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(The FASEB Journal. 2001;15:1886-1894.)
© 2001 FASEB

A novel cardioprotective role of RhoA: new signaling mechanism for adenosine

JANG EUN LEE, GARY BOKOCH* and BRUCE T. LIANG

Department of Medicine, Cardiovascular Division, and Department of Pharmacology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, 19104, USA; and
* Department of Immunology and Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA

1Correspondence: University of Pennsylvania Medical Center, Room 956, BRBII/III, 421 Curie BLVD, Philadelphia, PA 19104, USA. E-mail: liangb{at}mail.med.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine exerts a potent cardioprotective effect that is mediated by adenosine A1 and A3 receptors. The signaling pathways activated by the A1 and A3 receptors are distinct and involve selective coupling to phospholipases C and D, respectively. The objective of our study was to elucidate the signaling mechanism that mediates the coupling of each receptor to its respective phospholipase and to test the role of RhoA as a novel mediator leading from adenosine receptors to cardioprotection. C3 transferase and dominant negative RhoA (RhoAT19N) blocked the A3 receptor-mediated phospholipase D activation and cardioprotection but had no effect on A1 receptor-mediated phospholipase C activation or cardioprotection. In contrast, pertussis toxin treatment caused a greater inhibition of the diacylglycerol accumulation induced by the A1 agonist than by the A3 agonist, and it completely abrogated the A1 agonist-mediated cardioprotection. Thus, adenosine A1 and A3 receptors are linked to different G-proteins. The A3 receptor is coupled via RhoA to activate phospholipase D in exerting its cardioprotective effect, whereas the A1 receptor is linked via Gi to phospholipase C to produce cardioprotective responses. The present study identifies a novel role for RhoA and further suggests its importance in regulating cardiac cellular function.—Lee, J. E., Bokoch, G., and Liang, B. T. A novel cardioprotective role of RhoA: new signaling mechanism for adenosine.


Key Words: myocytes • ischemia • cytoprotection • phospholipase D • monomeric G-protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADENOSINE IS AN important regulatory agent that exerts potent protection against ischemia-induced cardiac myocyte injury in an autocrine and paracrine manner (1) . Furthermore, adenosine released during a brief ischemic episode is able to protect the heart against injury during a subsequent period of prolonged ischemia, resulting in a reduction in infarct size (2 3 4 5 6 7 8) . Brief exposure of the heart to adenosine, instead of to ischemia, can also induce a protective effect against subsequent ischemia-induced damage. This effect of adenosine, known as the preconditioning effect, has been the subject of much investigative interest (1 2 3 4 5 6 7 8 9 10) . Although activation of either the A1 or the A3 subtype of the adenosine receptor can mimic the cardioprotective effect of ischemic preconditioning, emerging evidence suggests that the two receptors mediate distinct cardioprotective functions (9 , 10) . Using a cardiac cell model for simulated ischemia and cardioprotection, our recent study showed that the two receptors are coupled via phospholipase C (PLC) and phospholipase D (PLD), respectively, to achieve their distinct protective effects (10) . The study provides the initial evidence for different signaling pathways that mediate the separate protective functions of the two receptors. However, the mechanism by which each receptor is coupled to its respective phospholipase remains unknown.

The objective here was to elucidate the signaling mechanism that mediates the coupling of each receptor to its respective phospholipase and to test the role of RhoA as a novel mediator in this signaling pathway. This study used a characterized cardiac cell model for cardioprotection (5 , 9 , 11 , 12) , which exhibits adenosine-induced protective effects similar to those apparent in the intact heart (4 , 6 7 8 , 13 , 14) . Specifically, the study was aimed at testing the hypothesis that the A3 receptor, but not the A1 receptor, signals via the low molecular weight GTPase RhoA to activate PLD in causing its cardioprotective effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of cultured cardiac ventricular myocytes and simulation of the cardioprotective effect of ischemic preconditioning
Atrial and ventricular myocytes were cultured from chick embryos 14 days in ovo as previously described (5 , 11) . Cells were maintained in culture and used on day 3, at which time they showed spontaneous beating. In simulating ischemia and ischemic preconditioning, the medium was changed to a glucose-free HEPES-buffered medium containing 139 mM NaCl, 4.7 mM KCl, 0.5 mM MgCl2, 0.9 mM CaCl2, 5 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and 2% FBS, pH 7.4, at 37°C. Ischemia was then simulated by incubating the cells in a hypoxic incubator (NuAire) for 90 min, during which time O2 was replaced by N2 as described previously (5 , 11) . Determination of myocyte injury was made at the end of the simulated ischemia, when aliquots of the media were collected for creatine kinase (CK) activity measurement. This was followed by quantitating the number of viable cells. The extent of myocyte injury was determined as the percentage of cells killed and as the amount of CK released. CK was measured as enzyme activity (U/mg), and increases in CK activity above the control level were determined. The percentage of cells killed was calculated as the number of cells obtained from the control group (representing cells not subjected to any hypoxia or drug treatment) minus the number of cells from the treatment group divided by the number of cells in the control group multiplied by 100%.

