influx. Transgenic cardiac overexpression of the human P2X4 receptor showed an in vitro phenotype of enhanced basal contractility. The objective here was to determine the in vivo cardiac physiological role of this receptor. Specifically, we tested the hypothesis that this receptor plays an important role in modulating heart failure progression. Transgenic cardiac overexpression of canine calsequestrin (CSQ) showed hypertrophy, heart failure, and premature death. Crossing the P2X4R mouse with the CSQ mouse more than doubled the lifespan (182 Ϯ 91 days for the binary CSQ/P2X4R mouse, n ϭ 35) of the CSQ mouse (71.3 Ϯ 25.4 days, n ϭ 50, P Ͻ 0.0001). The prolonged survival in the binary CSQ/P2X4R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored ␤-adrenergic responsiveness. The beneficial phenotype of the binary mouse was not associated with any downregulation of the CSQ level but correlated with improved left ventricular developed pressure and ϮdP/dt. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2X 4 receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure. cardiac failure; contraction; isoproterenol; purines; adenine nucleotide ATP HAS LONG BEEN DEMONSTRATED to stimulate cardiac myocyte contractility at submicromolar concentrations (3,4,5,18,23). Recent studies show that activation of the P2X receptor subtype of the extracellular ATP receptor family increases the contractility of both cardiac myocytes and the intact heart (3,4,5,9,17,18,23). P2X receptors are ligand-gated ion channels whose activation results in sodium and calcium entry (6,10,12,19,24). Transgenic overexpression of the P2X 4 subtype of this family of receptor channels exhibits a phenotype of enhanced basal cardiac contractility and output in a working heart model (9). While these data suggest that the P2X receptor subfamily may mediate the effect of ATP on cardiac contractility, the potential cardiac physiological role of this receptor channel is not known. Because of the enhanced cardiac contractile performance of the P2X 4 receptor (P2X 4 R) transgenic mouse, it is possible that P2X 4 receptor overexpression will modulate the progression of heart failure. To test this hypothesis, the P2X 4 R mouse was crossed with the calsequestrin (CSQ) mouse model of cardiac hypertrophy and heart failure. The CSQ model of severe heart failure is generated by overexpressing the sarcoplasmic reticulum (SR) calcium-binding protein CSQ, which disrupts coordinated regulation of calcium release. The model showed a phenotype of hypertrophy that progress to dilated cardiomyopathy, failure, and premature cardiac death by 16 wk of age (2, 11). Here, we tested whether the cardiac P2X 4 R mediates a beneficial effect on the progression to heart failure in the CSQ-overexpressing mouse. We find that the binary CSQ/P2...

Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X 4 receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X 4 R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X 4 R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5-(␣,␤-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5 region of cP2X 4 R abrogated the P2 agonist-stimulated 45 Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X 4 receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X 4 receptor, antibody against cP2X 4 R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X 4 receptor. The nonglycosylated 44-kDa P2X 4 receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins with N-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X 4 R showed that only the glycosylated 58-kDa P2X 4 receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor's localization on the plasma membrane. These data revealed a novel physiologic function of the P2X 4 receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility.P2X receptors are nonselective cation channels gated by ATP (for reviews, see Refs. 1-3). Seven isoforms of P2X receptors identified so far show 35-50% sequence identities based on comparison of the homologous region. Each receptor has two transmembrane domains and a large extracellular loop that contains a number of putative N-glycosylation sites and an ATP binding site. Recent human genome data suggested three additional possible P2X receptors in the human genome, and no P2X counterparts were found in the yeast or Drosophila genome (4). Both N-and C-intracellular termini have been shown to be important in determining the rate of desensitization of the P2X receptor (1-3). Biochemical and elctrophysiological studies suggested homomeric and heteromeric structures, possibly trimer, as the functional form of P2X receptors (1-3, 5-7).In vitro and in vivo studies indicated that P2X receptors ar...

No abstract

No abstract

No abstract