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A novel contractile phenotype with cardiac transgenic expression of the human P2X₄ receptor ¹

Department of Medicine, Cardiovascular Division, and the Department of Pharmacology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA; and
^* Nora Eccles Harrison Professor of Cardiology, Department of Medicine, University of Utah Medical Center, Salt Lake City, Utah 84132, USA

²Correspondence: 956 BRBII/III, University of Pennsylvania Medical Center, 421 Curie Blvd., Philadelphia, PA 19104, USA. E-mail: liangb{at}mail.med.upenn.edu

SPECIFIC AIM

A novel P2X receptor, the P2X₄ receptor, is expressed in the heart cell. The aim of the study was to elucidate its function.

PRINCIPAL FINDINGS

ATP is released as a cotransmitter from the sympathetic nerve endings and may be available from platelets, endothelium, and possibly from hypoxic myocardium. Although the biological effects of ATP are mediated by its receptors, also known as the P2 purinergic receptor, the physiological role of ATP and its receptor are incompletely understood. The P2 purinergic receptor class contains two subfamilies: the G protein-coupled P2Y receptor and the ligand-gated ion channel P2X receptor. In the cardiac cell, ATP stimulates a large increase in cytosolic calcium transient and myocyte contractile amplitude without an enhanced sensitivity of the myofilament to calcium. In rat and chick cardiac cells, the P2X receptor-selective agonist 2-methylthioATP (2-meSATP) was able to stimulate myocyte contractile amplitude with little or no effect on the phospholipase C activity, and this increase in contractile amplitude occurred in the presence of phospholipase C inhibitor U-73122. These data suggest that a cardiac P2X receptor likely mediates the increase in myocyte contractility in response to ATP. The identity and function of this P2X receptor are suggested by the findings presented here.

1. Functional features of the contractile response mediated by the native cardiac myocyte P2 receptor
Features of the increased contractility mediated by the native P2 receptor in the cardiac cell were characterized. The objective was to compare the pharmacological features of the native cardiac P2 receptor with those of the cloned P2 receptors. Among the P2X receptors, the P2X₄ receptor appeared to be important in mediating the increased cardiac contractility by the P2 agonist. First, the 2-meSATP-stimulated increase in mouse cardiac cell contractile amplitude was insensitive to blockade by the P2 receptor antagonist suramin. The percent of increase in response to 2-meSATP was 42 ± 7% (±SE, n=33 cells from 10 mice) whereas the percent of increase in the presence of 2-meSATP and suramin was 59 ± 7% (n=12 cells from 7 mice). Similar data were obtained in the rat cardiac cell (not shown). Insensitivity of the native cardiac P2 response to the P2 receptor antagonist is similar to that of the cloned P2X₄ receptor-mediated response to the same antagonist. Second, the P2 receptor agonist ,ß-methylene ATP is a weak agonist at stimulating the contractile amplitude in both mouse (percent stimulation of 13±2%, n=14 myocytes from six mice) and rat cardiac cells, similar to its weak agonist activity at the cloned mouse or rat P2X₄ receptor. Third, P2X₄ receptors are expressed in isolated cardiac cells, as determined by immunoblotting of homogenates from mouse and rat heart cells (not shown).

2. Cardiac transgenic expression of the P2X₄ receptor: characterization of the -MyHC P2X₄ receptor transgenic mouse
To elucidate the role of ATP and its receptor in the heart, transgenic mouse with cardiac-specific overexpression of the human P2X₄ receptor was developed and both intact heart and isolated cardiac cell functional studies were carried out. The human P2X₄ receptor was targeted to the heart using the -myosin heavy chain (-MyHC) promoter. Transgenic mice were generated in the B6SJL/F1 background, and mice containing the transgene were identified by PCR using an upstream primer 5'-GTCGACTGACTAACTAGAAGCT-3' complementary to the -MyHC linker region of the -MyHC vector and a downstream primer 5'-CTGAGCTGGTATCACATAATCC-3' complementary to human P2X₄ receptor (position 288–310) (not shown). The presence of the transgene was confirmed by Southern blotting of the genomic DNA (not shown). Overexpression of the P2X₄ receptor in the transgenic mouse was demonstrated by immunohistochemistry of the isolated cardiac cell (Fig. 1 ) and the intact heart section (not shown) as well as by immunoblotting of homogenates from isolated cardiac cells and intact hearts (not shown).

View larger version (142K): [in this window] [in a new window]   Figure 1. Cardiac transgenic expression of the human P2X4 receptor. Immunohistochemistry of isolated cardiac cells of transgenic and nontransgenic mice was shown and six nontransgenic mice.

3. Cardiac transgenic expression of the P2X₄ receptor showed the gain of an essential biological function, that of cardiac contractility
Transgenic overexpression of the P2X₄ receptor resulted in an enhanced basal cardiac performance. The P2X₄ receptor mouse exhibited a higher basal level of cardiac output (Fig. 2 A), contractility (+dP/dt), relaxation (-dP/dt) (Fig. 2B ), and left ventricular developed pressure (not shown) than the nontransgenic animal. The enhanced basal cardiac performance in the transgenic mouse is not associated with the development of cardiac hypertrophy or heart failure at < 6 months of observation. Transgenic mice showed a greater increase than nontransgenic animals in +dP/dt (Fig. 2C ) and -dP/dt (not shown) in response to the maximal concentration of 2-meSATP tested (1 µM). The heart weight and heart-to-body weight ratio are similar in the transgenic and nontransgenic mice as are the systolic blood pressure and heart rate obtained in conscious animals (not shown). Hematoxylin and eosin (H&E) staining of the transgenic mouse heart showed no apparent pathological changes and was similar to that obtained in the nontransgenic animal (Fig. 1C ).

