all con-
structs contained a peripheral tissue-like region (50–70 µm
thick) in which differentiated cardiac myocytes were orga-
nized in multiple layers in a 3-D configuration. Indexes of cell
size (protein/DNA) and metabolic activity (tetrazolium conver-
sion/DNA) were similar for constructs and neonatal rat
ventricles. Electrophysiological studies conducted using a
linear array of extracellular electrodes showed that the
peripheral region of constructs exhibited relatively homoge-
neous electrical properties and sustained macroscopically
continuous impulse propagation on a centimeter-size scale.
Electrophysiological properties of enriched constructs were
superior to those of regular constructs but inferior to those of
native ventricles. These results demonstrate that 3-D cardiac
muscle constructs can be engineered with cardiac-specific
structural and electrophysiological properties and used for in
vitro impulse propagation studies.
myocyte; impulse propagation; electrophysiology; three-
dimensional
CULTURED CARDIAC MYOCYTES offer many advantages for
developmental, physiological, and pharmacological stud-
ies of cardiac tissue because they allow for direct cell
manipulation and control of environmental parameters
without interference from the compensatory feedback
mechanisms that exist in vivo. Compared with mono-
layer cultures, it has been suggested that three-
dimensional (3-D) multilayered cultures of cardiac myo-
cytes more closely resemble intact cardiac tissue with
respect to cellular differentiation (8) and electrical
properties (38, 39). Three-dimensional cardiac myocyte
cultures could thus be used for in vitro studies of
cardiac tissue development and function and, if suffi-
ciently large and functional, for in vivo cardiac repair.
Impulse propagation studies in cultures of cardiac
myocytes can improve our understanding of the electro-
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/99 $5.00 Copyright r 1999 the American Physiological Society
physiological behavior of normal and pathological car-
diac tissues. Such studies are currently performed in
one-dimensional cardiac strands and two-dimensional
(2-D) isotropic, anisotropic, and photolithographically
patterned monolayers using optical mapping tech-
niques (9, 10, 27). Impulse propagation studies cannot
be performed in 3-D myocyte aggregates (17, 30) be-
cause of their small size (100–300 µm) and isopotential
nature. Other 3-D cultures of cardiac myocytes grown
on microcarrier beads (1, 31), collagen fibers (1), syn-
Downloaded from ajpheart.physiology.org on May 24, 2010
thetic, biodegradable polymeric templates (3, 12), or in
collagen gels (8) have not yet been evaluated electro-
physiologically.
The goal of the present work was to establish a 3-D in
vitro model system for impulse propagation studies in
cardiac muscle using tissue engineering principles.
This approach relies on the use of primary cells in
conjunction with biodegradable polymer scaffolds (13,
18) and tissue culture bioreactors (11, 12). The polymer
scaffold provides a 3-D substrate for cell attachment
and tissue formation, whereas the mixing of culture
medium in the bioreactor promotes mass transfer of
nutrients and gases to the forming tissue. Primary
neonatal rat ventricular cells were cultured on polymer
scaffolds in bioreactors to form tissue constructs, which
were characterized histologically, biochemically, and
electrophysiologically and compared with neonatal and
adult rat ventricular tissues.
MATERIALS AND METHODS
All experiments involving animals were performed accord-
ing to the Institutional Committee on Animal Care of the
Massachusetts Institute of Technology, which follows federal
and state guidelines.
Cardiac myocyte preparation. Primary cultures of cardiac
myocytes were prepared by enzymatic digestion of ventricles
obtained from neonatal (2 day old) Sprague-Dawley rats
(Taconic), as previously described (44). Briefly, ventricles (n ⫽
50, 5 litters in 3 independent studies) were incubated with
0.1% trypsin overnight and dissociated in four to five sequen-
tial steps using 0.1% collagenase. Isolated cells were resus-
pended in culture medium [DMEM, supplemented with 10%
fetal bovine serum (FBS), 50 U/ml penicillin and 10 mM
HEPES, all obtained from GIBCO-BRL].
Two experimental groups were established as follows (Fig.
1A): 1) a regular group of ventricular cells isolated as
described above and 2) an enriched group with a higher
fraction of cardiac myocytes, prepared from the regular group
by centrifugation at 600 rpm for 5 min, followed by two
preplatings, 75 min each (Fig. 1A); cells that remained
unattached after the second preplating were used. Cell yields
were ⬃6 ⫻ 106 and 5 ⫻ 106 cells/ventricle for the regular and
H433
H434 CARDIAC MUSCLE TISSUE ENGINEERING

Downloaded from ajpheart.physiology.org on May 24, 2010

Fig. 1. A: model system for tissue engineering. Cells from neonatal rat ventricles were seeded onto polymer
scaffolds and cultured for 7 days to form regular and cardiac myocyte-enriched constructs. B: electrophysiological
setup. Tissue constructs were studied using an extracellular microelectrode array (inset) under controlled
environmental conditions in a 37°C/5% CO2 perfused chamber. Stimulation was bipolar, and extracellular
recordings were unipolar with reference to a Ag-AgCl electrode placed 3.5 cm away from the microelectrode array.
 CARDIAC MUSCLE TISSUE ENGINEERING
enriched group, respectively. Cell viability was 91 ⫾ 3%, as
assessed by trypan blue exclusion.
Monolayer studies. Cells from the regular and enriched
groups were cultured in monolayers at a cell density of 1.3 ⫻
104 cells/cm2 in 12-well dishes, T75 flasks, and on glass
coverslips to assess spontaneous contractions and biochemi-
cal and immunohistochemical parameters, respectively. After
2 days of static culture, monolayers were placed on an orbital
shaker set to 75 rpm. Medium was completely replaced on day
3 and by 50% on day 5. Spontaneous contractions were
assessed by videomicroscopy, by manually counting the num-
ber of beats per minute using five randomly selected fields
(0.3 ⫻ 0.4 mm2 each) per plate and six plates per experimen-
tal group, on days 3, 5, and 7. Cells in T75 flasks were
removed after 7 days by a 5-min incubation with 0.05%
trypsin-EDTA (GIBCO-BRL) and counted, and a suspension
of 2 ⫻ 106 cells/ml was stored at ⫺20°C for determination of
DNA and protein contents and lactate dehydrogenase (LDH)
activity per cell. Cells on glass coverslips were fixed with
HistoCHOICE (Amresco) for immunohistochemical analysis.
