| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; and 2 Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California 94305
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
Rapid development of
transgenic and gene-targeted mice and acute genetic manipulation via
gene transfer vector systems have provided powerful tools for
cardiovascular research. To facilitate the phenotyping of genetically
engineered murine models at the cellular and subcellular levels and to
implement acute gene transfer techniques in single mouse
cardiomyocytes, we have modified and improved current enzymatic methods
to isolate a high yield of high-quality adult mouse myocytes (5.3 ± 0.5 × 105 cells/left ventricle, 83.8 ± 2.5%
rod shaped). We have also developed a technique to culture these
isolated myocytes while maintaining their morphological integrity for
2-3 days. The high percentage of viable myocytes after 1 day in
culture (72.5 ± 2.3%) permitted both physiological and
biochemical characterization. The major functional aspects of these
cells, including excitation-contraction coupling and receptor-mediated
signaling, remained intact, but the contraction kinetics were
significantly slowed. Furthermore, gene delivery via recombinant
adenoviral infection was highly efficient and reproducible. In adult
1/2-adrenergic receptor (AR)
double-knockout mouse myocytes, adenovirus-directed expression of
either 1- or 2-AR, which occurred in
100% of cells, rescued the functional response to -AR agonist
stimulation. These techniques will permit novel experimental settings
for cellular genetic physiology.
excitation-contraction coupling; -adrenergic signaling
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
RECENT ADVANCES in molecular genetics have heralded a new era of "genetic physiology" to study molecular function using genetically engineered animal models and gene-encoding vector-directed gene transfer systems. Since the first cardiac-specific transgenic mouse was created in 1988 (5), hundreds of transgenic or gene-targeted murine models have been generated for the overexpression, genetic ablation, or site-specific mutation of key proteins governing cardiac structure and function. The subsequent phenotypic characterization of these genetic manipulations in intact animals and isolated hearts has greatly enhanced our current knowledge of cardiac development, Ca2+ handling, excitation-contraction (EC) coupling, and receptor-mediated signal transduction (see Refs. 7 and 8 for review). However, cellular and subcellular phenotypic characterization of these murine models has still been a challenge, and gene transfer by viral transfection has not been well established in adult mouse cardiomyocytes.
Freshly isolated adult mouse myocytes usually maintain functional integrity for 6-8 h without culture and are suitable for acute studies in electrophysiology (1, 14, 24), intracellular Ca2+ dynamics, and cell contraction (1, 12, 20, 22-24). Because vector-mediated gene transfer and subsequent functional characterization require that the adult myocytes be healthy and functional for at least 24 h, the ability to culture the isolated mouse myocytes is essential to cellular "genetic physiology." However, the isolated adult mouse myocytes have a much lower viability (~50%) (3, 13, 22) than those isolated from other species (70-90% for rat, rabbit, and guinea pig) (17, 18) and could rarely survive an overnight culture (see RESULTS AND DISCUSSION). Little information is currently available on cultured adult mouse cardiomyocytes (13).
In the present study, we have modified current enzymatic methods to improve adult mouse cardiomyocyte yield and quality and have developed a practical and reliable method for short-term culture of these isolated myocytes that retains their morphological as well as physiological integrity. Moreover, we have demonstrated the feasibility of adenovirus-mediated gene transfer in such cultured myocytes from both wild-type and genetically engineered mouse hearts. These new technical developments provide a set of powerful tools for acute gene engineering in single cells, for phenotyping transgenic or knockout models at the cellular and subcellular levels, and for combining the approaches of whole animal and single-cell gene manipulations.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
Isolation of adult mouse ventricular myocytes.
Wild-type or gene-targeted mice (2-4 mo) of either sex and from
various genetic backgrounds (C57B16/J, 129SvJ, DBA/2, or FVB/N) were
anesthetized with pentobarbital sodium (100 mg/kg ip). The heart was
quickly removed from the chest and retrogradely aortic perfused at
constant pressure (100 cmH2O) at 37°C for ~3 min with a
Ca2+-free bicarbonate-based buffer containing (in mM) 120 NaCl, 5.4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5.6 glucose, 20 NaHCO3, 10 2,3-butanedione monoxime (BDM; Sigma), and 5 taurine (Sigma),
gassed with 95% O2-5% CO2 (see Table
1 and Fig.