View larger version (17K): [in this window] [in a new window]   Figure 2. P2X4 receptor transgenic mouse heart showed increased contractility. The various functional parameters of cardiac performance, such as left ventricular developed pressure, cardiac output, rate of contraction (the first derivative of rate of contraction over time), and rate of relaxation (first derivative of rate of relaxation over time), were quantitated using the working heart model. Data points under each basal condition are summarized as means ± SE from 28 transgenic and 26 nontransgenic animals; statistical differences were analyzed by Student’s t test (unpaired). The basal level of cardiac output (A) was significantly greater, as was the basal level of ± dP/dt (B), in the P2X4 receptor transgenic mouse than in the nontransgenic animal (P<0.001, Student’s t test). C) The increase in +dP/dt in response to 2-meSATP was also greater in TG than in NTG animals (P<0.05, Student’s t test; *significantly different from TG). Data are mean ± SE of seven mice from each group.

The mechanism that underlies the increased basal cardiac function was determined next. The hypothesis is that the endogenous ATP activates the overexpressed P2X₄ receptor and causes an enhanced cardiac contractility in the intact heart. This hypothesis would predict that, with dissipation of the endogenous ATP, cardiac ventricular cells isolated from the P2X₄ receptor transgenic animal would show similar basal contractile function as cells isolated from the nontransgenic heart. The data show this is indeed the case. The basal contractile function was determined by quantitating the amplitude of contraction and normalizing the amplitude to the length of the resting cell. In the chamber in which the contractile amplitude was measured, ATP was not detected in the agonist-free superfusing solution bathing for either nontransgenic (n=8 cells from four mice) or transgenic cardiac myocytes (n=9 cells from three mice). The basal contractile amplitude, expressed as percent of resting cell length, was similar in both transgenic and nontransgenic myocytes (not shown). In the nontransgenic group, the percent stimulation of contractile amplitude in response to 2-meSATP (1 µM) was 40 ± 4.3% (±SE, n=51 cells from 16 mice). The percent of stimulation in the transgenic group was 140 ± 16% (n=37 cardiac cells from 12 mice). Thus, although the basal contractile function is similar in both the transgenic and nontransgenic cardiac cells, the transgenic cells showed a threefold greater increase of myocyte contractility by a 2-meSATP than nontransgenic cells.

CONCLUSIONS AND SIGNIFICANCE

The importance of the P2X₄ receptor in mediating the contractile effect of the P2X agonist 2-meSATP was supported by its expression in the cardiac cell, by the insensitivity of the native myocyte P2 response to inhibition by known P2 antagonists such as suramin or PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, data not shown), and by the relative inability of ,ß-meATP to stimulate a positive inotropic response. The pharmacological features of this native cardiac P2 response are conserved in both rat and mouse cardiac cells and are similar to the features of cloned rat and mouse P2X₄ receptors. If the P2X₄ receptor is important in mediating the positive contractile effect of ATP, one may anticipate that cardiac transgenic overexpression of the receptor should result in a gain of its contractile function in the heart. The data showed that this is indeed the case. Thus, transgenic mice overexpressing the P2X₄ receptor have an elevated basal level of cardiac contractility and performance. All of the cardiac parameters measured, such as left ventricular developed pressure, cardiac output, and the indices of contractility and relaxation (±dP/dt), showed a greater basal level in the transgenic mouse. The transgenic heart also showed a greater increase of cardiac contractility in response to the P2X receptor agonist 2-meSATP than did the nontransgenic heart, consistent with overexpression of a functional P2X₄ receptor with a consequent increase in receptor-mediated cardiac contractility.

The increased basal cardiac contractility in the transgenic mouse is likely due to an enhanced activation of the P2X₄ receptor owing to a combination of receptor overexpression and the presence of endogenous ATP (Fig. 3 ).

View larger version (8K): [in this window] [in a new window]   Figure 3. A working model on the mechanism by which P2X4 receptor activation leads to increased cardiac contractility

The only other P2X receptor subtype that has features in response to P2 agonists and antagonists similar to the native cardiac P2 receptor is the P2X₆ receptor. However, cloned P2X₆ receptor cannot easily be expressed into a functional receptor in some heterologous, including Xenopus oocyte cells. Although mRNA for P2X₆ receptor is detected in the intact heart, whether it is expressed as a functional protein and/or localized on the cardiac cell is unknown. The potential biological function of the P2X₆ receptor remains to be determined. Overall, the present study provides strong evidence for the concept that the P2X₄ receptor plays an important role in mediating the contractile effect of ATP in the heart. Cardiac transgenic expression of the P2X₄ receptor resulted in the gain of an essential biological function, that of an enhanced cardiac performance. The study suggests a novel physiological role of the P2X₄ receptor in the heart, that of stimulating cardiac contractility.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0445fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0445fje

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This Article Full Text (PDF) All Versions of this Article: 15/14/2739 01-0445fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by HU, B. Articles by LIANG, B. T. Search for Related Content PubMed PubMed Citation Articles by HU, B. Articles by LIANG, B. T.