The preconditioning effect of the adenosine receptor agonist was determined by exposing the cells to agonist for 5 min, followed by their incubation in agonist-free medium before exposure to simulated ischemia for 90 min. Myocytes not subjected to preconditioning were exposed to 90-min ischemia only (nonpreconditioned cells). Uncoupling of Gi from the adenosine A1 and A3 receptor by pertussis toxin (PTX) was carried out according to the previously described method (15 , 16) . Prior treatment of the myocytes with pertussis toxin at 5 ng/ml for 18 h abolished the inhibition of adenylate cyclase activity by A1 receptor agonist (16) and by A3 receptor agonist (data not shown) and blocked the 32P-labeled ADP-ribosylation of Gi by [32P]NAD + in membranes of toxin-treated myocytes (16) .

Measurement of PLC and PLD activity
For measurement of diacylglycerol (DAG) and phosphatidylethanol (PEt), myocytes labeled with [3H]myristate for 24 h were exposed to receptor agonist. Quantitation of DAG and PEt was carried out as described previously (10 , 17 , 18) . For measurement of PLD activity, myocytes labeled with [3H]myristate for 24 h were exposed to receptor agonist in the presence of 0.5% (v/v) ethanol. The formation of [3H]PEt was an indication of the PLD activity. In brief, cultured myocytes labeled with [3H]myristate (49 Ci/mmol, 2 µCi/ml) for 24 h were exposed to receptor agonist. Lipids were extracted by the method of Bligh and Dyer (19) . The formation of [3H]DAG or [3H]PEt in cells was quantitated by separation of the labeled DAG or PEt from other phospholipids on thin-layer chromatography (petroleum ether/diethyl ether/acetic acid, 70:30:1, v/v/v for DAG separation; chloroform/methanol/H2O, 65:25:3, v/v/v for PEt) and scintillation counting of the 3H label in spots migrating to the same position as unlabeled DAG or Pet (20). The position of the phospholipids was determined visually by placing the thin-layer plate in an iodine chamber, and their levels were expressed as a percentage of total lipids. Data were expressed as the percent increase in the amount of DAG or PEt relative to that for unstimulated cells.

The PLC activity was determined as the increase in the total inositol phosphates after receptor agonist stimulation according to the previously described method (10 , 21) . The sum of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate represented the total inositol phosphates. Unless otherwise indicated, data were expressed as the percent increase in the amount of inositol phosphates relative to that for unstimulated cells.

