The role of sodium/phosphate (Na+/Pi)-cotransporter in myocardial contraction is gradually coming into focus. This symporter uses the electrochemical gradient of sodium to transport inorganic phosphate into the cardiomyocytes (1,2) and cells of various organs (3-9). Numerous studies in noncardiac tissues (3,10-13) and various cell lines (4,14,15) have shown that this symporter can be activated via the phosphoinositide pathway (PIP) and inhibited through the cyclic adenosine monophosphate (cAMP)-activated mechanism (15,16). Two studies from this laboratory (17,18) have shown that this symporter plays an important role in myocardial contraction. One of these studies (17) showed that a high concentration of Pi (10 mM) produced transient increase in myocardial contractility, whereas lower concentrations (2.5 and 5.0 mM) produced stable and nontransient increases in contractility. The second study (18) showed that Pi (3.5 mM) markedly potentiated myocardial positive inotropic responses to phenylephrine (PHE, and α1-adrenoceptor agonist and an agonist of PIP).
Our study was undertaken to address two questions that were raised by these earlier studies from this laboratory: (a) does Na/Pi-cotransporter have an effect on myocardial contraction that is independent of the α1-adrenoceptor? and (b) would exogenous α1-adrenoceptor agonist potentiate the myocardial biphasic contractile response to a high concentration of Pi? The former objective is of interest because it is not clear from earlier studies from this laboratory (17,18) and other laboratories (19) whether Pi-induced increase in myocardial contractility can be dissociated from the activity of α1-adrenergic receptor. In these studies, the influence of Pi on myocardial contraction was investigated by using the isolated perfused heart (18) or myocardial septum (19) under conditions in which the sympathetic nerve terminals to the myocardium could still be activated by electrical stimulation. In the later study, we tested the hypothesis that an α1-adrenoceptor agonist would potentiate myocardial biphasic contractile response to a high concentration of Pi. If this hypothesis is valid, then we would expect marked potentiation of the transient increase in myocardial contractility by a high concentration of Pi in the presence of an α1-adrenoceptor agonist: this would be followed by marked potentiation of Pi-induced myocardial depression. The interaction of α-adrenoceptor and Na/Pi-cotransporter is important because some conditions, such as ischemia, would simultaneously cause increased sympathetic tone of the heart and increased tissue and extracellular levels of Pi. The 10 mM Pi used in our study is reasonable because this concentration can produce the biphasic contractile response; second, plasma Pi levels of 10 mM have been reported in severe cases of hyperphos-phatemia (20,21).
Isolated heart preparation
The investigation conformed with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1985). Male Sprague-Dawley rats weighing 285-385 g were anesthetized intraperitoneally (i.p.) with sodium pentobarbital (30 mg/kg) and heparinized (500 U/kg i.p.). The heart of each rat was removed and perfused (Langendorff preparation) at 37°C by using a modified Krebs-Henseleit buffer at a constant flow rate of 8 ml/min. This buffer was equilibrated with 95% O2/5% CO2 at pH 7.3. Accumulation of fluid in the left ventricle was minimized or prevented by placing a polyethylene catheter (PE 50) in the ventricle via an apical stab wound. A latex balloon-tipped catheter was placed in the left ventricle via the left atrium so that left ventricular pressure was measured by using a pressure transducer connected to a physiograph. Through a sidearm of this catheter, water was introduced or withdrawn from the balloon to control the preload (left ventricular diastolic pressure), which was set and maintained at 15 mm Hg throughout each experiment. Next the right atrium was excised, and the heart was paced at a rate of 280 beats/min by using a voltage with an intensity 20% above threshold and a duration of 2 ms.
Buffer and general experimental protocol
Each perfusion buffer (with and without phosphate) had the same concentration of calcium (1.20 mM) and the same calcium activity (0.72 mM). Calcium activity in each medium was controlled by addition of ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA), and measurement with calcium electrode and an ion analyzer (Ion Analyzer 250, Corning, NY, U.S.A.). Appropriate amount of sodium chloride was added to each buffer so that sodium ion concentration was the same in the presence and absence of Pi(17). Perfusion of the isolated heart at a constant rate of 8 ml/min was initiated with the Pi-free buffer, and the heart was allowed to equilibrate in the medium for 30 min before initiation of treatment. Control values for the left ventricular pressure (LVP); myocardial contractility (maximal rate of LVP increase, +dP/dt); and maximal rate of LVP decay (−dP/dt) were taken at the end of equilibration. These +dP/dt and −dP/dt values were obtained from the electronically differentiated value of the LVP signal.
α1-Adrenoceptor-independent effect of Na+/Pi-cotransporter in myocardial contraction
The α1-adrenoceptor-independent influence of Na+/Pi-cotransporter on myocardial contraction was investigated with α1-adrenoceptor blocked with prazosin. Prazosin (7.87 × 10−5M) was infused at a rate of 0.206 ml/min when the heart was perfused with a medium that contained 5 mM Pi. This infusion rate would produce a prazosin concentration of 2 μM if uniform mixing were assumed.