1). To reduce bacterial or viral contamination, the perfusion setup was washed with 70% alcohol and
then rinsed three times with sterilized distilled water before cannulation. All the solutions were filtered (0.2-µm filter) and equilibrated with 95% O2-5% CO2 for at
least 20 min before use. The enzymatic digestion was initiated by
adding collagenase type B (0.5 mg/ml; Boehringer Manheim), collagenase
type D (0.5 mg/ml; Boehringer Manheim), and protease type XIV (0.02 mg/ml; Sigma) to the perfusion solution. When the heart became swollen
and hard after ~3 min of digestion, 50 µM Ca2+ was
added to the enzyme solution. About 7 min later, the left ventricle was
quickly removed, cut into several chunks, and further digested in a
shaker (60-70 rpm) for 10 min at 37°C in the same enzyme
solution. The supernatant containing the dispersed myocytes was
filtered into a sterilized tube and gently centrifuged at 500 rpm for 1 min. The cell pellet was then promptly resuspended in Ca solution
I (125 µM Ca2+; see Table 1). After the myocytes
were pelleted by gravity for ~10 min, the supernatant was aspirated
and the myocytes were resuspended in Ca solution II (250 µM Ca2+; Table 1). The final cell pellet was
suspended in Ca solution III (500 µM Ca2+;
Table 1). Meanwhile, the shake-harvest procedure was repeated several
times until all the chunks were digested. For experiments on freshly
isolated cells, myocytes were stored in HEPES-buffered solution
consisting of (in mM) 1 CaCl2, 137 NaCl, 5.4 KCl, 15 dextrose, 1.3 MgSO4, 1.2 NaH2PO4,
and 20 HEPES, adjusted to pH 7.4 with NaOH.
|
|
Culture of adult mouse cardiac myocytes. The entire culture procedure was performed in a class II flow hood. Culture dishes were precoated for 1 h with 10 µg/ml mouse laminin (GIBCO) in phosphate-buffered saline (PBS; GIBCO) with 1% penicillin-streptomycin (PS; GIBCO) at room temperature (see Ref. 16 for review). Freshly isolated cardiac myocytes were suspended in minimal essential medium (MEM; Sigma M1018) containing 1.2 mM Ca2+, 2.5% preselected fetal bovine serum (FBS; GIBCO), and 1% PS (pH 7.35-7.45). After the myocytes were pelleted by gravity for ~10 min, the supernatant was aspirated and the myocytes were washed two more times using the same protocol. The myocytes were then plated at 0.5-1 × 104 cells/cm2 in MEM containing 2.5% FBS and 1% PS. After 1 h of culture in a 5% CO2 incubator at 370C , the medium was changed to FBS-free MEM and this was changed every 48 h during culture.
Criteria for viable mouse myocytes were 1) rod shape, 2) clearly defined sarcomeric striations, and 3) quiescent state (no spontaneous contractile waves) for at least 5 min during observation. Functionally, the viable myocytes exhibited a clear negative contraction staircase during stimulation from rest at 0.5 Hz and a stable steady-state contraction amplitude for at least 10 min.Adenoviral infection. Adenovirus-directed gene transfer was implemented after 1 h of culture to achieve myocyte attachment. The culture medium was aspirated along with unattached myocytes, and a half-volume (e.g., 1 ml for a 35-mm petri dish) of the FBS-free MEM containing an appropriate titer of gene-carrying adenovirus was added to the dish. Another half-volume of the FBS-free MEM was added following an additional 1-2 h of culture.
To test the expression efficiency, myocytes were infected with an adenoviral vector carrying a marker gene,-galactosidase (Adeno--Gal), at a multiplicity of infection (MOI) of 1-1,000 plaque-forming units/cell. After 24 h of infection, myocytes were fixed in 0.5% glutaraldehyde for 5 min at room temperature. The myocytes were then washed twice with PBS and stained for 2 h at 37°C in PBS containing 1 mg/ml
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal),
5 mM K4Fe(CN)6, 5 mM
K3Fe(CN)6, and 1 mM MgCl2.
Adenoviral vectors carrying human 1- or
2-AR genes (Adeno--1-AR or
Adeno-2-AR) and Adeno--Gal were kindly provided by Dr. Robert Lefkowitz (Duke University, Durham, NC).
Measurement of cell contraction. Myocytes were placed on the stage of an inverted microscope (Zeiss model IM-35), superfused with HEPES-buffered solution at a flow rate of 1.8 ml/min, and electrically stimulated at 0.5 Hz at 23°C. Cell length was monitored from the bright-field image (650- to 750-nm red light illumination) by an optical edge-tracking method using a photodiode array (model 1024 SAQ, Reticon) with a 3-ms time resolution. The contraction amplitude was measured as the percentage of shortening of cell length; the half-relaxation time was measured as the interval from the peak contraction to 50% relaxation.
Ca2+ current measurement.