3-D tissue culture studies. Cells from the regular and
enriched groups were cultured on polyglycolic acid (PGA)
scaffolds, which are highly porous (97%) meshes of randomly
entangled 13-µm fibers formed as 5 ⫻ 2-mm (diameter ⫻
thickness) disks (Fig. 1A; Ref. 13). Briefly, scaffolds were
prewetted in culture medium, positioned on thin stainless
steel wires using segments of silicone tubing, and fixed to a
silicone stopper placed in the mouth of a spinner flask (8
scaffolds per flask) (12). Flasks were filled with 120 ml of
culture medium, placed in a humidified 37°C, 5% CO2 incuba-
tor with the side arm caps loosened to permit gas exchange,
and mixed at 50 rpm using a magnetic stir bar. After 24 h,
flasks were inoculated with cells (8 ⫻ 106 cells per scaffold).
Culture medium was replaced by 100% on day 3 and by 50%
on day 5. Cell-polymer constructs (n ⫽ 22, from 3 independent
studies) were harvested after 7 days for morphometric,
histological, biochemical, and electrophysiological assess-
ments.
Ventricular tissues. To verify the analytical methods, evalu-
ate the developmental state of cardiac myocytes in constructs,
and establish baseline values for parameters studied in
engineered constructs that were not readily found in the
literature, two control groups were examined. Adult ven-
tricles (n ⫽ 10) were obtained from 3- to 4-mo-old Sprague-
Dawley rats following anesthesia by intramuscular injection
of 65 mg/kg ketamine and 5 mg/kg xylazine (Sigma). Hearts
were rapidly removed, and ventricular sections were excised
from 1 mm below the atrioventricular groove to 1–2 mm
above the apex. For electrophysiological studies, full-thick-
ness pieces of the ventricular wall (⬃9 ⫻ 7 mm2, 2–4 mm
thick) were then prepared by making two longitudinal cuts
parallel to the base-apex line. Neonatal ventricles (n ⫽ 10,
from 3 litters) were obtained from 2-day-old rats following
decapitation. For electrophysiological studies, full-thickness
pieces of the ventricular wall (⬃6 ⫻ 4 mm2, 1.5–2.5 mm thick)
were prepared by bisecting the ventricle. Smaller pieces of
the adult and neonatal ventricles (7–13 mg wet wt) were used
for biochemical and histological assessments. The properties
of neonatal and adult ventricles were compared with those of
constructs without a priori assumption that the engineered
tissue resembled either of the native ventricular tissues.
Histological and immunohistochemical assessments. Cells
on glass coverslips were incubated for 30 min with mouse
antisarcomeric tropomyosin monoclonal antibody (clone CH1,
Sigma) diluted 1:100 in PBS containing 0.5% Tween 20 and
1.5% horse serum and then for 30 min with a secondary
antibody (Vectastain), diluted 1:200. Coverslips were then
H435
incubated with avidin-biotin complex reagent and 3,38-
diaminobenzidine (Sigma). Ten randomly selected fields (0.3 ⫻
0.4 mm2 each) from six coverslips from each group were
analyzed using videomicroscopy and NIH Image 1.60 soft-
ware to estimate cardiac myocyte fraction as a percentage of
cell area stained positively for tropomyosin.
Ventricles and 7-day constructs were fixed in 2% glutaralde-
hyde for 10 min, rinsed in PBS, and immersed in 10% neutral
buffered Formalin (Sigma). Samples were embedded in paraf-
fin, sectioned at 5 µm, and stained with hematoxylin and
eosin (H ⫹ E) for general evaluation and Masson’s trichrome
stain for collagen assessment. Immunohistochemical stain-
ing for tropomyosin was used to assess the fraction of cardiac
myocytes in constructs. Sections were incubated with 1 mg/ml
trypsin (Sigma) at 37°C for 15 min and 0.3% hydrogen
peroxide for 30 min, blocked with horse serum for 30 min, and
incubated with antisarcomeric tropomyosin as described
above. A humidified chamber was used for all incubation
steps. Sections were counterstained with Mayer’s hematoxy-
lin (Sigma) and coverslipped using glycerol mounting media
(Sigma). Specificity of staining for tropomyosin was con-
firmed by staining for sarcomeric ␣-actin, another myocyte-
Downloaded from ajpheart.physiology.org on May 24, 2010
specific protein, using otherwise identical methodology. Con-
struct macroscopic architecture was assessed from stained
tissue sections using videomicroscopy and NIH Image 1.60
software.
Transmission electron microscopy. Samples were fixed in
Karnovsky’s reagent (0.1 M sodium cacodylate with 2%
paraformaldehyde and 2.5% glutaraldehyde, pH ⫽ 7.4), post-
fixed in 2% osmium tetroxide, dehydrated in ethanol in
propylene oxide, and embedded in Poly/Bed812 (Poly-
sciences). Sections were cut at 60 nm, stained with lead
citrate and uranyl acetate, and examined using a transmis-
sion electron microscope (JEOL-100CX, JEOL).
Media analysis. Physiological ranges of PO2 (115–130
mmHg), PCO2 (48–55 mmHg), and pH (7.21–7.33) were
maintained for the duration of cultivation, as measured by a
blood gas analyzer (IL 1610, Instrumentation Laboratory).
Glucose and lactate concentrations were measured using a
glucose/lactate analyzer (2300 StatPlus, YSI). The activity of
LDH in the culture media was monitored using a LDH-L
reagent kit (Chiron Diagnostics). Media samples were soni-
cated using a Sonic Dismembrator (Vibra-Cell, Sonics and
Materials), and absorbance was measured at 340 nm (Spec-
tronic 1001⫹, Milton Roy) against cell-free medium. An LDH
activity of 1 U/l corresponded to 3,600 cells in monolayers.
DNA and protein assays. DNA and protein assays were
performed on engineered constructs and native ventricles
using modifications of previously described methods (7).
Samples were homogenized in buffer (1 N ammonium hydrox-
ide/2% Triton X-100, 0.04 ml/mg wet wt) for 1 min. For the
DNA assay, homogenates were incubated at 37°C for 10 min,
diluted with assay buffer (100 mM NaCl, 1 mM EDTA, 10 mM
Tris, pH 7.00), and centrifuged. DNA contents of superna-
tants were determined using a spectrofluorometer (PTI) and
calf thymus DNA as a standard (7). DNA contents measured
for regular and enriched monolayers were comparable (7.1 ⫾
0.2 pg/cell) and consistent with published values (7).
For protein assays, the viscosity of homogenates was
reduced by several passages through a 26-gauge needle. After
centrifugation, protein concentration was measured in the
supernatant using a Bio-Rad DC protein assay kit and a
microplate spectrophotometer (MR5000, Dynatech). Regular
and enriched monolayers had comparable protein contents
(290 pg/cell), resulting in protein-to-DNA ratios of 41 mg/mg
that were consistent with published values (29).
