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Cardiomyocyte death induced by ischaemic/hypoxic stress is differentially affected by distinct purinergic P2 receptors.

Cosentino S, Banfi C, Burbiel JC, Luo H, Tremoli E, Abbracchio MP - J. Cell. Mol. Med. (2012)

Bottom Line: Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown.In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone-associated-DNA-fragments, a marker of apoptosis.Both approaches indicated that the P2Y(2) and P2χ(7) receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y(4) receptor has a protective effect.

View Article: PubMed Central - PubMed

Affiliation: Centro Cardiologico Monzino IRCCS, Milan, Italy.

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Effect of P2Y2, P2Y4 and P2Y6 gene silencing on the induction of cardiomyocyte apoptosis after ischaemia/hypoxia. mRNA analysis of P2Y2 (A) and P2Y6 (B) by quantitative qRT-PCR in HL-1 cells transfected with either negative siRNA or with P2Y2 and P2Y6 siRNA. (C) Micrograph showing amplification of P2Y4 mRNA by standard RT-PCR in HL-1 cells transfected with either negative siRNA or with specific P2Y4 siRNA. Similar data have been obtained in three independent experiments analysis. (D–F) HL-1 cardiomyocytes transfected with either negative siRNA or with specific P2Y2, P2Y6, P2Y4 siRNA were exposed to normoxia or hypoxia. After 16 hrs, apoptosis was determined in hypoxic cells and set to 100%. Values are the mean ± S.E.M. from three individual experiments run in triplicate *P < 0.05 versus control siRNA-negative treated cells. H is the apoptosis value under hypoxic conditions after subtraction of the apoptosis value detected under normoxic conditions.
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fig05: Effect of P2Y2, P2Y4 and P2Y6 gene silencing on the induction of cardiomyocyte apoptosis after ischaemia/hypoxia. mRNA analysis of P2Y2 (A) and P2Y6 (B) by quantitative qRT-PCR in HL-1 cells transfected with either negative siRNA or with P2Y2 and P2Y6 siRNA. (C) Micrograph showing amplification of P2Y4 mRNA by standard RT-PCR in HL-1 cells transfected with either negative siRNA or with specific P2Y4 siRNA. Similar data have been obtained in three independent experiments analysis. (D–F) HL-1 cardiomyocytes transfected with either negative siRNA or with specific P2Y2, P2Y6, P2Y4 siRNA were exposed to normoxia or hypoxia. After 16 hrs, apoptosis was determined in hypoxic cells and set to 100%. Values are the mean ± S.E.M. from three individual experiments run in triplicate *P < 0.05 versus control siRNA-negative treated cells. H is the apoptosis value under hypoxic conditions after subtraction of the apoptosis value detected under normoxic conditions.

Mentions: To evaluate the role of P2Y receptor subtypes in the induction of apoptosis by the ischaemic/hypoxic stress, we used selective P2Y2 and P2Y6 antagonists. AR-C118925, the only known selective, heterocyclic antagonist of the P2Y2 receptor [27], significantly prevented the appearance of apoptosis by −50.8% ± 2 (Fig. 4). By contrast, the potent antagonist of P2Y6 nucleotide receptor diisothiocyanate derivative MRS2578 [28] did not affect hypoxia-induced apoptosis in HL-1 cardiomyocytes (Fig. 4). This pharmacological approach could not be adopted for the P2Y4 receptor, because no selective antagonists are currently available for this receptor subtype. Globally, these pharmacological data support a role for P2Y2, but not P2Y6, in ischaemic/hypoxic stress-induced apoptosis. To validate these results, we adopted a second approach based on the use of specific siRNAs against the various P2Y receptors. Analysis of P2Y2, and P2Y6 mRNAs after transfection of HL-1 cardiomyocytes with specific siRNAs showed a reduction of the corresponding receptor mRNAs of 70% ± 6.8 and 89% ± 3.6, for the P2Y2 and P2Y6 receptors, respectively (Fig. 5A and B). Semi-quantitative real-time analysis could not be performed for the P2Y4 receptor in silenced cells, due to the lack of efficacious oligonucleotide primers for this type of analysis [29]. However, standard RT-PCR in cardiomyocytes transfected with P2Y4 siRNA showed a dramatic reduction of P2Y4 amplification product at the expected 447 bp (Fig. 5C) with respect to cells transfected with corresponding negative siRNAs.


