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and -
isoforms
Department of Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preconditioning reduces
cardiomyocyte necrosis in vivo and in vitro, but it is unknown whether
preconditioning blocks apoptosis. We wanted to compare the
effects of preconditioning on necrosis and apoptosis in
cardiomyocytes. Necrosis was detected with propidium iodide, and
apoptosis was quantified by three complementary techniques: flow cytometry, TdT-mediated dUTP nick-end labeling assay, and DNA-laddering electrophoresis. Apoptosis increased with
simulated ischemia time (6 h, 19 ± 1%; 12 h,
27 ± 2%; 18 h, 40 ± 4%; 24 h, 54 ± 4%;
and 36 h, 83 ± 4%; n = 6 for each group).
Simulated ischemia and reoxygenation contributed equally to
apoptosis (12-h ischemia, 27 ± 2%,
n = 6; 12-h ischemia and 12-h reoxygenation, 51 ± 4%, n = 6; and 24-h ischemia,
54 ± 5%, n = 8). Necrosis occurred primarily
during reoxygenation; none was detected during simulated ischemia. Preconditioning with 10 min of simulated
ischemia reduced necrosis (18 ± 6%, n = 8) but had no effect on apoptosis. However, three 1-min cycles
of simulated ischemia separated by 5 min of reoxygenation
reduced necrosis and apoptosis similarly. The protein kinase C
(PKC) inhibitors Go6976 (0.1 µM) or chelerythrene (4 µM) abolished
the effect of preconditioning. Preconditioning selectively activated
PKC
but had no effect on PKC
and on total PKC enzyme activity.
Preconditioning protected against necrosis and apoptosis, but
the preconditioning ischemia required for blocking
apoptosis was less than that for reducing necrosis. Activation
of PKC isoform is important in mediating the protection.
hypoxia; cultured cardiomyocytes
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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APOPTOSIS, OR PROGRAMMED CELL DEATH, and necrosis are two different types of cell death. Apoptosis has been described in ischemic situations, including myocardial infarcts in animals and in humans (9, 14, 29) and is important in the pathogenesis of ischemia-reperfusion injury (5, 9, 14, 29). Apoptosis is a caspase-mediated proteolytic process, in which cell cleavage is deliberate and the immune response is not activated. While apoptotic cells shrink, the genome is progressively destroyed and cleaved into 180- to 200-bp fragments, giving rise to the typical "apoptotic DNA ladder" (20). It is unknown whether ischemia, reperfusion, or both are more critical to the development of cardiocyte apoptosis. Our objective was to examine the effects of various simulated ischemia and reoxygenation periods on the pathogenesis of cardiocyte apoptosis. Some in vivo studies (6, 24) found that preconditioning reduced apoptosis. We also wanted to determine whether preconditioning blocks apoptosis in isolated cultured cardiomyocytes.
The necrotic process elicits an inflammatory response, is detected mainly during reperfusion, and develops only minimally during ischemia (32, 33, 35). Preconditioning reduces myocardial necrosis in vivo and in vitro (12, 17, 18, 23).
Activation of protein kinase C (PKC) mediated ischemic
preconditioning to reduce necrosis in intact animals (16,
34) and in chick embryonic cardiomyocytes (17, 35).
Translocation of activated isoforms PKC and PKC has been detected
in preconditioned hearts (15, 25), and PKC activation was
responsible for preconditioning-attenuated apoptosis in vivo
(24). Gray et al. (10) were the first to develop a peptide to selectively antagonize PKC. Other studies have
further demonstrated the importance of PKC in preconditioning to
reduce necrosis both in vitro (10, 21, 36) and in vivo (13, 22). We wanted to see whether PKC and PKC would
be activated and whether apoptosis would be reduced with preconditioning.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiomyocyte preparation. Ventricular myocytes from 10-day-old chick embryos were prepared according to a method described by Barry et al. (2) and modified by Vanden Hoek et al. (32). Briefly, hearts were harvested and placed in Hanks' balanced salt solution lacking magnesium and calcium (Life Technologies; Grand Island, NY). Ventricles were minced, and myocytes were dissociated with the use of 4-6 repeats of trypsin degradation (0.025%, Life Technologies) at 37°C with gentle agitation. Isolated cells were then transferred to a solution with trypsin inhibitor for 8 min, filtered through a 100-µm mesh, centrifuged for 5 min at 1,200 rpm at 4°C, and finally resuspended in a medium described previously (32). Resuspended cells were placed in a petri dish in a humidified incubator (5% CO2-95% O2 at 37°C) for 45 min to promote early adherence of fibroblasts. Nonadherent cells were counted with a hemocytometer, and viability was measured with trypan blue (0.4%). Approximately 0.2 × 106 cells in nutritive medium were pipetted onto coverslips (25 mm) and incubated for 5-6 days, after which synchronous contractions of the monolayer were noted.