H436 CARDIAC MUSCLE TISSUE ENGINEERING
Metabolic activity assays. Metabolic activities of cells within
constructs and ventricular tissues were assessed by the
uptake and enzymatic reduction of the tetrazolium dye
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT; Sigma). Samples (2–15 mg wet wt) were rinsed with
PBS and incubated with MEM (GIBCO-BRL) without phenol
red and 0.5 mg/ml MTT for 4 h on an orbital shaker at 37°C
and 60 rpm. Medium was replaced with an equal volume of
0.1 N HCl in absolute isopropanol and pipetted directly
through the constructs to solubilize the resulting formazan
crystals. After 10 min of incubation at 37°C, the absorbance
was read at 570 nm, using a microplate spectrophotometer.
Electrophysiological assessment. An electrophysiological
system was custom-designed to enable stimulation and record-
ing of unipolar extracellular potentials in constructs and
ventricular tissues under controlled environmental condi-
tions using a linear array of microelectrodes (Fig. 1B). A
cylindrical Plexiglas chamber was tightly fitted inside an
electrically grounded brass casing placed on a 37°C heater
(VWR). The brass case distributed the heat evenly through
the chamber and served as an electrostatic shield. The
chamber was gassed with a prewarmed mixture of 5% CO2 in
air and filled with 50 ml of culture medium (DMEM with 15
mM HEPES, 4.5 g/l glucose), which was recirculated (at 60
ml/min for constructs and 120 ml/min for ventricular tissues)
using a pulseless gear pump (Cole-Parmer). Temperature and
pH were maintained at 37 ⫾ 0.1°C and 7.32 ⫾ 0.02, respec-
tively.
A photomicrograph of the microelectrode array is shown in
Fig. 1B. All microelectrodes were made of insulated tungsten
wire and had uninsulated tips with diameters of 50 ⫾ 6 µm
(Microprobe). Two electrodes for bipolar stimulation were
positioned 200 µm apart and connected to a programmable
cardiac stimulator (SEC-3102, Nihon Kohden). Eight record-
ing electrodes were positioned 500 µm apart in a linear array,
1.5 to 5 mm from the stimulating site. Exact distances
between electrodes were measured using a microscope and
NIH 1.60 image analysis software. Shielded cables connected
recording electrodes to bioelectric amplifiers (AB.601G, Ni-
hon Kohden). A reference Ag-AgCl electrode (WPI) was placed
in the medium 3.5 cm away from the microelectrode array.
Samples were placed in a tissue holder 2–3 mm under the
surface of the culture medium, secured using Teflon screws,
and left to equilibrate for 15 min. An XYZ mechanical
micropositioner (Taurusr, WPI) was used to gradually ad-
vance the microelectrode array toward either the top surface
of the construct or the epicardial surface of the ventricle, and
pacing impulses were simultaneously applied (3–5 V, 1-ms
pulses at a rate of 60 beats/min). The position of the array was
fixed at the point where the amplitudes of the recorded
responses appeared maximal, and a recording protocol was
performed as follows.
Spontaneous beating, if present, was recorded for 3–5 min.
After 15 min, monophasic pacing pulses (1-ms duration) were
Table 1. Morphometric and biochemical parameters in 7-day constructs
Construct Weight and Dimensions
Group Wet weight, mg Thickness, mm Circular area, mm2
Regular 35.45 ⫾ 3.33 1.34 ⫾ 0.11 20.14 ⫾ 0.82
Enriched 31.08 ⫾ 1.71 1.30 ⫾ 0.07 22.92 ⫾ 0.58*
Values are means ⫾ SE; n ⫽ 5 constructs. Parenthetical values are cumulative lactate dehydrogenase activity per construct, in U/l. Regular
and enriched groups were prepared as defined in MATERIALS AND METHODS. * Statistically significant difference between regular and enriched
groups.
applied at a rate of 60 beats/min, starting at a pacing voltage
of 0.1 V, which was then increased in 0.1-V increments until
the sample was captured (i.e., until each pacing impulse was
followed by a recorded tissue response). The corresponding
pacing voltage, defined as the excitation threshold, repre-
sented the lowest stimulus that produced a stable propaga-
tion (for at least 1 min at a rate of 60 beats/min) over the
length of the recording array. For the next 20–30 min, the
sample was continuously paced at 60 beats/min using pacing
amplitudes 1.5 times higher than the excitation threshold,
and responses were recorded every 4–5 min for a period of 1
min. The pacing rate was then increased every 5 min by 30
beats/min, and responses at each rate were recorded for the
last 40 s, similar to the protocol in Ref. 37. The maximum
pacing frequency at which the sample could be captured for at
least 5 min was defined as the maximum capture rate. After
reaching the maximum capture rate, stimulation was stopped
for 10 min and then reapplied at 30 and 60 beats/min for 5
min each to check for reproducibility of the recorded wave-
forms. At the end of the experiment, double and triple
extrastimuli and rapid stimulation at frequencies above the
maximum capture rate were applied in an attempt to induce
Downloaded from ajpheart.physiology.org on May 24, 2010
arrhythmia.
All recorded signals were amplified and band-pass filtered
between 0.3 and 1,000 Hz. The unfiltered noise level was 35
µV, peak to peak, with virtually no 60-Hz component. Analog
recordings were digitized at a sampling rate of 3 kHz using a
16-bit analog-to-digital board (AT-MIO-16X, National Instru-
ments), real-time displayed using LabView data acquisition
software, and stored and analyzed using MATLAB (The
Mathworks).
Activation times at each recording electrode were deter-
mined as the minima of five-point derivatives (2) of the
low-pass filtered signals. The stimulus-activation time inter-
vals at each electrode (conduction times) were plotted against
the corresponding distances and fitted by linear regression.
The conduction velocity of a propagated beat was calculated
as the inverse slope of the best linear fit (16). The peak-to-
peak (p-p) amplitudes of the responses were determined from
linearly detrended signals around the activation times. Re-
cording sites with very low or fractionated (polyphasic)
activity were ignored.
For each tissue sample, p-p amplitudes at each electrode
and conduction velocities were averaged from recordings
made during the initial 20 min of pacing at 60 beats/min (i.e.,
over at least 200 beats). Conduction velocity, maximum
amplitude, and average amplitude were calculated, respec-
tively, as the averages of conduction velocities, maximum p-p
amplitudes, and all p-p amplitudes from all samples within a
group. The maximum and average amplitudes, respectively,
represented local and spatially averaged properties of con-
structs or ventricles.
