Opposite effects of uracil and adenine nucleotides on the survival of murine cardiomyocytes

Abstract

We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X(2)), as well as the P2Y(2,4,6,14) subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-alpha, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect 'per se', but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-alpha, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-alpha contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be 'primed' by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible 'therapeutic' window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.

1

Murine HL-1 cardiomyocytes express P2X_1,3,4,5,6,7 and P2Y_2,4,6,14. The presence of P2 receptors was assessed using ad hoc designed RT-PCR primers for all cloned murine P2 receptors. For each receptor, amplification products in parallel with MW standards are reported. Various tissues or cell lines were analysed in parallel as a positive controls: trigeminal ganglia for P2X₂ and P2X₃, total brain for P2X₁, P2X₄, P2X₅, P2X₇, P2Y₁₃ and P2Y₁₄, heart for P2X₆, liver for P2Y₄, lung for P2Y₂ and N9 cells for P2Y₁, P2Y₆ and P2Y_12. No products were detected in samples that did not undergo retrotranscription (–).

2

Cardiomyocyte death induced by ATP or BzATP alone or in combination with TNF-α. After 24hrs treatment with either ATP (500 M) or the selective P2X-agonist BzATP (100 μM), cardiomyocytes were fixed, stained with the Hoechst 33258 dye and the number of adhering cells evaluated by fluorescence microscopy. Data are reported as % of control cell number (A). For evaluation of apoptosis and necrosis (B), cells were exposed to ATP or BzATP, alone or in combination with TNF-α (10ng/ml), as indicated. After 24 hrs, cells were collected and the percentage of apoptotic and necrotic cells measured by flow cytometric analysis of DNA fragmentation with Propidium Iodide. Data represent the mean ± S.E.M. of three independent experiments. *P<0.05 versus control, (P<0.05 versus TNF-α alone, and §P<0.05 versus ATP or BzATP alone, by one way ANOVA (Scheffe's F test). (C) Annexin V staining confirmed the induction of apoptotic cell death. Cells were exposed to ATP alone or in combination with TNF-α, as previously described. At the end of the incubation period, cells were collected, and stained with Annexin V to detect membrane signs of apoptotic death. Blank refers to unstained negative control cells, used to calibrate the instrument. The graph shows a representative experiments (percentages of Annexin V-positive cells: Control, 8.61%; ATP alone, 38.57%; ATP+TNF-α, 47.12%). Comparable results were obtained in four independent experiments.

3

Pharmacological characterization of adenine nucleotides-induced cardiomyocyte death. HL-1 cardiomyocytes were exposed to either ADP, ATPγS, 2MeSATP or 2MeSADP (all 500 μM) for 24 hrs, alone or in combination with TNF-α (10 ng/ml), as indicated (A), or to ATP (500 μM) and TNF-α (10 ng/ml), either alone or in combination with the P2 receptor antagonists RB2 or suramin, all utilized at a 100 μM concentration (B). For suramin, the original traces of a representative experiment are shown in C. The percentages of apoptotic and necrotic cells measured by flow cytometric analysis with hypotonic PI (A) or by Annexin V/isotonic PI (B) (see Materials and methods) are shown. In A and B, data represent the mean ± S.E.M.of three and four independent experiments, respectively.*P<0.05 versus control. In A, #P<0.05 versus TNF-α and §P<0.05 versus corresponding adenine nucleotide alone, and in B §P<0.05 versus ATP + TNF-α, by one way _ANOVA (Scheffe's F test).

4

Uracil nucleotides have no effect ‘per se’ on cardiomyocytic viability, but protect cells from ATP and ADP-induced cytotoxicity. To assess the effect of uracil nucleotides on HL-1 cardiomyocytic viability, cells were exposed to UTP, UDP or UDPglucose (500 μM) alone or in combination with TNF-α, and apoptosis and necrosis determined after 24 hrs by flow-cytometry. Parallel cultures were treated with ATP, here utilized as a positive control (A). The effect of TNF-α alone is shown in Figure 2B.*P <0.05 versus corresponding Control, by one-way _ANOVA (Scheffe's F test). To assess the ability of uracil nucleotides to counteract cardiomyocyte death induced by ATP+TNF-α, cells were treated with either vehicle (B), or ATP+TNF-α (500 μM and 10 ng/ml, respectively) in the absence (C) or presence of UDP (100 μM, D), UTP (25 μM) or UDPglucose (10 μM) (E). In these experiments, uracil nucleotides were added to cells 30 min before adenine nucleotides. After 24 hrs, micrographs of adhering cells were taken (scale bar: 50 μm) and flow cytometric analysis was performed on the total cell population. In cytograms, first and second numbers on the left represent the percentage of necrotic and apoptotic cells, respectively. Results from a typical experiment are shown in (B–E). Similar data were obtained in five independent experiments. UDP similarly protected cells from the apoptosis and necrosis induced by either ADP alone (500 μM) or ADP and TNF-α (10 ng/ml) (F). Annexin V staining of cells confirmed the complete protection exerted by uracil nucleotides. The percentages of Annexin V-positive cells in this representative experiment (out of three) were: Control, 8.1%; UDP alone, 8.4%; ATP+TNF-α, 56.4%; ATP+TNF-α+UDP, 11.3%. (G) *P <0.05 versus corresponding Control, §P<0.05 versus ADP alone, #P <0.05 versus ADP+TNF-α, by one-way _ANOVA (Scheffe's F test); the mean values ± S.E.M.of four independent experiments are shown.

5

Concentration dependence of ATP-induced cardiomyocytic death (apoptosis+necrosis) and of uracil nucleotides-mediated protection. HL-1 cardiomyocytes were exposed to graded concentrations of ATP (25–250 μM) alone or in combination with TNF-α (10 ng/ml, A). Cultures pre-treated with TNF-α and incubated with 250 μM ATP were exposed to graded (100 nM to 250 μM) concentrations of UTP (B), UDP (C), or UDPglucose (D). After 24 hrs, the percentage of cell death was evaluated by cytofluorimetric analysis of PI stained nuclei. Data represent the mean ± S.E.M.of three independent experiments. Curves were calculated using GRAPH PAD Prism 4.0 software. EC₅₀ values for induction of cell death by ATP or ATP+TNF-α and IC₅₀ values for uracil nucleotides protection are also shown in individual panels and summarized in Table 3.

6

Characterization of UDP-induced protection. HL-1 cardiomyocytes were exposed to either vehicle (Control, A), ATP (100 μM) and TNF-α (10 ng/ml) (B), as indicated, for 24 hrs. UDP (100 μM) was added to cultures either together with ATP (C) or 30 min after its addition (D). At the end of treatment, flow cytometric analysis was performed on the total cell population. In cytograms, first and second numbers on the left represent the percentage of necrotic and apoptotic cells, respectively.