L-type Ca2+ current (ICa) was
measured via the whole cell patch-clamp technique using an Axopatch 1D
amplifier (Axon Instruments, Foster City, CA). For selective
examination of ICa, myocytes were voltage
clamped at 
40 mV to inactivate Na+ and T-type
Ca2+ channels. Tetrodotoxin at 0.01 mM was also included in
the perfusion solution to ensure that the Na+ current was
completely eliminated. K+ currents were inhibited by
appropriate blockers in the extracellular HEPES buffer solution (4 mM
4-aminopyridine, 5.4 mM CsCl substituted for KCl in standard HEPES
buffer solution) and in the pipette (1-2 M
) solution containing
(in mM) 100 CsCl, 10 NaCl, 20 triethylammonium chloride, 10 HEPES, 5 MgATP, and 10 EGTA, adjusted to pH 7.2 with CsOH.
ICa was activated at 0.1 Hz by 300-ms pulses
from a holding potential of 40 mV to test potentials between 30 and
+50 mV in 10-mV increments. To monitor drug effects, we continuously recorded ICa elicited by a depolarization from
40 to 0 mV. The amplitude of ICa was measured
as the difference between the peak inward current and the current at
the end of the 300-ms pulse. All experiments were performed at room
temperature (23°C).
Confocal imaging of Ca2+ transients and cell morphology. Myocytes were placed on the stage of a Zeiss LSM-410 inverted confocal microscope (Carl Zeiss) and excited by the 488-nm line of an argon laser. Intracellular loading of the Ca2+ indicator fluo 3 was achieved by a 10-min incubation of the myocytes in HEPES buffer containing 10 µM fluo 3-AM (Molecular Probes), followed by a 15-min wash. All images were taken in the line-scan mode (2 ms/line, 0.1-0.3 µm/pixel), with the scan line oriented along the long axis of the myocyte, avoiding the nuclei of the cell. The microscope was equipped with a Zeiss Plan-Neofluar 40×, NA 1.3, oil-immersion objective, with an axial resolution of 1.1 µm. IDL software (Research Systems, Boulder, CO) was used for image processing, data analysis, and presentation.
To characterize morphological changes in the myocytes during culture, we stained the cells with 40 µM di-8-ANEPPS (Molecular Probes), a hydrophobic fluorescent indicator, in HEPES buffer for 30 min at room temperature. The sarcolemmal membrane system was visualized with confocal optical section images obtained under 488-nm excitation.Statistical analysis. Data are reported as means ± SE. Student's t-test was used to test for differences between freshly isolated cell and cultured cell groups, and a paired t-test was used for assessing the significance of drug effects. A value of P < 0.05 was considered to be statistically significant.
| |
RESULTS AND DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
Isolation of adult mouse cardiomyocytes.
Combining features of previously reported protocols (3,
13, 22) and our own modifications, we have
developed a simple and reliable myocyte isolation method that greatly
improves the yield, cell quality, and reproducibility of cardiomyocytes
isolated from adult mice (see METHODS for details). With
this method, we routinely obtained a high yield (5.3 ± 0.5 × 106 cells/left ventricle, n = 15) and
high percentage (83.8 ± 2.5%, n = 12) of
rod-shaped myocytes that were suitable not only for acute functional
studies but, more importantly, for short-term culture and gene transfer
(see Highly efficient adenovirus-directed gene
transfer). Figure 2 shows a
transillumination image of freshly isolated cardiomyocytes from the
left ventricle of a wild-type C57B16/J mouse. Most of these myocytes
were quiescent at physiological concentrations of extracellular
Ca2+ (1-2 mM) and, when electrically stimulated at 0.5 Hz at room temperature, generated a characteristic negative staircase
of contraction (data not shown), followed by a stable steady-state contraction (3.23 ± 0.21% of diastolic cell length,
n = 31). Similar results were obtained for myocytes
from adult mice (2-4 mo old) of other strains, including 129SvJ,
DBA/2, and FVB/N.
|
Culture of isolated mouse myocytes. We initially adopted previously reported culture methods for mouse (13) or rat (21) cells but observed that most adult mouse myocytes lost their rod shape and contractility, and even died after 24 h in culture. To solve this problem, we systematically screened several culture media [MEM, DMEM, or medium 199 (M199)] with or without supplements (insulin, creatine, L-carnitine, and taurine), employed various Ca2+ concentrations in the culture medium (0-1.8 mM), and explored different combinations of these maneuvers. After many rounds of trial and error, we found that the specific MEM (Sigma 1018) with a physiological concentration of Ca2+ (1.2 mM) produced the best cultured adult mouse myocytes. In addition, to minimize the dedifferentiation of the cultured adult myocytes, the FBS was withdrawn as soon as the myocytes attached to the culture dish (~1 h). Contamination with fibroblasts was negligible after the freshly isolated myocytes were further purified with BSA- and FBS-containing solutions. The growth of fibroblasts was imperceptible during the short-term culture with FBS-free MEM.
