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Role of direct RhoA-phospholipase D1 interaction in mediating adenosine-induced protection from cardiac ischemia¹

SUSAN MOZZICATO^*, BHALCHANDRA V. JOSHI, KENNETH A. JACOBSON and BRUCE T. LIANG^*,2

^* Department of Cardiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA; and
Molecular Recognition Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA

²Correspondence: Department of Cardiology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA. E-mail: bliang{at}uchc.edu

SPECIFIC AIM

The aim of the study was to elucidate the mechanism of protection from myocardial ischemia induced by the adenosine A₃ receptor.

PRINCIPAL FINDINGS

The adenosine A₃ receptor mediates anti-ischemic function in the heart and appears to signal selectively via RhoA and phospholipase D (PLD) to exert its protective effect, different from the cardioprotective mechanism of the adenosine A₁ receptor. While a direct RhoA-PLD1 interaction is important in stimulating PLD1 activity, the physiological role of this direct interaction in the intact cell—the cardiac myocyte—is unclear. In an established cardiac myocyte model of preconditioning using cultured chick embryo heart cells, overexpression of the RhoA-noninteracting PLD1 mutant I870R inhibited A₃ agonist-induced PLD activation and selectively blocked A₃ agonist but not A₁ agonist-induced cardioprotection. Atrial cardiac myocytes were rendered null for native adenosine receptors by treatment with irreversible A₁ antagonist m-DITC-XAC and selectively transfected with human adenosine A₁ or A₃ receptor cDNA individually or cotransfected with one of the receptors and I870R. I870R preferentially inhibited human A₃ receptor-mediated protection from ischemia. The study elucidated a novel physiologic role for direct RhoA-PLD1 interaction in the intact cell—that of potent protection from cardiac ischemia—and supported the existence of divergent signaling pathways that lead to cardioprotection.

1. Differential inhibition of the A₃ vs. A₁ receptor-mediated protection from ischemia after overexpression of the RhoA-noninteracting PLD1
Overexpressing the RhoA-noninteracting mutant I870R caused significant attenuation of Cl-IBMECA-induced anti-ischemic effect. A₃ agonist-induced protection was illustrated by a decrease in percentage of cells killed (Fig. 1 A) or the amount of CK released (not shown). The percentage of cells killed and amount of CK released were significantly higher in I870R-transfected cells than in mock-transfected cells in the presence of the same Cl-IBMECA concentration (P<0.01). Since the A₃ receptor is coupled to stimulation of PLD activity, overexpression of RhoA-noninteracting PLD1 should block A₃ receptor agonist-induced PLD activation. Data summarized in Fig. 1B showed this was the case. It is not known whether the RhoA-noninteracting PLD1 mutant could also block the A₁ agonist-induced cardioprotection. Overexpressing the I870R mutant had only a slight inhibitory effect on the CCPA-induced anti-ischemic response vs. its effect on Cl-IB-MECA-mediated cardioprotection. I870R caused a higher percentage of cells killed in the presence of a maximally protective concentration of Cl-IBMECA than in the presence of a maximal CCPA concentration (P<0.05). Thus, the RhoA-noninteracting mutant induced greater inhibition of A₃ agonist-mediated protection than of the A₁ agonist-induced cardioprotection. RhoA-noninteracting PLD affects the two receptor pathways differentially, showing a selective blockade of the A₃ receptor signaling pathway.

View larger version (11K): [in this window] [in a new window]   Figure 1. Differential effects of the RhoA-noninteracting mutant I870R on A1 vs. A3 agonist-mediated cardioprotection. A) Cardiac ventricular myocytes were cultured from chick embryos after 14 days in ovo and the effect of I870R on A1 receptor agonist CCPA- or the A3 receptor agonist Cl-IB-MECA-mediated cardioprotection was determined. The extent of myocyte injury was quantitated after 90 min by determining the % of cardiac cells killed and the amount of CK released. Data are means ± SE of values from 5 experiments. *In I870R-transfected myocytes, % of cells killed was higher in Cl-IBMECA-treated than in CCPA-treated myocytes (1-way ANOVA and post-test comparison, P<0.05). B) Cardiac ventricular myocytes were prepared and the effect of I870R on Cl-IBMECA-stimulated PLD activity was determined. 48 h after transfection with I870R cDNA, the ability of Cl-IBMECA to stimulate PLD activation was determined. The amount of phosphatidylethanol (PEt) are expressed as a % of total lipids and are means ± SE of 6 experiments. *P < 0.01 compared with value in cells not stimulated with Cl-IBMECA in mock-transfected myocytes (paired t test).

2. RhoA-noninteracting PLD inhibited the human adenosine A₃ receptor-mediated anti-ischemic response
Embryonic chick atrial cells lack native A₃ receptors. Transfection of atrial myocytes with the human A₃ adenosine receptor cDNA decreased the percent of myocytes killed during simulated ischemia and resulted in acquisition of an A₃ agonist-mediated preconditioning response. Atrial myocytes were transfected with hA₃R cDNA or cotransfected with human A₃ receptor (hA₃R) cDNA and I870R, and the extent of myocyte injury determined. Figure 2 A shows that transfection of the atrial myocyte with hA₃R cDNA reduced the percentage of cells killed during the ischemia. After cotransfection with I870R and hA₃R cDNA, the same cultured atrial myocytes showed a higher percentage of cells killed than did cells transfected with the hA₃R cDNA alone (P<0.05).