A simulated ischemia solution, composed of a buffered saline solution containing no glucose but with 2-deoxyglucose (20 mM) to inhibit glycolysis, was bubbled with a 20% CO2-80% N2 mixture for 0.5 h before the experiments. The preconditioning reoxygenation solution was the same as the culture medium with 6% serum (GIBCO-BRL). The reoxygenation solution used after prolonged simulated ischemia was RPMI-1640 (GIBCO-BRL) without serum. Cardiomyocytes were placed on dishes filled with simulated ischemia solution into a hypoxic chamber at 37°C, where the low oxygen pressure was confirmed with an oxygen probe (PO2 < 1%). The cells in reoxygenation media then were incubated at 37°C with 5% CO2. The pH of the perfusion solution was verified (RPMI-1640, 7.4 pH; simulated ischemic buffered saline solution, 6.8 pH).Necrosis assay. Fluorescent cell images were obtained with a ×10 objective lens. Data were acquired and analyzed with the use of Metamorph software. There were ~600 cardiomyocytes under the selected field for each experiment. Multiple fields were examined and compared before each study; the field with normal synchronous contraction was chosen and monitored throughout experiments. Cell viability was quantified with the nuclear stain propidium iodide (PI; 5 µM) (Molecular Probes; Eugene, OR), an exclusion fluorescent dye that binds to chromatin upon loss of membrane integrity. This method is similar in principle to trypan blue staining and has been reported (1) to predict the transition from reversible to irreversible cell injury in cultured cardiomyocytes. Isotonic PI is not toxic to cells over a course of 8 h, permitting its addition to the perfusate throughout the experiments. At the completion of each experiment, digitonin (300 µmol/l) was added to the perfusate for 1 h. Digitonin disrupted cell membrane integrity of all cells, thereby allowing PI to enter. Percent loss of viability (cell death) was then expressed relative to the maximum value after 1 h of digitonin exposure (100%).
Apoptosis assay.
After treatment, the cells were washed once with a phosphate-buffered
solution (pH 7.4) and digested by enzymes on a coverslip (0.5 mg/ml
collagenase type IA and 0.025% trypsin) (Sigma; St. Louis, MO) for
10-15 min at 37°C. The digestion was stopped with the
use of 10% serum. After the cells were centrifuged for 5 min at 1,300 rpm, they were resuspended in staining solution with 50 µg/ml PI
(Molecular Probes), 0.5% Triton X-100, and 0.1% sodium citrate. After
12 h, DNA fragmentation was quantified by flow cytometry. A
wavelength of 670 nm (FL3-H) was used to detect the fluorescence
intensity of PI with flowcytometry to quantify DNA fragmentation. In
Fig. 2, the y-axis is the number of cells counted (labled as
Counts). The x-axis is the DNA size and content for each
cell registered (labled as FL3-H). Cells with normal DNA will have
higher fluorescence intensity (the peak in Fig.
1). Apoptotic cells, which have more
fragmented DNA, will have lower fluorescence intensity. The M
region (left side of normal peak) is apoptotic. The number of cells
in the M region divided by the total cell count is expressed as the
percentage of apoptosis.
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80°C with a 2.5 volume of pure ethanol and 0.1 volume of 5 M NaCl. After being centrifuged at
12,000 rpm for 20 min, the pellet was washed once with 70% ethanol.
After the pellet was dried, the genome DNA was dissolved in a solution
(10 mM Tris · Cl and 1 mM EDTA, pH 8.0) and analyzed by
measuring the absorbance at a 260-nm ultraviolet wavelength. A
DNA sample of 0.5 µg was loaded to a 1.5% agarose gel containing 0.5 µg/ml of ethidium bromide. DNA electrophoretic patterns were visualized under ultraviolet light.