On average, 72.5 ± 2.3% of the mouse ventricular myocytes (n = 13) remained rod shaped and quiescent after 24 h of culture (Fig. 2, right); i.e., the loss of viable myocytes was ~13.2 ± 1.9% (n = 7) during the first 24 h. Because 1 × 106 mouse ventricular myocytes produced 1.5 ± 0.2 mg of total protein (n = 6) under our experimental conditions, the number of viable myocytes (4.6-4.8 × 105) from a single mouse left ventricle after 1 day in culture would be adequate for most biochemical and molecular biological assays, including cAMP measurement, receptor radioligand binding assay, or Northern or Southern blot analysis (data not shown). During a prolonged culture period (2-7 days), the rod-shaped myocytes were gradually lost at a rate of ~15% each additional day.Morphology and function of the cultured mouse cardiomyocytes.
Cultured adult mouse cardiomyocytes were observed for morphological
changes for 7 days. Figure 3 shows
typical images of both myocytes and their sarcolemmal membrane system
(including surface membrane and T tubules) taken from freshly isolated
cardiac myocytes (day 0) and myocytes cultured for different
times (days 2 and 7). Most myocytes retained
their rod-like appearance, clear striation, and well-organized T-tubule
system during the initial 2 days in culture (Fig. 3,
middle), but the cell edges became slightly rounded and the
cell length gradually shortened. After 24 h in culture, the
resting cell length was decreased by ~15%, i.e., from 129 ± 3.3 (n = 31) to 108 ± 4.5 µm (n = 26, P < 0.05), in agreement with previous
observations in cultured myocytes of other species (15).
Furthermore, the T tubules gradually recessed after culture for 2 days.
On day 7, the remaining myocytes had lost their striation and had become shrunken and rounded up, with either very few or no
discernible T tubules (Fig. 3, right).
|
|
-AR,
or Gi protein-coupled muscarinic receptors. Direct
activation of adenylyl cyclase by forskolin (10
6 M)
induced a robust increase in the ICa amplitude,
which could be completely blocked by 50 µM Cd2+, a
Ca2+-channel blocker (Fig.
5A). This result indicates
that cAMP signaling, one of the most important intracellular second
message systems, remained effective in regulating the downstream
effectors. Furthermore, Fig. 5B shows that in a
representative cultured cell, stimulation of -AR by isoproterenol
(Iso; 5 × 108 M) markedly increased the contraction
amplitude, and subsequent activation of muscarinic receptors by
carbachol (5 × 106 M) largely reversed this effect.
Taken together, these results suggest that the major components of
Gs and Gi protein-coupled receptor signaling
systems remain intact in myocytes cultured for 1 day in MEM.
|
Highly efficient adenovirus-directed gene transfer.
We next explored the feasibility of replication-deficient adenoviral
vector-mediated gene transfer in adult mouse myocytes isolated and
cultured using our method. As an example, expression of a marker gene,
-Gal, was conducted by infecting myocytes with Adeno--Gal. The
infection efficiency was determined by histochemical staining for
-Gal activity at 24 h after infection with Adeno--Gal. As
shown by the -Gal-driven color reaction (Fig.
6), 53% and 94% of myocytes were
positively stained at MOI 1 and 10, respectively (results of 2 experiments). The expression level varied from cell to cell at these
low MOI, as reflected in the nonuniform staining. However, both the
percentage and uniformity of -Gal expression improved at increased
MOI; uniform expression in all myocytes was achieved at MOI >30 (Fig.
6). These results indicate that adenovirus-directed gene expression in
adult mouse cardiomyocytes is highly efficient, a finding comparable
with that in rats or rabbits (4, 9-11).
In addition, the infection with adenovirus did not significantly affect
cell viability. The percentage of rod-shaped and quiescent myocytes was
still 69.8 ± 4.1% (n = 6, P > 0.05 vs. control) of the total cells after 24 h of culture with
Adeno--Gal at MOI 100.
|
Rescuing the phenotype of cardiomyocytes isolated from
1/2-AR knockout mice.
To further illustrate the potential application of our methods to
molecular and genetic physiology, we opted to characterize and rescue,
at the single-cell level, the phenotype of a
1/2-AR double-knockout (DKO) model,
recently developed in Brian Kobilka's laboratory (19). In
ventricular myocytes isolated from the DKO mice, Iso (106
M), a nonselective -AR agonist, had no effect on either cell contractility or ICa (Fig.
7), even though the downstream
cyclase-cAMP pathway was intact, as evidenced by a robust
ICa response to forskolin (106 M).