Statistics. Data were calculated as means ⫾ SE and
analyzed using either a paired t-test or one-way ANOVA
Cell Number Cell Number per Glucose Consumption Lactate Production
in Construct, Construct Lost During Rate per Construct, Rate per Construct,
⫻106 Cultivation, ⫻106 g · l⫺1 · day⫺1 g · l⫺1 · day⫺1
3.75 ⫾ 0.59 5.36 ⫾ 0.19 2.21 ⫾ 0.02 1.43 ⫾ 0.12
(20.95 ⫾ 0.65)
3.00 ⫾ 0.46 4.63 ⫾ 0.31 5.37 ⫾ 0.95* 2.51 ⫾ 0.11*
(17.68 ⫾ 1.12)
 CARDIAC MUSCLE TISSUE ENGINEERING
followed by Fisher’s protected least significant difference post
hoc test. To determine time-dependence trends for beating
rates in monolayer cultures, a univariate repeated-measures
ANOVA was used. Differences were considered statistically
significant when P ⬍ 0.05. All calculations were performed
using SuperANOVA III for Macintosh.
RESULTS
Monolayer cultures. After 24 h of culture, cardiac
myocytes from both the regular and enriched groups
started to contract spontaneously and by day 3–4
formed synchronously contracting networks. Rates of
contraction decreased significantly between culture
days 3 and 7 (P ⬍ 0.05) in monolayers from both groups.
At day 7, enriched monolayers had significantly higher
cardiac myocyte fractions and contraction rates than
regular monolayers (60.5 ⫾ 1.5 vs. 43.8 ⫾ 0.5% of the
culture area, P ⬍ 0.04, and 169 ⫾ 8 vs. 132 ⫾ 10
beats/min, P ⬍ 0.01), which is consistent with previous
reports (22).
Construct morphology. After 7 days of culture, cell-
polymer constructs appeared discoid [⬃5 ⫻ 1.3 mm
(diameter ⫻ thickness); Table 1]. The peripheral zone
was 50–70 µm thick (Fig. 2A) and consisted of more cell
H437
Fig. 2. Histology and immunohistochem-
istry of enriched constructs and native
ventricles. A: construct periphery (hema-
toxylin and eosin, H ⫹ E). B: construct
center (H ⫹ E). C: construct periphery
(tropomyosin). D: construct periphery
(tropomyosin). E: neonatal ventricle
(tropomyosin). F: adult ventricle (tropo-
myosin). A–C: magnification, ⫻400; bar,
Downloaded from ajpheart.physiology.org on May 24, 2010
50 µm. D–F: magnification, ⫻1,000; bar,
20 µm. Brown color indicates tropomyosin-
positive cells. Asterisks denote polymer
fibers; arrows point to cross striations.
layers in the enriched than in the regular group (7 ⫾ 1
vs. 5 ⫾ 1 layers, respectively). Cells in this outermost
zone formed a continuous, 3-D tissuelike structure by
attaching to other cells, spreading along the randomly
oriented PGA fibers, and forming bridges between the
fibers (Fig. 2, A and C). Distinct cardiac bundles,
spatially oriented groups of cells (⬎100 µm in size), and
interstitial collagen septa were not observed. Ran-
domly oriented cells in the peripheral zone exhibited a
variety of shapes, from elongated cells spread on the
polymer fibers to round unattached cells, as assessed
histologically. The majority of the cells expressed the
muscle-specific proteins sarcomeric tropomyosin (Fig.
2, C and D) and sarcomeric ␣-actin (data not shown).
Immediately below the peripheral zone was a 60- to
70-µm-thick region consisting mainly of cells that did
not express tropomyosin. At the construct center, cells
were sparsely distributed and either elongated, express-
ing tropomyosin, or round, with pyknotic nuclei and
acidophilic cytoplasm (Fig. 2B).
Cross striations were present in cells in the periph-
eral zone of the constructs as well as in neonatal and
adult ventricles, as assessed immunohistochemically
H438 CARDIAC MUSCLE TISSUE ENGINEERING
Fig. 3. Transmission electron micro-
graph of an enriched construct. A: well-
organized myofilaments (Mfl) with
clearly defined sarcomeres, z-lines (Z),
and abundant glycogen granules (Gly).
Magnification, ⫻26,000; bar, 1 µm. B:
several mitochondria (Mito) located be-
tween myofilaments. Magnification,
⫻36,000; bar, 400 nm. C: desmosomes
(Des) at lateral border of adjacent car-
diac myocytes. Magnification, ⫻18,000;
bar, 250 nm. D: intercalated disk (ID)
with visible desmosomes. Magnifica-
tion, ⫻18,000; bar, 250 nm.
(Fig. 2, D–F). The presence of subcellular elements
characteristic of cardiac myocytes, including myofila-
ments with well-defined sarcomeres, z-lines, glycogen
granules (Fig. 3A), and mitochondria (Fig. 3B) in the
outermost layer of constructs, was demonstrated by
transmission electron microscopy (TEM). Cell-to-cell
connections were demonstrated by the presence of
desmosomes (Fig. 3C) and intercalated disks (Fig. 3D).
Construct composition. After 3 days, the respective
numbers of viable cells present in enriched and regular
constructs were 66 and 57% of those seeded at time 0, as
calculated from medium LDH levels. LDH release be-
tween culture days 3 and 7 was one-third of that
between days 0 and 3, indicating that the cell death
rate decreased with cultivation time. At 7 days, cell
numbers in enriched and regular constructs were 38
and 47% of the respective numbers seeded at time 0, as
determined by the DNA content of constructs. For
comparison, 7-day cell monolayers from both groups
contained 61 ⫾ 6% of the initially plated cells. The
number of cells seeded at time 0 (8 million per PGA
disk) could be accounted for by summing cell numbers
in constructs at day 7 (determined from DNA content)
and in the medium over 7 days (calculated from cumu-
lative LDH activity/construct) (Table 1), implying that
no significant cell proliferation occurred during the
cultivation period. Glucose consumption and lactate
production rates were higher in enriched than in
regular constructs (P ⬍ 0.005, Table 1), whereas the
lactate-to-glucose molar ratios were similar for both
Downloaded from ajpheart.physiology.org on May 24, 2010
construct groups (1.00 ⫾ 0.20 and 1.30 ⫾ 0.11, respec-
tively).
Ventricular tissues from neonatal and adult rats had
respectively six- and threefold higher DNA contents per
unit wet weight (an index of cellularity) than engi-
neered constructs from either group (P ⬍ 0.01, Fig. 4A),
which is consistent with the relatively acellular appear-
ance of the construct centers (Fig. 2B). Relative cell
size, assessed from the ratio of total protein to DNA,
was comparable for cells in constructs, neonatal ven-
tricles, and monolayers and lower than for cells in adult
ventricles (P ⬍ 0.01) (Fig. 4B). This finding was consis-
tent with the relative cross-sectional areas of cells in
constructs and neonatal and adult ventricles observed
histologically (Fig. 2, D–F, respectively). The MTT
conversion per unit DNA (an index of metabolic activ-
ity) was similar for constructs and neonatal ventricles
and was slightly higher in adult ventricles (Fig. 4C).