The TdT-mediated dUTP nick-end labeling (TUNEL) staining of myocytes on
coverslip was performed with the use of a TdT-Blue Label
apoptosis detection kit (Trevigen; Gaithersburg, MD). The enzyme TdT was used to incorporate biotinylated-conjugated dUTP to the
ends of DNA fragments. The TUNEL sites were then achieved using
streptavidin-horseradish peroxidase and Colorimetric Substrates TACS
Blue Label. The results were visualized with the use of a microscope.
PKC enzyme assay.
Enzyme activity of total PKC (and its - and -isoforms) was
measured by a method described previously (8, 25). For
each experiment, 5,000,000 cells were collected in sample buffer
composed of 50 mmol/l Tris · HCl, pH 7.5; 5 mmol/l EDTA; 10 mmol/l each EGTA and benzamidine; 50 µg/ml phenylmethylsulfonyl
fluoride; 10 µg/ml each of aprotinin, leupeptin, and pepstatin A; and
0.3% 
-mercaptoethanol (Sigma). The collection was centrifuged at
45,000 g for 30 min and separated into cytosol and
particulate fractions. The particulate pellet was dissolved
ultrasonically in sample buffer. Protein concentration was determined
according to the Bradford method (3). Each fraction,
50-100 µg, was assayed for activity of total PKC and its
isoforms (assay kit, Amersham Pharmacia; Piscataway, NJ). For PKC
and PKC assay, proteins were immunoprecipitated overnight by PKC
and PKC monoclonal antibody (BD Transduction Lab) in an
immunoprecipitation buffer (pH 7.4) composed of the following: 150 mmol/l NaCl, 50 mmol/l Tris, 1 mmol/l EGTA, 1 mmol/l EDTA, 1% NP-40, 1 mmol/l sodium orthovanadate, 1 mmol/l phenylmethylsulfonyl fluoride, 16 µg/ml benzamidine-HCl, and 10 µg/ml each for phenanthroline,
aprotinin, leupeptin, and pepstatin A (Sigma) with protein A/G beads
(Santa Cruz Biotechnology). PKC- or PKC-specific substrate
(ERMRPRKRQGSVRRRV) (BioMol; Plymouth Meeting, PA) was used for
phosphorylation reaction with [32P]ATP (Amersham Pharmacia).
Statistical analysis. Data are means ± SE. Differences between groups for cell death and enzyme activity were compared with the use of a two-factor analysis of variance with repeated measures and Fisher's least significant difference test. Differences between groups were considered significant if P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiocyte apoptosis increased with longer ischemic time compared with control (see Fig. 1). At the completion of 12 h of simulated ischemia, apoptosis was 27% (n = 6), which increased progressively with reoxygenation (3 h, 30 ± 2%, n = 7; 6 h, 37 ± 4%, n = 6; 12 h, 54 ± 4%, n = 5; and 24 h, 75 ± 3%, n = 6). Apoptosis was confirmed with the use of DNA laddering electrophoresis. TUNEL assay showed that apoptotic cells were cardiocytes.
After 12 h of simulated ischemia, apoptosis was at
27%, and, after 24 h, apoptosis was at 54% (Fig.
2). Percentage of apoptosis was
similar after 12 h of simulated ischemia, followed by
12 h of reoxygenation and 24 h of simulated ischemia
alone. Data were verified with DNA laddering and TUNEL assay. Thus
simulated ischemia and reoxygenation were equally important in
development of cardiocyte apoptosis.
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Preconditioning, elicited with three cycles of 1-min simulated
ischemia separated by 5-min reoxygenation before 12-h simulated ischemia and 12-h reoxygenation, reduced apoptosis
(28 ± 3%, n = 10, vs. 51 ± 3%,
n = 6) (Fig. 3). The
protection of preconditioning was abolished with treatment of the PKC
inhibitors Go-6876 (0.1 µM) or chelerythrine (4 µM) (Fig. 3).
Go-6876 or chelerythrine alone had no effect on apoptosis
compared with controls.