These single-cell results are thus consistent with genotype and
phenotype data obtained in vivo (19), indicating that
1- and 2-AR are the predominant -AR
subtypes in the mouse heart.
|
-AR signaling in the DKO myocytes remains
intact, reexpression of -AR by the adenoviral gene transfection system should be able to restore the -AR agonist-mediated cardiac response. Indeed, in myocytes infected with Adeno-1-AR
for 24 h at MOI 100, both the contractile and
ICa responses to 1-AR stimulation
by norepinephrine (NE, 107 M) plus an 
1-AR
antagonist, prazosin (106 M) were fully restored (Fig.
7). Similarly, infection with Adeno-2-AR at MOI 100 restored the contraction and ICa responses to
2-AR stimulation by zinterol (105 M) in
DKO myocytes (Fig. 7). Thus expression of either -AR subtype via
adenoviral infection in DKO myocytes rescued -AR function and, more
importantly, created a genetically pure 1- or
2-AR biological system that would be ideal for future
studies of the functional roles and signaling mechanisms of specific
-AR subtype in cardiac myocytes.
In summary, we have developed practical and reliable methods to isolate
and culture adult mouse cardiomyocytes from wild-type or genetically
engineered mice and to implement acute gene expression in these
myocytes via adenoviral infection. With its greatly improved yield and
cell quality, our modified isolation method provides an essential tool
for physiological and biochemical phenotyping of transgenic or
gene-targeted murine models at the cellular or subcellular level.
Moreover, the feasibility of our adult mouse myocyte culture and gene
transfer techniques should greatly facilitate the studies of molecular
and cellular functions of important proteins using acute genetic
manipulations. The combination of genetically engineered murine models
with the adenoviral gene-transfer technique should further amplify the
power of these complementary genetic approaches and create a myriad of
novel and comprehensive experimental paradigms to rescue phenotypes of
animal models or to target multiple functionally related proteins.
| |
ACKNOWLEDGEMENTS |
|---|
We sincerely appreciate the excellent technical assistance of Drs. Sheng-Jun Zhang and Harold Spurgeon. We also thank Drs. Annarosa Leri and Piero Anversa at New York Medical College for helpful discussions on cell culture and are grateful to Dr. Robert Lefkowitz for providing the adenoviral vectors.
| |
FOOTNOTES |
|---|
Present address of Y.-Y. Zhou: New York Univ. Medical Center, Molecular and Cellular Research Laboratory, Tisch Hospital (TH-501), 560 First Ave., New York, NY 10016.
Address for reprint requests and other correspondence: R.-P. Xiao, Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224 (E-mail: xiaor{at}grc.nia.nih.gov).
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. §1734 solely to indicate this fact.
Received 21 October 1999; accepted in final form 4 January 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
1.
Adachi-Akahane, S,
Lu L,
Li Z,
Frank JS,
Philipson KD,
and
Morad M.
Calcium signaling in transgenic mice overexpressing cardiac Na+-Ca2+ exchanger.
J Gen Physiol
109:
717-729,
1997
2.
Berger, HJ,
Prasad SK,
Davidoff AJ,
Pimental D,
Ellingsen O,
Marsh JD,
Smith TW,
and
Kelly RA.
Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture.
Am J Physiol Heart Circ Physiol
266:
H341-H349,
1994
3.
Benndorf, K,
Boldt W,
and
Nilius B.
Sodium current in single myocardial mouse cells.
Pflügers Arch
404:
190-196,
1985[Web of Science][Medline].
4.
Drazner, MH,
Peppel KC,
Dyer S,
Grant AO,
Koch WJ,
and
Lefkowitz RJ.
Potentiation of -adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.
J Clin Invest
99:
288-296,
1997[Web of Science][Medline].
5.
Field, LJ.
Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice.
Science
239:
1029-1033,
1988
6.
Gwathmey, JK,
Hajjar RJ,
and
Solaro RJ.
Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium.
Circ Res
69:
1280-1292,
1991
7.
Izumo, S,
and
Shioi T.
Cardiac transgenic and gene-targeted mice as models of cardiac hypertrophy and failure: a problem of (new) riches.
J Card Fail
4:
263-270,
1998[Medline].
8.
Kadambi, VJ,
and
Kranias EG.
Genetically engineered mice: model systems for left ventricular failure.
J Card Fail
4:
349-361,
1998[Medline].
9.
Kass-Eisler, A,
Falck-Pedersen E,
Alvira M,
Rivera J,
Buttrick PM,
Wittenberg BA,
Cipriani L,
and
Leinwand LA.
Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.
Proc Natl Acad Sci USA
90:
11498-11502,
1993
10.