Construct electrophysiology. Spontaneous, macro-
scopic contractions of engineered constructs were visu-
ally observed in flasks between days 2 and 4 of cultiva-
tion, which indicated the presence of intercellular
communication. At day 7 the majority of constructs and
native ventricles exhibited transient spontaneous beat-
ing lasting for 1–10 contractions (Fig. 5A), which may
have resulted from reentrant or triggered activity (4).
Electrical stimulation resulted in impulse propagation
in the peripheral cardiac tissue-like zone of the con-
structs. In contrast, impulses failed to propagate when
the electrodes were advanced toward the central acellu-
 CARDIAC MUSCLE TISSUE ENGINEERING
Fig. 4. Cellularity, hypertrophy, and metabolic activity indexes of
constructs and ventricles. A: DNA content per unit wet weight (ww).
B: protein per unit DNA. C: MTT conversion per unit DNA. Data
represent means ⫾ SE of 5 samples. * Statistical difference between
constructs and native ventricles; † statistical difference between
neonatal and adult ventricles.
lar region of the constructs. All 7-day constructs were
electrically excitable and could be captured over a wide
range of pacing frequencies (up to 270 beats/min, Fig. 5,
B-D). Step increases in construct pacing frequency
resulted in transient decreases in conduction velocity to
steady-state values (data not shown). Rapid stimula-
tion induced short tachyarrhythmias with rates close to
the maximum capture rates in 3 of 6 enriched con-
structs, 2 of 6 regular constructs (Fig. 5E), 1 of 10 adult
ventricles, and 0 of 10 neonatal ventricles. A separate
experiment showed that constructs remained electri-
H439
cally excitable for up to 4 wk of culture (data not
shown).
Representative examples of impulse propagation in
an enriched construct, a neonatal ventricle, and an
adult ventricle are shown in Fig. 6, A–C, respectively.
Propagating extracellular waveforms in constructs and
native tissues showed fairly smooth, biphasic shapes
with distinct downward deflections that enabled confi-
dent determination of activation times and implied
nondecremental, macroscopically continuous propaga-
tion without wave collisions (34). Notches in extracellu-
lar waveforms occasionally observed in constructs (see
E3 in Fig. 6A) may have reflected asynchronous excita-
tion in adjacent groups of cells caused by the presence
of empty space, polymer fibers, and/or necrotic tissue
(36). Conduction times in ventricles and constructs
increased linearly with distance over 5 mm (Fig. 6D,
Downloaded from ajpheart.physiology.org on May 24, 2010
Fig. 5. Electrophysiological recordings. A: short transient spontane-
ous beating at a rate of 70 beats/min, which lost regularity after 6 s.
B, C, and D: steady-state responses after ⱖ4 min of pacing at rates of
80, 150, and 200 beats/min, respectively. E: short, 5-s tachyarrhyth-
mia at ⬃190 beats/min, apparently induced by 4 rapid stimuli at 250
beats/min. S and R indicate the stimulus spike and the construct
response, respectively. All tracings were recorded from enriched
constructs using the same electrode.
 Downloaded from ajpheart.physiology.org on May 24, 2010
CARDIAC MUSCLE TISSUE ENGINEERING
H440
 CARDIAC MUSCLE TISSUE ENGINEERING H441

Table 2. Electrophysiological parameters in 7-day constructs and native ventricles

Excitation Conduction Maximum Average Maximum Capture
Group n Threshold, V Velocity, cm/s Amplitude, mV Amplitude, mV Rate, beats/min

Constructs
Regular* 6 2.70 ⫾ 0.24 9.35 ⫾ 0.27 0.52 ⫾ 0.05 0.26 ⫾ 0.09 111.7 ⫾ 9.5
Enriched* 6 2.97 ⫾ 0.30 11.89 ⫾ 0.46† 0.90 ⫾ 0.14† 0.43 ⫾ 0.14 175.0 ⫾ 21.3†
Ventricles
Neonatal 10 0.74 ⫾ 0.20 21.82 ⫾ 1.48 31.91 ⫾ 3.53 18.34 ⫾ 4.31 475.0 ⫾ 25.0
Adult 10 1.34 ⫾ 0.17‡ 31.69 ⫾ 4.44‡ 25.82 ⫾ 2.81 14.62 ⫾ 3.59 281.2 ⫾ 21.0‡
Data represent means ⫾ SE; n ⫽ no. of constructs or ventricles. * Significant difference between constructs and ventricles; † significant
difference between enriched and regular constructs; ‡ significant difference between neonatal and adult ventricles.

regression coefficients ⬎ 0.98), implying similar conduc-
tion velocities between adjacent electrodes and thus
relatively homogeneous electrical properties through-
out the cardiac tissue-like zone.
Conduction velocities descended in the following
order: adult ventricles, neonatal ventricles, enriched
constructs, and regular constructs (Table 2, Fig. 6D)
(P ⬍ 0.001). Lower conduction velocities measured in
eral cardiac tissue-like zone in which differentiated
cardiac myocytes were organized in multiple layers and
attached to other cells and/or polymer fibers in a 3-D
configuration. Impulse propagation studies carried out
using an array of extracellular microelectrodes demon-
strated that the peripheral cardiac tissue-like zone of
constructs sustained macroscopically continuous im-

Downloaded from ajpheart.physiology.org on May 24, 2010

neonatal than in adult ventricles were consistent with
previously published values (40, 41). Conduction veloci-
ties measured in enriched constructs were ⬃30 and
50% as high as those in adult and neonatal ventricles,
respectively (P ⬍ 0.001) and were 27% higher than in
regular constructs (P ⬍ 0.02). Excitation thresholds
were higher in engineered constructs than in native
ventricles (P ⬍ 0.001, Table 2) and were lower in
neonatal than in adult ventricles (P ⬍ 0.01). Excitation
thresholds of constructs and ventricles thus varied
inversely with cellularity indexes (Table 2, Fig. 4A).
Maximum and average amplitudes were signifi-
cantly lower in constructs than in native ventricles
(P ⬍ 0.0001, Table 2). Maximum amplitudes were
1.7-fold higher in enriched than in regular constructs
(P ⬍ 0.002), whereas average amplitudes did not differ
significantly. The range of recorded amplitudes was
⬃3-fold higher in constructs than in native ventricles
(data not shown). Maximum capture rates differed
significantly (P ⬍ 0.001) among groups and descended
in the following order: neonatal ventricles, adult ven-
tricles, enriched constructs, and regular constructs
(Table 2). The higher maximum capture rates in neona-
tal ventricles than in adult ventricles were consistent
with the higher resting heart rates and higher toler-
ance to ischemia previously reported for neonatal ven-
tricles (47).