Effect of high concentration of Pi on myocardial contractile responses to PHE
The buffer for this study contained 1 μM propranolol, a β-adrenergic antagonist. This was used to remove or minimize the cAMP-stimulating influence of norepinephrine, which may arise from the electrical stimulation of the sympathetic nerve terminals to the heart. Numerous studies in noncardiac cells have shown that the Na+/Pi-cotransporter can be inhibited through the cAMP/protein kinase A pathway (5,15,19); it has not been shown whether cAMP can similarly inhibit cardiac Na+/Pi-cotransporter. The heart was perfused for 60 min in the presence or absence of phenylephrine (1.0 μM) by using buffer that contained 0, 2.5, 5.0, or 10 mM Pi.
Values are expressed as mean ± SEM. Data were analyzed by analysis of variance (one-way ANOVA or factorial ANOVA); a p value < 0.05 was considered significant.
Role of α1-adrenoceptor in myocardial inotropic response to Pi
Figure 1 presents data from the test of the hypothesis that Pi can give rise to increases in myocardial contractility independent of α1-adrenoceptors. The time-control group (open circle) showed no significant change in left ventricular developed pressure (LVDP) value during the 30-min study in which both the β- and α1-adrenoceptors were blocked. The treated group showed a significant increase in LVDP when 5 mM Pi was introduced (first arrow from the left). No change in LVDP value was noted when 1 μM PHE was added to verify that α1-adrenoceptor was blocked (second arrow). Similar responses were noted for +dP/dt and −dP/dt. Therefore the data support the contention that Pi has a positive isotropic effect independent of α1-adrenoceptor.
Effect of Pi on PHE-activated changes in myocardial contraction when the β-adrenoceptor is blocked with propranolol
The effect of Pi on PHE-activated changes in myocardial contraction in propranolol-containing medium is shown in Fig. 2. When the heart was perfused with Pi-free medium in the absence of PHE, no change in LVDP, +dP/dt, and −dP/dt was noted for the 60-min time-control study. When the heart was perfused with Pi-containing medium in the absence of PHE, Pi produced a dose-dependent increase in LVDP. Similar responses were noted for +dP/dt and −dP/dt. In the presence of PHE, marked increases in LVDP occurred for various concentrations of Pi. Potentiation of this increased LVDP, however, was noted only for 2.5 and 5.0 mM Pi: 10 mM Pi had no effect on myocardial inotropic response to PHE (i.e., 10 mM Pi had the same effect on LVDP as 0 mM Pi). The same type of response to PHE was noted for +dP/dt and −dP/dt. Therefore the data showed that Pi does not potentiate myocardial inotropic response to PHE in a dose-dependent manner. These data do not support the hypothesis that PHE will potentiate transient cardiac positive inotropic responses to a high concentration of Pi. They also suggest that β-adrenoceptor may contribute to myocardial depression by high concentrations of Pi.
Effect of Pi on myocardial contraction when both the β- and α-adrenoceptors are activated
We carried out another study in which both the β- and α-adrenoceptors were activated to see whether myocardial depression by 10 mM Pi would be potentiated by adrenergic stimulation. When the basal tone of the β-adrenoceptor was maintained (absence of propranolol), Pi-PHE interaction did not produce a remarkable effect. Pi-PHE interaction resulted in the depression of the initial transient increase in dP/dt (Fig. 3 from this study compared with data from our previous study; 17). Because the previous study under the basal tone of the β-adrenoceptor did not produce a definitive result, we repeated the study in the presence of norepinephrine, a condition that would simultaneously increase both the α- and the β-adrenoceptor tones. The data (Fig. 4) showed that 10 mM Pi produced a marked and precipitate decline in LVDP in the presence of norepinephrine (1 × 10−7M). This rapid decline in LVDP reflects marked potentiation of Pi-induced myocardial depression when the present data are compared with our earlier data (16), which was obtained under similar experimental conditions in the absence of adrenergic agents. The same type of response was noted for +dP/dt and −dP/dt; therefore the β-adrenoceptor plays a role in Pi-induced myocardial depression.
Prevention of the myocardial depression induced by the interaction between 10 mM Pi and norepinephrine
Another study was performed to determine whether phosphonoformate (PFA), a selective inhibitor of Na+/Pi-cotransporter, can reverse the myocardial depression from the combined effects of norepinephrine and Pi. A saline solution of PFA (43 mM) was infused into the perfusion line 3 cm above the heart throughout the entire experiment (60 min) at a rate of 0.494 ml/min, a rate that would produce 2.5 mM perfusion PFA concentration if uniform mixing is assumed. The cardiodepressive interaction between Pi and norepinephrine was abolished with the PFA treatment (Fig. 5).