Kirshenbaum, LA,
MacLellan WR,
Mazur W,
French BA,
and
Schneider MD.
Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.
J Clin Invest
92:
381-387,
1993.
11.
Kohout, TA,
O'Brian JJ,
Gaa ST,
Lederer WJ,
and
Rogers TB.
Novel adenovirus component system that transfects cultured cardiac cells with high efficiency.
Circ Res
78:
971-977,
1996
12.
Korzick, DH,
Xiao R-P,
Ziman BD,
Koch WJ,
Lefkowitz RJ,
and
Lakatta EG.
Transgenic manipulation of -adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine.
Am J Physiol Heart Circ Physiol
272:
H590-H596,
1997
13.
Kruppenbacher, JP,
May T,
Eggers HJ,
and
Piper HM.
Cardiomyocytes of adult mice in long-term culture.
Naturwissenschaften
80:
132-134,
1993[Web of Science][Medline].
14.
Masaki, H,
Sato Y,
Luo W,
Kranias EG,
and
Yatani A.
Phospholamban deficiency alters inactivation kinetics of L-type Ca2+ channels in mouse ventricular myocytes.
Am J Physiol Heart Circ Physiol
272:
H606-H612,
1997
15.
Mitcheson, JS,
Hancox JC,
and
Levi AJ.
Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture.
Pflügers Arch
431:
814-827,
1996[Web of Science][Medline].
16.
Mitcheson, JS,
Hancox JC,
and
Levi AJ.
Cultured adult cardiac myocytes: future applications, culture methods, morphological and electrophysiological properties.
Cardiovasc Res
39:
280-300,
1998
17.
Mitra, R,
and
Morad M.
A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates.
Am J Physiol Heart Circ Physiol
249:
H1056-H1060,
1985.
18.
Powell, T.
The isolation and characterization of calcium-tolerant myocytes.
Basic Res Cardiol
80, Suppl2:
15-18,
1985.
19.
Rohrer, DK,
Chruscinski A,
Schauble EH,
Bernstein D,
and
Kobilka BK.
Cardiovascular and metabolic alterations in mice lacking both 1- and 2-adrenergic receptors.
J Biol Chem
274:
16701-16708,
1999
20.
Santana, LF,
Kranias EG,
and
Lederer WJ.
Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocytes.
J Physiol (Lond)
503:
21-29,
1997
21.
Volz, A,
Piper HM,
Siegmund B,
and
Schwartz P.
Longevity of adult ventricular rat heart muscle cells in serum-free primary culture.
J Mol Cell Cardiol
23:
161-173,
1991[Web of Science][Medline].
22.
Wolska, BM,
and
Solaro RJ.
Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.
Am J Physiol Heart Circ Physiol
271:
H1250-H1255,
1996
23.
Xiao, R-P,
Avdonin P,
Zhou Y-Y,
Cheng H,
Akhter SA,
Eschenhagen T,
Lefkowitz RJ,
Koch WJ,
and
Lakatta EG.
Coupling of 2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.
Circ Res
84:
43-52,
1999
24.
Zhou, Y-Y,
Cheng H,
Song LS,
Wang D,
Lakatta EG,
and
Xiao R-P.
Spontaneous 2-adrenergic signaling fails to modulate L-type Ca2+ current in mouse ventricular myocytes.