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of I870R on human adenosine A1 or A3 receptor-mediated cardioprotection. A) Cultured cardiac atrial myocytes were transfected with pcDNA3/hA3R or pcCGN/I870R + pcDNA3/hA3R. The extent of ischemia-induced myocyte injury was determined as percent cardiac cells killed or as amount of CK released (data not shown). Data are means ± SE of values from 5 experiments. *P< 0.05 vs. the value obtained in mock-transfected myocytes or that obtained in myocytes transfected with pcDNA3/hA3R alone (ANOVA and post-test comparison). B) Cultured cardiac atrial myocytes were treated with the irreversible A1 receptor antagonist m-DITC-XAC and transfected with pcDNA3/hA1R to create recombinant myocytes expressing only the human adenosine A1 receptor. Atrial myocytes treated with m-DITC-XAC were transfected with pcCGN/I870R + pcDNA3/hA1R. The extent of ischemia-induced myocyte injury was determined as % of cardiac cells killed or amount of CK released (data not shown). Data are means ± SE of values from 5 experiments. *P < 0.05 vs. the value in myocytes transfected with pcDNA3/hA1R alone or that obtained in myocytes cotransfected with pCGN/I870R and pcDNA3/hA1R; % of cells killed in pcDNA3/hA1R-transfected myocytes was similar to that in pCGN/I870R/pcDNA3/hA1R-transfected myocytes (P>0.05) (ANOVA and post-test comparison).

The specificity of the A₃ receptor vs. the A₁ receptor coupling to the RhoA-dependent PLD1 was examined in recombinant atrial cardiac myocytes that were null for native adenosine receptors and engineered to express only the human A₁ receptor. Native adenosine A₁ receptors of the atrial myocytes were irreversibly inactivated by the alkylating A₁ receptor-selective antagonist M-DITC-XAC (MRS264), rendering such myocytes devoid of any endogenous adenosine receptor. Transfection of these adenosine receptor null myocytes with the human A₁ receptor cDNA resulted in a significant reduction in the percentage of cells killed (Fig. 2B ) and the amount of CK released (not shown), consistent with an anti-ischemic effect of the human A₁ receptor. After cotransfection with I870R and human A₁ receptor cDNA, the same atrial myocyte cultures showed no increase in the percentage of cells killed during ischemia over that obtained in myocytes transfected with human A₁ receptor cDNA (1-way ANOVA and post-test comparison, P>0.05). Thus, I870R had no inhibitory effect on the human A₁ receptor-mediated anti-ischemic response.

CONCLUSIONS AND SIGNIFICANCE

Activation of the cell surface adenosine receptor exerts a potent anti-ischemic effect in the heart. Two principal adenosine receptor subtypes, the A₁ and the A₃ receptors, have been shown to mediate this cardioprotective effect. Although both are activated by the common endogenous ligand adenosine, the underlying protective signaling mechanism for the two receptors appears to be different. That PLD is an important cardioprotective signaling molecule in the cardiac myocyte and a direct RhoA-PLD1 interaction is a critical mechanism that mediates the anti-ischemic effect of A₃ but not of A₁ receptors are supported by the following. First, the RhoA-noninteracting PLD1 mutant I870R, when overexpressed in the myocyte, caused a significant inhibition of A₃ receptor-mediated protection. Specificity of the direct RhoA-PLD1 interaction is shown only in the A₃ receptor-mediated signaling pathway. Second, overexpressing the RhoA-noninteracting PLD1 attenuated A₃ agonist-stimulated PLD activation in the intact cardiac myocyte. The inhibition of A₃ agonist-stimulated PLD activity paralleled that of the A₃ agonist-induced cardioprotective response. Third, overexpressing the RhoA-noninteracting PLD1 selectively inhibited the anti-ischemic effect induced by the human A₃ receptor. The I870R mutant did not inhibit the human A₁ receptor-mediated anti-ischemic effect. Finally, overexpressing the wild-type PLD1 protected the cardiac myocyte from ischemia-induced injury. The cardioprotective effect of PLD1 appears to be constitutive in that it is associated with increased basal PLD activity and the protective response is preserved even in the presence of maximal blockade at the A₁ or A₃ receptors (data not shown).

The present data suggest a novel physiological role for the direct RhoA-PLD1 interaction, that of potent protection of the cardiac myocyte from ischemia. The study further supported the existence of divergent cardioprotective signaling pathways and of different mechanisms mediating the anti-ischemic effect of adenosine A₁ and A₃ receptors.

View larger version (20K): [in this window] [in a new window]   Figure 3. A model of how adenosine causes protection from cardiac ischemia. Adenosine released from the heart during myocardial ischemia exerts potent cardioprotection. Although activation of the A1 and the A3 receptors can induce protection from ischemia, the signaling mechanism that mediates this protection appears to be different. A direct RhoA-PLD1 interaction selectively mediates the effect of adenosine A3 receptor on protein kinase C (PKC) activation. The A1 receptor does not appear to signal via such a direct RhoA-PLD1 interaction and achieves a less sustained activation of PKC (dotted line). PKC activation causes protection from ischemia.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0592fje;

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