Gene transfer into cardiac myocytes
Cardiac ventricular myocytes were transfected with pcDNA3 or with the recombinant pEF or pRK5M vector by using a modified calcium phosphate precipitate method (22) . cDNAs encoding the dominant negative mutants (N19RhoA or RhoAT19N, N17Cdc42 or Cdc42T17N) or the constitutively activated mutants (RhoAQ63L, Cdc42Q61L) of small GTPases were subcloned into the eukaryotic expression vector pRK5M (RhoA and Cdc42) in frame with the myc epitope tag (EQKLISEEDL). The C3 transferase cDNA subcloned in pEF vector containing the myc epitope was kindly provided by Dr. A. Hall (23) . Cardiac myocytes were maintained in culture for 24 h prior to being exposed to the calcium phosphate/DNA precipitates for 6 h at 37°C. Media were replaced with fresh growth media, and the myocytes were cultured for an additional 48 h. The expression of GTPase and C3 transferase cDNAs was determined by Western blotting of membranes from transfected myocytes with mouse monoclonal antibodies against c-Myc (9E10, Santa Cruz Biotechnoloy, Santa Cruz, CA). HeLa cell extracts, which express a high level of myc, served as the positive control (24) . Cell lysates of transfected myocytes were prepared, and equal amounts of proteins loaded in each lane of an 11% sodium dodecyl sulfate-polyacrylamide electrophoresis gel (SDS-PAGE) following solubilization in Laemmli buffer. Proteins were transferred onto nitrocellulose membranes. Membranes were blocked in PBS and 0.5% Tween 20 containing 5% condensed milk and were then incubated with monoclonal mouse antibodies against c-Myc in the same blocking buffer. After several washes, membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz). Enhanced chemiluminescence was then measured by using the ECL-Plus detection system (Amersham, Arlington Heights, IL). Ponceau staining of the nitrocellulose membrane was carried out to confirm equal protein loading.

In other experiments, atrial cardiac myocytes cultured from embryonic chick hearts 14 days in ovo were cotransfected with pcDNA3 plus pcDNA/hA3R (human adenosine A3 receptor cDNA subcloned in pcDNA3) or with pcDNA3/hA3R plus pRK5M/N19RhoA. The human adenosine A3 receptor cDNA was kindly provided by M. Atkinson, A. Townsend-Nicholson, and P. R. Schofield of the Garvan Institute for Medical Research, Sydney, Australia.

Materials
The adenosine analogue 2-chloro-N6-cyclopentyladenosine (CCPA) was from Research Biochemicals International (Natick, MA). Cl-IB-MECA was obtained from the National Institute of Mental Health Chemical Synthesis and Drug Supply Program (kindly supplied by Dr. Linda S. Brady). DAG and PEt were from Avanti Polar Lipids (Alabaster, AL). PTX was from List Biologicals (Campbell, CA). The vector pcDNA3 was obtained from Invitrogen (Carlsbad, CA). [3H]Myristate and myo-[3H]inositol were from New England Nuclear (Boston, MA). Embryonated chick eggs were from Spafas (Storrs, CT).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PTX-sensitive coupling of the adenosine A1 but not the A3 receptor to a cardioprotective response
Although both A1 and A3 receptors are coupled to a potent cardioprotective response, the signaling mechanism downstream of the two receptors remains incompletely understood. Both receptors are coupled to inhibition of adenylate cyclase via the PTX-sensitive Gi (5) . Uncoupling of Gi from the receptor by prior treatment of the myocyte with PTX completely abrogated the A1 receptor agonist-mediated cardioprotection (P<0.001, paired t test) (Fig. 1 ). However, the same PTX treatment had only a small inhibitory effect on the cardioprotection induced by the A3 receptor agonist Cl-IB-MECA (Fig. 1) . The A3 agonist-induced extent of cardioprotection was determined as the percent reduction in the number of cells injured during the ischemia. In the absence of prior PTX treatment, the A3 agonist caused a 62 ± 3% decrease in the ischemia-induced cell death (n=6, ±SE). After the toxin treatment, the A3 agonist still caused a significant 47 ± 2% decrease in cell death during the ischemia (n=5), although the extent of decrease in toxin-treated cells was less than that obtained in the absence of the PTX treatment (P<0.05, paired t test). Thus, although the A3 receptor can mediate a similar protection, the A3 effect is relatively insensitive to inhibition by PTX.



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  		Figure 1. Differential effects of PTX on A1 vs. A3 receptor agonist-stimulated cardioprotection. Cardiac ventricular myocytes were cultured from chick embryos after 14 days in ovo, and the ability of the A1 receptor agonist CCPA or the A3 receptor agonist Cl-IB-MECA to induce cardioprotection was determined by a preconditioning protocol as described in Materials and Methods. Briefly, myocytes without pretreatment or pretreated with PTX (5 ng/ml x 18 h) were incubated in the presence of 10 nM CCPA or Cl-IB-MECA for 5 min and were exposed to agonist-free medium for 10 min and then to simulated ischemia for 90 min. The extent of myocyte injury was quantitated at the end of the 90-min period by determining the percentage of cardiac cells killed and the amount of CK released. Data are means ± SE of values from five experiments. *P < 0.001 vs. the corresponding value for agonist-stimulated myocytes that had not been preincubated with PTX (paired t test).