Mol Pharmacol
56:
485-493,
1999
This article has been cited by other articles:
|
|
 |
|
  G. A. Fuller-Bicer, G. Varadi, S. E. Koch, M. Ishii, I. Bodi, N. Kadeer, J. N. Muth, G. Mikala, N. N. Petrashevskaya, M. A. Jordan, et al. Targeted disruption of the voltage-dependent calcium channel {alpha}2/{delta}-1-subunit Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H117 - H124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Bell, D. Lloyd, C. L. Curl, L. M. D. Delbridge, and M. J. Shattock Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume decrease and is this influenced by deletion of PLM? Exp Physiol, March 1, 2009; 94(3): 330 - 343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. De Arcangelis, D. Soto, and Y. Xiang Phosphodiesterase 4 and Phosphatase 2A Differentially Regulate cAMP/Protein Kinase A Signaling for Cardiac Myocyte Contraction under Stimulation of {beta}1 Adrenergic Receptor Mol. Pharmacol., November 1, 2008; 74(5): 1453 - 1462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger Designing Heart Performance by Gene Transfer Physiol Rev, October 1, 2008; 88(4): 1567 - 1651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Song, X.-Q. Zhang, J. Wang, E. Cheskis, T. O. Chan, A. M. Feldman, A. L. Tucker, and J. Y. Cheung Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1615 - H1625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Mizukami, K. Ono, C.-K. Du, T. Aki, N. Hatano, Y. Okamoto, Y. Ikeda, H. Ito, K. Hamano, and S. Morimoto Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion Cardiovasc Res, September 1, 2008; 79(4): 589 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. W. Raake, L. E. Vinge, E. Gao, M. Boucher, G. Rengo, X. Chen, B. R. DeGeorge Jr, S. Matkovich, S. R. Houser, P. Most, et al. G Protein-Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure Circ. Res., August 15, 2008; 103(4): 413 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Siddiqi, N. Gude, T. Hosoda, J. Muraski, M. Rubio, G. Emmanuel, J. Fransioli, S. Vitale, C. Parolin, D. D'Amario, et al. Myocardial Induction of Nucleostemin in Response to Postnatal Growth and Pathological Challenge Circ. Res., July 3, 2008; 103(1): 89 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Suleman, S. Somers, R. Smith, L. H. Opie, and S. C. Lecour Dual activation of STAT-3 and Akt is required during the trigger phase of ischaemic preconditioning Cardiovasc Res, July 1, 2008; 79(1): 127 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Y. Chung, M. Kang, and J. W. Walker Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2391 - H2399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Kabaeva, M. Zhao, and D. E. Michele Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1667 - H1674. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. S. O'Connell, J. D. Whitesell, and M. M. Tamkun Localization and mobility of the delayed-rectifer K+ channel Kv2.1 in adult cardiomyocytes Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H229 - H237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, N. Honbo, E. J. Goetzl, K. Chatterjee, J. S. Karliner, and M. O. Gray Signals from type 1 sphingosine 1-phosphate receptors enhance adult mouse cardiac myocyte survival during hypoxia Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3150 - H3158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Llamas, S. Belanger, S. Picard, and C. F. Deschepper Cardiac mass and cardiomyocyte size are governed by different genetic loci on either autosomes or chromosome Y in recombinant inbred mice Physiol Genomics, October 19, 2007; 31(2): 176 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. Means, C.-Y. Xiao, Z. Li, T. Zhang, J. H. Omens, I. Ishii, J. Chun, and J. H. Brown Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2944 - H2951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pavlovic, W. Fuller, and M. J. Shattock The intracellular region of FXYD1 is sufficient to regulate cardiac Na/K ATPase FASEB J, May 1, 2007; 21(7): 1539 - 1546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Clerk, T. J. Kemp, G. Zoumpoulidou, and P. H. Sugden Cardiac myocyte gene expression profiling during H2O2-induced apoptosis Physiol Genomics, April 24, 2007; 29(2): 118 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Tao, J. Zhang, D. A. Vessey, N. Honbo, and J. S. Karliner Deletion of the Sphingosine Kinase-1 gene influences cell fate during hypoxia and glucose deprivation in adult mouse cardiomyocytes Cardiovasc Res, April 1, 2007; 74(1): 56 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Tucker, J. Song, X.-Q. Zhang, J. Wang, B. A. Ahlers, L. L. Carl, J. P. Mounsey, J. R. Moorman, L. I. Rothblum, and J. Y. Cheung Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2199 - H2209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Thuerauf, M. Marcinko, N. Gude, M. Rubio, M. A. Sussman, and C. C. Glembotski Activation of the Unfolded Protein Response in Infarcted Mouse Heart and Hypoxic Cultured Cardiac Myocytes Circ. Res., August 4, 2006; 99(3): 275 - 282. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Takahashi, T.