Cardiomyocyte death induced by ischaemic/hypoxic stress is differentially affected by distinct purinergic P2 receptors.

Cosentino S, Banfi C, Burbiel JC, Luo H, Tremoli E, Abbracchio MP - J. Cell. Mol. Med. (2012)

Effect of P2Y2, P2Y4 and P2Y6 gene silencing on the induction of cardiomyocyte apoptosis after ischaemia/hypoxia. mRNA analysis of P2Y2 (A) and P2Y6 (B) by quantitative qRT-PCR in HL-1 cells transfected with either negative siRNA or with P2Y2 and P2Y6 siRNA. (C) Micrograph showing amplification of P2Y4 mRNA by standard RT-PCR in HL-1 cells transfected with either negative siRNA or with specific P2Y4 siRNA. Similar data have been obtained in three independent experiments analysis. (D–F) HL-1 cardiomyocytes transfected with either negative siRNA or with specific P2Y2, P2Y6, P2Y4 siRNA were exposed to normoxia or hypoxia. After 16 hrs, apoptosis was determined in hypoxic cells and set to 100%. Values are the mean ± S.E.M. from three individual experiments run in triplicate *P < 0.05 versus control siRNA-negative treated cells. H is the apoptosis value under hypoxic conditions after subtraction of the apoptosis value detected under normoxic conditions.
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fig05: Effect of P2Y2, P2Y4 and P2Y6 gene silencing on the induction of cardiomyocyte apoptosis after ischaemia/hypoxia. mRNA analysis of P2Y2 (A) and P2Y6 (B) by quantitative qRT-PCR in HL-1 cells transfected with either negative siRNA or with P2Y2 and P2Y6 siRNA. (C) Micrograph showing amplification of P2Y4 mRNA by standard RT-PCR in HL-1 cells transfected with either negative siRNA or with specific P2Y4 siRNA. Similar data have been obtained in three independent experiments analysis. (D–F) HL-1 cardiomyocytes transfected with either negative siRNA or with specific P2Y2, P2Y6, P2Y4 siRNA were exposed to normoxia or hypoxia. After 16 hrs, apoptosis was determined in hypoxic cells and set to 100%. Values are the mean ± S.E.M. from three individual experiments run in triplicate *P < 0.05 versus control siRNA-negative treated cells. H is the apoptosis value under hypoxic conditions after subtraction of the apoptosis value detected under normoxic conditions.
Mentions: To evaluate the role of P2Y receptor subtypes in the induction of apoptosis by the ischaemic/hypoxic stress, we used selective P2Y2 and P2Y6 antagonists. AR-C118925, the only known selective, heterocyclic antagonist of the P2Y2 receptor [27], significantly prevented the appearance of apoptosis by −50.8% ± 2 (Fig. 4). By contrast, the potent antagonist of P2Y6 nucleotide receptor diisothiocyanate derivative MRS2578 [28] did not affect hypoxia-induced apoptosis in HL-1 cardiomyocytes (Fig. 4). This pharmacological approach could not be adopted for the P2Y4 receptor, because no selective antagonists are currently available for this receptor subtype. Globally, these pharmacological data support a role for P2Y2, but not P2Y6, in ischaemic/hypoxic stress-induced apoptosis. To validate these results, we adopted a second approach based on the use of specific siRNAs against the various P2Y receptors. Analysis of P2Y2, and P2Y6 mRNAs after transfection of HL-1 cardiomyocytes with specific siRNAs showed a reduction of the corresponding receptor mRNAs of 70% ± 6.8 and 89% ± 3.6, for the P2Y2 and P2Y6 receptors, respectively (Fig. 5A and B). Semi-quantitative real-time analysis could not be performed for the P2Y4 receptor in silenced cells, due to the lack of efficacious oligonucleotide primers for this type of analysis [29]. However, standard RT-PCR in cardiomyocytes transfected with P2Y4 siRNA showed a dramatic reduction of P2Y4 amplification product at the expected 447 bp (Fig. 5C) with respect to cells transfected with corresponding negative siRNAs.

Bottom Line: Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown.In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone-associated-DNA-fragments, a marker of apoptosis.Both approaches indicated that the P2Y(2) and P2χ(7) receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y(4) receptor has a protective effect.

View Article: PubMed Central - PubMed

Affiliation: Centro Cardiologico Monzino IRCCS, Milan, Italy.

Show MeSH
Related in: MedlinePlus