Effect of α1-adrenoceptor on myocardial depression induced by Pi-norepinephrine interaction
We carried out another study to determine whether the induction of myocardial depression by Pi-norepinephrine interaction would still occur if the α1-adrenoceptor were blocked. The data (Fig. 6) show that 10 mM Pi and norepinephrine (1 × 10−7M) did not interact to produce myocardial depression. Therefore the data show that norepinephrine does not potentiate Pi-induced myocardial depression when the α1-adrenoceptor is blocked.
Phenylephrine-activated increase in myocardial contractility was potentiated by normal or near-normal physiologic levels of extracellular Pi; no change in the PHE-induced contractile response was noted in the presence of 10 mM Pi and a β-adrenoceptor blocker. Our results further showed that the α1-agonist does not potentiate Pi-induced transient increase in myocardial contractility by 10 mM Pi, but myocardial depression is accelerated by the simultaneous activation of the β- and α-adrenoceptors (by norepinephrine) in the presence of 10 mM Pi. This cardiodepressive interaction between 10 mM Pi and norepinephrine was inhibited by either the selective blocker of Na+/Pi-cotransporter (PFA), propranolol, or prazosin. These results portray a very complex role for Na+/Pi-cotransporter in modulating myocardial contraction (i.e., time, Pi concentration, and the intensity of the tone of the α- and the β-adrenoceptors are important determinants of myocardial contraction mediated through this symporter). Our data are consistent with the conclusion that an increase in myocardial contractility is the predominant effect of Pi-activated changes in myocardial contraction at normal or near-normal plasma Pi levels, whereas the cardiodepressive effect of Pi presumably overrides this stimulatory effect at high Pi concentrations.
The mechanisms by which Pi influences myocardial contraction are poorly understood. Some of the suggested or implied mechanisms for this influence have been discussed elsewhere (17,18), and they may be divided into three basic categories: (a) influence of Pi on myofibrils to sensitize (22-25) or desensitize (26-28) them to calcium; (b) Pi-associated increase in the level of intracellular free calcium (2,29); and (c) influence of Pi on energy metabolism, including Pi-associated impairment of mitochondrial metabolic activity (19,30). Myocardial contractile responses to Pi would reflect the interactions and net balances among these mechanisms.
It is well established that an increase in the activity of the β-adrenoceptor would cause an increase in the influx of calcium. Second, Na+/Pi-cotransporter-mediated increase in Pi influx has been reported in the presence of α1-agonists in noncardiac cells (4,14,15). Therefore it is likely that the deleterious effects of Pi on myocardial contractile activity in our study may have been exacerbated by conditions that simultaneously enhance the activity of Na+/Pi-cotransporter and cAMP-activated increase in calcium influx. This may hold significant implications for ischemia-associated myocardial depression. In ischemia the sympathetic tone of the heart is increased, and the levels of catecholamines in the heart can be increased (31). If cAMP is a weak inhibitor of the cardiac isoform of the Na+/Pi-cotransporter or if the α1-adrenoceptor activation of this symporter can readily override the inhibitory influence of cAMP, then Na+/Pi-cotransporter may contribute significantly to increased intracellular levels of Pi and calcium in the ischemic heart. Another factor in ischemia that may influence the Na+/Pi-cotransporter-associated Pi and calcium overload is acidosis. It has been shown that acidosis stimulates the activity of cardiac Na+/Pi-cotransporter (2), leading to increased Pi transport into the cardiomyocytes. It has also been shown that acidosis potentiates Pi-induced inhibition of Na+/K-ATPase (32), an effect that could result in the accumulation of intracellular calcium via the Na/Ca-exchanger (33-35). More studies are needed to explain the interaction between the adrenoceptors and Na+/Pi-cotransporter in ion homeostasis of the ischemic heart.
Pi can produce a positive inotropic effect independent of the α1-adrenergic receptor: on the other hand, Pi can potentiate myocardial contractile responses to PHE and interact with norepinephrine to produce myocardial depression. It is surprising that the interaction between norepinephrine and Pi would result in myocardial depression. Because various isoforms of Na+/Pi-cotransporter can be activated via the phosphoinositide pathway and inhibited through the cAMP/protein kinase A axis, we would expect the inhibitory tone (cAMP effect) of norepinephrine to be the predominant tone on this symporter so that ion overload and myocardial depression would be avoided. If the α1-adrenoceptor tone is the predominant tone of norepinephrine on this symporter, as our data suggest, then this symporter may be the heart's Achilles heel for ischemiainduced cardiomyopathy and contractile deterioration in heart failure. More studies are needed further to explain the biochemical mechanisms for Pi-induced changes in myocardial contraction. Our data support and strengthen the conclusion that Na+/Pi-cotransporter plays an important role in myocardial contraction.
Acknowledgment: We thank Drs. A. Askari and D. Wilkerson for their helpful suggestions during the course of this study. This study was supported by National Institutes of Health grant HL-36573, awarded by National Heart, Lung and Blood Institute, United States Public Health Service/Department of Health and Human Services.
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