DISCUSSION
The present study demonstrates that 3-D cardiac
muscle constructs with cardiac-specific structural and
electrophysiological properties can be engineered in
vitro using isolated cells and biodegradable polymer
scaffolds. In particular, constructs contained a periph-
pulse propagation (Fig. 6A) that depended on the
fraction of seeded cardiac myocytes (Table 2). Func-
tional constructs may thus enable in vitro electrophysi-
ological studies that may complement those currently
carried out using thin ventricular slices (5, 14, 35) and
monolayers of cardiac myocytes (9).
Structurally, constructs were 5 ⫻ 1.3-mm (diameter ⫻
thickness) disks and contained a 50- to 70-µm-thick
outer cardiac tissue-like zone composed of cells that
expressed sarcomeric tropomyosin (Fig. 2C) and con-
tained myofilaments, desmosomes, and intercalated
disks (Fig. 3D). For comparison, a recently reported (8)
heart muscle model system based on cardiac myocyte-
populated 3-D collagen gels (15 mm long ⫻ 8 mm
wide ⫻ 180 µm thick) contained several layers of
differentiated cells at the edges and less concentrated
cells centrally. The small thickness of the cardiac
tissue-like zone in constructs (Fig. 2A) and collagen
gels (8) can be attributed to the low survival rate of
metabolically demanding cardiac myocytes located more
than 50 µm from a source of gas exchange (15).
The molar ratios of lactate to glucose of 1.0–1.3
indicated aerobic cell metabolism in the constructs (21).
Compared with the regular group, enriched constructs
had higher glucose consumption rates (Table 1), prob-
ably due to the relatively higher fraction of myocytes.
The absence of cell proliferation in constructs (Table 1)
was consistent with the previous findings that neonatal
ventricular cardiac myocytes lose their ability to prolif-
erate after 2–3 days in vitro (45), whereas fibroblasts
proliferate slowly in 3-D cultures (20).
Electrophysiologically, impulse propagation in con-
structs was studied on a macroscopic level using a
linear array of extracellular electrodes (Fig. 1B). Inter-
electrode distances of 500 µm were selected on the basis

Fig. 6. Impulse propagation (representative 66 ms) in a construct from the enriched group (A), neonatal ventricle
(B), and adult ventricle (C). The extracellular waveform is shown propagating from the electrode closest to the
stimulus site (E1) toward the furthest electrode still in the sample (E6). Simultaneous deflections at the beginning
of traces (S) represent stimulus artifacts. Amplitude ranges for each electrode were adjusted to best display
recorded response waveforms. D: plot of conduction times vs. distances relative to E1, which was assigned the
coordinates (0,0). Conduction velocities were calculated as inverse slopes of best fit lines.
H442 CARDIAC MUSCLE TISSUE ENGINEERING
of previously reported in vivo and ex vivo epicardial
mapping studies (6, 46, 48). Bipolar point stimulation
and unipolar recording (16, 25) in the custom-designed
test chamber (Fig. 1B) did not adversely affect samples
with respect to their electrical properties (waveform
shapes were stable) or structure (no apparent tissue
damage was observed histologically). Automated data
analysis was facilitated by the high average signal-to-
noise ratios (of ⬃10 and 470 for constructs and native
ventricles, respectively). Whereas 1- to 5-V amplitude,
1-ms duration electrical pulses were sufficient to induce
impulse propagation in slices of ventricles and in the
peripheral zone of 7-day constructs, it was difficult to
overdrive 7-day confluent monolayers of neonatal car-
diac myocytes even when using stimuli of twice this
amplitude and duration. In addition, impulse propaga-
tion in monolayers could not be assessed using extracel-
lular electrodes because of fractionation and low ampli-
tudes of recorded waveforms. These findings may be
due to 3-D electrotonic interactions between cells (9)
and relatively high cell density around the stimulating
and recording electrodes in 3-D constructs compared
with 2-D monolayers.
The inferior electrophysiological properties of con-
structs compared with native ventricles (Table 2) can
be attributed to differences in their macroscopic tissue
architecture. In particular, the relatively high excita-
tion thresholds (24) and low response amplitudes were
associated with low construct cellularity (Fig. 4A). Low
maximum capture rates and conduction velocities in
constructs probably resulted from decreased cell cou-
pling, the presence of intercellular clefts, and geometric
current-to-load mismatches (due to tissue discontinui-
ties) (9, 26). Other mechanisms that could contribute to
inferior construct electrophysiological properties in-
clude cell depolarization, reduced excitability, and
slower repolarization resulting from injury during isola-
tion and/or cultivation (32, 39). Intracellular recordings
would be necessary to test the proposed mechanisms.
Compared with enriched constructs, lower conduc-
tion velocities, maximum capture rates, and ampli-
tudes in regular constructs probably resulted from 1)
the higher fraction of noncardiomyocytic cells, which
would be expected to form high-resistance junctions
with cardiac myocytes (28) and act as passive current
sinks (9), and 2) the thinner cardiac tissue-like zone
(Table 1). Lower maximum capture rates in the regular
than enriched constructs could also be due to the
relatively longer duration of cellular action potentials
(as previously observed in fibrotic compared with nor-
mal cardiac tissue; Ref. 42).
Neonatal and adult ventricular tissues did not ex-
hibit spontaneous beating ex vivo in a previous (39) or
the present study. In contrast, enzymatically isolated
ventricular cardiac myocytes cultured in monolayers
are known to revert to a less differentiated phenotype,
depolarize, and regain spontaneous contractile activity
for as yet unknown reasons (39). In the present study,
visible spontaneous contractions in constructs ceased
after 4 days of cultivation. This finding might be
attributed to gradual depolarization and decoupling of
cardiac myocytes due to injury during cultivation.
However, it is more likely that the cultivation of cardiac
myocytes on 3-D biomaterial scaffolds in tissue culture
bioreactors (Fig. 1A) promoted differentiated cellular
phenotype and function. In support of this hypothesis,
Sperelakis (38) showed that 3-D aggregates composed
of electrically differentiated cardiac myocytes did not
contract spontaneously but responded to electrical
stimulation.
The aim of the present study was to demonstrate
basic cardiac-specific features in constructs and to
evaluate construct structure and electrophysiological
properties on a macroscopic (tissue) level, rather than
on a cellular level. In ongoing work, we are expanding
our electrophysiological studies to include whole cell
clamp and sharp microelectrode intracellular record-
ings and assessment of the spatial distribution of the
gap junctional protein connexin 43 (23). We are also
attempting to culture constructs with a thicker cardiac
tissue-like zone by direct perfusion of constructs during
Downloaded from ajpheart.physiology.org on May 24, 2010
cultivation (to improve mass transfer) and by cocultur-
ing cardiac myocytes with microvascular endothelial
cells (as a first step toward inducing vascularization).