C3 transferase selectively inhibited the A3 receptor-mediated cardioprotection
The possibility that a small GTPase is involved in mediating the cardioprotective response to A3 receptor agonist was examined next. Transfection of the cardiac myocyte with cDNA encoding the C3 transferase markedly inhibited the Cl-IB-MECA-mediated cardioprotective response, as evidenced by an increased percentage of cells killed and a greater amount of CK released even in response to the same concentrations of Cl-IB-MECA (P<0.01, paired t test) (Fig. 2 ). However, expression of the C3 transferase had no effect on the CCPA-induced cardioprotective response (Fig. 2) . In response to 10 nM CCPA, the percentage of cells killed and the amount of CK released were similar whether the myocytes were transfected with the C3 transferase cDNA or pcDNA3 (P>0.1, paired t test). The data provide further evidence for the hypothesis that the two receptors signal differently to achieve their cardioprotective response and for the possibility that the A3 receptor signals via a small GTPase to cause protection.



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  		Figure 2. Effects of C3 transferase on the A1 vs. A3 receptor agonist-mediated cardioprotection: the enzyme selectively inhibited the A3 agonist-mediated cardioprotection. Ventricular myocytes were cultured as described in the legend to Fig. 1 and were mock-transfected with an empty vector pcDNA3 or were transfected with the full-length C3 transferase (pEF/C3T). Forty-eight hours after transfection, the ability of CCPA or Cl-IB-MECA (10 nM) to induce preconditioning was determined as described in the legend to Fig. 1 . *P < 0.01 vs. the corresponding value for each agonist in mock-transfected myocytes (paired t test).

The small GTPase RhoA coupled the A3 receptor to the cardioprotective response
Because C3 transferase inactivates RhoA (23 , 25 , 26) , data summarized in Fig. 2 are consistent with the hypothesis that RhoA couples the A3 receptor to its cardioprotective response. To test this directly, myocytes were transfected with the dominant negative RhoA (RhoAT19N), and the ability of Cl-IB-MECA to cause cardioprotection was determined. Overexpressing RhoAT19N blocked the Cl-IB-MECA-mediated decrease in the percentage of cardiac cells killed (Fig. 3 ). In response to the same Cl-IB-MECA concentration (10 nM), the percentage of cells killed and the amount of CK released were significantly greater in myocytes transfected with RhoAT19N than in myocytes transfected with pcDNA3 (P<0.01, paired t test). However, transfection of the same myocyte cultures had no effect on the CCPA-induced cardioprotection. In the presence of 10 nM CCPA, the percentage of cells killed and the amount of CK released were similar whether the myocytes were transfected with RhoAT19N or pcDNA3 (P>0.1, paired t test). Thus, RhoA is coupled selectively to the adenosine A3 receptor.



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  		Figure 3. Dominant negative RhoA (RhoAT19N) selectively inhibited the A3 agonist-mediated cardioprotection. Ventricular myocytes were cultured as described in the legend to Fig. 1 and were mock-transfected with an empty vector pcDNA3 or transfected with the dominant negative RhoA (pRK5M/N19RhoA). Forty-eight hours after transfection, the ability of CCPA or Cl-IB-MECA (10 nM) to induce preconditioning was determined as described in the legend to Fig. 1 . Data are means ± SE of values from five experiments. *P < 0.01 vs. the corresponding value for each agonist in mock-transfected myocytes (paired t test).

If RhoA is involved in coupling the A3 receptor to its cardioprotective response, overexpressing the constitutively active mutant of RhoA, RhoAQ63L, should mimic the protective effect of A3 receptor agonist. The present data show that this is indeed the case. Overexpressing RhoAQ63L caused a marked protective effect in the absence of any agonist exposure. Compared with transfection with pcDNA3, transfection with RhoAQ63L resulted in fewer cells killed and less CK released during the prolonged simulated ischemia. The percentage of cells killed and the amount of CK released were 27.9 ± 2.2% and 32 ± 2.8 U/mg (n=6) in pcDNA3-transfected myocytes, respectively, whereas myocytes overexpressing the RhoAQ63L showed 15.7 ± 1.4% cells killed and 19 ± 3 U/mg of CK released after the prolonged ischemia (P<0.01, paired t test).