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Preconditioning, initiated with three cycles of simulated
ischemia separated by 5 min of reoxygenation, markedly
increased the enzyme activity of the PKC isoform in the particulate
fraction but had no effect on the enzyme activity of total PKC and
PKC isoform compared with controls. In the cytosol fraction, no
difference was observed in the enzyme activity of total PKC, PKC, or
PKC isoforms (Fig. 4).
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One hour after preconditioning, which was initiated with either three
cycles of 1 min of simulated ischemia separated by 5 min of
reoxygenation (1' × 3 preconditioning) or 10 min of simulated ischemia (10' preconditioning), markedly increased the enzyme activity of the PKC isoform in the particulate fraction. The enzyme
activity of the PKC isoform was still elevated 12 h after the
cardiocytes were preconditioned with three cycles of 1-min simulated
ischemia separated by 5 min of reoxygenation (Fig.
5). In the cytosol fraction, no
difference was observed in the enzyme activity of total PKC, PKC, or
PKC isoforms (data not shown).
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By using a necrosis model of simulated ischemia and
reoxygenation described in our previous studies (33, 35),
we found that simulated ischemia had no effect on necrosis, and
reoxygenation produced 50 ± 5% (n = 8) cell
necrosis (Fig. 6A).
Preconditioning protected against both necrosis and apoptosis,
but less ischemic time was needed for protection against
apoptosis. A 10-min simulated ischemia as
preconditioning reduced necrosis but failed to prevent apoptosis (see Fig. 6). Three cycles of 1-min simulated
ischemia attenuated cardiocyte apoptosis and necrosis
(Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In our study, ischemic preconditioning blocked cardiocyte
apoptosis via activation of the PKC isoform in an in vitro
model of simulated ischemia and reoxygenation.
Apoptosis, which is prominent in the border zone of an
ischemic area (9), is documented in acute human
myocardial infarction (26). Death of heart muscle irreversibly compromises cardiac function and correlates with overall
morbidity and mortality (4, 28). Because adult cardiocytes are postmitotic, damaged heart muscle cannot be regenerated through cell division. Blocking cardiocyte apoptosis and identifying
opportunities for intervention have significant clinical implications.
Discrepancy of simulated ischemia and reoxygenation-induced apoptosis and necrosis. Apoptotic cardiocytes increased as simulated ischemia was prolonged. After 12 h of simulated ischemia, apoptosis was at 27%, which increased progressively with longer reoxygenation. Interestingly, with 12 h of simulated ischemia, followed by 12 h of reoxygenation and 24 h of simulated ischemia, cardiocyte apoptosis was at 54%. Although we could not rule out the possibility that apoptotic process or genes were activated during 12 h of simulated ischemia and persisted during the reoxygenation period, these results suggest that simulated ischemia and reoxygenation are equally important in the pathogenesis of cardiocyte apoptosis. Others (6, 9) have reported apoptosis only during reperfusion after cardiomyocytes had been subjected to 45 min of ischemia. We found no significant apoptosis even when cardiocytes had been subjected to 1 h of simulated ischemia. In reports by Yao et al. (33), Zhang and Yao (35), and Vanden Hoek et al. (32), necrosis was primarily associated with reperfusion injury. Necrotic injury is attributed to oxidant stress at reperfusion (19, 31). Both necrosis and apoptosis develop during simulated ischemia and reoxygenation, but they are two distinct processes in the pathogenesis of injury. Our results suggest that therapy should be targeted at both necrosis and apoptosis.
Preconditioning attenuates necrosis and apoptosis. Ischemic preconditioning reduces necrosis in vivo (11-12) and in vitro (17, 33). Ten minutes of simulated ischemia was sufficient to precondition cardiomyocytes against necrosis (Fig. 6). This result was consistent with the reports by Zhang and Yao (35) and Liang (17). However, preconditioning with 10 min of simulated ischemia failed to prevent cardiocyte apoptosis. Yet a shorter preconditioning stimulus, three cycles of simulated ischemia for 1 min separated by 5 min of reoxygenation, attenuated apoptosis and reduced necrosis (Fig. 6). Necrosis and apoptosis are two distinct processes, both of which can be attenuated with preconditioning. The underlining mechanisms for blocking apoptosis and reducing necrosis, however, may be different.