In conclusion, cardiac-specific features of engineered
cardiac muscle constructs were demonstrated structur-
ally and electrophysiologically and were related to the
cellular composition of constructs. The 3-D multilayer
structure in conjunction with macroscopic impulse
propagation in engineered constructs can offer advan-
tages for in vitro studies of cardiac muscle. In addition,
structurally and functionally improved 3-D engineered
cardiac muscle constructs could be eventually applied
in vivo. To date, attempts to regenerate cardiac tissue
have involved the injection of different muscle cell
types (33, 43) or small tissue fragments (19) into the
heart. Implantation of cardiac muscle constructs with a
defined shape instead of isolated cells could potentially
improve the efficiency and localization of tissue repair.
N. Bursac and M. Papadaki contributed equally to this study.
We thank R. Langer for advice, R. Padera for help with animal
surgery, H. Shing for carrying out the transmission electron micros-
copy, Y. Lee for help establishing the electrophysiological recording
system, and J. Merok, H. Cho, and P. Gupta for help with biochemical
assays.
This work was supported by National Aeronautics and Space
Administration Grant NAG9-836.
Address for reprint requests and other correspondence: L. E.
Freed, Massachusetts Institute of Technology, Div. of Health Science
and Technology, MIT, Bldg. E25–342, Cambridge, MA 02139 (E-mail:
lfreed@mit.edu).
Received 7 October 1998; accepted in final form 8 March 1999.
REFERENCES
1. Akins, R. E., N. A. Schroedl, S. R. Gonda, and C. R. Hartzell.
Neonatal rat heart cells cultured in simulated microgravity. In
Vitro Cell Dev. Biol. 33: 337–343, 1997.
2. Blanchard, S., W. Smith, R. Damiano, D. Molter, R. Ideker,
and J. Lowe. Four digital algorithms for activation detection
from unipolar epicardial electrograms. IEEE Trans. Biomed.
Eng. 36: 256–261, 1989.
3. Carrier, R., M. Papadaki, M. Rupnick, F. J. Schoen, N.
Bursac, R. Langer, L. E. Freed, and G. Vunjak-Novakovic.
Cardiac tissue engineering: cell seeding, cultivation parameters
 CARDIAC MUSCLE TISSUE ENGINEERING
and tissue construct characterization. Biotechnol Bioeng. In
press.
4. Cranefield, P., and R. Aronson. Cardiac Arrhythmias: The
Role of Triggered Activity and Other Mechanisms. Mount Kisco,
NY: Futura, 1988.
5. Davidenko, J., R. Salomonsz, A. Pertsov, W. Baxter, and J.
Jalife. Effects of pacing on stationary reentrant activity: theoreti-
cal and experimental study. Circ. Res. 77: 1166–1179, 1995.
6. De Bakker, J., F. van Cappele, and M. Janse. Slow conduc-
tion in the infarcted human heart: ‘‘zigzag’’ course of activation.
Circulation 88: 915–926, 1993.
7. Downs, T., and M. Wilfinger. Fluorometric quantification of
DNA in cells and tissue. Anal. Biochem. 131: 538–547, 1983.
8. Eschenhagen, T., C. Fink, U. Remmers, H. Scholz, J.
Wattchow, J. Weil, W. Zimmerman, H. Dohmen, H. Schafer,
N. Bishopric, T. Wakatsuki, and E. Elson. Three-dimensional
reconstitution of embryonic cardiomyocytes in a collagen matrix:
a new heart muscle model system. FASEB J. 11: 683–694, 1997.
9. Fast, V., B. Darrow, J. Saffitz, and A. Kleber. Anisotropic
activation spread in heart cell monolayers assessed by high-
resolution optical mapping. Role of tissue discontinuities. Circ.
Res. 79: 115–127, 1996.
10. Fast, V., and A. Kleber. Microscopic conduction in cultured
cardiac strands of neonatal rat heart cells measured with
voltage-sensitive dyes. Circ. Res. 73: 914–925, 1993.
11. Freed, L. E., and G. Vunjak-Novakovic. Culture of organized
cell communities. Adv. Drug Delivery Res. 33: 15–30, 1998.
12. Freed, L., and G. Vunjak-Novakovic. Microgravity tissue
engineering. In Vitro Cell Dev. Biol. 33: 381–385, 1997.
13. Freed, L. E., G. Vunjak-Novakovic, R. Biron, D. Eagles, D.
Lesnoy, S. Barlow, and R. Langer. Biodegradable polymer
scaffolds for tissue engineering. Biotechnology 12: 689–693,
1994.
14. Girouard, S., K. Laurita, and D. Rosenbaum. Unique proper-
ties of cardiac action potentials recorded with voltage-sensitive
dyes. J. Cardiovasc. Electrophysiol. 7: 1024–1037, 1996.
15. Guyton, A. Transport of oxygen and carbon dioxide in the blood
and body fluids. In: Textbook of Medical Physiology, edited by A.
Guyton. Philadelphia, PA: Saunders, 1986, p. 493–503.
16. Knisley, S., and B. Hill. Effects of bipolar point and line
stimulation in anisotropic rabbit epicardium: assessment of the
critical radius of curvature for longitudinal block. IEEE Trans.
Biomed. Eng. 42: 957–966, 1995.
17. Kunysz, A., A. Shrier, and L. Glass. Overdrive suppression of
spontaneously beating chick heart cell aggregates: experiment
and theory. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1153–
H1164, 1995.
18. Langer, R., and J. Vacanti. Tissue engineering. Science 260:
920–926, 1993.
19. Leor, J., M. Patterson, M. Quinones, L. Kedes, and R.
Kloner. Transplantation of fetal myocardial tissue into the
infarcted myocardium of rat. Circulation 94: II-322–II-336, 1996.
20. Mauch, C., A. Hatamochi, K. Scharffetter, and T. Krieg.
Regulation of collagen synthesis in fibroblasts within a three-
dimensional collagen gel. Exp. Cell Res. 178: 493–503, 1988.
21. Opie, L. Fuels: aerobic and anaerobic metabolism. In: The Heart:
Physiology, from Cell to Circulation (3rd ed.), edited by L. Opie.
Philadelphia, PA: Lippincott-Raven, 1998, p. 295–342.
22. Orita, H., M. Fukasawa, S. Hirooka, H. Uchino, K. Fukui,
and M. Washio. Modulation of cardiac myocyte beating rate and
hypertrophy by cardiac fibroblasts isolated from neonatal rat
ventricle. Jpn. Circ. J. 57: 912–920, 1993.
23. Papadaki, M., N. Bursac, R. Langer, F. Schoen, G. Vunjak-
Novakovic, and L.E. Freed. Towards a functional engineered
cardiac muscle: effects of cell culture substrate and medium
serum concentration. In: Society for Biomaterials, 25th Annual
Meeting Transactions. Minneapolis, MN: Soc. Biomaterials, 1999,
p. 359.