To further show the specificity of adenosine A3 receptor-RhoA coupling, the effect of overexpressing another small GTPase, Cdc42, was determined. Transfection with either the dominant negative Cdc42 (Cdc42T17N) or the constitutively active Cdc42Q61L had no effect on the ischemia-induced myocyte injury. The extent of injury incurred during the prolonged ischemia was similar whether myocytes were transfected with pcDNA3 (26±1.3% cells killed and 23.5±2.5 U/mg of CK released, n=5), with Cdc42T17N (25.4±2.3% and 25±3.9 U/mg, n=5), or with Cdc42Q61L (26.7±2.2% and 24±4 U/mg, n=5) (one-way ANOVA and post-test comparison, P>0.1). Overexpressing Cdc42 T17N or Cdc42Q61L also had no effect on the A3 agonist-stimulated DAG accumulation or cardioprotective response (data not shown). The expression of the myc-tagged mutants of RhoA (Fig. 4 ) and Cdc42 and of C3 transferase cDNA (not shown) was confirmed by Western blotting of membranes prepared from transfected myocytes.



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  		Figure 4. Expression of RhoA immunoreactivity in lysates of myocytes transfected with RhoA T19N cDNA. After solubilization of vector-transfected (CON) and RhoAT19N cDNA-transfected (RhoA N19) cell lysates, proteins were separated via SDS-PAGE, electrotransfered onto a nitrocellulose membrane, and probed with antibodies against the coexpressed epitope c-myc. The presence of RhoA mutant was detected by using enhanced chemiluminescence as described in Materials and Methods. The autoradiograph was typical of four similar experiments.

Specificity of RhoA in coupling the adenosine A3 receptor to DAG accumulation
The A1 and A3 receptors are selectively coupled to phospholipases C and D respectively, which in turn act as downstream effectors to mediate the cardioprotective response by each receptor (10) . Because RhoA couples the A3 receptor to its cardioprotective effect, the question arises regarding whether RhoA is an intermediate signaling molecule between the A3 receptor and PLD. Furthermore, the role of RhoA and Gi in coupling the A1 receptor to PLC needs to be determined. Figure 5 A shows that PTX treatment blocked most of the A1 agonist-stimulated DAG accumulation but only modestly inhibited the A3 agonist-stimulated DAG increase. PTX treatment caused a 70 ± 3% inhibition of CCPA-mediated DAG accumulation (n=6, ±SE), which was significantly greater than the percentage of inhibition of Cl-IB-MECA-induced DAG increase by the same toxin treatment (38±3%, n=6, P<0.05, t test). To further test the role of Gi in coupling of A1 receptor to PLC, the effect of PTX treatment on the CCPA-induced PLC activation was examined. CCPA stimulated PLC activity in cells not treated with PTX (percent increase relative to unstimulated cells was 30±2%, n=4, P<0.05, paired t test) but did not increase PLC activity after the toxin treatment (P>0.1, paired t test) (Fig. 5B ). These data are consistent with a role of Gi in coupling the A1 receptor to PLC activation and to the PLC-derived DAG accumulation.



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  		Figure 5. Differential effects of PTX on A1 vs. A3 agonist-stimulated DAG accumulation. Myocytes pretreated with or without PTX (5 ng/ml for 18 h) were labeled with either (A) [3H]myristate or (B) myo-[3H]inositol (21 Ci/mmol, 5 µCi/ml) for 24 h. A) Cells prelabeled with [3H]myristate were then exposed for 5 min to 30 nM CCPA or Cl-IB-MECA, after which the amount of DAG was measured. Data were expressed as the percent increase in the amount of DAG relative to that for unstimulated cells and are the means ± SE of values from five experiments. B) Cells prelabeled with myo-[3H]inositol were stimulated for 30 min with 30 nM CCPA. The production of total inositol phosphates was determined by the sum of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate as described (10) and served as a measure of PLC activity. Data are expressed as the total inositol phosphates in cpm and are the means ± SE of triplicates of an experiment that is representative of four others.