Preconditioning attenuates apoptosis via activation of
PKC.
The PKC inhibitors Go-6976 or chelerythrine, added during simulated
ischemia and reoxygenation, abolished the effects of
preconditioning on apoptosis. Okamura and co-workers
(24) showed that ischemic preconditioning
attenuated apoptosis in intact rat hearts and that this
protection was abolished by inhibition of PKC. Several isoforms of PKC,
i.e., , , and 
, have been suggested as mediators of
preconditioning (15, 25). We found that preconditioning selectively activated the PKC isoform in the particulate fraction without changing its activity in cytosol. The activity of total PKC and
its -isoform in both compartments was not affected by preconditioning. In addition, preconditioning with three cycles of
1-min simulated ischemia separated by 5-min reoxygenation
increased the activity of PKC, which persisted for at least 12 h; however, the elevated enzyme activity with 10-min simulated
ischemia as preconditioning returns to baseline level within
12 h. Interestingly, these effects correlate with protection
against apoptosis. In isolated cardiomyocytes, preconditioning
attenuates apoptosis via activation of the PKC enzyme in the
particulate fraction.
plays a critical role in preconditioning to limit
necrosis in cultured neonatal rat ventricular myocytes (10) and in anesthetized rats (21, 22). Ping
et al. (25) showed that activation and translocation of
PKC isoform-mediated preconditioning to reduce cardiocyte necrosis
in conscious rabbits. PKC may exert its cardioprotection via
regulation of cardiac calcium channels (13).
PKC is important in attenuating both necrosis and apoptosis,
but the mechanism by which preconditioning activates the PKC isoform
is less clear. It is known that preconditioning generates oxygen
radicals (30). They activate PKC (7, 8),
mediating preconditioning to limit necrosis (17, 27), and
block apoptosis during simulated ischemia and reoxygenation.
In conclusion, ischemic preconditioning attenuates
apoptosis and necrosis via stimulation of the PKC isoform in
isolated cultured cardiomyocytes. Although preconditioning protects
against both necrosis and apoptosis, these two responses to
simulated ischemia and reoxygenation stress are distinct.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-03881-02.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Z. Yao, Dept. of Anesthesia and Critical Care, Univ. of Chicago, 5841 S. Maryland Ave., MC 4028, Chicago, IL 60637 (E-mail: zyao{at}aims.unc.edu).
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.
Received 13 October 2000; accepted in final form 22 March 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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X. Dong, J. Liu, H. Zheng, J. W. Glasford, W. Huang, Q. H. Chen, N. R. Harden, F. Li, A. M. Gerdes, and X. Wang In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1417 - H1425. [Abstract] [Full Text] [PDF]
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M. Mayr, Y.-L. Chung, U. Mayr, E. McGregor, H. Troy, G. Baier, M. Leitges, M. J. Dunn, J. R. Griffiths, and Q. Xu Loss of PKC-{delta} alters cardiac metabolism Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H937 - H945. [Abstract] [Full Text] [PDF]
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J. D. McCully, H. Wakiyama, Y.-J. Hsieh, M. Jones, and S. Levitsky Differential contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1923 - H1935. [Abstract] [Full Text] [PDF]
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D. Garcia-Dorado, M. Ruiz-Meana, F. Padilla, A. Rodriguez-Sinovas, and M. Mirabet Gap junction-mediated intercellular communication in ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 456 - 465. [Abstract] [Full Text] [PDF]
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H. Liu, H. Y. Zhang, X. Zhu, Z. Shao, and Z. Yao Preconditioning blocks cardiocyte apoptosis: role of KATP channels and PKC-epsilon Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1380 - H1386. [Abstract] [Full Text] [PDF]
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C. Song, T. M. Vondriska, G.-W. Wang, J. B. Klein, X. Cao, J. Zhang, Y. J. Kang, S. D'Souza, and P. Ping Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H1166 - H1171. [Abstract] [Full Text] [PDF]
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Z. Yang, R. J. Cerniway, A. M. Byford, S. S. Berr, B. A. French, and G. P. Matherne Cardiac overexpression of A1-adenosine receptor protects intact mice against myocardial infarction Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H949 - H955. [Abstract] [Full Text] [PDF]
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