The absence of myocardial calcium-independent phospholipase A2γ results in impaired prostaglandin E2 production and decreased survival in mice with acute Trypanosoma cruzi infection

Abstract

Cardiomyopathy is a serious complication of Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi. The parasite often infects cardiac myocytes, causing the release of inflammatory mediators, including eicosanoids. A recent study from our laboratory demonstrated that calcium-independent phospholipase A2γ (iPLA2γ) accounts for the majority of PLA2 activity in rabbit ventricular myocytes and is responsible for arachidonic acid (AA) and prostaglandin E2 (PGE2) release. Thus, we hypothesized that cardiac iPLA2γ contributes to eicosanoid production in T. cruzi infection. Inhibition of the isoform iPLA2γ or iPLA2β, with the R or S enantiomer of bromoenol lactone (BEL), respectively, demonstrated that iPLA2γ is the predominant isoform in immortalized mouse cardiac myocytes (HL-1 cells). Stimulation of HL-1 cells with thrombin, a serine protease associated with microthrombus formation in Chagas' disease and a known activator of iPLA2, increased AA and PGE2 release, accompanied by platelet-activating factor (PAF) production. Similarly, T. cruzi infection resulted in increased AA and PGE2 release over time that was inhibited by pretreatment with (R)-BEL. Further, T. cruzi-infected iPLA2γ-knockout (KO) mice had lower survival rates and increased tissue parasitism compared to wild-type (WT) mice, suggesting that iPLA2γ-KO mice were more susceptible to infection than WT mice. A significant increase in iPLA2 activity was observed in WT mice following infection, whereas iPLA2γ-KO mice showed no alteration in cardiac iPLA2 activity and produced less PGE2. In summary, these studies demonstrate that T. cruzi infection activates cardiac myocyte iPLA2γ, resulting in increased AA and PGE2 release, mediators that may be essential for host survival during acute infection. Thus, these studies suggest that iPLA2γ plays a cardioprotective role during the acute stage of Chagas' disease.

Fig 1

Inhibition of iPLA₂ activity in mouse cardiac myocytes treated with either (R)- or (S)-BEL (10-min pretreatment). Cardiac myocytes were incubated with BEL (10 min). PLA₂ activity was measured using 100 μM (16:0, [³H]18:1) plasmenylcholine in the presence of 4 mM EGTA. *, P < 0.05, and **, P < 0.01, compared to activity measured in the absence of BEL. The values shown are means ± SEM for 4 separate cell cultures.

Fig 2

(A) Increase in arachidonic acid release from T. cruzi (MOI, 0.2)-infected cardiac myocytes in the presence or absence of pretreatment with (R)- or (S)-BEL (2 μM; 10 min). **, P < 0.01 compared to unstimulated release, and ++, P < 0.01 when comparing results for T. cruzi-infected myocytes in the presence or absence of BEL. The values shown are means ± SEM for 4 separate cell cultures. (B) Increase in arachidonic acid release from thrombin-stimulated (0.1 IU/ml) mouse cardiac myocytes with or without pretreatment (10 min) with (R)- or (S)-BEL (2 μM). **, P < 0.01 compared to unstimulated release, and ++, P < 0.01 when comparing results for thrombin-stimulated myocytes in the presence or absence of (R)-BEL. The values shown are means ± SEM for 4 separate cell cultures.

Fig 3

(A) Increase in PGE₂ release from T. cruzi-infected (MOI, 0.2) mouse cardiac myocytes with or without pretreatment with (R)- or (S)-BEL (2 μM; 10 min). **, P < 0.01 compared to unstimulated release. and ++, P < 0.01 when comparing results for T. cruzi-infected myocytes in the presence or absence of (R)-BEL. The values shown are means ± SEM for 6 separate cell cultures. (B) Increase in PGE₂ release from thrombin-stimulated (0.1 IU/ml) mouse cardiac myocytes with or without pretreatment with (R)- or (S)-BEL (2 μM; 10 min). **, P < 0.01 compared to unstimulated release, and ++, P < 0.01 when comparing results for thrombin-stimulated myocytes in the presence or absence of (R)-BEL. The values shown are means ± SEM for 6 separate cell cultures.

Fig 4

(A) PAF production in mouse cardiac myocytes infected with T. cruzi (MOI, 0.2) with or without (R)- or (S)-BEL pretreatment (2 μM; 10 min). The values shown are means ± SEM for 6 separate cell cultures. (B) PAF production in mouse cardiac myocytes stimulated with thrombin (1 IU/ml) with or without pretreatment with (R)- or (S)-BEL (2 μM; 10 min). *, P < 0.05, and **, P < 0.01 compared with values for unstimulated myocytes, and ++, P < 0.01 when comparing values in the presence or absence of BEL. The values shown are means ± SEM for 8 separate cell cultures.

Fig 5

(A) RAW 264.7 cell adherence to mouse cardiac myocytes infected with T. cruzi (MOI, 0.2) with or without pretreatment with (R)- or (S)-BEL (2 μM; 10 min). Where indicated, RAW 264.7 cells were incubated with the PAF receptor antagonist CV3988 (10 μM; 10 min). (B) RAW 264.7 cell adherence to mouse cardiac myocytes stimulated with thrombin (1 IU/ml) with or without pretreatment with (R)- or (S)-BEL (2 μM; 10 min). Where indicated, RAW 264.7 cells were incubated with the PAF receptor antagonist CV3988 (10 μM; 10 min). *, P < 0.05 compared to unstimulated control; *, P < 0.05 compared to unstimulated RAW 264.7 cell adherence. The values shown are means ± SEM for 4 separate cell cultures.

Fig 6

Survival curve (%) for WT (n = 13) and iPLA₂γ-KO (n = 19) mice following infection with the Tulahuén strain of T. cruzi (5,000 BFT; subcutaneous injection).

Fig 7

(A) Parasitemia at day 14 in WT and iPLA₂γ-KO mice following infection with the Tulahuén strain of T. cruzi (5,000 BFT; subcutaneous injection). *, P < 0.05 compared to the WT. The values shown are means and SEM; n = 6. (B) T. cruzi molecular equivalents (TcME), quantified using T. cruzi-specific quantitative PCR (qPCR), from DNA isolated from the hearts of WT and iPLA₂γ-KO mice 14 days postinfection. *, P < 0.05 compared to the WT. The values shown are means and SEM; n = 6.

Fig 8

iPLA₂ activity in the hearts of WT and iPLA₂γ-KO mice 14 days postinfection with T. cruzi. Shown is PLA₂ activity, measured using 100 μM (16:0, [³H]18:1) plasmenylcholine in the presence of 4 mM EGTA. **, P < 0.01 compared to activity measured in the uninfected heart. The values shown are means and SEM; n = 6.

Fig 9

PGE₂ content in the hearts of WT and iPLA₂γ-KO mice 14 days postinfection with T. cruzi. +, P < 0.05, and ++, P < 0.01, compared to activity measured in the WT heart. The values shown are means and SEM; n = 6.