However, overexpressing the C3 transferase and RhoAT19N resulted in significant inhibition of the A3 agonist-stimulated, but not the A1 agonist-stimulated, DAG accumulation. Figure 6 shows that myocytes transfected with cDNA encoding the C3 transferase or the dominant negative RhoA showed little DAG response to Cl-IB-MECA. The percentage of stimulation by A3 agonist in C3 transferase- or RhoAT19N-transfected myocytes was significantly less than the extent of stimulation in pcDNA3-transfected myocytes (one-way ANOVA and post-test comparison, P<0.01). C3 transferase did not affect the A1 agonist-stimulated increase in DAG level. The percent increase in DAG in response to CCPA, relative to unstimulated cells, was 26.2 ± 3.5% (n=9) in pcDNA3-transfected cells, as compared with the 40±15% increase (n=5) in C3 transferase-transfected myocytes (P>0.1, paired t test). Consistent with a role of RhoA in coupling to PLD activation, RhoAQ63L-transfected myocytes showed an increased basal DAG level as compared with pcDNA3-transfected myocytes. The basal DAG level in RhoAQ63L-transfected myocytes was 41.3 ± 7% higher compared with the basal DAG level in pcDNA3-transfected myocytes (n=9, P<0.01, paired t test). Providing further evidence for a specific A3 receptor-PLD coupling, RhoAT19N also blocked the Cl-IB-MECA-mediated stimulation of PLD activity but had no effect on the ability of CCPA to stimulate PLC activity (data not shown).



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  		Figure 6. C3 transferase and RhoAT19N inhibited the A3 agonist-stimulated DAG accumulation. Cultured ventricular myocytes were mock-transfected with pcDNA3 or were transfected with cDNA encoding the C3 transferase or RhoAT19N. The ability of Cl-IB-MECA (30 nM) to stimulate DAG accumulation was determined as described in the legend to Fig. 4 . Data were expressed as the percent increase in the amount of DAG relative to that for unstimulated cells and are means ± SE of values from five experiments. *P < 0.01 vs. the corresponding value for the agonist effect obtained in C3 transferase- or RhoAT19N-transfected myocytes (one-way ANOVA and post-test comparison, P<0.01).

RhoA transduced the human adenosine A3 receptor-mediated cardioprotection
Previous studies showed that embryonic chick atrial cells lack native A3 receptor (9) . Transfection of these myocytes with the human adenosine A3 receptor cDNA resulted in the appearance of a potent cardioprotective response to A3 agonist (9 , 10) . To further examine the specificity of adenosine A3 receptor-RhoA coupling, atrial myocytes were cotransfected with both hA3R cDNA and RhoAT19N, and the ability of the A3 agonist to cause cardioprotection was determined. Figure 7 shows that the dominant negative RhoA inhibited the protection mediated via the human adenosine A3 receptor. After exposure to the same concentration of Cl-IB-MECA, the percentage of cells killed and the amount of CK released were significantly higher in cells cotransfected with RhoAT19N and hA3R than in cells transfected with hA3R alone (P<0.01, paired t test). Prior treatment of hA3R cDNA-transfected myocytes with PTX had no effect on the human A3 receptor-mediated protective effect. After exposure of cells to Cl-IB-MECA, the percentage of cells killed and the amount of CK released were similar in myocytes transfected with hA3R cDNA that were preincubated with the toxin as compared with those obtained in the same hA3R cDNA-transfected myocytes without prior toxin treatment (P>0.1, paired t test).