-S. Li, R. Suzuki, T. Kobayashi, H. Ito, Y. Ikeda, M. Matsuzaki, and K. Hamano Cytokines produced by bone marrow cells can contribute to functional improvement of the infarcted heart by protecting cardiomyocytes from ischemic injury Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H886 - H893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, E. A. Oestreich, N. Maekawa, T. A. Bullard, K. L. Vikstrom, R. T. Dirksen, G. G. Kelley, B. C. Blaxall, and A. V. Smrcka Phospholipase C {epsilon} Modulates {beta}-Adrenergic Receptor- Dependent Cardiac Contraction and Inhibits Cardiac Hypertrophy Circ. Res., December 9, 2005; 97(12): 1305 - 1313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Tuteja, D. Xu, V. Timofeyev, L. Lu, D. Sharma, Z. Zhang, Y. Xu, L. Nie, A. E Vazquez, J. N. Young, et al. Differential expression of small-conductance Ca2+-activated K+ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2714 - H2723. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rubio, I. Bodi, G. A. Fuller-Bicer, H. S. Hahn, M. Periasamy, and A. Schwartz Sarcoplasmic Reticulum Adenosine Triphosphatase Overexpression in the L-type Ca2+ Channel Mouse Results in Cardiomyopathy and Ca2+-Induced Arrhythmogenesis Journal of Cardiovascular Pharmacology and Therapeutics, October 1, 2005; 10(4): 235 - 249. [Abstract] [PDF]
|
|
|
|
|
|
T. Dieterle, M. Meyer, Y. Gu, D. D. Belke, E. Swanson, M. Iwatate, J. Hollander, K. L. Peterson, J. Ross Jr., and W. H. Dillmann Gene transfer of a phospholamban-targeted antibody improves calcium handling and cardiac function in heart failure Cardiovasc Res, September 1, 2005; 67(4): 678 - 688. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-Z. Zhu, K. Chakir, S. Zhang, D. Yang, C. Lavoie, M. Bouvier, T. E. Hebert, E. G. Lakatta, H. Cheng, and R.-P. Xiao Heterodimerization of {beta}1- and {beta}2-Adrenergic Receptor Subtypes Optimizes {beta}-Adrenergic Modulation of Cardiac Contractility Circ. Res., August 5, 2005; 97(3): 244 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Warrier, A. E. Belevych, M. Ruse, R. L. Eckert, M. Zaccolo, T. Pozzan, and R. D. Harvey {beta}-Adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor Am J Physiol Cell Physiol, August 1, 2005; 289(2): C455 - C461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wang, W. Zhu, S. Wang, D. Yang, M. T. Crow, R.-P. Xiao, and H. Cheng Sustained {beta}1-Adrenergic Stimulation Modulates Cardiac Contractility by Ca2+/Calmodulin Kinase Signaling Pathway Circ. Res., October 15, 2004; 95(8): 798 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Smith, N. Suleman, L. Lacerda, L. H. Opie, S. Akira, K. R. Chien, and M. N. Sack Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning Cardiovasc Res, September 1, 2004; 63(4): 611 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chen, Z. Zsengeller, C.-Y. Xiao, and C. Szabo Mitochondrial-to-nuclear translocation of apoptosis-inducing factor in cardiac myocytes during oxidant stress: potential role of poly(ADP-ribose) polymerase-1 Cardiovasc Res, September 1, 2004; 63(4): 682 - 688. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Liu and P. A. Hofmann Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2204 - H2212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. Rossow, E. Minami, E. G. Chase, C. E. Murry, and L.F. Santana NFATc3-Induced Reductions in Voltage-Gated K+ Currents After Myocardial Infarction Circ. Res., May 28, 2004; 94(10): 1340 - 1350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Potapova, A. Plotnikov, Z. Lu, P. Danilo Jr, V. Valiunas, J. Qu, S. Doronin, J. Zuckerman, I. N. Shlapakova, J. Gao, et al. Human Mesenchymal Stem Cells as a Gene Delivery System to Create Cardiac Pacemakers Circ. Res., April 16, 2004; 94(7): 952 - 959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Valiunas, S. Doronin, L. Valiuniene, I. Potapova, J. Zuckerman, B. Walcott, R. B. Robinson, M. R. Rosen, P. R. Brink, and I. S. Cohen Human mesenchymal stem cells make cardiac connexins and form functional gap junctions J. Physiol., March 15, 2004; 555(3): 617 - 626. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sasaki, M. Murata, Y. Guo, S.-H. Jo, A. Ohler, M. Akao, B. O'Rourke, R.-P. Xiao, R. Bolli, and E. Marban MCC-134, a Single Pharmacophore, Opens Surface ATP-Sensitive Potassium Channels, Blocks Mitochondrial ATP-Sensitive Potassium Channels, and Suppresses Preconditioning Circulation, March 4, 2003; 107(8): 1183 - 1188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-S. Song, A. Guia, J. N. Muth, M. Rubio, S.-Q. Wang, R.-P. Xiao, I. R. Josephson, E. G. Lakatta, A. Schwartz, and H. Cheng Ca2+ Signaling in Cardiac Myocytes Overexpressing the {alpha}1 Subunit of L-Type Ca2+ Channel Circ. Res., February 8, 2002; 90(2): 174 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shizukuda, M. E. Reyland, and P. M. Buttrick Protein kinase C-delta modulates apoptosis induced by hyperglycemia in adult ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1625 - H1634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-S. Song, A. Guia, J. N. Muth, M. Rubio, S.-Q. Wang, R.-P. Xiao, I. R. Josephson, E. G. Lakatta, A. Schwartz, and H. Cheng Ca2+ Signaling in Cardiac Myocytes Overexpressing the {alpha}1 Subunit of L-Type Ca2+ Channel Circ. Res., February 8, 2002; 90(2): 174 - 181. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