24. Ramza, B., R. Tan, T. Osaka, and R. Joyner. Cellular mecha-
nism of the functional refractory period in ventricular muscle.
Circ. Res. 66: 147–162, 1990.
H443
25. Rogers, J., P. Bayly, R. Ideker, and W. Smith. Quantitative
techniques for analyzing high-resolution cardiac mapping data.
IEEE Eng. Med. Biol. Mag. 17: 62–72, 1998.
26. Rohr, S., and B. Salzberg. Characterization of impulse propa-
gation at the microscopic level across geometrically defined
expansions of excitable tissue: multiple site optical recording of
transmembrane voltage (MSORTV) in patterned growth heart
cell cultures. J. Gen. Physiol. 104: 287–309, 1994.
27. Rohr, S., and B. Salzberg. Multiple site optical recording of
transmembrane voltage (MSORTV) in patterned growth heart
cell cultures: assessing electrical behavior, with microsecond
resolution, on a cellular and subcellular scale. Biophys. J. 67:
1301–1315, 1994.
28. Rook, M., A. Van Ginneken, B. De Jonge, A. El Aoumari, D.
Gros, and H. Jongsma. Differences in gap junction channels
between cardiac myocytes, fibroblasts, and heterologous pairs.
Am. J. Physiol. 263 (Cell Physiol. 32): C959–C977, 1992.
29. Schroedl, N., and C. Hartzell. Myocytes and fibroblasts exhibit
functional synergism in mixed cultures of neonatal rat heart
cells. J. Cell. Physiol. 117: 326–332, 1983.
30. Schubert, B., R. Bodewei, S. Hering, and A. Wollenberger.
Cell-attached patch clamp measurement of macroscopic rapid
inward sodium current in cultured heart cell reaggregates. J.
Mol. Cell. Cardiol. 19: 1129–1139, 1987.
Downloaded from ajpheart.physiology.org on May 24, 2010
31. Shahar, A., S. Reuveny, Y. David, C. Budu, and A. Shain-
burg. Cerebral neurons, skeletal myoblasts, and cardiac muscle
cells cultured on macroporous beads. Biotechnol. Bioeng. 43:
826–831, 1994.
32. Shaw, R., and Y. Rudy. Electrophysiological effects of acute
myocardial ischemia: a theoretical model of altered excitability
and action potential duration. Cardiovasc. Res. 35: 256–272,
1997.
33. Soonpa, M., A. Daud, G. Koh, M. Klug, K. Kim, H. Wang, and
L. Field. Potential approaches for myocardial regeneration.
Ann. NY Acad. Sci. 752: 446–454, 1995.
34. Spach, M., R. Bar, and G. Serwer. Collision of excitation waves
in the dog Purkinje system. Circ. Res. 29: 499–511, 1971.
35. Spach, M., P. Dolber, and J. Heidlage. Properties of discontinu-
ous anisotropic propagation at a microscopic level. Ann. NY
Acad. Sci. 591: 62–74, 1990.
36. Spach, M., P. Dolber, and J. Heidlage. Resolution of discontinu-
ous versus continuous propagation: microscopic mapping of the
derivatives of extracellular potential waveforms. In: Cardiac
Electrophysiology: From Cell to Bedside, edited by D. Zipes and J.
Jalife. Philadelphia, PA: Saunders, 1990, p. 139–148.
37. Spach, M., J. Kootsey, and J. Sloan. Active modulation of
electrical coupling between cardiac cells of the dog: a mechanism
for transient and steady state variations in conduction velocity.
Circ. Res. 51: 347–362, 1982.
38. Sperelakis, N. Cultured heart cell reaggregate model for study-
ing cardiac toxicology. Environ. Health Perspect. 26: 243–267,
1978.
39. Sperelakis, N., and G. Haddad. Developmental changes in
membrane electrical properties of the heart. In: Physiology and
Pathophysiology of the Heart (3rd ed.), edited by N. Sperelakis.
Norwell, MA: Kluwer Academic, 1995, p. 669–700.
40. Sun, L., M. Legato, T. Rosen, S. Steinberg, and M. Rosen.
Sympathetic innervation modulates ventricular impulse propaga-
tion and repolarisation in immature rat heart. Cardiovasc. Res.
27: 459–463, 1993.
41. Suzuki, J., H. Tsubone, and S. Sugano. Characteristics of
ventricular activation and recovery patterns in the rat. J. Vet.
Med. Sci. 54: 711–716, 1992.
42. Swynghedauw, B., B. Chevalier, D. Charlemagne, P. Man-
sier, and F. Carre. Cardiac hypertrophy, arrhythmogenicity and
the new myocardial phenotype. II. The cellular adaptational
process. Cardiovasc. Res. 35: 6–12, 1997.
43. Taylor, D., B. Atkins, P. Hungspreugs, T. Jones, M. Reedy,
K. Hutcheson, D. Glower, and W. Kraus. Regenerating func-
tional myocardium: improved performance after skeletal myo-
blast transplantation. Nat. Med. 4: 929–933, 1998.
44. Toraason, M., M. Luken, M. Breitenstein, J. Krueger, and
R. Biagini. Comparative toxicity of allylamine and acrolein in
H444 CARDIAC MUSCLE TISSUE ENGINEERING
cultured myocytes and fibroblasts from neonatal rat heart.
Toxicology 56: 107–113, 1989.
45. Undo, H., M. B. Perryman, R. Roberts, and M. D. Schneider.
Differentiation of cardiac myocytes after mitogen withdrawal
exhibits three sequential states of the ventricular growth re-
sponse. J. Cell Biol. 107: 1911–1918, 1988.
46. Wit, A., S. Dillon, J. Coromilas, A. Saltman, and B. Wal-
decker. Anisotropic reentry in the epicardial border zone of
myocardial infarcts. Ann. NY Acad. Sci. 591: 86–108, 1990.
47. Xie, L., M. Takano, and A. Noma. Development of inwardly
rectifying K⫹ channel family in rat ventricular myocytes. Am. J.
Physiol. 272 (Heart Circ. Physiol. 41): H1741–H1750, 1997.
48. Yin, H., N. El-Sherif, E. B. Caref, G. Ndrepepa, R. Levin,
N. Isber, K. Stergiopolus, M. Assadi, W. Gough, and M.
Restivo. Actions of lidocaine on reentrant ventricular rhythms
in the subacute myocardial infarction period in dogs. Am. J.
Physiol. 272 (Heart Circ. Physiol. 41): H299–H309, 1997.
Downloaded from ajpheart.physiology.org on May 24, 2010