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  		Figure 7. Effect of RhoAT19N on the human adenosine A3 receptor-mediated cardioprotection: RhoA transduced this cardioprotective response. Cultured ventricular myocytes were transfected with pcDNA3/hA3R or with pRK5M/RhoAT19N plus pcDNA3/hA3R. The ability of Cl-IB-MECA (10 nM) to cause cardioprotection was determined as described in the legend to Fig. 1 . Data are means ± SE of values from five experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adenosine exerts a potent cardioprotective effect in the heart (1 2 3 4 5 6 7 8) . This protective response can be mediated by activation of the cell surface A1 receptor on the cardiac myocyte. Our previous studies showed the presence of a novel cardioprotective adenosine receptor, the A3 receptor, on the cardiac myocyte (5 , 9) . Activation of this receptor mediates a cardioprotective function distinct from that caused by activation of the adenosine A1 receptor. The A3 but not the A1 receptor signals selectively via PLD to induce its protective function, whereas PLC selectively couples the A1 receptor to its protective effect (10) . However, the signaling mechanisms that couple the two receptors to stimulation of their phospholipases are unknown. Growing evidence suggests a potentially important function of RhoA in regulating cellular functions (for reviews, see refs 18 , 27 28 29 30 31 ), including its emerging role in the cardiac myocyte (32 , 33) . The objective of the present study was to specifically test the role of RhoA as a novel mediator in the signaling pathway leading from adenosine receptors to their cardioprotective effects. Using a well-characterized cardiac myocyte model of adenosine-mediated cardioprotection and a myocyte transfection approach, the present study showed that the adenosine A3 receptor is selectively coupled to RhoA, which in turn acts upstream of PLD to mediate the cardioprotective response to A3 agonist. The A1 receptor, in contrast, is not coupled to RhoA. Instead, Gi links the A1 receptor to activation of the PLC and to induction of its cardioprotective effect.

RhoA and Rho kinase have been shown to link various membrane receptors to the assembly of actin-myosin filaments in both muscle and nonmuscle cells (27 , 28 , 32 33 34) . In smooth muscle cells, Rho kinase activation increases the phosphorylation of myosin light chain, which promotes actin-myosin interaction (35 , 36) . In neonatal rat ventricular myocytes, Rho and Rho kinase transduce the {alpha}1-adrenergic receptor-mediated atrial natriuretic factor expression and organization of actin myofibrils (32 , 33) . Overexpression of activated Rho in these cardiac myocytes resulted in a marked increase in the assembly of sarcomeres. Cardiac-specific overexpression of RhoA led to dysfunction of the sinus and atrioventricular nodes, followed by the development of ventricular dilation and failure (37) . These data provided the first evidence that RhoA may mediate an important regulatory function under pathophysiological conditions such as those leading to the development of atrial cardiac conduction and ventricular dysfunction. However, RhoA can also regulate a variety of biochemical pathways, including the transcriptional factors serum response factor and nuclear factor-{kappa}B, the enzymes phosphatidylinositol 3-kinase, PLD, c-jun N-terminal kinase, p38 mitogen-activated protein kinase, and the phagocytic NADPH oxidase complex, among a growing list of cellular functions (for review, see refs 18 , 27 28 29 30 31 ). Thus, RhoA may mediate other important regulatory functions in the cardiac myocyte.

The novel cardioprotective role of RhoA and the specificity of adenosine A3 receptor-RhoA coupling are supported by a number of lines of evidence. First, uncoupling of the A3 receptor from Gi by PTX treatment had little effect on the A3 agonist-stimulated DAG accumulation, whereas uncoupling of the A1 receptor-Gi linkage blocked the A1 agonist at the DAG level. Similarly, uncoupling from Gi had only a small effect on the A3 receptor-mediated cardioprotective response, whereas such uncoupling completely blocked the protection by the A1 receptor agonist. Thus, Gi, in contrast to its role in mediating the A1 effects, is less important in coupling the A3 receptor to its stimulatory effect at the DAG level or its cardioprotective response. Studies with the C3 transferase provided a second type of evidence. C3 transferase inactivates the small GTPase RhoA (23 , 25 , 26) , and its expression in the cardiac cells inhibited the A3 agonist-stimulated DAG increase as well as the A3 agonist-mediated cardioprotective effect. In contrast, C3 transferase had no effect on the ability of A1 agonist to stimulate DAG accumulation or to induce its cardioprotective response. These data implicate Rho in coupling the A3 but not the A1 receptor to cardioprotection as well as the accumulation of DAG that mediates such protective response.

To test the role of RhoA in the A3 receptor-PLD-cardioprotection linkage directly, the effects of dominant negative and constitutively active mutants of RhoA were determined. Transfection of the myocytes with RhoAT19N inhibited the A3 agonist-stimulated DAG accumulation. Similarly, RhoAT19N also blocked the A3 receptor-mediated cardioprotective response. The extent of injury determined in the presence of A3 agonist was similar to that determined in the absence of A3 agonist after RhoAT19N transfection. The nearly complete inhibition of the A3 receptor-mediated cardioprotection by RhoATN is somewhat surprising given that the transfection efficiency averages more than 50% (22) . Several explanations are possible. First, the calculation of transfection efficiency was based on quantitation of ß-galactosidase-positive cells in which only the dark blue cells were counted. The lightly stained cells were not quantitated. Thus, it is possible that the transfection efficiency is more than 50%. Second, RhoATN caused a marked inhibition of the A3 agonist-stimulated DAG accumulation. Because DAG may diffuse through the gap junction connecting the myocytes, a large decrease in the A3 agonist-mediated DAG accumulation in cells expressing the RhoATN may translate into a moderate decrease in the surrounding untransfected cells. This would then lead to a blockade of the A3 agonist-induced cardioprotection. Another possibility is that overexpressing RhoA may lead to release of a substance that can inhibit the protective effect of A3 agonist.

To provide further evidence for a role of RhoA in coupling the A3 receptor to PLD, RhoAQ63L was able to stimulate the basal DAG level and protect the myocytes against injury induced by the prolonged ischemia. RhoAT19N blocked the A3 effect on PLD activity but had no effect on the PLC activation by the A1 agonist. Transfection with the activated or inhibitory mutant of another small GTPase Cdc42 had no effect on the ischemia-induced myocyte injury, or on the A3 agonist-mediated DAG accumulation and cardioprotection. This finding further supports a specific role of RhoA in the A3 receptor-PLD-cardioprotection linkage.

Data from a final series of experiments on myocytes transfected with the human adenosine A3 receptor provided additional evidence for the link between the A3 receptor and RhoA. Atrial myocytes cultured from embryonic chick hearts, which lack native A3 receptor, were used to study the specificity of the adenosine A3 receptor-RhoA coupling. Previous studies showed that these myocytes can be efficiently transfected and that transfection with human A3 receptor cDNA resulted in the appearance of an A3 agonist-stimulated increase in DAG level, PLD activity, and a sustained cardioprotective response to A3 agonist (9 , 10) . Cotransfection of human A3 receptor cDNA with the dominant negative RhoA cDNA inhibited the A3 agonist-induced cardioprotective effect. The specificity of A3 receptor-RhoA coupling was further supported by the lack of a PTX effect on the human A3 receptor-mediated cardioprotection. Expression of the transgenes in the myocytes is supported by the detection of the myc epitope that is coexpressed with the C3 transferase and the small GTPases. The effects on the A3 response after transfection with C3 transferase, RhoAT19N, and RhoAQ63L were similar whether DAG level or myocyte injury was used as the functional end point. Such parallel effects of the transgenes provide more evidence for their expression as functional proteins.

Together, the data suggest that RhoA selectively transduces signals from adenosine A3 receptors to PLD activation and mediates a subsequent protective effect against ischemia (Fig. 8 ). The A1 receptor is linked via Gi to activate PLC to induce its cardioprotection. Activation of RhoA is capable of exerting potent protection against myocardial ischemia. The present study identifies a novel role for RhoA and further suggests its importance in regulating cardiac cellular function.



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  		Figure 8. Distinctive cardioprotective pathways via the adenosine A1 and A3 receptors. In this model, the A1 receptor is coupled via the PTX-sensitive Gi to stimulate PLC, whereas the A3 receptor is coupled via RhoA to activate PLD. The pathways then converge at the level of protein kinase C (PKC) to stimulate the mitochondrial KATP channel and cause cardioprotection.


   ACKNOWLEDGMENTS
 
This work was supported by an Established Investigatorship Award from the American Heart Association and a RO1 grant (HL48225) from the National Institutes of Health awarded to Dr. B. Liang.

Received for publication March 23, 2001. Revision received May 23, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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